本申请要求2020年12月22日提交的美国临时申请No.63/129,395；2021年2月3日提交的PCT申请No.PCT/EP2021/052455；2021年7月14日提交的PCT申请No.PCT/EP2021/0699626和2021年7月14日提交的PCT申请No.PCT/EP2021/0069632的优先权。上述每项申请均通过引用并入本文。This application claims priority to U.S. Provisional Application No. 63/129,395, filed December 22, 2020; PCT Application No. PCT/EP2021/052455, filed February 3, 2021; PCT Application No. PCT/EP2021/0699626, filed July 14, 2021, and PCT Application No. PCT/EP2021/0069632, filed July 14, 2021. Each of the above applications is incorporated herein by reference.
背景技术Background Art
本发明特别涉及适用于治疗或预防SARS-CoV-2变体的感染或与这类感染相关的障碍的RNA，所述变体包括但不限于：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。本发明还涉及组合物、多肽和疫苗。组合物和疫苗优选包含至少一种所述RNA序列，优选与脂质纳米颗粒(LNP)结合的RNA。The present invention particularly relates to RNA suitable for treating or preventing infection by SARS-CoV-2 variants or disorders associated with such infection, including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia). The present invention also relates to compositions, polypeptides and vaccines. Compositions and vaccines preferably contain at least one of the RNA sequences, preferably RNA bound to lipid nanoparticles (LNPs).
列表List
本发明还涉及RNA、组合物、疫苗和试剂盒的第一和第二医药用途，以及涉及治疗或预防由SARS-Cov-2变体引起的SARS-CoV-2感染的方法，所述变体包括但不限于：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。The present invention also relates to first and second medical uses of RNA, compositions, vaccines and kits, as well as methods for treating or preventing SARS-CoV-2 infection caused by SARS-Cov-2 variants, including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
冠状病毒是冠状病毒科的高度接触传染性的、包膜的、正单链人畜共患的RNA病毒。Coronaviruses are highly contagious, enveloped, positive-single-stranded zoonotic RNA viruses of the Coronaviridae family.
冠状病毒在遗传上高度可变，且单个病毒物种有可能通过克服物种屏障而感染几个宿主物种。一种新型冠状病毒被命名为“严重急性呼吸综合征冠状病毒2”(SARS-CoV-2)。SARS-CoV-2引起的病毒感染(也称为COVID-19病)的典型症状包括发烧、咳嗽、气短和肺炎，且老年人群的死亡率高。2020年3月，WHO宣布SARS-CoV-2爆发为大流行。此外，一些个体在感染后数周至数月内会受到COVID-19感染的影响。这一群体被称为“长Covid”。随着时间的推移，常见的体征和症状包括：疲劳、气短或呼吸困难、咳嗽、关节疼痛、胸痛、记忆力、注意力或睡眠问题、肌肉疼痛或头痛、心跳加快或剧烈跳动、嗅觉或味觉丧失、抑郁或焦虑、发烧、站立时头晕、身体或精神活动后症状恶化。Coronaviruses are genetically highly variable, and a single virus species has the potential to infect several host species by overcoming species barriers. A new coronavirus has been named "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2). Typical symptoms of viral infection caused by SARS-CoV-2 (also known as COVID-19 disease) include fever, cough, shortness of breath, and pneumonia, with a high mortality rate in the elderly population. In March 2020, the WHO declared the SARS-CoV-2 outbreak a pandemic. In addition, some individuals are affected by COVID-19 infection weeks to months after infection. This group is called "long Covid". Common signs and symptoms over time include: fatigue, shortness of breath or difficulty breathing, cough, joint pain, chest pain, memory, concentration or sleeping problems, muscle pain or headache, fast or pounding heartbeat, loss of smell or taste, depression or anxiety, fever, dizziness when standing, and worsening of symptoms after physical or mental activity.
在大流行期间，出现了新的SARS-CoV-2变异株，这些变异株通常比原始SARS-CoV-2株更具传染性或致病性。这种新出现的SARS-CoV-2株可能会导致针对原始SARS-CoV-2株开发的第一代疫苗的效率降低。此外，尚不清楚在接种了针对原始SARS-CoV-2株疫苗的受试者中，使用专门针对新出现的SARS-CoV-2株设计的疫苗加强接种是否会导致针对新出现的SARS-CoV-2株的保护性免疫反应。During the pandemic, new SARS-CoV-2 variants have emerged that are often more transmissible or pathogenic than the original SARS-CoV-2 strain. Such emerging SARS-CoV-2 strains may render first-generation vaccines developed against the original SARS-CoV-2 strain less effective. In addition, it is unclear whether boosting vaccination with a vaccine designed specifically for an emerging SARS-CoV-2 strain will result in a protective immune response against the emerging SARS-CoV-2 strain in subjects vaccinated against the original SARS-CoV-2 strain.
发明内容Summary of the invention
因此，本发明的一个目的是提供针对SARS-CoV-2感染的基于RNA的疫苗，特别是由新出现的SARS-CoV-2变异株引起的SARS-CoV-2感染。这种新出现的株包括但不限于：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。基于RNA的疫苗接种是最有前景的用于针对新出现的SARS-CoV-2病毒的新疫苗的技术之一。RNA可以通过基因工程改造并适应新出现的SARS-CoV-2株，并施用于人类受试者，其中转染的细胞直接产生由RNA提供的编码的抗原，其产生免疫反应。Therefore, it is an object of the present invention to provide RNA-based vaccines against SARS-CoV-2 infection, in particular SARS-CoV-2 infection caused by emerging SARS-CoV-2 variants. Such emerging strains include, but are not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta RNA-based vaccination is one of the most promising technologies for new vaccines against the emerging SARS-CoV-2 virus. The RNA can be genetically engineered and adapted to newly emerging strains of SARS-CoV-2 and administered to human subjects, where the transfected cells directly produce the encoded antigens provided by the RNA, which generates an immune response.
如权利要求和以下说明书中进一步限定的，特别是通过提供包含至少一个编码序列的RNA实现了这些目的，所述编码序列编码至少一种源自SARS-CoV-2的抗原肽或蛋白质，例如，包含至少一个源自SARS-CoV-2株的突变，该SARS-CoV-2株包括但不限于：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。As further defined in the claims and the following description, these objects are achieved, in particular, by providing an RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from SARS-CoV-2, for example, comprising at least one mutation derived from a SARS-CoV-2 strain, the SARS-CoV-2 strain including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在本发明的优选实施方式中，RNA和基于RNA的疫苗包含编码至少一种源自SARS-CoV-2刺突蛋白的抗原肽的RNA，例如，包含源自包括但不限于以下的SARS-CoV-2株的刺突蛋白：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(DeltaPlus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a preferred embodiment of the present invention, RNA and RNA-based vaccines comprise RNA encoding at least one antigenic peptide derived from the SARS-CoV-2 spike protein, for example, comprising spike proteins derived from SARS-CoV-2 strains including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.5 (India), and AY.6 (India). DeltaPlus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示了接种均在LNP中配制的mRNA疫苗CV2CoV和CV2CoV.351的组在第14天(图1A-D)和第21天(图1E-H)对祖先SARS-CoV-2的受体结合结构域(RBD)和B.1.351变体的RBD的显著IgG1和IgG2a结合抗体反应。在第14天，图1A显示了对于所有组的相当IgG1反应(祖先SARS-CoV-2受体结合结构域(RBD)蛋白衣壳)和图1B显示了对于所有疫苗接种设计的相当IgG2a滴度(祖先RBD蛋白衣壳)。在第14天，图1C显示了对于所有疫苗接种设计的相当IgG1反应(RBD B.1.351变体K417N、E484K、N501Y蛋白衣壳)和图1D显示了对于所有接种设计的相当IgG2a滴度(RBD B.1351变体K417N、E484K、N501Y蛋白衣壳)。在第21天，图1E显示了对于所有疫苗接种设计的相当IgG1反应(祖先SARS-CoV-2受体结合结构域(RBD)蛋白衣壳)和图1F显示了对于所有接种设计的相当IgG2a滴度(祖先SARS-CoV-2受体结合结构域(RBD)蛋白衣壳)。在第21天，图1G显示了对于所有疫苗接种设计的相当IgG1反应(RBDB.1.351变体K417N、E484K、N501Y蛋白衣壳)和图1H显示了对于所有疫苗接种设计的相当IgG2a滴度(RBD B.1351变体K417N、E484K、N501Y蛋白衣壳)。Figure 1 shows the significant IgG1 and IgG2a binding antibody responses of the groups vaccinated with mRNA vaccines CV2CoV and CV2CoV.351, both formulated in LNP, on the 14th day (Figure 1A-D) and the 21st day (Figure 1E-H) to the receptor binding domain (RBD) of the ancestral SARS-CoV-2 and the RBD of the B.1.351 variant. On the 14th day, Figure 1A shows the equivalent IgG1 response (ancestral SARS-CoV-2 receptor binding domain (RBD) protein capsid) for all groups and Figure 1B shows the equivalent IgG2a titer (ancestral RBD protein capsid) for all vaccination designs. On the 14th day, Figure 1C shows the equivalent IgG1 response (RBD B.1.351 variant K417N, E484K, N501Y protein capsid) for all vaccination designs and Figure 1D shows the equivalent IgG2a titer (RBD B.1351 variant K417N, E484K, N501Y protein capsid) for all vaccination designs. At day 21, Figure 1E shows the equivalent IgG1 response for all vaccination designs (ancestral SARS-CoV-2 receptor binding domain (RBD) protein capsid) and Figure 1F shows the equivalent IgG2a titers for all vaccination designs (ancestral SARS-CoV-2 receptor binding domain (RBD) protein capsid). At day 21, Figure 1G shows the equivalent IgG1 response for all vaccination designs (RBDB.1.351 variant K417N, E484K, N501Y protein capsid) and Figure 1H shows the equivalent IgG2a titers for all vaccination designs (RBD B.1351 variant K417N, E484K, N501Y protein capsid).
图2显示了使用祖先SARS-CoV-2在基于细胞病变效应(CPE)的测定中评估的病毒中和滴度(VNT)的显著诱导。第14天的图2A和第21天的图2B显示了对于所有组B-H的VNT的增加。将mRNA疫苗CV2CoV和CV2CoV.351共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第14天和第21天产生针对两种疫苗变体的反应。第42天的图2C显示了对于所有组(B-H组)的VNT水平的增加。将两种疫苗变体共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第42天产生针对两种变体的反应。Fig. 2 shows the significant induction of virus neutralization titer (VNT) evaluated in the assay based on cytopathic effect (CPE) using ancestral SARS-CoV-2. Fig. 2A on the 14th day and Fig. 2B on the 21st day show the increase of VNT for all groups B-H. The mRNA vaccine CV2CoV and CV2CoV.351 are co-delivered to the same leg (Group F and Group G) or different legs (Group H) and can produce the reaction for two vaccine variants at the 14th and 21st days. Fig. 2C on the 42nd day shows the increase of VNT level for all groups (Group B-H). The two vaccine variants are co-delivered to the same leg (Group F and Group G) or different legs (Group H) and can produce the reaction for two variants at the 42nd day.
图3显示了使用B.1.351变体SARS-CoV-2在基于CPE的测定中评估的VNT的显著诱导。第14天的图3A和第21天的图3B显示了对于所有组B-H的VNT的增加。将mRNA疫苗CV2CoV和CV2CoV.351共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第14天和第21天产生针对两种疫苗变体的反应。第42天的图3C显示了对于所有组(B-H组)的VNT水平的增加。将两种疫苗变体共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第42天产生针对两种变体的反应。Fig. 3 shows the significant induction of VNT evaluated in CPE-based assays using B.1.351 variant SARS-CoV-2. Fig. 3A on the 14th day and Fig. 3B on the 21st day show the increase of VNT for all groups B-H. Co-delivery of mRNA vaccines CV2CoV and CV2CoV.351 to the same leg (Group F and Group G) or different legs (Group H) can produce responses for both vaccine variants on the 14th and 21st days. Fig. 3C on the 42nd day shows the increase of VNT levels for all groups (Group B-H). Co-delivery of two vaccine variants to the same leg (Group F and Group G) or different legs (Group H) can produce responses for both variants on the 42nd day.
图4显示了使用B.1.1.7变体SARS-CoV-2(图4A)或P.1(B.1.1.28)(图4B)在基于CPE的测定中评估的VNT的显著诱导。第42天的图4A显示了对于所有组(B-H组)使用B.1.1.7变体的VNT水平的增加。将两种疫苗变体共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第42天产生针对两种变体的反应。第42天的图4B显示了对于所有组(B-H组)使用B.1.1.28P.1变体的VNT水平的增加。将两种疫苗变体共同递送到同一腿(F组和G组)或不同的腿(H组)中可以在第42天产生针对两种变体的反应。Figure 4 shows the significant induction of VNTs evaluated in CPE-based assays using B.1.1.7 variant SARS-CoV-2 (Figure 4A) or P.1 (B.1.1.28) (Figure 4B). Figure 4A on the 42nd day shows the increase in VNT levels for all groups (B-H groups) using the B.1.1.7 variant. Co-delivery of the two vaccine variants to the same leg (F and G groups) or different legs (H group) can produce a response to the two variants at day 42. Figure 4B on the 42nd day shows the increase in VNT levels for all groups (B-H groups) using the B.1.1.28P.1 variant. Co-delivery of the two vaccine variants to the same leg (F and G groups) or different legs (H group) can produce a response to the two variants at day 42.
图5显示了疫苗接种CV2CoV和CV2CoV.351的组在第14天(图5A-D)和第21天(图5E-H)的显著IgG1和IgG2a结合抗体反应。第14天，图5A(IgG1滴度)和5B(IgG2a滴度)显示了使用0.5μg、2μg、8μg和40μg剂量的结合抗体滴度的剂量依赖性水平，并且在用40μg疫苗接种的组(祖先SARS-CoV-2受体结合结构域(RBD)蛋白衣壳)中达到饱和。第14天，图5C(IgG1滴度)和5D(IgG2a滴度)显示了使用0.5μg、2μg、8μg和40μg剂量的结合抗体滴度的剂量依赖性水平，并且在用40μg疫苗接种的组(RBD B.1.351变体K417N、E484K、N501Y蛋白衣壳)中达到饱和。第21天，图5E(IgG1滴度)和5F(IgG2a滴度)显示了对于CV2CoV.351疫苗接种的所有量的结合抗体滴度的剂量依赖性水平及饱和的IgG1滴度和IgG2a滴度，以及对于CV2CoV(祖先SARS-CoV-2受体结合结构域(RBD)蛋白衣壳)的>8μg量的饱和(I组)。第21天，图5G(IgG1滴度)和5H(IgG2a滴度)显示了在用CV2CoV.351疫苗接种时对于所有量的结合抗体滴度的剂量依赖性水平及饱和的IgG1和IgG2a滴度，并且对于CV2CoV(RBD B.1.351变体K417N、E484K、N501Y蛋白衣壳)的mRNA疫苗的>8μG量的饱和(I组)。Figure 5 shows the significant IgG1 and IgG2a binding antibody responses of the groups vaccinated with CV2CoV and CV2CoV.351 on day 14 (Figures 5A-D) and day 21 (Figures 5E-H). On day 14, Figures 5A (IgG1 titer) and 5B (IgG2a titer) show the dose-dependent levels of binding antibody titers using 0.5μg, 2μg, 8μg, and 40μg doses, and saturation is reached in the group vaccinated with 40μg (ancestral SARS-CoV-2 receptor binding domain (RBD) protein capsid). On day 14, Figures 5C (IgG1 titer) and 5D (IgG2a titer) show the dose-dependent levels of binding antibody titers using 0.5μg, 2μg, 8μg, and 40μg doses, and saturation is reached in the group vaccinated with 40μg (RBD B.1.351 variants K417N, E484K, N501Y protein capsid). On day 21, Figures 5E (IgG1 titer) and 5F (IgG2a titer) show dose-dependent levels of binding antibody titers for all amounts of CV2CoV.351 vaccination and saturated IgG1 titers and IgG2a titers, as well as saturation (Group I) of >8 μg for CV2CoV (ancestral SARS-CoV-2 receptor binding domain (RBD) protein capsid). On day 21, Figures 5G (IgG1 titer) and 5H (IgG2a titer) show dose-dependent levels of binding antibody titers for all amounts of CV2CoV.351 vaccination and saturated IgG1 and IgG2a titers, and saturation (Group I) of >8 μG for mRNA vaccines of CV2CoV (RBD B.1.351 variants K417N, E484K, N501Y protein capsid).
图6显示了在第14天、第21天和第42天使用祖先SARS-CoV-2(CV2CoV，图6A、6C和6E)或B.1.351变体SARS-CoV-2(CV2CoV.351，图6B、6D和6F)在基于CPE的测定中评估的VNT的显著诱导。图6还显示了在第42天使用B.1.1.7变体SARS-CoV-2(图6G)或B.1.1.28P.1变体(图6H)在基于CPE中评估的VNT的显著诱导。图6A显示了在所有剂量组中，B.1.351变体疫苗CV2CoV.351在第14天诱导针对祖先SARS-CoV-2的剂量依赖性VNT(异源反应)。与CV2CoV疫苗接种后的反应(同源反应)相比，CV2CoV.351接种组中的VNT在第14天降低了约2倍。图6B显示了在所有剂量组中，CV2CoV.351在第14天诱导针对B.1.351SARS-CoV-2的剂量依赖性VNT(同源反应)。CV2CoV.351疫苗接种引发了针对同源病毒的高水平VNT，其与针对祖先病毒的异源VNT相比，在第14天增加了45x(所有剂量组的平均差异)。与用CV2CoV疫苗接种相比，CV2CoV.351诱导的VNT在第14天增加了41倍(所有剂量组的平均差异)。图6C显示了在所有剂量组中，B.1.351变体疫苗CV2CoV.351在第21天诱导针对祖先SARS-CoV-2的剂量依赖性VNT(异源反应)。与疫苗接种CV2CoV后的反应(同源反应)相比，CV2CoV.351接种组的VNT在第21天降低了约2倍。图6D显示了在所有剂量组中CV2CoV.351在第21天诱导针对B.1.351SARS-CoV-2的轻微剂量依赖性VNT(同源反应)。CV2CoV.351疫苗接种引发了针对同源病毒的高水平VNT，其与针对祖先病毒的异源VNT相比，在第21天增加了35倍(所有剂量组的平均差异)。与CV2CoV疫苗接种相比，CV2CoV.351诱导的VNT在第21天增加了42倍(所有剂量组的平均差异)。图6E显示了在所有剂量组中，B.1.351变体疫苗CV2CoV.351在第42天诱导了针对祖先SARS-CoV-2的剂量依赖性VNT(异源反应)。CV2CoV疫苗接种后，除所有0.5μg疫苗接种(F组)外，显示反应略高(同源反应)。图6F显示了在所有剂量组中CV2CoV.351在第42天诱导针对B.1.351 SARS-CoV-2的剂量依赖性VNT(同源反应)。与CV2CoV疫苗接种相比，CV2CoV.351诱导的VNT在第42天增加。图6G显示了在所有剂量组中，B.1.351变体疫苗CV2CoV.351在第42天诱导针对B.1.1.7变体SARS-CoV-2的剂量依赖性VNT(异源反应)。除了0.5μg疫苗接种(F组)外，H组在用CV2CoV疫苗接种后显示出类似的反应(同源反应)。图6H显示了在所有剂量组中，CV2CoV.351在第42天诱导针对B.1.1.28P.1变体SARS-CoV-2的剂量依赖性VNT(同源反应)。接种CV2CoV后观察至较低反应(同源反应)。Figure 6 shows the significant induction of VNTs assessed in a CPE-based assay using ancestral SARS-CoV-2 (CV2CoV, Figures 6A, 6C, and 6E) or B.1.351 variant SARS-CoV-2 (CV2CoV.351, Figures 6B, 6D, and 6F) on days 14, 21, and 42. Figure 6 also shows the significant induction of VNTs assessed in a CPE-based assay using B.1.1.7 variant SARS-CoV-2 (Figure 6G) or B.1.1.28P.1 variant (Figure 6H) on day 42. Figure 6A shows that in all dose groups, the B.1.351 variant vaccine CV2CoV.351 induced dose-dependent VNTs (heterologous responses) against ancestral SARS-CoV-2 on day 14. Compared to the response after CV2CoV vaccination (homologous response), VNTs in the CV2CoV.351-vaccinated group were reduced by about 2-fold on day 14. Figure 6B shows that in all dose groups, CV2CoV.351 induced dose-dependent VNTs (homologous responses) against B.1.351 SARS-CoV-2 on day 14. CV2CoV.351 vaccination triggered high levels of VNTs against homologous viruses, which increased by 45x on day 14 compared to heterologous VNTs against the ancestral virus (mean difference across all dose groups). CV2CoV.351-induced VNTs increased 41-fold on day 14 compared to vaccination with CV2CoV (mean difference across all dose groups). Figure 6C shows that in all dose groups, the B.1.351 variant vaccine CV2CoV.351 induced dose-dependent VNTs (heterologous responses) against the ancestral SARS-CoV-2 on day 21. Compared to the response after vaccination with CV2CoV (homologous response), the VNTs of the CV2CoV.351-vaccinated group were reduced by about 2-fold on day 21. Figure 6D shows that CV2CoV.351 induced a slight dose-dependent VNT (homologous response) against B.1.351 SARS-CoV-2 on day 21 in all dose groups. CV2CoV.351 vaccination triggered high levels of VNT against the homologous virus, which increased 35-fold on day 21 compared to heterologous VNT against the ancestral virus (average difference across all dose groups). CV2CoV.351-induced VNT increased 42-fold on day 21 compared to CV2CoV vaccination (average difference across all dose groups). Figure 6E shows that in all dose groups, the B.1.351 variant vaccine CV2CoV.351 induced a dose-dependent VNT (heterologous response) against the ancestral SARS-CoV-2 on day 42. After CV2CoV vaccination, all but one 0.5μg vaccination (Group F) showed a slightly higher response (homologous response). Figure 6F shows that CV2CoV.351 induced dose-dependent VNT (homologous response) against B.1.351 SARS-CoV-2 on day 42 in all dose groups. Compared with CV2CoV vaccination, CV2CoV.351-induced VNT increased on day 42. Figure 6G shows that in all dose groups, the B.1.351 variant vaccine CV2CoV.351 induced dose-dependent VNT (heterologous response) against B.1.1.7 variant SARS-CoV-2 on day 42. In addition to 0.5μg vaccination (Group F), Group H showed a similar response (homologous response) after vaccination with CV2CoV. Figure 6H shows that in all dose groups, CV2CoV.351 induced dose-dependent VNT (homologous response) against B.1.1.28P.1 variant SARS-CoV-2 on day 42. Lower responses (homologous responses) were observed after vaccination with CV2CoV.
图7显示了对于在LNP中配制的二价mRNA疫苗组合物CV2CoV+CV2CoV.351接种的组，在第14天显示出显著的IgG1和IgG2a结合抗体反应(图7A-D)。第14天，图7A(IgG1滴度)和7B(IgG2a滴度)显示了使用0.5μg、2μg和8μg剂量(SARS-CoV-2祖先受体结合结构域(RBD)蛋白衣壳)的结合抗体滴度的剂量依赖性水平。第14天，图7C(IgG1滴度)和7D(IgG2a滴度)显示了使用0.5μg、2μg和8μg剂量(B.1.351RBD变体K417N、E484K、N501Y蛋白衣壳)的结合抗体滴度的剂量依赖性水平。使用祖先SARS-CoV-2(图7E、7F和7I)或B.1.351变体SARS-CoV-2(图7G、7H和7J)分别在第14天、第21天和第42天显示了在基于CPE的测定中评估的VNT的显著诱导。图7K和7L还显示了在第42天使用1.1.7变体SARS-CoV-2(图7K)或B.1.1.28P.1变体(图7L)在基于CPE的测定中评估的VNT的显著诱导。Figure 7 shows that for the bivalent mRNA vaccine composition CV2CoV + CV2CoV.351 vaccinated group formulated in LNP, significant IgG1 and IgG2a binding antibody responses were shown on day 14 (Figures 7A-D). On day 14, Figures 7A (IgG1 titer) and 7B (IgG2a titer) show the dose-dependent levels of binding antibody titers using 0.5μg, 2μg, and 8μg doses (SARS-CoV-2 ancestral receptor binding domain (RBD) protein capsid). On day 14, Figures 7C (IgG1 titer) and 7D (IgG2a titer) show the dose-dependent levels of binding antibody titers using 0.5μg, 2μg, and 8μg doses (B.1.351RBD variants K417N, E484K, N501Y protein capsid). Significant induction of VNTs assessed in a CPE-based assay was shown using either ancestral SARS-CoV-2 (Figures 7E, 7F, and 7I) or B.1.351 variant SARS-CoV-2 (Figures 7G, 7H, and 7J) at days 14, 21, and 42, respectively. Figures 7K and 7L also show significant induction of VNTs assessed in a CPE-based assay using either 1.1.7 variant SARS-CoV-2 (Figure 7K) or B.1.1.28P.1 variant (Figure 7L) at day 42.
图8显示了使用祖先SARS-CoV-2(图8A)或使用B.1.351变体SARS-CoV-2(图8B)在基于CPE的测定中评估的VNT。用CV2CoV或B.1.351变体疫苗CV2CoV.351加强显示出针对祖先SARS-CoV-2和B.1.351变体SARS-CoV-2的同源和异源反应的强大的增强能力。B、D和F组的同源反应如图8A所示，C、E和G组的同源反应如图8B所示。C、E和G组的异源反应如图8A所示，B、D和F组的异源反应如图8B所示。在第119天加强后(图8C-8F)14天测试了针对祖先SARS-CoV-2以及针对SARS-CoV-2 B.1.1.7(alpha)、B.1.351(beta)和P.1(gamma)变体的病毒中和反应(针对祖先SARS-CoV-2(图8C)、针对SARS-CoV-2 B.1.351(图8D)、SARS-CoV-2B.1.1.7(图8E)和针对P.1(图8F)的VNT)。Figure 8 shows VNTs evaluated in a CPE-based assay using either ancestral SARS-CoV-2 (Figure 8A) or using B.1.351 variant SARS-CoV-2 (Figure 8B). Boosting with CV2CoV or the B.1.351 variant vaccine CV2CoV.351 showed strong enhancement of homologous and heterologous responses against ancestral SARS-CoV-2 and B.1.351 variant SARS-CoV-2. Homologous responses of groups B, D, and F are shown in Figure 8A, and homologous responses of groups C, E, and G are shown in Figure 8B. Heterologous responses of groups C, E, and G are shown in Figure 8A, and heterologous responses of groups B, D, and F are shown in Figure 8B. Virus neutralization responses against ancestral SARS-CoV-2 and against SARS-CoV-2 B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) variants were tested 14 days after the boost on day 119 ( Figures 8C-8F ) (VNTs against ancestral SARS-CoV-2 ( Figure 8C ), against SARS-CoV-2 B.1.351 ( Figure 8D ), against SARS-CoV-2 B.1.1.7 ( Figure 8E ), and against P.1 ( Figure 8F )).
图9显示了对于所有组在第14天对祖先SARS-CoV-2RBD(图9A)和B.1.351RBD变体K417N、E484K、N501Y(图9B)的显著总IgG刺突结合抗体反应。随着时间的推移，针对不同SARS-CoV-2变体的VNT的诱导如图9C-F所示(图9C：祖先；图9D：B.1.1.7；图9E：B.1.351；图F：P1)。图9G-J显示了使用胞内细胞因子染色测定，用祖先SARS-CoV-2肽文库(图9G和H)的混合物刺激的或用B.1.351 SARS-CoV-2肽文库(图9I和J)的混合物刺激的小鼠中CD8(图9G和9I)和CD4(图9H和9J)阳性T细胞的细胞免疫反应。Figure 9 shows significant total IgG spike binding antibody responses for all groups on day 14 to the ancestral SARS-CoV-2 RBD (Figure 9A) and the B.1.351 RBD variants K417N, E484K, N501Y (Figure 9B). Over time, the induction of VNTs against different SARS-CoV-2 variants is shown in Figures 9C-F (Figure 9C: ancestral; Figure 9D: B.1.1.7; Figure 9E: B.1.351; Figure F: P1). Figures 9G-J show the cellular immune responses of CD8 (Figures 9G and 9I) and CD4 (Figures 9H and 9J) positive T cells in mice stimulated with a mixture of the ancestral SARS-CoV-2 peptide library (Figures 9G and H) or with a mixture of the B.1.351 SARS-CoV-2 peptide library (Figures 9I and J) using intracellular cytokine staining assays.
图10显示了使用胞内细胞因子染色测定，用祖先SARS-CoV-2肽文库的混合物刺激的小鼠中CD8(图10B)和CD4(图10A)阳性T细胞的细胞免疫反应。Figure 10 shows the cellular immune response of CD8 (Figure 10B) and CD4 (Figure 10A) positive T cells in mice stimulated with a mixture of the ancestral SARS-CoV-2 peptide library, as measured by intracellular cytokine staining.
图11显示了在大鼠中用CVnCoV或CV2CoV进行初次和加强疫苗接种及二价CV2CoV+CV2CoV.351疫苗组合物的第三疫苗接种后随时间直至第一次疫苗接种后第133天的VNT(图11A：针对祖先SARS-CoV-2的VNT，图11B：针对SARS-CoV-2 B.1.351的VNT)。在第119天不仅针对祖先和B.1.351 SARS-CoV-2，而且也针对B.1.1.7和P.1 SARS-CoV-2变体诱导了稳定而高的VNT(图11C：祖先，图11D：B.1.351，图11E：B.1.1.7，图11F：P.1)。Figure 11 shows the VNT after primary and booster vaccination with CVnCoV or CV2CoV and the third vaccination of the bivalent CV2CoV+CV2CoV.351 vaccine composition over time until day 133 after the first vaccination in rats (Figure 11A: VNT against ancestral SARS-CoV-2, Figure 11B: VNT against SARS-CoV-2 B.1.351). Stable and high VNT was induced not only against ancestral and B.1.351 SARS-CoV-2, but also against B.1.1.7 and P.1 SARS-CoV-2 variants on day 119 (Figure 11C: ancestral, Figure 11D: B.1.351, Figure 11E: B.1.1.7, Figure 11F: P.1).
图12显示了大鼠中用编码delta变体SARS-CoV-2 B1.617.2的稳定刺突(S_stabpp)的不同mRNA形式的疫苗组合物疫苗接种后的抗体反应(图12A和图12B：分别在第14天和第42天通过ELISA检测针对delta B.1.617.2变体RBD的刺突结合抗体；图12C、图12D、图12E：分别在第14天、第21天和第42天检测针对SARS-CoV-2B.1.617.2的VNT)。不仅针对同源的SARS-CoV-2变体(B.1.617.2)，而且针对异源的SARS-CoV-2祖先和SARS-CoV-2变体B.1.351和P.1诱导稳定的VNT(图12F：祖先，图G：B.1.351，图12H：P.1)。Figure 12 shows the antibody response after vaccination in rats with vaccine compositions of different mRNA forms encoding the stable spike (S_stabpp) of delta variant SARS-CoV-2 B1.617.2 (Figures 12A and 12B: spike binding antibodies against delta B.1.617.2 variant RBD were detected by ELISA on days 14 and 42, respectively; Figures 12C, 12D, 12E: VNTs against SARS-CoV-2 B.1.617.2 were detected on days 14, 21, and 42, respectively). Stable VNTs were induced not only against the homologous SARS-CoV-2 variant (B.1.617.2), but also against the heterologous SARS-CoV-2 ancestor and SARS-CoV-2 variants B.1.351 and P.1 (Figure 12F: ancestor, Figure G: B.1.351, Figure 12H: P.1).
图13显示了大鼠中用编码不同mRNA构建体(其编码不同变体SARS-CoV-2的S_stabpp)的疫苗组合物疫苗接种后第14天的早期抗体反应(总IgG)。此外，二价途径将化学修饰的mRNA与未修饰的mRNA进行比较(图13A：祖先RBD；图13B：delta RBD(L452R，T478K)；图13C：beta RBD(K417N，E484K，N501Y)。Figure 13 shows the early antibody response (total IgG) on day 14 after vaccination with a vaccine composition encoding different mRNA constructs (which encode S_stabpp of different variant SARS-CoV-2) in rats. In addition, the bivalent approach compared chemically modified mRNA with unmodified mRNA (Figure 13A: ancestral RBD; Figure 13B: delta RBD (L452R, T478K); Figure 13C: beta RBD (K417N, E484K, N501Y).
图14显示了通过用SARS-CoV-2变体B.1.351或SARS-CoV-2变体B.1.627.2激发小鼠的疫苗效力。激发的小鼠的存活显示于图14A：用B.1.351激发，和图14B：用B.1.617.2激发。平均体重百分变化显示于图14C：用B.1.351激发，和图14D：用B.1.617.2激发。唾液中的病毒RNA载量显示于图14E：B.1.351激发组，和图14F：B.1.617.2激发组。上呼吸道(URT)(鼻甲)中的病毒载量显示于图14G(B.1.351激发组)和图14H(B.617.2激发组)，且下呼吸道(LRT)(肺)中的病毒载量显示于图14I(B.1.351激发)和图14J(B.617.2激发)。脑中的病毒载量显示于图14K至图14N(图14K和L用于小脑，图14M和N用于大脑(对于激发组B.1.351：图14K和M，对于B.1.617.2：图14L和N)。抗RBD总免疫球蛋白的诱导显示于图14O：激发组B.1.351和图14P：激发组B.1.617.2，以及VNT显示于图14Q：激发后组B.1.351，图14R：激发前组B.1.617.2，和图14S：激发后组B.1.617.2。Figure 14 shows the vaccine efficacy by stimulating mice with SARS-CoV-2 variant B.1.351 or SARS-CoV-2 variant B.1.627.2. The survival of stimulated mice is shown in Figure 14A: stimulated with B.1.351, and Figure 14B: stimulated with B.1.617.2. The average percent change in body weight is shown in Figure 14C: stimulated with B.1.351, and Figure 14D: stimulated with B.1.617.2. The viral RNA load in saliva is shown in Figure 14E: B.1.351 stimulated group, and Figure 14F: B.1.617.2 stimulated group. The viral load in the upper respiratory tract (URT) (nasal concha) is shown in Figure 14G (B.1.351 challenge group) and Figure 14H (B.617.2 challenge group), and the viral load in the lower respiratory tract (LRT) (lung) is shown in Figure 14I (B.1.351 challenge) and Figure 14J (B.617.2 challenge). Viral load in the brain is shown in Figures 14K to 14N (Figures 14K and L for cerebellum, Figures 14M and N for cerebrum (for challenge group B.1.351: Figures 14K and M, for B.1.617.2: Figures 14L and N). Induction of anti-RBD total immunoglobulins is shown in Figure 14O: challenge group B.1.351 and Figure 14P: challenge group B.1.617.2, and VNT is shown in Figure 14Q: post-challenge group B.1.351, Figure 14R: pre-challenge group B.1.617.2, and Figure 14S: post-challenge group B.1.617.2.
图15显示了SARS-CoV-2变体B.1.351激发的仓鼠中的疫苗效力。图15A显示了以挑战感染后天数计的百分体重变化。唾液中的病毒RNA载量显示于图15B，而肺组织中的病毒RNA载量显示于图15C。图15D证明了抗RBD总免疫球蛋白(Ig)的诱导，而图15E显示了VNT。Figure 15 shows the vaccine efficacy in hamsters stimulated by SARS-CoV-2 variant B.1.351. Figure 15A shows the percent weight change in days after challenge infection. The viral RNA load in saliva is shown in Figure 15B, and the viral RNA load in lung tissue is shown in Figure 15C. Figure 15D demonstrates the induction of anti-RBD total immunoglobulin (Ig), while Figure 15E shows VNT.
具体实施方式DETAILED DESCRIPTION
定义definition
为了清晰易读，提供了以下定义。对于这些定义提及的任何技术特征可以在本发明的各个或每个实施方式上阅读。可以在这些实施方式的上下文中具体提供附加的定义和解释。For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions can be read on each or each embodiment of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments.
数量情况中的百分比应理解为相对于相应项目的总数。在其他情况下，除非上下文另有规定，否则百分比应理解为重量百分比(wt.-％)。Percentages in quantitative cases are to be understood as relating to the total amount of the respective item. In other cases, percentages are to be understood as percentages by weight (wt.-%), unless the context dictates otherwise.
约：当决定因素或值不需要相同，即100％相同时，使用术语“约”。因此，“约”是指决定因素或值可能相差0.1％至20％，或0.1％至10％，包括这些范围内的任何点；例如相差0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％。本领域技术人员将知道，例如，某些参数或决定因素可能基于如何确定参数的方法而略有变化。例如，如果某些决定因素或值在本文中被定义为具有例如“约1000个核苷酸”的长度，则该长度可以相差0.1％至20％，或0.1％至10％，包括这些范围内的任何点；例如相差0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.7％、0.8％、0.9％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％。因此，本领域技术人员将知道，在该特定实例中，长度可以相差1至200个核苷酸，或相差1至100个核苷酸；特别地，相差1、2、3、4、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200个核苷酸，或该范围内的任何整数值。About : The term "about" is used when the Determinants or values need not be identical, i.e., 100% identical. Thus, "about" means that the Determinants or values may differ by 0.1% to 20%, or 0.1% to 10%, including any point within these ranges; for example, by 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. One skilled in the art will appreciate that certain parameters or Determinants may vary slightly based on, for example, the method by which the parameters are determined. For example, if certain Determinants or values are defined herein as having a length of, for example, "about 1000 nucleotides," the length may vary from 0.1% to 20%, or from 0.1% to 10%, including any points within these ranges; for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. Thus, one skilled in the art will appreciate that, in this particular example, the lengths may differ by 1 to 200 nucleotides, or by 1 to 100 nucleotides; in particular, by 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides, or any integer value within this range.
适应性免疫反应：本文中使用的术语“适应性免疫反应”被本领域普通技术人员认识和理解，并且例如旨在指免疫系统(适应性免疫系统)的抗原特异性反应。抗原特异性允许产生针对特定病原体或病原体感染细胞定制的反应。产生这些定制反应的能力通常由“记忆细胞”(B细胞)在体内维持。在本发明的上下文中，抗原由编码至少一种源自SARS-CoV-2的抗原性肽或蛋白质的RNA提供，例如包括但不限于的SARS-CoV-2株：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。优选地，抗原由编码至少一种源自SARS-CoV-2刺突蛋白的抗原肽的RNA来提供，例如，包含源自包括，但不限于以下SAR-Cov-2株的刺突蛋白：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。Adaptive immune response : The term "adaptive immune response" as used herein is recognized and understood by those of ordinary skill in the art and is intended, for example, to refer to the antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows for the generation of responses tailored to specific pathogens or pathogen-infected cells. The ability to generate these tailored responses is typically maintained in vivo by "memory cells" (B cells). In the context of the present invention, the antigen is provided by RNA encoding at least one antigenic peptide or protein derived from SARS-CoV-2, such as, but not limited to, SARS-CoV-2 strains: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia). Preferably, the antigen is provided by RNA encoding at least one antigenic peptide derived from the SARS-CoV-2 spike protein, for example, comprising a spike protein derived from, but not limited to, the following SAR-Cov-2 strains: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
抗原：如本文使用的术语“抗原”将被本领域技术人员认识和理解，并且例如旨在表示可以被免疫系统识别，优选被适应性免疫系统识别，并且能够例如通过形成作为适应性免疫反应一部分的抗体和/或抗原特异性T细胞来引发抗原特异性免疫反应的物质。通常，抗原可以是或可以包含肽或蛋白质，其可以通过MHC递呈给T细胞。此外，包含至少一个表位的源自例如SARS-CoV-2的刺突蛋白的肽或蛋白质的片段、变体或衍生物理解为本发明背景中的抗原，例如源自包括但不限于以下SARS-Cov-2株：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。在本发明的情况中，抗原可以是如本文所述的所提供的RNA的翻译产物。Antigen : The term "antigen" as used herein will be recognized and understood by those skilled in the art, and is intended, for example, to mean a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of eliciting an antigen-specific immune response, for example by forming antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or can comprise a peptide or protein, which can be presented to T cells via MHC. In addition, fragments, variants or derivatives of peptides or proteins derived from, for example, the spike protein of SARS-CoV-2, which contain at least one epitope, are understood as antigens in the context of the present invention, for example derived from, but not limited to, the following SARS-Cov-2 strains: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta), In some embodiments, the antigens of the present invention may be selected from the group consisting of B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines), and/or B.1.621 (Mu, Colombia). In the context of the present invention, the antigen may be a translation product of a provided RNA as described herein.
抗原肽或蛋白质：术语“抗原肽或蛋白质”或“免疫原性肽或蛋白质”被本领域普通技术人员认识和理解，并且例如旨在是指源自刺激身体的适应性免疫系统以提供适应性免疫反应的(抗原性或免疫原性)蛋白质的肽、蛋白质。因此，抗原性/免疫原性肽或蛋白质包含其源自(例如，SARS-CoV-2的刺突蛋白(S))的蛋白质的至少一个表位或抗原，例如，源自包括但不限于以下SARS-Cov-2株的刺突蛋白(S)：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。Antigenic peptide or protein : The term "antigenic peptide or protein" or "immunogenic peptide or protein" is recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a peptide, protein derived from an (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Thus, the antigenic/immunogenic peptide or protein comprises at least one epitope or antigen of a protein from which it is derived (e.g., the spike protein (S) of SARS-CoV-2), for example, the spike protein (S) derived from SARS-Cov-2 strains including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
阳离子的：除非从具体上下文中明确不同的含义，否则术语“阳离子的”是指相应的结构携带正电荷，无论是永久性的还是非永久性的，但对如pH的特定条件有反应。因此，术语“阳离子”涵盖“永久性阳离子的”和“可阳离子化的(Cationisable)”两者。Cationic : Unless a different meaning is clear from the specific context, the term "cationic" means that the corresponding structure carries a positive charge, whether permanent or non-permanent, but responsive to specific conditions such as pH. Thus, the term "cationic" encompasses both "permanently cationic" and "cationisable".
可阳离子化的：如本文使用的术语“可阳离子化的”是指化合物，或基团或原子在其环境的较低pH下带正电，和在较高pH下不带电。同样，在无法确定pH值的非水性环境中，可阳离子化的化合物、基团或原子在高氢离子浓度下带正电，而在低氢离子浓度或活性下不带电。它取决于可阳离子化或多阳离子化化合物的单个性质，特别是相应的可阳离子化基团或原子的pKa，在该pH或氢离子浓度下它带电或不带电。在稀释的水性环境中，带正电荷的可阳离子化化合物、基团或原子的分数可以使用本领域技术人员熟知的所谓Henderson-Hasselbalch方程来估计。例如，在一些实施方式中，如果化合物或部分是可阳离子化的，则优选其在约1至9，优选4至9，5至8或甚至6至8的pH值下，更优选9或以下、8或以下、7或以下的pH值，最优选在生理pH值下，例如约7.3至7.4，即在生理条件下，特别是在体内细胞的生理盐条件下，带正电荷。在其它实施方式中，优选地可阳离子化化合物或部分在生理pH值(例如约7.0-7.4)下主要是中性的，但在较低pH值下变得带正电。在一些实施方式中，可阳离子化化合物或部分的pKa的优选范围为约5至约7。Cationizable : As used herein, the term "cationizable" refers to a compound, or a group or atom that is positively charged at the lower pH of its environment, and uncharged at a higher pH. Likewise, in a non-aqueous environment where the pH cannot be determined, a cationizable compound, group or atom is positively charged at high hydrogen ion concentrations, and uncharged at low hydrogen ion concentrations or activities. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the corresponding cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In a dilute aqueous environment, the fraction of positively charged cationizable compounds, groups or atoms can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, in some embodiments, if the compound or moiety is cationizable, it is preferably positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably 9 or below, 8 or below, 7 or below, and most preferably at a physiological pH, such as about 7.3 to 7.4, i.e., under physiological conditions, particularly physiological salt conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or moiety is predominantly neutral at physiological pH (e.g., about 7.0-7.4), but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the cationizable compound or moiety is from about 5 to about 7.
编码序列/编码区：如本文使用的术语“编码序列”或“编码区”和相应的缩写“cds”被本领域普通技术人员认识和了解，并且例如旨在表示几个核苷酸三联体的序列，其可以翻译成肽或蛋白质。在本发明的上下文中，编码序列可以是由可以被按三个分割的多个核苷酸组成的RNA序列，其以起始密码子开始并且优选以终止密码子终止。Coding sequence/coding region : The term "coding sequence" or "coding region" and the corresponding abbreviation "cds" as used herein are recognized and understood by those of ordinary skill in the art and are intended, for example, to mean a sequence of several nucleotide triplets, which can be translated into a peptide or protein. In the context of the present invention, a coding sequence may be an RNA sequence consisting of a plurality of nucleotides that may be divided in three, which begins with a start codon and preferably ends with a stop codon.
源自：在核酸的情况中整个本说明书中使用的术语“源自”，即，对于“源自”(另一个)核酸的核酸，表示其源自(另一个)核酸的核酸与从其衍生的核酸享有例如至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性。技术人员认识到序列同一性通常针对相同类型的核酸来计算，即，针对DNA序列或针对RNA序列。因此，应理解在如果DNA“源自”RNA或如果RNA“源自”DNA，则在第一步中将RNA序列转化成相应的DNA序列(特别是在整个序列中用胸腺嘧啶(T)替代尿嘧啶(U))，或者相反，将DNA序列转化成相应的RNA序列(特别是在整个序列中用U替代T)。此后，确定DNA序列的序列同一性或RNA序列的序列同一性。优选地，“源自”核酸的核酸也称为核酸，其与从其衍生的核酸相比是修饰的，例如，甚至进一步提高RNA稳定性和/或延长和/或增加蛋白质产生。在氨基酸序列(例如，抗原肽或蛋白质)的情况中，术语“源自”表示源自(另一个)氨基酸序列的氨基酸序列与从其衍生的氨基酸序列共有例如至少60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性。Derived from : The term "derived from" as used throughout this specification in the context of nucleic acids, i.e., for a nucleic acid "derived from" (another) nucleic acid, means that the nucleic acid derived from (another) nucleic acid shares, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid derived therefrom. The skilled person recognizes that sequence identity is usually calculated for nucleic acids of the same type, i.e., for DNA sequences or for RNA sequences. Thus, it will be understood that if DNA is "derived from" RNA or if RNA is "derived from" DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (particularly replacing uracil (U) with thymine (T) throughout the sequence), or conversely, the DNA sequence is converted into the corresponding RNA sequence (particularly replacing T with U throughout the sequence). In some embodiments, the term "derived from" refers to a nucleic acid that is derived from a nucleic acid that is modified compared to a nucleic acid derived therefrom, such as to further improve RNA stability and/or extend and/or increase protein production. In the case of an amino acid sequence (such as an antigenic peptide or protein), the term "derived from" refers to an amino acid sequence that is derived from (another) amino acid sequence that has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence derived therefrom.
表位：如本文使用的术语“表位”(本领域也称为“抗原决定簇”)将被本领域普通技术人员认识和理解，并且例如旨在表示T细胞表位和B细胞表位。T细胞表位或者抗原肽或蛋白质的一部分可以包含优选具有约6至约20或甚至更多个氨基酸长度的片段，例如，由MHCI类分子加工和呈递的片段，优选具有约8至约10个氨基酸的长度，例如，8、9或10个(或甚至11或12个氨基酸)，或由MHC II类分子加工和呈递的片段，优选具有约13至约20或甚至更多个氨基酸的长度。这些片段通常以由肽片段和MHC分子组成的复合物的形式由T细胞识别，即，通常不以其天然形式被识别的片段。B细胞表位通常是位于(天然)蛋白质或肽抗原的外表面上的片段，优选具有5至15个氨基酸，更优选具有5至12个氨基酸，甚至更优选具有6至9个氨基酸，其可以被抗体识别，即，以其天然形式被识别。这样的蛋白质或肽的表位可以进一步选自本文提及的此类蛋白质或肽的变体中的任一个。在该情况中，表位可以是构象的或不连续的表位，其可以由如本文定义的蛋白质或肽的区段组成，其在如本文定义的蛋白质或肽的氨基酸序列中是不连续的，而是在三维结构中汇合在一起，或是由单个多肽链组成的连续或线性的表位。Epitope : The term "epitope" (also referred to in the art as "antigenic determinant") as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to represent T cell epitopes and B cell epitopes. A portion of a T cell epitope or antigenic peptide or protein may comprise a fragment preferably having a length of about 6 to about 20 or even more amino acids, for example, a fragment processed and presented by an MHC I class molecule, preferably having a length of about 8 to about 10 amino acids, for example, 8, 9 or 10 (or even 11 or 12 amino acids), or a fragment processed and presented by an MHC II class molecule, preferably having a length of about 13 to about 20 or even more amino acids. These fragments are usually recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, i.e., a fragment that is not usually recognized in its native form. A B cell epitope is usually a fragment located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which can be recognized by an antibody, i.e., recognized in its native form. Such an epitope of a protein or peptide may further be selected from any of the variants of such a protein or peptide mentioned herein. In this case, the epitope may be a conformational or discontinuous epitope, which may consist of a segment of a protein or peptide as defined herein, which is discontinuous in the amino acid sequence of the protein or peptide as defined herein, but converges together in a three-dimensional structure, or a continuous or linear epitope consisting of a single polypeptide chain.
片段：在核酸序列(例如，RNA或DNA)或氨基酸序列的情况中，在整个本说明书中使用的术语“片段”通常可以是例如核酸序列或氨基酸序列的全长序列的较短部分，同时仍然保留其预期的功能。因此，片段通常由全长序列内与相应链段相同的序列组成。本发明情况中优选的序列片段由连续的实体链希组成，如对应于该片段所来源的分子中连续实体链段的核苷酸或氨基酸，其代表该片段所来源的总(即，全长)分子(例如，SARS-CoV-2的刺突蛋白(S)，例如，来自括但不限于SARS-Cov-2株的刺突蛋白(S)：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚))的至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％。在蛋白质或肽的情况中，在整个本说明书中使用的术语“片段”可以通常包含如本文定义的蛋白质或肽的序列，与初始蛋白质的氨基酸序列相比，其关于氨基酸序列，N-末端和/或C-末端截短。这种截短因此可以发生在氨基酸水平或相应地核酸水平。关于如本文定义的这样的片段的序列同一性因此可以优选是指如本文定义的整个蛋白质或肽或这样的蛋白质或肽的整个(编码)核酸分子。蛋白质或肽的片段可以包含那些蛋白质或肽的至少一个表位。Fragment : In the case of nucleic acid sequences (e.g., RNA or DNA) or amino acid sequences, the term "fragment" as used throughout this specification generally can be a shorter portion of the full-length sequence, such as a nucleic acid sequence or an amino acid sequence, while still retaining its intended function. Thus, a fragment generally consists of the same sequence as the corresponding segment within the full-length sequence. In the context of the present invention, the preferred sequence fragments consist of a continuous entity chain, such as nucleotides or amino acids corresponding to a continuous entity chain segment in the molecule from which the fragment is derived, which represents the total (i.e., full-length) molecule from which the fragment is derived (e.g., the spike protein (S) of SARS-CoV-2, for example, the spike protein (S) from SARS-Cov-2 strains including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India) , B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia)). At least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. In the case of proteins or peptides, the term "fragment" used throughout this specification may generally comprise a sequence of a protein or peptide as defined herein, which is truncated with respect to the amino acid sequence, N-terminus and/or C-terminus compared to the amino acid sequence of the original protein. Such truncation may therefore occur at the amino acid level or, respectively, at the nucleic acid level. Sequence identity with respect to such fragments as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or the entire (encoding) nucleic acid molecule of such a protein or peptide.Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides.
异源的：在核酸序列或氨基酸序列的情况中，在整个本说明书中使用的术语“异源”或“异源序列”是指必须理解为源自另一个基因、另一个等位基因或例如另一个物种或病毒的序列的序列(例如，RNA、DNA、氨基酸)。如果不是可源自同一基因或同一等位基因，则通常将两个序列理解为“异源的”。即，尽管异源序列本质上可源自同一生物体或病毒，但它们没有发生在同一核酸或蛋白质中。Heterologous : In the context of nucleic acid sequences or amino acid sequences, the term "heterologous" or "heterologous sequence" as used throughout this specification refers to a sequence (e.g., RNA, DNA, amino acid) that must be understood as being derived from another gene, another allele, or, for example, another species or virus. Two sequences are generally understood to be "heterologous" if they are not derived from the same gene or the same allele. That is, although heterologous sequences may essentially be derived from the same organism or virus, they do not occur in the same nucleic acid or protein.
体液免疫反应：术语“体液免疫”或“体液免疫反应”将被本领域普通技术人员认识和理解，并且例如旨在表示B-细胞介导的抗体产生和任选表示伴随抗体产生的辅助过程。体液免疫反应通常特征可以在于例如Th2激活和细胞因子产生、生发中心形成和同种型转换、亲和力成熟和记忆细胞产生。体液免疫也可能是指抗体的效应子功能，包括病原体和毒素中和、经典的补体激活以及吞噬和病原体清除的调理素促进。Humoral immune response : The term "humoral immunity" or "humoral immune response" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to B-cell mediated antibody production and optionally to the auxiliary processes that accompany antibody production. The humoral immune response can be generally characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell production. Humoral immunity may also refer to the effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen clearance.
(序列的)同一性：在核酸序列或氨基酸序列的情况中，在整个本说明书中使用的术语“同一性”将被本领域普通技术人员认识和理解，并且例如旨在表示两个序列在其完全/整个长度上或在其特定的指定部分、区域或结构域上相同的百分比，例如，在其完全/整个长度上或在其特定的指定部分、区域或结构域上存在至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同一性。为了确定两个序列相同的百分比，例如，如本文定义的核酸序列或氨基酸(aa)序列，优选地由如本文定义的核酸序列编码的aa序列或本身的aa序列，可以对序列进行比对以随后彼此进行比较。因此，例如，第一序列的位置可以与第二序列的相应位置进行比较。如果第一序列中的某个位置被第二序列中的位置处相同的残基占据，则两个序列在这个位置是相同的。如果不是这样的情况，则序列在这个位置是不同的。如果与第一序列相比，在第二序列中发生了插入，可以将缺口插入第一序列中以允许进一步的对准。如果与第一序列相比，在第二序列中发生了缺失，则可以将缺口插入第二序列中以允许进一步的对准。然后两个序列相同的百分比是相同位置的数量除以位置总数(包括仅在一个序列中占据的那些位置)的函数。可以使用算法，例如，整合在BLAST程序中的算法，来确定两个序列相同的百分比。(Sequence) identity : In the context of nucleic acid sequences or amino acid sequences, the term "identity" as used throughout this specification will be recognized and understood by those of ordinary skill in the art and is intended, for example, to indicate the percentage of identity between two sequences over their complete/entire length or over a specific designated portion, region or domain thereof, for example, at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identity over their complete/entire length or over a specific designated portion, region or domain thereof. In order to determine the percentage of identity between two sequences, for example, a nucleic acid sequence as defined herein or an amino acid (aa) sequence, preferably an aa sequence encoded by a nucleic acid sequence as defined herein or the aa sequence itself, the sequences can be aligned for subsequent comparison to each other. Thus, for example, the position of the first sequence can be compared with the corresponding position of the second sequence. If a certain position in the first sequence is occupied by the same residue at the position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences are different at this position. If an insertion has occurred in the second sequence compared to the first sequence, a gap can be inserted into the first sequence to allow further alignment. If a deletion has occurred in the second sequence compared to the first sequence, a gap can be inserted into the second sequence to allow further alignment. Then the percentage of the two sequences being identical is a function of the number of identical positions divided by the total number of positions (including those positions occupied only in one sequence). An algorithm can be used, for example, an algorithm integrated in the BLAST program, to determine the percentage of the two sequences being identical.
免疫原，免疫原性的：术语“免疫原”或“免疫原性的”将被本领域普通技术人员认识和理解，并且例如旨在表示能够刺激/诱导免疫反应的化合物。优选地，免疫原可以是肽、多肽或蛋白质。本发明意义上的免疫原是提供的RNA的翻译产物，所述RNA包含至少一个编码至少一个源自SARS-CoV-2的刺突蛋白的抗原肽、蛋白质的编码序列，例如，源自包括但不限于以下SARS-Cov-2株的刺突蛋白的蛋白质：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)，如本文中定义的。通常，免疫原引发适应性免疫反应。Immunogen, immunogenic : The term "immunogen" or "immunogenic" will be recognized and understood by those of ordinary skill in the art and is intended, for example, to mean a compound capable of stimulating/inducing an immune response. Preferably, the immunogen may be a peptide, polypeptide or protein. An immunogen in the sense of the present invention is a translation product of a provided RNA, wherein the RNA comprises at least one coding sequence for an antigenic peptide or protein that encodes at least one spike protein derived from SARS-CoV-2, for example, a protein derived from a spike protein including but not limited to the following SARS-Cov-2 strains: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia), as defined herein. Typically, an immunogen elicits an adaptive immune response.
免疫反应：术语“免疫反应”将被本领域普通技术人员认识和理解，并且例如旨在表示适应性免疫系统对特定抗原的特异性反应(所谓的特异性或适应性免疫反应)或先天性免疫系统的非特异性反应(所谓的非特异性或先天性免疫反应)，或其组合。合适的疫苗在施用疫苗的正常健康接受者中诱导有效免疫反应。随着有效的免疫反应，一次疫苗接种将导致病毒中和抗体滴度。另外地，或替代地，有效免疫反应将引发适应性免疫反应。在一些实施方式中，相对于未接种的对照受试者的中和抗体滴度，有效免疫反应将冠状病毒感染降低至少50％。在一些实施方式中，有效免疫反应将是其中中和抗体滴度和/或T细胞免疫反应相对于未接种的对照受试者的中和抗体滴度足以降低无症状病毒感染率的免疫反应。有效免疫反应也可以是其中中和抗体滴度和/或T细胞免疫反应足以防止受试者中的病毒潜伏和/或中和抗体滴度足以阻断病毒与受试者的上皮细胞融合的免疫反应。在一些实施方式中，有效免疫反应是其中治疗有效量的核酸、组合物、多肽或疫苗施用于受试者诱导受试者中对抗冠状病毒的T细胞免疫反应的免疫反应。在优选的实施方式中，T细胞免疫反应包括CD4+T细胞免疫反应和/或CD8+T细胞免疫反应。在进一步方面中，有效免疫反应是其中免疫反应保护受试者免于严重的COVID-19疾病至少约6个月和/或与未接种的人员相比降低住院发生率的免疫反应。与未接种疫苗的病毒感染者相比，有效免疫反应也可以减少病毒的传播。有效免疫反应也可以被认为是由于异源免疫反应导致的对变体提供一些保护的免疫反应。Immune response : The term "immune response" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to represent a specific response of the adaptive immune system to a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response), or a combination thereof. Suitable vaccines induce an effective immune response in normal healthy recipients to whom the vaccine is administered. With an effective immune response, a vaccination will result in a virus neutralizing antibody titer. Additionally, or alternatively, an effective immune response will trigger an adaptive immune response. In some embodiments, an effective immune response reduces coronavirus infection by at least 50% relative to the neutralizing antibody titer of an unvaccinated control subject. In some embodiments, an effective immune response will be an immune response in which the neutralizing antibody titer and/or T cell immune response is sufficient to reduce the asymptomatic viral infection rate relative to the neutralizing antibody titer of an unvaccinated control subject. An effective immune response may also be an immune response in which the neutralizing antibody titer and/or T cell immune response is sufficient to prevent viral latency in a subject and/or the neutralizing antibody titer is sufficient to block viral fusion with epithelial cells of the subject. In some embodiments, an effective immune response is an immune response in which a therapeutically effective amount of a nucleic acid, a composition, a polypeptide or a vaccine is administered to a subject to induce a T cell immune response against coronavirus in the subject. In a preferred embodiment, the T cell immune response includes a CD4+T cell immune response and/or a CD8+T cell immune response. In a further aspect, an effective immune response is an immune response in which the immune response protects the subject from severe COVID-19 disease for at least about 6 months and/or reduces the incidence of hospitalization compared to unvaccinated personnel. An effective immune response can also reduce the spread of the virus compared to unvaccinated virus-infected persons. An effective immune response can also be considered as an immune response that provides some protection to variants due to a heterologous immune response.
免疫系统：术语“免疫系统”将被本领域普通技术人员认识和理解，并且例如旨在表示可以保护生物体免受感染的生物体的系统。如果病原体成功穿过生物体的物理屏障并进入这个生物体，则先天性免疫系统提供即时的但非特异性的反应。如果病原体逃避了这种先天性反应，脊椎动物具有第二层保护，适应性免疫系统。在此，免疫系统在感染期间调整其反应以提高其对病原体的识别。在病原体已经消除后，这种改进的反应然后以免疫记忆的形式得到保留，并允许适应性免疫系统在每次遇到这种病原体时开启更快且更强的攻击。据此，免疫系统包括先天性和适应性免疫系统。这两个部分中的每一个通常含有所谓的体液和细胞组分。Immune system : The term "immune system" will be recognized and understood by those of ordinary skill in the art and is intended, for example, to mean the system of an organism that can protect the organism from infection. If a pathogen successfully crosses the physical barriers of an organism and enters this organism, the innate immune system provides an immediate but non-specific response. If a pathogen evades this innate response, vertebrates have a second layer of protection, the adaptive immune system. Here, the immune system adjusts its response during infection to improve its recognition of the pathogen. After the pathogen has been eliminated, this improved response is then retained in the form of immunological memory and allows the adaptive immune system to launch a faster and stronger attack each time this pathogen is encountered. Accordingly, the immune system includes an innate and an adaptive immune system. Each of these two parts generally contains so-called humoral and cellular components.
先天性免疫系统：术语“先天性免疫系统”(也称为非特异性或无特异性免疫系统)被本领域普通技术人员认识和理解，并且例如旨在表示通常包括以非特异性方式保护宿主免受其他生物体感染的细胞及机制。这意味着先天性系统的细胞可以以通用的方式识别并对病原体作出反应，但与适应性免疫系统不同，它不赋予宿主长效或保护性免疫。先天性免疫系统可以通过模式识别受体的配体来激活，例如为Toll-样受体、NOD-样受体或RIG-I样受体等。Innate immune system : The term "innate immune system" (also called the nonspecific or aspecific immune system) is recognized and understood by those of ordinary skill in the art and is intended, for example, to mean cells and mechanisms that generally protect the host from infection by other organisms in a nonspecific manner. This means that the cells of the innate system can recognize and respond to pathogens in a universal manner, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system can be activated by ligands of pattern recognition receptors, such as Toll-like receptors, NOD-like receptors, or RIG-I-like receptors, among others.
类脂化合物：类脂化合物，也称为类脂，是脂质样化合物，即，具有脂质样物理性质的两亲性化合物。在本发明的情况中，术语脂质认为包括类脂化合物。Lipid compounds : Lipid compounds, also called lipids, are lipid-like compounds, ie amphiphilic compounds having lipid-like physical properties. In the context of the present invention, the term lipid is considered to include lipid compounds.
永久性阳离子的：如本文使用的术语“永久性阳离子的”被本领域普通技术人员认识和理解，并且例如表示相应的化合物，或基团，或原子在其环境的任何pH值或氢离子活性下带有正电荷。通常，正电荷由季氮原子的存在引起。在化合物带有多个这样的正电荷的情况下，其可以称为永久性多阳离子的。Permanent cationic : The term "permanent cationic" as used herein is recognized and understood by those of ordinary skill in the art and means, for example, that the corresponding compound, or group, or atom carries a positive charge at any pH value or hydrogen ion activity of its environment. Typically, the positive charge is caused by the presence of a quaternary nitrogen atom. In the case where a compound carries multiple such positive charges, it may be referred to as permanently polycationic.
RNA序列：术语“RNA序列”将被本领域普通技术人员认识和理解，并且例如是指其连续核糖核苷酸的特定的、单个的顺序。RNA sequence : The term "RNA sequence" will be recognized and understood by those of ordinary skill in the art, and refers, for example, to a specific, individual order of consecutive ribonucleotides thereof.
稳定的RNA：术语“稳定的RNA”是指经修饰以使得与未经所述修饰的RNA相比对崩解或降解更稳定的RNA，例如，由环境因素或酶消化引起的，如通过核酸外切酶或核酸内切酶降解。优选地，本发明情况中的稳定的RNA是在细胞中稳定的，如原核或真核细胞，优选在哺乳动物细胞中，如人细胞。稳定效果也可以在细胞外发挥，例如，在缓冲液中等，例如，用于储存包含稳定RNA的组合物。Stable RNA : The term "stable RNA" refers to RNA that has been modified so as to be more stable to disintegration or degradation, e.g., caused by environmental factors or enzymatic digestion, such as degradation by exonucleases or endonucleases, than RNA that has not been modified as described. Preferably, the stable RNA in the context of the present invention is stable in cells, such as prokaryotic or eukaryotic cells, preferably in mammalian cells, such as human cells. The stabilizing effect can also be exerted outside the cell, e.g., in a buffer, etc., e.g., for storing a composition comprising the stabilized RNA.
T细胞反应：如本文使用的术语“细胞免疫”或“细胞免疫反应”或“细胞T细胞反应”被本领域普通技术人员认识和理解，并且例如旨在表示响应抗原的巨噬细胞、自然杀伤细胞(NK)、抗原特异性细胞毒性T-淋巴细胞的激活以及各种细胞因子的释放。在更一般的术语中，细胞免疫不是基于抗体，而是基于免疫系统的细胞的激活。通常，细胞免疫反应可以例如通过激活抗原特异性细胞毒性T-淋巴细胞来表征，其能够诱导细胞的凋亡，例如，特异性免疫细胞，如树突细胞或其他细胞，在其表面上展示外来抗原的表位。T cell response : The terms "cellular immunity" or "cellular immune response" or "cellular T cell response" as used herein are recognized and understood by those of ordinary skill in the art and are intended, for example, to refer to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes in response to antigens, and the release of various cytokines. In more general terms, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. In general, a cellular immune response can be characterized, for example, by the activation of antigen-specific cytotoxic T-lymphocytes, which are capable of inducing apoptosis of cells, for example, specific immune cells, such as dendritic cells or other cells, displaying epitopes of foreign antigens on their surfaces.
UTR：术语“未翻译区域”或“UTR”或“UTR元件”被本领域普通技术人员认识和理解，并且例如旨在表示通常位于编码序列的5’或3’侧的核酸分子的部分。UTR不翻译成蛋白质。UTR可以是核酸(例如，DNA或RNA)的部分。UTR可以包含用于控制基因表达的元件，也称为调控元件。这样的调控元件可以是例如核糖体结合位点、miRNA-结合位点等。UTR : The term "untranslated region" or "UTR" or "UTR element" is recognized and understood by those of ordinary skill in the art and is intended, for example, to refer to the portion of a nucleic acid molecule that is typically located on the 5' or 3' side of a coding sequence. A UTR is not translated into protein. A UTR can be part of a nucleic acid (e.g., DNA or RNA). A UTR can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA-binding sites, etc.
3’-UTR：术语“3’-未翻译区”或“3’-UTR”或“3’-UTR元件”被本领域普通技术人员认识和理解，并且例如旨在表示位于编码序列的3’侧(即，下游)且不翻译成蛋白质的核酸分子的部分。3’-UTR可以是RNA的部分，其位于编码序列和(任选的)聚(A)序列之间。3’-UTR可以包含用于控制基因表达的元件，也称为调控元件。这样的调控元件可以是例如核糖体结合位点、miRNA-结合位点等。3'-UTR : The term "3'-untranslated region" or "3'-UTR" or "3'-UTR element" is recognized and understood by a person skilled in the art and is intended, for example, to mean the portion of a nucleic acid molecule that is located 3' to (i.e., downstream of) a coding sequence and that is not translated into protein. The 3'-UTR may be a portion of an RNA that is located between the coding sequence and the (optional) poly(A) sequence. The 3'-UTR may comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA-binding sites, etc.
5’-UTR：术语“5’-未翻译区”或“5’-UTR”或“5’-UTR元件”被本领域普通技术人员认识和理解，并且例如旨在表示位于编码序列的5’侧(即，上游)且不翻译成蛋白质的核酸分子的部分。5’-UTR可以是RNA的部分，其位于编码序列和(任选的)5’帽之间。5’-UTR可以包含用于控制基因表达的元件，也称为调控元件。这样的调控元件可以是例如核糖体结合位点、miRNA-结合位点等。5'-UTR : The term "5'-untranslated region" or "5'-UTR" or "5'-UTR element" is recognized and understood by a person of ordinary skill in the art and is intended, for example, to mean the portion of a nucleic acid molecule that is located 5' to (i.e., upstream of) a coding sequence and that is not translated into protein. The 5'-UTR may be the portion of an RNA that is located between the coding sequence and the (optional) 5' cap. The 5'-UTR may comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA-binding sites, etc.
(序列的)变体：在核酸序列的情况中，在整个本说明书中使用的术语“变体”被本领域普通技术人员认识和理解，并且例如旨在表示源自另一个核酸序列的核酸序列的变体。例如，核酸序列的变体与变体从其衍生的核酸序列相比，可以呈现出一个或多个核苷酸缺失、插入、添加和/或取代。核酸序列的变体可以与变体由其衍生的核酸序列至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％相同。变体在保留变体由其的衍生的序列的至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或更高的功能的意义上是功能性变体。在一个实施方式中，核酸序列的“变体”在该核酸序列的至少10、20、30、50、75或100个核苷酸的链上具有至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％的核苷酸同一性。Variant (of a sequence) : In the case of a nucleic acid sequence, the term "variant" as used throughout this specification is recognized and understood by those of ordinary skill in the art, and is intended, for example, to represent a variant of a nucleic acid sequence derived from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may be at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the nucleic acid sequence from which the variant is derived. A variant is a functional variant in the sense that it retains at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of the function of the sequence from which the variant is derived. In one embodiment, a "variant" of a nucleic acid sequence has at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% nucleotide identity over a strand of at least 10, 20, 30, 50, 75, or 100 nucleotides of the nucleic acid sequence.
在蛋白质或肽的情况中，在整个本说明书中使用的术语“变体”例如旨在表示具有与初始序列相差一个或多个突变/置换(如一个或多个置换的、插入的和/或缺失的氨基酸)的氨基酸序列的蛋白质或肽变体。例如，在一些方面中，蛋白质序列中的插入包括1至10个氨基酸的插入，如1、2、3、4、5、6、7、8、9或10个连续的氨基酸。优选地，这些片段和/或变体可以具有相同或相当的特定抗原特性(免疫原性变体、抗原性变体)。插入和置换是可能的，特别是在不对三维结构引起改变或不影响结合区的那些序列位置处。例如，可以使用CD谱(圆二色谱)容易地确定由插入或缺失引起的三维结构的改变。蛋白质或肽的“变体”可以在至少10、20、30、50、75或100个氨基酸的链上或在此类蛋白质或肽的整个长度上具有至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％的氨基酸同一性。优选地，蛋白质的变体可以包括蛋白质的功能性片段，这在本发明的情况中表示变体发挥出与其衍生的蛋白质基本上相同，或至少40％、50％、60％、70％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或更高的免疫原性。In the case of proteins or peptides, the term "variant" used throughout this specification is intended to represent, for example, a protein or peptide variant having an amino acid sequence that differs from the original sequence by one or more mutations/displacements (such as one or more substituted, inserted and/or deleted amino acids). For example, in some aspects, the insertion in the protein sequence includes the insertion of 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 continuous amino acids. Preferably, these fragments and/or variants can have the same or comparable specific antigenic properties (immunogenic variants, antigenic variants). Insertion and displacement are possible, particularly at those sequence positions that do not cause changes to the three-dimensional structure or do not affect the binding zone. For example, CD spectra (circular dichroism spectra) can be used to easily determine the changes in the three-dimensional structure caused by insertion or deletion. "Variants" of a protein or peptide may have at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% amino acid identity over a chain of at least 10, 20, 30, 50, 75 or 100 amino acids, or over the entire length of such protein or peptide. Preferably, variants of a protein may include functional fragments of a protein, which in the context of the present invention means that the variant exerts substantially the same immunogenicity as the protein from which it is derived, or at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more.
本发明的简述Brief description of the invention
本发明部分地基于编码源自SARS-CoV-2变体的刺突蛋白的RNA可以在人细胞中有效地表达并在动物中诱导广泛地中和不同SARS-CoV-2变体的抗体反应的发现，所述SARS-CoV-2变体例如为包括但不限于以下的SARS-CoV-2株：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。此外，编码不同SARS-CoV-2刺突蛋白变体的RNA的混合物在产生对一系列SARS-CoV-2变体的中和抗体中也显示出是有效的。这些发现提供了用于新的基于RNA的冠状病毒疫苗的基础。The present invention is based in part on the discovery that RNA encoding spike proteins derived from SARS-CoV-2 variants can be efficiently expressed in human cells and induce antibody responses in animals that broadly neutralize different SARS-CoV-2 variants, such as SARS-CoV-2 strains including but not limited to the following: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia). In addition, a mixture of RNA encoding different SARS-CoV-2 spike protein variants was also shown to be effective in generating neutralizing antibodies against a range of SARS-CoV-2 variants. These findings provide the basis for new RNA-based coronavirus vaccines.
如本文所述的RNA序列、组合物或疫苗具有至少一些以下的有利特征：The RNA sequences, compositions or vaccines as described herein have at least some of the following advantageous features:
·RNA在注射/疫苗接种部位(例如，肌肉)处的翻译；Translation of RNA at the injection/vaccination site (e.g., muscle);
·在非常低的剂量和给药方案下非常有效地诱导抗编码的SARS-CoV-2蛋白的抗原特异性免疫反应；Very effective in inducing antigen-specific immune responses against encoded SARS-CoV-2 proteins at very low doses and dosing schedules;
·适合于婴儿和/或新生儿或老年人的疫苗接种，特别是老年人；Suitable for vaccination of infants and/or newborns or the elderly, especially the elderly;
·组合物/疫苗用于肌内施用的合适性；The suitability of the composition/vaccine for intramuscular administration;
·诱导抗SARS-CoV-2变体的特异性和功能性体液免疫反应；Induce specific and functional humoral immune responses against SARS-CoV-2 variants;
·诱导抗SARS-CoV-2变体的广泛的、功能性的细胞性T细胞反应；Induce broad, functional cellular T-cell responses against SARS-CoV-2 variants;
·诱导抗SARS-CoV-2变体的特异性B细胞记忆；Induce specific B cell memory against SARS-CoV-2 variants;
·诱导可以有效地中和SARS-CoV-2病毒变体的功能性抗体；Induce functional antibodies that can effectively neutralize SARS-CoV-2 virus variants;
·诱导也可以有效地中和初始SARS-CoV-2病毒的功能性抗体；Induce functional antibodies that can also effectively neutralize the initial SARS-CoV-2 virus;
·通过诱导粘膜IgA抗体引发粘膜IgA免疫；· Triggering mucosal IgA immunity by inducing mucosal IgA antibodies;
·诱导充分平衡的B细胞和T细胞反应；Induce a well-balanced B-cell and T-cell response;
·诱导抗SARS-CoV-2变体的保护性免疫；Induce protective immunity against SARS-CoV-2 variants;
·抗SARS-CoV-2变体的免疫保护的快速起效；Rapid onset of immune protection against SARS-CoV-2 variants;
·诱导的抗SARS-CoV-2变体的免疫反应的持久性；Durability of induced immune responses against SARS-CoV-2 variants;
·没有增强由于疫苗接种或免疫病理作用引起的SARS-CoV-2感染；No enhancement of SARS-CoV-2 infection due to vaccination or immunopathological effects;
·没有由基于RNA的SARS-CoV-2疫苗引起的抗体依赖性增强(ADE)；No antibody-dependent enhancement (ADE) caused by RNA-based SARS-CoV-2 vaccines;
·应用疫苗(其可能在疫苗接种时导致不合需要的高反应原性)后没有过度诱导全身性细胞因子或趋化因子反应；The absence of excessive induction of systemic cytokine or chemokine responses following vaccine administration (which could result in undesirably high reactogenicity at the time of vaccination);
·疫苗充分的耐受性，无副作用，非毒性；The vaccine is well tolerated, has no side effects, and is non-toxic;
·基于RNA的疫苗的有利稳定性特征；Favorable stability characteristics of RNA-based vaccines;
·SARS-CoV-2变体疫苗产生的速度、适应性、简单性和可扩展性；The speed, adaptability, simplicity, and scalability of vaccine generation for SARS-CoV-2 variants;
·对于充分保护仅需要一次或两次接种的有利接种方案；· Favorable vaccination schedules that require only one or two doses for full protection;
·对于充分保护仅需要低疫苗剂量的有利接种方案；· Favorable vaccination schedules that require only low vaccine doses for adequate protection;
·对于充分保护仅需要低组合物/疫苗剂量的有利接种方案，这允许提供用于多价疫苗的RNA的不同抗原的组合；Favorable vaccination schedules requiring only low composition/vaccine doses for adequate protection, which allows providing combinations of different antigens of RNA for multivalent vaccines;
·现有抗SARS-CoV-2免疫的可加强性，优选诱导另外的抗SARS-CoV-2变体的免疫反应；· The ability to boost existing anti-SARS-CoV-2 immunity, preferably to induce additional immune responses against SARS-CoV-2 variants;
·在已经暴露于不同株或已经接种针对不同株的疫苗的受试者中诱导不同的、SARS-CoV-2株特异性的免疫反应；Inducing distinct, SARS-CoV-2 strain-specific immune responses in subjects who have been exposed to different strains or who have been vaccinated against different strains;
·诱导各种不同SARS-CoV-2变体上的广泛免疫反应；Induce broad immune responses against a variety of different SARS-CoV-2 variants;
在第一方面中，本发明提供了编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的RNA，其中相对于SEQ ID NO:1的序列，SARS-CoV-2刺突蛋白在对应于H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485,F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；Q506；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；V1176；T859；S247；Y248；L249；T250；P251；G252；G75；T76；D950；E154；G769；S254；Q613；F157；R158；Q957；D253；T95；F888；Q677；A67；Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187；W258；T19；V126；H245；S12；A899；G142；E156；K558；和/或Q52的位置处包含至少一个氨基酸置换、缺失或插入，其中RNA包含至少一个异源未翻译区。在某些实施方式中，RNA编码在来自SARS-CoV-2变体刺突蛋白(例如，来自包括但不限于以下的SARS-Cov-2株：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚))的位置处包含至少一个氨基酸置换、缺失或插入的SARS-CoV-2刺突蛋白。In a first aspect, the invention provides an RNA encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein, relative to the sequence of SEQ ID NO:1, the SARS-CoV-2 spike protein corresponds to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q4 93; S494; P499; T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118; L18; 138; R190; H655; T1027; S13; W1 52; L452; R346; P384; G447; G502; T748; A522; V1176; T859; S247; Y248; L249; T250; P251; G252; G75; T76; D950; ;Q957; D253; T95; F888; Q677; The RNA comprises at least one amino acid substitution, deletion or insertion at positions A67; Q414; N450; V483; G669; T732; Q949; Q1071; E1092; H1101; N1187; W258; T19; V126; H245; S12; A899; G142; E156; K558; and/or Q52, wherein the RNA comprises at least one heterologous untranslated region. In certain embodiments, the RNA encodes a spike protein from a SARS-CoV-2 variant (e.g., from SARS-Cov-2 strains including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia)).
在第二方面中，本发明提供了包含至少一个第一方面的RNA的组合物，优选免疫原性组合物。合适地，所述组合物包含至少一个在基于脂质的载体(优选脂质纳米颗粒(LNP))中配制的第一方面的RNA。在优选的实施方式中，第二方面涉及多价组合物，如包含具有不同氨基酸编码序列的编码SARS-CoV-2刺突蛋白的RNA的组合物(例如，来自超过一个SARS-CoV-2株的刺突蛋白，包括超过一个SARS-CoV-2变体株，例如，来自超过一个多个包括但不限于以下的SARS-Cov-2株的刺突蛋白：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚))。In a second aspect, the invention provides a composition, preferably an immunogenic composition, comprising at least one RNA of the first aspect. Suitably, the composition comprises at least one RNA of the first aspect formulated in a lipid-based carrier, preferably a lipid nanoparticle (LNP). In a preferred embodiment, the second aspect relates to a multivalent composition, such as a composition comprising RNA encoding SARS-CoV-2 spike proteins with different amino acid coding sequences (e.g., spike proteins from more than one SARS-CoV-2 strain, including more than one SARS-CoV-2 variant strain, for example, spike proteins from more than one SARS-Cov-2 strain including but not limited to the following: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia)).
在第三方面中，本发明提供了SARS-CoV-2变体疫苗，其中疫苗包含至少一个第一方面的RNA，或至少一种第二方面的组合物。在优选实施方式中，第二方面涉及多价SARS-CoV-2疫苗。在优选实施方式中，第三方面涉及SARS-CoV-2变体加强疫苗。SARS-CoV-2变体加强疫苗可以是针对一个或多个SARS-Cov-2株，包括但不限于：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a third aspect, the present invention provides a SARS-CoV-2 variant vaccine, wherein the vaccine comprises at least one RNA of the first aspect, or at least one composition of the second aspect. In a preferred embodiment, the second aspect relates to a multivalent SARS-CoV-2 vaccine. In a preferred embodiment, the third aspect relates to a SARS-CoV-2 variant booster vaccine. The SARS-CoV-2 variant booster vaccine can be for one or more SARS-Cov-2 strains, including but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在第四方面中，本发明提供了包含至少一个第一方面的RNA，和/或至少一个第二方面的组合物，和/或至少一个第三方面的SARS-CoV-2变体疫苗的试剂盒或部件套装。In a fourth aspect, the present invention provides a kit or a set of parts comprising at least one RNA of the first aspect, and/or at least one composition of the second aspect, and/or at least one SARS-CoV-2 variant vaccine of the third aspect.
在第五方面中，本发明提供了包含至少两种独立组分的组合，其中所述至少两种独立组分各自是第一方面的RNA种类，和/或第二方面的组合物，和/或第三方面的SARS-CoV-2变体疫苗，即，每种组分是针对不同SARS-Cov-2的RNA种类、组合物和/或SARS-Cov-2变体疫苗，其中所述两种独立组分可以各自针对包括但不限于以下的SARS-Cov-2变体：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a fifth aspect, the present invention provides a combination comprising at least two independent components, wherein each of the at least two independent components is an RNA species of the first aspect, and/or a composition of the second aspect, and/or a SARS-CoV-2 variant vaccine of the third aspect, i.e., each component is an RNA species, composition and/or SARS-Cov-2 variant vaccine against a different SARS-Cov-2, wherein the two independent components may each be against SARS-Cov-2 variants including but not limited to the following: C.1.2 (South Africa) , B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
本发明的更多方面涉及治疗或预防受试者的SARS-CoV-2感染的方法，核酸、组合物和疫苗的第一和第二医药用途。还提供了制备核酸、组合物或疫苗的方法。Further aspects of the invention relate to methods of treating or preventing SARS-CoV-2 infection in a subject, first and second medical uses of nucleic acids, compositions and vaccines. Methods of preparing nucleic acids, compositions or vaccines are also provided.
本发明的详细说明Detailed description of the invention
本申请与电子形式的序列表一起提交，该序列表是本申请说明书的一部分(WIPO标准ST.25)。序列表中包含的信息通过引用整体并入本文。本文中提及“SEQ ID NO”时，指序列表中具有相应标识符的相应核酸序列或氨基酸(aa)序列。对于许多序列，序列表还提供了额外的详细信息，例如关于某些结构特征、序列优化、GenBank(NCBI)或GISAID(epi)标识符，或者关于其编码能力的附加详细信息。特别地，此类信息是在WIPO标准ST.25序列表中的数字标识符＜223＞下提供的。因此，在所述数字标识符＜223＞下提供的信息被明确地整体包括在本文中，并且必须被理解为基础发明的说明书的整体部分。This application is submitted together with a sequence listing in electronic form, which is part of the specification of this application (WIPO Standard ST.25). The information contained in the sequence listing is incorporated herein by reference in its entirety. When "SEQ ID NO" is mentioned herein, it refers to the corresponding nucleic acid sequence or amino acid (aa) sequence with the corresponding identifier in the sequence listing. For many sequences, the sequence listing also provides additional detailed information, such as additional details about certain structural features, sequence optimization, GenBank (NCBI) or GISAID (epi) identifiers, or about its coding capabilities. In particular, such information is provided under the digital identifier <223> in the WIPO Standard ST.25 sequence listing. Therefore, the information provided under the digital identifier <223> is explicitly included in this article as a whole and must be understood as an integral part of the specification of the basic invention.
适用于SARS-CoV-2变体疫苗的RNA：RNA for SARS-CoV-2 variant vaccines :
在第一方面中，本发明涉及适用于SARS-CoV-2变体疫苗的RNA。In a first aspect, the invention relates to RNA suitable for use in SARS-CoV-2 variant vaccines.
必须注意到本发明的第一方面的内容中描述的特定特征和实施方式，即本发明的RNA，同样适用于第二方面(本发明的组合物)、第三方面(本发明的疫苗)、第四方面(本发明的试剂盒或部件套装)、第五方面(本发明的组合物)，或更多方面，包括医疗用途和治疗方法。It must be noted that the specific features and embodiments described in the context of the first aspect of the invention, i.e., the RNA of the invention, are also applicable to the second aspect (the composition of the invention), the third aspect (the vaccine of the invention), the fourth aspect (the kit or kit of parts of the invention), the fifth aspect (the composition of the invention), or more aspects, including medical uses and methods of treatment.
第一方面的RNA形成用于基于RNA的组合物或疫苗的基础。通常，基于蛋白质的疫苗，或减活疫苗，对于在发展中国家的使用是次佳的，因为它们的生产成本高。此外，基于蛋白质的疫苗，或减活疫苗需要的研发时间长并且不适于大流行病毒爆发的快速反应，如2019/2020年的SARS-CoV-2爆发。相反，根据本发明的基于RNA的疫苗允许非常快速和经济的生产。因此，与已知疫苗相比，基于本发明的RNA的疫苗可以显著更便宜且更快地生产，这特别是对于在发展中国家的使用是非常有利的。基于RNA的疫苗的再一个优势是其与基于蛋白质或肽的疫苗相比的温度稳定性。The RNA of the first aspect forms the basis for RNA-based compositions or vaccines. Typically, protein-based vaccines, or attenuated vaccines, are suboptimal for use in developing countries because of their high production costs. In addition, protein-based vaccines, or attenuated vaccines require long development times and are not suitable for rapid response to pandemic virus outbreaks, such as the SARS-CoV-2 outbreak in 2019/2020. In contrast, RNA-based vaccines according to the present invention allow very rapid and economical production. Therefore, compared to known vaccines, vaccines based on the RNA of the present invention can be significantly cheaper and faster to produce, which is particularly advantageous for use in developing countries. Another advantage of RNA-based vaccines is their temperature stability compared to protein- or peptide-based vaccines.
在特别优选的实施方式中，本发明的第一方面涉及包含至少一个编码至少一种来自SARS-CoV-2刺突蛋白的抗原肽或蛋白质或其免疫原性片段或免疫原性变体的编码序列的RNA，其中RNA包含至少一个异源未翻译区(UTR)和其中SARS-CoV-2刺突蛋白包含至少一个选自SARS-CoV-2变体的氨基酸置换和任选地SARS-Co-2株的稳定突变。In a particularly preferred embodiment, the first aspect of the invention relates to an RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the RNA comprises at least one heterologous untranslated region (UTR) and wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution selected from a SARS-CoV-2 variant and optionally a stabilizing mutation of a SARS-Co-2 strain.
本文的术语“来自SARS-CoV-2刺突蛋白的抗原肽或蛋白质”表示(i)作为具有与SARS-CoV-2变体蛋白(或其片段)相同的抗原肽或蛋白质(或其片段)的氨基酸序列的SARS-CoV-2刺突蛋白的抗原，或(ii)源自具有与相应的SARS-CoV-2变体蛋白(或其片段)不同的抗原肽或蛋白质(或其片段)的氨基酸序列的SARS-CoV-2刺突蛋白的抗原。例如，相应的SARS-CoV-2刺突蛋白可以包含至少一个选自SARS-CoV-2变体的氨基酸置换、插入或缺失和/或至少一个融合前稳定突变。The term "antigenic peptide or protein from the SARS-CoV-2 spike protein" herein refers to (i) an antigen of the SARS-CoV-2 spike protein having the same amino acid sequence of an antigenic peptide or protein (or a fragment thereof) as a SARS-CoV-2 variant protein (or a fragment thereof), or (ii) an antigen of the SARS-CoV-2 spike protein having an amino acid sequence of an antigenic peptide or protein (or a fragment thereof) different from that of the corresponding SARS-CoV-2 variant protein (or a fragment thereof). For example, the corresponding SARS-CoV-2 spike protein may comprise at least one amino acid substitution, insertion or deletion selected from a SARS-CoV-2 variant and/or at least one prefusion stabilizing mutation.
本文的术语“免疫原性片段”或“免疫原性变体”表示能够在受试者中引发免疫反应的相应SARS-CoV-2抗原的任何片段/变体。优选地，第一方面的RNA的肌内或皮内施用导致编码的SARS-CoV-2刺突蛋白在受试者中的表达。The term "immunogenic fragment" or "immunogenic variant" herein refers to any fragment/variant of the corresponding SARS-CoV-2 antigen that is capable of eliciting an immune response in a subject. Preferably, intramuscular or intradermal administration of the RNA of the first aspect results in expression of the encoded SARS-CoV-2 spike protein in a subject.
如本文使用的术语“表达”是指SARS-CoV-2刺突蛋白的产生，其中所述SARS-CoV-2刺突蛋白由第一方面的RNA的编码序列来提供。例如，RNA的“表达”是指经由RNA翻译成多肽来产生蛋白质(例如，将所述RNA施用于细胞或受试者后)，例如，翻译成是或源自SARS-CoV-2冠状病毒的肽或蛋白质。术语“表达”和术语“产生”在本文可互换使用。此外，术语“表达”优选涉及在RNA施用于细胞或生物体后某些肽或蛋白质的产生。As used herein, the term "expression" refers to the production of a SARS-CoV-2 spike protein, wherein the SARS-CoV-2 spike protein is provided by the coding sequence of the RNA of the first aspect. For example, "expression" of RNA refers to the production of a protein (e.g., after the RNA is administered to a cell or subject) via translation of the RNA into a polypeptide, e.g., translation into a peptide or protein that is or is derived from the SARS-CoV-2 coronavirus. The term "expression" and the term "production" are used interchangeably herein. In addition, the term "expression" preferably relates to the production of certain peptides or proteins after the RNA is administered to a cell or an organism.
在优选的实施方式中，本发明的RNA适用于SARS-CoV-2变体疫苗。In a preferred embodiment, the RNA of the present invention is suitable for use in a SARS-CoV-2 variant vaccine.
SARS-CoV-2刺突蛋白是I型病毒融合蛋白，其作为三聚体存在于病毒表面上，每个单体由头(S1)和茎(S2)组成。单个前体S多肽形成同型二聚体并且在高尔基体内经历糖基化以及加工以除去信号肽，并被细胞蛋白酶切割以生成单独的S1和S2多肽链，其作为同型三聚体内的S1/S2原聚体保持结合并且因此是杂二聚体的三聚体。刺突糖蛋白的S1结构域包括(最可能)与血管紧张素转换酶2受体啮合并介导病毒融合至宿主细胞中的受体结合结构域(RBD)、可能与靶细胞形成最初接触的N-末端结构域和2个子结构域，其全部都对中和抗体敏感。S2结构域由六个螺旋束融合芯组成，其涉及与宿主内含体膜的膜融合以及也是中和的靶标。S2亚基还包含两个七肽重复序列(HR1和HR2)和融合糖蛋白典型的中心螺旋、跨膜结构域和胞质尾结构域。The SARS-CoV-2 spike protein is a type I viral fusion protein that exists as a trimer on the surface of the virus, with each monomer consisting of a head (S1) and a stem (S2). The single precursor S polypeptide forms a homodimer and undergoes glycosylation and processing in the Golgi to remove the signal peptide, and is cleaved by cellular proteases to generate separate S1 and S2 polypeptide chains, which remain bound as S1/S2 protomers within the homotrimer and are therefore trimers of heterodimers. The S1 domain of the spike glycoprotein includes (most likely) a receptor binding domain (RBD) that engages with the angiotensin-converting enzyme 2 receptor and mediates viral fusion into host cells, an N-terminal domain that may form initial contact with target cells, and 2 subdomains, all of which are sensitive to neutralizing antibodies. The S2 domain consists of a six-helix bundle fusion core, which is involved in membrane fusion with the host endosomal membrane and is also a target for neutralization. The S2 subunit also contains two heptad repeat sequences (HR1 and HR2) and the central helix, transmembrane domain and cytoplasmic tail domain typical of fusion glycoproteins.
在本发明的情况中，可以使用选自或源自SARS-CoV-2变体并且包含与SEQ ID NO:1相比的至少一个氨基酸置换、缺失或插入的任何刺突蛋白并且可以合适地由第一方面的编码序列或RNA来编码。在本发明的范围中，进一步地至少一个抗原肽或蛋白质可以包含合成工程化的或人工的SARS-CoV-2刺突蛋白或由其组成。术语“合成工程化的”SARS-CoV-2刺突蛋白，或术语“人工的SARS-CoV-2刺突蛋白”或术语“重组的”SARS-CoV-2刺突蛋白涉及不是天然出现的蛋白质。因此，“人工的SARS-CoV-2刺突蛋白”或“合成工程化的SARS-CoV-2刺突蛋白”或术语“重组的”SARS-CoV-2刺突蛋白可以例如与天然产生的SARS-CoV-2刺突蛋白相比至少一个氨基酸不同(例如，与天然产生的SARS-CoV-2刺突蛋白相比，包含一个或多个异源/引入的氨基酸)，和/或可以包含另外的异源肽或蛋白质元件，和/或可以是N-末端或C-末端延伸的或截短的。In the context of the present invention, any spike protein selected from or derived from a SARS-CoV-2 variant and comprising at least one amino acid substitution, deletion or insertion compared to SEQ ID NO: 1 may be used and may be suitably encoded by the coding sequence or RNA of the first aspect. In the context of the present invention, further at least one antigenic peptide or protein may comprise or consist of a synthetically engineered or artificial SARS-CoV-2 spike protein. The term "synthetically engineered" SARS-CoV-2 spike protein, or the term "artificial SARS-CoV-2 spike protein" or the term "recombinant" SARS-CoV-2 spike protein relates to a protein that does not occur naturally. Thus, an “artificial SARS-CoV-2 spike protein” or “synthetically engineered SARS-CoV-2 spike protein” or the term “recombinant” SARS-CoV-2 spike protein can, for example, differ in at least one amino acid compared to the naturally occurring SARS-CoV-2 spike protein (e.g., comprise one or more heterologous/introduced amino acids compared to the naturally occurring SARS-CoV-2 spike protein), and/or can comprise additional heterologous peptide or protein elements, and/or can be N-terminally or C-terminally extended or truncated.
在下文中，详细描述了由本发明的RNA提供的优选抗原肽或蛋白质序列。In the following, preferred antigenic peptide or protein sequences provided by the RNA of the present invention are described in detail.
应当注意，在提及SARS-CoV-2刺突蛋白(S)中的氨基酸(aa)残基及其位置的情况下，本文使用的任何编号-除非另外指出-涉及根据SEQ ID NO:1的原始SARS-CoV-2冠状病毒分离株EPI_ISL_402128的相应刺突蛋白(S)中的相应氨基酸残基的位置。在整个公开中，相应的氨基酸位置示例性地指示原始SARS-CoV-2冠状病毒分离株EPI_ISL_402128的刺突蛋白(S)(SEQ ID NO:1)。It should be noted that, in the context of referring to amino acid (aa) residues and their positions in the SARS-CoV-2 spike protein (S), any numbering used herein - unless otherwise indicated - relates to the position of the corresponding amino acid residue in the corresponding spike protein (S) of the original SARS-CoV-2 coronavirus isolate EPI_ISL_402128 according to SEQ ID NO: 1. Throughout the disclosure, the corresponding amino acid positions exemplarily indicate the spike protein (S) of the original SARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1).
如本文使用的蛋白质注释涉及作为参考蛋白的SEQ ID NO:1。原始SARS-CoV-2冠状病毒参考蛋白的全长刺突蛋白(S)具有1273个氨基酸残基，并且包含以下元件：Protein annotation as used herein refers to SEQ ID NO: 1 as a reference protein. The full-length spike protein (S) of the original SARS-CoV-2 coronavirus reference protein has 1273 amino acid residues and contains the following elements:
-分泌信号肽：氨基酸位置aa 1至aa 15(参见SEQ ID NO:28)- Secretory signal peptide: amino acid positions aa 1 to aa 15 (see SEQ ID NO: 28)
-刺突蛋白片段S1：氨基酸位置aa 1至aa 681(参见SEQ ID NO:27)- Spike protein fragment S1: amino acid positions aa 1 to aa 681 (see SEQ ID NO: 27)
-S1-N-末端结构域(S1-NTD)：氨基酸位置aa 13至aa 303(参见SEQ ID NO:26992- S1-N-terminal domain (S1-NTD): amino acid positions aa 13 to aa 303 (see SEQ ID NO: 26992
-受体结合结构域(RBD)：氨基酸位置aa 319至aa 541(参见SEQ ID NO:13243)- Receptor binding domain (RBD): amino acid positions aa 319 to aa 541 (see SEQ ID NO: 13243)
-临界中和结构域(critical neutralization domain)(CND)：氨基酸位置aa 329至aa 529(参见SEQ ID NO:13310)- Critical neutralization domain (CND): amino acid positions aa 329 to aa 529 (see SEQ ID NO: 13310)
-刺突蛋白片段S2：氨基酸位置aa 682至aa 1273(参见SEQ ID NO:30)- Spike protein fragment S2: amino acid positions aa 682 to aa 1273 (see SEQ ID NO: 30)
-跨膜结构域(TM)：氨基酸位置aa 1212至aa 1273(参见SEQ ID NO:49)- Transmembrane domain (TM): amino acid positions aa 1212 to aa 1273 (see SEQ ID NO: 49)
-跨膜结构域(TMflex)：氨基酸位置aa 1148至aa 1273(参见SEQ ID NO:13176)- Transmembrane domain (TMflex): amino acid positions aa 1148 to aa 1273 (see SEQ ID NO: 13176)
-Furine切割位点区(S1/S2)：氨基酸位置aa 681至aa 685(参见SEQ ID NO:26994)-Furine cleavage site region (S1/S2): amino acid positions aa 681 to aa 685 (see SEQ ID NO: 26994)
应当注意，氨基酸水平上的变化在源自不同的SARS-CoV-2分离株或SARS-CoV-2变体的刺突蛋白之间天然发生。在本发明的情况中，这样的氨基酸变化可以应用于如本文所述的源自刺突蛋白的抗原肽或蛋白质。合适地，以以下方式选择氨基酸变化或突变：1)诱导抗取代或突变由其衍生的SARS-CoV-2病毒变体的免疫反应和/或(2)产生对于诱导免疫反应所需的抗原肽或蛋白质(例如，源自刺突蛋白的并且是融合前形式的抗原肽或蛋白质)。It should be noted that variations at the amino acid level occur naturally between spike proteins derived from different SARS-CoV-2 isolates or SARS-CoV-2 variants. In the context of the present invention, such amino acid changes can be applied to antigenic peptides or proteins derived from the spike protein as described herein. Suitably, the amino acid changes or mutations are selected in such a way as to: 1) induce an immune response against the SARS-CoV-2 viral variant from which the substitution or mutation is derived and/or (2) produce an antigenic peptide or protein (e.g., an antigenic peptide or protein derived from the spike protein and in a pre-fusion form) required for inducing an immune response.
因此，在特别优选的实施方式，本发明的RNA包含至少一个编码至少一种SARS-CoV-2刺突蛋白，或其免疫原性片段或免疫原性变体的编码序列，其中SARS-CoV-2刺突蛋白包含至少一个选自SARS-CoV-2变体的氨基酸置换、缺失或插入。Therefore, in a particularly preferred embodiment, the RNA of the present invention comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein, or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion selected from a SARS-CoV-2 variant.
在该情况中，本文的术语“至少一个选自SARS-CoV-2变体的氨基酸置换、缺失或插入”表示SARS-CoV-2刺突蛋白(或其片段)中与原始SARS-CoV-2刺突蛋白(根据SEQ ID NO:1参考株)不同的至少一个氨基酸位置。In this context, the term "at least one amino acid substitution, deletion or insertion selected from a SARS-CoV-2 variant" herein refers to at least one amino acid position in the SARS-CoV-2 spike protein (or a fragment thereof) that is different from the original SARS-CoV-2 spike protein (according to the reference strain of SEQ ID NO: 1).
在优选实施方式中，SARS-CoV-2变体选自或源自以下SARS-CoV-2谱系：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a preferred embodiment, the SARS-CoV-2 variant is selected from or derived from the following SARS-CoV-2 lineages: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在特别优选的实施方式中，SARS-CoV-2变体选自或源自以下SARS-CoV-2谱系：B.1.351(南非)、P.1(巴西)、B.1.617.1(印度)、B.1.617.2(印度)、B.1.617.3(印度)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529,BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)。In a particularly preferred embodiment, the SARS-CoV-2 variant is selected from or derived from the following SARS-CoV-2 lineages: B.1.351 (South Africa), P.1 (Brazil), B.1.617.1 (India), B.1.617.2 (India), B.1.617.3 (India), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
因此，本文提供的并考虑为本发明情况中的合适抗原的每种刺突蛋白可以具有一个或多个以下的氨基酸变异或突变(根据参考SEQ ID NO:1的氨基酸位置)，如列表1中所提供的。以下提供的变异或突变源自新出现的SARS-CoV-2病毒变体，并且可以整合至由本发明的RNA编码的刺突蛋白中：Thus, each of the spike proteins provided herein and considered to be suitable antigens in the context of the present invention may have one or more of the following amino acid variations or mutations (according to the amino acid positions with reference to SEQ ID NO: 1), as provided in List 1. The variations or mutations provided below are derived from emerging SARS-CoV-2 virus variants and may be incorporated into the spike protein encoded by the RNA of the present invention:
列表1A：用于置换、缺失和/或插入的氨基酸位置Table 1A: Amino Acid Positions for Substitutions, Deletions and/or Insertions
H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485,F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；Q506；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；V1176；T859；S247；Y248；L249；T250；P251；G252；G75；T76；D950；E154；G769；S254；Q613；F157；R158；Q957；D253；T95；F888；Q677；A67；Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187；W258；T19；V126；H245；S12；A899；G142；E156；K558；G339；P9；C136；Y449；L24；P25；P26；A27；V213；S371；T376；D405；A701；I210；D936；S939；R357；R682；R683；A684；R685；V143、Y144、Y145、N211、L212、R214、E241、G339、S371、S373、S375、N440、G496、Q498、Y505、T547、D614、N679、P681、N764、D796、N856、Q954、N969、L981或Q52(相对于SEQ ID NO:1的序列)。H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; S494; P499; T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982 ;D1118;L18;D80;D215;L242;A243;L244;R246;A701;T20;P26;D138;R190;H655;T1027;S13;W152;L452;R346;P384;G447;G502;T748;A522;V11 76 ;T859;S247;Y248;L249;T250;P251;G252;G75;T76;D950;E154;G769;S254; Q613; F157; R158; Q957; D253; T95; F888; Q677; A67; Q414; N450; V483; G669; T732; Q949; Q1071; E1092; H1101; N1187; W258; T19; V126; H245; S12; A8 99; G142; E156; K558; G339; P9; C136; Y449; L24; P25; P26; A27; V213; S371; T37 6; D405; A701; I210; D936; S939; R357; R682; R683; A684; R685; V143, Y144, Y145, N211, L212, R214, E241, G339, S371, S373, S375, N440, G496, Q498, Y505, T547, D614, N679, P681, N764, D796, N856, Q954, N969, L981 or Q52 (relative to the sequence of SEQ ID NO: 1).
列表1B：氨基酸置换、缺失或插入List 1B: Amino Acid Substitutions, Deletions or Insertions
H69del；V70del；A222V；Y453F；S477N；I692V；R403K；K417N；N437S；N439K；V445A；V445I；V445F；G446V；G446S；G446A；L455F；F456L；K458N；A475V；G476S；G476A；S477I；S477R；S477G；S477T；T478I；T478K；T478R；T478A；E484Q；E484K；E484A；E484D；G485R；G485S,F486L；N487I；Y489H；F490S；F490L；Q493L；Q493K；S494P；S494L；P499L；T500I；N501Y；N501T；N501S；V503F；V503I；G504D；Y505W；Q506K；Q506H；Y144del；A570D；P681H；T716I；S982A；D1118H；L18F；D80A；D215G；L242del；A243del；L244del；L242del；A243del；L244del；R246I；A701V；T20N；P26S；D138Y；R190S；H655Y；T1027I；S13I；W152C；L452R；R346T；P384L；L452M；F456A；F456K；F456V；E484P；K417T；G447V；L452Q；A475S；F486I；F490Y；Q493R；S494A；P499H；P499S；G502V；T748K；A522S；V1176F；T859N；S247del；Y248del；L249del；T250del；P251del；G252del；R246del；S247del；Y248del；L249del；T250del；P251del；G252del；G75V；T76I；G75V；T76I；D950N；P681R；E154K；G769V；S254F；Q613H；F157L；F157del；R158del；Q957R；D253G；T95I；F888L；Q677H；A67V；Q414K；N450K；V483A；G669S；T732A；Q949R；Q1071H；E1092K；H1101Y；N1187D；W258L；V70F；T19R；T19I；Y144T；Y145S；ins145N；R346K；R346S；V126A；H245Y；ins214TDR；S12F；W152R；A899S；G142D；E156G；K558N；P9L；C136F；Y449H；L24del；P25del；P26del；A27S；V213G；S371F；T376A；D405N；D253N；Y144S；I210T；D936N；S939F；W152L；T20I；R357K；D796H；Y145H；R682del；R683del；A684del；R685del；A701V；V143del,Y144del,Y145del,Y145N；N211del、L212del、L212I、ins214EPE、E241del、G339D、S371L、S373P、S375F、N440K、G496S、Q498R、Y505H；T547K、D614G、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F或Q52R(相对于SEQ ID NO:1的序列)。H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T4 78R; T478A; E484Q; E484K; E484A; E484D; G485R; G485S, F486L; N487I; Y489H; F490S; F490L; T; N501S; V503F; V503I; G504D; Y505W; Q506K; Q506H; Y144del ;A570D;P681H;T716I;S982A;D1118H;L18F;D80A;D215G;L242del;A243del;L244del;L242del;A243del;L244del;R246I;A701V;T20N;P26S;D138Y;R190S;H65 5Y; T1027I; S13I; W152C; L452R ;R346T;P384L; L452M; F456A; F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; P499H; P499S; 247del; Y248del; L249del; T250del; P251del; G252del; R246 del; S247del; Y248del; L249del; T250del; P251del; G252del; G75V; T76I; G75V; T76I; D950N; P681R; G; T95I; F888L; Q677H; A67V; Q414K; N4 50K; V483A; G669S; T732A; Q949R; Q1071H; E1092K; H1101Y; N1187D; W258L; V70F; T19R; T19I; 4TDR; S12F; W152R; A899S; G142D; E156G; K558N; P9L; C13 6F; Y449H; L24del; P25del; P26del; A27S; V213G; S371F; T376A; D405N; D253N; Y144S; 3del;A684del;R685del;A701V; V143del,Y144del, Y145del,Y145N; N211del, L212del, L212I, ins214EPE, E241del, G339D, S371L, S373P, S375F, N440K, G496S, Q498R, Y505H; T547K, D614G, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F or Q52R (sequence relative to SEQ ID NO: 1).
在优选的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQID NO:1的序列在对应于H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485,F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；Q506；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；V1176；T859；S247；Y248；L249；T250；P251；G252；G75；T76；D950；E154；G769；S254；Q613；F157；R158；Q957；D253；T95；F888；Q677；A67；Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187；W258；T19；V126；H245；S12；A899；G142；E156；K558；和/或Q52的位置处包含至少一个氨基酸置换、缺失或插入，其中RNA包含至少一个异源未翻译区。在某些方面中，RNA不包含包括SEQ ID NO:268序列的3’UTR。在某些方面中，RNA包含包括SEQ ID NO:268的序列的3’UTR。In a preferred embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein corresponds to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P496; 9; T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118; L18; D80; D215; L242; A243; 5;T1027;S13;W152;L452 ; R346; P384; G447; G502; T748; A522; V1176; T859; S247; Y248; L249; T250; P251; G252; G75; T76; D950; 253;T95;F888;Q677;A67; Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187；W258；T19；V126；H245；S12；A899；G142；E156；K558；And/or Q52 comprises at least one amino acid substitution, deletion or insertion, wherein the RNA comprises at least one heterologous untranslated region. In some aspects, the RNA does not comprise a 3'UTR comprising the sequence of SEQ ID NO:268. In some aspects, the RNA comprises a 3'UTR comprising the sequence of SEQ ID NO:268.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于H69del；V70del；A222V；Y453F；S477N；I692V；R403K；K417N；N437S；N439K；V445A；V445I；V445F；G446V；G446S；G446A；L455F；F456L；K458N；A475V；G476S；G476A；S477I；S477R；S477G；S477T；T478I；T478K；T478R；T478A；E484Q；E484K；E484A；E484D；G485R；G485S,F486L；N487I；Y489H；F490S；F490L；Q493L；Q493K；S494P；S494L；P499L；T500I；N501Y；N501T；N501S；V503F；V503I；G504D；Y505W；Q506K；Q506H；Y144del；A570D；P681H；T716I；S982A；D1118H；L18F；D80A；D215G；L242del；A243del；L244del；L242del；A243del；L244del；R246I；A701V；T20N；P26S；D138Y；R190S；H655Y；T1027I；S13I；W152C；L452R；R346T；P384L；L452M；F456A；F456K；F456V；E484P；K417T；G447V；L452Q；A475S；F486I；F490Y；Q493R；S494A；P499H；P499S；G502V；T748K；A522S；V1176F；T859N；S247del；Y248del；L249del；T250del；P251del；G252del；R246del；S247del；Y248del；L249del；T250del；P251del；G252del；G75V；T76I；G75V；T76I；D950N；P681R；E154K；G769V；S254F；Q613H；F157L；F157del；R158del；Q957R；D253G；T95I；F888L；Q677H；A67V；Q414K；N450K；V483A；G669S；T732A；Q949R；Q1071H；E1092K；H1101Y；N1187D；W258L；V70F；T19R；Y144T；Y145S；ins145N；R346K；R346S；V126A；H245Y；ins214TDR；S12F；W152R；A899S；G142D；E156G；K558N；和/或Q52R的位置处包含至少一个氨基酸置换、缺失或插入。In a particularly preferred embodiment, the SARS-CoV-2 spike protein corresponds to H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V ;G476S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; F490L; Q493L; Q493K; S494P; S494L; P499L; T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; Q506H; 8F;D80A; D215G; L242del; A243del; L244del; L242del; A243del; L244del; R246I; A701V; T20N; P26S; D138Y; R190S; 2M; F456A; F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; P499H; P499S; 7del; Y248del; L249del; T250del; P251del; G252del; R246del; S247del; Y248del; L249del; T250del; S254F; Q613H; F157L; F157del; R158del; Q957R; D253G; T95I; F888L; Q677H; A67V; Q414K; N450K; V483A; G669S; T732A; Q949R; Q1071H; E1092K; H1101Y; N1 187D;W 258L; V70F; T19R; Y144T; Y145S; ins145N; R346K; R346S; V126A; H245Y; ins214TDR; S12F; W152R; A899S; G142D; E156G; K558N; and/or Q52R contain at least one amino acid substitution, deletion or insertion.
在某些实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQ IDNO:1的序列在对应于H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485、F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；和/或Q506的位置处包含至少一个氨基酸置换、缺失或插入。因此，在一些实施方式中，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于H69del；V70del；A222V；Y453F；S477N；I692V；R403K；K417N；N437S；N439K；V445A；V445I；V445F；G446V；G446S；G446A；L455F；F456L；K458N；A475V；G476S；G476A；S477I；S477R；S477G；S477T；T478I；T478K；T478R；T478A；E484Q；E484K；E484A；E484D；G485R；G485S、F486L；N487I；Y489H；F490S；F490L；Q493L；Q493K；S494P；S494L；P499L；T500I；N501Y；N501T；N501S；V503F；V503I；G504D；Y505W；Q506K；和/或Q506H的位置处包含至少一个氨基酸置换、缺失或插入。In certain embodiments, an RNA is provided that comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion, or insertion relative to the sequence of SEQ ID NO: 1 at a position corresponding to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499; T500; N501; V503; G504; Y505; and/or Q506. Thus, in some embodiments, the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion, or insertion relative to the sequence of SEQ ID NO: 1 at a position corresponding to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499; T500; N501; V503; G504; Y505; and/or Q506. The sequence of NO:1 corresponds to H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T478R ; T478A; E484Q; E484K; E484A; E484D; G485R; G485S, F486L; N487I; Y489H; F490S; F490L; Q493L; Q493K; S494P; S494L; P499L; T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; and/or Q506H contain at least one amino acid substitution, deletion or insertion.
在进一步的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQID NO:1的序列在对应于H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485,F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；Q506；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；或V1176的位置处包含至少一个氨基酸置换、缺失或插入。因此，在一些实施方式，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于H69del；V70del；A222V；Y453F；S477N；I692V；R403K；K417N；N437S；N439K；V445A；V445I；V445F；G446V；G446S；G446A；L455F；F456L；K458N；A475V；G476S；G476A；S477I；S477R；S477G；S477T；T478I；T478K；T478R；T478A；E484Q；E484K；E484A；E484D；G485R；G485S,F486L；N487I；Y489H；F490S；F490L；Q493L；Q493K；S494P；S494L；P499L；T500I；N501Y；N501T；N501S；V503F；V503I；G504D；Y505W；Q506K；Q506H；Y144del；A570D；P681H；T716I；S982A；D1118H；L18F；D80A；D215G；L242del；A243del；L244del；L242del；A243del；L244del；R246I；A701V；T20N；P26S；D138Y；R190S；H655Y；T1027I；S13I；W152C；L452R；R346T；P384L；L452M；F456A；F456K；F456V；E484P；K417T；G447V；L452Q；A475S；F486I；F490Y；Q493R；S494A；P499H；P499S；G502V；T748K；A522S；和/或V1176F的位置处包含至少一个氨基酸置换、缺失或插入。In a further embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein corresponds to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499; T500; N501; V503; G504; Y505; Q506; 6；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；or V1176. Thus, in some embodiments, the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion relative to SEQ ID The sequence of NO:1 corresponds to H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477 7T; T478I; T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; L; T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y5 05W; Q506K; Q506H; Y144del; A570D; P681H; T716I; S982A; D1118H; L18F; D80A; D215G; L242del; P26S; D138Y; R190S; H655Y; T1027I; S 13I; W152C; L452R; R346T; P384L; L452M; F456A; F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; P499H; P499S; G502V; T748K; A522S; and/or V1176F contain at least one amino acid substitution, deletion or insertion.
在进一步优选的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于T859；R246；S247；Y248；L249；T250；P251；G252；G75；T76；D950；E154；G769；S254；Q613；F157；Q957；D253；T95；F888；Q677；A67；Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187；F157；R158；W258；T19；H245；S12；A899；G142；E156；K558和/或Q52的位置处包含至少一个氨基酸置换、缺失或插入。因此，在一些实施方式中，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于T859N；S247del；Y248del；L249del；T250del；P251del；G252del；R246del；S247del；Y248del；L249del；T250del；P251del；G252del；G75V；T76I；G75V；T76I；D950N；P681R；E154K；G769V；S254F；Q613H；F157L；Q957R；D253G；T95I；F888L；Q677H；A67V；Q414K；N450K；V483A；G669S；T732A；Q949R；Q1071H；E1092K；H1101Y；N1187D；F157del；R158del；W258L；V70F；T19R；Y144T；Y145S；ins145N；R346K；R346S；V126A；H245Y；ins214TDR；S12F；W152R；A899S；G142D；E156G；K558N和/或Q52R的位置处包含至少一个氨基酸置换、缺失或插入。In a further preferred embodiment, an RNA comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof is provided, wherein the SARS-CoV-2 spike protein is SEQ ID The sequence of NO:1 comprises at least one amino acid substitution, deletion or insertion at a position corresponding to T859; R246; S247; Y248; L249; T250; P251; G252; G75; T76; D950; E154; G769; S254; Q613; F157; Q957; D253; T95; F888; Q677; A67; Q414; N450; V483; G669; T732; Q949; Q1071; E1092; H1101; N1187; F157; R158; W258; T19; H245; S12; A899; G142; E156; K558 and/or Q52. Thus, in some embodiments, the SARS-CoV-2 spike protein is present in the sequence corresponding to T859N; S247del; Y248del; L249del; T250del; P251del; G252del; R246del; S247del; Y248del; L249del; T250del; P251del; G252del; G75V; T76I; G75V; T76I; D950N; P681R; E154K; G769V; S254F; Q613H; F157L; Q957R; D253G; T95I; F888L; Q677H; A67 V; Q414K; N450K; V483A; G669S; T732A; Q949R; Q1071H; E1092K; H1101Y; N1187D; F157del; R158del; W258L; V70F; T19R; Y144T; Y145S; ins145N; R346K; R346S; V126A; H245Y; ins214TDR; S12F; W152R; A899S; G142D; E156G; K558N and/or Q52R contain at least one amino acid substitution, deletion or insertion.
在再进一步的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于D614；H49；V367；P1263；V483；S939；S943；L5；L8；S940；C1254；Q239；M153；V1040；A845；Y145；A831；和/或M1229的位置处包含至少一个氨基酸置换、缺失或插入。因此，在一些实施方式中，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于D614G；H49Y；V367F；P1263L；V483A；S939F；S943P；L5F；L8V；S940F；C1254F；Q239K；M153T；V1040F；A845S；Y145H；A831V；和/或M1229I的位置处包含至少一个氨基酸置换、缺失或插入。In yet a further embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion at a position corresponding to D614; H49; V367; P1263; V483; S939; S943; L5; L8; S940; C1254; Q239; M153; V1040; A845; Y145; A831; and/or M1229 relative to the sequence of SEQ ID NO:1. Thus, in some embodiments, the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion, or insertion relative to the sequence of SEQ ID NO:1 at a position corresponding to D614G; H49Y; V367F; P1263L; V483A; S939F; S943P; L5F; L8V; S940F; C1254F; Q239K; M153T; V1040F; A845S; Y145H; A831V; and/or M1229I.
在再进一步的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于H69；V70；A222；Y453；S477；I692；R403；K417；N437；N439；V445；G446；L455；F456；K458；A475；G476；T478；E484；G485,F486；N487；Y489；F490；Q493；S494；P499；T500；N501；V503；G504；Y505；Q506；Y144；A570；P681；T716；S982；D1118；L18；D80；D215；L242；A243；L244；R246；A701；T20；P26；D138；R190；H655；T1027；S13；W152；L452；R346；P384；G447；G502；T748；A522；V1176；T859；S247；Y248；L249；T250；P251；G252；G75；T76；D950；E154；G769；S254；Q613；F157；Q957；D253；T95；F888；Q677；A67；Q414；N450；V483；G669；T732；Q949；Q1071；E1092；H1101；N1187和/或Q52的位置处包含至少一个氨基酸置换或缺失。因此，在一些实施方式中，SARS-CoV-2刺突蛋白在对应于H69del；V70del；A222V；Y453F；S477N；I692V；R403K；K417N；N437S；N439K；V445A；V445I；V445F；G446V；G446S；G446A；L455F；F456L；K458N；A475V；G476S；G476A；S477I；S477R；S477G；S477T；T478I；T478K；T478R；T478A；E484Q；E484K；E484A；E484D；G485R；G485S,F486L；N487I；Y489H；F490S；F490L；Q493L；Q493K；S494P；S494L；P499L；T500I；N501Y；N501T；N501S；V503F；V503I；G504D；Y505W；Q506K；Q506H；Y144del；A570D；P681H；T716I；S982A；D1118H；L18F；D80A；D215G；L242del；A243del；L244del；L242del；A243del；L244del；R246I；A701V；T20N；P26S；D138Y；R190S；H655Y；T1027I；S13I；W152C；L452R；R346T；P384L；L452M；F456A；F456K；F456V；E484P；K417T；G447V；L452Q；A475S；F486I；F490Y；Q493R；S494A；P499H；P499S；G502V；T748K；A522S；V1176F；T859N；S247del；Y248del；L249del；T250del；P251del；G252del；R246del；S247del；Y248del；L249del；T250del；P251del；G252del；G75V；T76I；G75V；T76I；D950N；P681R；E154K；G769V；S254F；Q613H；F157L；F157del；R158del；Q957R；D253G；T95I；F888L；Q677H；A67V；Q414K；N450K；V483A；G669S；T732A；Q949R；Q1071H；E1092K；H1101Y；N1187D；W258L；V70F；T19R；Y144T；Y145S；R346K；R346S；V126A；H245Y；S12F；W152R；A899S；G142D；E156G；K558N；和/或Q52R的位置处包含至少一个氨基酸置换或缺失。In yet a further embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein corresponds to H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490; 490; Q493; S494; P499; T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118; L18; D80; ;P26 ;D138;R190;H655;T1027;S13;W152;L452;R346;P384;G447;G502;T748;A522;V1176;T859;S247;Y248;L249;T250;P251;G252;G75;T76;D950;E154;G769;S254;Q613;F157;Q957;D253;T95;F888;Q677;A67;Q414;N450;V483;G669;T732;Q949;Q1071;E1092;H1101;N1187 and/or Q52 contain at least one amino acid substitution or deletion. Thus, in some embodiments, the SARS-CoV-2 spike protein is in the region corresponding to H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; G485S, F486L; N487I; Y489H ;F490S; F490L; Q493L; Q493K; S494P; S494L; P499L; T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; Q506H; T716I;S98 2A; D1118H; L18F; D80A; D215G; L242del; A243del; L244del; L242del; A243del; L244del; R246I; A701V; T20N; P26S; D138Y; R190S; H655Y; T1027I; S13I; W152C; L 452R; R346T; P384L; L452M; F456A; F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; 22S; V1176F; T859N; S247del; Y248del; L249del; T250del; P251del; G252del; R246del; S247del; Y248del; L249del; 0N; P681R; E154K; G769V; S254F; Q613H; F157L; F157del; R158del; Q957R; D253G; T95I; F888L; Q677H; A67V; Q414K; N450K; V483A; G669S; Q1071H;E 1092K; H1101Y; N1187D; W258L; V70F; T19R; Y144T; Y145S; R346K; R346S; V126A; H245Y; S12F; W152R; A899S; G142D; E156G; K558N; and/or Q52R contain at least one amino acid substitution or deletion.
在优选的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列的RNA，其中SARS-CoV-2刺突蛋白相对于SEQID NO:1的序列在对应于T19I；L24del；P25del；P26del；A27S；A67V；H69del；V70del；T95I；G142D；V143del；Y144del；Y145del；N211del；L212I；V213G；ins214EPE；G339D；S371L；S371F；S373P；S375F；T376A；D405N；K417N；N440K；G446S；S477N；T478K；E484A；Q493R；G496S；Q498R；N501Y；Y505H；T547K；D614G；H655Y；N679K；P681H；A701V；N764K；D796Y；N856K；Q954H；N969K；L981F的位置处包含至少一个氨基酸置换、缺失或插入。因此，在一些实施方式中，SARS-CoV-2刺突蛋白相对于SEQ ID NO:1的序列在对应于T19I；L24del；P25del；P26del；A27S；A67V；H69del；V70del；T95I；G142D；V143del；Y144del；Y145del；N211del；L212I；V213G；ins214EPE；G339D；S371L；S371F；S373P；S375F；T376A；D405N；K417N；N440K；G446S；S477N；T478K；E484A；Q493R；G496S；Q498R；N501Y；Y505H；T547K；D614G；H655Y；N679K；P681H；A701V；N764K；D796Y；N856K；Q954H；N969K；L981F的位置处包含至少一个氨基酸置换、缺失或插入。在某些实施方式中，SARS-CoV-2刺突蛋白为与SEQ IDNO:10的氨基酸序列90％相同并且相对于SEQ ID NO:1的序列包含至少3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40个选自T19I；L24del；P25del；P26del；A27S；A67V；H69del；V70del；T95I；G142D；V143del；Y144del；Y145del；N211del；L212I；V213G；ins214EPE；G339D；S371L；S371F；S373P；S375F；T376A；D405N；K417N；N440K；G446S；S477N；T478K；E484A；Q493R；G496S；Q498R；N501Y；Y505H；T547K；D614G；H655Y；N679K；P681H；A701V；N764K；D796Y；N856K；Q954H；N969K；L981F的氨基酸置换、缺失或插入。In a preferred embodiment, an RNA is provided comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein corresponds to T19I; L24del; P25del; P26del; A27S; A67V; H69del; V70del; T95I; G142D; V143del; Y144del; Y145del; N211del; L212I; V213G; ins214EPE; G339D; S371L; S371F; S373P; S375F; T376A; D 405N; K417N; N440K; G446S; S477N; T478K; E484A; Q493R; G496S; Q498R; N501Y; Y505H; T547K; D614G; H655Y; N679K; P681H; A701V; N764K; D796Y; N856K; Q954H; N969K; L981F. Thus, in some embodiments, the SARS-CoV-2 spike protein comprises at least one amino acid substitution, deletion or insertion relative to SEQ ID The sequence of NO:1 corresponds to T19I; L24del; P25del; P26del; A27S; A67V; H69del; V70del; T95I; G142D; V143del; Y144del; Y145del; N211del; L212I; V213G; ins214EPE; G339D; S371L; S371F; S373P; S375F; T376A; D 405N; K417N; N440K; G446S; S477N; T478K; E484A; Q493R; G496S; Q498R; N501Y; Y505H; T547K; D614G; H655Y; N679K; P681H; A701V; N764K; D796Y; N856K; Q954H; N969K; L981F. In certain embodiments, the SARS-CoV-2 spike protein is 90% identical to the amino acid sequence of SEQ ID NO: 10 and is 90% identical to the amino acid sequence of SEQ ID NO: 11. The sequence of NO:1 comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 selected from T19I; L24del; P25del; P26del; A27S; A67V; H69del; V70del; T95I; G142D; V143del; Y144del; Y145del; N211del; Amino acid substitutions, deletions or insertions of L212I; V213G; ins214EPE; G339D; S371L; S371F; S373P; S375F; T376A; D405N; K417N; N440K; G446S; S477N; T478K; E484A; Q493R; G496S; Q498R; N501Y; Y505H; T547K; D614G; H655Y; N679K; P681H; A701V; N764K; D796Y; N856K; Q954H; N969K; L981F.
还在进一步的实施方式中，提供了包含至少一个编码至少一种SARS-CoV-2刺突蛋白或其免疫原性片段的编码序列的RNA，其中SARS-CoV-2刺突蛋白包含至少一个对应于A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F的氨基酸置换、插入或缺失。In a further embodiment, an RNA comprising at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment thereof is provided, wherein the SARS-CoV-2 spike protein comprises at least one corresponding to A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins2 Amino acid substitutions, insertions or deletions of 14EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F.
在优选的实施方式中，SARS-CoV-2刺突蛋白在位于RBD结构域(氨基酸位置aa319至aa541；氨基酸位置根据参考SEQ ID NO:1)或CND结构域(氨基酸位置aa329至aa529；氨基酸位置根据参考SEQ ID NO:1)中的位置处包含氨基酸置换。不希望受到理论的束缚，CND结构域中的氨基酸置换或突变可以帮助新出现的SARS-CoV-2变体逃避接种第一代疫苗(针对原始SARS-CoV-2株设计)的受试者中诱导的或感染原始SARS-CoV-2株后的受试者中诱导的一些类型的抗体的抗体探测。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at a position located in the RBD domain (amino acid positions aa319 to aa541; amino acid positions according to reference SEQ ID NO: 1) or the CND domain (amino acid positions aa329 to aa529; amino acid positions according to reference SEQ ID NO: 1). Without wishing to be bound by theory, amino acid substitutions or mutations in the CND domain may help emerging SARS-CoV-2 variants evade antibody detection by some types of antibodies induced in subjects vaccinated with first generation vaccines (designed against the original SARS-CoV-2 strain) or in subjects infected with the original SARS-CoV-2 strain.
因此，在优选的实施方式中，本发明的第一方面涉及包含至少一个编码至少一种来自SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的抗原肽或蛋白质的编码序列的RNA，其中RNA包含至少一个异源未翻译区(UTR)和其中SARS-CoV-2刺突蛋白在位于RBD结构域(氨基酸位置aa319至aa541；氨基酸位置根据参考SEQ ID NO:1)或CND结构域(氨基酸位置aa329至aa529；氨基酸位置根据参考SEQ ID NO:1)中的位置处包含至少一个氨基酸置换。Therefore, in a preferred embodiment, the first aspect of the invention relates to an RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the RNA comprises at least one heterologous untranslated region (UTR) and wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution at a position located in the RBD domain (amino acid positions aa319 to aa541; amino acid positions are according to reference SEQ ID NO: 1) or the CND domain (amino acid positions aa329 to aa529; amino acid positions are according to reference SEQ ID NO: 1).
在某些优选的实施方式中，SARS-CoV-2刺突蛋白在至少一个以下的位置处包含氨基酸置换、插入或缺失：R346；V367,P384；R403；K417；N437；N439；V445；G446；G447；N450；L452；Y453；L455；F456；A475；G476；S477；T478；E484；G485；F486；N487；Y489；F490；Q493；S494；P499；T500；N501；G502；V503；G504；Y505；Q506；A522(氨基酸位置根据参考SEQ IDNO:1)。In certain preferred embodiments, the SARS-CoV-2 spike protein comprises an amino acid substitution, insertion or deletion at at least one of the following positions: R346; V367, P384; R403; K417; N437; N439; V445; G446; G447; N450; L452; Y453; L455; F456; A475; G476; S477; T478; E484; G485; F486; N487; Y489; F490; Q493; S494; P499; T500; N501; G502; V503; G504; Y505; Q506; A522 (amino acid positions are according to reference SEQ ID NO: 1).
因此，在某些优选的实施方式中，本发明的第一方面涉及包含至少一个编码至少一种来自SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的抗原肽或蛋白质的编码序列的RNA，其中SARS-CoV-2刺突蛋白在选自K417；L452；T478；E484；N501和/或P681的位置处(氨基酸位置根据参考SEQ ID NO:1)包含至少一个氨基酸置换和其中RNA包含至少一个异源未翻译区(UTR)。Therefore, in certain preferred embodiments, the first aspect of the invention relates to an RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof, wherein the SARS-CoV-2 spike protein comprises at least one amino acid substitution at a position selected from K417; L452; T478; E484; N501 and/or P681 (amino acid position according to reference SEQ ID NO: 1) and wherein the RNA comprises at least one heterologous untranslated region (UTR).
不希望受到理论的束缚，位置E484处的氨基酸置换可以帮助SARS-CoV-2病毒变体逃避接种第一代疫苗(针对原始SARS-CoV-2株设计)的受试者中诱导的或感染原始SARS-CoV-2株后的受试者中诱导的一些类型的抗体的抗体探测。N501的突变/置换存在于冠状病毒刺突的顶部附近，其在那可以改变蛋白质的形状，这可能有助于逃避一些类型的冠状病毒抗体。这样的SARS-CoV-2在整个本发明中称为SARS-CoV-2E484变体并且包括例如SARS-CoV-2 B.1.351(南非)、SARS-CoV-2 B.1.617(印度)、P.1(巴西)或B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)。Without wishing to be bound by theory, amino acid substitutions at position E484 can help SARS-CoV-2 virus variants escape antibody detection of some types of antibodies induced in subjects induced in subjects who have received first-generation vaccines (designed for original SARS-CoV-2 strains) or infected with original SARS-CoV-2 strains. The mutation/substitution of N501 is present near the top of the coronavirus spike, where it can change the shape of the protein, which may help to escape some types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2E484 variants throughout the present invention and includes, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.617 (India), P.1 (Brazil) or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
因此，在一些实施方式中，可能有利的是本发明的RNA提供包含位置E484的置换的SARS-CoV-2刺突蛋白，以允许诱导有效的抗病毒SARS-CoV-2E484变体的免疫反应。Therefore, in some embodiments, it may be advantageous that the RNA of the invention provides a SARS-CoV-2 spike protein comprising a substitution at position E484, to allow for the induction of an effective immune response against the viral SARS-CoV-2 E484 variant.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置484的氨基酸置换，其中氨基酸E484被K、P、Q、A或D取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含E484K、E484P、E484Q、E484A、E484D氨基酸置换。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position 484, wherein the amino acid E484 is substituted with K, P, Q, A or D (amino acid position according to reference SEQ ID NO: 1). Thus, an antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises E484K, E484P, E484Q, E484A, E484D amino acid substitutions.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白包含位置E484的氨基酸置换，其中氨基酸E484被K或Q取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含E484K或E484Q氨基酸置换。在某些优选的实施方式中，SARS-CoV-2刺突蛋白包含E484K氨基酸置换。In a particularly preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position E484, wherein amino acid E484 is substituted with K or Q (amino acid position according to reference SEQ ID NO: 1). Thus, an antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises an E484K or E484Q amino acid substitution. In certain preferred embodiments, the SARS-CoV-2 spike protein comprises an E484K amino acid substitution.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置N501的氨基酸置换，其中氨基酸N501被不同的氨基酸取代(氨基酸位置根据参考SEQ ID NO:1)。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position N501, wherein amino acid N501 is replaced by a different amino acid (amino acid position according to reference SEQ ID NO: 1).
不希望受到理论的束缚，位置N501处的氨基酸置换可以帮助SARS-CoV-2病毒变体逃避接种第一代疫苗(针对原始SARS-CoV-2株设计)的受试者中诱导的或感染原始SARS-CoV-2株后的受试者中诱导的一些类型的抗体的抗体探测。N501的突变/置换发生在冠状病毒刺突的顶部附近，其在那可以改变蛋白质的形状，这可能有助于逃避一些类型的冠状病毒抗体。这样的SARS-CoV-2在整个本发明中称为SARS-CoV-2N501变体并且包括例如SARS-CoV-2 B.1.351(南非)、SARS-CoV-2 B.1.1.7(UK)、P.1(巴西)或B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)。Without wishing to be bound by theory, amino acid replacement at position N501 can help SARS-CoV-2 virus variants escape antibody detection of some types of antibodies induced in subjects induced in subjects after infecting original SARS-CoV-2 strains by first generation vaccines (designed for original SARS-CoV-2 strains). The mutation/replacement of N501 occurs near the top of coronavirus spikes, where it can change the shape of protein, which may help to escape some types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2N501 variants throughout the present invention and includes, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.1.7 (UK), P.1 (Brazil) or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
因此，可能有利的是本发明的RNA提供了在位置N501包含突变的SARS-CoV-2刺突蛋白，以允许诱导有效的抗病毒SARS-CoV-2N501变体的免疫反应。Therefore, it may be advantageous that the RNA of the invention provides a SARS-CoV-2 spike protein comprising a mutation at position N501 to allow the induction of an effective immune response against the viral SARS-CoV-2 N501 variant.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置N501的氨基酸置换，其中氨基酸N501被Y、T、S取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含N501Y、N501T、N501S氨基酸置换。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position N501, wherein amino acid N501 is substituted by Y, T, S (amino acid position according to reference SEQ ID NO: 1). Therefore, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises N501Y, N501T, N501S amino acid substitutions.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白包含位置N501的氨基酸置换，其中氨基酸N501被Y取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含N501Y氨基酸置换。In a particularly preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position N501, wherein the amino acid N501 is substituted with Y (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises an N501Y amino acid substitution.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置K417的氨基酸置换，其中氨基酸K417被不同的氨基酸取代(氨基酸位置根据参考SEQ ID NO:1)。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position K417, wherein amino acid K417 is replaced by a different amino acid (amino acid position according to reference SEQ ID NO: 1).
不希望受到理论的束缚，位置K417处的氨基酸置换可以帮助SARS-CoV-2病毒变体逃避接种使用SEQ ID NO:1针对原始SARS-CoV-2株设计的疫苗的受试者中诱导的或感染包含SEQ ID NO:1的原始SARS-CoV-2株后的受试者中诱导的一些类型的抗体的抗体探测。K417的突变/置换发生在冠状病毒刺突的顶部附近，其在此处可以改变蛋白质的形状，这可能有助于逃避一些类型的冠状病毒抗体。这样的SARS-CoV-2在整个本发明中称为SARS-CoV-2K417变体并且包括例如SARS-CoV-2 B.1.351(南非)、SARS-CoV-2 B.1.1.7(UK)、P.1(巴西)、AY.1/AY.2或B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)。Without wishing to be bound by theory, amino acid substitutions at position K417 may help SARS-CoV-2 virus variants evade antibody detection by some types of antibodies induced in subjects vaccinated with a vaccine designed against the original SARS-CoV-2 strain using SEQ ID NO: 1 or induced in subjects following infection with the original SARS-CoV-2 strain comprising SEQ ID NO: 1. The mutation/substitution of K417 occurs near the top of the coronavirus spike, where it may change the shape of the protein, which may help evade some types of coronavirus antibodies. Such SARS-CoV-2 is referred to as SARS-CoV-2K417 variant throughout the present invention and includes, for example, SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2 B.1.1.7 (UK), P.1 (Brazil), AY.1/AY.2 or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
因此，可能有利的是本发明的RNA提供了包含位置K417的置换的SARS-CoV-2刺突蛋白，以允许诱导有效的抗病毒SARS-CoV-2K417变体的免疫反应。Therefore, it may be advantageous that the RNA of the invention provides a SARS-CoV-2 spike protein comprising a substitution at position K417 to allow the induction of an effective immune response against the viral SARS-CoV-2 K417 variant.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置K417处的氨基酸置换，其中氨基酸N501被S、T、Q或N取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含K417S、K417T、K417Q或K417N氨基酸置换。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position K417, wherein amino acid N501 is substituted with S, T, Q or N (amino acid position according to reference SEQ ID NO: 1). Thus, an antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises a K417S, K417T, K417Q or K417N amino acid substitution.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白包含位置N501处的氨基酸置换，其中氨基酸K417被T或N取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含K417T或K417N氨基酸置换。在某些优选的实施方式中，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含K417N氨基酸置换。In a particularly preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position N501, wherein the amino acid K417 is substituted with T or N (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises a K417T or K417N amino acid substitution. In certain preferred embodiments, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises a K417N amino acid substitution.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置L452处的氨基酸置换，其中氨基酸L452被不同的氨基酸取代(氨基酸位置根据参考SEQ ID NO:1)。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position L452, wherein amino acid L452 is replaced by a different amino acid (amino acid position according to reference SEQ ID NO: 1).
不希望受理论的束缚，位置L452处的氨基酸置换可以帮助SARS-CoV-2病毒变体逃避接种基于SEQ ID NO:1针对原始SARS-CoV-2株设计的疫苗的受试者中诱导的或感染具有SEQ ID NO:1的原始SARS-CoV-2株后的受试者中诱导的一些类型的抗体的抗体探测。L452的突变/置换发生在冠状病毒刺突的顶部附件，其在此处可以改变蛋白质的形状，这可能有助于逃避一些类型的冠状病毒抗体。这样的SARS-CoV-2在整个本发明中称为SARS-CoV-2L452变体并且包括例如SARS-CoV-2 B.1.617.1(印度)、SARS-CoV-2B.1.617.2(印度)或SARS-CoV-2 B.1.617.3(印度)。Without wishing to be bound by theory, amino acid substitutions at position L452 may help SARS-CoV-2 virus variants evade antibody detection by some types of antibodies induced in subjects vaccinated with a vaccine designed based on SEQ ID NO: 1 against the original SARS-CoV-2 strain or induced in subjects infected with the original SARS-CoV-2 strain having SEQ ID NO: 1. The mutation/substitution of L452 occurs at the top appendage of the coronavirus spike, where it can change the shape of the protein, which may help evade some types of coronavirus antibodies. Such SARS-CoV-2 is referred to throughout the present invention as SARS-CoV-2 L452 variants and includes, for example, SARS-CoV-2 B.1.617.1 (India), SARS-CoV-2 B.1.617.2 (India), or SARS-CoV-2 B.1.617.3 (India).
因此，可能有利的是本发明的RNA提供了包含位置L452的置换的SARS-CoV-2刺突蛋白，以允许诱导有效的抗病毒SARS-CoV-2L452变体的免疫反应。Therefore, it may be advantageous that the RNA of the invention provides a SARS-CoV-2 spike protein comprising a substitution at position L452, to allow the induction of an effective immune response against the viral SARS-CoV-2 L452 variant.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置L452处的氨基酸置换，其中氨基酸L452被R或Q取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含L452R或L452Q氨基酸置换。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position L452, wherein the amino acid L452 is substituted by R or Q (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises an L452R or L452Q amino acid substitution.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白包含位置L452处的氨基酸置换，其中氨基酸L452被R取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含L452R氨基酸置换。In a particularly preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position L452, wherein the amino acid L452 is substituted by R (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises an L452R amino acid substitution.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位于弗林蛋白酶切割位点(氨基酸位置aa 681至685；氨基酸位置根据参考SEQ ID NO:1)中的位置处的氨基酸置换。据信该序列链段(SEQ ID NO:1中的PRRAR)作为弗林蛋白酶切割的识别位点。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid replacement at a position in the furin cleavage site (amino acid positions aa 681 to 685; amino acid positions are based on reference SEQ ID NO: 1). It is believed that this sequence segment (PRRAR in SEQ ID NO: 1) serves as a recognition site for furin cleavage.
不希望束缚于理论，弗林蛋白酶切割位点中的氨基酸置换或突变可以有助于新出现的SARS-CoV-2变体具有增加的膜融合并因此导致增加的传播性。Without wishing to be bound by theory, amino acid substitutions or mutations in the furin cleavage site may contribute to emerging SARS-CoV-2 variants having increased membrane fusion and thus resulting in increased transmissibility.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含弗林蛋白酶切割位点中的位置P681处的氨基酸置换。合适地，氨基酸P681被不同的氨基酸(优选提高弗林蛋白酶切割的氨基酸)取代(氨基酸位置根据参考SEQ ID NO:1)。这样的SARS-CoV-2在整个本发明中被称为SARS-CoV-2P681变体并且包括例如SARS-CoV-2 B.1.617.1(印度)、SARS-CoV-2 B.1.617.2(印度)，或SARS-CoV-2 B.1.617.3(印度)、SARS-CoV-2 B.1.1.7(UK)、SARS-CoV-2A.23.1(乌干达)，或B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid replacement at position P681 in the furin cleavage site. Suitably, amino acid P681 is replaced by a different amino acid (preferably an amino acid that improves furin cleavage) (amino acid position according to reference SEQ ID NO: 1). Such SARS-CoV-2 is referred to as SARS-CoV-2P681 variant throughout the present invention and includes, for example, SARS-CoV-2 B.1.617.1 (India), SARS-CoV-2 B.1.617.2 (India), or SARS-CoV-2 B.1.617.3 (India), SARS-CoV-2 B.1.1.7 (UK), SARS-CoV-2A.23.1 (Uganda), or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
因此，可能有利的是本发明的RNA提供了包含位置P681的置换的SARS-CoV-2刺突蛋白，以允许诱导有效的抗病毒SARS-CoV-2P681变体的免疫反应。Therefore, it may be advantageous that the RNA of the invention provides a SARS-CoV-2 spike protein comprising a substitution at position P681 to allow the induction of an effective immune response against the viral SARS-CoV-2 P681 variant.
在优选的实施方式中，SARS-CoV-2刺突蛋白包含位置P681处的氨基酸置换，其中氨基酸P681被R或H取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含P681R或P681H氨基酸置换。In a preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position P681, wherein the amino acid P681 is substituted with R or H (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises a P681R or P681H amino acid substitution.
在特别优选的实施方式中，SARS-CoV-2刺突蛋白包含位置P681处的氨基酸置换，其中P681被R取代(氨基酸位置根据参考SEQ ID NO:1)。因此，选自或源自SARS-CoV-2刺突蛋白的抗原肽或蛋白质包含P681R氨基酸置换。In a particularly preferred embodiment, the SARS-CoV-2 spike protein comprises an amino acid substitution at position P681, wherein P681 is substituted by R (amino acid position according to reference SEQ ID NO: 1). Thus, the antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein comprises a P681R amino acid substitution.
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置L452处的氨基酸置换，优选L452R，以及如本文定义的位置P681处的氨基酸置换，优选P681R(氨基酸位置根据参考SEQ ID NO:1)。In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position P681 as defined herein, preferably P681R (amino acid positions according to reference SEQ ID NO: 1).
在另一个特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置L452处的氨基酸置换，优选L452R，以及如本文定义的位置P681处的氨基酸置换，优选P681R(氨基酸位置根据参考SEQ ID NO:1)。在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置L452处的氨基酸置换，优选L452R，如本文定义的位置P681处的氨基酸置换，优选P681R，和如本文定义的位置D614处的氨基酸置换，优选D614G(氨基酸位置根据参考SEQ ID NO:1)。In another particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position P681 as defined herein, preferably P681R (amino acid positions according to reference SEQ ID NO: 1). In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, an amino acid substitution at position P681 as defined herein, preferably P681R, and an amino acid substitution at position D614 as defined herein, preferably D614G (amino acid positions according to reference SEQ ID NO: 1).
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置N501处的氨基酸置换，优选N501Y，以及如本文定义的位置E484处的氨基酸置换，优选E484K(氨基酸位置根据参考SEQ ID NO:1)。In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution at position N501 as defined herein, preferably N501Y, and an amino acid substitution at position E484 as defined herein, preferably E484K (amino acid positions according to reference SEQ ID NO: 1).
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置L452处的氨基酸置换，优选L452R，以及如本文定义的位置E484处的氨基酸置换，优选E484Q(氨基酸位置根据参考SEQ ID NO:1)。In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution at position L452 as defined herein, preferably L452R, and an amino acid substitution at position E484 as defined herein, preferably E484Q (amino acid positions according to reference SEQ ID NO: 1).
在优选的实施方式中，除了以上定义的置换(在位置E484、N501、L452和任选P681处)外，SARS-CoV-2刺突蛋白包含至少一个，特别是1、2、3、4、5、6、7、8、9或10个选自列表1A或列表1B的另外的氨基酸置换、插入或缺失。In a preferred embodiment, in addition to the substitutions defined above (at positions E484, N501, L452 and optionally P681), the SARS-CoV-2 spike protein comprises at least one, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 further amino acid substitutions, insertions or deletions selected from List 1A or List 1B.
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含如本文定义的位置H69处的氨基酸置换或缺失，优选H69del，以及如本文定义的位置V70处的氨基酸置换或缺失，优选V70del(氨基酸位置根据参考SEQ ID NO:1)。在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含H69和V70两者处的缺失。In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution or deletion at position H69 as defined herein, preferably H69del, and an amino acid substitution or deletion at position V70 as defined herein, preferably V70del (amino acid positions according to reference SEQ ID NO: 1). In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises deletions at both H69 and V70.
在优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个选自以下SARS-CoV-2分离株的进一步氨基酸置换或缺失：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention comprises at least one further amino acid substitution or deletion selected from the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含选自以下的氨基酸置换或缺失(相对于SEQ ID NO:1)：In a preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises an amino acid substitution or deletion selected from the following (relative to SEQ ID NO: 1):
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v1)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q49 3R, G496S, Q498R, N501Y, Y505H , T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v1);
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、K417N、N440K、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v0)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S47 7N, T478K, E484A, Q493R, G496S , Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v0);
·K986P、V987P、A67V、T95I、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、D796Y、N856K、Q954H、N969K、L981F(SA，B.1.1.529)；·K986P, V987P, A67V, T95I, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P6 81H, D796Y, N856K, Q954H, N969K , L981F (SA, B.1.1.529);
·K986P、V987P、T19I、L24del、P25del、P26del、A27S、G142D、V213G、G339D、S371F、S373P、S375F、T376A、D405N、S477N、T478K、E484A、Q493R、Q498R、N501Y、Y505H、D614G、H655Y、N679K、P681H、D796Y、Q954H、N969K(SA，BA.2)；·K986P, V987P, T19I, L24del, P25del, P26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, S477N, T478K, E484A, Q493R, Q498R, N501Y , Y505H, D614G, H655Y, N679K , P681H, D796Y, Q954H, N969K (SA, BA.2);
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、N440K、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v2)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, N440K, S477N, T478K, E48 4A, Q493R, G496S, Q498R, N501Y , Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v2);
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v3)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q49 3R, G496S, Q498R, N501Y, Y505H , T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v3);
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、A701V、N764K、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v4)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q49 3R, G496S, Q498R, N501Y, Y505H , T547K, D614G, H655Y, N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v4);
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F(SA，BA.1_v5)；·K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S, S477N, T478K, E48 4A, Q493R, G496S, Q498R, N501Y , Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v5);
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；(SA；B.1.351)·E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G and A701V; (SA; B.1.351)
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V；(SA；B.1.351)·E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G and A701V; (SA; B.1.351)
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y和T1027I；(巴西；P1)E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I; (Brazil; P1)
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I和V1176F；(巴西，P1)E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F; (Brazil, P1)
·L452R、P681R和D614G；(B.1.617.1；印度)L452R, P681R and D614G; (B.1.617.1; India)
·L452R、E484Q、P681R、E154K、D614G和Q1071H；(B.1.617.2；印度)L452R, E484Q, P681R, E154K, D614G, and Q1071H; (B.1.617.2; India)
·L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N；(B.1.617.2；印度)L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N; (B.1.617.2; India)
·T19R、L452R、E484Q、D614G、P681R和D950N；(B.1.617.3；印度)T19R, L452R, E484Q, D614G, P681R and D950N; (B.1.617.3; India)
·G75V、T76I、S247del、Y248del、L249del、T250del、P251del、G252del、D253del、L452Q、F490S、D614G和T859N；(C.37.1；秘鲁)G75V, T76I, S247del, Y248del, L249del, T250del, P251del, G252del, D253del, L452Q, F490S, D614G, and T859N; (C.37.1; Peru)
·T95I、Y145N、R346K、E484K、N501Y、D614G、P681H和D950N；(B.1.1.621)T95I, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N; (B.1.1.621)
·T95I、Y144T、Y145S、ins145N、R346K、E484K、N501Y、D614G、P681H和D950N；(B.1.1.621)T95I, Y144T, Y145S, ins145N, R346K, E484K, N501Y, D614G, P681H, and D950N; (B.1.1.621)
·H69del、V70del、Y144del、E484K、N501Y、A570D、D614G、P681H、T716I、S982A和D1118H；(B.1.1.7-E484K)H69del, V70del, Y144del, E484K, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H; (B.1.1.7-E484K)
·S13I、W152C、L452R和D614G；(B.1.429)S13I, W152C, L452R and D614G; (B.1.429)
·L452R；和D614G；(B.1.429)L452R; and D614G; (B.1.429)
·H69del；V70del；N439K；D614G；(B.1.258)·H69del; V70del; N439K; D614G; (B.1.258)
·T95I；E484K；D614G；和A701V；(B.1.526)T95I; E484K; D614G; and A701V; (B.1.526)
·L5F、T95I、D253G、E484K、D614G和A701V；(B.1.526)L5F, T95I, D253G, E484K, D614G, and A701V; (B.1.526)
·L5F、T95I、D253G、S477N、D614G和Q957R；(B.1.526)·L5F, T95I, D253G, S477N, D614G and Q957R; (B.1.526)
·F157L；V367F；Q613H；和P681R(A.23.1)F157L; V367F; Q613H; and P681R (A.23.1)
·S254F；D614G；P681R；和G769V(A.23.1)S254F; D614G; P681R; and G769V (A.23.1)
·T478K；D614G；P681H；和T732A(B.1.1.519；墨西哥)T478K; D614G; P681H; and T732A (B.1.1.519; Mexico)
·P26S、H69del、V70del、V126A、Y144del、L242del、A243del、L244del、H245Y、S477N、E484K、D614G、P681H、T1027I和D1118H；(B.1.620；非洲)P26S, H69del, V70del, V126A, Y144del, L242del, A243del, L244del, H245Y, S477N, E484K, D614G, P681H, T1027I, and D1118H; (B.1.620; Africa)
·ins214TDR、Q414K、N450K、D614G和T716I；(B.1.214.2)ins214TDR, Q414K, N450K, D614G and T716I; (B.1.214.2)
·S12F、H69del、V70del、W152R、R346S、L452R、D614G、Q677H和A899S；(C.36.3；泰国)S12F, H69del, V70del, W152R, R346S, L452R, D614G, Q677H, and A899S; (C.36.3; Thailand)
·E484K、D614G和V1176F；(P2)E484K, D614G and V1176F; (P2)
·Q52R；A67V；H69del；V70del；F157del；R158del；E484K；D614G；Q677H和F888L；(B.1.525)Q52R; A67V; H69del; V70del; F157del; R158del; E484K; D614G; Q677H and F888L; (B.1.525)
·Q52R；A67V；H69del；V70del；Y144del；E484K；D614G；Q677H和F888L；(B.1.525)Q52R; A67V; H69del; V70del; Y144del; E484K; D614G; Q677H and F888L; (B.1.525)
·A67V；H69del；V70del；Y144del；E484K；D614G；Q677H和F888L；(B.1.525)A67V; H69del; V70del; Y144del; E484K; D614G; Q677H and F888L; (B.1.525)
·T19R；T95I；G142D、E156G、F157del；R158del；W258L；K417N；L452R；T478K；K558N、D614G；P681R；和D950N；(AY.1)·T19R; T95I; G142D, E156G, F157del; R158del; W258L; K417N; L452R; T478K; K558N, D614G; P681R; and D950N; (AY.1)
·T19R；V70F；G142D、E156G、F157del；R158del；A222V、K417N；L452R；T478K；D614G；P681R；和D950N；(AY.2)T19R; V70F; G142D, E156G, F157del; R158del; A222V, K417N; L452R; T478K; D614G; P681R; and D950N; (AY.2)
·T19R；T95I；F157del；R158del；W258L；K417N；L452R；T478K；D614G；P681R；和D950N；或(AY.1)T19R; T95I; F157del; R158del; W258L; K417N; L452R; T478K; D614G; P681R; and D950N; or (AY.1)
·T19R；V70F；F157del；R158del；A222V；K417N；L452R；T478K；D614G；P681R；和D950N；(AY.2)T19R; V70F; F157del; R158del; A222V; K417N; L452R; T478K; D614G; P681R; and D950N; (AY.2)
·H69del、V70del和D614G；H69del, V70del and D614G;
·D614G和M1229I；D614G and M1229I;
·A222V和D614G；A222V and D614G;
·S477N和D614G；S477N and D614G;
·N439K和D614G；N439K and D614G;
·H69del、V70del、Y453F、D614G和I692I；H69del, V70del, Y453F, D614G, and I692I;
·Y453F和D614G；Y453F and D614G;
·D614G和I692V；D614G and I692V;
·H69del、V70del、A222V、Y453F、D614G和I692I；H69del, V70del, A222V, Y453F, D614G, and I692I;
·N501Y和D614G；N501Y and D614G;
·K417N；E484K；N501Y和D614G；或K417N; E484K; N501Y and D614G; or
·E484K和D614G。E484K and D614G.
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含以下的氨基酸置换或缺失(相对于SEQ ID NO:1)：In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises the following amino acid substitutions or deletions (relative to SEQ ID NO: 1):
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V;
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y和T1027I；E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I和V1176F；E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F;
·L452R、P681R和D614G；L452R, P681R and D614G;
·L452R、E484Q、P681R、E154K、D614G和Q1071H；或L452R, E484Q, P681R, E154K, D614G, and Q1071H; or
·L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N。L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.
在甚至更优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含以下的氨基酸置换或缺失(相对于SEQ ID NO:1)：In an even more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises the following amino acid substitutions or deletions (relative to SEQ ID NO: 1):
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；或E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V。E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V.
在进一步特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含以下的氨基酸置换或缺失(相对于SEQ ID NO:1)：In a further particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises the following amino acid substitutions or deletions (relative to SEQ ID NO: 1):
·L452R、P681R和D614G；L452R, P681R and D614G;
·L452R、E484Q、P681R、E154K、D614G和Q1071H；L452R, E484Q, P681R, E154K, D614G, and Q1071H;
·L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N；或L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N; or
·T19R、L452R、E484Q、D614G、P681R和D950N。T19R, L452R, E484Q, D614G, P681R, and D950N.
在甚至更优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:1、10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中的任一个或这些中任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:1、10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588或28917-28920中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588或28917-28920中的任一个相同。关于所述氨基酸序列的更多信息也提供于表1中，以及在ST25序列表的相应序列SEQID NO的<223>标识符下。In an even more preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. Further information about the amino acid sequence is also provided in Table 1, and under the <223> identifier of the corresponding sequence SEQ ID NO in the ST25 sequence listing.
在一些实施方式中，如本文定义的刺突蛋白(S)的片段可以由本发明的RNA来编码，其中所述片段可以是N-末端截短的，从而缺少全长SARS-CoV-2变体蛋白质的N-末端氨基酸1至最多100，和/或其中所述片段可以是C-末端截短的，从而缺少全长SARS-CoV-2变体蛋白的C-末端氨基酸(aa)531至最多aa 1273。这样的“刺突蛋白(S)的片段”可以另外包含氨基酸置换(如本文所述的)和可以另外包含至少一个异源肽或蛋白质元件(如本文所述的)。在优选的实施方式中，刺突蛋白(S)的片段可以是C-末端截短的，由此缺少C-末端跨膜结构域(即，缺少aa 1212至aa 1273或缺少aa 1148至aa1273)(氨基酸位置根据参考SEQ IDNO:1)。In some embodiments, a fragment of the spike protein (S) as defined herein may be encoded by the RNA of the invention, wherein the fragment may be N-terminally truncated, thereby lacking the N-terminal amino acid 1 to a maximum of 100 of the full-length SARS-CoV-2 variant protein, and/or wherein the fragment may be C-terminally truncated, thereby lacking the C-terminal amino acid (aa) 531 to a maximum of aa 1273 of the full-length SARS-CoV-2 variant protein. Such a "fragment of the spike protein (S)" may additionally comprise an amino acid substitution (as described herein) and may additionally comprise at least one heterologous peptide or protein element (as described herein). In a preferred embodiment, the fragment of the spike protein (S) may be C-terminally truncated, thereby lacking the C-terminal transmembrane domain (i.e., lacking aa 1212 to aa 1273 or lacking aa 1148 to aa 1273) (amino acid positions according to reference SEQ ID NO: 1).
在其他实施方式中，编码的源自SARS-CoV-2的刺突蛋白(S)缺少跨膜结构域(TM)(氨基酸位置aa 1212至aa 1273根据参考SEQ ID NO:1)。在实施方式中，编码的源自SARS-CoV-2的刺突蛋白(S)缺少跨膜结构域的延伸部分(TMflex)(根据参考SEQ ID NO:1的氨基酸位置aa 1148至aa 1273)。不希望束缚于理论，如本文定义的缺少跨膜结构域(TM或TMflex)的刺突蛋白(S)可以适用于疫苗，因为这样的蛋白质将是可溶的并且没有锚定在细胞膜中。因此，在施用于受试者时可以以更高浓度产生(即翻译)可溶性蛋白，从而导致提高的免疫反应。In other embodiments, the encoded spike protein (S) derived from SARS-CoV-2 lacks a transmembrane domain (TM) (amino acid positions aa 1212 to aa 1273 according to reference SEQ ID NO: 1). In an embodiment, the encoded spike protein (S) derived from SARS-CoV-2 lacks an extended portion of the transmembrane domain (TMflex) (according to amino acid positions aa 1148 to aa 1273 of reference SEQ ID NO: 1). Without wishing to be bound by theory, a spike protein (S) lacking a transmembrane domain (TM or TMflex) as defined herein may be suitable for use in a vaccine because such a protein will be soluble and not anchored in the cell membrane. Therefore, a soluble protein may be produced (i.e., translated) at a higher concentration when administered to a subject, resulting in an enhanced immune response.
不希望束缚于理论，如参考SEQ ID NO:1的氨基酸位置，RBD(aa 319至aa 541)和CND(aa 329至aa 529)结构域对于免疫原性可能是关键的。两个区域位于刺突蛋白的S1片段。因此，在本发明的情况中，可能合适的是抗原肽或蛋白质包含刺突蛋白的S1片段或其免疫原性片段或免疫原性变体或由其组成。合适地，这样的S1片段可以至少包含如上定义的RBD和/或CND结构域。在某些实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白CND结构域包含至少一个与SEQ ID NO:27051-27086中的任一个或这些中任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白CND结构域与SEQ ID NO:27051-27086中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白CND结构域与SEQ ID NO:27051-27086中的任一个相同。关于所述氨基酸序列的更多信息也提供于表1中，以及在ST25序列表的相应序列SEQ ID NO的<223>标识符下。Without wishing to be bound by theory, as referenced to the amino acid positions of SEQ ID NO: 1, the RBD (aa 319 to aa 541) and CND (aa 329 to aa 529) domains may be critical for immunogenicity. Both regions are located in the S1 fragment of the spike protein. Therefore, in the context of the present invention, it may be appropriate that the antigenic peptide or protein comprises or consists of the S1 fragment of the spike protein or an immunogenic fragment or immunogenic variant thereof. Suitably, such an S1 fragment may comprise at least the RBD and/or CND domains as defined above. In certain embodiments, the SARS-CoV-2 spike protein CND domain encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27051-27086 or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein CND domain is at least 95% identical to any one of SEQ ID NOs: 27051-27086. In certain embodiments, the SARS-CoV-2 spike protein CND domain is identical to any one of SEQ ID NOs: 27051-27086. Further information on the amino acid sequence is also provided in Table 1, and under the <223> identifier of the corresponding sequence SEQ ID NO in the ST25 sequence listing.
在优选的实施方式中，编码的至少一个抗原肽或蛋白质包含受体结合结构域(RBD；aa 319至aa 541)或由其组成，其中RBD包含刺突蛋白片段，或其免疫原性片段或免疫原性变体，或由其组成。在某些实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白RBD结构域包含至少一个与SEQ ID NO:27007-27046中的任一个或这些中任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白RBD结构域与SEQ ID NO:27007-27046中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白RBD结构域与SEQ ID NO:27007-27046中的任一个相同。关于所述氨基酸序列的更多信息也提供于表1中，以及在ST25序列表的相应序列SEQ ID NO的<223>标识符下。In a preferred embodiment, at least one antigenic peptide or protein encoded comprises or consists of a receptor binding domain (RBD; aa 319 to aa 541), wherein the RBD comprises or consists of a spike protein fragment, or an immunogenic fragment or immunogenic variant thereof. In certain embodiments, the SARS-CoV-2 spike protein RBD domain encoded by the RNA of the present invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27007-27046 or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein RBD domain is at least 95% identical to any one of SEQ ID NOs: 27007-27046. In certain embodiments, the SARS-CoV-2 spike protein RBD domain is identical to any one of SEQ ID NOs: 27007-27046. Further information about the amino acid sequence is also provided in Table 1, and under the <223> identifier of the corresponding sequence SEQ ID NO in the ST25 sequence listing.
在进一步优选的实施方式中，编码的至少一个抗原肽或蛋白质包含截短的受体结合结构域(truncRBD；aa 334至aa 528)或由其组成，其中RBD包含刺突蛋白片段，或其免疫原性片段或免疫原性片段，或由其组成。In a further preferred embodiment, at least one encoded antigenic peptide or protein comprises or consists of a truncated receptor binding domain (truncRBD; aa 334 to aa 528), wherein the RBD comprises or consists of a spike protein fragment, or an immunogenic fragment or immunogenic fragment thereof.
这种“刺突蛋白(S)的片段”(RBD；aa 319至aa 541；或truncRBD，aa 334至aa 528)可以另外包含氨基酸置换(如本文所述的)和可以另外包含至少一个异源肽或蛋白质元件(如本文所述的)。This "fragment of the spike protein (S)" (RBD; aa 319 to aa 541; or truncRBD, aa 334 to aa 528) may further comprise amino acid substitutions (as described herein) and may further comprise at least one heterologous peptide or protein element (as described herein).
在特别优选的实施方式中，编码的至少一个抗原肽或蛋白质包含刺突蛋白(S)或由其组成，其中刺突蛋白(S)包含刺突蛋白片段S1，或其免疫原性片段或免疫原性变体，或由其组成。In a particularly preferred embodiment, the encoded at least one antigenic peptide or protein comprises or consists of the spike protein (S), wherein the spike protein (S) comprises or consists of the spike protein fragment S1, or an immunogenic fragment or immunogenic variant thereof.
在优选的实施方式中，编码的至少一个抗原肽或蛋白质包含刺突蛋白片段S1并缺少至少70％、80％、81％、82％、83％,84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的刺突蛋白片段S2(aa 682至aa 1273)。这样的实施方式可能是有益的，因为S1片段包含中和表位。In a preferred embodiment, the encoded at least one antigenic peptide or protein comprises the spike protein fragment S1 and lacks at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the spike protein fragment S2 (aa 682 to aa 1273). Such an embodiment may be beneficial because the S1 fragment contains a neutralizing epitope.
不希望束缚于理论，可能合适的是抗原肽或蛋白质包含刺突蛋白片段S1和刺突蛋白片段S2(至少其片段)或由其组成，因为可以促进免疫原性刺突蛋白的形成。Without wishing to be bound by theory, it may be appropriate that the antigenic peptide or protein comprises or consists of the spike protein fragment S1 and the spike protein fragment S2 (at least fragments thereof) because the formation of an immunogenic spike protein may be promoted.
因此，在特别优选的实施方式，编码的至少一个抗原肽或蛋白质包含刺突蛋白(S)或由刺突蛋白(S)组成，其中刺突蛋白(S)包含刺突蛋白S1或其免疫原性片段或免疫原性变体及刺突蛋白片段S2或其免疫原性片段或免疫原性变体，或由其组成。Therefore, in a particularly preferred embodiment, at least one encoded antigenic peptide or protein comprises a spike protein (S) or consists of a spike protein (S), wherein the spike protein (S) comprises, or consists of, a spike protein S1 or an immunogenic fragment or an immunogenic variant thereof and a spike protein fragment S2 or an immunogenic fragment or an immunogenic variant thereof.
在可选的优选实施方式中，编码的至少一个抗原肽或蛋白质包含全长刺突蛋白或这些中任一个的免疫原性片段或免疫原性变体，或由其组成。In an alternative preferred embodiment, the encoded at least one antigenic peptide or protein comprises or consists of the full-length Spike protein or an immunogenic fragment or immunogenic variant of any of these.
术语“全长刺突蛋白”必须理解为具有对应于基本上完整刺突蛋白的氨基酸序列的源自SARS-CoV-2的刺突蛋白。因此，“全长刺突蛋白”可以包含aa 1至aa 1273(参考蛋白：SEQ ID NO:1)。因此，全长刺突蛋白通常可以包含分泌信号肽、刺突蛋白片段S1、刺突蛋白片段S2、受体结合结构域(RBD)和临界中和结构域CND和跨膜结构域。值得注意的是，术语“全长刺突蛋白”还包括包含某些氨基酸置换(例如，用于允许S蛋白的融合前稳定)或天然产生的氨基酸缺失的变体。The term "full-length spike protein" must be understood as a spike protein derived from SARS-CoV-2 having an amino acid sequence corresponding to a substantially complete spike protein. Thus, a "full-length spike protein" may comprise aa 1 to aa 1273 (reference protein: SEQ ID NO: 1). Thus, a full-length spike protein may generally comprise a secretion signal peptide, a spike protein fragment S1, a spike protein fragment S2, a receptor binding domain (RBD) and a critical neutralization domain CND and a transmembrane domain. It is noteworthy that the term "full-length spike protein" also includes variants comprising certain amino acid substitutions (e.g., for allowing pre-fusion stabilization of the S protein) or naturally occurring amino acid deletions.
在特别优选的实施方式中，由第一方面的RNA编码的刺突蛋白(S)进行设计或适应以将抗原稳定在融合前构象。融合前构象在有效的冠状病毒疫苗的情况中特别有利，因为几个潜在的用于中和抗体的表位只在所述融合前蛋白构象中可及。此外，保持蛋白质在融合前构象中目的是避免免疫病理学效应，例如增强的疾病和/或抗体依赖性增强(ADE)。In a particularly preferred embodiment, the spike protein (S) encoded by the RNA of the first aspect is designed or adapted to stabilize the antigen in a prefusion conformation. The prefusion conformation is particularly advantageous in the case of an effective coronavirus vaccine because several potential epitopes for neutralizing antibodies are only accessible in the prefusion protein conformation. In addition, the purpose of keeping the protein in the prefusion conformation is to avoid immunopathological effects, such as enhanced disease and/or antibody-dependent enhancement (ADE).
在优选的实施方式中，将编码融合前稳定刺突蛋白的RNA(或组合物或疫苗)施用于受试者引发刺突蛋白中和抗体，但不引发疾病增强抗体。特别地，将编码融合前稳定刺突蛋白的核酸(或组合物或疫苗)施用于受试者不引发免疫病理学效应，例如增强的疾病和/或抗体依赖性增强(ADE)。In a preferred embodiment, administration of RNA (or composition or vaccine) encoding a pre-fusion stabilized spike protein to a subject elicits spike protein neutralizing antibodies, but does not elicit disease enhancing antibodies. In particular, administration of a nucleic acid (or composition or vaccine) encoding a pre-fusion stabilized spike protein to a subject does not elicit immunopathological effects, such as enhanced disease and/or antibody-dependent enhancement (ADE).
因此，在优选的实施方式中，本发明的RNA包含至少一个编码至少一种选自或源自SARS-CoV-2刺突蛋白(S)的抗原肽或蛋白质的编码序列，其中SARS-CoV-2刺突蛋白(S)是融合前稳定的刺突蛋白(S_stab)。合适地，所述融合前稳定的刺突蛋白包含至少一个融合前稳定突变。Therefore, in a preferred embodiment, the RNA of the present invention comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein (S), wherein the SARS-CoV-2 spike protein (S) is a pre-fusion stabilized spike protein (S_stab). Suitably, the pre-fusion stabilized spike protein comprises at least one pre-fusion stabilizing mutation.
如本文使用的术语“融合前构象”涉及由SARS-CoV-2 S蛋白的胞外结构域在分泌系统中加工成成熟SARS-CoV-2 S蛋白后并且在引发导致SARS-CoV-2 S转变成融合后构象的融合事件前采取的结构构象。The term "prefusion conformation" as used herein relates to the structural conformation adopted by the extracellular domain of the SARS-CoV-2 S protein after processing into the mature SARS-CoV-2 S protein in the secretion system and before initiating the fusion event that leads to the transition of SARS-CoV-2 S to the postfusion conformation.
如本文所述的“融合前稳定的刺突蛋白(S_stab)”与天然SARS-CoV-2 S序列相比包含一个或多个氨基酸置换、缺失或插入，其与从相应的天然SARS-CoV-2 S序列形成的SARS-CoV-2 S胞外结构域三聚体相比，提供了增加的融合前构象的保持。通过一个或多个氨基酸置换、缺失或插入“稳定”融合前构象可以是例如能量稳定(例如，相对于融合后开放构象，降低融合前构象的能量)和/或动力学稳定(例如，降低从融合前构象到融合后构象的转变率)。此外，融合前构象的SARS-CoV-2 S胞外结构域三聚体的稳定可以包括与相应的天然SARS-CoV-2 S序列相比提高对变性的抗性。As described herein, the "pre-fusion stabilized spike protein (S_stab)" comprises one or more amino acid substitutions, deletions or insertions compared to the native SARS-CoV-2 S sequence, which provides increased retention of the pre-fusion conformation compared to the SARS-CoV-2 S extracellular domain trimer formed from the corresponding native SARS-CoV-2 S sequence. "Stabilizing" the pre-fusion conformation by one or more amino acid substitutions, deletions or insertions can be, for example, energy stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and/or kinetic stabilization (e.g., reducing the transition rate from the pre-fusion conformation to the post-fusion conformation). In addition, the stabilization of the SARS-CoV-2 S extracellular domain trimer in the pre-fusion conformation can include increased resistance to denaturation compared to the corresponding native SARS-CoV-2 S sequence.
因此，在优选的实施方式中，SARS-CoV-2刺突蛋白包括一个或多个将S蛋白稳定在融合前构象中的氨基酸置换，例如，稳定融合前构象的S蛋白的膜远端部分(包括N-末端区)的置换。Therefore, in a preferred embodiment, the SARS-CoV-2 spike protein includes one or more amino acid substitutions that stabilize the S protein in a prefusion conformation, for example, substitutions of the membrane distal portion (including the N-terminal region) of the S protein that stabilizes the prefusion conformation.
可以通过用稳定融合前构象的刺突蛋白的氨基酸来置换位置K986和/或V987处的至少一个氨基酸来获得SARS-CoV-2冠状病毒刺突蛋白的稳定(氨基酸位置根据参考SEQ IDNO:1)。Stabilization of the SARS-CoV-2 coronavirus spike protein can be achieved by replacing at least one amino acid at position K986 and/or V987 with an amino acid that stabilizes the spike protein in the pre-fusion conformation (amino acid positions according to reference SEQ ID NO: 1).
在优选的实施方式中，融合前稳定突变包括位置K986和V987的氨基酸置换，其中用选自A、I、L、M、F、V、G或P的氨基酸置换氨基酸K986和/或V987(氨基酸位置根据参考SEQID NO:1)。In a preferred embodiment, the pre-fusion stabilizing mutation comprises amino acid substitutions at positions K986 and V987, wherein amino acids K986 and/or V987 are substituted with amino acids selected from A, I, L, M, F, V, G or P (amino acid positions according to reference SEQ ID NO: 1).
优选地，通过在刺突蛋白中的残基K986和V987处引入两个连续的脯氨酸置换来获得融合前构象的稳定(氨基酸位置根据参考SEQ ID NO:1)。Preferably, stabilization of the prefusion conformation is obtained by introducing two consecutive proline substitutions at residues K986 and V987 in the spike protein (amino acid positions according to reference SEQ ID NO: 1).
因此，在优选的实施方式中，融合前稳定的刺突蛋白(S_stab)包含至少一个融合前稳定突变，其中至少一个融合前稳定突变包括以下氨基酸置换：K986P和V987P(氨基酸位置根据参考SEQ ID NO:1)。Therefore, in a preferred embodiment, the pre-fusion stabilized spike protein (S_stab) comprises at least one pre-fusion stabilizing mutation, wherein at least one pre-fusion stabilizing mutation comprises the following amino acid replacements: K986P and V987P (amino acid positions according to reference SEQ ID NO: 1).
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白是包含至少一个融合前稳定K986P和V987P突变和另外包含以下氨基酸置换或缺失的融合前稳定的刺突蛋白(S_stab)(氨基酸位置根据参考SEQ ID NO:1)：In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention is a prefusion stabilized spike protein (S_stab) comprising at least one prefusion stabilizing K986P and V987P mutation and additionally comprising the following amino acid substitutions or deletions (amino acid positions according to reference SEQ ID NO: 1):
·E484K、N501Y，和任选L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G、A701V；E484K, N501Y, and optionally L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, A701V;
·E484K、N501Y，和任选L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G、A701V；E484K, N501Y, and optionally L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, A701V;
·N501Y、P681H，和任选H69del、V70del、Y144del、A570D、D614G、T716I、S982A、D1118H；N501Y, P681H, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H;
·N501Y、P681H、E484K，和任选H69del、V70del、Y144del、A570D、D614G、T716I、S982A、D1118H；N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H;
·E484K、N501Y，和任选L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I；E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I;
·E484K、N501Y，和任选L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I、V1176F；E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, V1176F;
·N501Y、P681H、E484K，和任选H69del、V70del、Y144del、A570D、D614G、T716I、S982A、D1118H；N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D, D614G, T716I, S982A, D1118H;
·L452R，和任选S13I、W152C、D614G；L452R, and optionally S13I, W152C, D614G;
·L452R、D614D和任选P681R；L452R, D614D and optional P681R;
·L452R、D614D、P681R和任选E484Q、E154K、Q1071H；L452R, D614D, P681R and optional E484Q, E154K, Q1071H;
·L452R、D614D、P681R和任选T19R、L452R、D950N；L452R, D614D, P681R and optional T19R, L452R, D950N;
·L452R、D614D、P681R和任选T19R、F157del、T478K、D950N；L452R, D614D, P681R and optionally T19R, F157del, T478K, D950N;
·E484K，和任选Q52R、A67V、H69del、V70del、delY144、D614G、Q677H、F888L；E484K, and optionally Q52R, A67V, H69del, V70del, delY144, D614G, Q677H, F888L;
·E484K，和任选A67V、H69del、V70del、Y144del、D614G、Q677H、F888L；E484K, and optionally A67V, H69del, V70del, Y144del, D614G, Q677H, F888L;
·E484K，和任选L5F、T95I、D253G、D614G、A701V；E484K, and optionally L5F, T95I, D253G, D614G, A701V;
·P681R，和任选F157L、V367F、Q613H；P681R, and optionally F157L, V367F, Q613H;
·P681R，和任选S254F、D614G、G769V；P681R, and optionally S254F, D614G, G769V;
·L452R、P681R，和任选D614G；L452R, P681R, and optionally D614G;
·L452R、E484Q、P681R，和任选E154K、D614G、Q1071H；L452R, E484Q, P681R, and optionally E154K, D614G, Q1071H;
·L452R、P681R，和任选T19R、F157del、R158del、T478K、D614G、D950N；L452R, P681R, and optionally T19R, F157del, R158del, T478K, D614G, D950N;
·E484K，和任选D614G、V1176F；E484K, and optionally D614G, V1176F;
·L452Q，和任选G75V、T76I、R246del、S247del、Y248del、L249del、T250del、P251del、G252del、F490S、D614G、T859N；L452Q, and optionally G75V, T76I, R246del, S247del, Y248del, L249del, T250del, P251del, G252del, F490S, D614G, T859N;
·K417N，和任选P681R；K417N, and optionally P681R;
·K417N、P681R，和任选D614G；K417N, P681R, and optionally D614G;
·K417N、L452R、P681R，和任选D614G；K417N, L452R, P681R, and optionally D614G;
·K417N、T478K、P681R，和任选D614G；K417N, T478K, P681R, and optionally D614G;
·K417N、D950N、P681R，和任选D614G.K417N, D950N, P681R, and optionally D614G.
·K417N、D614G、P681R，和任选T478K；K417N, D614G, P681R, and optionally T478K;
·K417N、D614G、P681R，和任选L452R；K417N, D614G, P681R, and optionally L452R;
·K417N、D614G、P681R、L452R和任选T478K；K417N, D614G, P681R, L452R and optional T478K;
·S247del、Y248del、L249del、T250del、P251del、G252del、D253del和任选D614G；S247del, Y248del, L249del, T250del, P251del, G252del, D253del, and optionally D614G;
·S247del、Y248del、L249del、T250del、P251del、G252del、D253del和任选L452Q、D614G；S247del, Y248del, L249del, T250del, P251del, G252del, D253del and optionally L452Q, D614G;
·H69del、V70del和任选D614G；H69del, V70del and optionally D614G;
·H69del、V70del、E484K和任选D614G；H69del, V70del, E484K and optionally D614G;
·H69del、V70del、N501Y和任选D614G；或H69del, V70del, N501Y and optionally D614G; or
·H69del、V70del、N501Y、E484K和任选P681H。H69del, V70del, N501Y, E484K and optionally P681H.
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白是包含至少一个融合前稳定K986P和V987P突变和另外包含以下氨基酸置换或缺失的融合前稳定的刺突蛋白(S_stab)(或其片段或变体)(氨基酸位置根据参考SEQ ID NO:1)：In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention is a prefusion stabilized spike protein (S_stab) (or a fragment or variant thereof) comprising at least one prefusion stabilizing K986P and V987P mutation and further comprising the following amino acid substitutions or deletions (amino acid positions according to reference SEQ ID NO: 1):
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V;
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y和T1027I；E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I、V1176F；·E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, V1176F;
·L452R、P681R和D614G；L452R, P681R and D614G;
·L452R、E484Q、P681R、E154K、D614G和Q1071H；或L452R, E484Q, P681R, E154K, D614G, and Q1071H; or
·L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N。L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.
在特别优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白是包含选自以下的氨基酸置换或缺失的融合前稳定的刺突蛋白(S_stab)(或其片段或变体)(氨基酸位置根据参考SEQ ID NO:1)：In a particularly preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the present invention is a pre-fusion stabilized spike protein (S_stab) (or a fragment or variant thereof) comprising an amino acid substitution or deletion selected from the following (amino acid positions are based on reference SEQ ID NO: 1):
·K986P、V987P、E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；或K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or
·K986P、V987P、E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V。K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V.
必须强调，在本发明上下文的实施方式中，如本文定义的任何SARS-CoV-2冠状病毒刺突蛋白可以如上所述进行突变(针对参考蛋白SEQ ID NO:1举例说明)以将刺突蛋白稳定在融合前构象中。It must be emphasized that in the context of the present invention, any SARS-CoV-2 coronavirus spike protein as defined herein can be mutated as described above (exemplified for reference protein SEQ ID NO: 1) to stabilize the spike protein in a pre-fusion conformation.
根据各个实施方式，本发明的RNA编码至少一种如本文定义的抗原SARS-CoV-2刺突蛋白和另外地至少一个异源肽或蛋白质元件。According to various embodiments, the RNA of the invention encodes at least one antigenic SARS-CoV-2 spike protein as defined herein and additionally at least one heterologous peptide or protein element.
合适地，至少一个异源肽或蛋白质元件可以促进或提高所编码的抗原SARS-CoV-2刺突蛋白的分泌(例如，通过分泌信号序列)、促进或提高所编码的SARS-CoV-2刺突蛋白在浆膜中的锚定(例如，通过跨膜元件)、促进或提高抗原复合物的形成(例如，通过多聚化结构域或抗原成簇元件)或者促进或提高病毒样颗粒形成(VLP形成序列)。此外，第一方面的RNA可以另外编码肽接头元件、自我切割肽、免疫性辅助序列或树突细胞靶向序列。Suitably, at least one heterologous peptide or protein element can promote or increase the secretion of the encoded antigen SARS-CoV-2 spike protein (e.g., via a secretion signal sequence), promote or increase the anchoring of the encoded SARS-CoV-2 spike protein in the plasma membrane (e.g., via a transmembrane element), promote or increase the formation of antigen complexes (e.g., via a multimerization domain or antigen clustering element), or promote or increase virus-like particle formation (VLP forming sequence). In addition, the RNA of the first aspect may additionally encode a peptide linker element, a self-cleaving peptide, an immunogenic auxiliary sequence, or a dendritic cell targeting sequence.
合适的多聚化结构域可以选自根据WO2017/081082的SEQ ID NO:1116-1167的氨基酸序列的列表，或这些序列的片段或变体。合适的跨膜元件可以选自根据WO2017/081082的SEQ ID NO:1228-1343的氨基酸序列的列表，或这些序列的片段或变体。合适的VLP形成序列可以选自根据专利申请WO2017/081082的SEQ ID NO:1168-1227的氨基酸序列的列表，或这些序列的片段或变体。合适的肽结构可以选自根据专利申请WO2017/081082的SEQ IDNO:1509-1565的氨基酸序列的列表，或这些序列的片段或变体。合适的自我切割肽可以选自根据专利申请WO2017/081082的SEQ ID NO:1434-1508的氨基酸序列的列表，或这些序列的片段或变体。合适的免疫性辅助序列可以选自根据专利申请WO2017/081082的SEQ IDNO:1360-1421的氨基酸序列的列表，或这些序列的片段或变体。合适的树突细胞(DC)靶向序列可以选自根据专利申请WO2017/081082的SEQ ID NO:1344-1359的氨基酸序列的列表，或这些序列的片段或变体。合适的分泌信号肽可以选自根据公开的PCT专利申请WO2017/081082的SEQ ID NO:1-1115和SEQ ID NO:1728的氨基酸序列的列表，或这些序列的片段或变体。Suitable multimerization domains can be selected from the list of amino acid sequences of SEQ ID NO: 1116-1167 according to WO2017/081082, or fragments or variants of these sequences. Suitable transmembrane elements can be selected from the list of amino acid sequences of SEQ ID NO: 1228-1343 according to WO2017/081082, or fragments or variants of these sequences. Suitable VLP-forming sequences can be selected from the list of amino acid sequences of SEQ ID NO: 1168-1227 according to patent application WO2017/081082, or fragments or variants of these sequences. Suitable peptide structures can be selected from the list of amino acid sequences of SEQ ID NO: 1509-1565 according to patent application WO2017/081082, or fragments or variants of these sequences. Suitable self-cleaving peptides can be selected from the list of amino acid sequences of SEQ ID NO: 1434-1508 according to patent application WO2017/081082, or fragments or variants of these sequences. Suitable immunogenic auxiliary sequences can be selected from the list of amino acid sequences of SEQ ID NO: 1360-1421 according to patent application WO2017/081082, or fragments or variants of these sequences. Suitable dendritic cell (DC) targeting sequences can be selected from the list of amino acid sequences of SEQ ID NO: 1344-1359 according to patent application WO2017/081082, or fragments or variants of these sequences. Suitable secretion signal peptides can be selected from the list of amino acid sequences of SEQ ID NO: 1-1115 and SEQ ID NO: 1728 according to the published PCT patent application WO2017/081082, or fragments or variants of these sequences.
在优选的实施方式中，编码至少一个抗原SARS-CoV-2刺突蛋白的RNA另外编码至少一个异源分泌信号序列和/或三聚化元件，和/或抗原成簇元件，和/或VLP形成序列。In a preferred embodiment, the RNA encoding at least one antigenic SARS-CoV-2 spike protein additionally encodes at least one heterologous secretion signal sequence and/or trimerization element, and/or antigen clustering element, and/or VLP forming sequence.
因此，在优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中任一个或这些中任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588或28917-28920中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ IDNO:22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588或28917-28920中的任一个相同。关于所述氨基酸序列的更多信息也提供于表1中，以及在ST25序列表的相应序列SEQ ID NO的<223>标识符下。Thus, in a preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises at least one amino acid sequence or composition thereof that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. Further information about the amino acid sequence is also provided in Table 1, and under the <223> identifier of the corresponding sequence SEQ ID NO in the ST25 sequence listing.
因此，在优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:27093-27095、28552-28558中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27093-27095、28552-28558中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27093-27095、28552-28558中的任一个相同。Thus, in preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NOs: 27093-27095, 28552-28558.
在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:27095、28552-28557中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27095、28552-28557中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27095、28552-28557中的任一个相同。In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27095, 28552-28557, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 27095, 28552-28557. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NOs: 27095, 28552-28557.
在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:27095中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27095中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27095中的任一个相同。In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 27095 or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 27095. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 27095.
在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:23090、23091、22960、22961、28540中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或氨基酸编码序列，或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:23090、23091、22960、22961、28540中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ IDNO:23090、23091、22960、22961、28540中的任一个相同。In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence or amino acid coding sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 23090, 23091, 22960, 22961, 28540, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 23090, 23091, 22960, 22961, 28540. In some embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 23090, 23091, 22960, 22961, 28540.
在再进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:27096、28545中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27096、28545中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:27096、28545中的任一个相同。In yet further preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 27096, 28545. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NOs: 27096, 28545.
在再进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:22959中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:22959中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:22959中的任一个相同。In yet further preferred embodiments, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959 or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 22959. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NO: 22959.
在进一步优选的实施方式中，由本发明的RNA编码的SARS-CoV-2刺突蛋白包含至少一个与SEQ ID NO:28541-28544、28917-28920中任一个或这些任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列或由其组成。因此，在一些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:28541-28544、28917-28920中的任一个至少95％相同。在某些实施方式中，SARS-CoV-2刺突蛋白与SEQ ID NO:28541-28544、28917-28920中的任一个相同。In a further preferred embodiment, the SARS-CoV-2 spike protein encoded by the RNA of the invention comprises or consists of at least one amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920, or an immunogenic fragment or immunogenic variant of any of these. Thus, in some embodiments, the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920. In certain embodiments, the SARS-CoV-2 spike protein is identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
如本文定义的源自SARS-CoV-2的优选抗原肽或蛋白质提供于表1中。其中，每行对应于合适的SARS-CoV-2蛋白构建体。表1的列A提供了合适的抗原构建体的简短描述。表1的列B提供相应抗原构建体的蛋白质(氨基酸)SEQ ID NO。表1的列C提供了相应的G/C优化的核酸编码序列(opt1，gc)的SEQ ID NO。表1的列D提供了相应的G/C含量改变的核酸编码序列(opt10，gc mod)的SEQ ID NO(对于“编码序列”的详细描述，参见段落“合适的编码序列”)。Preferred antigenic peptides or proteins derived from SARS-CoV-2 as defined herein are provided in Table 1. Therein, each row corresponds to a suitable SARS-CoV-2 protein construct. Column A of Table 1 provides a brief description of a suitable antigenic construct. Column B of Table 1 provides the protein (amino acid) SEQ ID NO of the corresponding antigenic construct. Column C of Table 1 provides the SEQ ID NO of the corresponding G/C optimized nucleic acid coding sequence (opt1, gc). Column D of Table 1 provides the SEQ ID NO of the corresponding nucleic acid coding sequence with a changed G/C content (opt10, gc mod) (for a detailed description of the "coding sequence", see the paragraph "suitable coding sequence").
值得注意的是，本发明的描述明确包括了本申请的ST25序列表的<223>标识符下提供的信息。包含表1的编码序列的优选RNA构建体，例如，包含表1的编码序列的mRNA序列，提供于表2中。It is noteworthy that the description of the present invention explicitly includes the information provided under the <223> identifier of the ST25 sequence listing of the present application. Preferred RNA constructs comprising the coding sequence of Table 1, for example, mRNA sequences comprising the coding sequence of Table 1, are provided in Table 2.
表1：优选的SARS-CoV-2构建体(氨基酸序列和核酸编码序列)：Table 1: Preferred SARS-CoV-2 constructs (amino acid sequences and nucleic acid coding sequences):
w/oFCS：缺失的弗林蛋白酶切割位点w/oFCS: missing furin cleavage site
合适的编码序列：Suitable coding sequences :
根据优选的实施方式，本发明的RNA包含至少一个编码至少一种选自或源自SARS-CoV-2刺突蛋白(优选如上定义的)或其片段和变体的抗原肽或蛋白质的编码序列。在该情况中，任何编码至少一种如本文定义的抗原蛋白SARS-CoV-2刺突蛋白或其片段和变体的编码序列可以理解为合适的编码序列并且因此可以包含在本发明的RNA中。According to a preferred embodiment, the RNA of the present invention comprises at least one coding sequence encoding at least one antigenic peptide or protein selected from or derived from the SARS-CoV-2 spike protein (preferably as defined above) or fragments and variants thereof. In this case, any coding sequence encoding at least one antigenic protein SARS-CoV-2 spike protein or fragments and variants thereof as defined herein can be understood as a suitable coding sequence and can therefore be included in the RNA of the present invention.
在优选的实施方式中，第一方面的RNA可以包含至少一个编码至少一种来自如本文定义的SARS-CoV-2的抗原肽或蛋白质的编码序列，或由其组成，优选编码SEQ ID NO:1、10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中的任一个，或其片段或变体。必须理解，在核酸水平上，可以选择任何编码与SEQ ID NO:116、136、137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637、28916、28921-28924中任一个或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的RNA序列，并可以因此理解为本发明的合适的编码序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:116、136、137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637、28916或28921-28924中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect may comprise or consist of at least one coding sequence encoding at least one antigenic peptide or protein from SARS-CoV-2 as defined herein, preferably encoding any one of SEQ ID NO: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. It must be understood that at the nucleic acid level, any coding sequence that is similar to SEQ ID NO: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920 may be selected. NO:116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916, 28921-28924, or a fragment or variant thereof, or an RNA sequence having an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and can therefore be understood as a suitable coding sequence of the present invention. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916, or 28921-28924.
在优选的实施方式中，第一方面的RNA可以包含至少一个编码至少一种来自如本文定义的SARS-CoV-2的抗原肽或蛋白质的编码序列，或由其组成，优选编码SEQ ID NO:10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中的任一个，或其片段或变体。必须理解，在核酸水平上，可以选择任何编码与SEQ ID NO:137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、23095-23112、27110-27247、28589-28637、28921-28924中任一个或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的RNA序列，并可以因此理解为本发明的合适的编码序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247 28589-28637或28921-28924中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect may comprise or consist of at least one coding sequence encoding at least one antigenic peptide or protein from SARS-CoV-2 as defined herein, preferably encoding any one of SEQ ID NO: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. It must be understood that at the nucleic acid level, any coding sequence that is similar to SEQ ID NO: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920 may be selected. NO:137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 23095-23112, 27110-27247, 28589-28637, 28921-28924, or a fragment or variant thereof, or an RNA sequence having an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and can therefore be understood as a suitable coding sequence of the present invention. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247 28589-28637 or 28921-28924.
在优选的实施方式中，第一方面的RNA可以包含至少一个编码至少一种来自如本文定义的SARS-CoV-2的抗原肽或蛋白质的编码序列，或由所述编码序列组成，优选编码SEQID NO:10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中的任一个，或其片段或变体。必须理解，在核酸水平上，可以选择任何编码与SEQ ID NO:137、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、27110-27201、28589-28637、28921-28924中任一个或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的RNA序列，并可以因此理解为本发明的合适的编码序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:137、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、27110-27201、28589-28637或28921-28924中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect may comprise or consist of at least one coding sequence encoding at least one antigenic peptide or protein from SARS-CoV-2 as defined herein, preferably encoding any one of SEQ ID NO: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. It must be understood that at the nucleic acid level, any RNA sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, 28921-28924, or a fragment or variant thereof, can be selected and can therefore be understood as a suitable coding sequence of the present invention. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, or 28921-28924.
在优选的实施方式中，第一方面的RNA可以包含至少一个编码至少一种来自如本文定义的SARS-CoV-2的抗原肽或蛋白质的编码序列，或由所述编码序列组成，优选编码SEQID NO:10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920中的任一个，或其片段或变体。必须理解，在核酸水平上，可以选择任何编码与SEQ ID NO:146、23150-23184、27202-27247中任一个或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的RNA序列，并可以因此理解为本发明的合适的编码序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:146、23150-23184或27202-27247中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect may comprise or consist of at least one coding sequence encoding at least one antigenic peptide or protein from SARS-CoV-2 as defined herein, preferably encoding any one of SEQ ID NO: 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or a fragment or variant thereof. It must be understood that at the nucleic acid level, any RNA sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 146, 23150-23184, 27202-27247, or a fragment or variant thereof, can be selected and can therefore be understood as a suitable coding sequence for the present invention. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 146, 23150-23184 or 27202-27247.
在优选的实施方式中，第一方面的RNA包含编码序列，所述编码序列包含至少一个与根据SEQ ID NO:22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637、28921-28924的序列或这些序列中任一个的片段或者片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637或28921-28924中的任一个至少95％相同。关于这些合适的核酸序列中每一个的更多信息也可以源自序列表，特别是来自标识符<223>下本文提供的详细内容。In a preferred embodiment, the RNA of the first aspect comprises a coding sequence comprising at least one nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence according to SEQ ID NO: 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28921-28924, or a fragment or fragment or variant of any of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NOs: 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, or 28921-28924. Further information about each of these suitable nucleic acid sequences can also be derived from the sequence listing, in particular from the details provided herein under identifier <223>.
在优选的实施方式中，第一方面的RNA包含编码序列，所述编码序列包含至少一个与根据SEQ ID NO:22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23094、27110-27132、28589-28637或28921-28924的序列或这些序列中任一个的片段或者片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23094、27110-27132、28589-28637或28921-28924中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect comprises a coding sequence comprising at least one nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence according to SEQ ID NO: 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23094, 27110-27132, 28589-28637 or 28921-28924, or a fragment or fragment or variant of any of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any one of SEQ ID NO: 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23094, 27110-27132, 28589-28637, or 28921-28924.
在优选的实施方式中，第一方面的RNA包含编码序列，所述编码序列包含至少一个与根据SEQ ID NO:23150-23166或27202-27224的序列或这些序列中任一个的片段或片段变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:23150-23166或27202-27224中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect comprises a coding sequence comprising at least one nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence according to SEQ ID NO: 23150-23166 or 27202-27224, or a fragment or fragment variant of any of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any of SEQ ID NO: 23150-23166 or 27202-27224.
在优选的实施方式中，第一方面的RNA包含编码序列，所述编码序列包含至少一个与根据SEQ ID NO:23150-23166、27202-27224、23114-23130或27156-27178的序列或这些序列中任一个的片段或片段变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:23114-23130或27156-27178中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect comprises a coding sequence comprising at least one nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence according to SEQ ID NO: 23150-23166, 27202-27224, 23114-23130 or 27156-27178, or a fragment or fragment variant of any of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any of SEQ ID NO: 23114-23130 or 27156-27178.
在优选的实施方式中，第一方面的RNA包含编码序列，所述编码序列包含至少一个与根据SEQ ID NO:23150-23166、27202-27224、23167-23184或27225-27247的序列或这些序列中任一个的片段或片段变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在某些实施方式中，编码SARS-CoV-2刺突蛋白的RNA序列与SEQ ID NO:23167-23184或27225-27247中的任一个至少95％相同。In a preferred embodiment, the RNA of the first aspect comprises a coding sequence comprising at least one nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence according to SEQ ID NO: 23150-23166, 27202-27224, 23167-23184 or 27225-27247, or a fragment or fragment variant of any of these sequences. In certain embodiments, the RNA sequence encoding the SARS-CoV-2 spike protein is at least 95% identical to any of SEQ ID NO: 23167-23184 or 27225-27247.
在优选的实施方式中，第一方面的RNA是人工RNA。In a preferred embodiment, the RNA of the first aspect is an artificial RNA.
如本文使用的术语“人工RNA”旨在表示不是天然产生的RNA。换句话说，人工RNA可以理解为非天然RNA分子。这样的RNA分子可以是由于其单独序列(例如，G/C含量改变的编码序列，UTR)和/或由于其他修饰(例如，核苷酸的结构修饰)而非天然的。通常，人工RNA可以通过遗传工程化至对应于所需的人工核苷酸序列来设计和/或产生。在这种情况中，人工RNA是可能非天然产生的序列，即，与野生型序列/天然产生序列至少一个核苷酸不同的序列。术语“人工RNA”不限于表示“一个单一RNA分子”，而是理解为包含基本上相同的RNA分子的集合。因此，可以涉及多个基本上相同的RNA分子。As used herein, the term "artificial RNA" is intended to represent RNA that is not naturally produced. In other words, artificial RNA can be understood as non-natural RNA molecules. Such RNA molecules can be due to their individual sequences (e.g., coding sequences that G/C content changes, UTR) and/or due to other modifications (e.g., structural modifications of nucleotides) and non-natural. Generally, artificial RNA can be designed and/or produced by genetic engineering to the corresponding desired artificial nucleotide sequence. In this case, artificial RNA is a sequence that may be non-naturally produced, that is, a sequence different from at least one nucleotide of a wild-type sequence/naturally produced sequence. The term "artificial RNA" is not limited to representing "a single RNA molecule", but is understood to comprise a set of substantially identical RNA molecules. Therefore, multiple substantially identical RNA molecules can be related to.
在优选的实施方式中，第一方面的RNA是修饰和/或稳定的RNA，优选修饰和/或稳定的人工RNA。In a preferred embodiment, the RNA of the first aspect is a modified and/or stabilized RNA, preferably a modified and/or stabilized artificial RNA.
根据优选的实施方式，本发明的RNA因此可以作为“稳定的人工RNA”或“稳定的编码RNA”来提供，显示出提高的对体内降解的抗性的RNA和/或显示出提高的体内稳定性的RNA，和/或显示出提高的体内可翻译性的RNA。在下文中，描述了这种情况中特定的合适修饰/适应，其合适地“稳定”RNA。优选地，本发明的RNA可以作为“稳定的RNA”或“稳定的编码RNA”来提供。According to a preferred embodiment, the RNA of the present invention can therefore be provided as a "stable artificial RNA" or a "stable coding RNA", an RNA showing increased resistance to in vivo degradation and/or an RNA showing increased in vivo stability, and/or an RNA showing increased in vivo translatability. In the following, specific suitable modifications/adaptations in this case are described, which suitably "stabilize" the RNA. Preferably, the RNA of the present invention can be provided as a "stable RNA" or a "stable coding RNA".
这样的稳定可以通过提供“干燥的RNA”和/或“纯化的RNA”来实现，如下面进一步说明的。可选地，或另外地，这样的稳定可以例如通过本发明的RNA的修饰的磷酸酯主链来实现。与本发明相关的主链修饰是其中核酸所包含的核苷酸主链的磷酸酯被化学修饰的修饰。可以在这方面使用的核苷酸包含例如硫代磷酸酯修饰的磷酸酯主链，优选磷酸酯主链中含有的至少一个磷酸酯氧被硫原子替代。稳定的RNA可以进一步包括，例如：非离子磷酸酯类似物，如，例如烷基和芳基膦酸酯，其中带电的膦酸酯氧被烷基或芳基替代，或磷酸二酯和烷基磷酸三酯，其中带电荷的氧残基以烷基化形式存在。这种主链修饰通常包括选自甲基膦酸酯、氨基磷酸酯和硫代磷酸酯(例如胞苷-5’-O-(1-硫代磷酸酯))的修饰，但不表示任何限制。Such stabilization can be achieved by providing "dry RNA" and/or "purified RNA", as further described below. Alternatively, or additionally, such stabilization can be achieved, for example, by the modified phosphate backbone of the RNA of the present invention. The backbone modification associated with the present invention is a modification in which the phosphate of the nucleotide backbone contained in the nucleic acid is chemically modified. The nucleotides that can be used in this regard include, for example, thiophosphate-modified phosphate backbones, preferably at least one phosphate oxygen contained in the phosphate backbone is replaced by a sulfur atom. Stable RNA can further include, for example: nonionic phosphate analogs, such as, for example, alkyl and aryl phosphonates, wherein the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiester and alkyl phosphotriester, wherein the charged oxygen residue exists in an alkylated form. This backbone modification generally includes modifications selected from methylphosphonate, phosphoramidate and thiophosphate (for example cytidine-5'-O-(1-thiophosphate)), but does not represent any limitation.
在下文中，描述了合适的修饰，其能够“稳定”本发明的RNA。In the following, suitable modifications are described, which are able to "stabilize" the RNA of the invention.
在优选的实施方式中，RNA包含至少一个密码子修饰的编码序列。In a preferred embodiment, the RNA comprises a coding sequence in which at least one codon is modified.
在优选的实施方式中，RNA的至少一个编码序列是密码子修饰的编码序列，其中由至少一个密码子修饰的编码序列所编码的氨基酸序列优选与由相应的野生型编码序列或参考编码序列编码的氨基酸序列相比是未修饰的。In a preferred embodiment, at least one coding sequence of the RNA is a codon-modified coding sequence, wherein the amino acid sequence encoded by the at least one codon-modified coding sequence is preferably unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence or a reference coding sequence.
术语“密码子修饰的编码序列”涉及与相应的野生型或参考编码序列相比至少一个密码子(编码一个氨基酸的核苷酸三联体)不同的编码序列。合适地，本发明情况中的密码子修饰的编码序列可以显示出提高的对体内降解的抗性和/或提高的体内稳定性和/或提高的体内可翻译性。最宽泛含义的密码子修饰利用遗传密码的简并性，其中多个密码子可以编码相同的氨基酸并且可以互换使用来优化/修饰用于体内应用的编码序列。The term "codon-modified coding sequence" relates to a coding sequence that differs in at least one codon (nucleotide triplet encoding one amino acid) compared to a corresponding wild-type or reference coding sequence. Suitably, the codon-modified coding sequence in the context of the present invention may exhibit increased resistance to in vivo degradation and/or increased in vivo stability and/or increased in vivo translatability. Codon modification in the broadest sense utilizes the degeneracy of the genetic code, wherein multiple codons can encode the same amino acid and can be used interchangeably to optimize/modify coding sequences for in vivo applications.
术语“参考编码序列”涉及作为待修饰和/或优化的原始序列的编码序列。The term "reference coding sequence" relates to a coding sequence which is the original sequence to be modified and/or optimized.
在优选的实施方式中，RNA的至少一个编码序列是密码子修饰的编码序列，其中密码子修饰的编码序列选自C最大化的编码序列、CAI最大化的编码序列、人密码子使用适应的编码序列、G/C含量改变的编码序列和G/C优化的编码序列，或其任意组合。In a preferred embodiment, at least one coding sequence of the RNA is a codon-modified coding sequence, wherein the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage-adapted coding sequence, a G/C content-altered coding sequence, and a G/C-optimized coding sequence, or any combination thereof.
在优选的实施方式中，RNA的至少一个编码序列具有至少约50％、55％或60％的G/C含量。在特定的实施方式中，组分A的RNA的至少一个编码序列具有至少约50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％或70％的G/C含量。In preferred embodiments, at least one coding sequence of the RNA has a G/C content of at least about 50%, 55%, or 60%. In specific embodiments, at least one coding sequence of the RNA of component A has a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
转染至哺乳动物宿主细胞中时，包含密码子修饰的编码序列的RNA具有12-18小时，或大于18小时，例如，24、36、48、60、72，或大于72小时的稳定性，并且能够被哺乳动物宿主细胞(例如，肌肉细胞)表达。When transfected into a mammalian host cell, the RNA comprising the codon modified coding sequence has a stability of 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours, and is capable of being expressed by the mammalian host cell (e.g., muscle cell).
转染至哺乳动物宿主细胞中时，包含密码子修饰的编码序列的RNA翻译成蛋白质，其中蛋白质的量至少与通过转染至哺乳动物宿主细胞中的天然产生的或野生型或参考编码序列获得的蛋白质的量相当，或优选比通过转染至哺乳动物宿主细胞中的天然产生的或野生型或参考编码序列获得的蛋白质量多至少10％，或多至少20％，或多至少30％，或多至少40％，或多至少50％，或多至少100％，或多至少200％。When transfected into a mammalian host cell, RNA comprising a codon-modified coding sequence is translated into a protein, wherein the amount of protein is at least comparable to the amount of protein obtained by transfecting a naturally occurring or wild-type or reference coding sequence into a mammalian host cell, or preferably at least 10% more, or at least 20% more, or at least 30% more, or at least 40% more, or at least 50% more, or at least 100% more, or at least 200% more than the amount of protein obtained by transfecting a naturally occurring or wild-type or reference coding sequence into a mammalian host cell.
在一些实施方式中，RNA可以是修饰的，其中与相应的野生型或参考编码序列的C含量相比，至少一个编码序列的C含量可以增加，优选最大化(在本文中称为“C最大化的编码序列”)。可以合适地使用根据WO2015/062738的修饰方法进行C最大化核酸序列的产生。在这种情况中，WO2015/062738的公开内容在此通过引用包括。In some embodiments, the RNA may be modified, wherein the C content of at least one coding sequence may be increased, preferably maximized, compared to the C content of the corresponding wild-type or reference coding sequence (referred to herein as a "C-maximized coding sequence"). The generation of a C-maximized nucleic acid sequence may be suitably performed using a modification method according to WO2015/062738. In this case, the disclosure of WO2015/062738 is hereby included by reference.
在优选的实施方式中，RNA可以是修饰的，其中与相应的野生型或参考编码序列的G/C含量相比，至少一个编码序列的G/C含量可以是优化的(在本文称为“G/C含量优化的编码序列”)。在该情况中的“优化的”是指其中G/C含量优选增加至基本上最高可能的G/C含量的编码序列。可以使用根据WO2002/098443的方法来进行G/C含量优化的RNA序列的产生。在这种情况中，将WO2002/098443的公开内容以其全部范围包括在本发明中。在整个说明书中，包括序列表的<223>标识符，通过缩写“opt1”或“gc”来表示G/C优化的编码序列。In a preferred embodiment, the RNA may be modified, wherein the G/C content of at least one coding sequence may be optimized (referred to herein as "G/C content optimized coding sequence") compared to the G/C content of the corresponding wild-type or reference coding sequence. "Optimized" in this case refers to a coding sequence in which the G/C content is preferably increased to substantially the highest possible G/C content. The generation of RNA sequences optimized for G/C content may be performed using the method according to WO2002/098443. In this case, the disclosure of WO2002/098443 is included in the present invention to its full extent. Throughout the specification, including the <223> identifier of the sequence listing, G/C optimized coding sequences are represented by the abbreviations "opt1" or "gc".
在优选的实施方式中，RNA可以是修饰的，其中至少一个编码序列中的密码子可以适应于人密码子使用(在本文称为“人密码子使用适应的编码序列”)。编码相同氨基酸的密码子在人中以不同频率出现。因此，核酸的编码序列优选是修饰的，使得编码相同氨基酸的密码子的频率对应于该密码子根据人密码子使用的天然产生的频率。例如，在氨基酸Ala的情况中，野生型或参考编码序列优选采用密码子“GCC”以0.40的频率使用、密码子“GCT”以0.28的频率使用、密码子“GCA”以0.22的频率使用和密码子“GCG”以0.10的频率使用等的方式来适应。因此，这样的程序(如针对Ala举例说明的)适用于由核酸的编码序列编码的每个氨基酸，以获得适应于人密码子使用的序列。在整个说明书中，包括序列表中的<223>标识符，通过缩写“opt3”或“人”来表示人密码子使用适应的编码序列。In a preferred embodiment, the RNA may be modified, wherein the codons in at least one coding sequence may be adapted to human codon usage (referred to herein as "human codon usage adapted coding sequences"). Codons encoding the same amino acid occur at different frequencies in humans. Therefore, the coding sequence of the nucleic acid is preferably modified so that the frequency of codons encoding the same amino acid corresponds to the frequency of the codons naturally produced according to human codon usage. For example, in the case of the amino acid Ala, the wild-type or reference coding sequence is preferably adapted in such a way that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" is used at a frequency of 0.28, the codon "GCA" is used at a frequency of 0.22, and the codon "GCG" is used at a frequency of 0.10. Therefore, such a procedure (as exemplified for Ala) is applied to each amino acid encoded by the coding sequence of the nucleic acid to obtain a sequence adapted to human codon usage. Throughout the specification, including the <223> identifier in the sequence listing, coding sequences adapted to human codon usage are represented by the abbreviations "opt3" or "human".
在一些实施方式中，RNA可以是修饰的，其中与相应的野生型或参考编码序列的G/C含量相比，至少一个编码序列的G/C含量可以是改变的(在本文称为“G/C含量改变的编码序列”)。在这种情况中，术语“G/C优化”或“G/C含量改变”涉及与相应的野生型或参考编码序列相比，包含改变的(优选增加的)鸟苷和/或胞嘧啶核苷酸数量的核酸。这种增加的数量可以通过用含有鸟苷或胞嘧啶核苷酸的密码子替代含有腺苷或胸腺嘧啶核苷酸的密码子来产生。有利地，与具有增加的A/U的序列相比，具有增加的G/C含量的核酸序列更稳定或显示出更好的表达。优选地，与相应野生型或参考核酸序列的编码序列的G/C含量相比，核酸的编码序列的G/C含量增加至少10％、20％、30％，优选增加至少40％(在本文称为“opt 10”或“gc mod”)。例如，相对于SEQ ID NO:28916的G/C含量，核酸的编码序列的G/C含量优选增加至少10％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％或25％。In some embodiments, the RNA may be modified, wherein the G/C content of at least one coding sequence may be altered compared to the G/C content of the corresponding wild-type or reference coding sequence (referred to herein as a "coding sequence with altered G/C content"). In this case, the term "G/C optimization" or "G/C content alteration" relates to a nucleic acid comprising an altered (preferably increased) number of guanosine and/or cytosine nucleotides compared to the corresponding wild-type or reference coding sequence. This increased number can be generated by replacing codons containing adenosine or thymine nucleotides with codons containing guanosine or cytosine nucleotides. Advantageously, nucleic acid sequences with increased G/C content are more stable or show better expression compared to sequences with increased A/U. Preferably, the G/C content of the coding sequence of the nucleic acid is increased by at least 10%, 20%, 30%, preferably by at least 40% (referred to herein as "opt 10" or "gc mod") compared to the G/C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence. For example, relative to the G/C content of SEQ ID NO: 28916, the G/C content of the coding sequence of the nucleic acid is preferably increased by at least 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%.
在一些实施方式中，RNA可以是修饰的，其中在至少一个编码序列中的密码子适应指数(CAI)可以增加，或优选最大化(在本文称为“CAI最大化的编码序列”)。优选地例如在人中相对稀少的所有野生型或参考核酸序列的密码子交换为例如在人中频繁的相应密码子，其中频繁的密码子编码与相对稀少的密码子相同的氨基酸。合适地，最频繁的密码子用于编码的蛋白质的每个氨基酸。合适地，RNA包含至少一个编码序列，其中该至少一个编码序列的密码子适应指数(CAI)为至少0.5、至少0.8、至少0.9或至少0.95。最优选地，至少一个编码序列的密码子适应指数(CAI)是1(CAI＝1)。例如，在氨基酸Ala的情况下，野生型或参考编码序列可以以最频繁的人密码子“GCC”总是用于所述氨基酸的方式来适应。因此，这样的程序(如针对Ala举例说明的)可以应用于由核酸的编码序列编码的每个氨基酸以获得CAI最大化的编码序列。In some embodiments, RNA can be modified, wherein the codon adaptation index (CAI) in at least one coding sequence can be increased, or preferably maximized (referred to herein as "CAI maximized coding sequence"). Preferably, the codons of all wild-type or reference nucleic acid sequences that are relatively rare, such as in humans, are exchanged for corresponding codons that are frequent, such as in humans, wherein the frequent codons encode the same amino acids as the relatively rare codons. Suitably, the most frequent codons are used for each amino acid of the encoded protein. Suitably, RNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of at least one coding sequence is 1 (CAI=1). For example, in the case of amino acid Ala, the wild-type or reference coding sequence can be adapted in such a way that the most frequent human codon "GCC" is always used for the amino acid. Therefore, such a program (as exemplified for Ala) can be applied to each amino acid encoded by the coding sequence of the nucleic acid to obtain a coding sequence that maximizes CAI.
在特别优选的实施方式中，核酸的至少一个编码序列是密码子修饰的编码序列，其中密码子修饰的编码序列是G/C优化的编码序列。In a particularly preferred embodiment, at least one coding sequence of the nucleic acid is a codon-modified coding sequence, wherein the codon-modified coding sequence is a G/C-optimized coding sequence.
在特别优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由G/C优化的编码序列组成的编码序列，其与选自SEQ ID NO:137、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、27110-27201、28589-28637、28921-28924或这些序列的任一个的片段或变体的G/C优化的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。In a particularly preferred embodiment, the RNA of the first aspect comprises at least one coding sequence comprising or consisting of a G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein, which is selected from SEQ ID NO:137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, 28921-28924 or a fragment or variant of any of these sequences is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the G/C optimized nucleic acid sequence.
在特别优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2的G/C优化的编码序列或由其组成的编码序列，其与选自SEQ ID NO:146、23150-23184、27202-27247或这些序列的任一个的片段或变体的G/C优化的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。In a particularly preferred embodiment, the RNA of the first aspect comprises at least one coding sequence comprising or consisting of a G/C optimized coding sequence encoding SARS-CoV-2 as defined herein, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a G/C optimized nucleic acid sequence selected from SEQ ID NO: 146, 23150-23184, 27202-27247 or a fragment or variant of any of these sequences.
在甚至更优选的实施方式中，第一方面的RNA包含至少一个包含码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由其组成的编码序列，其与选自SEQ ID NO:23090、23108、23126、23144、23162、23180、23091、23109、23127、23145、23163、23181、28589(B.1.315；C.1.2)的密码子修饰的核酸序列或这些序列的任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:23090、23108、23126、23144、23162、23180、23091、23109、23127、23145、23163、23181或28589至少95％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ IDNO:23090-23091、23162-23163或28589至少95％相同。In an even more preferred embodiment, the RNA of the first aspect comprises at least one G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein, or consisting of a coding sequence, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a codon modified nucleic acid sequence selected from SEQ ID NO: 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109, 23127, 23145, 23163, 23181, 28589 (B.1.315; C.1.2), or a fragment or variant of any of these sequences. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109, 23127, 23145, 23163, 23181, or 28589. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 23090-23091, 23162-23163, or 28589.
在甚至更优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由其组成的编码序列，其与选自27116、27139、27162、27185、27208、27231、27117、27140、27163、27186、27209、27232、27118、27141、27164、27187、27210、27233、28601-28607(B.1.617；B.1.617.1；B.1.617.2；AY.1；AY.2；AY.4；AY.4.2；B.1.617.3)的密码子修饰的核酸序列或这些序列的任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27116、27139、27162、27185、27208、27231、27117、27140、27163、27186、27209、27232、27118、27141、27164、27187、27210、27233或28601-28607至少95％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27116-27118、27208-27210或28601-28607至少95％相同。In an even more preferred embodiment, the RNA of the first aspect comprises at least one G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein or consisting of a coding sequence selected from 27116, 27139, 27162, 27185, 27208, 27231, 27117, 27140, 27163, 27186, 27209, 27232, 27118, 27141, 27164, 27187, 27210, 2 7233, 28601-28607 (B.1.617; B.1.617.1; B.1.617.2; AY.1; AY.2; AY.4; AY.4.2; B.1.617.3) codon-modified nucleic acid sequences or fragments or variants of any of these sequences are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27116, 27139, 27162, 27185, 27208, 27231, 27117, 27140, 27163, 27186, 27209, 27232, 27118, 27141, 27164, 27187, 27210, 27233, or 28601-28607. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27116-27118, 27208-27210, or 28601-28607.
在甚至更优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由其组成的编码序列，其与选自SEQ ID NO:27118、27141、27164、27187、27210、27233或28601-28606(B.1.617.2；AY.1；AY.2；AY.4；AY.4.2)的密码子修饰的核酸序列或这些序列的任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27118、27141、27164、27187、27210、27233或28601-28606至少95％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27118、27210或28601-28606至少95％相同。In an even more preferred embodiment, the RNA of the first aspect comprises at least one G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein, or consisting of a coding sequence, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a codon-modified nucleic acid sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233 or 28601-28606 (B.1.617.2; AY.1; AY.2; AY.4; AY.4.2), or a fragment or variant of any of these sequences. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233, or 28601-28606. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27118, 27210, or 28601-28606.
在甚至更优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由其组成的编码序列，其与选自SEQ ID NO:27118、27141、27164、27187、27210或27233(B.1.617.2)的密码子修饰的核酸序列或这些序列的任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27118、27141、27164、27187、27210或27233至少95％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:27118或27210至少95％相同。In an even more preferred embodiment, the RNA of the first aspect comprises at least one G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein or consisting of a coding sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a codon-modified nucleic acid sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210 or 27233 (B.1.617.2) or a fragment or variant of any of these sequences. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27118, 27141, 27164, 27187, 27210 or 27233. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 27118 or 27210.
在进一步优选的实施方式中，第一方面的RNA包含至少一个包含编码如本文定义的SARS-CoV-2抗原的G/C优化的编码序列或由其组成的编码序列，其与选自SEQ ID NO:28590-28593、28921-28924(B.1.1.529，Omicron)的密码子修饰的核酸序列或这些序列的任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:28590-28593至少95％相同。在一些方面中，至少一个编码SARS-CoV-2抗原的编码序列与SEQ ID NO:28590-28593、28921-28924至少95％相同。In a further preferred embodiment, the RNA of the first aspect comprises at least one G/C optimized coding sequence encoding a SARS-CoV-2 antigen as defined herein or consisting of a coding sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a codon-modified nucleic acid sequence selected from SEQ ID NO: 28590-28593, 28921-28924 (B.1.1.529, Omicron) or a fragment or variant of any of these sequences. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NO: 28590-28593. In some aspects, at least one coding sequence encoding a SARS-CoV-2 antigen is at least 95% identical to SEQ ID NOs: 28590-28593, 28921-28924.
UTR：UTR :
在优选的实施方式中，本发明的RNA包含至少一个编码至少一种如本文定义的SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体的编码序列，其中RNA包含至少一个异源未翻译区(UTR)。在一些方面中，该实施方式的RNA不包括包含SEQ ID NO:268的序列的3’UTR。在某些方面中，该实施方式的RNA包括包含SEQ ID NO:268的序列的3’UTR。In a preferred embodiment, the RNA of the invention comprises at least one coding sequence encoding at least one SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof as defined herein, wherein the RNA comprises at least one heterologous untranslated region (UTR). In some aspects, the RNA of this embodiment does not include a 3'UTR comprising the sequence of SEQ ID NO: 268. In certain aspects, the RNA of this embodiment includes a 3'UTR comprising the sequence of SEQ ID NO: 268.
在优选的实施方式中，本发明的RNA包含蛋白质编码区(“编码序列”或“cds”)，和5’-UTR和/或3’-UTR。值得注意的是，UTR可以带有调控序列元件，其确定核酸，例如，RNA周转、稳定性和定位。此外，UTR可以带有增强翻译的序列元件。在医疗应用中，RNA翻译成至少一种肽或蛋白质对治疗功效是极为重要的。3’-UTR和/或5’-UTR的某些组合可以增强可操作连接的编码本发明的肽或蛋白质的编码序列的表达。带有所述UTR组合的RNA分子有利地能够在施用于受试者后，优选肌内施用后，快速和瞬时表达抗原肽或蛋白质。因此，包含如本文提供的某些3’-UTR和/或5’-UTR组合的RNA特别适用于作为疫苗施用，特别是适用于施用至受试者的肌肉、真皮或表皮中。In a preferred embodiment, the RNA of the present invention comprises a protein coding region ("coding sequence" or "cds"), and a 5'-UTR and/or a 3'-UTR. It is noteworthy that the UTR may carry regulatory sequence elements that determine nucleic acid, e.g., RNA turnover, stability and positioning. In addition, the UTR may carry sequence elements that enhance translation. In medical applications, the translation of RNA into at least one peptide or protein is extremely important for therapeutic efficacy. Certain combinations of 3'-UTR and/or 5'-UTR can enhance the expression of operably linked coding sequences encoding peptides or proteins of the present invention. The RNA molecule with the UTR combination is advantageously able to rapidly and transiently express antigenic peptides or proteins after administration to a subject, preferably after intramuscular administration. Therefore, RNA comprising certain 3'-UTR and/or 5'-UTR combinations as provided herein is particularly suitable for administration as a vaccine, particularly suitable for administration to the muscle, dermis or epidermis of a subject.
合适地，本发明的RNA包含至少一个异源5’-UTR和/或至少一个异源3’-UTR。所述异源5’-UTR或3’-UTR可以源自天然产生的基因或可以合成地工程化。在优选的实施方式中，RNA包含至少一个如本文定义的编码序列，其可操作地连接于至少一个(异源)3’-UTR和/或至少一个(异源)5’-UTR。Suitably, the RNA of the present invention comprises at least one heterologous 5'-UTR and/or at least one heterologous 3'-UTR. The heterologous 5'-UTR or 3'-UTR may be derived from a naturally occurring gene or may be synthetically engineered. In a preferred embodiment, the RNA comprises at least one coding sequence as defined herein, which is operably linked to at least one (heterologous) 3'-UTR and/or at least one (heterologous) 5'-UTR.
在优选的实施方式中，RNA包含至少一个异源3’-UTR，其中RNA不包括包含SEQ IDNO:268序列的3’-UTR。优选地，RNA包含3’-UTR，其可以源自与具有增强的半衰期的RNA相关的基因(即，提供稳定的RNA)。In a preferred embodiment, the RNA comprises at least one heterologous 3'-UTR, wherein the RNA does not include a 3'-UTR comprising the sequence of SEQ ID NO: 268. Preferably, the RNA comprises a 3'-UTR that can be derived from a gene associated with an RNA having an enhanced half-life (i.e., providing a stable RNA).
在一些实施方式中，3’-UTR包含一个或多个聚腺苷酸化信号、用于影响核酸在细胞中的稳定性或定位的蛋白质的结合位点或者一个或多个miRNA或用于miRNA的结合位点。In some embodiments, the 3'-UTR comprises one or more polyadenylation signals, binding sites for proteins that affect the stability or localization of the nucleic acid in the cell, or one or more miRNAs or binding sites for miRNAs.
微RNA(或miRNA)是19-25个核苷酸长的非编码RNA，其结合核酸分子的3’-UTR并通过降低核酸分子稳定性或通过抑制翻译下调基因表达。例如，已知微RNA调控RNA，并且由此调控蛋白质表达，例如，肝脏(miR-122)、心脏(miR-ld、miR-149)、内皮细胞(miR-17-92、miR-126)、脂肪组织(let-7、miR-30c)、肾脏(miR-192、miR-194、miR-204)、髓样细胞(miR-142-3p、miR-142-5p、miR-16、miR-21、miR-223、miR-24、miR-27)、肌肉(miR-133、miR-206、miR-208)和肺上皮细胞(let-7、miR-133、miR-126)中。RNA可以包含一个或多个微RNA靶序列、微RNA序列或微RNA种子。这样的序列可以例如对应于任何已知的微RNA，如US2005/0261218和US2005/0059005中教导的那些。MicroRNA (or miRNA) is a non-coding RNA of 19-25 nucleotides in length, which binds to the 3'-UTR of nucleic acid molecules and downregulates gene expression by reducing nucleic acid molecule stability or by inhibiting translation. For example, it is known that microRNA regulates RNA, and thus regulates protein expression, for example, in liver (miR-122), heart (miR-ld, miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-194, miR-204), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133, miR-206, miR-208) and lung epithelial cells (let-7, miR-133, miR-126). RNA can comprise one or more microRNA target sequences, microRNA sequences or microRNA seeds.Such sequences can for example correspond to any known microRNA, such as those taught in US2005/0261218 and US2005/0059005.
因此，如本文定义的miRNA，或用于miRNA的结合位点可以从3’-UTR移除或引入3’-UTR中，以将RNA表达对于所需细胞类型或组织(例如，肌肉细胞)定制。Thus, a miRNA as defined herein, or a binding site for a miRNA, can be removed from or introduced into a 3'-UTR to tailor RNA expression to a desired cell type or tissue (e.g., muscle cells).
在优选的实施方式中，RNA包含至少一个异源3’-UTR，其包含源自选自PSMB3、ALB7、CASP1、COX6B1、GNAS、NDUFA1和RPS9的基因的3’-UTR，或这些基因的任一个的同源物、片段或变体的核酸序列，或由其组成。在一些实施方式中，RNA包含至少一个异源3’-UTR，其中该至少一个异源3’-UTR包含源自选自PSMB3、ALB7、CASP1、COX6B1、GNAS、NDUFA1和RPS9的基因的3’-UTR，或这些基因的任一个的同源物、片段或变体的核酸序列，优选根据与SEQ IDNO:253-266、22902-22905、22876-22895、26996-26999、28528-28539或这些中任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在该情况中特别优选的核酸序列可以源自公开的PCT申请WO2019/077001A1，特别是WO2019/077001A1的权利要求9。WO2019/077001A1的权利要求9的相应3’-UTR序列在此按引用并入(例如，WO2019/077001A1的SEQID NO:23-34，或其片段或变体)。In a preferred embodiment, the RNA comprises at least one heterologous 3'-UTR comprising or consisting of a nucleic acid sequence derived from the 3'-UTR of a gene selected from the group consisting of PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any of these genes. In some embodiments, the RNA comprises at least one heterologous 3'-UTR, wherein the at least one heterologous 3'-UTR comprises a nucleic acid sequence derived from the 3'-UTR of a gene selected from the group consisting of PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any of these genes, preferably according to a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 253-266, 22902-22905, 22876-22895, 26996-26999, 28528-28539 or a fragment or variant of any of these. Particularly preferred nucleic acid sequences in this context may be derived from the published PCT application WO2019/077001A1, in particular claim 9 of WO2019/077001A1. The corresponding 3'-UTR sequences of claim 9 of WO2019/077001A1 are hereby incorporated by reference (e.g., SEQ ID NOs: 23-34 of WO2019/077001A1, or fragments or variants thereof).
在进一步的实施方式中，RNA包含源自RSP9基因的3’-UTR。所述源自RPS9基因的3’-UTR可以包含与SEQ ID NO:263或264、22894、22895、22904、22905或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a further embodiment, the RNA comprises a 3'-UTR derived from the RSP9 gene. The 3'-UTR derived from the RPS9 gene may comprise a nucleic acid sequence identical to SEQ ID NO: 263 or 264, 22894, 22895, 22904, 22905 or a fragment or variant thereof, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consist of it.
在优选的实施方式中，RNA包含源自PSMB3基因的3’-UTR。所述源自PSMB3基因的3’-UTR可以包含与SEQ ID NO:253或254、22892、22893、22903、26996-26999、28528-28539或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA comprises a 3'-UTR derived from a PSMB3 gene. The 3'-UTR derived from a PSMB3 gene may comprise a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 253 or 254, 22892, 22893, 22903, 26996-26999, 28528-28539 or a fragment or variant thereof, or consists of.
在其他实施方式中，RNA包含3’-UTR，其包含与SEQ ID NO:22876-22891、28526、28527或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In other embodiments, the RNA comprises a 3'-UTR comprising, or consisting of, a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22876-22891, 28526, 28527, or a fragment or variant thereof.
在其他实施方式中，RNA可以包含WO2016/107877中所述的3’-UTR，将WO2016/107877中关于3’-UTR的公开内容在此按引用并入。合适的3’-UTR是WO2016/107877的SEQID NO:1-24和SEQ ID NO:49-318，或这些序列的片段或变体。在其他实施方式中，核酸包含WO2017/036580中所述的3’-UTR，将WO2017/036580中关于3’-UTR序列的公开内容在此按引用并入。合适的3’-UTR是WO2017/036580的SEQ ID NO:152-204，或这些序列的片段或变体。在其他实施方式中，核酸包含WO2016/02914中所述的3’-UTR，将WO2016/02914中关于3’-UTR序列的公开内容在此按引用并入。特别优选的3’-UTR是根据WO2016/02914的SEQ IDNO:20-36的核酸序列，或这些序列的片段或变体。In other embodiments, the RNA may comprise a 3'-UTR as described in WO2016/107877, the disclosure of which in WO2016/107877 is incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016/107877, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 3'-UTR as described in WO2017/036580, the disclosure of which in WO2017/036580 is incorporated herein by reference for 3'-UTR sequences. Suitable 3'-UTRs are SEQ ID NOs: 152-204 of WO2017/036580, or fragments or variants thereof. In other embodiments, the nucleic acid comprises a 3'-UTR as described in WO2016/02914, the disclosure of which in WO2016/02914 is incorporated herein by reference for 3'-UTR sequences. Particularly preferred 3'-UTRs are nucleic acid sequences according to SEQ ID NO: 20-36 of WO2016/02914, or fragments or variants of these sequences.
在优选的实施方式中，RNA包含至少一个异源5’-UTR。In a preferred embodiment, the RNA comprises at least one heterologous 5'-UTR.
术语“5’-未翻译区”或“5’-UTR”或“5’-UTR元件”由本领域普通技术人员认识和理解，并且例如旨在表示位于编码序列5’侧(即，“上游”)的RNA分子的部分且其不翻译成蛋白质。5’-UTR可以是位于编码序列5’的核酸序列的部分。通常，5’-UTR从转录起始位点开始并在编码序列的起始密码子前结束。5’-UTR可以包含用于控制基因表达的元件，也称为调控元件。这样的调控元件可以是例如核糖体结合位点、miRNA结合位点等。5’-UTR可以例如转录后修饰，通过酶或翻译后添加5’-帽结构(例如，对于mRNA)。The terms "5'-untranslated region" or "5'-UTR" or "5'-UTR element" are recognized and understood by those of ordinary skill in the art, and are intended, for example, to refer to the portion of an RNA molecule that is located 5' to the coding sequence (i.e., "upstream") and which is not translated into protein. The 5'-UTR can be the portion of a nucleic acid sequence that is located 5' to the coding sequence. Typically, the 5'-UTR starts at the transcription start site and ends before the start codon of the coding sequence. The 5'-UTR can contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'-UTR can be modified, for example, post-transcriptionally, by enzymatic or post-translational addition of a 5'-cap structure (e.g., for mRNA).
优选地，RNA包含可以源自与具有增加的半衰期的RNA(即，提供稳定的RNA)相关的基因的5’-UTR。Preferably, the RNA comprises a 5'-UTR that may be derived from a gene associated with an RNA having an increased half-life (i.e., providing a stable RNA).
在一些实施方式中，5’-UTR包含一个或多个用于影响细胞中的RNA稳定性或RNA定位的蛋白质的结合位点，或一个或多个miRNA或用于miRNA的结合位点(如以上定义的)。In some embodiments, the 5'-UTR comprises one or more binding sites for proteins that affect RNA stability or RNA localization in the cell, or one or more miRNAs or binding sites for miRNAs (as defined above).
因此，如上定义的miRNA或用于miRNA的结合位点可以从5’-UTR除去或引入5’-UTR中，以将核酸表达对于所需的细胞类型或组织(例如，肌肉细胞)定制。Thus, a miRNA or a binding site for a miRNA as defined above may be removed from or introduced into a 5'-UTR to tailor nucleic acid expression to a desired cell type or tissue (e.g., muscle cells).
在优选的实施方式中，RNA包含至少一个异源5’-UTR，其中至少一个异源5’-UTR包含源自选自HSD17B4、RPL32、ASAH1、ATP5A1、MP68、NDUFA4、NOSIP、RPL31、SLC7A3、TUBB4B和UBQLN2的基因的5’-UTR，或这些基因的同源物、片段或变体的核酸序列，其根据与SEQ IDNO:231-252、22870-22875或这些中任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。在该情况中特别优选的核酸序列可以选自公开的PCT申请WO2019/077001A1，特别是WO2019/077001A1的权利要求9。WO2019/077001A1的权利要求9的相应5’-UTR序列在此按引用并入(例如，WO2019/077001A1的SEQ ID NO:1-20，或其片段或变体)。In a preferred embodiment, the RNA comprises at least one heterologous 5'-UTR, wherein the at least one heterologous 5'-UTR comprises a nucleic acid sequence derived from the 5'-UTR of a gene selected from the group consisting of HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homolog, fragment or variant of these genes, according to a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 231-252, 22870-22875 or a fragment or variant of any of these. Particularly preferred nucleic acid sequences in this context can be selected from the published PCT application WO2019/077001A1, in particular claim 9 of WO2019/077001A1. The corresponding 5'-UTR sequences of claim 9 of WO2019/077001A1 are hereby incorporated by reference (e.g., SEQ ID NOs: 1-20 of WO2019/077001A1, or fragments or variants thereof).
在优选的实施方式中，RNA包含源自RPL31基因的5’-UTR，其中所述源自RPL31基因的5’-UTR包含与SEQ ID NO:243、244、22872、22873或片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA comprises a 5'-UTR derived from the RPL31 gene, wherein the 5'-UTR derived from the RPL31 gene comprises a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 243, 244, 22872, 22873 or a fragment or variant, or consists of.
在其它实施方式中，RNA包含源自SLC7A3基因的5’-UTR，其中所述源自SLC7A3基因的5’-UTR包含与SEQ ID NO:245、246、22874、22875或片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In other embodiments, the RNA comprises a 5'-UTR derived from the SLC7A3 gene, wherein the 5'-UTR derived from the SLC7A3 gene comprises, or consists of, a nucleic acid sequence that is identical, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 245, 246, 22874, 22875, or a fragment or variant.
在特别优选的实施方式中，RNA包含源自HSD17B4基因的5’-UTR，其中所述源自HSD17B4基因的5’-UTR包含与SEQ ID NO:231、232、22870、22871或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a particularly preferred embodiment, the RNA comprises a 5'-UTR derived from the HSD17B4 gene, wherein the 5'-UTR derived from the HSD17B4 gene comprises a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 231, 232, 22870, 22871 or a fragment or variant thereof, or consists of the same.
在其他实施方式中，RNA包含5’-UTR，其包含与SEQ ID NO:22848-22867、28522-28525或其片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In other embodiments, the RNA comprises a 5'-UTR comprising, or consisting of, a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22848-22867, 28522-28525, or a fragment or variant thereof.
在其他实施方式中，RNA包含如WO2013/143700中所述的5’-UTR，将WO2013/143799中关于5’-UTR序列的公开内容在此按引用并入。特别优选的5’-UTR是源自WO2013/143700的SEQ ID NO:1-1363、SEQ ID NO:1395、SEQ ID NO:1421和SEQ ID NO:1422的核酸序列，或这些序列的片段或变体。在其他实施方式中，RNA包含如WO2016/107877中所述的5’-UTR，将WO2016/107877中关于5’-UTR序列的公开内容在此按引用并入。特别优选的5’-UTR是根据WO2016/107877的SEQ ID NO:25-30和SEQ ID NO:319-382的核酸序列，或这些序列的片段或变体。在其他实施方式中，RNA包含如WO2017/036580中所述的5’-UTR，将WO2017/036580中关于5’-UTR序列的公开内容在此按引用并入。特别优选的5’-UTR是根据WO2017/036580的SEQ ID NO:1-151的核酸序列，或这些序列的片段或变体。在其他实施方式中，核酸序列包含如WO2016/022914中所述的5’-UTR，将WO2016/022914中关于5’-UTR序列的公开内容在此按引用并入。特别优选的5’-UTR是根据WO2016/022914的SEQ ID NO:3-19的核酸序列，或这些序列的片段或变体。In other embodiments, the RNA comprises a 5'-UTR as described in WO2013/143700, and the disclosure of WO2013/143799 regarding 5'-UTR sequences is incorporated herein by reference. Particularly preferred 5'-UTRs are nucleic acid sequences of SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 derived from WO2013/143700, or fragments or variants of these sequences. In other embodiments, the RNA comprises a 5'-UTR as described in WO2016/107877, and the disclosure of WO2016/107877 regarding 5'-UTR sequences is incorporated herein by reference. Particularly preferred 5'-UTRs are nucleic acid sequences of SEQ ID NO: 25-30 and SEQ ID NO: 319-382 according to WO2016/107877, or fragments or variants of these sequences. In other embodiments, the RNA comprises a 5'-UTR as described in WO2017/036580, the disclosure of which in WO2017/036580 is incorporated herein by reference for 5'-UTR sequences. Particularly preferred 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 of WO2017/036580, or fragments or variants of these sequences. In other embodiments, the nucleic acid sequence comprises a 5'-UTR as described in WO2016/022914, the disclosure of which in WO2016/022914 is incorporated herein by reference for 5'-UTR sequences. Particularly preferred 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 of WO2016/022914, or fragments or variants of these sequences.
合适地，在优选的实施方式中，RNA包含至少一个编码至少一个如本文定义的抗原蛋白，优选源自SARS-CoV-2的如本文具体说明的编码序列，其可操作地连接选自以下的5’UTR/3’UTR组合(“也称为UTR设计”)的3’-UTR和/或5’-UTR：a-1(HSD17B4/PSMB3)、a-2(NDUFA4/PSMB3)、a-3(SLC7A3/PSMB3)、a-4(NOSIP/PSMB3)、a-5(MP68/PSMB3)、b-1(UBQLN2/RPS9)、b-2(ASAH1/RPS9)、b-3(HSD17B4/RPS9)、b-4(HSD17B4/CASP1)、b-5(NOSIP/COX6B1)、c-1(NDUFA4/RPS9)、c-2(NOSIP/NDUFA1)、c-3(NDUFA4/COX6B1)、c-4(NDUFA4/NDUFA1)、c-5(ATP5A1/PSMB3)、d-1(Rpl31/PSMB3)、d-2(ATP5A1/CASP1)、d-3(SLC7A3/GNAS)、d-4(HSD17B4/NDUFA1)、d-5(Slc7a3/Ndufa1)、e-1(TUBB4B/RPS9)、e-2(RPL31/RPS9)、e-3(MP68/RPS9)、e-4(NOSIP/RPS9)、e-5(ATP5A1/RPS9)、e-6(ATP5A1/COX6B1)、f-1(ATP5A1/GNAS)、f-2(ATP5A1/NDUFA1)、f-3(HSD17B4/COX6B1)、f-4(HSD17B4/GNAS)、f-5(MP68/COX6B1)、g-1(MP68/NDUFA1)、g-2(NDUFA4/CASP1)、g-3(NDUFA4/GNAS)、g-4(NOSIP/CASP1)、g-5(RPL31/CASP1)、h-1(RPL31/COX6B1)、h-2(RPL31/GNAS)、h-3(RPL31/NDUFA1)、h-4(Slc7a3/CASP1)、h-5(SLC7A3/COX6B1)、i-1(SLC7A3/RPS9)、i-2(RPL32/ALB7)、i-2(RPL32/ALB7)。Suitably, in a preferred embodiment, the RNA comprises at least one coding sequence encoding at least one antigenic protein as defined herein, preferably derived from SARS-CoV-2 as specifically described herein, operably linked to a 3'-UTR and/or 5'-UTR selected from the following 5'UTR/3'UTR combinations ("also referred to as UTR designs"): a-1 (HSD17B4/PSMB3), a-2 (NDUFA4/PSMB3), a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RP3), S9), b-2(ASAH1/RPS9), b-3(HSD17B4/RPS9), b-4(HSD17B4/CASP1), b-5(NOSIP/COX6B1), c-1(NDUFA4/RPS9), c-2(NOSIP/NDUFA1), c-3(NDUFA4/COX6B1), c-4(NDUFA4/NDUFA1) , c-5(ATP5A1/PSMB3), d-1(Rpl31/PSMB3), d-2(ATP5A1/CASP1), d-3(SLC7A3/GNAS), d-4(HS D17B4/NDUFA1), d-5(Slc7a3/Ndufa1), e-1(TUBB4B/RPS9), e-2(RPL31/RPS9), e-3(MP68/RPS9), e-4(NOSIP/RPS9), e-5(ATP5A1/RPS9), e-6(ATP5A1/COX6B1), f-1(ATP5A1 /GNAS), f-2(ATP5A1/NDUFA1), f-3(HSD17B4/COX6B1), f-4(HSD17B4/GNAS), f-5(MP68/COX6B1), g-1(MP68/NDUFA1), g-2(NDUFA4/CASP1), g-3(NDUFA4/GNAS), g-4(NOSIP/CASP1), g-5(RPL31/CASP1), h-1(RPL31/COX6B1), h-2(RPL31/GNAS), h-3(RPL31/NDUFA1), h-4(Slc7a3/ CASP1), h-5(SLC7A3/COX6B1), i-1(SLC7A3/RPS9), i-2(RPL32/ALB7), i-2(RPL32/ALB7).
在特别优选的实施方式中，RNA包含至少一个编码至少一种源自SARS-CoV-2的抗原蛋白的如本文具体说明的编码序列，其中所述编码序列可操作地连接HSD17B4 5’-UTR和PSMB3 3’-UTR(HSD17B4/PSMB3(UTR设计a-1))。In a particularly preferred embodiment, the RNA comprises at least one coding sequence encoding at least one antigenic protein derived from SARS-CoV-2 as specifically described herein, wherein the coding sequence is operably linked to HSD17B4 5'-UTR and PSMB3 3'-UTR (HSD17B4/PSMB3 (UTR design a-1)).
本发明人已经证明了这个实施方式对于诱导针对SARS-CoV-2的免疫反应特别有益。在这种情况中，显示了一次接种已经足以导致病毒中和抗体滴度。The inventors have demonstrated that this embodiment is particularly beneficial for inducing an immune response against SARS-CoV-2. In this case, it was shown that a single vaccination was sufficient to induce virus neutralizing antibody titers.
在进一步优选的实施方式中，核酸包含至少一个编码至少一种如本文定义的抗原蛋白的如本文具体说明的编码序列，优选源自SARS-CoV-2(nCoV-2019)冠状病毒，其中所述编码序列可操作地连接SLC7A3 5’-UTR和PSMB3 3’-UTR(SLC7A3/PSMB3(UTR设计a-3))。In a further preferred embodiment, the nucleic acid comprises at least one coding sequence as specifically described herein encoding at least one antigenic protein as defined herein, preferably derived from the SARS-CoV-2 (nCoV-2019) coronavirus, wherein the coding sequence is operably linked to SLC7A3 5'-UTR and PSMB3 3'-UTR (SLC7A3/PSMB3 (UTR design a-3)).
在进一步优选的实施方式中，核酸包含至少一个编码至少一种如本文定义的抗原蛋白的如本文具体说明的编码序列，优选源自SARS-CoV-2(nCoV-2019)冠状病毒，其中所述编码序列可操作地连接RPL31 5’-UTR和RPS9 3’-UTR(RPL31/RPS9(UTR设计e-2))。In a further preferred embodiment, the nucleic acid comprises at least one coding sequence as specifically described herein encoding at least one antigenic protein as defined herein, preferably derived from the SARS-CoV-2 (nCoV-2019) coronavirus, wherein the coding sequence is operably linked to RPL31 5'-UTR and RPS9 3'-UTR (RPL31/RPS9 (UTR design e-2)).
在一些实施方式中，RNA可以是单顺反子、双顺反子或多顺反子的。In some embodiments, the RNA can be monocistronic, bicistronic, or polycistronic.
术语“单顺反子的”被本领域普通技术人员认识和理解，并且例如旨在表示仅包含一个编码序列的核酸。术语“双顺反子的”或“多顺反子的”由本领域普通技术人员认识和理解，并且例如旨在表示可以包含两个(双顺反子)或多个(多顺反子)的编码序列的核酸。The term "monocistronic" is known and understood by those of ordinary skill in the art and is intended, for example, to refer to a nucleic acid comprising only one coding sequence. The term "bicistronic" or "polycistronic" is known and understood by those of ordinary skill in the art and is intended, for example, to refer to a nucleic acid that may comprise two (bicistronic) or more (polycistronic) coding sequences.
在优选的实施方式中，第一方面的RNA是单顺反子的。In a preferred embodiment, the RNA of the first aspect is monocistronic.
在其他实施方式中，RNA是单顺反子的并且所述核酸的编码序列编码至少两种源自SARS-CoV-2的不同的抗原肽或蛋白质。因此，所述编码序列可以编码至少两种、三种、四种、五种、六种、七种、八种或更多种源自SARS-CoV-2的抗原肽或蛋白质，其用氨基酸接头序列连接或不连接，其中所述接头序列可以包含刚性接头、柔性接头、可切割接头或其组合。这样的构建体在本文中称为“多抗原构建体”。In other embodiments, the RNA is monocistronic and the coding sequence of the nucleic acid encodes at least two different antigenic peptides or proteins derived from SARS-CoV-2. Thus, the coding sequence can encode at least two, three, four, five, six, seven, eight or more antigenic peptides or proteins derived from SARS-CoV-2, which are connected or not connected with an amino acid linker sequence, wherein the linker sequence can comprise a rigid linker, a flexible linker, a cleavable linker or a combination thereof. Such a construct is referred to herein as a "multi-antigen construct".
在进一步的实施方式中，RNA可以是双顺反子或多顺反子的并且包含至少两个编码序列，其中至少两个编码序列编码两种或更多种源自SARS-CoV-2的不同抗原肽或蛋白质。因此，双顺反子或多顺反子核酸中的编码序列合适地编码如本文定义的不同的抗原蛋白或肽或其免疫原性片段或免疫原性变体。优选地，所述双顺反子或多顺反子构建体中的编码序列可以由至少一个IRES(内部核糖体进入位点)序列隔开。因此，术语“编码两种或更多种抗原肽或蛋白质”可以表示双顺反子或多顺反子核酸编码例如不同SARS-CoV-2分离株的至少两种、三种、四种、五种、六种或更多种(优选不同的)抗原肽或蛋白质，但不限于此。或者，双顺反子或多顺反子核酸可以编码例如源自相同的SARS-CoV-2的至少两种、三种、四种、五种、六种或更多种(优选不同的)抗原肽或蛋白质。在这种情况中，合适的IRES序列可以选自根据专利申请WO2017/081082的SEQ ID NO:1566-1662的氨基酸序列的列表，或这些序列的片段或变体。在这种情况中，将WO2017/081082中关于IRES序列的公开内容在此按引用并入。In a further embodiment, the RNA can be a bicistronic or polycistronic and comprises at least two coding sequences, wherein at least two coding sequences encode two or more different antigenic peptides or proteins derived from SARS-CoV-2. Therefore, the coding sequence in the bicistronic or polycistronic nucleic acid suitably encodes different antigenic proteins or peptides as defined herein or their immunogenic fragments or immunogenic variants. Preferably, the coding sequence in the bicistronic or polycistronic construct can be separated by at least one IRES (internal ribosome entry site) sequence. Therefore, the term "encoding two or more antigenic peptides or proteins" can represent that the bicistronic or polycistronic nucleic acid encodes at least two, three, four, five, six or more (preferably different) antigenic peptides or proteins of, for example, different SARS-CoV-2 isolates, but is not limited thereto. Alternatively, the bicistronic or polycistronic nucleic acid can encode, for example, at least two, three, four, five, six or more (preferably different) antigenic peptides or proteins derived from the same SARS-CoV-2. In this case, suitable IRES sequences can be selected from a list of amino acid sequences of SEQ ID NO: 1566-1662 according to patent application WO2017/081082, or fragments or variants of these sequences. In this case, the disclosure of IRES sequences in WO2017/081082 is incorporated herein by reference.
必须理解，在本发明的情况中，编码序列的某些组合可以通过单顺反子、双顺反子或多顺贩子RNA构建体和/或多抗原构建体的任意组合来产生，以获得编码多个如本文定义的抗原肽或蛋白质的核酸。It must be understood that in the context of the present invention, certain combinations of coding sequences can be generated by any combination of monocistronic, bicistronic or polycistronic RNA constructs and/or multi-antigenic constructs to obtain nucleic acids encoding multiple antigenic peptides or proteins as defined herein.
在优选的实施方式中，与其相应野生型或参考RNA的核糖体结合位点的环境中的A/Y(A/T)含量相比，RNA的核糖体结合位点的环境中的A/U(A/T)含量可以增加。这种改变(核糖体结合位点周围增加的A/U(A/T)含量)增加了核糖体与RNA结合的效率。有效的核糖体与核糖体结合位点的结合转而对有效的RNA翻译具有效果。In a preferred embodiment, the A/U (A/T) content in the environment of the ribosome binding site of the RNA can be increased compared to the A/Y (A/T) content in the environment of the ribosome binding site of its corresponding wild-type or reference RNA. This change (increased A/U (A/T) content around the ribosome binding site) increases the efficiency of ribosome binding to the RNA. Effective ribosome binding to the ribosome binding site in turn has an effect on effective RNA translation.
因此，在特别优选的实施方式中，RNA包含核糖体结合位点，也称为“Kozak序列”，其与SEQ ID NO:180、181、22845-22847的任一序列，或其片段或变体相同或至少80％、85％、90％、95％相同。Therefore, in a particularly preferred embodiment, the RNA comprises a ribosome binding site, also known as a "Kozak sequence", which is identical or at least 80%, 85%, 90%, 95% identical to any one of SEQ ID NOs: 180, 181, 22845-22847, or a fragment or variant thereof.
在优选的实施方式中，RNA包含至少一个聚(N)序列，例如，至少一个聚(A)序列、至少一个聚(U)序列、至少一个聚(C)序列或其组合。In a preferred embodiment, the RNA comprises at least one poly (N) sequence, e.g., at least one poly (A) sequence, at least one poly (U) sequence, at least one poly (C) sequence, or a combination thereof.
在优选的实施方式中，本发明的RNA包含至少一个聚(A)序列。In a preferred embodiment, the RNA of the invention comprises at least one poly(A) sequence.
如本文使用的术语“聚(A)序列”、“聚(A)尾”或“3’-聚(A)尾”由本领域普通技术人员认识和理解，并且例如旨在是多达约1000个腺苷核苷酸的腺苷核苷酸序列，通常位于线性RNA的3’-端(或在环状RNA中)。优选地，所述聚(A)序列基本上是同型多聚的，例如，100个腺苷核苷酸的聚(A)序列具有基本上100个核苷酸的长度。在其他实施方式中，聚(A)序列由至少一个不同于腺苷核苷酸的核苷酸打断，例如，100个腺苷核苷酸的聚(A)序列可以具有超过100个核苷酸的长度(包括100个腺苷核苷酸和另外的所述至少一个不同于腺苷核苷酸的核苷酸或核苷酸链)。The terms "poly (A) sequence", "poly (A) tail" or "3'-poly (A) tail" as used herein are recognized and understood by those of ordinary skill in the art, and are intended, for example, to be an adenosine nucleotide sequence of up to about 1000 adenosine nucleotides, typically located at the 3'-end of a linear RNA (or in a circular RNA). Preferably, the poly (A) sequence is substantially homopolymeric, for example, a poly (A) sequence of 100 adenosine nucleotides has a length of substantially 100 nucleotides. In other embodiments, the poly (A) sequence is interrupted by at least one nucleotide other than adenosine nucleotides, for example, a poly (A) sequence of 100 adenosine nucleotides may have a length of more than 100 nucleotides (including 100 adenosine nucleotides and the additional at least one nucleotide or nucleotide chain other than adenosine nucleotides).
聚(A)序列可以包含约10至约500个腺苷核苷酸，约10至约200个腺苷核苷酸，约40至约200个腺苷核苷酸，或约40至约150个腺苷核苷酸。合适地，聚(A)序列的长度可以为至少约或甚至超过约10、50、64、75、100、200、300、400或500个腺苷核苷酸。在某些实施方式中，RNA包含至少一个聚(A)序列，其包含30至200个腺苷核苷酸，其中所述RNA的3’末端核苷酸是腺苷。The poly(A) sequence may comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. Suitably, the length of the poly(A) sequence may be at least about or even more than about 10, 50, 64, 75, 100, 200, 300, 400 or 500 adenosine nucleotides. In certain embodiments, the RNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides, wherein the 3' terminal nucleotide of the RNA is an adenosine.
在优选的实施方式中，本发明的RNA包含至少一个聚(A)序列，其包含约30至约200个腺苷核苷酸。在特别优选的实施方式中，聚(A)序列包含约64个腺苷核苷酸(A64)。在特别优选的实施方式中，聚(A)序列包含约100个腺苷核苷酸(A100)。在其他实施方式中，聚(A)序列包含约150个腺苷核苷酸。In a preferred embodiment, the RNA of the invention comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides. In a particularly preferred embodiment, the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In a particularly preferred embodiment, the poly(A) sequence comprises about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides.
在进一步的实施方式中，本发明的RNA包含至少一个聚(A)序列，其包含约100个腺苷核苷酸，其中聚(A)序列被非腺苷核苷酸打断，优选被10个非腺苷核苷酸打断(A30-N10-A70)。In a further embodiment, the RNA of the invention comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, preferably by 10 non-adenosine nucleotides (A30-N10-A70).
如本文定义的聚(A)序列可以直接位于RNA的3’末端，优选直接位于RNA的3’末端。The poly(A) sequence as defined herein may be located directly at the 3' end of the RNA, is preferably located directly at the 3' end of the RNA.
在优选的实施方式中，3’-末端核苷酸(即多核苷酸链中最后一个3’-末端核苷酸)是至少一个聚(A)序列的3’-末端A核苷酸。术语“直接位于3’-末端”必须理解为就位于3’末端-换句话说，核酸的3’末端由终止于A核苷酸的聚(A)序列组成。In a preferred embodiment, the 3'-terminal nucleotide (i.e., the last 3'-terminal nucleotide in the polynucleotide chain) is the 3'-terminal A nucleotide of at least one poly(A) sequence. The term "directly at the 3'-terminus" must be understood as being located right at the 3'-terminus - in other words, the 3'-terminus of the nucleic acid consists of a poly(A) sequence that terminates with an A nucleotide.
发明人已经显示了这个实施方式对于诱导针对SARS-CoV-2的免疫反应特别有益。在这种情况中，显示了一次接种就已经足以导致病毒中和抗体滴度。The inventors have shown that this embodiment is particularly beneficial for inducing an immune response against SARS-CoV-2. In this case, it was shown that a single vaccination was sufficient to induce virus neutralizing antibody titers.
在特别优选的实施方式中，RNA序列包含至少70个腺苷核苷酸的聚(A)序列，其中3’-末端核苷酸是腺苷核苷酸。In a particularly preferred embodiment, the RNA sequence comprises a poly(A) sequence of at least 70 adenosine nucleotides, wherein the 3'-terminal nucleotide is an adenosine nucleotide.
在这种情况中，已经显示了终止于腺苷核苷酸降低了RNA疫苗对IFNα的诱导。这是特别重要的，因为认为IFNα的诱导是诱发接种疫苗的受试者中发烧的主要因素，这当然必须避免。In this context, it has been shown that termination with an adenosine nucleotide reduces the induction of IFNα by RNA vaccines. This is particularly important since the induction of IFNα is considered to be the main factor in inducing fever in vaccinated subjects, which of course must be avoided.
在优选的实施方式中，RNA的聚(A)序列获自RNA在体外转录期间的DNA模板。在其他实施方式中，聚(A)序列通过常用的化学合成方法在体外获得，不是必需从DNA模板转录。在其他实施方式中，使用商购的聚腺苷酸化试剂盒和本领域已知的相应实验方案通过RNA的酶促聚腺苷酸化(在RNA体外转录后)，或替代地，通过使用固定化的聚(A)聚合酶，例如，使用WO2016/174271中所述的方法和方式(其完整内容在此按引用并入)，来产生聚(A)序列。In a preferred embodiment, the poly (A) sequence of the RNA is obtained from a DNA template during in vitro transcription of the RNA. In other embodiments, the poly (A) sequence is obtained in vitro by conventional chemical synthesis methods, and is not necessarily transcribed from a DNA template. In other embodiments, the poly (A) sequence is produced by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding experimental protocols known in the art, or alternatively, by using an immobilized poly (A) polymerase, for example, using the methods and means described in WO2016/174271 (the entire contents of which are hereby incorporated by reference).
在一些实施方式中，RNA包含通过酶促聚腺苷酸化获得的聚(A)序列，其中大部分的RNA分子包含约100(+/-20)至约500(+/-50)个，优选约250(+/-20)个腺苷核苷酸。In some embodiments, the RNA comprises a poly(A) sequence obtained by enzymatic polyadenylation, wherein a majority of the RNA molecules comprise about 100 (+/- 20) to about 500 (+/- 50), preferably about 250 (+/- 20) adenosine nucleotides.
在其他实施方式中，RNA包含源自模板DNA的聚(A)序列和至少一个另外的通过酶促聚腺苷酸化生成的聚(A)序列，例如，如WO2016/091391中所述的，将其完整内容在此按引用并入。In other embodiments, the RNA comprises a poly(A) sequence derived from the template DNA and at least one additional poly(A) sequence generated by enzymatic polyadenylation, for example, as described in WO2016/091391, the entire contents of which are hereby incorporated by reference.
在进一步的实施方式中，RNA包含至少一个聚(C)序列。In a further embodiment, the RNA comprises at least one poly(C) sequence.
如本文使用的术语“聚(C)序列”旨在是多达约200个胞嘧啶核苷酸的胞嘧啶核苷酸序列。在优选的实施方式中，聚(C)序列包含约10至约200个胞嘧啶核苷酸、约10至约100个胞嘧啶核苷酸、约20至约70个胞嘧啶核苷酸、约20至约60个胞嘧啶核苷酸，或约10至约40个胞嘧啶核苷酸。在特别优选的实施方式中，聚(C)序列包含约30个胞嘧啶核苷酸。As used herein, the term "poly (C) sequence" is intended to be a cytosine nucleotide sequence of up to about 200 cytosine nucleotides. In preferred embodiments, the poly (C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In particularly preferred embodiments, the poly (C) sequence comprises about 30 cytosine nucleotides.
在优选的实施方式中，本发明的RNA包含至少一个组蛋白茎-环(hSL)。In a preferred embodiment, the RNA of the invention comprises at least one histone stem-loop (hSL).
术语“组蛋白茎-环”(缩写为“hSL”，例如，在序列表中)旨在表示形成主要在组蛋白mRNA中发现的茎-环二级结构的核酸序列。The term "histone stem-loop" (abbreviated "hSL", eg, in the sequence listing) is intended to mean a nucleic acid sequence that forms a stem-loop secondary structure predominantly found in histone mRNA.
组蛋白茎-环序列/结构可以合适地选自如WO2012/019780中公开的组蛋白茎-环序列，将其完整内容在此按引用并入，与组蛋白茎-环序列/组蛋白茎-环结构相关的公开内容在此按引用并入。可以在本发明内使用的组蛋白茎-环序列可以优选源自WO2012/019780的式(I)或(II)。根据进一步优选的实施方式，RNA包含至少一个源自专利申请WO2012/0197780的特定式(Ia)或(IIa)中的至少一个的组蛋白茎-环序列。The histone stem-loop sequence/structure may be suitably selected from the histone stem-loop sequences disclosed in WO2012/019780, the entire contents of which are incorporated herein by reference, and the disclosures related to histone stem-loop sequences/histone stem-loop structures are incorporated herein by reference. The histone stem-loop sequence that can be used in the present invention may preferably be derived from formula (I) or (II) of WO2012/019780. According to a further preferred embodiment, the RNA comprises at least one histone stem-loop sequence derived from at least one of the specific formulas (Ia) or (IIa) of patent application WO2012/0197780.
在优选的实施方式中，本发明的RNA包含至少一个组蛋白茎-环结构，其中所述组蛋白茎-环(hSL)包含与SEQ ID NO:178或179或其片段或变体相同或至少70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA of the present invention comprises at least one histone stem-loop structure, wherein the histone stem-loop (hSL) comprises a nucleic acid sequence that is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 178 or 179 or a fragment or variant thereof, or consists of the same.
在其他实施方式中，RNA不包含如本文定义的组蛋白茎-环。In other embodiments, the RNA does not comprise a histone stem-loop as defined herein.
在各种实施方式中，RNA包含3’-末端序列元件。所述3’-末端序列元件包含聚(A)序列和任选地组蛋白茎-环序列。因此，本发明的RNA包含至少一个3’-末端序列元件，其包含与SEQ ID NO:254、22893、22903、26997、26999、28529、28531、28533、28535、28537、28539或其片段或变体相同或至少70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In various embodiments, the RNA comprises a 3'-terminal sequence element. The 3'-terminal sequence element comprises a poly (A) sequence and optionally a histone stem-loop sequence. Therefore, the RNA of the present invention comprises at least one 3'-terminal sequence element comprising a nucleic acid sequence identical to SEQ ID NO: 254, 22893, 22903, 26997, 26999, 28529, 28531, 28533, 28535, 28537, 28539 or a fragment or variant thereof, or consisting of the same or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
在优选的实施方式中，RNA包含3’-末端序列元件。所述3’-末端序列元件包含聚(A)序列。因此，本发明的核酸包含至少一个3’-末端序列元件，其包含与SEQ ID NO:254、22903、26999、28531、28525、28539或其片段或变体相同或至少70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA comprises a 3'-terminal sequence element. The 3'-terminal sequence element comprises a poly (A) sequence. Thus, the nucleic acid of the present invention comprises at least one 3'-terminal sequence element comprising a nucleic acid sequence that is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 254, 22903, 26999, 28531, 28525, 28539 or a fragment or variant thereof, or consists of.
在优选的实施方式中，RNA包含3’-末端序列元件。所述3’-末端序列元件包含聚(A)序列和组蛋白-茎-环序列。因此，本发明的核酸包含至少一个3’-末端序列元件，其包含与SEQ ID NO:254、22893、26997、28529、28533、28537或其片段或变体相同或至少70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA comprises a 3'-terminal sequence element. The 3'-terminal sequence element comprises a poly (A) sequence and a histone-stem-loop sequence. Therefore, the nucleic acid of the present invention comprises at least one 3'-terminal sequence element, which comprises a nucleic acid sequence that is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 254, 22893, 26997, 28529, 28533, 28537 or a fragment or variant thereof, or consists of it.
在各种实施方式中，RNA可以包含根据SEQ ID NO:176、177或2840-22844或其片段或变体的5’-末端序列元件。In various embodiments, the RNA may comprise a 5'-terminal sequence element according to SEQ ID NO: 176, 177 or 2840-22844 or a fragment or variant thereof.
在进一步的实施方式中，RNA可以包含根据SEQ ID NO:176、177或2840-22844或其片段或变体的5’-末端序列元件。这样的5’-末端序列元件包含例如用于T7 RNA聚合酶的结合位点。此外，所述5’-末端起始序列的第一核苷酸可以优选包含2’O甲基化，例如，2’O甲基化的鸟苷或2’O甲基化的腺苷。In a further embodiment, the RNA may comprise a 5'-terminal sequence element according to SEQ ID NO: 176, 177 or 2840-22844 or a fragment or variant thereof. Such a 5'-terminal sequence element comprises, for example, a binding site for T7 RNA polymerase. In addition, the first nucleotide of the 5'-terminal start sequence may preferably comprise a 2'O-methylation, for example, a 2'O-methylated guanosine or a 2'O-methylated adenosine.
在优选的实施方式中，包含至少一个异源5’-UTR和至少一个异源3’-UTR，所述5’-UTR包含源自来自HSD17B4的5’-UTR的核酸序列或由其组成，所述3’-UTR包含源自PSMB3的3’-UTR的核酸序列或由其组成。在某些实施方式中，来自HSD17B4的5’-UTR与SEQ ID NO:232至少约95％、96％、97％、98％至99％相同。在一些实施方式中，PSMB3的3’-UTR与SEQ IDNO:254至少约95％、96％、97％、98％至99％相同。在特别优选的实施方式中，从5’至3’，RNA包含：i)5’-帽1结构；ii)源自HSD17B4基因的5’-UTR的5’-UTR，优选根据SEQ ID NO:232；iii)至少一个编码序列(编码实施方式的SARS-CoV刺突抗原)；iv)源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:254；v)任选地，组蛋白茎-环序列；和vi)包含约100个A核苷酸的聚(A)序列，其中所述RNA的3’末端核苷酸是腺苷。In a preferred embodiment, at least one heterologous 5'-UTR and at least one heterologous 3'-UTR are included, wherein the 5'-UTR comprises or consists of a nucleic acid sequence derived from the 5'-UTR from HSD17B4, and the 3'-UTR comprises or consists of a nucleic acid sequence derived from the 3'-UTR of PSMB3. In certain embodiments, the 5'-UTR from HSD17B4 is at least about 95%, 96%, 97%, 98% to 99% identical to SEQ ID NO: 232. In some embodiments, the 3'-UTR of PSMB3 is at least about 95%, 96%, 97%, 98% to 99% identical to SEQ ID NO: 254. In a particularly preferred embodiment, from 5' to 3', the RNA comprises: i) a 5'-cap 1 structure; ii) a 5'-UTR derived from the 5'-UTR of the HSD17B4 gene, preferably according to SEQ ID NO: 232; iii) at least one coding sequence (encoding a SARS-CoV spike antigen of an embodiment); iv) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene, preferably according to SEQ ID NO: 254; v) optionally, a histone stem-loop sequence; and vi) a poly(A) sequence comprising about 100 A nucleotides, wherein the 3' terminal nucleotide of the RNA is an adenosine.
优选地，RNA包含约50至约20000个核苷酸，或约500至约10000个核苷酸，或约1000至约10000个核苷酸，或优选约1000至约5000个核苷酸，或甚至更优选约2000至约5000个核苷酸。Preferably, the RNA comprises about 50 to about 20,000 nucleotides, or about 500 to about 10,000 nucleotides, or about 1,000 to about 10,000 nucleotides, or preferably about 1,000 to about 5,000 nucleotides, or even more preferably about 2,000 to about 5,000 nucleotides.
根据特别优选的实施方式，RNA是编码RNA。在优选的实施方式中，编码RNA可以选自mRNA、(编码)自我复制RNA、(编码)环状RNA、(编码)病毒RNA或(编码)复制子RNA。According to a particularly preferred embodiment, the RNA is a coding RNA. In a preferred embodiment, the coding RNA can be selected from mRNA, (coding) self-replicating RNA, (coding) circular RNA, (coding) viral RNA or (coding) replicon RNA.
在其他实施方式中，编码RNA是环状RNA。如本文使用的，“环状RNA”或“环RNA”必须理解为编码至少一个如本文定义的抗原肽或蛋白质的环状多核苷酸构建体。优选地，这样的环RNA是单链RNA分子。在优选的实施方式中，所述环RNA包含至少一个编码至少一种来自SARS-CoV-2冠状病毒的抗原蛋白或其免疫原性片段或免疫原性变体的编码序列。In other embodiments, the coding RNA is a circular RNA. As used herein, "circular RNA" or "circRNA" must be understood as a circular polynucleotide construct encoding at least one antigenic peptide or protein as defined herein. Preferably, such a circular RNA is a single-stranded RNA molecule. In a preferred embodiment, the circular RNA comprises at least one coding sequence encoding at least one antigenic protein from SARS-CoV-2 coronavirus or an immunogenic fragment or immunogenic variant thereof.
在进一步的实施方式中，编码RNA是复制子RNA。术语“复制子RNA”由本领域普通技术人员认识和理解，并且例如旨在是优化的自我复制RNA。这样的构建体可以包括源自例如α病毒(例如，SFV、VEE或RRV)的复制酶元件以及目标核酸(即，编码SARS-CoV-2冠状病毒的抗原肽或蛋白的编码序列)对结构病毒蛋白的取代。或者，复制酶可以在独立的编码RNA构建体或编码DNA构建体上提供。复制酶的下游可以是控制复制子RNA复制的亚基因组启动子。In a further embodiment, the coding RNA is a replicon RNA. The term "replicon RNA" is recognized and understood by those of ordinary skill in the art, and is intended to be, for example, an optimized self-replicating RNA. Such a construct may include a replicase element derived from, for example, an alpha virus (e.g., SFV, VEE or RRV) and a target nucleic acid (i.e., a coding sequence for an antigenic peptide or protein encoding a SARS-CoV-2 coronavirus) to replace structural viral proteins. Alternatively, a replicase may be provided on an independent coding RNA construct or a coding DNA construct. The downstream of the replicase may be a subgenomic promoter controlling the replication of the replicon RNA.
在特别优选的实施方式中，至少一个核酸不是复制子RNA或自我复制RNA。In a particularly preferred embodiment, at least one nucleic acid is not a replicon RNA or a self-replicating RNA.
在特别优选的实施方式中，本发明的RNA是mRNA。In a particularly preferred embodiment, the RNA of the invention is mRNA.
优选地，mRNA不包含复制酶元件(例如，编码复制酶的核酸)。Preferably, the mRNA does not contain a replicase element (eg, a nucleic acid encoding a replicase).
术语“RNA”和“mRNA”由本领域普通技术人员认识和理解，并且例如旨在是核糖核酸分子，即，由核苷酸组成的聚合物。这些核苷酸通常是沿着所谓的主链彼此连接的腺苷单磷酸、尿苷单磷酸、鸟苷单磷酸和胞苷单磷酸单体。主链通过第一单体的糖(即，核糖)和第二邻近单体的磷酸酯部分之间的磷酸二酯键形成。特定的连续单体称为RNA序列。mRNA(信使RNA)提供了可以翻译成特定肽或蛋白质的氨基酸序列的核苷酸编码序列。The terms "RNA" and "mRNA" are known and understood by those of ordinary skill in the art, and are intended, for example, to be ribonucleic acid molecules, i.e., polymers consisting of nucleotides. These nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers linked to one another along a so-called backbone. The backbone is formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. Specific continuous monomers are referred to as RNA sequences. mRNA (messenger RNA) provides a nucleotide coding sequence that can be translated into an amino acid sequence of a specific peptide or protein.
在本发明的情况中，RNA，优选mRNA，提供了至少一个编码来自如本文定义的SARS-CoV-2刺突蛋白的抗原蛋白的编码序列，其在施用后(例如，施用于受试者后，例如，人受试者)翻译成(功能性)抗原。In the context of the present invention, the RNA, preferably mRNA, provides at least one coding sequence encoding an antigenic protein from the SARS-CoV-2 Spike protein as defined herein, which is translated into a (functional) antigen upon administration (e.g., upon administration to a subject, e.g., a human subject).
在优选的实施方式中，RNA，优选mRNA适用于SARS-CoV-2疫苗，优选针对至少一种以下的SARS-CoV-2分离株的SARS-CoV-2疫苗：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。In a preferred embodiment, the RNA, preferably mRNA, is suitable for use in a SARS-CoV-2 vaccine, preferably a SARS-CoV-2 vaccine against at least one of the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta). Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在特别优选的实施方式中，RNA，优选mRNA适用于SARS-CoV-2疫苗，优选针对B.1.351(Beta，南非)或B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)的SARS-CoV-2疫苗。In a particularly preferred embodiment, RNA, preferably mRNA, is suitable for a SARS-CoV-2 vaccine, preferably a SARS-CoV-2 vaccine against B.1.351 (Beta, South Africa) or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).
合适地，RNA可以通过添加5’-帽结构来修饰，其优选稳定RNA和/或增强编码的抗原的表达和/或降低先天性免疫系统的刺激(在施用于受试者后)。5’-帽结构在其中RNA是线性编码RNA的实施方式中是特别重要的，例如，线性mRNA或线性编码复制子RNA。Suitably, the RNA may be modified by the addition of a 5'-cap structure, which preferably stabilizes the RNA and/or enhances expression of the encoded antigen and/or reduces stimulation of the innate immune system (upon administration to a subject). The 5'-cap structure is particularly important in embodiments where the RNA is a linear coding RNA, e.g., a linear mRNA or a linear coding replicon RNA.
因此，在优选的实施方式中，RNA，特别是mRNA，包含5’-帽结构，优选帽0、帽1、帽2、修饰的帽0或修饰的帽1结构。Therefore, in a preferred embodiment, the RNA, in particular the mRNA, comprises a 5'-cap structure, preferably a cap 0, cap 1, cap 2, a modified cap 0 or a modified cap 1 structure.
如本文使用的术语“5’-帽结构”由本领域普通技术人员认识和理解并且例如旨在表示5’修饰的核苷酸，特别是鸟苷核苷酸，其位于RNA(例如，mRNA)的5’-端。优选地，5’-帽结构经由5’-5’-三磷酸键连接于RNA。The term "5'-cap structure" as used herein is recognized and understood by those of ordinary skill in the art and is intended, for example, to refer to a 5'-modified nucleotide, particularly a guanosine nucleotide, which is located at the 5'-end of an RNA (e.g., mRNA). Preferably, the 5'-cap structure is attached to the RNA via a 5'-5'-triphosphate bond.
在本发明的情况中合适的5’-帽结构是帽0(第一核碱基的甲基化，例如，m7GpppN)、帽1(m7GpppN的邻近核苷酸的核糖的附加甲基化)、帽2(m7GpppN下游的第2核苷酸的核糖的附加甲基化)、帽3(m7GpppN下游的第3核苷酸的核糖的附加甲基化)、帽4(m7GpppN下游的第4核苷酸的核糖的附加甲基化)、ARCA(抗反向帽类似物)、修饰的ARCA(例如，硫代磷酸酯修饰的ARCA)、肌苷、N1-甲基-鸟苷、2’-氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷和2-叠氮基-鸟苷。Suitable 5'-cap structures in the context of the present invention are cap 0 (methylation of the first nucleobase, e.g., m7GpppN), cap 1 (additional methylation of the ribose of the adjacent nucleotide to m7GpppN), cap 2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.
5’-帽(帽0或帽1)结构可以在化学RNA合成或使用帽类似物的RNA体外转录(共-转录加帽)中形成。The 5'-cap (cap 0 or cap 1) structure can be formed during chemical RNA synthesis or in vitro transcription of RNA using a cap analog (co-transcriptional capping).
如本文使用的术语“帽类似物”由本领域普通技术人员认识和理解，并且例如旨在表示具有帽功能性的不可聚合的二核苷酸或三核苷酸，因为其掺入核酸分子的5’-端时，促进翻译或定位，和/或防止核酸分子(特别是RNA分子)的降解。不可聚合表示帽类似物仅在5’-末端掺入，因为其不具有5’三磷酸并且因此不能通过模板依赖性聚合酶在3’-方向上延伸，特别地，通过模板依赖性RNA聚合酶。帽类似物的实例包括，但不限于，选自m7GpppG、m7GpppA、m7GpppC的化学结构；未甲基化的帽类似物(例如，GpppG)；二甲基化的帽类似物(例如，m2,7GpppG)、三甲基化的帽类似物(例如，m2,2,7GpppG)、二甲基化对称帽类似物(例如，m7Gpppm7G)或抗反向帽类似物(例如，ARCA；m7,2’OmeGpppG、m7,2’dGpppG、m7,3’OmeGpppG、m7,3’dGpppG及其四磷酸酯衍生物)。之前已经描述了进一步的帽类似物(WO2008/016473、WO2008/157688、WO2009/149253、WO2011/015347和WO2013/059475)。在该情况中，进一步合适的帽类似物描述于WO2017/066793、WO2017/066781、WO2017/066791、WO2017/066789、WO2017/053297、WO2017/066782、WO2018/075827和WO2017/066797，其中涉及帽类似物的公开内容在此按引用并入。The term "cap analogue" as used herein is recognized and understood by those of ordinary skill in the art, and is intended, for example, to mean a non-polymerizable dinucleotide or trinucleotide having a cap functionality, because when incorporated into the 5'-end of a nucleic acid molecule, it promotes translation or localization, and/or prevents degradation of a nucleic acid molecule, particularly an RNA molecule. Non-polymerizable means that the cap analogue is incorporated only at the 5'-end, because it does not have a 5' triphosphate and therefore cannot be extended in the 3'-direction by a template-dependent polymerase, in particular, by a template-dependent RNA polymerase. Examples of cap analogs include, but are not limited to, chemical structures selected from m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and tetraphosphate derivatives thereof). Further cap analogs have been described previously (WO2008/016473, WO2008/157688, WO2009/149253, WO2011/015347 and WO2013/059475). In this context, further suitable cap analogs are described in WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/053297, WO2017/066782, WO2018/075827 and WO2017/066797, the disclosures of which concerning cap analogs are hereby incorporated by reference.
在一些实施方式中，使用如WO2017/053297、WO2017/066793、WO2017/066781、WO2017/066791、WO2017/066789、WO2017/066782、WO2018/075827和WO2017/066797中公开的三核苷酸帽类似物生成修饰的帽1结构，将上述PCT申请的全部内容在此按引用并入。特别地，任何可源自WO2017/053297的权利要求1-5中公开的结构的帽结构可以合适地用于共转录地生成修饰的帽1结构。此外，任何可源自WO2018/075827的权利要求1或权利要求2中定义的结构的帽结构可以合适地用于共转录地生成修饰的帽1结构。In some embodiments, a modified cap 1 structure is generated using a trinucleotide cap analog as disclosed in WO2017/053297, WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/066782, WO2018/075827, and WO2017/066797, the entire contents of the above PCT applications are hereby incorporated by reference. In particular, any cap structure that can be derived from the structure disclosed in claims 1-5 of WO2017/053297 can be suitably used to co-transcriptionally generate a modified cap 1 structure. In addition, any cap structure that can be derived from the structure defined in claim 1 or claim 2 of WO2018/075827 can be suitably used to co-transcriptionally generate a modified cap 1 structure.
在优选的实施方式中，RNA，特别是mRNA，包含帽1结构。In a preferred embodiment, the RNA, in particular the mRNA, comprises a cap 1 structure.
在优选的实施方式中，可以在如本文定义的RNA体外转录反应中使用如本文定义的三核苷酸帽类似物通过共转录合适地添加5’-帽结构。In a preferred embodiment, the 5'-cap structure may be suitably added by co-transcription in an RNA in vitro transcription reaction as defined herein using a trinucleotide cap analogue as defined herein.
在优选的实施方式中，使用三核苷酸帽类似物m7G(5’)ppp(5’)(2’OMeA)pG或m7G(5’)ppp(5’)(2’OMeG)pG利用共转录加帽来形成本发明的编码RNA的帽1结构。该情况中优选的帽1类似物是m7G(5’)ppp(5’)(2’OMeA)pG。In a preferred embodiment, the cap 1 structure of the coding RNA of the present invention is formed by co-transcriptional capping using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. The preferred cap 1 analog in this case is m7G(5')ppp(5')(2'OMeA)pG.
在其他优选的实施方式中，使用三核苷酸帽类似物3’OMe-m7G(5’)ppp(5’)(2’OMeA)pG利用共转录加帽来形成本发明的RNA的帽1结构。In other preferred embodiments, the cap 1 structure of the RNA of the present invention is formed by co-transcriptional capping using the trinucleotide cap analog 3’OMe-m7G(5’)ppp(5’)(2’OMeA)pG.
在其他实施方式中，使用帽类似物3’OMe-m7G(5’)ppp(5’)G利用共转录加帽来形成本发明的RNA的帽0结构。In other embodiments, the cap analog 3'OMe-m7G(5')ppp(5')G is used to form the cap 0 structure of the RNA of the present invention by co-transcriptional capping.
在其他实施方式中，使用加帽酶(例如，牛痘病毒加帽酶和/或帽依赖性2’-O甲基转移酶)通过酶促加帽来形成5’-帽结构，以产生帽0或帽1或帽2结构。可以使用WO2016/193226中公开的方法和方式，使用固定化的加帽酶和/或帽依赖性2’-O甲基转移酶来添加5’-帽结构(帽0或帽1)，将其全部内容在此按引用并入。In other embodiments, a 5'-cap structure is formed by enzymatic capping using a capping enzyme (e.g., a vaccinia virus capping enzyme and/or a cap-dependent 2'-O methyltransferase) to produce a cap 0, cap 1, or cap 2 structure. The 5'-cap structure (cap 0 or cap 1) can be added using an immobilized capping enzyme and/or a cap-dependent 2'-O methyltransferase using the methods and means disclosed in WO2016/193226, the entire contents of which are hereby incorporated by reference.
在优选的实施方式中，约70％、75％、80％、85％、90％、95％的RNA(物质)包含帽1结构，如使用加帽测定确定的。在优选的实施方式中，低于约20％、15％、10％、5％、4％、3％、2％、1％的RNA(物质)不含帽1结构，如使用加帽测定确定的。在其他优选的实施方式中，约70％、75％、80％、85％、90％、95％的RNA(物质)包含帽0结构，如使用加帽测定确定的。在优选的实施方式中，低于约20％、15％、10％、5％、4％、3％、2％、1％的RNA(物质)不含帽0结构，如使用加帽测定确定的。In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (substance) comprises a cap 1 structure, as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (substance) does not contain a cap 1 structure, as determined using a capping assay. In other preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (substance) comprises a cap 0 structure, as determined using a capping assay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (substance) does not contain a cap 0 structure, as determined using a capping assay.
术语“RNA物质”不限于表示“一个单一分子”，而是理解为包含基本上相同的RNA分子的集合。因此，可以涉及多个基本上相同的(编码)RNA分子。The term "RNA substance" is not limited to meaning "a single molecule", but is to be understood as comprising a collection of substantially identical RNA molecules. Thus, a plurality of substantially identical (coding) RNA molecules may be involved.
为了确定帽0或帽1结构的存在/不存在，可以使用如公开的PCT申请WO2015/101416中所述的加帽测定，将其完整内容在此按引用并入；特别地，如公开的PCT申请WO2015/101416的权利要求27至46中所述的。可以用于确定RNA的帽0或帽1结构的存在/不存在的其他加帽测定描述于PCT/EP2018/08667，或公开的PCT申请WO2014/152673和WO2014/152659，将上述PCT申请的全部内容在此按引用并入。To determine the presence/absence of cap 0 or cap 1 structures, a capping assay as described in published PCT application WO2015/101416, the entire contents of which are hereby incorporated by reference, may be used; in particular, as described in claims 27 to 46 of published PCT application WO2015/101416. Other capping assays that can be used to determine the presence/absence of cap 0 or cap 1 structures of RNA are described in PCT/EP2018/08667, or published PCT applications WO2014/152673 and WO2014/152659, the entire contents of which are hereby incorporated by reference.
在优选的实施方式中，RNA包含m7G(5')ppp(5')(2'OMeA)帽结构。在这样的实施方式中，编码RNA包含5’-末端m7G帽，以及m7GpppN的相邻核苷酸的核糖的额外甲基化，在这种情况下，2’O-甲基化的腺苷。优选地，约70％、75％、80％、85％、90％、95％的RNA(物质)包含这种帽1结构，如使用加帽测定确定的。In a preferred embodiment, the RNA comprises a m7G(5')ppp(5')(2'OMeA) cap structure. In such an embodiment, the encoding RNA comprises a 5'-terminal m7G cap, and additional methylation of the ribose of the adjacent nucleotide of the m7GpppN, in this case, a 2'O-methylated adenosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (substance) comprises this cap 1 structure, as determined using a capping assay.
在其他优选的实施方式中，RNA包含m7G(5')ppp(5')(2'OMeG)帽结构。在这样的实施方式中，编码RNA包含5’-末端m7G帽，以及相邻核苷酸的核糖的额外甲基化，在这种情况下，2’O-甲基化的鸟苷。优选地，约70％、75％、80％、85％、90％、95％的编码RNA(物质)包含这种帽1结构，如使用加帽测定确定的。In other preferred embodiments, the RNA comprises a m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the encoding RNA comprises a 5'-terminal m7G cap, as well as additional methylation of the ribose of the adjacent nucleotide, in this case, a 2'O-methylated guanosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the encoding RNA (substance) comprises this cap 1 structure, as determined using a capping assay.
因此，所述RNA或mRNA序列的第一核苷酸，即，m7G(5')ppp结构下游的核苷酸，可以是2’O-甲基化的鸟苷或2’O-甲基化的腺苷。Therefore, the first nucleotide of the RNA or mRNA sequence, i.e., the nucleotide downstream of the m7G(5')ppp structure, can be 2'O-methylated guanosine or 2'O-methylated adenosine.
根据一些实施方式，RNA是修饰的RNA，其中修饰是指包括主链修饰以及糖修饰或碱基修饰的化学修饰。According to some embodiments, the RNA is a modified RNA, wherein the modification refers to chemical modifications including backbone modifications and sugar modifications or base modifications.
修饰的RNA可以包含核苷酸类似物/修饰，例如，主链修饰、糖修饰或碱基修饰。本发明情况中的主链修饰是其中RNA的核苷酸主链的磷酸酯是化学修饰的。本发明情况中的糖修饰是RNA的核苷酸的糖的化学修饰。此外，本发明的情况中的碱基修饰是RNA的核苷酸的碱基部分的化学修饰。在这种情况中，核苷酸类似物或修饰优选选自适用于转录和/或翻译的核苷酸类似物。Modified RNA can include nucleotide analogs/modifications, for example, backbone modifications, sugar modifications or base modifications. The backbone modification in the present invention is that the phosphate of the nucleotide backbone of the RNA is chemically modified. The sugar modification in the present invention is a chemical modification of the sugar of the nucleotide of the RNA. In addition, the base modification in the present invention is a chemical modification of the base part of the nucleotide of the RNA. In this case, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and/or translation.
在特别优选的实施方式中，可以掺入如本文所述的修饰的RNA中的核苷酸类似物/修饰优选选自2-氨基-6-氯嘌呤核苷-5'-三磷酸、2-氨基嘌呤-核苷-5'-三磷酸；2-氨基腺苷-5'-三磷酸、2'-氨基-2'-脱氧胞苷-三磷酸、2-硫胞苷-5'-三磷酸、2-硫尿苷-5'-三磷酸、2'-氟胸苷-5'-三磷酸、2'-O-甲基-鸟苷-5'-三磷酸盐4-硫尿苷5'-三磷酸、5-氨基烯丙基胞苷-5'-三磷酸、5-氨基烯丙基尿苷-5'-三磷酸、5-溴胞苷-5'-三磷酸、5-溴尿苷-5'-三磷酸，5-溴-2'-脱氧尿苷-5'-三磷酸，5-溴-2'-脱氧尿苷-5'-三磷酸、5-碘胞苷-5'-三磷酸、5-碘-2'-脱氧尿苷-5'三磷酸、5-碘尿苷-5'-三磷酸、5-碘-2'-脱氧尿苷-5'-三磷酸、5-甲基胞苷-5'-三磷酸、5-甲基尿苷-5'-三磷酸、5-丙炔基-2'-脱氧胞苷-5'-三磷酸、5-丙炔基-2'-脱氧尿苷-5'-三磷酸、6-氮胞苷-5'-三磷酸、6-氮尿苷-5'-三磷酸腺苷、6-氯嘌呤核苷-5'-三磷酸、7-脱氮腺苷-5'-三磷酸、7-脱氮鸟苷-5'-三磷酸、8-氮腺苷-5'-三磷酸、8-叠氮腺苷-5'-三磷酸、苯并咪唑-核苷5'-三磷酸、N1-甲基腺苷-5'-三磷酸、N1-甲基鸟苷-5'-三磷酸、N6-甲基腺苷-5'-三磷酸、O6-甲基鸟苷-5'-三磷酸、假尿苷-5'-三磷酸或嘌呤霉素-5'-三磷酸、黄苷-5'-三磷酸盐。特别优选的是用于碱基修饰的核苷酸，选自由5-甲基胞苷-5’-三磷酸、7-脱氮鸟苷-5’-三磷酸、5-溴胞苷-5’-三磷酸和假尿苷-5’-三磷酸、吡啶-4-酮核糖核苷、5-氮杂-尿苷、2-硫-5-氮杂-尿苷、2-硫尿苷、4-硫-假尿苷、2-硫-假尿苷、5-羟基尿苷、3-甲基尿苷、5-羧甲基-尿苷、1-羧甲基-假尿苷、5-丙炔基尿苷、1-丙炔甲基-假尿苷、5-牛磺酸甲基尿苷、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫-尿苷、1-牛磺酸甲基-4-硫-尿苷、5-甲基-尿苷、1-甲基-假尿苷、4-硫-1-甲基-假尿苷、2-硫-1-甲基-假尿苷、1-甲基-1-脱氮-假尿苷、2-硫-1-甲基-1-脱氮-假尿苷、二氢尿苷、二氢假尿苷、2-硫-二氢尿苷、2-硫-二氢假尿苷、2-甲氧基尿苷、2-甲氧基-4-硫-尿苷、4-甲氧基-假尿苷和4-甲氧基-2-硫-假尿苷、5-氮杂-胞苷、假异胞苷、3-甲基-胞苷、N4-乙酰基胞苷、5-甲酰基胞苷、N4-甲基胞苷、5-羟甲基胞苷、1-甲基-假异胞苷、吡咯基-胞苷、吡咯基-假异胞苷、2-硫-胞苷、2-硫-5-甲基-胞苷、4-硫-假异胞苷、4-硫-1-甲基-假异胞苷、4-硫-1-甲基-1-脱氮-假异胞苷、1-甲基-1-脱氮-假异胞苷、泽布拉啉(zebularine)、5-氮杂-泽布拉啉、5-甲基-泽布拉啉、5-氮杂-2-硫-泽布拉啉、2-硫-泽布拉啉、2-甲氧基-胞苷、2-甲基-5-甲基-胞苷、4-甲氧基-假异胞苷和4-甲氧基-1-甲基-假异胞苷、2-氨基嘌呤、2,6-二氨基嘌呤、7-脱氮-腺嘌呤、7-脱氮-8-氮杂-腺嘌呤、7-脱氮-2-氨基嘌呤、7-脱氮-8-氮杂-2-氨基嘌呤、7-脱氮-2,6-二氨基嘌呤，7-脱氮-8-氮杂-2,6-二氨基嘌呤、1-甲基腺苷、N6-甲基腺苷、N6-异戊烯基腺苷、N6-(顺式-羟基异戊烯基)腺苷、2-甲基硫-N6-(顺式-羟基异戊烯)腺苷、N6-甘氨酰基氨甲酰基腺苷、N6-苏氨酰基氨甲酰基腺苷、2-甲基硫-N6-苏氨酰基氨甲酰基腺苷、N6,N6-二甲基腺苷、7-甲基腺苷、2-甲基硫-腺苷和2-甲氧基-腺苷、肌苷、1-甲基-肌苷、怀俄苷(wyosine)、怀丁苷(wybutosine)、7-脱氮-鸟苷、7-脱氮-8-氮杂-鸟苷、6-硫-鸟苷、6-硫-7-脱氮-鸟苷、6-硫-7-脱氮-8-氮杂-鸟苷、7-甲基-鸟苷、6-硫-7-甲基-鸟苷、7-甲基肌苷、6-甲氧基-鸟苷、1-甲基鸟苷、N2-甲基鸟苷、N2,N2-二甲基鸟苷、8-氧代-鸟苷、7-甲基-8-氧代-鸟苷、1-甲基-6-硫-鸟苷、N2-甲基-6-硫-鸟苷和N2,N2-二甲基-6-硫-鸟苷、5'-O-(1-硫代磷酸酯)-腺苷、5'-O-(1-硫代磷酸酯)-胞苷、5'-O-(1-硫代磷酸酯)-鸟苷、5'-O-(1-硫代磷酸酯)-尿苷、5'-O-(1-硫代磷酸酯)-假尿苷、6-氮杂-胞苷、2-硫-胞苷、α-硫-胞苷、假-异-胞苷、5-氨基烯丙基-尿苷、5-碘-尿苷、N1-甲基-假尿苷、5,6-二氢尿苷、α-硫-尿苷、4-硫-尿苷、6-氮杂-尿苷、5-羟基-尿苷、脱氧-胸苷、5-甲基-尿苷、吡咯-胞苷、肌苷、α-硫-鸟苷、6-甲基-鸟苷、5-甲基-胞苷、8-氧代-鸟苷，7-脱氮-鸟苷、N1-甲基-腺苷、2-氨基-6-氯-嘌呤、N6-甲基-2-氨基-嘌呤、假-异-胞苷、6-氯-嘌呤、N6-甲基-腺苷、α-硫-腺苷、8-叠氮-腺苷、7-脱氮-腺苷组成的碱基修饰的核苷酸的组。In a particularly preferred embodiment, the nucleotide analogs/modifications that can be incorporated into the modified RNA as described herein are preferably selected from 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2' -O-methyl-guanosine-5'-triphosphate 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate Acid, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-adenosine triphosphate, 6-chloropurine nucleoside-5'-triphosphate, 7-deazaadenosine-5 '-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside 5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred are nucleotides for base modification selected from the group consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl uridine, 1-propynylmethyl-pseudouridine, 5-taurine methyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolyl-cytidine, pyrrolyl-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebulariline (zebulari ne), 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methyl-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza- Adenosine, 8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine and 2-methoxy-adenosine, inosine, 1-methyl-inosine, wyosine (wy osine), wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6 -thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-phosphorothioate)-adenosine, 5'-O-(1-phosphorothioate)-cytidine, 5'-O-(1-phosphorothioate)-guanosine, 5'-O-(1-phosphorothioate)-uridine, 5'-O-(1-phosphorothioate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio -uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrole-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.
在一些实施方式中，至少一种修饰的核苷酸选自假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫假尿苷、4’-硫假尿苷、5-甲基胞苷、5-甲基尿苷、2-硫-1-甲基-1-脱氮-假尿苷、2-硫-1-甲基-假尿苷、2-硫-5-氮杂-尿苷、2-硫-二氢假尿苷、2-硫-二氢尿苷、2-硫-假尿苷、4-甲氧基-2-硫-假尿苷、4-甲氧基-假尿苷、4-硫-1-甲基-假尿苷、4-硫-假尿苷、5-氮杂-尿苷、二氢假尿苷、5-甲氧基尿苷和2’-O-甲基尿苷。In some embodiments, at least one modified nucleotide is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiopseudouridine, 4'-thiopseudouridine, 5-methylcytidine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.
在一些实施方式中，如本文定义的编码序列中的100％尿嘧啶具有化学修饰，优选化学修饰在尿嘧啶的5-位。In some embodiments, 100% of the uracils in the coding sequence as defined herein have a chemical modification, preferably the chemical modification is at the 5-position of uracil.
在本发明的情况中特别优选的是假尿苷(ψ)、N1-甲基假尿苷(m1ψ)、5-甲基胞苷和5-甲氧基尿苷。Particularly preferred in the context of the present invention are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytidine and 5-methoxyuridine.
然而，在一些实施方式中，本发明的RNA不包括任何N1-甲基假尿苷(m1ψ)取代的位置。在进一步方面中，实施方式的RNA不包括任何假尿苷(ψ)、N1-甲基假尿苷(m1ψ)、5-甲基胞苷和5-甲氧基尿苷取代的位置。在再进一步的实施方式中，本发明的RNA包含仅有G、C、A和U核苷酸组成的编码序列。However, in some embodiments, the RNA of the present invention does not include any N1-methylpseudouridine (m1ψ) substituted position. In further aspects, the RNA of the embodiment does not include any pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytidine and 5-methoxyuridine substituted position. In further embodiments, the RNA of the present invention comprises a coding sequence consisting of only G, C, A and U nucleotides.
将修饰的核苷酸(如假尿苷(ψ)、N1-甲基假尿苷(m1ψ)、5-甲基胞苷和/或5-甲氧基尿苷)掺入RNA的编码序列中可能是有利的，因为可以调节或减少(如果需要)不合需要的先天性免疫反应(施用编码RNA或疫苗后)。Incorporation of modified nucleotides such as pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytidine and/or 5-methoxyuridine into the coding sequence of an RNA may be advantageous because undesirable innate immune responses (following administration of the encoding RNA or vaccine) may be modulated or reduced (if necessary).
在一些实施方式中，RNA包含至少一个编码如本文定义的SARS-CoV-2抗原蛋白的编码序列，其中所述编码序列包含至少一个选自假尿苷(ψ)和N1-甲基假尿苷(m1ψ)的修饰核苷酸，优选其中所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In some embodiments, the RNA comprises at least one coding sequence encoding a SARS-CoV-2 antigenic protein as defined herein, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA不包含N1-甲基假尿苷(m1ψ)取代的位置。在进一步的实施方式中，RNA不包含假尿苷(ψ)、N1-甲基假尿苷(m1ψ)、5-甲基胞苷和5-甲氧基尿苷取代的位置。In a preferred embodiment, the RNA does not contain a position substituted with N1-methylpseudouridine (m1ψ). In a further embodiment, the RNA does not contain a position substituted with pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytidine, and 5-methoxyuridine.
在优选的实施方式中，RNA包含仅有G、C、A和U核苷酸组成的编码序列并且因此不包含修饰的核苷酸(除了5’末端帽结构，例如，帽1)。In a preferred embodiment, the RNA comprises a coding sequence consisting of only G, C, A and U nucleotides and therefore contains no modified nucleotides (except for the 5' terminal cap structure, e.g., Cap 1).
适用于冠状病毒疫苗的核酸，优选mRNA构建体：Nucleic acids suitable for coronavirus vaccines, preferably mRNA constructs:
在各种不同的实施方式中，RNA，优选mRNA包含以下元件，优选以5’-至3’-方向：In various embodiments, the RNA, preferably mRNA, comprises the following elements, preferably in 5'- to 3'-direction:
A)5’-帽结构，优选如本文具体说明的；A) a 5'-cap structure, preferably as specified herein;
B)5’-末端起始元件，优选如本文具体说明的；B) a 5'-terminal initiation element, preferably as specified herein;
C)任选，5’-UTR，优选如本文具体说明的；C) optionally, a 5'-UTR, preferably as specified herein;
D)核糖体结合位点，优选如本文具体说明的；D) a ribosome binding site, preferably as specified herein;
E)至少一个编码序列，优选如本文具体说明的；E) at least one coding sequence, preferably as specified herein;
F)3’-UTR，优选如本文具体说明的；F) 3'-UTR, preferably as specified herein;
G)任选，聚(A)序列，优选如本文具体说明的；G) optionally, a poly(A) sequence, preferably as specified herein;
H)任选，聚(C)序列，优选如本文具体说明的；H) optionally, a poly(C) sequence, preferably as specified herein;
I)任选，组蛋白茎-环，优选如本文具体说明的；I) optionally, a histone stem-loop, preferably as specified herein;
J)任选，3’-末端序列元件，优选如本文具体说明的。J) Optionally, a 3'-terminal sequence element, preferably as specified herein.
在特别优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a particularly preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from HSD17B4 as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:116、136、137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637、28916、28921-28924或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916, 28921-28924, or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
E)选自SEQ ID NO:178或179的组蛋白茎-环；E) a histone stem-loop selected from SEQ ID NO: 178 or 179;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在进一步优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a further preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from HSD17B4 as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:116、136、137、146、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、23089-23148、23150-23184、27110-27247、28589-28637、28916、28921-28924或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 116, 136, 137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916, 28921-28924, or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在特别优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a particularly preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:27118、27141、27164、27187、27210、27233、28601-28606或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233, 28601-28606, or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
E)选自SEQ ID NO:178或179的组蛋白茎-环；E) a histone stem-loop selected from SEQ ID NO: 178 or 179;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在进一步优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a further preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:27118、27141、27164、27187、27210、27233、28601-28606或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233, 28601-28606, or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在特别优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a particularly preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:27118、27141、27164、27187、27210、27233或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233 or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:2 53或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
E)选自SEQ ID NO:178或179的组蛋白茎-环；E) a histone stem-loop selected from SEQ ID NO: 178 or 179;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在进一步优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a further preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:27118、27141、27164、27187、27210、27233或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NO: 27118, 27141, 27164, 27187, 27210, 27233 or a fragment or variant thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在特别优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a particularly preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:28590-28593、28921-28924或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NOs: 28590-28593, 28921-28924, or fragments or variants thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
E)选自SEQ ID NO:178或179的组蛋白茎-环；E) a histone stem-loop selected from SEQ ID NO: 178 or 179;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
在进一步优选的实施方式中，核酸，优选mRNA，以5’-至3’-方向包含以下元件：In a further preferred embodiment, the nucleic acid, preferably mRNA, comprises the following elements in 5'- to 3'-direction:
A)如本文定义的帽1结构；A) a Cap 1 structure as defined herein;
B)如本文定义的源自HSD17B4基因的5’-UTR，优选根据SEQ ID NO:231或232；B) a 5'-UTR derived from the HSD17B4 gene as defined herein, preferably according to SEQ ID NO: 231 or 232;
C)选自SEQ ID NO:28590-28593、28921-28924或其片段或变体的编码序列；C) a coding sequence selected from SEQ ID NOs: 28590-28593, 28921-28924, or fragments or variants thereof;
D)如本文定义的源自PSMB3基因的3’-UTR的3’-UTR，优选根据SEQ ID NO:253或254；D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein, preferably according to SEQ ID NO: 253 or 254;
F)包含约100个A核苷酸的聚(A)序列，优选代表3’末端。F) A poly(A) sequence comprising about 100 A nucleotides, preferably representing the 3' end.
本发明的优选的RNA序列，优选mRNA序列，提供于表2中。其中，每行表示本发明的特别合适的SARS-CoV-2构建体，其中表2的列A(Col A)显示了SARS CoV-2主链构建体的说明，并且相应的RNA序列，特别是包含优选的编码序列的mRNA序列提供于列C-G(Col.C-G)中。表2a提供了具有HSD17B4/PSMB3-hSL-A100(a-1)的RNA序列。表2b具有HSD17B4/PSMB3-A100(a-1)。Preferred RNA sequences, preferably mRNA sequences, of the present invention are provided in Table 2. Therein, each row represents a particularly suitable SARS-CoV-2 construct of the present invention, wherein Column A (Col A) of Table 2 shows the description of the SARS CoV-2 backbone construct, and the corresponding RNA sequences, in particular mRNA sequences comprising preferred coding sequences are provided in Columns C-G (Col. C-G). Table 2a provides RNA sequences with HSD17B4/PSMB3-hSL-A100 (a-1). Table 2b has HSD17B4/PSMB3-A100 (a-1).
表2a：适用于疫苗的核酸，优选mRNA构建体Table 2a: Nucleic acids suitable for vaccines, preferably mRNA constructs
表2b：适用于冠状病毒疫苗的核酸，优选mRNA构建体Table 2b: Nucleic acids suitable for coronavirus vaccines, preferably mRNA constructs
在某些实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:148、149、151、162、163、165、22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、22819、22821、22823、22825、22827、22829、22831、22833、22835、22837、22839、23309-23368、23370-23404、23529-23588、23590-23624、24837-24944、27248-27907、28638-28915、28925-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。在某些实施方式中，所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In certain embodiments, the RNA, preferably mRNA, comprises a polypeptide selected from the group consisting of SEQ ID NOs: 148, 149, 151, 162, 163, 165, 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835, 22837, 22839, 23309-23368, 23370-23371. 404, 23529-23588, 23590-23624, 24837-24944, 27248-27907, 28638-28915, 28925-28940, or a fragment or variant of any of these sequences, or a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methyl pseudouridine (m1ψ) nucleotides.
在某些实施方式中，RNA，优选mRNA，包含与选自表2a或表2b的列B-G中提供的SEQID NO的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。在某些实施方式中，所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In certain embodiments, RNA, preferably mRNA, comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs provided in columns B-G of Table 2a or Table 2b, or a fragment or variant of any of these sequences. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methyl pseudouridine (m1ψ) nucleotides.
在某些实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27256、27279、27302、27325、27348、27371、27394、27417、27440、27463、27486、27509、27532、27555、27578、27601、27624、27647、27688、27729、27770、27811、27852、27893、28650-28655、28699-28704、28762、28789-28794、28852、28879-28884的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。在某些实施方式中，所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In certain embodiments, the RNA, preferably mRNA, comprises a polypeptide selected from the group consisting of SEQ ID NOs: 27256, 27279, 27302, 27325, 27348, 27371, 27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601, 27624, 27647, 27688, 27729, 27770, 27811, 27852, 27893, 28650-28654. 655, 28699-28704, 28762, 28789-28794, 28852, 28879-28884, or a fragment or variant of any of these sequences, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consists of. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methyl pseudouridine (m1ψ) nucleotides.
在某些实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27256、27279、27302、27325、27348、27371、27394、27417、27440、27463、27486、27509、27532、27555、27578、27601、27624、27647、27688、27729、27770、27811、27852、27893、28762、28852的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。在某些实施方式中，所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In certain embodiments, the RNA, preferably mRNA, comprises a polypeptide selected from the group consisting of SEQ ID NOs: 27256, 27279, 27302, 27325, 27348, 27371, 27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601, 27624, 27647, 27688, 27729, 27770, 27771, 27772, 27773, 27774, 27775, 27776, 27777, 27778, 27779, 27780, 27781, 27782, 27783, 27784, 27785, 27786, 27787, 27786, 27787, 27788, 27789, 27781, 27782, 27783, 27784, 27786, 27787 ...6, 27786, 27786, 27786, 27786, 27786, 27786, 27786, 2 811, 27852, 27893, 28762, 28852 or a fragment or variant of any of these sequences is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, or consists of it. In certain embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methyl pseudouridine (m1ψ) nucleotides.
在某些实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:28639-28642、28778-28781、28688-28691、28868-28871、28925-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。在某些实施方式中，所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In some embodiments, RNA, preferably mRNA, comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a nucleic acid sequence selected from SEQ ID NO: 28639-28642, 28778-28781, 28688-28691, 28868-28871, 28925-28940, or a fragment or variant of any of these sequences. In some embodiments, at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methyl pseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。关于相应核酸序列的更多信息提供于序列表中相应SEQ ID NO的<223>标识符下，以及表2中(特别是参见列B-G)。In a preferred embodiment, the RNA, preferably mRNA, comprises a polypeptide selected from the group consisting of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776 , 28638-28686, 28777-28825, 28925-28928, 28933-28936, or a fragment or variant of any of these sequences, or a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. More information about the corresponding nucleic acid sequences is provided in the sequence listing under the <223> identifier of the corresponding SEQ ID NO, and in Table 2 (see in particular columns B-G).
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27394、27417、27486、28762、28650-28655、28789-28794的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27394, 27417, 27486, 28762, 28650-28655, 28789-28794, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27394、27417、27486、28762的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27394, 27417, 27486, 28762, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:28639-28642、28778-28781、28925-28928、28933-28936的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 28639-28642, 28778-28781, 28925-28928, 28933-28936, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27532、27555、27624、28852、28699-28704、28879-28884的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27532, 27555, 27624, 28852, 28699-28704, 28879-28884, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27532、27555、27624、28852的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27532, 27555, 27624, 28852, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:28688-28691、28868-28871、28929-28932、28937-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 28688-28691, 28868-28871, 28929-28932, 28937-28940, or a fragment or variant of any of these sequences.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises a polypeptide selected from the group consisting of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 2892 5-28928, 28933-28936, or a fragment or variant of any of these sequences, or a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consists of, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27394、27417、27486、28762、28650-28655、28789-28794的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27394, 27417, 27486, 28762, 28650-28655, 28789-28794, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27394、27417、27486、28762的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27394, 27417, 27486, 28762, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:28639-28642、28778-28781、28925-28928、28933-28936的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 28639-28642, 28778-28781, 28925-28928, 28933-28936, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises a nucleic acid sequence selected from SEQ ID NO: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940, or any of these sequences. Fragments or variants are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid sequences, or consist of them, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27532、27555、27624、28852、28699-28704、28879-28884的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27532, 27555, 27624, 28852, 28699-28704, 28879-28884, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:27532、27555、27624、28852的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 27532, 27555, 27624, 28852, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在优选的实施方式中，RNA，优选mRNA，包含与选自SEQ ID NO:28688-28691、28868-28871、28929-28932、28937-28940的核酸序列或这些序列任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中所述RNA序列中的至少一个，优选所有尿嘧啶核苷酸被假尿苷(ψ)核苷酸和/或N1-甲基假尿苷(m1ψ)核苷酸替代。In a preferred embodiment, the RNA, preferably mRNA, comprises or consists of a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 28688-28691, 28868-28871, 28929-28932, 28937-28940, or a fragment or variant of any of these sequences, wherein at least one, preferably all, uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.
在特定的实施方式中，使用本领域已知的任何方法，包括化学合成，如，例如，固相RNA合成，以及体外方法，如RNA体外转录反应，来制备本发明的RNA。因此，在优选的实施方式中，通过RNA体外转录获得RNA。In a specific embodiment, any method known in the art is used, including chemical synthesis, such as, for example, solid phase RNA synthesis, and in vitro methods, such as RNA in vitro transcription reactions, to prepare the RNA of the present invention. Therefore, in a preferred embodiment, RNA is obtained by RNA in vitro transcription.
因此，在优选的实施方式中，本发明的RNA优选是体外转录的RNA。Therefore, in a preferred embodiment, the RNA of the present invention is preferably in vitro transcribed RNA.
术语“RNA体外转录”或“体外转录”涉及其中RNA在无细胞系统中(体外)合成的过程。可以通过合适的DNA模板的DNA依赖性体外转录获得RNA，其根据本发明是线性化质粒DNA模板或PCR扩增的DNA模板。用于控制RNA体外转录的启动子可以是用于任何DNA依赖性RNA聚合酶的任何启动子。DNA依赖性RNA聚合酶的特定实例是T7、T3、SP6或Syn5 RNA聚合酶。在本发明的优选实施方式中，在进行RNA体外转录前，DNA模板用合适的限制酶将线性化。The term "RNA in vitro transcription" or "in vitro transcription" refers to a process in which RNA is synthesized in a cell-free system (in vitro). RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which according to the present invention is a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter used to control RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6 or Syn5 RNA polymerases. In a preferred embodiment of the present invention, before performing RNA in vitro transcription, the DNA template is linearized with a suitable restriction enzyme.
RNA体外转录中使用的试剂通常包括：DNA模板(线性化质粒DNA或PCR产物)与对其相应RNA聚合酶具有高结合亲和力的启动子序列，所述RNA聚合酶如噬菌体编码的RNA聚合酶(T7、T3、SP6或Syn5)；对于四种碱基(腺嘌呤、胞嘧啶、鸟嘌呤和尿嘧啶)的核糖核苷酸三磷酸(NTP)；任选地，如本文定义的帽类似物；任选地，如本文定义的进一步修饰的核苷酸；能够结合DNA模板内的启动子序列的DNA依赖性RNA聚合酶(例如，T7、T3、SP6或Syn5RNA聚合酶)；任选地，核糖核酸酶(RNA酶)抑制剂以灭活任何潜在污染的RNA酶；任选地，焦磷酸酶以降解焦磷酸盐，其可能抑制RNA体外转录；MgCl2，其提供Mg2+离子作为聚合酶的辅因子；缓冲剂(TRIS或HEPES)以维持合适的pH值，其也可以含有抗氧化剂(例如，DTT)和/或最佳浓度的聚胺(如亚精胺)，例如，WO2017/109161中公开的包含TRIS-柠檬酸盐的缓冲系统。The reagents used in RNA in vitro transcription generally include: a DNA template (linearized plasmid DNA or PCR product) and a promoter sequence with high binding affinity to its corresponding RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analog as defined herein; optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase (e.g., e.g., T7, T3, SP6 or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potential contaminating RNases; optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit RNA transcription in vitro; MgCl2, which provides Mg2+ ions as a cofactor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which may also contain an antioxidant (e.g., DTT) and/or an optimal concentration of a polyamine (e.g., spermidine), for example, a buffer system comprising TRIS-citrate disclosed in WO2017/109161.
在优选的实施方式中，使用三核苷酸帽类似物m7G(5’)ppp(5’)(2’OMeA)pG或m7G(5’)ppp(5’)(2’OMeG)pG，采用共转录加帽来形成本发明的RNA的帽1结构。在制造本发明的编码RNA中可以合适地使用的优选帽1类似物是m7G(5’)ppp(5’)(2’OMeG)pG。In a preferred embodiment, the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG is used to form the cap 1 structure of the RNA of the present invention by co-transcriptional capping. A preferred cap 1 analog that can be suitably used in making the coding RNA of the present invention is m7G(5')ppp(5')(2'OMeG)pG.
在特别优选的实施方式中，使用三核苷酸帽类似物3’OMe-m7G(5’)ppp(5’)(2’OMeA)pG，利用共转录加帽形成本发明的RNA的帽1结构。In a particularly preferred embodiment, the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG is used to form the cap 1 structure of the RNA of the present invention by co-transcriptional capping.
在其他实施方式中，使用帽类似物3’OMe-m7G(5’)ppp(5’)G，利用共转录加帽形成本发明的RNA的帽O结构。In other embodiments, the cap analog 3'OMe-m7G(5')ppp(5')G is used to form the cap O structure of the RNA of the present invention by co-transcriptional capping.
在另外的实施方式中，RNA体外转录中使用的核苷酸混合物可以另外包含如本文定义的修饰的核苷酸。在该情况中，优选的修饰的核苷酸可以选自假尿苷(ψ)、N1-甲基假尿苷(m1ψ)、5-甲基胞苷和5-甲氧基尿苷。在特别的实施方式中，核苷酸混合物中的尿嘧啶核苷酸(部分或全部)被假尿苷(ψ)和/或N1-甲基假尿苷(m1ψ)替代，以获得修饰的RNA。In other embodiments, the nucleotide mixture used in RNA in vitro transcription may further comprise modified nucleotides as defined herein. In this case, preferred modified nucleotides may be selected from pseudouridine (ψ), N1-methyl pseudouridine (m1ψ), 5-methylcytidine and 5-methoxyuridine. In a particular embodiment, the uracil nucleotides (partially or entirely) in the nucleotide mixture are replaced by pseudouridine (ψ) and/or N1-methyl pseudouridine (m1ψ) to obtain modified RNA.
在优选的实施方式中，RNA体外转录中使用的核苷酸混合物不包含如本文定义的修饰的核苷酸。在优选的实施方式中，RNA体外转录中使用的核苷酸混合物仅包含G、C、A和U核苷酸，和任选地如本文定义的帽类似物。In a preferred embodiment, the nucleotide mixture used in RNA in vitro transcription does not contain modified nucleotides as defined herein. In a preferred embodiment, the nucleotide mixture used in RNA in vitro transcription contains only G, C, A and U nucleotides, and optionally a cap analog as defined herein.
在优选的实施方式中，用于RNA体外转录反应的核苷酸混合物(即，混合物中每种核苷酸的分数)可以针对给定的RNA序列优化，优选如WO2015/188933中所述的，将其完整内容按引用并入本文中的。In a preferred embodiment, the nucleotide mixture (i.e., the fraction of each nucleotide in the mixture) used for the RNA in vitro transcription reaction can be optimized for a given RNA sequence, preferably as described in WO2015/188933, the entire contents of which are incorporated herein by reference.
在这种情况中，在序列优化的核苷酸混合物和任选的帽类似物的存在下进行体外转录，优选其中序列优化的核苷酸混合物不包含化学修饰的核苷酸。In this case, in vitro transcription is performed in the presence of a sequence-optimized nucleotide mixture and optionally a cap analog, preferably wherein the sequence-optimized nucleotide mixture does not contain chemically modified nucleotides.
在这种情况中，序列优化的核苷三磷酸(NTP)混合物是用于包含四种核苷三磷酸(NTP)GTP、ATP、CTP和UTP的给定序列的RNA分子的体外转录反应中的核苷三磷酸(NTP)的混合物，其中序列优化的核苷三磷酸(NTP)混合物中的四种核苷三磷酸(NTP)的每个的分数对应于所述RNA分子中各自核苷酸的分数。如果某个核糖核苷酸不存在于RNA分子中，相应的核苷酸三磷酸也不存在于序列优化的核苷三磷酸(NTP)混合物中。In this case, the sequence-optimized nucleoside triphosphate (NTP) mixture is a mixture of nucleoside triphosphates (NTPs) used in an in vitro transcription reaction of an RNA molecule of a given sequence comprising the four nucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, wherein the fraction of each of the four nucleoside triphosphates (NTPs) in the sequence-optimized nucleoside triphosphate (NTP) mixture corresponds to the fraction of each nucleotide in the RNA molecule. If a certain ribonucleotide is not present in the RNA molecule, the corresponding nucleotide triphosphate is also not present in the sequence-optimized nucleoside triphosphate (NTP) mixture.
在其中必须产生超过一种如本文定义的不同的RNA的实施方式中，例如，其中必须产生2、3、4、5、6、7、8、9、10或甚至更多种不同的RNA(参见第二方面)的情况下，合适地可以使用WO2017/109134中所述的程序。In embodiments in which more than one different RNA as defined herein has to be produced, for example, in cases in which 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNAs have to be produced (see the second aspect), the procedure described in WO2017/109134 may suitably be used.
在基于RNA的疫苗生产的情况中，可能需要提供GMP级核酸，例如，GMP级RNA。可以使用由监管结构批准的制造方法来产生GMP级RNA。因此，在特别优选的实施方式中，在现行良好生产规范(GMP)下进行RNA生产，在DNA(模板)和RNA水平上实施各种质量控制步骤，优选根据WO2016/180430。在优选的实施方式中，本发明的RNA是GMP级RNA，特别是GMP级mRNA。因此，用于疫苗的RNA优选是GPM级RNA。In the case of RNA-based vaccine production, it may be necessary to provide GMP-grade nucleic acids, for example, GMP-grade RNA. GMP-grade RNA can be produced using manufacturing methods approved by regulatory agencies. Therefore, in a particularly preferred embodiment, RNA production is carried out under current good manufacturing practices (GMP), and various quality control steps are implemented at the DNA (template) and RNA levels, preferably according to WO2016/180430. In a preferred embodiment, the RNA of the present invention is GMP-grade RNA, particularly GMP-grade mRNA. Therefore, the RNA used for the vaccine is preferably GPM-grade RNA.
所获得的RNA产物优选使用(CureVac，Tübingen，德国；根据WO2008/077592的RP-HPLC)和/或切线流过滤(如WO2016/1932-6中所述的)和/或寡d(T)纯化(参见WO2016/180430)来进行纯化。The RNA product obtained is preferably used Purification was performed by ELISA (CureVac, Tübingen, Germany; RP-HPLC according to WO2008/077592) and/or tangential flow filtration (as described in WO2016/1932-6) and/or oligo-d(T) purification (see WO2016/180430).
在进一步优选的实施方式中，将RNA冻干(例如，根据WO2016/165831或WO2011/069586，将两篇PCT申请的完整内容在此按引用并入)以产生温度稳定的干燥的RNA(粉末)。RNA也可以使用喷雾干燥或喷雾冷冻干燥(例如，根据WO2016/184575或WO2016/184576)来干燥以产生如本文定义的温度稳定的RNA(粉末)。因此，在制造和纯化核酸的情况中，特别是RNA，将WO2017/109161、WO2015/188933、WO2016/180430、WO2008/077592、WO2016/193206、WO2016/165831、WO2011/069586、WO2016/184575和WO2016/184576的公开内容在此按引用并入。In a further preferred embodiment, the RNA is lyophilized (e.g., according to WO2016/165831 or WO2011/069586, the entire contents of both PCT applications are hereby incorporated by reference) to produce a temperature-stable dry RNA (powder). The RNA can also be dried using spray drying or spray freeze drying (e.g., according to WO2016/184575 or WO2016/184576) to produce a temperature-stable RNA (powder) as defined herein. Therefore, in the context of the production and purification of nucleic acids, in particular RNA, the disclosures of WO2017/109161, WO2015/188933, WO2016/180430, WO2008/077592, WO2016/193206, WO2016/165831, WO2011/069586, WO2016/184575 and WO2016/184576 are hereby incorporated by reference.
因此，在优选的实施方式中，RNA是干燥的RNA。Thus, in a preferred embodiment, the RNA is dried RNA.
如本文使用的术语“干燥的RNA”必须理解为如上定义的冻干、或喷雾干燥或喷雾冷冻干燥的RNA，以获得温度稳定的干燥的RNA(粉末)。The term "dried RNA" as used herein has to be understood as lyophilized, or spray-dried or spray-freeze-dried RNA as defined above, to obtain a temperature-stable dried RNA (powder).
在优选的实施方式中，本发明的核酸是纯化的核酸，特别是纯化的RNA。In a preferred embodiment, the nucleic acid of the invention is a purified nucleic acid, in particular a purified RNA.
如本文使用的术语“纯化的核酸”应当理解为在某些纯化步骤后具有比起始材料更高纯度的核酸。通常基本上不存在于纯化的核酸中的杂质包括肽或蛋白质、亚精胺、BSA、失败的核酸序列、核酸片段、游离核苷酸、细菌杂质或源自纯化程序的杂质。因此，在这一点上，对于“核酸纯度的程度”理想的是尽可能地接近100％。对于核酸纯度的程度，还希望的是全长核酸的量尽可能地接近100％。因此，如本文使用的“纯化的核酸”具有超过75％、80％、85％纯度的程度，非常特别地是90％、91％、92％、93％、94％、95％、96％、97％、98％和最优选99％或更高。纯度的程度例如可以通过分析HPLC来确定，其中以上提供的百分比对应于靶核酸峰面积与表示副产物的所有峰的总面积的比率。或者，纯度的程度可以例如通过分析琼脂糖凝胶电泳或毛细管凝胶电泳来确定。As used herein, the term "purified nucleic acid" should be understood as a nucleic acid with a higher purity than the starting material after some purification steps. Impurities that are generally not present in the purified nucleic acid include peptides or proteins, spermidine, BSA, failed nucleic acid sequences, nucleic acid fragments, free nucleotides, bacterial impurities or impurities derived from purification procedures. Therefore, at this point, it is desirable to be as close to 100% as possible for "the degree of nucleic acid purity". For the degree of nucleic acid purity, it is also desirable that the amount of full-length nucleic acid is as close to 100% as possible. Therefore, as used herein, "purified nucleic acid" has a degree exceeding 75%, 80%, 85% purity, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most preferably 99% or higher. The degree of purity can be determined, for example, by analyzing HPLC, wherein the percentage provided above corresponds to the ratio of the target nucleic acid peak area to the total area of all peaks representing by-products. Alternatively, the degree of purity can be determined, for example, by analyzing agarose gel electrophoresis or capillary gel electrophoresis.
在优选的实施方式中，本发明的核酸是纯化的RNA。In a preferred embodiment, the nucleic acid of the invention is purified RNA.
如本文使用的术语“纯化的RNA”或“纯化的mRNA”应当理解为在某些纯化步骤(例如，HPLC、TFF、寡g(T)纯化、沉淀步骤)后比起始材料(例如，体外转录的RNA)具有更高纯度的RNA。通常基本上不存在于纯化的RNA中的杂质包括肽或蛋白质(例如，源自DNA依赖性RNA体外转录的酶，例如，RNA聚合酶、RNA酶、焦磷酸酶、限制性核酸内切酶、DNA酶)、亚精胺、BSA、失败的RNA序列、RNA片段(短双链RNA片段、失败的序列等)、游离核苷酸(修饰的核苷酸、常规NTP、帽类似物)、模板DNA片段、缓冲液组分(HEPES、TRIS、MgCl2)等。可能源自例如发酵程序的其他潜在的杂质包括细菌杂质(生物负载，细菌DNA)或源自纯化程序的杂质(有机溶剂等)。因此，在这点上，对于“RNA纯度的程度”理想的是尽可能地接近100％。对于RNA纯度的程度，全长RNA转录物的量尽可能地接近100％也是希望的。因此，如本文使用的“纯化的RNA”具有超过75％、80％、85％纯度的程度，非常特别是90％、91％、92％、93％、94％、95％、96％、97％、98％和最适当地99％或更高。纯度的程度例如可以通过分析HPLC来确定，其中以上提供的百分比对应于靶RNA的峰面积与表示副产物的所有峰的总面积的比率。或者，纯度的程度可以例如通过分析琼脂糖凝胶电泳或毛细管凝胶电泳来确定。As used herein, the term "purified RNA" or "purified mRNA" should be understood as RNA having a higher purity than the starting material (e.g., in vitro transcribed RNA) after certain purification steps (e.g., HPLC, TFF, oligo g (T) purification, precipitation steps). Impurities that are generally not substantially present in purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, e.g., RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNA enzyme), spermidine, BSA, failed RNA sequences, RNA fragments (short double-stranded RNA fragments, failed sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that may originate from, for example, fermentation procedures include bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents, etc.). Therefore, in this regard, it is ideal for the "degree of RNA purity" to be as close to 100% as possible. With respect to the degree of RNA purity, it is also desirable that the amount of full-length RNA transcript is as close to 100% as possible. Thus, "purified RNA" as used herein has a degree of purity exceeding 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most suitably 99% or more. The degree of purity can be determined, for example, by analytical HPLC, wherein the percentages provided above correspond to the ratio of the peak area of the target RNA to the total area of all peaks representing by-products. Alternatively, the degree of purity can be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.
在特别优选的实施方式中，RNA通过RP-HPLC和/或TFF纯化，以除去双链RNA、未加帽的RNA和/或RNA片段。In particularly preferred embodiments, the RNA is purified by RP-HPLC and/or TFF to remove double-stranded RNA, uncapped RNA and/or RNA fragments.
在例如RNA体外转录期间作为副产物的双链RNA的形成可以导致先天性免疫反应的诱导，特别是IFNα，其是在接种疫苗的受试者中导致发烧的主要因素，这当然是不合需要的副作用。目前用于dsRNA免疫印迹的技术(例如，通过斑点印迹、血清学特异性电子显微术(SSEM)或ELISA)用于检测来自核酸混合物的dsRNA物质并测定其大小。The formation of double-stranded RNA as a byproduct during, for example, RNA in vitro transcription can lead to the induction of innate immune responses, particularly IFNα, which is a major factor leading to fever in vaccinated subjects, which is of course an undesirable side effect. Current techniques for dsRNA immunoblotting (e.g., by dot blotting, serological specific electron microscopy (SSEM) or ELISA) are used to detect dsRNA species from nucleic acid mixtures and determine their size.
合适地，本发明的RNA已经通过如本文所述的RP-HPLC和/或TFF纯化以减少dsRNA的量。Suitably, the RNA of the invention has been purified by RP-HPLC and/or TFF as described herein to reduce the amount of dsRNA.
优选地，使用RP-HPLC，优选使用具有大孔苯乙烯/二乙烯苯柱(例如，粒径30μm，孔径)的反相高压液相色谱(RP-HPLC)，和另外使用具有分子量截止约100kDa的基于纤维素的膜的过滤盒纯化根据本发明的RNA。Preferably, RP-HPLC is used, preferably using a macroporous styrene/divinylbenzene column (e.g., particle size 30 μm, pore size ) and additionally purifying the RNA according to the invention using a filtration cassette based on a cellulose membrane with a molecular weight cut-off of about 100 kDa.
在这种情况中，特别优选的是纯化的RNA通过RP-HPLC和/或TFF纯化，其导致与未经RP-HPLC和/或TFF纯化的RNA相比少约5％、10％或20％的双链RNA副产物。因此，与未经RP-HPLC和/或TFF纯化的RNA相比，本发明的RNA包含少约5％、10％或20％的双链RNA副产物。In this case, it is particularly preferred that the purified RNA is purified by RP-HPLC and/or TFF, which results in about 5%, 10% or 20% less double-stranded RNA byproducts compared to RNA that has not been purified by RP-HPLC and/or TFF. Thus, the RNA of the present invention contains about 5%, 10% or 20% less double-stranded RNA byproducts compared to RNA that has not been purified by RP-HPLC and/or TFF.
或者，与使用寡dT纯化、沉淀、过滤和/或阴离子交换色谱纯化的RNA相比，通过RP-HPLC和/或TFF纯化的经纯化RNA包含少约5％、10％或20％的双链RNA副产物。因此，与使用寡dT纯化、沉淀、过滤和/或AEX纯化的RNA相比，本发明的RP-HPLC和/或TFF纯化的RNA包含少约5％、10％或20％的双链RNA副产物。Alternatively, the purified RNA purified by RP-HPLC and/or TFF contains about 5%, 10%, or 20% less double-stranded RNA byproducts than RNA purified using oligo dT purification, precipitation, filtration, and/or anion exchange chromatography. Thus, the RP-HPLC and/or TFF purified RNA of the present invention contains about 5%, 10%, or 20% less double-stranded RNA byproducts than RNA purified using oligo dT purification, precipitation, filtration, and/or AEX purification.
在实施方式中，用于进行RNA体外转录的自动化装置可以用于产生和纯化本发明的核酸。这样的装置也可以用于产生组合物或疫苗(参见方面2和3)。优选地，可以合适地使用如WO2020/002598(将其完整内容在此按引用并入)中所述的装置，特别地，如WO2020/002598的权利要求1至59和/或68至76(以及图1-18)中所述的装置。In an embodiment, an automated device for performing in vitro transcription of RNA can be used to produce and purify the nucleic acid of the present invention. Such a device can also be used to produce a composition or vaccine (see aspects 2 and 3). Preferably, a device as described in WO2020/002598 (the entire contents of which are incorporated herein by reference) can be suitably used, in particular, a device as described in claims 1 to 59 and/or 68 to 76 (and Figures 1-18) of WO2020/002598.
本文所述的方法可以优选应用于如下进一步详细描述的产生RNA组合物或疫苗的方法。The methods described herein may preferably be applied to methods of producing RNA compositions or vaccines as described in further detail below.
组合物、药物组合物：Composition, pharmaceutical composition :
第二方面涉及包含至少一个第一方面的RNA的组合物。The second aspect relates to a composition comprising at least one RNA of the first aspect.
值得注意的是，涉及第二方面的组合物的实施方式同样可以阅读并理解为第三方面的疫苗的合适实施方式。此外，涉及第三方面的疫苗的实施方式同样可以阅读并理解为第二方面的组合物(包含至少一个第一方面的RNA)的合适的实施方式。此外，第一方面(本发明的RNA)的情况中所述的特征和实施方式必须阅读并且必须理解为第二方面的组合物的合适的实施方式。It is noteworthy that the embodiments of the composition related to the second aspect can also be read and understood as suitable embodiments of the vaccine of the third aspect. In addition, the embodiments of the vaccine related to the third aspect can also be read and understood as suitable embodiments of the composition of the second aspect (comprising at least one RNA of the first aspect). In addition, the features and embodiments described in the context of the first aspect (RNA of the present invention) must be read and must be understood as suitable embodiments of the composition of the second aspect.
在优选的实施方式中，组合物包含至少一个编码至少一种抗原肽或蛋白质(其是或源自SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体)的根据第一方面的RNA。In a preferred embodiment, the composition comprises at least one RNA according to the first aspect encoding at least one antigenic peptide or protein (which is or is derived from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof).
在优选的实施方式中，组合物包含至少一个根据第一方面的编码至少一个抗原肽或蛋白质(其选自或源自SARS-CoV-2刺突蛋白或其免疫原性片段或免疫原性变体)的RNA，其中所述组合物将优选地肌内或皮内施用。In a preferred embodiment, the composition comprises at least one RNA according to the first aspect encoding at least one antigenic peptide or protein (which is selected from or derived from the SARS-CoV-2 spike protein or an immunogenic fragment or immunogenic variant thereof), wherein the composition is preferably administered intramuscularly or intradermally.
优选地，所述组合物的肌内或皮内施用导致所编码的SARS-CoV-2刺突蛋白构建体在受试者中的表达。在优选的实施方式中，组合物的施用导致RNA的翻译并在受试者中产生编码的SARS-CoV-2刺突蛋白。Preferably, intramuscular or intradermal administration of the composition results in expression of the encoded SARS-CoV-2 spike protein construct in the subject. In a preferred embodiment, administration of the composition results in translation of the RNA and production of the encoded SARS-CoV-2 spike protein in the subject.
优选地，第二方面的组合物适合于疫苗，特别地，适用于SARS-CoV-2疫苗，优选针对至少一种以下的SARS-CoV-2分离株的SARS-CoV-2疫苗：C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)。Preferably, the composition of the second aspect is suitable for a vaccine, in particular, for a SARS-CoV-2 vaccine, preferably a SARS-CoV-2 vaccine against at least one of the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在特别优选的实施方式中，第二方面的组合物适用于针对B.1.351(Beta，南非)的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for a SARS-CoV-2 vaccine against B.1.351 (Beta, South Africa).
在特别优选的实施方式中，第二方面的组合物适用于针对B.1.617.2、AY.1、AY.2、AY.4或AY.4.2的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for a SARS-CoV-2 vaccine against B.1.617.2, AY.1, AY.2, AY.4 or AY.4.2.
在特别优选的实施方式中，第二方面的组合物适用于针对B.1.617.2的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for a SARS-CoV-2 vaccine against B.1.617.2.
在特别优选的实施方式中，第二方面的组合物适用于针对B.1.1.529、B.1.1.529/BA.1(Omicron)和/或B.1.1.529/BA.2的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for a SARS-CoV-2 vaccine against B.1.1.529, B.1.1.529/BA.1 (Omicron) and/or B.1.1.529/BA.2.
在特别优选的实施方式中，第二方面的组合物适用于针对BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4和/或BA.1_v5的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for SARS-CoV-2 vaccines against BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4 and/or BA.1_v5.
在特别优选的实施方式中，第二方面的组合物适用于针对B.1.1.529和B.1.617.2的SARS-CoV-2疫苗。In a particularly preferred embodiment, the composition of the second aspect is suitable for a SARS-CoV-2 vaccine against B.1.1.529 and B.1.617.2.
在本发明的情况中，“组合物”是指其中可以掺入指定的成分(例如，编码至少一种选自或源自SARS-CoV-2的抗原肽或蛋白质的RNA，例如，与基于脂质的载体结合)的任何类型的组合物，任选地与任何进一步的成分一起，通常是与至少一种药学上可接受的载体或赋形剂一起。组合物可以是干燥组合物，如粉末或颗粒，或固体单位如冻干形式。或者，组合物可以是液体形式，并且每种成分可以独立地以溶解或分散(例如，悬浮或乳化)的形式掺入。In the context of the present invention, a "composition" refers to any type of composition into which a specified component (e.g., an RNA encoding at least one antigenic peptide or protein selected from or derived from SARS-CoV-2, for example, in combination with a lipid-based carrier) can be incorporated, optionally together with any further components, typically together with at least one pharmaceutically acceptable carrier or excipient. The composition can be a dry composition, such as a powder or granules, or a solid unit such as a lyophilized form. Alternatively, the composition can be in liquid form, and each component can be incorporated independently in dissolved or dispersed (e.g., suspended or emulsified) form.
在第二方面的优选实施方式中，组合物包含至少一个第一方面的RNA，和任选地，至少一种药学上可接受的载体或赋形剂。In a preferred embodiment of the second aspect, the composition comprises at least one RNA of the first aspect, and optionally, at least one pharmaceutically acceptable carrier or excipient.
如本文使用的术语“药学上可接受的载体”或“药学上可接受的赋形剂”优选包括用于施用的组合物的液体或非液体基质。如果以液体形式提供组合物，载体可以是水，例如，无热原水；等渗盐水或缓冲的(水性)溶液，例如，磷酸盐、柠檬酸盐等缓冲溶液。可以使用水，或优选缓冲液，更优选水性缓冲液，其含有钠盐，优选至少50mM的钠盐、钙盐，优选至少0.01mM的钙盐，和任选地钾盐，优选至少3mM的钾盐。根据优选的实施方式，钠、钙和任选地钾盐可以以其卤化物的形式存在，例如，氯化物、碘化物或溴化物，以其氢氧化物、碳酸盐、碳酸氢盐或硫酸盐等的形式。钠盐的实例包括NaCl、NaI、NaBr、Na2CO3、NaHCO3、Na2SO4，任选的钾盐的实例包括KCl、KI、KBr、K2CO3、KHCO3、K2SO4，和钙盐的实例包括CaCl2、CaI2、CaBr2、CaCO3、CaSO4、Ca(OH)2。As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" preferably includes a liquid or non-liquid matrix for the composition to be administered. If the composition is provided in liquid form, the carrier can be water, for example, pyrogen-free water; isotonic saline or a buffered (aqueous) solution, for example, a buffer solution such as phosphate, citrate, etc. Water can be used, or preferably a buffer, more preferably an aqueous buffer, which contains a sodium salt, preferably at least 50mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt, and optionally a potassium salt, preferably at least 3mM of a potassium salt. According to a preferred embodiment, sodium, calcium and optionally potassium salts can be present in the form of their halides, for example, chlorides, iodides or bromides, in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include NaCl, NaI, NaBr,Na2CO3 ,NaHCO3 ,Na2SO4 , examples of optional potassium salts include KCl, KI, KBr,K2CO3,KHCO3 ,K2SO4, and examples of calcium salts includeCaCl2 ,CaI2 ,CaBr2,CaCO3 ,CaSO4 ,Ca (OH)2 .
此外，上述阳离子的有机阴离子可以在缓冲液中。因此，在实施方式中，核酸组合物可以包含药学上可接受的载体或赋形剂，其使用一种或多种药学上可接受的载体或赋形剂来例如增加稳定性、增加细胞转染、允许持续的或延迟的、增加所编码的冠状病毒蛋白在体内的翻译和/或改变所编码的冠状病毒蛋白在体内的释放特征。除了传统赋形剂，如任何和所有溶剂、分散介质、稀释剂或其他液体溶媒、分散或悬浮助剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂之外，本发明的赋形剂可以包括但不限于类脂、脂质体、脂质纳米颗粒、聚合物、脂质复合物、核-壳纳米颗粒、肽、蛋白质、用多核苷酸转染的细胞、透明质酸酶、纳米颗粒模拟物及其组合。在实施方式中，也可以使用一种或多种相容的固体或液体填充剂或稀释剂或包封化合物，其适合施用于受试者。如本文所用的术语“相容的”意指组合物的成分能够以不发生相互作用的方式与组合物的至少一种核酸和任选地多种核酸混合，所述相互作用在典型的使用条件(例如，肌内或皮内施用)下显著降低组合物的生物活性或药物有效性。药学上可接受的载体或赋形剂必须具有足够高的纯度和足够低的毒性，以使它们适于施用于待治疗的受试者。可用作药学上可接受的载体或赋形剂的化合物可以是糖，如，例如乳糖、葡萄糖、海藻糖、甘露糖和蔗糖；淀粉，如，例如玉米淀粉或马铃薯淀粉；右旋糖；纤维素及其衍生物，如，例如羧甲基纤维素钠、乙基纤维素、乙酸纤维素；粉末状黄蓍胶；麦芽；明胶；牛脂；固体助流剂，如，例如硬脂酸、硬脂酸镁；硫酸钙；植物油，如，例如花生油、棉籽油、芝麻油、橄榄油、玉米油和来自可可属植物的油；多元醇，如，例如聚丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸。In addition, the organic anion of the above-mentioned cation can be in buffer.Therefore, in an embodiment, nucleic acid composition can comprise pharmaceutically acceptable carrier or excipient, and it uses one or more pharmaceutically acceptable carrier or excipient to for example increase stability, increase cell transfection, allow lasting or delay, increase the translation of encoded coronavirus protein in vivo and/or change the release characteristics of encoded coronavirus protein in vivo.Except traditional excipients, such as any and all solvents, dispersion medium, diluent or other liquid solvents, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, excipients of the present invention can include but are not limited to lipid, liposome, lipid nanoparticle, polymer, lipid complex, core-shell nanoparticle, peptide, protein, cell, hyaluronidase, nanoparticle mimics and combination thereof with polynucleotide transfection.In an embodiment, one or more compatible solid or liquid fillers or diluents or encapsulated compounds can also be used, and it is suitable for being applied to the experimenter. The term "compatible" as used herein means that the components of the composition are capable of being mixed with at least one nucleic acid and optionally multiple nucleic acids of the composition in a manner that does not interact, which interaction significantly reduces the biological activity or pharmaceutical effectiveness of the composition under typical conditions of use (e.g., intramuscular or intradermal administration). Pharmaceutically acceptable carriers or excipients must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the subject to be treated. Compounds that can be used as pharmaceutically acceptable carriers or excipients can be sugars, such as, for example, lactose, glucose, trehalose, mannose and sucrose; starches, such as, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from the genus Theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
组合物的至少一种药学上可接受的载体或赋形剂可以优选选择为适用于肌内或皮内递送/施用所述组合物。因此，组合物优选是药物组合物，合适地用于肌内施用的组合物。At least one pharmaceutically acceptable carrier or excipient of the composition may preferably be selected to be suitable for intramuscular or intradermal delivery/administration of the composition. Thus, the composition is preferably a pharmaceutical composition, a composition suitable for intramuscular administration.
施用组合物(优选药物组合物)的受试者考虑包括但不限于人类和/或其他灵长类；哺乳动物，包括商业上相关的哺乳动物，如牛、猪、马、绵羊、猫、狗、小鼠和/或大鼠；和/或鸟类，包括商业上相关的鸟类，如家禽、鸡、鸭、鹅和/或火鸡。Subjects to which a composition (preferably a pharmaceutical composition) is administered are contemplated to include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese and/or turkeys.
本发明的药物组合物可以合适地是无菌的和/或无热原的。The pharmaceutical compositions of the invention may suitably be sterile and/or pyrogen-free.
本发明的多价组合物：The multivalent composition of the present invention:
在实施方式中，如本文定义的组合物(例如，多价组合物)可以包含多种或至少超过一种如本发明第一方面的情况中定义的RNA物质。优选地，如本文定义的组合物可以包含2、3、4、5、6、7、8、9或10种不同的RNA物质，各自第一方面的情况中定义。In an embodiment, a composition as defined herein (e.g., a multivalent composition) may comprise multiple or at least more than one RNA species as defined in the context of the first aspect of the invention. Preferably, a composition as defined herein may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNA species, each defined in the context of the first aspect.
在实施方式中，组合物(例如，多价组合物)包含至少2、3、4、5、6、7、8、9、10种或甚至更多种不同的第一方面的情况中定义的RNA物质，各自编码至少一个不同的SARS-CoV-2刺突蛋白(第一方面的情况中所定义的)。In an embodiment, a composition (e.g., a multivalent composition) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNA species as defined in the context of the first aspect, each encoding at least one different SARS-CoV-2 spike protein (as defined in the context of the first aspect).
在这种情况中，进一步优选的是不同的SARS-CoV-2刺突蛋白或融合前稳定的刺突蛋白具有包括以下的刺突蛋白中的氨基酸变化：In this case, it is further preferred that the different SARS-CoV-2 spike proteins or pre-fusion stabilized spike proteins have amino acid changes in the spike proteins that include:
·K986、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F；K986, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、K417N、N440K、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、A67V、T95I、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, T95I, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、T19I、L24del、P25del、P26del、A27S、G142D、V213G、G339D、S371F、S373P、S375F、T376A、D405N、S477N、T478K、E484A、Q493R、Q498R、N501Y、Y505H、D614G、H655Y、N679K、P681H、D796Y、Q954H和N969K；K986P, V987P, T19I, L24del, P25del, P26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, D796Y, Q954H, and N969K;
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、N440K、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, N440K, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、A701V、N764K、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, and L981F;
·K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F；K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V;
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V；E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y和T1027I；E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, and T1027I;
·E484K、N501Y、L18F、T20N、P26S、D138Y、R190S、K417T、D614G、H655Y、T1027I和V1176F；E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, T1027I, and V1176F;
·L452R、P681R和D614G；L452R, P681R and D614G;
·L452R、E484Q、P681R、E154K、D614G和Q1071H；或L452R, E484Q, P681R, E154K, D614G, and Q1071H; or
·L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N。L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.
在这种情况中，甚至更优选的是不同的SARS-CoV-2刺突蛋白或融合前稳定的刺突蛋白具有包含以下的刺突蛋白中的氨基酸改变：In this case, it is even more preferred that the different SARS-CoV-2 spike protein or the pre-fusion stabilized spike protein has amino acid changes in the spike protein comprising:
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、R246I、K417N、D614G和A701V；或E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I, K417N, D614G, and A701V; or
·E484K、N501Y、L18F、D80A、D215G、L242del、A243del、L244del、K417N、D614G和A701V。E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N, D614G, and A701V.
在这种情况中，甚至更优选的是不同SARS-CoV-2刺突蛋白或融合前稳定的刺突蛋白具有包含以下的刺突蛋白中的氨基酸改变：In this case, it is even more preferred that the different SARS-CoV-2 spike protein or the pre-fusion stabilized spike protein has amino acid changes in the spike protein comprising:
至少一个具有以下刺突蛋白中的氨基酸改变的SARS-CoV-2刺突蛋白或融合前稳定的刺突蛋白：A SARS-CoV-2 spike protein or prefusion stabilized spike protein with at least one of the following amino acid changes in the spike protein:
K986P、V987P、A67V、H69del、V70del、T95I、G142D、V143del、Y144del、Y145del、N211del、L212I、ins214EPE、G339D、S371L、S373P、S375F、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K和L981F；和K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; and
至少一个具有以下刺突蛋白中的氨基酸改变的SARS-CoV-2刺突蛋白或融合前稳定的刺突蛋白：A SARS-CoV-2 spike protein or prefusion stabilized spike protein with at least one of the following amino acid changes in the spike protein:
L452R、E484Q、P681R、E154K、D614G和Q1071H；或L452R, E484Q, P681R, E154K, D614G, and Q1071H; or
L452R、P681R、T19R、F157del、R158del、T478K、D614G和D950N。L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.
在优选的实施方式中，组合物(例如，多价组合物)包含2、3、4或5种RNA物质和任选地至少一种药学上可接受的载体或赋形剂，其中所述RNA物质包含与选自SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中2、3、4或5种核酸物质中的每一种编码不同的SARS-CoV-2刺突蛋白。In a preferred embodiment, the composition (e.g., a multivalent composition) comprises 2, 3, 4 or 5 RNA species and optionally at least one pharmaceutically acceptable carrier or excipient, wherein the RNA species comprises a polypeptide selected from the group consisting of SEQ ID NO: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences, or consist of, wherein each of 2, 3, 4 or 5 nucleic acid species encodes a different SARS-CoV-2 spike protein.
在优选的实施方式中，组合物(例如，多价组合物)包含2、3、4或5种RNA物质和任选至少一种药学上可接受的载体或赋形剂，其中所述RNA物质包含与选自SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成，其中2、3、4或5种核酸物质中的每一种编码不同的SARS-CoV-2刺突蛋白。In a preferred embodiment, the composition (e.g., a multivalent composition) comprises 2, 3, 4 or 5 RNA species and optionally at least one pharmaceutically acceptable carrier or excipient, wherein the RNA species comprises a polypeptide selected from the group consisting of SEQ ID NO:24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28937-28940, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences, or consisting of such nucleic acid sequences, wherein each of the 2, 3, 4 or 5 nucleic acid species encodes a different SARS-CoV-2 spike protein.
在下文中，提供了特别优选的多价组合物的实施方式。In the following, particularly preferred embodiments of the multivalent compositions are provided.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的进一步RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10, wherein the multivalent composition further comprises at least 2, 3, 4 further RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540; and/or
iii)一种RNA物质包含编码与SEQ ID NO:27093-27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558; and/or
vii)一种RNA物质包含编码与SEQ ID NO:27095任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 27095; and/or
viii)一种RNA物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。viii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:27093-27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的其他RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558, wherein the multivalent composition further comprises at least 2, 3, 4 other RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540; and/or
iii)一种RNA物质包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的其他RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558, wherein the multivalent composition further comprises at least 2, 3, 4 other RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540; and/or
iii)一种RNA物质包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:27095任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的其他RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 27095, wherein the multivalent composition further comprises at least 2, 3, 4 other RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540; and/or
iii)一种RNA物质包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的其他RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540, wherein the multivalent composition further comprises at least 2, 3, 4 other RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10; and/or
iii)一种RNA物质包含编码与SEQ ID NO:27093-27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558; and/or
vii)一种RNA物质包含编码与SEQ ID NO:27095任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 27095; and/or
viii)一种RNA物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。viii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
在优选的实施方式中，多价组合物包含一种RNA物质，其包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物另外包含至少2、3、4种选自以下的其他RNA物质：In a preferred embodiment, the multivalent composition comprises one RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920, wherein the multivalent composition further comprises at least 2, 3, 4 other RNA species selected from the group consisting of:
i)一种RNA物质包含编码与SEQ ID NO:27108-27109任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27108-27109; and/or
ii)一种RNA物质包含编码与SEQ ID NO:10任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10; and/or
iii)一种RNA物质包含编码与SEQ ID NO:27093-27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558; and/or
iv)一种RNA物质包含编码与SEQ ID NO:27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iv) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or
v)一种RNA物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或v) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959; and/or
vi)一种RNA物质包含编码与SEQ ID NO:27095、28552-28558任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vi) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558; and/or
vii)一种RNA物质包含编码与SEQ ID NO:27095任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或vii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 27095; and/or
viii)一种RNA物质包含编码与SEQ ID NO:22960-22961、28540任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。viii) an RNA species comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540.
在优选的实施方式中，组合物，优选多价组合物，适用于针对C.1.2(南非)、B.1.1.529(Omicron，南非)(包括BA.1_v1、BA.1_v0、B.1.1.529、BA.2、BA.1_v2、BA.1_v3、BA.1_v4、BA.1_v5)、C.36.3(泰国)、B.1.619(喀麦隆)、R.1(肯塔基州，US)、B.1.1.176(加拿大)、AZ.3、AY.1(印度)、AY.2(印度)、AY.4(印度)、AY.4.2(Delta Plus，印度)、B.1.617.3(印度)、B.1.351(Beta，南非)、B.1.1.7(Alpha，UK)、P.1(Gamma，巴西)、B.1.427/B.1.429(Epsilon，加利福尼亚，US)、B.1.525(Eta，尼日利亚)、B.1.258(捷克共和国)、B.1.526(Jota，纽约，US)、A.23.1(乌干达)、B.1.617.1(Kappa，印度)、B.1.617.2(Delta，印度)、P.2(Zeta，巴西)、C37.1(Lambda，秘鲁)、P.3(Theta，菲律宾)和/或B.1.621(Mu，哥伦比亚)的疫苗。In a preferred embodiment, the composition, preferably a multivalent composition, is suitable for use against C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czech Republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru), P.3 (Theta, Philippines) and/or B.1.621 (Mu, Colombia).
在实施方式中，作为包含在组合物中的RNA以约100ng至约500ug的量、以约1ug至约200ug的量、以约1ug至约100ug的量、以约5ug至约100ug的量，优选以约10ug至约50ug的量，具体地，以约1ug、2ug、3ug、4ug、5ug、6ug、7ug、8ug、9ug、10ug、11ug、12ug、13ug、14ug、15ug、20ug、25ug、30ug、35ug、40ug、45ug、50ug、55ug、60ug、65ug、70ug、75ug、80ug、85ug、90ug、95ug或100ug的量来提供。In an embodiment, the RNA contained in the composition is provided in an amount of about 100 ng to about 500 ug, in an amount of about 1 ug to about 200 ug, in an amount of about 1 ug to about 100 ug, in an amount of about 5 ug to about 100 ug, preferably in an amount of about 10 ug to about 50 ug, specifically, in an amount of about 1 ug, 2 ug, 3 ug, 4 ug, 5 ug, 6 ug, 7 ug, 8 ug, 9 ug, 10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 20 ug, 25 ug, 30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80 ug, 85 ug, 90 ug, 95 ug or 100 ug.
在组合物包含多种或至少超过一种如本文定义的RNA物质(多价组合物)的情况下，用于每种RNA物质的RNA的量以约100ng至约500ug的量、以约1ug至约200ug的量、以约1ug至约100ug的量、以约5ug至约100ug的量，优选以约10ug至约50ug的量，具体地，以约1ug、2ug、3ug、4ug、5ug、6ug、7ug、8ug、9ug、10ug、11ug、12ug、13ug、14ug、15ug、20ug、25ug、30ug、35ug、40ug、45ug、50ug、55ug、60ug、65ug、70ug、75ug、80ug、85ug、90ug、95ug或100ug的量来提供。In the case where the composition comprises a plurality of or at least more than one RNA species as defined herein (multivalent composition), the amount of RNA for each RNA species is provided in an amount of about 100 ng to about 500 ug, in an amount of about 1 ug to about 200 ug, in an amount of about 1 ug to about 100 ug, in an amount of about 5 ug to about 100 ug, preferably in an amount of about 10 ug to about 50 ug, specifically, in an amount of about 1 ug, 2 ug, 3 ug, 4 ug, 5 ug, 6 ug, 7 ug, 8 ug, 9 ug, 10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 20 ug, 25 ug, 30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80 ug, 85 ug, 90 ug, 95 ug or 100 ug.
在一些实施方式中，用于每种RNA物质的RNA的量在质量上基本上相等。在其他实施方式中，用于每种RNA物质的量选择为等摩尔的。In some embodiments, the amount of RNA used for each RNA species is substantially equal in quality. In other embodiments, the amount used for each RNA species is selected to be equimolar.
复合complex
在第二方面的优选实施方式中，至少一种RNA，优选至少一种mRNA，与进一步的化合物复合或结合，以获得复合的配制组合物。复合的制剂可以具有转染剂的功能。复合的配制组合物还可以具有保护RNA和/或mRNA免于降解的功能。In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, is compounded or combined with a further compound to obtain a composite formulation composition. The composite formulation may function as a transfection agent. The composite formulation composition may also function to protect RNA and/or mRNA from degradation.
在第二方面的优选实施方式中，至少一种RNA，优选至少一种mRNA，和任选至少一种另外的RNA，与一种或多种阳离子或多阳离子化合物(优选阳离子或多阳离子聚合物、阳离子或多阳离子多糖、阳离子或多阳离子脂质、阳离子或多阳离子蛋白质、阳离子或多阳离子肽或其任意组合)复合或结合，或至少部分复合或部分结合。In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, and optionally at least one additional RNA, is complexed or bound, or at least partially complexed or partially bound, to one or more cationic or polycationic compounds (preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides or any combination thereof).
如本文使用的术语“阳离子或多阳离子化合物”被本领域普通技术人员认识和理解，并且例如旨在表示带电荷的分子，其在约1至9范围的pH值，约3至8范围的pH值，约4至8范围的pH值，约5至8范围的pH值，更优选约6至8范围的pH值，甚至更优选约7至8范围的pH值，最优选在生理pH下，例如，约7.2至约7.5的范围下是带正电荷的。因此，阳离子组分，例如，阳离子肽、阳离子蛋白质、阳离子聚合物、阳离子多糖、阳离子脂质，可以是任何带正电的化合物或在生理条件下带正电的聚合物。“阳离子或多阳离子肽或蛋白质”可以含有至少一个带正电荷的氨基酸，或超过一个带正电荷的氨基酸，例如，选自Arg、His、Lys或Orn。因此，在给定条件下呈现超过一个正电荷的“多阳离子”组分也在该范围内。The term "cationic or polycationic compound" as used herein is recognized and understood by those of ordinary skill in the art, and is intended, for example, to mean a charged molecule that is positively charged at a pH value in the range of about 1 to 9, a pH value in the range of about 3 to 8, a pH value in the range of about 4 to 8, a pH value in the range of about 5 to 8, more preferably a pH value in the range of about 6 to 8, even more preferably a pH value in the range of about 7 to 8, and most preferably at physiological pH, for example, in the range of about 7.2 to about 7.5. Thus, a cationic component, for example, a cationic peptide, a cationic protein, a cationic polymer, a cationic polysaccharide, a cationic lipid, can be any positively charged compound or a polymer that is positively charged under physiological conditions. A "cationic or polycationic peptide or protein" can contain at least one positively charged amino acid, or more than one positively charged amino acid, for example, selected from Arg, His, Lys or Orn. Thus, a "polycationic" component that exhibits more than one positive charge under given conditions is also within the scope.
在这种情况中特别优选的阳离子或多阳离子化合物可以选自以下阳离子或多阳离子肽或蛋白质或其片段的列表：鱼精蛋白、核仁蛋白、精胺或亚精胺，或其他阳离子肽或蛋白质，如聚-L-赖氨酸(PLL)、聚精氨酸、碱性多肽、细胞穿透肽(CPP)，包括HIV结合肽、HIV-1Tat(HIV)、Tat衍生肽、渗透蛋白、VP22衍生或类似肽、HSV VP22(单纯疱疹)、MAP、KALA或蛋白质转导结构域(PTD)、PpT620，富含脯氨酸的肽、富含精氨酸的肽、富含赖氨酸的肽、MPG肽、Pep-1、L-寡聚物、降钙素肽、触足衍生肽、pAntp、pIsl、FGF、乳铁蛋白、Transportan、Buforin-2、Bac715-24、SynB、SynB(1)、pVEC、hCT衍生肽、SAP或组蛋白。更优选，核酸(例如DNA或RNA)，例如编码RNA，优选mRNA，与一种或多种聚阳离子复合，优选与鱼精蛋白或oligofectamine复合，最优选与鱼精蛋白复合。Particularly preferred cationic or polycationic compounds in this context may be selected from the following list of cationic or polycationic peptides or proteins or fragments thereof: protamine, nucleolar protein, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetrating peptides (CPP), including HIV binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, permeabilin, VP22-derived or similar peptides, HSV VP22 (herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptides, Pep-1, L-oligomers, calcitonin peptides, antennapedia-derived peptides, pAntp, pIsl, FGF, lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP or histones. More preferably, the nucleic acid (eg DNA or RNA), eg coding RNA, preferably mRNA, is complexed with one or more polycations, preferably with protamine or oligofectamine, most preferably with protamine.
可以用作转染或复合剂的进一步优选的阳离子或多阳离子化合物可以包括阳离子多糖，例如壳聚糖、聚凝胺(polybrene)等；阳离子脂质，例如DOTMA、DMRIE、二-C14-脒、DOTIM、SAINT、DC-Chol、BGTC、CTAP、DOPC、DODAP、DOPE：二油基磷脂酰乙醇胺、DOSPA、DODAB、DOIC、DMEPC、DOGS、DIMRI、DOTAP、DC-6-14、CLIP1、CLIP6、CLIP9、oligofectamine；或阳离子或多阳离子聚合物，例如改性的聚氨基酸，如β-氨基酸聚合物或反向聚酰胺等，改性的聚乙烯，如PVP等，改性的丙烯酸酯，如pDMAEMA等，改性的酰胺基胺，如pAMAM等，改性聚β-氨基酯(PBAE)，如二胺末端改性的1,4-丁二醇二丙烯酸酯-共-5-氨基-1-戊醇聚合物等，树枝状聚合物，如聚丙基胺树枝状聚合物或基于pAMAM的树枝状聚合物等，聚酰亚胺，如PEI、聚(丙烯亚胺)等，聚烯丙基胺，基于糖主链的聚合物，如基于环糊精的聚合物、基于葡聚糖的聚合物等，基于硅烷骨架的聚合物，如PMOXA-PDMS共聚物等，由一个或多个阳离子嵌段(例如选自如上所述的阳离子聚合物)和一个或多个亲水或疏水嵌段(如聚乙二醇)的组合组成的嵌段聚合物；等。Further preferred cationic or polycationic compounds that can be used as transfection or complexing agents can include cationic polysaccharides, such as chitosan, polybrene, etc.; cationic lipids, such as DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or polycationic polymers, such as modified polyamino acids, such as β-amino acid polymers or reverse polyamides, modified polyethylene, etc. Such as PVP, modified acrylates, such as pDMAEMA, modified amidoamines, such as pAMAM, modified poly β-amino esters (PBAE), such as diamine-terminated 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers, etc., dendritic polymers, such as polypropylamine dendritic polymers or pAMAM-based dendritic polymers, etc., polyimides, such as PEI, poly(propyleneimine), etc., polyallylamine, polymers based on sugar backbones, such as cyclodextrin-based polymers, dextran-based polymers, etc., polymers based on silane backbones, such as PMOXA-PDMS copolymers, etc., block polymers composed of a combination of one or more cationic blocks (e.g., selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (such as polyethylene glycol); etc.
根据各种不同实施方式，本发明的组合物包含至少一个第一方面情况中定义的RNA，优选至少一个mRA，和聚合载体。According to various embodiments, the composition of the present invention comprises at least one RNA as defined in the first aspect, preferably at least one mRA, and a polymer carrier.
如本文使用的术语“聚合载体”被本领域普通技术人员认识和理解，并且例如旨在表示促进另一种化合物(例如，负载核酸)转运和/或复合的化合物。聚合载体通常是由聚合物形成的载体。聚合载体可以通过共价或非共价相互作用与其货物(例如，DNA或RNA)结合。聚合物可以基于不同的亚单元，如共聚物。The term "polymeric carrier" as used herein is recognized and understood by those of ordinary skill in the art and is intended, for example, to refer to a compound that facilitates the transport and/or complexation of another compound (e.g., a loaded nucleic acid). A polymeric carrier is typically a carrier formed of a polymer. A polymeric carrier can be bound to its cargo (e.g., DNA or RNA) by covalent or non-covalent interactions. Polymers can be based on different subunits, such as copolymers.
在该情况中合适的聚合载体可以包括，例如，聚丙烯酸酯、聚烷基氰基丙烯酸酯、聚乳酸、聚乳酸-聚乙交酯共聚物、聚己内酯、右旋糖酐、白蛋白、明胶、藻酸盐、胶原蛋白、壳聚糖、环糊精、鱼精蛋白、PEG化鱼精蛋白、PEG化PLL和聚乙烯亚胺(PEI)、二硫代双(琥珀酰亚胺基丙酸酯)(DSP)、二甲基-3,3’-二硫代双丙亚胺酸酯(DTBP)、聚(乙烯亚胺)双氨基甲酸酯(PEIC)，聚(L-赖氨酸)(PLL)、组氨酸修饰的PLL、聚(N-乙烯基吡咯烷酮))(PVP)、聚(丙烯亚胺)(PPI)、聚(酰胺胺)(PAMAM)、聚(酰胺基乙烯亚胺)(SS-PAEI)、三亚乙基四胺(TETA)、聚(β-氨基酯)、聚(4-羟基-L-脯氨酸酯)(PHP)、聚(烯丙胺)、聚(α-[4-氨基丁基]-L-乙醇酸)(PAGA)、聚(D,L-乳酸-共-乙醇酸)(PLGA)、聚(N-乙基-4-乙烯基溴化吡啶)、聚(磷腈)(PPZ)、聚(磷酸酯)(PPE)、聚(磷酰胺)(PPA)、聚(N-2-羟基丙基甲基丙烯酰胺)(pHPMA)、聚(2-(二甲基氨基)乙基甲基丙烯酸酯)(pDMAEMA)、聚(2-氨基乙基丙烯磷酸酯)(PPE_EA)、半乳糖基化壳聚糖、N-十二烷基化壳聚糖、组蛋白、胶原蛋白和右旋糖酐-精胺。在一个实施方式中，聚合物可以是惰性聚合物，如但不限于PEG。在一个实施方式中，聚合物可以是阳离子聚合物，如但不限于PEI、PLL、TETA、聚(烯丙胺)、聚(N-乙基-4-乙烯基溴化吡啶)、pHPMA和pDMAEMA。在一个实施方式中，聚合物可以是可生物降解的PEI，如但不限于DSP、DTBP和PEIC。在一个实施方式中，聚合物可以是可生物降解的，如但不限于组氨酸修饰的PLL、SS-PAEI、聚(β-氨基酯)、PHP、PAGA、PLGA、PPZ、PPE、PPA和PPE-EA。Suitable polymeric carriers in this case may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactic acid, polylactic acid-co-glycolide, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, protamine, PEGylated protamine, PEGylated PLL and polyethyleneimine (PEI), dithiobis(succinimidyl propionate) (DSP), dimethyl-3,3'-dithiobispropionimidate (DTBP), poly(ethyleneimine) biscarbamate (PEIC), poly(L-lysine) (PLL), histidine-modified PLL, poly(N-vinylpyrrolidone)) (PVP), poly(propyleneimine) (PPI), poly(amidoamine) (PAMAM), poly(amidoethyleneimine) (SS-P AEI), triethylenetetramine (TETA), poly(β-amino ester), poly(4-hydroxy-L-proline ester) (PHP), poly(allylamine), poly(α-[4-aminobutyl]-L-glycolic acid) (PAGA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene) (PPZ), poly(phosphoester) (PPE), poly(phosphamide) (PPA), poly(N-2-hydroxypropylmethacrylamide) (pHPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethyl propylene phosphate) (PPE_EA), galactosylated chitosan, N-dodecyl chitosan, histones, collagen, and dextran-spermine. In one embodiment, the polymer can be an inert polymer such as, but not limited to, PEG. In one embodiment, the polymer can be a cationic polymer, such as, but not limited to, PEI, PLL, TETA, poly(allylamine), poly(N-ethyl-4-vinylpyridinium bromide), pHPMA, and pDMAEMA. In one embodiment, the polymer can be a biodegradable PEI, such as, but not limited to, DSP, DTBP, and PEIC. In one embodiment, the polymer can be biodegradable, such as, but not limited to, histidine-modified PLL, SS-PAEI, poly(β-amino esters), PHP, PAGA, PLGA, PPZ, PPE, PPA, and PPE-EA.
在LNP中的包封/复合Encapsulation/complexation in LNPs
在第二方面的优选实施方式中，至少一种RNA，优选至少一种mRNA，和任选至少一种另外的RNA，与一种或多种脂质(例如，阳离子脂质和/或中性脂质)复合、包封、部分包封或结合，由此形成基于脂质的载体，如脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体。In a preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, and optionally at least one additional RNA, is complexed, encapsulated, partially encapsulated or associated with one or more lipids (e.g., cationic lipids and/or neutral lipids), thereby forming a lipid-based carrier such as a liposome, lipid nanoparticle (LNP), lipoplex and/or nanoliposome.
脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体-掺入RNA可以完全或部分位于脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体的内部空间中，位于脂质层/膜内，或与脂质层/膜的外表面缔合。The liposome, lipid nanoparticle (LNP), lipoplex and/or nanoliposome-incorporated RNA can be completely or partially located in the interior space of the liposome, lipid nanoparticle (LNP), lipoplex and/or nanoliposome, located within the lipid layer/membrane, or associated with the outer surface of the lipid layer/membrane.
将RNA掺入脂质体/LNP中在本文也称为“包封”，其中RNA完全包含在脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体的内部空间中。将RNA掺入脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体中的目的是保护RNA免受可能含有降解核酸的酶或化学物质或条件和/或引起核酸快速分泌的系统或受体的影响。此外，将RNA掺入脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体中可以促进RNA的摄取，并且因此可以增强编码抗原性SARS-CoV-2刺突蛋白的RNA的治疗效果。因此，将至少一种RNA掺入脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体中可能特别适用于SARS-CoV-2疫苗，例如，用于肌内和/或皮内施用。Incorporation of RNA into liposomes/LNPs is also referred to herein as "encapsulation", wherein the RNA is completely contained within the interior space of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes. The purpose of incorporating RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes is to protect the RNA from enzymes or chemicals or conditions that may degrade nucleic acids and/or systems or receptors that cause rapid secretion of nucleic acids. In addition, incorporation of RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes can promote the uptake of RNA, and thus can enhance the therapeutic effect of RNA encoding antigenic SARS-CoV-2 spike protein. Therefore, incorporation of at least one RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes may be particularly suitable for SARS-CoV-2 vaccines, for example, for intramuscular and/or intradermal administration.
在这种情况中，术语“复合的”或“结合的”是指RNA与一种或多种脂质在没有共价结合的情况下基本上稳定地组合成较大的复合物或组装物。In this context, the term "complexed" or "associated" refers to the substantial stable association of the RNA with one or more lipids into a larger complex or assembly without covalent bonding.
术语“脂质纳米颗粒”，也称为“LNP”，不限于任何颗粒形态，并且包括阳离子脂质和任选一种或多种其他脂质例如在水性环境中和/或在RNA存在下组合时生成的任何形态。例如，脂质体、脂质复合体、脂质复合物等在脂质纳米颗粒(LNP)的范围内。The term "lipid nanoparticle", also referred to as "LNP", is not limited to any particle morphology, and includes any morphology generated when a cationic lipid and optionally one or more other lipids are combined, for example, in an aqueous environment and/or in the presence of RNA. For example, liposomes, lipoplexes, lipoplexes, etc. are within the scope of lipid nanoparticles (LNP).
脂质体、脂质纳米颗粒(LNP)、脂质复合物和/或纳米脂质体可以是不同大小的，如，但不限于，多层囊泡(MLV)，其可以是数百纳米的直径并且可以含有一系列由窄的水性隔室分开的同心双层，小的单室囊泡(SUV)，其直径可以小于50nm，和大的单层囊泡(LUV)，其直径可以为50nm至500nm。Liposomes, lipid nanoparticles (LNPs), lipoplexes and/or nanoliposomes can be of different sizes, such as, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be 50 nm to 500 nm in diameter.
本发明的LNP可以适当地表征为具有通过一个或多个双层的膜与外部介质隔离的内部水空间的微观囊泡。LNP的双层膜通常由两亲性分子如合成或天然来源的脂质形成，其包括空间分离的亲水性和疏水性结构域。脂质体的双层膜也可以由亲两性聚合物和表面活性剂(例如，聚合物泡囊、泡囊等)形成。在本发明的情况中，LNP通常用于将至少一种RNA运输到靶组织。LNP of the present invention can be suitably characterized as the microscopic vesicle of the internal water space that is isolated from the external medium by one or more double-layered membranes.The double-layer membrane of LNP is usually formed by amphipathic molecules such as synthetic or natural lipids, which include spatially separated hydrophilic and hydrophobic domains.The double-layer membrane of liposome can also be formed by amphipathic polymers and surfactants (e.g., polymer vesicles, vesicles, etc.).In the case of the present invention, LNP is usually used for at least one RNA transport to target tissue.
因此，在第二方面的优选实施方式中，至少一个RNA与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)。优选地，所述LNP特别适用于肌内和/或皮内施用。LNP通常包含阳离子脂质和一种或多种选自中性脂质、带电荷的脂质、类固醇和聚合物缀合的脂质(例如，PEG化的脂质)的赋形剂。至少一个RNA可以包封在LNP的脂质部分或由LNP的一些或整个脂质部分包围的水性空间中。RNA或其一部分还可以与LNP缔合和复合。LNP可以包含能够形成RNA与其连接的颗粒的任何脂质，或其中包封一种或多种RNA物质。优选地，包含RNA的LNP包含一种或多种阳离子脂质，和一种或多种稳定脂质。稳定脂质包括中性脂质和PEG化的脂质。Therefore, in a preferred embodiment of the second aspect, at least one RNA is complexed with one or more lipids to form lipid nanoparticles (LNPs). Preferably, the LNP is particularly suitable for intramuscular and/or intradermal administration. LNP generally comprises cationic lipids and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer-conjugated lipids (e.g., PEGylated lipids). At least one RNA can be encapsulated in the lipid portion of LNP or in an aqueous space surrounded by some or all of the lipid portions of LNP. RNA or a portion thereof can also associate and compound with LNP. LNP can comprise any lipid that can form a particle to which RNA is connected, or wherein encapsulates one or more RNA materials. Preferably, the LNP comprising RNA comprises one or more cationic lipids, and one or more stable lipids. Stable lipids include neutral lipids and PEGylated lipids.
优选地，本发明的LNP包含Preferably, the LNP of the present invention comprises
(i)至少一种阳离子脂质；(i) at least one cationic lipid;
(ii)至少一种中性脂质；(ii) at least one neutral lipid;
(iii)至少一种类固醇或类固醇类似物，优选胆固醇；和(iii) at least one steroid or steroid analog, preferably cholesterol; and
(iv)至少一种聚合物缀合的脂质，优选PEG-脂质；(iv) at least one polymer-conjugated lipid, preferably a PEG-lipid;
其中(i)至(iv)的摩尔比为约20-60％阳离子脂质、5-25％中性脂质、25-55％固醇和0.5-15％聚合物缀合的脂质。wherein the molar ratio of (i) to (iv) is about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol and 0.5-15% polymer-conjugated lipid.
LNP的阳离子脂质可以是可阳离子化的，即，随着pH降至低于脂质的可离子化基团的pK，其变成质子化的，但在较高pH值下逐渐变得更中性。在低于pK的pH值下，脂质随后能够与带负电荷的核酸缔合。在某些实施方式中，阳离子脂质包含在pH降低时呈正电荷的两性离子脂质。The cationic lipid of LNP can be cationizable, that is, as the pH drops to below the pK of the ionizable group of the lipid, it becomes protonated, but gradually becomes more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged when the pH is reduced.
这样的脂质包括，但不限于，DSDMA、N,N-二油烯基-N,N-二甲基氯化铵(DODAC)、N,N-二硬脂酰基-N,N-二甲基溴化铵(DDAB)、1,2-二油酰基三甲基丙烷氯化铵(DOTAP)(也称为N-(2,3-二油酰氧基)丙基)-N,N,N-三甲基氯化铵和1,2-二油酰氧基-3-三甲基氨基丙烷氯化物盐)、N-(1-(2,3-二油酰氧基)丙基)-N,N,N-3-三甲基氯化铵(DOTMA)、N,N-二甲基-2,3-二油酰氧基)丙胺(DODMA)、ckk-E12、ckk、1,2-二亚油酰氧基-N,N-二甲基氨基丙烷(DLinDMA)、1,2-二亚麻酰氧基-N,N-二甲基氨基丙烷(DLenDMA)、1,2-二-γ-亚麻酰氧基-N,N-二甲基氨基丙烷(γ-DLenDMA)、98N12-5、1,2-二亚油基氨基甲酰氧基-3-二甲基氨基丙烷(DLin-C-DAP)、1,2-二亚油酰氧基-3-(二甲基氨基)乙酰氧基丙烷(DLin-DAC)、1,2-二亚油酰氧基-3-吗啉丙烷(DLin-MA)、1,2-二亚油酰基-3-二甲基氨基丙烷(DLinDAP)、1,2-二亚油酰基硫代-3-二甲基氨基丙烷(DLin-S-DMA)、1-亚油酰基-2-亚油酰氧基-3-二甲基氨基丙烷(DLin-2-DMAP)、1,2-二亚油酰氧基-3-三甲基氨基丙烷氯化物盐(DLin-TMA.Cl)、ICE(基于咪唑的)、HGT5000、HGT5001、DMDMA、CLinDMA、CpLinDMA、DMOBA、DOcarbDAP、DLincarbDAP、DLinCDAP、KLin-K-DMA、DLin-K-XTC2-DMA、XTC(2,2-二亚油基-4-二甲基氨乙基-[1,3]-二氧戊环)HGT4003、1,2-二亚油酰基-3-三甲基氨基丙烷氯化物盐(DLin-TAP.Cl)、1,2-二亚油酰氧基-3-(N-甲基哌嗪基)丙烷(DLin-MPZ)或3-(N,N-二亚油基氨基)-1,2-丙二醇(DLinAP)、3-(N,N-二油烯基氨基)-1,2-丙二醇(DOAP)、1,2-二亚油基氧代-3-(2-N,N-二甲基氨基)乙氧基丙烷(DLin-EG-DMA)、2,2-二亚油基-4-二甲基氨甲基-[1,3]-二氧戊环(DLin-K-DMA)或其类似物、(3aR,5s,6aS)-N,N-二甲基-2,2-二((9Z,12Z)-十八碳-9,12-二烯基)四氢-3aH-环戊并[d][1,3]二氧杂环戊烯-5-胺、(6Z,9Z,28Z,31Z)-三十七碳-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(MC3)、ALNY-100((3aR,5s,6aS)-N,N-二甲基-2,2-二((9Z,12Z)-十八碳-9,12-二烯基)四氢-3aH-环戊并[d][1,3]二氧杂环戊烯-5-胺))、1,1’-(2-(4-(2-((2-(双(2-羟基十二烷基)氨基)乙基)(2-羟基十二烷基)氨基)乙基)哌嗪-1-基)乙基氮烷二基)双十二烷-2-醇(C12-200)、2,2-二亚油基-4-(2-二甲基氨乙基)-[1,3]-二氧戊环(DLin-K-C2-DMA)、2,2-二亚油基-4-二甲基氨甲基-[1,3]-二氧戊环(DLin-K-DMA)、NC98-5(4,7,13-三(3-氧代-3-(十一烷基氨基)丙基)-N1,N16-双十一烷基-4,7,10,13-四氮杂十六烷-1,16-二酰胺)、(6Z,9Z,28Z,31Z)-三十七碳-6,9,28,31-四烯-19-基-4-(二甲基氨基)丁酸酯(DLin-M-C3-DMA)、3-((6Z,9Z,28Z,31Z)-三十七碳-6,9,28,31-四烯-19-基氧基)-N,N-二甲基丙-1-胺(MC3醚)、4-((6Z,9Z,28Z,31Z)-三十七碳-6,9,28,31-四烯-19-基氧基)-N,N-二甲基丁-1-胺(MC4醚)、(可商购的阳离子脂质体，其包含DOTMA和1,2-二油酰基-sn-3-磷酸乙醇胺，来自GIBCO/BRL，Grand Island，N.Y.)；(可商购的阳离子脂质体，其包含N-(1-(2,3-二油酰氧基)丙基)-N-(2-(精胺羧酰胺基)乙基)-N,N-二甲基三氟乙酸铵(DOSPA)和(DOPE)，来自GIBCO/BRL)；和(可商购的阳离子脂质，其包含乙醇中的双十八烷基酰胺基甘氨酰基羧精胺(DOGS)，来自Promega Corp.，Madison，Wis.)或上述任何脂质的任一组合。其他用于本发明的组合物和方法的合适阳离子脂质包括国际专利申请公开WO2010/053572(且特别地，第[00225]段所述的CI 2-200)和WO2012/170930中所述的那些，二者在此按引用并入，HGT4003、HGT5000、HGTS001、HGT5001、HGT5002(参见US20150140070A1)。Such lipids include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylpropane ammonium chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleoyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-3-trimethylammonium chloride (DOTMA), N,N-dimethyl 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-γ-linolenoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleoyloxy-3-(dimethylamino)acetoxy Propane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), ICE (imidazole-based), HGT5000, HGT500 1. DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003, 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ) or 3- (N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH -cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriacont ... (2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1 ,16-diamide), (6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-triacontria-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine from GIBCO/BRL, Grand Island, NY); (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO/BRL); and (commercially available cationic lipids comprising dioctadecylamido glycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the above lipids. Other suitable cationic lipids for the compositions and methods of the present invention include those described in International Patent Application Publication WO2010/053572 (and in particular, CI 2-200 described in paragraph [00225]) and WO2012/170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1).
在多种实施方式中，阳离子脂质可以是氨基脂质。In various embodiments, the cationic lipid can be an amino lipid.
代表性氨基脂质包括，但不限于，1,2-二亚油酰氧基-3-(二甲基氨基)乙酰氧基丙烷(DLin-DAC)、1,2-二亚油酰氧基-3-吗啉丙烷(DLin-MA)、1,2-二亚油酰基-3-二甲基氨基丙烷(DLinDAP)、1,2-二亚油基硫代-3-二甲基氨基丙烷(DLin-S-DMA)、1-亚油酰基-2-亚油酰氧基-3-二甲基氨基丙烷(DLin-2-DMAP)、1,2-二亚油酰氧基-3-三甲基氨基丙烷氯化物盐(DLin-TMA.Cl)、1,2-二亚油酰基-3-三甲基氨基丙烷氯化物盐(DLin-TAP.Cl)、1,2-二亚油酰氧基-3-(N-甲基哌嗪基)丙烷(DLin-MPZ)、3-(N,N-二亚油基氨基)-1,2-丙二醇(DlinAP)、3-(N,N-二油烯基氨基)-1,2-丙二醇(DOAP)、1,2-二亚油基氧代-3-(2-N,N-二甲基氨基)乙氧基丙烷(DLin-EG-DMA)和2,2-二亚油基-4-二甲基氨甲基-[1,3]-二氧戊环(DLin-K-DMA)、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧戊环(DLin-KC2-DMA)；二亚油基-甲基-4-二甲基氨基丁酸酯(DLin-MC3-DMA)；MC3(US20100324120)。Representative amino lipids include, but are not limited to, 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-trimethylaminopropane (DLin-S-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane (DLin-TMA.Cl), 1,2-dilinoleoyloxy-3-trimethylaminopropane (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-trimethylaminopropane (DLin-TAP.Cl). (N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DlinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA) and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).
在多种实施方式中，阳离子脂质可以是氨基醇类脂。In various embodiments, the cationic lipid can be an amino alcohol lipid.
可以用于本发明中的氨基醇类脂可以通过美国专利No.8,450,298中所述的方法来制备，在此将其全部按引用并入。合适的(可离子化)脂质也可以是WO2017/075531A1的表1、2和3中公开的以及权利要求1-24中限定的化合物，在此按引用并入。Amino alcohol lipids that can be used in the present invention can be prepared by the method described in U.S. Patent No. 8,450,298, which is incorporated herein by reference in its entirety. Suitable (ionizable) lipids can also be compounds disclosed in Tables 1, 2 and 3 of WO2017/075531A1 and defined in Claims 1-24, which are incorporated herein by reference.
在另一个实施方式中，合适的脂质也可以是如WO2015/074085A1(即，ATX-001至ATX-032或权利要求1-26中具体说明的化合物)、美国申请No.61/905,724和15/614,499或美国专利No.9,593,077和9,567,296中公开的化合物，在此将其全部按引用并入。In another embodiment, suitable lipids may also be compounds as disclosed in WO2015/074085A1 (i.e., ATX-001 to ATX-032 or compounds specified in claims 1-26), U.S. Application Nos. 61/905,724 and 15/614,499, or U.S. Patent Nos. 9,593,077 and 9,567,296, all of which are incorporated herein by reference.
在其他实施方式中，合适的阳离子脂质也可以是如WO2017/117530A1中公开的化合物(即，脂质13、14、15、16、17、18、19、20或权利要求中具体说明的化合物)，在此将其全部按引用并入。In other embodiments, suitable cationic lipids may also be compounds as disclosed in WO2017/117530A1 (ie, lipids 13, 14, 15, 16, 17, 18, 19, 20 or compounds specifically described in the claims), which are all incorporated herein by reference.
在优选的实施方式中，可离子化或阳离子脂质也可以选自WO2018/0708053A1中公开的脂质(即衍生自WO2018/078053A1的式I、II和III的脂质，或WO2018/0780053A1的权利要求1至12中具体说明的脂质)，在此将WO2018/0788053A1的公开内容全部按引用并入。在该情况下，WO2018/0708053A1的表7中公开的脂质(例如衍生自式I-1至I-41的脂质)和WO2018/078053A1的表8中公开的脂质(例如衍生自式II-1至II-36的脂质)可以适当地用于本发明的情况中。因此，WO2018/078053A1的式I-1至式I-41和式II-1至式II-36及与其相关的具体公开内容在此按引用并入。In a preferred embodiment, the ionizable or cationic lipid may also be selected from the lipids disclosed in WO2018/0708053A1 (i.e., lipids derived from formulas I, II and III of WO2018/078053A1, or lipids specifically described in claims 1 to 12 of WO2018/0780053A1), the disclosure of which is incorporated herein by reference in its entirety. In this case, lipids disclosed in Table 7 of WO2018/0708053A1 (e.g., lipids derived from formulas I-1 to I-41) and lipids disclosed in Table 8 of WO2018/078053A1 (e.g., lipids derived from formulas II-1 to II-36) may be appropriately used in the context of the present invention. Therefore, Formula I-1 to Formula I-41 and Formula II-1 to Formula II-36 of WO2018/078053A1 and the specific disclosures related thereto are incorporated herein by reference.
在优选的实施方式中，阳离子脂质可衍生自公开的PCT专利申请WO2018/078053A1的式III。因此，WO2018/078053A1的式III及与其相关的具体公开内容在此按引用并入。In a preferred embodiment, the cationic lipid can be derived from Formula III of the published PCT patent application WO2018/078053A1. Therefore, Formula III of WO2018/078053A1 and the specific disclosures related thereto are incorporated herein by reference.
在特别优选的实施方式中，组合物的至少一种RNA，优选至少一种mRNA，与一种或多种脂质复合，由此形成LNP，其中LNP的阳离子脂质选自公开的PCT专利申请WO2018/078053A1的表9的结构III-1至III-36。因此，WO2018/078053A1的结构III-1至III-36及与其相关的具体公开内容在此按引用并入。In a particularly preferred embodiment, at least one RNA, preferably at least one mRNA, of the composition is complexed with one or more lipids to form LNPs, wherein the cationic lipid of the LNP is selected from structures III-1 to III-36 of Table 9 of the published PCT patent application WO2018/078053A1. Therefore, structures III-1 to III-36 of WO2018/078053A1 and the specific disclosures related thereto are hereby incorporated by reference.
在第二方面特别优选的实施方式中，至少一种RNA，优选至少一种mRNA，与一种或多种脂质复合，由此形成LNP，其中LNP包含根据式III-3的阳离子脂质：In a particularly preferred embodiment of the second aspect, at least one RNA, preferably at least one mRNA, is complexed with one or more lipids to form LNPs, wherein the LNPs comprise a cationic lipid according to formula III-3:
如本文合适地使用的式III-3的脂质具有化学术语((4-羟丁基)氮烷二基)双(己烷-6,1-二基)双(2-己基癸酸酯)，也称为ALC-0315。The lipid of formula III-3 as suitably used herein has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315.
在某些实施方式中，本文定义的阳离子脂质，更优选阳离子脂质化合物III-3，以相对于LNP的总脂质含量约30至约95摩尔％的量存在于LNP中。如果在LNP中掺入了超过一种阳离子脂质，这些百分比适用于组合的阳离子脂质。In certain embodiments, the cationic lipids defined herein, more preferably cationic lipid compound III-3, are present in the LNP in an amount of about 30 to about 95 mol % relative to the total lipid content of the LNP. If more than one cationic lipid is incorporated into the LNP, these percentages apply to the combined cationic lipids.
在多种实施方式中，阳离子脂质以约30至约70摩尔％的量存在于LNP中。在一个实施方式中，阳离子脂质以约40至约60摩尔％的量存在于LNP中，如分别约40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59或60摩尔％。在多种实施方式中，阳离子脂质以约47至约48摩尔％的量存在于LNP中，如分别约47.0、47.1、47.2、47.3、47.4、47.5、47.6、47.7、47.8、47.9、50.0摩尔％，其中47.7摩尔％是特别优选的。In various embodiments, cationic lipid is present in LNP with the amount of about 30 to about 70 % by mole. In one embodiment, cationic lipid is present in LNP with the amount of about 40 to about 60 % by mole, such as respectively about 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 or 60 % by mole. In various embodiments, cationic lipid is present in LNP with the amount of about 47 to about 48 % by mole, such as respectively about 47.0,47.1,47.2,47.3,47.4,47.5,47.6,47.7,47.8,47.9,50.0 % by mole, wherein 47.7 % by mole is particularly preferred.
在一些实施方式中，阳离子脂质以LNP中存在的总脂质的约20摩尔％至约70或75摩尔％或约45至约65摩尔％或约20、25、30、35、40、45、50、55、60、65或约70摩尔％的比率存在。在进一步的实施方式中，LNP包含以摩尔计约25％至约75％的阳离子脂质，例如以摩尔计(基于脂质纳米颗粒中脂质的100％总摩尔)约20％至约70％、约35％至约65％、约45％至约65％，约60％、约57.5％、约57.1％、约50％或约40％。在一些实施方式中，阳离子脂质与RNA的比率为约3至约15，如约5至约13或约7至约11。In some embodiments, cationic lipid is present in the ratio of about 20 mol % to about 70 or 75 mol % or about 45 to about 65 mol % or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or about 70 mol % of the total lipid present in LNP. In further embodiments, LNP comprises about 25% to about 75% cationic lipid by mole, for example, about 20% to about 70%, about 35% to about 65%, about 45% to about 65% by mole (based on 100% total mole of lipid in lipid nanoparticle), about 60%, about 57.5%, about 57.1%, about 50% or about 40%. In some embodiments, the ratio of cationic lipid to RNA is about 3 to about 15, such as about 5 to about 13 or about 7 to about 11.
其他合适的(阳离子或可离子化的)脂质公开于WO2009/086558、WO2009/127060、WO2010/048536、WO2010/054406、WO2010/088537、WO2010/129709、WO2011/153493、WO2013/063468、US2011/0256175、US2012/0128760、US2012/0027803、US8158601、WO2016/118724、WO2016/118725、WO2017/070613、WO2017/070620、WO2017/099823、WO2012/040184、WO2011/153120、WO2011/149733、WO2011/090965、WO2011/043913、WO2011/022460、WO2012/061259、WO2012/054365、WO2012/044638、WO2010/080724、WO2010/21865、WO2008/103276、WO2013/086373、WO2013/086354、US专利No.7,893,302、7,404,969、8,283,333、8,466,122和8,569,256以及US专利公开No.US2010/0036115、US2012/0202871、US2013/0064894、US2013/0129785、US2013/0150625、US2013/0178541、US2013/0225836、US2014/0039032和WO2017/112865。在该情况中，将WO2009/086558、WO2009/127060、WO2010/048536、WO2010/054406、WO2010/088537、WO2010/129709、WO2011/153493、WO 2013/063468、US2011/0256175、US2012/0128760、US2012/0027803、US8158601、WO2016/118724、WO2016/118725、WO2017/070613、WO2017/070620、WO2017/099823、WO2012/040184、WO2011/153120、WO2011/149733、WO2011/090965、WO2011/043913、WO2011/022460、WO2012/061259、WO2012/054365、WO2012/044638、WO2010/080724、WO2010/21865、WO2008/103276、WO2013/086373、WO2013/086354、US专利No.7,893,302、7,404,969、8,283,333、8,466,122和8,569,256以及US专利公开No.US2010/0036115、US2012/0202871、US2013/0064894、US2013/0129785、US2013/0150625、US2013/0178541、US2013/0225836和US2014/0039032以及WO2017/112865具体涉及适用于LNP的(阳离子)脂质的公开内容在此按引用并入。Other suitable (cationic or ionizable) lipids are disclosed in WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493, WO2013/063468, US2011/0256175, US2012/0128760, US2012/0027803, US8158601, WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823, WO2012/040184, WO2011/153120, WO2011/14 9733、WO2011/090965、WO2011/043913 、WO2011/022460、WO2012/061259、WO2012/054365、WO2012/044638、WO2010/080724、WO2010/21865、WO2008/103276、WO2013/086373、WO2013/086354、US Patent No.7,893,302、7,404,969、8,283,333、 8,466,122 and 8,569,256 and US Patent Publication Nos. US2010/0036115, US2012/0202871, US2013/0064894, US2013/0129785, US2013/0150625, US2013/0178541, US2013/0225836, US2014/0039032 and WO2017/112865. In this case, WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493, WO 2013/063468, US2011/0256175, US2012/0128760, US2012/0027803, US8158601, WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823, WO20 12/040184、WO2011/153120、WO2011/149733、WO2011/090965、WO2011/043913、WO2011/022460、WO2012/061259、WO2012/054365、WO2012/044638、WO2010/0 80724,WO201 0/21865, WO2008/103276, WO2013/086373, WO2013/086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication Nos. US2010/0036115, US2012/02 The disclosures of US2013/02871, US2013/0064894, US2013/0129785, US2013/0150625, US2013/0178541, US2013/0225836 and US2014/0039032 and WO2017/112865 specifically relating to (cationic) lipids suitable for LNPs are hereby incorporated by reference.
在多种实施方式中，本文定义的氨基或阳离子脂质具有至少一个可质子化或可去质子化基团，使得脂质在生理pH或低于生理pH(例如pH 7.4)的pH下带正电，且在第二pH，优选在生理pH或高于生理pH下为中性。当然，应理解，随着pH变化添加或去除质子是一个平衡过程，并且提及带电或中性脂质是指主要物质的性质，并且不要求所有脂质必须以带电或中性形式存在。In various embodiments, the amino or cationic lipids defined herein have at least one protonatable or deprotonatable group such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH. Of course, it will be understood that adding or removing protons as pH changes is an equilibrium process, and reference to charged or neutral lipids refers to the nature of the primary species, and does not require that all lipids must exist in a charged or neutral form.
不排除具有一个以上可质子化或可去质子化基团的脂质，或作为两性离子的脂质，并且同样适用于本发明的情况中。在一些实施方式中，可质子化脂质具有约4至约11的范围内的可质子化基团的pKa，例如约5至约7的pKa。Lipids with more than one protonatable or deprotonatable group, or lipids that are zwitterions, are not excluded and are equally applicable in the context of the present invention. In some embodiments, the protonatable lipid has a pKa of a protonatable group in the range of about 4 to about 11, such as a pKa of about 5 to about 7.
LNP可包含本文所定义的两种或更多种(不同的)阳离子脂质。可以选择阳离子脂质以促成不同的有利性质。例如，可以在LNP中使用性质不同的阳离子脂质，所述性质如胺pKa、化学稳定性、循环半衰期、组织中半衰期、组织中净累积或毒性。特别地，可以选择阳离子脂质，使得混合的LNP的性质比单个脂质的单一LNP的性质更合乎需要。LNP can comprise two or more (different) cationic lipids defined herein. Cationic lipids can be selected to facilitate different favorable properties. For example, cationic lipids with different properties can be used in LNP, such as amine pKa, chemical stability, circulation half-life, half-life in tissue, net accumulation or toxicity in tissue. Especially, cationic lipids can be selected so that the property of the LNP mixed is more desirable than the property of the single LNP of single lipid.
可以考虑核酸货物的量来选择永久阳离子脂质或类脂的量。在一个实施方式中，选择这些量以使得纳米颗粒或组合物的N/P比率在约0.1至约20的范围内。在这种情况中，N/P比率定义为脂质或类脂的碱性含氮基团的氮原子(“N”)与用作货物的核酸的磷酸酯基团(“P”)的摩尔比。N/P比率可以基于例如1ug RNA通常包含约3nmol磷酸残基来计算，条件是RNA表现出碱基的统计学分布。脂质或类脂的“N”值可以根据其分子量和永久阳离子的和(如果存在的话)可阳离子化的基团的相对含量来计算。Can consider the amount of nucleic acid goods to select the amount of permanent cationic lipid or lipid.In one embodiment, select these amounts so that the N/P ratio of nanoparticle or composition is in the range of about 0.1 to about 20.In this case, the N/P ratio is defined as the nitrogen atom (" N ") of the basic nitrogen-containing group of lipid or lipid and the phosphate group (" P ") of the nucleic acid used as goods.The N/P ratio can be calculated based on for example 1ug RNA usually comprising about 3nmol phosphate residues, and condition is that RNA shows the statistical distribution of bases.The " N " value of lipid or lipid can be calculated according to its molecular weight and permanent cationic and (if present) the relative content of cationizable group.
LNP的体内特性和行为可以通过向LNP表面添加亲水性聚合物涂层(例如聚乙二醇(PEG))来改性，以赋予空间稳定性。此外，LNP可以通过将配体(例如抗体、肽和碳水化合物)连接于其表面或连接的PEG链的末端(例如通过PEG化的脂质或PEG化的胆固醇)来用于特异性靶向。The in vivo properties and behavior of LNPs can be modified by adding hydrophilic polymer coatings (e.g., polyethylene glycol (PEG)) to the LNP surface to impart steric stability. In addition, LNPs can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of attached PEG chains (e.g., via PEGylated lipids or PEGylated cholesterol).
在一些实施方式中，LNP包含聚合物缀合的脂质。术语“聚合物缀合的脂质”是指包含脂质部分和聚合物部分的分子。聚合物缀合的脂质的实例是PEG化的脂质。术语“PEG化的脂质”是指包含脂质部分和聚乙二醇部分的分子。PEG化的脂质是本领域已知的，并且包括1-(单甲氧基-聚乙二醇)-2,3-二肉豆蔻基甘油(PEG-s-DMG)等。In some embodiments, LNP comprises polymer-conjugated lipids. The term "polymer-conjugated lipids" refers to a molecule comprising a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristyl glycerol (PEG-s-DMG) and the like.
如本文定义的聚合物缀合的脂质，例如，PEG-脂质，可以用作聚集减少的脂质。A polymer-conjugated lipid as defined herein, e.g., a PEG-lipid, may be used as an aggregation-reducing lipid.
在某些实施方式中，LNP包含稳定脂质，其为聚乙二醇-脂质(PEG化的脂质)。合适的聚乙二醇-脂质包括PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺(例如PEG-CerC14或PEG-CerC20)、PEG-修饰的二烷基胺、PEG-修饰的二酰基甘油、PEG-修饰的二烷基甘油。代表性的聚乙二醇-脂质包括PEG-c-DOMG、PEG-c-DMA和PEG-s-DMG。在一个实施方式中，聚乙二醇-脂质是N-[(甲氧基聚(乙二醇)2000)氨基甲酰基]-1,2-二肉豆蔻酰氧基丙基-3-胺(PEG-c-DMA)。在优选的实施方式中，聚乙二醇-脂质是PEG-2000-DMG。在一个实施方式中，聚乙二醇-脂质是PEG-c-DOMG)。在其它实施方式中，LNP包含PEG化的二酰基甘油(PEG-DAG)，如1-(单甲氧基-聚乙二醇)-2,3-二肉豆蔻基酰甘油(PEG-DMG)、PEG化的磷脂酰乙醇胺(PEG-PE)、PEG琥珀酸酯二酰基甘油(PEG-S-DAG)，如4-O-(2',3'-二(十四酰氧基)丙基-1-O-(ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG)、PEG化神经酰胺(PEG-cer)或PEG二烷氧基丙基氨基甲酸酯，如ω-甲氧基(聚乙氧基)乙基-N-(2,3-二(十四烷氧基)丙基)氨基甲酸酯或2,3-二(十四烷氧基)丙基-N-(ω-甲氧基(聚乙氧基)乙基)氨基甲酸酯。In certain embodiments, LNP comprises stable lipid, it is polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipid includes PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (such as PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Representative polyethylene glycol-lipid includes PEG-c-DOMG, PEG-c-DMA and PEG-s-DMG. In one embodiment, polyethylene glycol-lipid is N-[(methoxy poly (ethylene glycol) 2000) aminoformyl] -1,2- dimyristoyloxypropyl -3-amine (PEG-c-DMA). In a preferred embodiment, polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNP comprises a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.
在优选的实施方式中，PEG化的脂质优选衍生自公开的PCT专利申请WO2018/078053A1的式(IV)。因此，将衍生自公开的PCT专利申请WO2018/078053A1的式(IV)的PEG化脂质及与其相关的相应公开内容在此按引用并入。In a preferred embodiment, the PEGylated lipid is preferably derived from the formula (IV) of the disclosed PCT patent application WO2018/078053A1. Therefore, the PEGylated lipid of formula (IV) derived from the disclosed PCT patent application WO2018/078053A1 and the corresponding disclosures related thereto are incorporated herein by reference.
在特别优选的实施方式中，组合物的至少一种RNA与一种或多种脂质复合，由此形成LNP，其中LNP包含PEG化的脂质，其中PEG脂质优选衍生自公开的PCT专利申请WO2018/078053A1的式(IVa)。因此，将衍生自公开的PCT专利申请WO2018/078053A1的式(IVa)的PEG化脂质及与其相关的相应公开内容在此按引用并入。In a particularly preferred embodiment, at least one RNA of the composition is complexed with one or more lipids to form LNPs, wherein the LNPs comprise PEGylated lipids, wherein the PEG lipids are preferably derived from the formula (IVa) of the published PCT patent application WO2018/078053A1. Therefore, the PEGylated lipids of formula (IVa) derived from the published PCT patent application WO2018/078053A1 and the corresponding disclosures related thereto are incorporated herein by reference.
在特别优选的实施方式中，至少一种RNA与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含PEG化的脂质/PEG脂质。优选地，所述PEG脂质是式(IVa)：In a particularly preferred embodiment, at least one RNA is complexed with one or more lipids to form lipid nanoparticles (LNPs), wherein the LNPs comprise PEGylated lipids/PEG lipids. Preferably, the PEG lipids are of formula (IVa):
其中n的平均值范围为30至60，如约30±2、32±2、34±2、36±2、38±2、40±2、42±2、44±2、46±2、48±2、50±2、52±2、54±2、56±2、58±2或60±2。在最优选的实施方式中，n为约49。在进一步优选的方面中，所述PEG脂质具有式(IVa)，其中n是选择以使得PEG脂质的平均分子量为约2000g/mol至约3000g/mol或约2300g/mol至大约2700g/mol，甚至更优选约2500g/mol的整数。wherein n has an average value ranging from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2 or 60±2. In a most preferred embodiment, n is about 49. In a further preferred aspect, the PEG lipid has formula (IVa), wherein n is an integer selected so that the average molecular weight of the PEG lipid is from about 2000 g/mol to about 3000 g/mol or from about 2300 g/mol to about 2700 g/mol, even more preferably about 2500 g/mol.
在本文中合适地使用的式IVa的脂质具有化学术语2-[(聚乙二醇)-2000]-N,N-二-十四烷基乙酰胺，也称为ALC-0159。The lipid of formula IVa suitably used herein has the chemical designation 2-[(polyethylene glycol)-2000]-N,N-di-tetradecanoylacetamide, also known as ALC-0159.
在该情况中合适的PEG-脂质的进一步实例提供于US2015/0376115A1和WO2015/199952中，将每篇全部按引用并入。Further examples of suitable PEG-lipids in this context are provided in US 2015/0376115 A1 and WO 2015/199952, each of which is incorporated by reference in its entirety.
在一些实施方式中，基于LNP中脂质的总摩尔数，LNP包括小于约3、2或1摩尔％的PEG或PEG修饰的脂质。在进一步的实施方式中，LNP包含以摩尔计约0.1％至约20％的PEG修饰的脂质，例如以摩尔计(基于LNP中脂质的100％总摩尔)约0.5％至约10％、约0.5％至约5％、约10％、约5％、约3.5％、约3％、约2.5％、约2％、约1.5％、约1％、约0.5％或约0.3％。在优选的实施方式中，LNP包含以摩尔计约1.0％至约2.0％的PEG修饰的脂质，例如约1.2至约1.9％、约1.2至约1.8％、约1.3至约1.8％，约1.4至约1.8％和约1.5至约1.8％、约1.6至约1.8％，特别是约1.4％、约1.5％、约1.6％、约1.7％、约1.8％、约1.9％，最优选1.7％(基于LNP中脂质的100％总摩尔数)。在各种实施方式中，阳离子脂质与PEG化脂质的摩尔比范围为约100:1至约25:1。In some embodiments, based on the total moles of lipid in LNP, LNP includes less than about 3, 2 or 1 mol % of PEG or PEG-modified lipid. In further embodiments, LNP comprises about 0.1% to about 20% PEG-modified lipid by mole, for example, about 0.5% to about 10%, about 0.5% to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.5% or about 0.3% by mole (based on 100% total moles of lipid in LNP). In preferred embodiments, LNP comprises about 1.0% to about 2.0% PEG-modified lipids by mole, such as about 1.2 to about 1.9%, about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about 1.8% and about 1.5 to about 1.8%, about 1.6 to about 1.8%, particularly about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most preferably 1.7% (based on 100% total moles of lipid in LNP). In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.
在优选的实施方式中，LNP包含一种或多种另外的脂质，其在颗粒形成过程中或在制造过程中稳定颗粒的形成(例如，中性脂质和/或一种或多种类固醇或类固醇类似物)。In preferred embodiments, the LNPs comprise one or more additional lipids that stabilize the formation of the particle during particle formation or during the manufacturing process (eg, a neutral lipid and/or one or more steroids or steroid analogs).
在第二方面的优选实施方式中，至少一种RNA与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含一种或多种中性脂质和/或一种或多种类固醇或类固醇类似物。In a preferred embodiment of the second aspect, the at least one RNA is complexed with one or more lipids, thereby forming a lipid nanoparticle (LNP), wherein the LNP comprises one or more neutral lipids and/or one or more steroids or steroid analogs.
合适的稳定脂质包括中性脂质和阴离子脂质。术语“中性脂质”是指在生理pH下以不带电的或中性两性离子的形式存在的多种脂质物质中的任一种。代表性的中性脂质包括二酰基磷脂酰胆碱、二酰基磷脂酰乙醇胺、神经酰胺、鞘磷脂、二氢鞘磷脂、脑磷脂和脑苷脂。Suitable stable lipids include neutral lipids and anionic lipids. The term "neutral lipid" refers to any of a variety of lipid substances that exist in the form of uncharged or neutral zwitterions at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.
在第二方面的实施方式中，LNP包含一种或多种中性脂质，其中中性脂质选自二硬脂酰磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰油酰磷脂酰胆碱(POPC)、棕榈酰油酰基磷脂酰乙醇胺(POPE)和二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(DOPE-mal)、二棕榈酰磷脂酰乙醇酰胺(DPPE)、二肉豆蔻酰磷酸乙醇胺(DMPE)、二硬脂酰磷脂酰醇胺(DSPE)、16-O-单甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂酰-2-油酰基磷脂酰乙醇胺(SOPE)和1,2-二反油酰-sn-甘油-3-磷酸乙醇胺(transDOPE)或其混合物。In an embodiment of the second aspect, the LNP comprises one or more neutral lipids, wherein the neutral lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine (4-phosphatidylethanolamine). -(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamide (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE) or a mixture thereof.
在一些实施方式中，LNP包含选自DSPC、DPPC、DMPC、DOPC、POPC、DOPE和SM的中性脂质。在各种不同的实施方式中，阳离子脂质与中性脂质的摩尔比范围为约2:1至约8:1。In some embodiments, LNP comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of cationic lipid to neutral lipid ranges from about 2:1 to about 8:1.
在优选的实施方式中，中性脂质是1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)。阳离子脂质与DSPC的摩尔比可以在约2:1至约8:1的范围内。In a preferred embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The molar ratio of the cationic lipid to DSPC may be in the range of about 2:1 to about 8:1.
在优选的实施方式中，类固醇是胆固醇。阳离子脂质与胆固醇的摩尔比可以在约2:1至约1:1的范围内。在一些实施方式中，胆固醇可以是PEG化的。In a preferred embodiment, the steroid is cholesterol. The molar ratio of the cationic lipid to cholesterol may be in the range of about 2: 1 to about 1: 1. In some embodiments, the cholesterol may be PEGylated.
固醇可以是脂质颗粒的约10摩尔％至约60摩尔％或约25摩尔％至约40摩尔％。在一个实施方式中，固醇为脂质颗粒中存在的总脂质的约10、15、20、25、30、35、40、45、50、55或约60摩尔％。在另一个实施方式中，LNP包括以摩尔计约5％至约50％的固醇，例如以摩尔计(基于脂质纳米颗粒中脂质的100％总摩尔)约15％至约45％、约20％至约40％、约48％、约40％、约38.5％、约35％、约34.4％、约31.5％或约31％。Sterol can be about 10 mol % to about 60 mol % or about 25 mol % to about 40 mol % of lipid particles. In one embodiment, sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or about 60 mol % of the total lipid present in the lipid particles. In another embodiment, LNP includes about 5% to about 50% sterol by mole, for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% by mole (based on 100% total mole of lipid in lipid nanoparticles).
优选地，脂质纳米颗粒(LNP)包含：(a)至少一种第一方面的RNA，(b)阳离子脂质，(c)聚集减少剂(如聚乙二醇(PEG)脂质或PEG修饰的脂质)，(d)任选地非阳离子脂质(如中性脂质)，和(e)任选地固醇。Preferably, the lipid nanoparticle (LNP) comprises: (a) at least one RNA of the first aspect, (b) a cationic lipid, (c) an aggregation reducing agent (such as a polyethylene glycol (PEG) lipid or a PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally a sterol.
在一些实施方式中，阳离子脂质(如上定义)、非阳离子脂质(如上定义)、胆固醇(如上定义)和/或PEG修饰的脂质(如上定义)可以以各种相对的摩尔比组合。例如，阳离子脂质与非阳离子脂质与基于胆固醇的脂质与PEG修饰的脂质的比率可以在约30-60:20-35:20-30:1-15之间，或分别为约40:30:25:5、50:22:20:5、50:27:20:3、40:30:20:10、40:32:20:8、40:3:25:3或40:33:25:2的比率，或为约50:25:20:5、50:20:25:5、50:27:20:3、40:30:20:10、40:30:25:5或40:32:22:8、40:32:25:3或40:33:25:20的比率。In some embodiments, the cationic lipid (as defined above), the non-cationic lipid (as defined above), cholesterol (as defined above) and/or the PEG-modified lipid (as defined above) can be combined in various relative molar ratios. For example, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEG-modified lipid can be between about 30-60:20-35:20-30:1-15, or a ratio of about 40:30:25:5, 50:22:20:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:3:25:3, or 40:33:25:2, respectively, or a ratio of about 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:30:25:5, or 40:32:22:8, 40:32:25:3, or 40:33:25:20.
在一些实施方式中，LNP包含式(III)的脂质、至少一种本文定义的NRA、中性脂质、类固醇和PEG化的脂质。在优选的实施方式中，式(III)的脂质是脂质化合物III-3(ALC-0315)，中性脂质是DSPC，类固醇是胆固醇，以及PEG化的脂质是式(IVa)的化合物(ALC-0159)。In some embodiments, the LNP comprises a lipid of formula (III), at least one NRA as defined herein, a neutral lipid, a steroid, and a PEGylated lipid. In a preferred embodiment, the lipid of formula (III) is lipid compound III-3 (ALC-0315), the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is a compound of formula (IVa) (ALC-0159).
在第二方面的优选实施方式中，LNP基本上由(i)至少一种阳离子脂质；(ii)中性脂质；(iii)固醇，例如，胆固醇；和(iv)PEG-脂质，例如，PEG-DMG或PEG-cDMA组成，其为约20-60％阳离子脂质:5-25％中性脂质:25-55％固醇；0.5-15％ PEG-脂质的比率。In a preferred embodiment of the second aspect, the LNP consists essentially of (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.
在特别优选的实施方式中，至少一种RNA与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含In a particularly preferred embodiment, at least one RNA is complexed with one or more lipids to form a lipid nanoparticle (LNP), wherein the LNP comprises
(i)至少一种本文定义的阳离子脂质，优选式(III)的脂质，更优选脂质III-3(ALC-0315)；(i) at least one cationic lipid as defined herein, preferably a lipid of formula (III), more preferably lipid III-3 (ALC-0315);
(ii)至少一种本文定义的中性脂质，优选1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)；(ii) at least one neutral lipid as defined herein, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
(iii)至少一种本文定义的类固醇或类固醇类似物，优选胆固醇；和(iii) at least one steroid or steroid analogue as defined herein, preferably cholesterol; and
(iv)至少一种本文定义的PEG-脂质，例如，PEG-DMG或PEG-cDMA，优选是或源自式(IVa)的PEG化脂质(ALC-0159)。(iv) at least one PEG-lipid as defined herein, e.g. PEG-DMG or PEG-cDMA, preferably being or derived from the PEGylated lipid of formula (IVa) (ALC-0159).
在特别优选的实施方式中，至少一种RNA与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含约20-60％阳离子脂质:5-25％中性脂质:25-55％固醇:0.5-15％ PEG-脂质摩尔比的(i)至(iv)。In a particularly preferred embodiment, at least one RNA is complexed with one or more lipids to form a lipid nanoparticle (LNP), wherein the LNP comprises (i) to (iv) in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol: 0.5-15% PEG-lipid.
在一个优选的实施方式中，脂质纳米颗粒包含：具有式(III)的阳离子脂质和/或具有式(IV)的PEG脂质，任选中性脂质，优选1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)和任选地类固醇，优选胆固醇，其中阳离子脂质与DSPC的摩尔比任选在约2:1至8:1的范围内，其中阳离子脂质与胆固醇的摩尔比任选在约2:1至1:1的范围内。In a preferred embodiment, the lipid nanoparticles comprise: a cationic lipid having formula (III) and/or a PEG lipid having formula (IV), optionally a neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally a steroid, preferably cholesterol, wherein the molar ratio of the cationic lipid to DSPC is optionally in the range of about 2:1 to 8:1, wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range of about 2:1 to 1:1.
在特别优选的实施方式中，包含至少一种RNA的第二方面的组合物包含脂质纳米颗粒(LNP)，其具有大约50:10:38.5:1.5，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的摩尔比(即，阳离子脂质(优选脂质III-3(ALC-0315))、DSPC、胆固醇和PEG-脂质(优选式(IVa)的PEG-脂质，n＝49，甚至更优选式(IVa)的PEG-脂质，n＝45(ALC-0159))的比例(mol％)；溶解在乙醇中)。In a particularly preferred embodiment, the composition of the second aspect comprising at least one RNA comprises lipid nanoparticles (LNP) having a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e., the ratio (mol %) of cationic lipid (preferably lipid III-3 (ALC-0315)), DSPC, cholesterol and PEG-lipid (preferably PEG-lipid of formula (IVa) with n=49, even more preferably PEG-lipid of formula (IVa) with n=45 (ALC-0159)); dissolved in ethanol).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% identical to the nucleic acid sequence of SEQ ID NO: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, the lipid nanoparticles having a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27532、27555、27624、28852、28699-28704、28879-28884的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) having the same sequence as SEQ ID NO:27532, 27555, 27624, 28852, 28699-28704, 28879-28884 are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of NO:27532, 27555, 27624, 28852, 28699-28704, 28879-28884, and the lipid nanoparticles have a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27532、27555、27624、28852的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27532, 27555, 27624, 28852 formulated in lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:28688-28691、28868-28871、28929-28932、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) having the same sequence as SEQ ID NO:28688-28691, 28868-28871, 28929-28932, 28937-28940 are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, and the lipid nanoparticles have a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) that is identical to or at least 70% identical to the nucleic acid sequence of SEQ ID NO: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936 The lipid nanoparticles are preferably cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)) in a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27394、27417、27486、28762、28650-28655、28789-28794的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) having the same sequence as SEQ ID NO:27394, 27417, 27486, 28762, 28650-28655, 28789-28794 are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, and the lipid nanoparticles have a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27394、27417、27486、28762的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27394, 27417, 27486, 28762, wherein the lipid nanoparticle has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
优选地，第二方面的组合物包含至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:28639-28642、28778-28781、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。Preferably, the composition of the second aspect comprises at least one RNA formulated in a lipid nanoparticle (LNP) having the same sequence as SEQ ID NO:28639-28642, 28778-28781, 28925-28928, 28933-28936 are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, and the lipid nanoparticles have a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)).
在其中组合物是如上定义的多价组合物的实施方式中，多价组合物的RNA物质，优选mRNA物质可以单独配制，例如，可以在脂质体或LNP中单独配制。合适地，多价组合物的RNA物质在LNP中单独配制，所述LNP具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45)。用于多价组合物的核酸物质优选如上限定地进行选择(参见“本发明的多价组合物”部分)。In embodiments where the composition is a multivalent composition as defined above, the RNA material, preferably the mRNA material, of the multivalent composition can be formulated separately, for example, can be formulated separately in liposomes or LNPs. Suitably, the RNA material of the multivalent composition is formulated separately in LNPs having a molar ratio (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7. The nucleic acid material used in the multivalent composition is preferably selected as defined above (see the section "Multivalent Compositions of the Invention ").
在该情况中，组合物可以包含In this case, the composition may comprise
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:149的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 149 formulated in a lipid nanoparticle (LNP) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA formulated in a lipid nanoparticle (LNP) that is identical to SEQ ID NO: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-2893 6 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of formula (IVa), the lipid nanoparticle having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)); and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27394、27417、27486、28762、28650-28655、28789-28794的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27394, 27417, 27486, 28762, 28650-28655, 28789-28794 formulated in a lipid nanoparticle (LNP) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27394、27417、27486、28762的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27394, 27417, 27486, 28762 formulated in a lipid nanoparticle (LNP) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:28639-28642、28778-28781、28925-28928、28933-28936的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))。- at least one RNA formulated in a lipid nanoparticle (LNP) which is identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 28639-28642, 28778-28781, 28925-28928, 28933-28936, wherein the lipid nanoparticle has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)).
在该情况中，组合物可以包含In this case, the composition may comprise
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:24837的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 24837 formulated in lipid nanoparticles (LNPs) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA formulated in a lipid nanoparticle (LNP) that is identical to or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, %, 96%, 97%, 98% or 99% identical, the lipid nanoparticles having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)); and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27532、27555、27624、28852、28699-28704、28879-28884的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27532, 27555, 27624, 28852, 28699-28704, 28879-28884 formulated in a lipid nanoparticle (LNP) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:27532、27555、27624、28852的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))；和/或- at least one RNA identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27532, 27555, 27624, 28852 formulated in a lipid nanoparticle (LNP) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7; and/or
-至少一种配制在脂质纳米颗粒(LNP)中的RNA，其与SEQ ID NO:28688-28691、28868-28871、28929-28932、28937-28940的核酸序列相同或至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，所述脂质纳米颗粒具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45(ALC-0159))。- at least one RNA formulated in a lipid nanoparticle (LNP) which is identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 28688-28691, 28868-28871, 28929-28932, 28937-28940, wherein the lipid nanoparticle has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45 (ALC-0159)).
在其中组合物是如上定义的多价组合物的实施方式中，多价组合物的核酸物质(例如，DNA或RNA)，优选RNA物质可以共同配制，优选在脂质体或LNP中共同配制。合适地，多价组合物的RNA物质在LNP中共同配制，所述LNP具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49或n＝45)。用于多价组合物的核酸物质优选如上限定地进行选择(参见“本发明的多价组合物”部分)。In embodiments where the composition is a multivalent composition as defined above, the nucleic acid material (e.g., DNA or RNA), preferably RNA material of the multivalent composition can be co-formulated, preferably co-formulated in liposomes or LNPs. Suitably, the RNA material of the multivalent composition is co-formulated in LNPs having a molar ratio (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49 or n=45) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7. The nucleic acid material used in the multivalent composition is preferably selected as defined above (see the "Multivalent Compositions of the Invention " section).
脂质纳米颗粒中的RNA的总量可以改变并且根据例如核酸与总脂质w/w比率来限定。在本发明的一个实施方式中，核酸(特别是RNA)与总脂质的比率小于0.06w/w，优选在0.03w/w和0.04w/w之间。The total amount of RNA in the lipid nanoparticle can vary and is defined according to, for example, the w/w ratio of nucleic acid to total lipid. In one embodiment of the invention, the ratio of nucleic acid (particularly RNA) to total lipid is less than 0.06 w/w, preferably between 0.03 w/w and 0.04 w/w.
在一些实施方式中，脂质纳米颗粒(LNP)，其仅由三种脂质组分组成，即咪唑胆固醇酯(ICE)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和1,2-二肉豆蔻酰基-sn-甘油、甲氧基聚乙二醇(DMG-PEG-2k)。In some embodiments, lipid nanoparticles (LNPs) are composed of only three lipid components, namely, imidazolyl cholesteryl ester (ICE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol (DMG-PEG-2k).
在一个实施方式中，组合物的脂质纳米颗粒包含阳离子脂质、类固醇；中性脂质；和聚合物缀合的脂质，优选PEG化的脂质。优选地，聚合物缀合的脂质是PEG化的脂质或PEG-脂质。在特定的实施方式中，脂质纳米颗粒包含由阳离子脂质SS-EC(曾用名：SS-33/4PE-15；NOF Corporation，东京，日本)表示的阳离子脂质，根据下式：In one embodiment, the lipid nanoparticles of the composition comprise a cationic lipid, a steroid; a neutral lipid; and a polymer-conjugated lipid, preferably a PEGylated lipid. Preferably, the polymer-conjugated lipid is a PEGylated lipid or a PEG-lipid. In a specific embodiment, the lipid nanoparticles comprise a cationic lipid. SS-EC (formerly known as SS-33/4PE-15; NOF Corporation, Tokyo, Japan) represents a cationic lipid according to the following formula:
如以下进一步描述的，那些脂质纳米颗粒称为“GN01”。As described further below, those lipid nanoparticles are referred to as "GN01".
此外，在特定的实施方式中，GN01脂质纳米颗粒包含由结构1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(DPhyPE)：Furthermore, in certain embodiments, the GN01 lipid nanoparticles comprise a 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE) having the structure:
此外，在特定的实施方式中，GN01脂质纳米颗粒包含聚合物缀合的脂质，优选PEG化的脂质，为1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG 2000)，具有以下结构：Furthermore, in a specific embodiment, the GN01 lipid nanoparticles comprise a polymer-conjugated lipid, preferably a PEGylated lipid, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), having the following structure:
如本领域所用的，“DMG-PEG 2000”认为是～97:3比率的1,2-DMG-PEG 2000和1,3-DMG-PEG 2000的混合物。As used in the art, "DMG-PEG 2000" is considered a mixture of 1,2-DMG-PEG 2000 and 1,3-DMG-PEG 2000 in a ratio of -97:3.
因此，根据优选实施方式之一的GN01脂质纳米颗粒(GN01-LNP)包含SS-EC阳离子脂质、中性脂质DPhyPE、胆固醇和聚合物缀合的脂质(PEG化的脂质)1,2-二肉豆蔻酰基-rac-甘油-3-甲氧基聚乙二醇(PEG-DMG)。Therefore, according to one of the preferred embodiments, the GN01 lipid nanoparticles (GN01-LNP) comprise SS-EC cationic lipids, neutral lipids DPhyPE, cholesterol and polymer-conjugated lipids (PEGylated lipids) 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG).
在优选的实施方式中，GN01 LNP包含：In a preferred embodiment, the GN01 LNP comprises:
(a)45-65mol％量的阳离子脂质SS-EC(曾用名：SS-33/4PE-15；NOF Corporation，日本，东京)；(a) 45-65 mol% of the cationic lipid SS-EC (formerly known as SS-33/4PE-15; NOF Corporation, Tokyo, Japan);
(b)25-45mol％量的胆固醇；(b) 25-45 mol% cholesterol;
(c)8-12mol％量的DPhyPE；和(c) DPhyPE in an amount of 8-12 mol%; and
(d)1-3mol％量的PEG-DMG 2000；(d) 1-3 mol% PEG-DMG 2000;
每个量相对于GN01脂质纳米颗粒的所有脂质赋形剂的总摩尔量。Each amount is relative to the total molar amount of all lipid excipients of GN01 lipid nanoparticles.
在进一步优选的实施方式中，如本文所述的GN01脂质纳米颗粒包含59mol％阳离子脂质，10mol％中性脂质，29.3mol％类固醇和1.7mol％聚合物缀合的脂质，优选PEG化的脂质。在最优选的实施方式中，如本文所述的GN01脂质纳米颗粒包含59mol％阳离子脂质SS-EC，10mol％DPhyPE，29.3mol％胆固醇和1.7mol％DMG-PEG 2000。In a further preferred embodiment, the GN01 lipid nanoparticles as described herein comprise 59 mol% cationic lipids, 10 mol% neutral lipids, 29.3 mol% steroids and 1.7 mol% polymer-conjugated lipids, preferably PEGylated lipids. In a most preferred embodiment, the GN01 lipid nanoparticles as described herein comprise 59 mol% cationic lipids SS-EC, 10 mol% DPhyPE, 29.3 mol% cholesterol and 1.7 mol% DMG-PEG 2000.
相对于GN01脂质纳米颗粒中的核酸量的阳离子脂质的量也可以表示为重量比(缩写，例如“m/m”)。例如，GN01脂质纳米颗粒包含至少一种核酸，优选至少一种RNA，其量如以获得约20至约60，或约10至约50范围内的脂质与RNA重量比。在其他实施方式中，阳离子脂质与核酸或RNA的比率为约3至约15，如约5至约13，约4至约8或约7至约11。在本发明非常优选的实施方式中，总脂质/RNA质量比为约40或40，即，约40倍或40倍质量过量，以确保RNA包封。另一个优选的RNA/脂质比为约1至约10，约2至约5，约2至约4，或优选约3。The amount of cationic lipid relative to the amount of nucleic acid in the GN01 lipid nanoparticles can also be expressed as a weight ratio (abbreviation, such as "m/m"). For example, the GN01 lipid nanoparticles contain at least one nucleic acid, preferably at least one RNA, in an amount such as to obtain a lipid to RNA weight ratio in the range of about 20 to about 60, or about 10 to about 50. In other embodiments, the ratio of cationic lipid to nucleic acid or RNA is about 3 to about 15, such as about 5 to about 13, about 4 to about 8 or about 7 to about 11. In a very preferred embodiment of the present invention, the total lipid/RNA mass ratio is about 40 or 40, that is, about 40 times or 40 times the mass excess to ensure RNA encapsulation. Another preferred RNA/lipid ratio is about 1 to about 10, about 2 to about 5, about 2 to about 4, or preferably about 3.
此外，可以考虑核酸货物如RNA化合物的量的量来选择阳离子脂质的量。在一个实施方式中，N/P比率可以在约1至约50的范围内。在另一个实施方式中，范围为约1至约20，约1至约10，约1至约5。在一个优选的实施方式中，选择这些量以获得在约10至约20的范围内的GN01脂质纳米颗粒或组合物的N/P比率。在进一步非常优选的实施方式中，N/P是14(即，14倍mol过量的正电荷，以确保核酸包封)。In addition, the amount of cationic lipids can be selected taking into account the amount of nucleic acid cargo, such as the amount of RNA compound. In one embodiment, the N/P ratio can be in the range of about 1 to about 50. In another embodiment, the range is about 1 to about 20, about 1 to about 10, about 1 to about 5. In a preferred embodiment, these amounts are selected to obtain an N/P ratio of the GN01 lipid nanoparticle or composition in the range of about 10 to about 20. In a further very preferred embodiment, N/P is 14 (i.e., 14 times molar excess positive charge to ensure nucleic acid encapsulation).
在优选的实施方式中，GN01脂质纳米颗粒包含59mol％阳离子脂质SS-EC(曾用名：SS-33/4PE-15，如从实施例部分清楚的；NOFCorporation，东京，日本)，29.3mol％胆固醇作为类固醇，10mol％DPhyPE作为中性脂质/磷脂和1.7mol％DMG-PEG 2000作为聚合物缀合的脂质。与使用DPhyPE关联的本发明的进一步优点是由于其庞大的尾巴而具有高的促融合能力，由此其能够以高水平与内体脂质融合。对于“GN01”，N/P(脂质与核酸(例如RNA)的mol比)优选为14，且总脂质/RNA质量比优选为40(m/m)。In a preferred embodiment, GN01 lipid nanoparticles contain 59 mol% cationic lipids SS-EC (formerly known as: SS-33/4PE-15, as is clear from the Examples section; NOF Corporation, Tokyo, Japan), 29.3 mol% cholesterol as steroid, 10 mol% DPhyPE as neutral lipid/phospholipid and 1.7 mol% DMG-PEG 2000 as polymer-conjugated lipid. A further advantage of the present invention associated with the use of DPhyPE is its high fusogenic ability due to its bulky tail, whereby it is able to fuse with endosomal lipids at a high level. For "GN01", the N/P (mol ratio of lipid to nucleic acid (e.g., RNA)) is preferably 14, and the total lipid/RNA mass ratio is preferably 40 (m/m).
在其他实施方式中，至少一种RNA，优选至少一种mRNA，与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含In other embodiments, at least one RNA, preferably at least one mRNA, is complexed with one or more lipids to form lipid nanoparticles (LNPs), wherein the LNPs comprise
I 至少一种阳离子脂质；I at least one cationic lipid;
Ii 至少一种中性脂质；Ii at least one neutral lipid;
Iii 至少一种类固醇或类固醇类似物；和iii. at least one steroid or steroid analog; and
Iiii 至少一种本文定义的PEG-脂质，iii at least one PEG-lipid as defined herein,
其中阳离子脂质是Dlin-KC2-DMA(50mol％)或Dlin-MC3-DMA(50mol％)、中性脂质是DSPC(10mol％)、PEG脂质是PEG-DOMG(1.5mol％)和结构脂质是胆固醇(38.5mol％)。The cationic lipid is Dlin-KC2-DMA (50 mol%) or Dlin-MC3-DMA (50 mol%), the neutral lipid is DSPC (10 mol%), the PEG lipid is PEG-DOMG (1.5 mol%) and the structural lipid is cholesterol (38.5 mol%).
在其他实施方式中，至少一种RNA，优选至少一种mRNA，与一种或多种脂质复合，由此形成脂质纳米颗粒(LNP)，其中LNP包含以50/38.5/10/1.5mol％的SS15/Chol/DOPE(或DOPC)DSG-5000。In other embodiments, at least one RNA, preferably at least one mRNA, is complexed with one or more lipids to form lipid nanoparticles (LNPs), wherein the LNPs comprise SS15/Chol/DOPE (or DOPC) DSG-5000 at 50/38.5/10/1.5 mol%.
在其他实施方式中，本发明的RNA可以配制在脂质体中，例如，如WO2019/222424、WO2019/226925、WO2019/232095、WO2019/232097或WO2019/232208中所述的脂质体，将WO2019/222424、WO2019/226925、WO2019/232095、WO2019/232097或WO2019/232208关于脂质体或基于脂质的载体分子的公开内容在此按引用并入。In other embodiments, the RNA of the invention can be formulated in liposomes, for example, as described in WO2019/222424, WO2019/226925, WO2019/232095, WO2019/232097 or WO2019/232208, the disclosures of WO2019/222424, WO2019/226925, WO2019/232095, WO2019/232097 or WO2019/232208 regarding liposomes or lipid-based carrier molecules are hereby incorporated by reference.
在各种实施方式中，合适地包封本发明的至少一种RNA的LNP具有约50nm至约200nm的平均直径，约60nm至约200nm，约70nm至约200nm，约80nm至约200nm，约90nm至约200nm，约90nm至约190nm，约90nm至约180nm，约90nm至约170nm，约90nm至约160nm，约90nm至约150nm，约90nm至约140nm、约90nm至约130nm、约90nm至约120nm、约90nm至约100nm、约70nm至约90nm、约80nm至约90nm、约70nm至约80nm，或约30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm、150nm、160nm，170nm、180nm、190nm或200nm，并且基本上是非毒性的。如本文使用的，平均直径可以通过z-平均来表示，如通过本领域通常已知的动态光散射来测定。In various embodiments, LNPs that suitably encapsulate at least one RNA of the invention have an average diameter of about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm, about 90 nm to about 200 nm, about 90 nm to about 190 nm, about 90 nm to about 180 nm, about 90 nm to about 170 nm, about 90 nm to about 160 nm, about 90 nm to about 150 nm, about 90 nm to about 140 nm, about 90 nm to about 130 nm, about 90 nm to about 120 nm, about 90 nm to about 100 nm. As used herein, mean diameter can be from about 10 nm to about 20 nm, from about 50 nm to about 80 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or from about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm, and is substantially non-toxic. As used herein, mean diameter can be represented by z-average, as measured by dynamic light scattering generally known in the art.
纳米颗粒的多分散指数(PDI)通常在0.1至0.5的范围内。在特定的实施方式中，PDI低于0.2。通常，PDI通过动态光散射来确定。The polydispersity index (PDI) of the nanoparticles is typically in the range of 0.1 to 0.5. In a particular embodiment, the PDI is below 0.2. Typically, the PDI is determined by dynamic light scattering.
在本发明的另一个优选实施方式中，脂质纳米颗粒分别具有约50nm至约300nm，或约60nm至约250nm，约60nm至约150nm，或约60nm至约120nm的流体动力学直径。In another preferred embodiment of the present invention, the lipid nanoparticles have a hydrodynamic diameter of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, about 60 nm to about 150 nm, or about 60 nm to about 120 nm, respectively.
在本发明的另一个优选实施方式中，脂质纳米颗粒分别具有约50nm至约300nm，或约60nm至约250nm，约60nm至约150nm，或约60nm至约120nm的流体动力学直径。In another preferred embodiment of the present invention, the lipid nanoparticles have a hydrodynamic diameter of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, about 60 nm to about 150 nm, or about 60 nm to about 120 nm, respectively.
在其中超过一种或多种，例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15种，本发明的RNA物质包含在组合物中的实施方式中，所述超过一种或所述多种，例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15种，本发明的RNA物质可以与一种或多种脂质复合，由此形成包含超过一种或多种，例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15种，不同的RNA物质的LNP。In embodiments where more than one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, RNA species of the invention are included in the composition, the more than one or the plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, RNA species of the invention can be complexed with one or more lipids to form LNPs comprising more than one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, different RNA species.
根据优选的实施方式，优选包封或包含RNA的LNP通过至少一个纯化步骤来纯化，优选通过至少一个TFF步骤和/或至少一个澄清步骤和/或至少一个过滤步骤。这种纯化特别地导致降低组合物中乙醇的量，乙醇是用于脂质配制的。According to a preferred embodiment, the LNPs preferably encapsulating or comprising RNA are purified by at least one purification step, preferably by at least one TFF step and/or at least one clarification step and/or at least one filtration step. This purification particularly results in reducing the amount of ethanol in the composition, which is used for lipid formulation.
在这种情况中，特别优选的是组合物在纯化后包含少于约500ppM乙醇，优选少于约50ppM乙醇，更优选少于约5ppM乙醇。In this case, it is particularly preferred that the composition, after purification, comprises less than about 500 ppM ethanol, preferably less than about 50 ppM ethanol, and more preferably less than about 5 ppM ethanol.
在实施方式中，本文所述的LNP可以冻干，以提高制剂和/或RNA的储存稳定性。在实施方式中，本文所述的LNP可以喷雾干燥，以提高制剂和/或核酸的储存稳定性。用于冻干和或喷雾干燥的冻干保护剂可以选自海藻糖、蔗糖、甘露糖、葡聚糖和菊粉。优选的冻干保护剂是蔗糖，任选包含进一步的冻干保护剂。进一步优选的冻干保护剂是海藻糖，任选包含进一步的冻干保护剂。In embodiments, the LNPs described herein can be lyophilized to improve the storage stability of the formulation and/or RNA. In embodiments, the LNPs described herein can be spray dried to improve the storage stability of the formulation and/or nucleic acid. The lyoprotectant for lyophilization and/or spray drying can be selected from trehalose, sucrose, mannose, dextran and inulin. A preferred lyoprotectant is sucrose, optionally comprising a further lyoprotectant. A further preferred lyoprotectant is trehalose, optionally comprising a further lyoprotectant.
因此，组合物，例如，包含LNP的组合物冻干(例如，根据WO2016/165831或WO2011/1069586，在此将其各自全部按引用并入)，以产生如本文定义的温度稳定的干燥的核酸(粉末)组合物(例如，RNA或DNA)。组合物，例如，包含LNP的组合物也可以使用喷雾干燥或喷雾冷冻干燥来干燥(例如，根据WO2016/184575或WO2016/184576)，以产生如本文定义的温度稳定的组合物(粉末)。Thus, compositions, e.g., compositions comprising LNPs are freeze-dried (e.g., according to WO2016/165831 or WO2011/1069586, each of which is hereby incorporated by reference in its entirety), to produce a temperature-stable dry nucleic acid (powder) composition (e.g., RNA or DNA) as defined herein. Compositions, e.g., compositions comprising LNPs can also be dried using spray drying or spray freeze drying (e.g., according to WO2016/184575 or WO2016/184576), to produce a temperature-stable composition (powder) as defined herein.
因此，在优选的实施方式中，组合物是干燥的组合物。Thus, in a preferred embodiment, the composition is a dry composition.
如本文使用的术语“干燥的组合物”必须理解为如上定义的已经冻干或喷雾干燥或喷雾冷冻干燥的组合物以获得温度稳定的干燥组合物(粉末)，例如，包含LNP复合的RNA(如上定义的)。The term "dried composition" as used herein has to be understood as a composition as defined above which has been lyophilized or spray-dried or spray-freeze-dried to obtain a temperature-stable dry composition (powder), e.g. comprising LNP-complexed RNA (as defined above).
根据进一步的实施方式，第二方面的组合物可以包含至少一种佐剂。According to a further embodiment, the composition of the second aspect may comprise at least one adjuvant.
合适地，优选添加佐剂以增强组合物的免疫刺激特性。Suitably, an adjuvant is preferably added to enhance the immunostimulatory properties of the composition.
如本文所使用的术语“佐剂”由本领域普通技术人员认识和理解，且例如旨在表示可修饰(例如增强)其他药剂的功效或可适合于支持组合物的施用和递送的药理学和/或免疫学药剂。术语“佐剂”是指广谱的物质。通常，这些物质能够增加抗原的免疫原性。例如，佐剂可由先天性免疫系统识别且例如可引发先天性免疫反应(即非特异性免疫反应)。“佐剂”通常不诱发适应性免疫反应。在本发明的情况中，佐剂可增强由核酸提供的抗原肽或蛋白质的功效。在该情况中，至少一种佐剂可选自本领域技术人员已知且适合于本发明情况(即支持受试者(例如，人受试者)中的免疫反应的诱导)的任何佐剂。The term "adjuvant" as used herein is recognized and understood by those of ordinary skill in the art, and is intended to represent, for example, a pharmacological and/or immunological agent that can modify (e.g., enhance) the efficacy of other medicaments or can be suitable for supporting the administration and delivery of the composition. The term "adjuvant" refers to a broad spectrum of substances. Typically, these substances can increase the immunogenicity of an antigen. For example, an adjuvant can be recognized by the innate immune system and, for example, can induce an innate immune response (i.e., a nonspecific immune response). "Adjuvant" does not usually induce an adaptive immune response. In the context of the present invention, an adjuvant can enhance the efficacy of an antigenic peptide or protein provided by a nucleic acid. In this case, at least one adjuvant can be selected from any adjuvant known to those skilled in the art and suitable for the context of the present invention (i.e., supporting the induction of an immune response in a subject (e.g., a human subject)).
因此，第二方面的组合物可包含至少一种佐剂，其中该至少一种佐剂可合适地选自WO2016/203025中所提供的任何佐剂，在此将其按引用并入。WO2016/203025的权利要求2至17中任一项所公开的佐剂，优选WO2016/203025的权利要求17中所公开的佐剂是尤其适合的，将与其相关的特定内容在此按引用并入。佐剂可适当地使用且包含于第二方面的组合物或第四方面的疫苗中，例如以减少针对所编码蛋白质的足够免疫反应所需的核酸量和/或提高组合物/疫苗用于老年人的治疗/疫苗接种的功效。在冠状病毒组合物或疫苗的情况中合适的佐剂(特别是针对包含第三方面的多肽的组合物)可以是Toll样受体9(TLR9)激动剂佐剂，CpG 1018TM。Therefore, the composition of the second aspect may include at least one adjuvant, wherein the at least one adjuvant may be suitably selected from any adjuvant provided in WO2016/203025, which is incorporated herein by reference. The adjuvant disclosed in any one of claims 2 to 17 of WO2016/203025, preferably the adjuvant disclosed in claim 17 of WO2016/203025, is particularly suitable, and the specific content related thereto is incorporated herein by reference. The adjuvant may be appropriately used and included in the composition of the second aspect or the vaccine of the fourth aspect, for example to reduce the amount of nucleic acid required for a sufficient immune response to the encoded protein and/or to improve the efficacy of the composition/vaccine for treatment/vaccination of the elderly. A suitable adjuvant in the case of a coronavirus composition or vaccine (particularly for a composition comprising a polypeptide of the third aspect) may be a Toll-like receptor 9 (TLR9) agonist adjuvant, CpG 1018TM.
除本文所指定的组分外，第二方面的组合物可包含至少一种其他组分，该组分可选自：进一步的抗原(例如以肽或蛋白质的形式，优选衍生自冠状病毒)或进一步的抗原编码核酸(优选编码肽或蛋白质，优选衍生自冠状病毒)；进一步的免疫治疗剂；一种或多种辅助物质(细胞因子，如单核因子、淋巴因子、白介素或趋化因子)；或任何进一步的化合物，其由于与人类Toll样受体的结合亲和力(作为配体)而已知为免疫刺激的；和/或佐剂核酸，优选免疫刺激性RNA(isRNA)，例如CpG-RNA等。In addition to the components specified herein, the composition of the second aspect may comprise at least one further component which may be selected from: a further antigen (e.g. in the form of a peptide or protein, preferably derived from a coronavirus) or a further antigen encoding nucleic acid (preferably encoding a peptide or protein, preferably derived from a coronavirus); a further immunotherapeutic agent; one or more auxiliary substances (cytokines, such as monokines, lymphokines, interleukins or chemokines); or any further compound which is known to be immunostimulatory due to its binding affinity to human Toll-like receptors (as ligands); and/or an adjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA), such as CpG-RNA or the like.
在优选的实施方式中，包封至少一种RNA的包含基于脂质的载体(例如，LNP)的组合物作为液体在储存后是稳定的，例如，在约5℃的温度下作为液体储存至少2周后是稳定的。In a preferred embodiment, the composition comprising a lipid-based carrier (eg, LNP) encapsulating at least one RNA is stable upon storage as a liquid, for example, stable upon storage as a liquid at a temperature of about 5°C for at least 2 weeks.
如本文使用的，“稳定的”是指包封RNA的包含基于脂质的载体(例如，LNP)的液体组合物，其中对于各种物理化学参数的测量值在储存后在限定范围内。在一个实施方式中，分析包封RNA的包含基于脂质的载体的液体组合物，以根据各种参数评估稳定性。合适的稳定性参数包括，但不限于，RNA完整性、Z-平均颗粒大小、多分散性指数(PDI)、液体组合物中游离RNA的量、RNA的包封效率(结合基于脂质的载体的以百分比计的RNA比例)、包封RNA的基于脂质的载体的形状和形态、pH、摩尔渗透压浓度或浊度。此外，“稳定的”是指包封RNA的包含基于脂质的载体的组合物，其中针对各种功能性参数的测量值在储存后在限定的范围内。在一个实施方式中，分析包封RNA的包含基于脂质的载体的液体组合物以评估液体组合物的效能，包括例如编码的肽或蛋白质的表达、特定抗体滴度的诱导、中和抗体滴度的诱导、T细胞的诱导、液体组合物的反应原性，包括例如先天性免疫反应的诱导等。As used herein, "stable" refers to a liquid composition comprising a lipid-based carrier (e.g., LNP) of an encapsulated RNA, wherein the measured values for various physicochemical parameters are within a limited range after storage. In one embodiment, the liquid composition comprising a lipid-based carrier of an encapsulated RNA is analyzed to assess stability according to various parameters. Suitable stability parameters include, but are not limited to, RNA integrity, Z-average particle size, polydispersity index (PDI), the amount of free RNA in the liquid composition, the encapsulation efficiency of RNA (in combination with the RNA ratio in percentage based on a lipid-based carrier), the shape and form, pH, molar osmotic pressure concentration or turbidity of the lipid-based carrier of the encapsulated RNA. In addition, "stable" refers to a composition comprising a lipid-based carrier of an encapsulated RNA, wherein the measured values for various functional parameters are within a limited range after storage. In one embodiment, the liquid composition comprising a lipid-based carrier of an encapsulated RNA is analyzed to assess the effectiveness of the liquid composition, including the expression of, for example, encoded peptides or proteins, the induction of specific antibody titers, the induction of neutralizing antibody titers, the induction of T cells, the reactivity of the liquid composition, including the induction of, for example, innate immune responses, etc.
在优选的实施方式中，术语“稳定的”是指RNA完整性。In a preferred embodiment, the term "stable" refers to RNA integrity.
术语“RNA完整性”通常描述液体组合物中是否存在完整的RNA序列。低的RNA完整性可能是由于特别是RNA降解、RNA断裂、不正确或不完整的RNA化学合成、不正确的碱基配对、修饰的核苷酸的整合或已经整合的核苷酸的修饰、缺少加帽或不完整的加帽、缺少聚腺苷酸化或不完整的聚腺苷酸化或者不完整的RNA体外转录。RNA是脆弱的分子，其可以容易地降解，其可以由例如温度、核糖核酸酶、pH或其他因素(例如，亲核性攻击、水解等)引起，这可以降低RNA完整性并且因此降低RNA的功能性。The term "RNA integrity" generally describes whether there is a complete RNA sequence in a liquid composition. Low RNA integrity may be due to, inter alia, RNA degradation, RNA fragmentation, incorrect or incomplete RNA chemical synthesis, incorrect base pairing, integration of modified nucleotides or modification of already integrated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation or incomplete RNA in vitro transcription. RNA is a fragile molecule that can be easily degraded, which can be caused by, for example, temperature, ribonucleases, pH or other factors (e.g., nucleophilic attack, hydrolysis, etc.), which can reduce RNA integrity and therefore reduce the functionality of the RNA.
在优选的实施方式中，组合物的RNA具有至少约50％，优选至少约60％，更优选至少约70％，最优选至少约80％或约90％的RNA完整性。合适地使用分析性HPLC，优选分析性RP-HPLC测定了RNA。In preferred embodiments, the RNA of the composition has an RNA integrity of at least about 50%, preferably at least about 60%, more preferably at least about 70%, most preferably at least about 80% or about 90%. The RNA is suitably assayed using analytical HPLC, preferably analytical RP-HPLC.
本领域技术人员可以从多种不同的色谱或电泳方法选择用于测定RNA完整性。色谱和电泳方法是本领域公知的。在使用色谱法(例如，RP-HPLC)的情况下，RNA完整性的分析可以基于确定相应色谱图中全长RNA的峰面积(或“峰下的面积”)。峰面积可以通过任何合适的软件来测定，其评价检测器系统的信号。测定峰面积的过程也称为积分。代表全长RNA的峰面积通常相对于相应样品中总RNA的峰面积来设定。RNA完整性可以以％RNA完整性来表示。Those skilled in the art can choose from a variety of different chromatographic or electrophoretic methods for determining RNA integrity. Chromatographic and electrophoretic methods are well known in the art. When using chromatography (e.g., RP-HPLC), the analysis of RNA integrity can be based on determining the peak area (or "area under the peak") of the full-length RNA in the corresponding chromatogram. The peak area can be determined by any suitable software that evaluates the signal of the detector system. The process of determining the peak area is also referred to as integration. The peak area representing the full-length RNA is usually set relative to the peak area of the total RNA in the corresponding sample. RNA integrity can be expressed as % RNA integrity.
在本发明的这些方面的情况中，RNA完整性可以使用分析性(RP)-HPLC来测定。通常，可以用去污剂(例如，约2％ Triton X100)处理包封RNA的包含基于脂质的载体的液体组合物的测试样品，以解离基于脂质的载体并释放包封的RNA。使用合适的结合化合物，例如，Agencourt AMPure XP珠(Beckman Coulter，Brea，CA，USA)，基本上根据制造商的说明，捕获释放的RNA。制备RNA样品后，可以进行分析性(RP)HPLC来测定RNA的完整性。通常，为了测定RNA完整性，使用例如注射用水(WFI)，将RNA样品稀释至0.1g/l的浓度。约10μl稀释的RNA样品可以注入HPLC柱(例如，单体聚(苯乙烯-二乙烯苯)基质)中。可使用标准条件进行分析性(RP)HPLC，例如：梯度1：缓冲液A(0.1M TEAA(pH 7.0))；缓冲液B(含有25％乙腈的0.1M TEAA(pH 7.0))。以30％缓冲液B开始，梯度在2min内扩展至32％缓冲液B，随后在15分钟内以1ml/min的流动速率扩展至55％缓冲液B。HPLC色谱图通常在260nm的波长下记录。所获得的色谱图可使用软件评估且以百分比(％)测定相对峰面积可，如本领域通常已知的。相对峰面积指示具有100％ RNA完整性的RNA的量。由于注射至HPLC中的RNA的量通常为已知的，因此相对峰面积的分析提供关于RNA完整性的信息。因此，如果注射总共例如100ngRNA，且100ng测定为相对峰面积，则RNA完整性将为100％。如果例如相对峰面积对应于80ng，则RNA完整性为80％。因此，在本发明的情况中，RNA完整性使用分析性HPLC，优选分析性RP-HPLC来测定。In the case of these aspects of the present invention, RNA integrity can be measured using analytical (RP)-HPLC. Typically, a test sample of a liquid composition comprising a lipid-based carrier that encapsulates RNA can be treated with a detergent (e.g., about 2% Triton X100) to dissociate the lipid-based carrier and release the encapsulated RNA. Use a suitable binding compound, for example, Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), substantially according to the manufacturer's instructions, to capture the released RNA. After preparing the RNA sample, analytical (RP) HPLC can be performed to measure the integrity of the RNA. Typically, in order to measure RNA integrity, using, for example, water for injection (WFI), the RNA sample is diluted to a concentration of 0.1 g/l. About 10 μl of diluted RNA samples can be injected into an HPLC column (e.g., a monomer poly (styrene-divinylbenzene) matrix). Analytical (RP) HPLC can be performed using standard conditions, for example: Gradient 1: Buffer A (0.1 M TEAA (pH 7.0)); Buffer B (0.1 M TEAA (pH 7.0) containing 25% acetonitrile). Starting with 30% buffer B, the gradient is extended to 32% buffer B in 2 min, followed by 55% buffer B in 15 minutes at a flow rate of 1 ml/min. HPLC chromatograms are typically recorded at a wavelength of 260 nm. The chromatograms obtained can be evaluated using software and the relative peak areas can be determined as percentages (%), as is generally known in the art. The relative peak area indicates the amount of RNA with 100% RNA integrity. Since the amount of RNA injected into the HPLC is generally known, analysis of the relative peak area provides information about RNA integrity. Thus, if a total of, for example, 100 ng RNA is injected, and 100 ng is determined as the relative peak area, the RNA integrity will be 100%. If, for example, the relative peak area corresponds to 80 ng, the RNA integrity is 80%. Therefore, in the context of the present invention, RNA integrity is determined using analytical HPLC, preferably analytical RP-HPLC.
在优选的实施方式中，液体组合物中包含的80％的RNA是包封的，优选地组合物中包含的85％的RNA是包封的，组合物中包含的90％的RNA是包封的，最优选组合物中95％或更多的RNA是包封的。包封的百分比可以通过Ribogreen测定来确定，如本领域已知的。In a preferred embodiment, 80% of the RNA contained in the liquid composition is encapsulated, preferably 85% of the RNA contained in the composition is encapsulated, 90% of the RNA contained in the composition is encapsulated, and most preferably 95% or more of the RNA in the composition is encapsulated. The percentage of encapsulation can be determined by Ribogreen assay, as known in the art.
在实施方式中，组合物包含至少一种RNA探测模式识别受体的至少一种拮抗剂。这样的拮抗剂可以优选共同配制在如本文定义的基于脂质的载体中。In an embodiment, the composition comprises at least one antagonist of at least one RNA probing pattern recognition receptor.Such antagonists may preferably be co-formulated in a lipid-based carrier as defined herein.
至少一种RNA探测模式识别受体的合适的拮抗剂公开于PCT专利申请PCT/EP2020/072516中，在此将其全部公开内容按引用并入。特别地，将涉及PCT/EP2020/072516的权利要求1至94任一项中限定的至少一种RNA探测模式识别受体的合适的拮抗剂的公开内容并入。Suitable antagonists of at least one RNA detection pattern recognition receptor are disclosed in PCT patent application PCT/EP2020/072516, the entire disclosure of which is hereby incorporated by reference. In particular, the disclosure relating to suitable antagonists of at least one RNA detection pattern recognition receptor as defined in any one of claims 1 to 94 of PCT/EP2020/072516 is incorporated.
在优选的实施方式中，组合物包含选自Toll样受体，优选TLR7和/或TLR8的至少一种RNA探测模式识别受体的至少一种拮抗剂。In a preferred embodiment, the composition comprises at least one antagonist of at least one RNA detecting pattern recognition receptor selected from the group consisting of Toll-like receptors, preferably TLR7 and/or TLR8.
在实施方式中，至少一种RNA探测模式识别受体的至少一种拮抗剂选自核苷酸、核苷酸类似物、核酸、肽、蛋白质、小分子、脂质，或这些中任何一种的片段、变体或衍生物。In an embodiment, the at least one antagonist of at least one RNA probing pattern recognition receptor is selected from a nucleotide, a nucleotide analog, a nucleic acid, a peptide, a protein, a small molecule, a lipid, or a fragment, variant or derivative of any of these.
在优选的实施方式中，至少一种RNA探测模式识别受体的至少一种拮抗剂是单链寡核苷酸，优选单链RNA寡核苷酸。In a preferred embodiment, the at least one antagonist of at least one RNA detecting pattern recognition receptor is a single stranded oligonucleotide, preferably a single stranded RNA oligonucleotide.
在实施方式中，至少一种RNA探测模式识别受体的拮抗剂是单链寡核苷酸，其包含与选自PCT/EP2020/0752516的SEQ ID NO:85-212的核酸序列，或这些序列中任一个的片段相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In an embodiment, the antagonist of at least one RNA detection pattern recognition receptor is a single-stranded oligonucleotide comprising a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO:85-212 of PCT/EP2020/0752516, or a fragment of any of these sequences, or consists of the same.
在优选的实施方式中，至少一种RNA探测模式识别受体的拮抗剂是单链寡核苷酸，其包含与选自PCT/EP2020/0752516的SEQ ID NO:85-87、149-212的核酸序列，或这些序列中任一个的片段相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列，或由其组成。In a preferred embodiment, the antagonist of at least one RNA detection pattern recognition receptor is a single-stranded oligonucleotide comprising a nucleic acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence selected from SEQ ID NO: 85-87, 149-212 of PCT/EP2020/0752516, or a fragment of any of these sequences, or consists of the same.
本发明的情况中至少一种RNA探测模式识别受体的的特别优选的拮抗剂是5’-GAGCGmG CCA-3’(PCT/EP2020/0752516的SEQ ID NO:85)，或其片段。A particularly preferred antagonist of at least one RNA-detecting pattern recognition receptor in the context of the present invention is 5'-GAGCGmG CCA-3' (SEQ ID NO: 85 of PCT/EP2020/0752516), or a fragment thereof.
在实施方式中，如本文定义的至少一种RNA探测模式识别受体的至少一种拮抗剂与至少一种核酸(优选如本文定义的编码SRAS-CoV-2抗原肽或蛋白质的RNA)的摩尔比范围合适地为约1:1至约100:1，或约20:1至约80:1的范围。In an embodiment, the molar ratio of at least one antagonist of at least one RNA detection pattern recognition receptor as defined herein to at least one nucleic acid (preferably an RNA encoding a SRAS-CoV-2 antigenic peptide or protein as defined herein) is suitably in the range of about 1:1 to about 100:1, or about 20:1 to about 80:1.
在实施方式中，其中如本文定义的至少一种RNA探测模式识别受体的至少一种拮抗剂与至少一种核酸(优选如本文定义的编码SRAS-CoV-2抗原肽或蛋白质的RNA)的重量与重量比率范围合适地为约1:1至约1:30，或约1:2至约1:10的范围。In an embodiment, the weight to weight ratio of at least one antagonist of at least one RNA detection pattern recognition receptor as defined herein to at least one nucleic acid (preferably an RNA encoding a SRAS-CoV-2 antigenic peptide or protein as defined herein) is suitably in the range of about 1:1 to about 1:30, or about 1:2 to about 1:10.
疫苗：vaccine :
在第三方面中，本发明提供了一种疫苗，例如，针对引起COVID-19疾病的SARS-CoV-2(之前为nCoV-2019)冠状病毒的疫苗。该疫苗针对多种SARS-CoV-2冠状病毒可以是有效的。疫苗针对一种或多种SARS-CoV-2冠状病毒和一种或多种非冠状病毒也是有效的(例如，疫苗针对SARS-CoV-2病毒和流感病毒可以是有效的)。In a third aspect, the invention provides a vaccine, for example, a vaccine for the SARS-CoV-2 (formerly nCoV-2019) coronavirus that causes the COVID-19 disease. The vaccine may be effective against a variety of SARS-CoV-2 coronaviruses. The vaccine may also be effective against one or more SARS-CoV-2 coronaviruses and one or more non-coronaviruses (e.g., the vaccine may be effective against SARS-CoV-2 virus and influenza virus).
在第四方面的优选实施方式中，疫苗包含至少一种核酸(例如，DNA或RNA)，优选至少一种第一方面的RNA，或第二方面的组合物。In a preferred embodiment of the fourth aspect, the vaccine comprises at least one nucleic acid (eg DNA or RNA), preferably at least one RNA of the first aspect, or the composition of the second aspect.
在其他实施方式中，疫苗包含至少一种第三方面中限定的多肽。In other embodiments, the vaccine comprises at least one polypeptide as defined in the third aspect.
在其他实施方式中，疫苗包含至少一种第一方面中限定的质粒DNA或腺病毒DNA。In other embodiments, the vaccine comprises at least one plasmid DNA or adenoviral DNA as defined in the first aspect.
值得注意的是，涉及第二方面的组合物的实施方式同样可以作为第四方面的疫苗的合适的实施方式来阅读和理解。此外，涉及第四方面的疫苗的实施方式同样可以作为第二方面的组合物的合适的实施方式来阅读和理解。此外，第一方面(本发明的核酸)的情况中描述的特征和实施方式必须作为第四方面的组合物的合适的实施方式来阅读和理解。It should be noted that the embodiment of the composition relating to the second aspect can be read and understood as the suitable embodiment of the vaccine of the fourth aspect equally. In addition, the embodiment of the vaccine relating to the fourth aspect can be read and understood as the suitable embodiment of the composition of the second aspect equally. In addition, the features and embodiments described in the situation of the first aspect (nucleic acid of the present invention) must be read and understood as the suitable embodiment of the composition of the fourth aspect.
术语“疫苗”由本领域普通技术人员来认识和理解，并且例如旨在是提供至少一个表位或抗原(优选免疫原)的预防或治疗材料。在本发明的情况中，由本发明第一方面的核酸(所述核酸包含编码源自SARS-CoV-2冠状病毒的抗原肽或蛋白质的编码序列)或第二方面的组合物(包含至少一个第一方面的核酸)合适地提供抗原或抗原功能。在其他实施方式中，由本发明第三方面的多肽来提供抗原或抗原功能。The term "vaccine" is recognized and understood by those of ordinary skill in the art, and is intended, for example, to be a preventive or therapeutic material that provides at least one epitope or antigen (preferably an immunogen). In the context of the present invention, the antigen or antigenic function is suitably provided by the nucleic acid of the first aspect of the present invention (the nucleic acid comprises a coding sequence encoding an antigenic peptide or protein derived from the SARS-CoV-2 coronavirus) or the composition of the second aspect (comprising at least one nucleic acid of the first aspect). In other embodiments, the antigen or antigenic function is provided by the polypeptide of the third aspect of the present invention.
在优选的实施方式中，第二方面的疫苗或组合物引发适应性免疫反应，优选针对冠状病毒，优选针对SARS-CoV-2冠状病毒的适应性免疫反应。In a preferred embodiment, the vaccine or composition of the second aspect elicits an adaptive immune response, preferably against a coronavirus, preferably against the SARS-CoV-2 coronavirus.
在特别优选的实施方式中，第二方面的疫苗或组合物引发可以有效地中和病毒(优选SARS-CoV-2冠状病毒)的功能性抗体。In a particularly preferred embodiment, the vaccine or composition of the second aspect elicits functional antibodies that can effectively neutralize a virus, preferably a SARS-CoV-2 coronavirus.
在进一步优选的实施方式中，第二方面的疫苗或组合物通过诱导粘膜IgA抗体引发粘膜IgA免疫。In a further preferred embodiment, the vaccine or composition of the second aspect elicits mucosal IgA immunity by inducing mucosal IgA antibodies.
在特别优选的实施方式中，第二方面的疫苗或组合物引发可以有效地中和病毒(优选SARS-CoV-2冠状病毒)的功能性抗体。In a particularly preferred embodiment, the vaccine or composition of the second aspect elicits functional antibodies that can effectively neutralize a virus, preferably a SARS-CoV-2 coronavirus.
在进一步特别优选的实施方式中，第二方面的疫苗或组合物诱导针对冠状病毒，优选针对SARS-CoV-2冠状病毒的广泛的、功能性细胞T-细胞反应。In a further particularly preferred embodiment, the vaccine or composition of the second aspect induces a broad, functional cellular T-cell response against a coronavirus, preferably against the SARS-CoV-2 coronavirus.
在进一步特别优选的实施方式中，第二方面的疫苗或组合物诱导针对冠状病毒，优选针对SARS-CoV-2冠状病毒的良好平衡的B细胞和T细胞反应。In a further particularly preferred embodiment, the vaccine or composition of the second aspect induces a well-balanced B cell and T cell response against a coronavirus, preferably against the SARS-CoV-2 coronavirus.
根据优选的实施方式，如本文定义的疫苗可以进一步包含药学上可接受的载体和任选至少一种第二方面的情况中具体说明的佐剂。According to a preferred embodiment, the vaccine as defined herein may further comprise a pharmaceutically acceptable carrier and optionally at least one adjuvant as specified in the context of the second aspect.
该情况中合适的佐剂可以选自WO2016/203025的权利要求17中公开的佐剂。Suitable adjuvants in this case may be selected from the adjuvants disclosed in claim 17 of WO2016/203025.
在优选的实施方式中，疫苗是单价疫苗。In a preferred embodiment, the vaccine is a monovalent vaccine.
术语“一价疫苗”、“一价组合物”、“单价疫苗”或“单价组合物”由本领域普通技术人员认识和理解，并且例如旨在表示仅包含一种来自病原体的抗原或抗原构建体的组合物或疫苗。因此，所述疫苗或组合物仅包含一种编码单一生物体的单一抗原或抗原构建体的核酸物质。术语“单价疫苗”包括针对单一价的免疫。在本发明的情况中，单价SARS-CoV-2冠状病毒疫苗或组合物将包含至少一种编码源自一种特定的SARS-CoV-2冠状病毒的一种单一抗原肽或蛋白质的核酸。The terms "monovalent vaccine", "monovalent composition", "monovalent vaccine" or "monovalent composition" are recognized and understood by a person of ordinary skill in the art and are intended, for example, to mean a composition or vaccine comprising only one antigen or antigenic construct from a pathogen. Thus, the vaccine or composition comprises only one nucleic acid material encoding a single antigen or antigenic construct of a single organism. The term "monovalent vaccine" includes immunization against a single valency. In the context of the present invention, a monovalent SARS-CoV-2 coronavirus vaccine or composition will comprise at least one nucleic acid encoding a single antigenic peptide or protein derived from one specific SARS-CoV-2 coronavirus.
在实施方式中，疫苗是包含多种或至少超过一种第一方面的情况中限定的核酸物质的多价疫苗。涉及第二方面的情况中公开的多价组合物的实施方式同样可以作为多价疫苗的合适的实施方式来阅读和理解。In an embodiment, the vaccine is a multivalent vaccine comprising multiple or at least more than one nucleic acid material defined in the context of the first aspect. The embodiments of the multivalent composition disclosed in the context of the second aspect can also be read and understood as suitable embodiments of the multivalent vaccine.
术语“多价(polyvalent)疫苗”、“多价(polyvalent)组合物”、“多重价(multivalent)疫苗”或“多重价(multivalent)组合物”由本领域普通技术人员来认识和理解，并且例如旨在表示包含来自超过一种病毒(例如，不同的SARS-CoV-2冠状病毒分离株)的抗原，或包含同一SARS-CoV-2冠状病毒的不同抗原或抗原构建体，或其任意组合的组合物或疫苗。该术语描述所述疫苗或组合物具有超过一个价。在本发明的情况中，多价SARS-CoV-2冠状病毒疫苗将包含编码源自几种不同的SARS-CoV-2冠状病毒(例如，不同的SARS-CoV-2冠状病毒分离株)的抗原肽或蛋白质的核酸序列，或包含编码来自同一SARS-CoV-2冠状病毒的不同抗原或抗原构建体的核酸序列，或其组合。The terms "polyvalent vaccine", "polyvalent composition", "multivalent vaccine" or "multivalent composition" are recognized and understood by those of ordinary skill in the art, and are intended, for example, to refer to a composition or vaccine comprising antigens from more than one virus (e.g., different SARS-CoV-2 coronavirus isolates), or comprising different antigens or antigenic constructs of the same SARS-CoV-2 coronavirus, or any combination thereof. The term describes that the vaccine or composition has more than one valency. In the context of the present invention, a multivalent SARS-CoV-2 coronavirus vaccine will comprise nucleic acid sequences encoding antigenic peptides or proteins derived from several different SARS-CoV-2 coronaviruses (e.g., different SARS-CoV-2 coronavirus isolates), or nucleic acid sequences encoding different antigens or antigenic constructs from the same SARS-CoV-2 coronavirus, or a combination thereof.
在优选的实施方式中，多价(polyvalent)或多重价(multivalent)疫苗包含至少一种第二方面中限定的多价组合物。特别优选的是如“本发明的多价组合物”部分中限定的多价组合物。In a preferred embodiment, the polyvalent or multivalent vaccine comprises at least one multivalent composition as defined in the second aspect. Particularly preferred aremultivalent compositions as defined in the section "Multivalent compositions of the invention ".
在一些实施方式中，疫苗包含第二方面中限定的至少一种RNA探测模式识别受体的至少一种拮抗剂。In some embodiments, the vaccine comprises at least one antagonist of at least one RNA probing pattern recognition receptor as defined in the second aspect.
疫苗通常包含安全且有效量的核酸(例如，DNA或RNA)，优选第一方面的RNA或第二方面的组合物(或第三方面的多肽)。如本文使用的，“安全且有效的量”表示足以显著诱导与冠状病毒(优选SARS-CoV-2冠状病毒)感染相关的疾病或失调的正向改变。同时，“安全且有效的量”足够小以避免严重的副作用。涉及本发明的核酸、组合物或疫苗，表述“安全且有效的量”优选表示适用于刺激适应性免疫系统对抗冠状病毒的核酸、组合物或疫苗的量，其方式使得没有过量或损伤性免疫反应(例如，先天性免疫反应)。Vaccines generally comprise a safe and effective amount of nucleic acid (e.g., DNA or RNA), preferably the RNA of the first aspect or the composition of the second aspect (or the polypeptide of the third aspect). As used herein, a "safe and effective amount" means a positive change sufficient to significantly induce a disease or disorder associated with a coronavirus (preferably a SARS-CoV-2 coronavirus) infection. At the same time, a "safe and effective amount" is small enough to avoid serious side effects. Relating to nucleic acids, compositions or vaccines of the present invention, the expression "safe and effective amount" preferably means the amount of nucleic acids, compositions or vaccines suitable for stimulating the adaptive immune system against coronaviruses, in a manner such that there is no excessive or damaging immune response (e.g., innate immune response).
如上限定的核酸、组合物或疫苗的“安全且有效的量”将结合待治疗的特定病症以及待治疗的患者的年龄和身体状况、病症的严重程度、治疗的持续时间、伴随治疗的性质、所用的特定的药学上可接受的载体和技术人员知识和经验内的相似因素而改变。此外，核酸、组合物或疫苗的“安全且有效的量”可以取决于施用/递送途径(皮内、肌内、鼻内)、施用装置(射流注射、针注射、微针贴剂、电穿孔装置)和/或复合/配制(鱼精蛋白复合或LNP包封，DNA或RNA)。此外，核酸、组合物或疫苗的“安全且有效的量”可以取决于待治疗的受试者(婴儿、妊娠女性、免疫受损的人类受试者等)的身体状况。"Safe and effective amount" of nucleic acid, composition or vaccine as defined above will be changed in conjunction with age and physical condition of specific illness to be treated and patient to be treated, the severity of illness, the duration of treatment, the nature of concomitant therapy, specific pharmaceutically acceptable carrier used and similar factors in technical personnel's knowledge and experience. In addition, "safe and effective amount" of nucleic acid, composition or vaccine can depend on administration/delivery route (intradermal, intramuscular, intranasal), application device (jet injection, needle injection, microneedle patch, electroporation device) and/or compound/preparation (protamine compound or LNP encapsulation, DNA or RNA). In addition, "safe and effective amount" of nucleic acid, composition or vaccine can depend on the physical condition of experimenter to be treated (infant, pregnant women, immunocompromised human subjects etc.).
根据本发明，疫苗可以用于人类医学目的以及也可以用于兽医医学目的(哺乳动物、脊椎动物或鸟类物种)。According to the invention, the vaccines can be used for human medical purposes as well as for veterinary medical purposes (mammalian, vertebrate or avian species).
本文使用的药学上可接受的载体优选包括疫苗的液体或非液体基质。如果疫苗以液体形式提供，载体将是水，通常是无热原水；等渗盐水或缓冲(水性)溶液，例如，磷酸盐、柠檬酸盐等缓冲液。优选地，将Ringer-乳酸盐溶液用作根据本发明的疫苗或组合物的液体基质，如WO2006/122828中所述的，在此将涉及合适的缓冲溶液的公开内容按引用并入。其他优选的用作疫苗或组合物的液体基质的溶液，特别是用于包含LNP的组合物/疫苗，包括蔗糖和/或海藻糖。Pharmaceutically acceptable carriers used herein preferably include liquid or non-liquid matrices of vaccines. If the vaccine is provided in liquid form, the carrier will be water, typically pyrogen-free water; isotonic saline or buffered (aqueous) solutions, for example, buffers such as phosphates, citrates, etc. Preferably, Ringer-lactate solutions are used as liquid matrices for vaccines or compositions according to the present invention, as described in WO2006/122828, the disclosure of which is incorporated herein by reference for suitable buffer solutions. Other preferred solutions for use as liquid matrices for vaccines or compositions, particularly for compositions/vaccines containing LNPs, include sucrose and/or trehalose.
如本文定义的药学上可接受的载体的选择原则上借由施用根据本发明的药物组合物或疫苗的方式来确定。疫苗优选局部施用。局部施用途径一般包括例如局部施用途径，但也包括皮内、经皮、皮下或肌内注射或病灶内、颅内、肺内、心内、关节内及舌下注射。更优选地，根据本发明的组合物或疫苗可通过皮内、皮下或肌内途径，优选通过注射施用，注射可为无针和/或针注射。在本发明的情况中，优选的是肌内注射。组合物/疫苗因此优选配制成液体或固体形式。待施用的根据本发明的疫苗或组合物的合适量可以通过常规实验，例如通过使用动物模型来测定。此类模型包括兔、绵羊、小鼠、大鼠、狗和非人类灵长类动物模型，但不意味着任何限制。优选的用于注射的单位剂型包括水、生理盐水或其混合物的无菌溶液。此类溶液的pH应调节至约7.4。The selection of a pharmaceutically acceptable carrier as defined herein is determined in principle by administering the pharmaceutical composition or vaccine according to the present invention. The vaccine is preferably administered topically. Local administration routes generally include, for example, topical administration routes, but also include intradermal, percutaneous, subcutaneous or intramuscular injections or intralesional, intracranial, intrapulmonary, intracardiac, intraarticular and sublingual injections. More preferably, the composition or vaccine according to the present invention can be administered by intradermal, subcutaneous or intramuscular routes, preferably by injection, and the injection can be needle-free and/or needle injection. In the case of the present invention, intramuscular injection is preferred. The composition/vaccine is therefore preferably formulated into a liquid or solid form. The appropriate amount of the vaccine or composition to be administered according to the present invention can be determined by routine experiments, for example, by using an animal model. Such models include rabbits, sheep, mice, rats, dogs and non-human primate models, but do not imply any limitation. Preferred unit dosage forms for injection include sterile solutions of water, saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4.
如本文定义的疫苗或组合物可以包含一种或多种如上限定的辅助物质或佐剂，以进一步增加免疫原性。优选由此获得组合物/疫苗中所包含的核酸和辅助物质(其可以任选与上述的疫苗或组合物共同配制(或分开配制))的协同作用。此类免疫原性增加剂或化合物可以单独提供(不与疫苗或组合物共同配制)并单独施用。Vaccines or compositions as defined herein may include one or more auxiliary substances or adjuvants as defined above to further increase immunogenicity. Preferably, the synergistic effect of the nucleic acid and auxiliary substances (which may optionally be co-formulated (or separately formulated) with the above-mentioned vaccine or composition) contained in the composition/vaccine is thus obtained. Such immunogenicity increasing agents or compounds may be provided separately (not co-formulated with the vaccine or composition) and administered separately.
疫苗优选以冻干或喷雾干燥形式来提供(第二方面的情况中所述的)。这样的冻干或喷雾干燥的疫苗通常包含海藻糖和/或蔗糖并且在施用于受试者前在合适的液体中重建。在一些方面中，实施方式的冻干疫苗包含与LNP复合的实施方式的mRNA。在一些方面中，冻干的组合物具有低于约10％的水含量。例如，冻干的组合物可以具有约0.1％至10％，0.1％至7.5％，或0.5％至7.5％的水含量，优选地冻干的组合物具有约0.5％至约5.0％的水含量。The vaccine is preferably provided in a freeze-dried or spray-dried form (described in the case of the second aspect). Such freeze-dried or spray-dried vaccines generally contain trehalose and/or sucrose and are reconstituted in a suitable liquid before being administered to a subject. In some aspects, the freeze-dried vaccine of the embodiment comprises the mRNA of the embodiment compounded with LNP. In some aspects, the freeze-dried composition has a water content lower than about 10%. For example, the freeze-dried composition can have a water content of about 0.1% to 10%, 0.1% to 7.5%, or 0.5% to 7.5%, preferably the freeze-dried composition has a water content of about 0.5% to about 5.0%.
在优选的实施方式中，将治疗有效量的核酸、组合物、多肽、疫苗施用于受试者诱导受试者中抗SARS-CoV-2冠状病毒的中和抗体滴度。In a preferred embodiment, a therapeutically effective amount of a nucleic acid, composition, polypeptide, or vaccine is administered to a subject to induce neutralizing antibody titers against the SARS-CoV-2 coronavirus in the subject.
在一些实施方式中，中和抗体滴度为至少100个中和单位/毫升(NU/mL)，至少500NU/mL，或至少1000NU/mL。In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units/milliliter (NU/mL), at least 500 NU/mL, or at least 1000 NU/mL.
在一些实施方式中，在施用核酸、组合物、多肽或疫苗后约1至约72小时在受试者中产生了可检测的冠状病毒抗原水平。In some embodiments, detectable levels of coronavirus antigen are produced in a subject from about 1 to about 72 hours after administration of the nucleic acid, composition, polypeptide, or vaccine.
在一些实施方式中，在施用核酸、组合物、多肽或疫苗后约1天至约72天在受试者的血清中产生至少100NU/ml，至少500NU/ml或至少1000NU/ml的(抗冠状病毒的)中和抗体滴度。In some embodiments, a neutralizing antibody titer (against coronavirus) of at least 100 NU/ml, at least 500 NU/ml or at least 1000 NU/ml is produced in the serum of the subject from about 1 day to about 72 days after administration of the nucleic acid, composition, polypeptide or vaccine.
在一些实施方式中，相对于未接种疫苗的对照受试者的中和抗体滴度或相对于接种了活减毒病毒疫苗、灭活的病毒疫苗或蛋白质亚基病毒疫苗的受试者的中和抗体滴度，中和抗体滴度足以将冠状病毒感染减少至少50％。In some embodiments, the neutralizing antibody titer is sufficient to reduce coronavirus infection by at least 50% relative to the neutralizing antibody titer of an unvaccinated control subject or relative to the neutralizing antibody titer of a subject vaccinated with a live attenuated virus vaccine, an inactivated virus vaccine, or a protein subunit virus vaccine.
在一些实施方式中，相对于未接种疫苗的对照受试者的中和抗体滴度，中和抗体滴度和/或T细胞免疫反应足以降低无症状病毒感染率。In some embodiments, the neutralizing antibody titer and/or T cell immune response is sufficient to reduce the asymptomatic viral infection rate relative to the neutralizing antibody titer in unvaccinated control subjects.
在一些实施方式中，中和抗体滴度和/或T细胞免疫反应足以防止受试者的病毒潜伏。In some embodiments, the neutralizing antibody titer and/or T cell immune response is sufficient to prevent viral latency in the subject.
在一些实施方式中，中和抗体滴度足以阻断病毒与受试者的表皮细胞的融合。In some embodiments, the neutralizing antibody titer is sufficient to block viral fusion to epidermal cells of the subject.
在一些实施方式中，在单次1ug-100ug剂量的核酸、组合物、多肽或疫苗后20天内，或在第二1ug-100μg剂量的核酸、组合物、多肽或疫苗后40天内，诱导了中和抗体滴度。In some embodiments, neutralizing antibody titers are induced within 20 days after a single 1 ug-100 ug dose of a nucleic acid, composition, polypeptide, or vaccine, or within 40 days after a second 1 ug-100 μg dose of a nucleic acid, composition, polypeptide, or vaccine.
在优选的实施方式中，将治疗有效量的核酸、组合物、多肽或疫苗施用于受试者在受试者中诱导针对冠状病毒的T细胞免疫反应。在优选的实施方式中，T细胞免疫反应包括CD4+T细胞免疫反应和/或CD8+T细胞免疫反应。In a preferred embodiment, a therapeutically effective amount of nucleic acid, composition, polypeptide or vaccine is administered to a subject to induce a T cell immune response against coronavirus in the subject. In a preferred embodiment, the T cell immune response includes a CD4+T cell immune response and/or a CD8+T cell immune response.
试剂盒或部件套装、应用、医疗用途、治疗方法：Kit or set of parts, application, medical use, method of treatment :
在第四方面中，本发明提供了适用于治疗或预防冠状病毒感染的试剂盒或部件套装。优选地，所述试剂盒或部件套装适用于治疗或预防冠状病毒，优选SARS-CoV-2(之前为nCoV-2-2019)冠状病毒感染。In a fourth aspect, the present invention provides a kit or a kit of parts suitable for treating or preventing coronavirus infection. Preferably, the kit or kit of parts is suitable for treating or preventing coronavirus, preferably SARS-CoV-2 (formerly nCoV-2-2019) coronavirus infection.
值得注意的是，涉及第一方面的核酸、第二方面的组合物、第三方面的多肽和第四方面的疫苗的实施方式同样可以作为本发明第五方面的试剂盒或部件套装的合适的实施方式来阅读和理解。It is noteworthy that the embodiments involving the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect and the vaccine of the fourth aspect can also be read and understood as suitable embodiments of the kit or component set of the fifth aspect of the present invention.
在优选的实施方式中，试剂盒或部件套装包含至少一种核酸(例如，RNA或DNA)，优选至少一种第一方面的RNA、至少一种第二方面的组合物，和/或至少一种第三方面的多肽，和/或至少一种第四方面的疫苗。In a preferred embodiment, the kit or set of parts comprises at least one nucleic acid (e.g., RNA or DNA), preferably at least one RNA of the first aspect, at least one composition of the second aspect, and/or at least one polypeptide of the third aspect, and/or at least one vaccine of the fourth aspect.
在实施方式中，试剂盒或部件套装包含至少一种第一方面中限定的DNA，例如，至少一种质粒DNA和/或至少一种腺病毒DNA。In an embodiment, the kit or kit of parts comprises at least one DNA as defined in the first aspect, for example at least one plasmid DNA and/or at least one adenoviral DNA.
在实施方式中，试剂盒或部件套装包含至少一种第三方面中限定的多肽。In an embodiment, the kit or set of parts comprises at least one polypeptide as defined in the third aspect.
此外，试剂盒或部件套装可以包含用于增溶的液体媒剂，和/或提供关于组分的施用和剂量的信息的技术说明。In addition, the kit or set of parts may contain a liquid vehicle for solubilization, and/or instructions providing information on the administration and dosage of the components.
试剂盒可以进一步包含第二方面的组合物和/或第四方面的疫苗的情况中描述的其他组分。The kit may further comprise the other components described in the context of the composition of the second aspect and/or the vaccine of the fourth aspect.
所述试剂盒的技术说明可以含有关于施用和剂量和患者组的信息。这样的试剂盒，优选部件套装，可以应用于例如本文提及的任何应用或使用，优选用于第一方面的核酸、第二方面的组合物、第三方面的多肽或第四方面的疫苗的使用，用于治疗或预防由冠状病毒(优选SARS-CoV-2冠状病毒)引起的感染或疾病或与其相关的失调。The technical description of the kit may contain information about administration and dosage and patient groups. Such a kit, preferably a kit of parts, can be applied to any application or use mentioned herein, for example, preferably for the use of the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect or the vaccine of the fourth aspect, for the treatment or prevention of infection or disease caused by a coronavirus (preferably a SARS-CoV-2 coronavirus) or a disorder associated therewith.
优选地，在试剂盒的单独部分中提供核酸、组合物、多肽或疫苗，其中核酸、组合物、多肽或疫苗优选是冻干的。Preferably, the nucleic acid, composition, polypeptide or vaccine is provided in separate parts of the kit, wherein the nucleic acid, composition, polypeptide or vaccine is preferably lyophilized.
试剂盒可以进一步包含媒剂(例如，缓冲液)作为部分，其用于溶解核酸、组合物、多肽或疫苗。The kit may further comprise as part a vehicle (eg, a buffer) for dissolving the nucleic acid, composition, polypeptide or vaccine.
在优选的实施方式中，如本文定义的试剂盒或部件套装包含Ringer乳酸盐溶液。In a preferred embodiment, the kit or set of parts as defined herein comprises Ringer's lactate solution.
在优选的实施方式中，如本文定义的试剂盒或部件套装包含用于施用组合物/疫苗的多剂量容器。In a preferred embodiment, the kit or set of parts as defined herein comprises multiple dose containers for administration of the composition/vaccine.
以上任一试剂盒可以用于如本文限定的治疗或预防中。更优选地，以上任一试剂盒可以用作疫苗，优选针对由冠状病毒引起的(优选由SARS-CoV-2冠状病毒引起的)感染的疫苗。Any of the above kits may be used in treatment or prevention as defined herein. More preferably, any of the above kits may be used as a vaccine, preferably a vaccine for infection caused by a coronavirus (preferably caused by the SARS-CoV-2 coronavirus).
在优选的实施方式中，试剂盒或部件套装包含以下组分：In a preferred embodiment, the kit or kit of parts comprises the following components:
a)至少一个包含如本文定义的组合物或SARS-CoV-2疫苗的容器或小瓶，其中组合物或SARS-CoV-2疫苗具有核酸浓度，优选约100μg/ml至约1mg/ml范围的RNA浓度，优选约100μg/ml至约500μg/ml的范围，例如，约270μg/ml。a) at least one container or vial comprising a composition or SARS-CoV-2 vaccine as defined herein, wherein the composition or SARS-CoV-2 vaccine has a nucleic acid concentration, preferably an RNA concentration in the range of about 100 μg/ml to about 1 mg/ml, preferably in the range of about 100 μg/ml to about 500 μg/ml, for example, about 270 μg/ml.
b)至少一个包含无菌稀释缓冲液的稀释容器或小瓶，合适地包含NaCl的缓冲液，任选包含防腐剂；b) at least one dilution container or vial comprising a sterile dilution buffer, suitably a buffer comprising NaCl, optionally containing a preservative;
c)至少一个用于将组合物或疫苗从储存容器转移到稀释容器的装置；和c) at least one device for transferring the composition or vaccine from a storage container to a dilution container; and
d)至少一个用于将最终稀释的组合物或疫苗施用于受试者的注射器，优选配制用于肌内施用于人类受试者，其中最终稀释的组合物或疫苗具有核酸浓度，优选约10μg/ml至约100μg/ml范围的RNA浓度，优选约10μg/ml至约50μg/ml的范围，例如，约24μg/ml。d) at least one syringe for administering the final diluted composition or vaccine to a subject, preferably formulated for intramuscular administration to a human subject, wherein the final diluted composition or vaccine has a nucleic acid concentration, preferably a RNA concentration in the range of about 10 μg/ml to about 100 μg/ml, preferably in the range of about 10 μg/ml to about 50 μg/ml, for example, about 24 μg/ml.
在实施方式中，试剂盒或部件套装包含超过一种基于mRNA的SARS-CoV-2组合物/疫苗，优选地In an embodiment, the kit or set of parts comprises more than one mRNA-based SARS-CoV-2 composition/vaccine, preferably
-至少一种在第一小瓶或容器中提供的如本文定义的疫苗，其中疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:163、149或24837的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one vaccine as defined herein provided in a first vial or container, wherein the vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 163, 149 or 24837, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7. Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical to the nucleic acid sequence of SEQ ID NO: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936 or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:27394、27417、27486、28762、28650-28655、28789-28794的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27394, 27417, 27486, 28762, 28650-28655, 28789-28794, preferably in a lipid nanoparticle (LNP) ), which has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:27394、27417、27486、28762的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, corresponding to SEQ ID NO: 27394, 27417, 27486, 28762 or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:28639-28642、28778-28781、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的。- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 28639-28642, 28778-28781, 28925-28928, 28933-28936, preferably in a lipid nanoparticle (LNP) ), which has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified.
在实施方式中，试剂盒或部件套装包含超过一种基于mRNA的SARS-CoV-2组合物/疫苗，优选地In an embodiment, the kit or set of parts comprises more than one mRNA-based SARS-CoV-2 composition/vaccine, preferably
-至少一种在第一小瓶或容器中提供的如本文定义的疫苗，其中疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:163、149或24837的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one vaccine as defined herein provided in a first vial or container, wherein the vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 163, 149 or 24837, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)) of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7. Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical to the nucleic acid sequence of SEQ ID NO: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940 or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 3%, 94%, 95%, 96%, 97%, 98% or 99% identical, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:27532、27555、27624、28852、28699-28704、28879-28884的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 27532, 27555, 27624, 28852, 28699-28704, 28879-28884, preferably in a lipid nanoparticle (LNP) ), which has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:27532、27555、27624、28852的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的；和/或- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, corresponding to SEQ ID NO: 27532, 27555, 27624, 28852 or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence, preferably formulated in lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified; and/or
-至少一种在第一小瓶或容器中提供的如本文定义的进一步的疫苗，其中组合物/疫苗包含至少一种核酸，优选RNA，其与SEQ ID NO:28688-28691、28868-28871、28929-28932、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同，优选在脂质纳米颗粒(LNP)中配制，其具有大约50:10:38.5:1.5的摩尔比，优选47.5:10:40.8:1.7或更优选47.4:10:40.9:1.7的比例(mol％)的阳离子脂质III-3(ALC-0315)、DSPC、胆固醇和式(IVa)的PEG-脂质(n＝49，或n＝45(ALC-0159))。优选地，核酸，优选mRNA，不是化学修饰的。- at least one further vaccine as defined herein provided in the first vial or container, wherein the composition/vaccine comprises at least one nucleic acid, preferably RNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 28688-28691, 28868-28871, 28929-28932, 28937-28940, preferably in a lipid nanoparticle (LNP) ), which has a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (mol%) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (n=49, or n=45 (ALC-0159)). Preferably, the nucleic acid, preferably mRNA, is not chemically modified.
在实施方式中，试剂盒或部件套装包含两种不同的SARS-CoV-2疫苗，其用于初免接种和加强接种：In an embodiment, the kit or set of parts comprises two different SARS-CoV-2 vaccines for a primary vaccination and a booster vaccination:
-至少一种在第一小瓶或容器中提供的如本文定义的初免疫苗，其中该疫苗是如本文定义的基于mRNA的SARS-CoV-2疫苗；和- at least one priming vaccine as defined herein provided in a first vial or container, wherein the vaccine is an mRNA-based SARS-CoV-2 vaccine as defined herein; and
-至少一种在第一小瓶或容器中提供的如本文定义的加强疫苗，其中该组合物/疫苗是如本文定义的基于腺病毒的SARS-CoV-2疫苗。- At least one booster vaccine as defined herein provided in a first vial or container, wherein the composition/vaccine is an adenovirus-based SARS-CoV-2 vaccine as defined herein.
在实施方式中，试剂盒或部件套装包含两种不同的SARS-CoV-2疫苗，其用于初免接种和加强接种：In an embodiment, the kit or set of parts comprises two different SARS-CoV-2 vaccines for a primary vaccination and a booster vaccination:
-至少一种在第一小瓶或容器中提供的如本文定义的加强疫苗，其中该疫苗是如本文定义的基于mRNA的SARS-CoV-2疫苗；和- at least one booster vaccine as defined herein provided in a first vial or container, wherein the vaccine is an mRNA-based SARS-CoV-2 vaccine as defined herein; and
-至少一种在第一小瓶或容器中提供的如本文定义的初免疫苗，其中该组合物/疫苗是如本文定义的基于腺病毒的SARS-CoV-2疫苗。- At least one priming vaccine as defined herein provided in a first vial or container, wherein the composition/vaccine is an adenovirus-based SARS-CoV-2 vaccine as defined herein.
组合：combination :
第五方面涉及至少两种第一方面中限定的核酸序列、至少两种第二方面的情况中限定的组合物、至少两种第三方面中限定的多肽、至少两种第四方面的情况中限定的疫苗或至少两种第五方面中限定的试剂盒的组合。The fifth aspect relates to a combination of at least two nucleic acid sequences defined in the first aspect, at least two compositions defined in the second aspect, at least two polypeptides defined in the third aspect, at least two vaccines defined in the fourth aspect, or at least two kits defined in the fifth aspect.
在本发明的情况中，术语“组合”优选表示至少两种组分的组合存在，优选地至少两种第一方面中限定的核酸序列、至少两种第二方面的情况中限定的组合物、至少两种第三方面中限定的多肽、至少两种第四方面的情况中限定的疫苗或至少两种第五方面中限定的试剂盒。这种组合的组分可以作为单独的实体出现。因此，组合的组分的施用可以同时地或在时间上错开进行，在同一施用部位或在不同的施用部位。In the context of the present invention, the term "combination" preferably means the presence of a combination of at least two components, preferably at least two nucleic acid sequences defined in the first aspect, at least two compositions defined in the context of the second aspect, at least two polypeptides defined in the third aspect, at least two vaccines defined in the context of the fourth aspect, or at least two kits defined in the fifth aspect. The components of this combination may appear as separate entities. Therefore, the administration of the components of the combination may be performed simultaneously or staggered in time, at the same application site or at different application sites.
值得注意的是，涉及第一方面的核酸、第二方面的组合物、第三方面的多肽和第四方面的疫苗，或第五方面的试剂盒或部件套装的实施方式同样可以作为第六方面的组合的组分的合适的实施方式来阅读和理解。It is noteworthy that the embodiments involving the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect and the vaccine of the fourth aspect, or the kit or component set of the fifth aspect can also be read and understood as suitable embodiments of the components of the combination of the sixth aspect.
在实施方式中，组合可以包含多种或至少超过一种核酸物质，例如，如本发明的第一方面的情况中限定的RNA物质，其中核酸物质作为分开的组分来提供。In embodiments, the combination may comprise a plurality or at least more than one nucleic acid species, for example an RNA species as defined in the context of the first aspect of the invention, wherein the nucleic acid species are provided as separate components.
优选地，如本文定义的组合可以包含2、3、4、5、6、7、8、9或10或更多种不同的核酸，例如，本发明的第一方面的情况中限定的RNA物质；2、3、4、5、6、7、8、9或10种不同的如本发明的第二方面的情况中限定的组合物；2、3、4、5、6、7、8、9或10种不同的如本发明的第三方面的情况中限定的多肽；2、3、4、5、6、7、8、9或10种不同的如本发明的第三方面的情况中限定的疫苗，其中核酸物质、组合物、多肽、疫苗作为单独的组分来提供。Preferably, the combination as defined herein may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different nucleic acids, e.g., RNA substances as defined in the context of the first aspect of the invention; 2, 3, 4, 5, 6, 7, 8, 9 or 10 different compositions as defined in the context of the second aspect of the invention; 2, 3, 4, 5, 6, 7, 8, 9 or 10 different polypeptides as defined in the context of the third aspect of the invention; 2, 3, 4, 5, 6, 7, 8, 9 or 10 different vaccines as defined in the context of the third aspect of the invention, wherein the nucleic acid substances, compositions, polypeptides, vaccines are provided as separate components.
在实施方式中，组合包含2、3、4或5种包含在单独的组分中的RNA，优选RNA物质，其中所述核酸物质包含与选自SEQ ID NO:149、22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、23529-23534、27386-27408、23535-23552、27409-27431、23590-23606、27478-27500、28736-28776、28638-28686、28777-28825、28925-28928、28933-28936的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列或由其组成，和任选地，至少一种药学上可接受的载体或赋形剂，其中2、3、4或5种核酸物质的每一种编码SARS-CoV-2冠状病毒的不同的抗原肽或蛋白质。In an embodiment, the combination comprises 2, 3, 4 or 5 RNA species contained in separate components, preferably RNA species, wherein the nucleic acid species comprises a nucleic acid species selected from the group consisting of SEQ ID NO: 149, 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936 are identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences or consist of them, and optionally, at least one pharmaceutically acceptable carrier or excipient, wherein each of the 2, 3, 4 or 5 nucleic acid substances encodes a different antigenic peptide or protein of the SARS-CoV-2 coronavirus.
在实施方式中，组合包含2、3、4或5种包含在单独的组分中的RNA，优选RNA物质，其中所述核酸物质包含与选自SEQ ID NO:24837-24854、27524-27546、24855-24872、27547-27569、24909-24926、27616-27638、28827-28866、28687-28735、28867-28915、28929-28932、28937-28940的核酸序列相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列或由其组成，和任选地，至少一种药学上可接受的载体或赋形剂，其中2、3、4或5种核酸物质的每一种编码SARS-CoV-2冠状病毒的不同的抗原肽或蛋白质。In an embodiment, the combination comprises 2, 3, 4 or 5 RNA species contained in separate components, preferably RNA species, wherein the nucleic acid species comprises a nucleic acid sequence that is identical to or at least 70% identical to a nucleic acid sequence selected from SEQ ID NO: 24837-24854, 27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940. %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences, or consisting of them, and optionally, at least one pharmaceutically acceptable carrier or excipient, wherein each of the 2, 3, 4 or 5 nucleic acid substances encodes a different antigenic peptide or protein of the SARS-CoV-2 coronavirus.
在下文中，提供了特别优选的组合的实施方式，其中组合的每种组分作为分开的实体来提供。In the following, particularly preferred embodiments of the combination are provided, wherein each component of the combination is provided as a separate entity.
优选地，组合的至少2、3、4、5、6、7、8、9、10或甚至更多种不同的核酸物质、组合物、疫苗各自编码不同的融合前稳定的刺突蛋白(如第一方面中限定的)。优选地，通过在刺突蛋白的残基K986和V987处引入两个连续的脯氨酸置换(氨基酸位置根据参考SEQ ID NO:1)获得融合前构象的稳定。因此，在优选的实施方式中，至少2、3、4、5、6、7、8、9、10个融合前稳定的刺突蛋白(S_stab)各自包含至少一个融合前稳定突变，其中该至少一个融合前稳定突变包含以下氨基酸置换：K986P和V987P(氨基酸位置根据参考SEQ ID NO:1)。Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different nucleic acid substances, compositions, vaccines in combination each encode a different pre-fusion stabilized spike protein (as defined in the first aspect). Preferably, stabilization of the pre-fusion conformation is obtained by introducing two consecutive proline substitutions at residues K986 and V987 of the spike protein (amino acid positions according to reference SEQ ID NO: 1). Therefore, in a preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 pre-fusion stabilized spike proteins (S_stab) each comprise at least one pre-fusion stabilizing mutation, wherein the at least one pre-fusion stabilizing mutation comprises the following amino acid substitutions: K986P and V987P (amino acid positions according to reference SEQ ID NO: 1).
因此，组合的至少2、3、4、5、6、7、8、9、10种或甚至更多种不同的核酸物质、组合物、疫苗各自编码不同的融合前稳定的刺突蛋白，其中至少2、3、4、5、6、7、8、9、10种或甚至更多种稳定的刺突蛋白选自与SEQ ID NO:10、22738、22740、22742、22744、22746、22748、22750、22752、22754、22756、22758、22959-22964、27087-27109、28540-28588、28917-28920的任一个或这些中任一个的免疫原性片段或免疫原性变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列。Thus, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different nucleic acid substances, compositions, vaccines in combination each encode a different pre-fusion stabilized spike protein, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more stabilized spike proteins are selected from the group consisting of SEQ ID NO:10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920, or an immunogenic fragment or immunogenic variant of any of these, or an amino acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
在优选的实施方式中，组合包含一种核酸物质、组合物、疫苗，其包含编码与SEQID NO:10的任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列，其中多价组合物任选包含至少2、3、4种选自以下的进一步RNA物质：In a preferred embodiment, the combination comprises a nucleic acid substance, composition, vaccine comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 10, wherein the multivalent composition optionally comprises at least 2, 3, 4 further RNA substances selected from the group consisting of:
i)一种核酸物质包含编码与SEQ ID NO:22961任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或i) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22961; and/or
ii)一种核酸物质包含编码与SEQ ID NO:22960任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或ii) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22960; and/or
iii)一种核酸物质包含编码与SEQ ID NO:22963任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或iii) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22963; and/or
iv)一种核酸物质包含编码与SEQ ID NO:22959任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；iv) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22959;
v)一种核酸物质包含编码与SEQ ID NO:27070、27093任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；v) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27070, 27093;
vi)一种核酸物质包含编码与SEQ ID NO:27071、27094任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；vi) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27071, 27094;
vii)一种核酸物质包含编码与SEQ ID NO:27072、27095任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；vii) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27072, 27095;
viii)一种核酸物质包含编码与SEQ ID NO:27073、27096、28545任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列；和/或viii) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 27073, 27096, 28545; and/or
ix)一种核酸物质包含编码与SEQ ID NO:22964任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。ix) a nucleic acid agent comprising a coding sequence encoding an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 22964.
x)一种核酸物质包含编码与SEQ ID NO:28541-28544、28917-28920任一个相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的编码序列。x) a nucleic acid agent comprising a coding sequence that encodes an amino acid sequence that is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920.
优选地，组合的至少2、3、4、5、6、7、8、9、10种或甚至更多种不同的核酸物质、组合物、疫苗包含核酸编码序列，其各自编码不同的融合前稳定的刺突蛋白，其中至少2、3、4、5、6、7、8、9、10种或甚至更多种核酸编码序列选自与SEQ ID NO:116、136、137、146、148、149、151、162、163、165、22765、22767、22769、22771、22773、22775、22777、22779、22781、22783、22785、22792、22794、22796、22798、22800、22802、22804、22806、22808、22810、22812、22819、22821、22823、22825、22827、22829、22831、22833、22835、22837、22839、28916、23089-23148、23150-23184、23309-23368、23370-23404、23529-23588、23590-23624、24837-24944、27110-27907、28589-28915、28916、28921-28940任一个或这些中任一个的片段或变体相同或至少70％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的核酸序列。优选地，每种mRNA物质包含帽1结构，和任选地，每种mRNA物质不包含修饰的核苷酸。Preferably, the combined at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different nucleic acid substances, compositions, vaccines comprise nucleic acid coding sequences, each of which encodes a different pre-fusion stabilized spike protein, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more nucleic acid coding sequences are selected from the group consisting of SEQ ID NO:116, 136, 137, 146, 148, 149, 151, 162, 163, 165, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 22792, 22794, 22 796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835, 22837 , 22839, 28916, 23089-23148, 23150-23184, 23309-23368, 23370-23404, 23529-23588, 23590-23624, 24837-24944, 27110-27907, 28589-28915, 28916, 28921-28940, or a fragment or variant of any of these, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. Preferably, each mRNA species comprises a Cap 1 structure, and optionally, each mRNA species does not comprise modified nucleotides.
在特定的实施方式中，组合的第一组分包含病毒载体疫苗/组合物，如基于腺病毒载体的疫苗，例如，ADZ1222或Ad26.COV-2.S，和第二组分包含基于核酸的疫苗/组合物，优选如本文限定的基于mRNA的疫苗。In a specific embodiment, the first component of the combination comprises a viral vector vaccine/composition, such as an adenoviral vector-based vaccine, e.g., ADZ1222 or Ad26.COV-2.S, and the second component comprises a nucleic acid-based vaccine/composition, preferably an mRNA-based vaccine as defined herein.
第一和第二/进一步医疗用途：First and second/further medical use :
进一步的方面涉及所提供的核酸、组合物、疫苗、试剂盒或组合的第一医疗用途。A further aspect relates to a first medical use of a provided nucleic acid, composition, vaccine, kit or combination.
值得注意的是，涉及第一方面的核酸、第二方面的组合物、第三方面的多肽和第四方面的疫苗，或第五方面的试剂盒或部件套装，或组合的实施方式同样可以作为本发明的医疗用途的合适的实施方式来阅读和理解。It is worth noting that the embodiments involving the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect and the vaccine of the fourth aspect, or the kit or component set of the fifth aspect, or the combination can also be read and understood as suitable embodiments of the medical use of the present invention.
因此，本发明提供了用作药物的至少一种第一方面中限定的核酸(例如，DNA或RNA)，优选RNA，用作药物的第二方面中限定的组合物，用作药物的第三方面中限定的多肽，用作药物的第四方面中限定的疫苗，和用作药物的第五方面中限定的试剂盒或部件套装，及组合。Thus, the present invention provides at least one nucleic acid (e.g., DNA or RNA), preferably RNA, as defined in the first aspect for use as a medicament, a composition as defined in the second aspect for use as a medicament, a polypeptide as defined in the third aspect for use as a medicament, a vaccine as defined in the fourth aspect for use as a medicament, and a kit or set of parts as defined in the fifth aspect for use as a medicament, and combinations thereof.
此外，本发明提供了核酸、组合物、多肽、疫苗或试剂盒，或者组合的几种应用和用途。In addition, the present invention provides several applications and uses of the nucleic acid, composition, polypeptide, vaccine or kit, or combination.
特别地，核酸(优选RNA)、组合物、多肽、疫苗或试剂盒，或者组合可以用于人类医学目的以及用于兽医学目的，优选用于人类医学目的。In particular, the nucleic acid (preferably RNA), composition, polypeptide, vaccine or kit, or the combination can be used for human medical purposes as well as for veterinary purposes, preferably for human medical purposes.
特别地，核酸(优选RNA)、组合物、多肽、疫苗或试剂盒或部件套装或者组合用作人类医学目的的药物，其中所述核酸(优选RNA)、组合物、多肽、疫苗或试剂盒或部件套装可以适用于年幼婴儿、新生儿、免疫受损的接受者以及妊娠和母乳喂养的女性和老年人。特别地，核酸(优选RNA)、组合物、多肽、疫苗或试剂盒或部件套装用作人类医学目的的药物，其中所述核酸(优选RNA)、组合物、多肽、疫苗或试剂盒或部件套装特别适用于老年人受试者。In particular, nucleic acids (preferably RNA), compositions, polypeptides, vaccines or kits or kits of parts or combinations are used as medicaments for human medical purposes, wherein the nucleic acids (preferably RNA), compositions, polypeptides, vaccines or kits of parts or kits of parts may be suitable for young infants, newborns, immunocompromised recipients, and pregnant and breast-feeding women and the elderly. In particular, nucleic acids (preferably RNA), compositions, polypeptides, vaccines or kits of parts or kits are used as medicaments for human medical purposes, wherein the nucleic acids (preferably RNA), compositions, polypeptides, vaccines or kits of parts or kits of parts are particularly suitable for elderly subjects.
所述核酸(优选RNA)、组合物、多肽、疫苗或试剂盒或组合用作人类医学目的的药物，其中所述RNA、组合物、疫苗，或试剂盒或部件套装特别适用于肌内注射或皮内注射。The nucleic acid (preferably RNA), composition, polypeptide, vaccine or kit or combination is used as a medicament for human medical purposes, wherein the RNA, composition, vaccine, or kit or set of parts is particularly suitable for intramuscular or intradermal injection.
在再另一方面中，本发明涉及所提供的核酸、组合物、多肽、疫苗或试剂盒或者组合的第二医疗用途。In yet another aspect, the present invention relates to a second medical use of the provided nucleic acid, composition, polypeptide, vaccine or kit or combination.
因此，本发明提供了至少一种第一方面中限定的核酸，优选RNA，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)感染，或与所述感染相关的障碍或疾病，如COVID-19；第二方面中限定的组合物，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)感染，或与所述感染相关的障碍或疾病，如COVID-19；第三方面中限定的多肽，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)的感染，或与所述感染相关的障碍或疾病，如COVID-19；第四方面中限定的疫苗，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)的感染，或与所述感染相关的障碍或疾病，如COVID-19；第五方面中限定的试剂盒或部件套装，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)的感染，或与所述感染相关的障碍或疾病，如COVID-19；第六方面中限定的组合，用于治疗或预防冠状病毒(优选SARS-CoV-2冠状病毒)的感染，或与所述感染相关的障碍或疾病，如COVID-19。Thus, the present invention provides at least one nucleic acid, preferably RNA, as defined in the first aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19; a composition as defined in the second aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19; a polypeptide as defined in the third aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19; a vaccine as defined in the fourth aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19; a kit or component set as defined in the fifth aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19; and a combination as defined in the sixth aspect, for treating or preventing infection by a coronavirus (preferably SARS-CoV-2 coronavirus), or a disorder or disease associated with said infection, such as COVID-19.
在实施方式中，第一方面的核酸(优选RNA)、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合，用于冠状病毒(优选SARS-CoV-2冠状病毒)感染的治疗或预防。In an embodiment, the nucleic acid (preferably RNA) of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, or the kit or component set of the fifth aspect, or the combination of the sixth aspect is used to treat or prevent coronavirus (preferably SARS-CoV-2 coronavirus) infection.
特别地，第一方面的核酸(优选RNA)、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合可以用于由冠状病毒(优选SARS-CoV-2冠状病毒)引起的感染的预防性(暴露前预防或暴露后预防)和/或治疗性处理的方法中。In particular, the nucleic acid (preferably RNA) of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, or the kit or set of parts of the fifth aspect, or the combination of the sixth aspect can be used in a method for prophylactic (pre-exposure prophylaxis or post-exposure prophylaxis) and/or therapeutic treatment of infection caused by a coronavirus (preferably SARS-CoV-2 coronavirus).
特别地，第一方面的核酸(优选RNA)、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合可以用于由SARS-CoV-2冠状病毒感染引起的COVID-19疾病的预防性(暴露前预防或暴露后预防)和/或治疗性处理的方法中。In particular, the nucleic acid (preferably RNA) of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, or the kit or component set of the fifth aspect, or the combination of the sixth aspect can be used in a method for preventive (pre-exposure prophylaxis or post-exposure prophylaxis) and/or therapeutic treatment of COVID-19 disease caused by SARS-CoV-2 coronavirus infection.
核酸、组合物、多肽或疫苗，或者组合可以优选局部施用。特别地，组合物或多肽或疫苗或组合可以通过皮内、皮下、鼻内或肌内途径施用。在实施方式中，本发明的核酸、组合物、多肽、疫苗可以通过常规针注射或无针射流注射来施用。在该情况中，优选的是肌内注射。Nucleic acid, composition, polypeptide or vaccine, or combination can be preferably topically applied.Especially, composition or polypeptide or vaccine or combination can be applied by intradermal, subcutaneous, intranasal or intramuscular route.In an embodiment, nucleic acid, composition, polypeptide, vaccine of the present invention can be applied by conventional needle injection or needle-free jet injection.In this case, preferably intramuscular injection.
在其中使用质粒DNA并包含在组合物或疫苗或组合中的实施方式中，组合物/疫苗/组合可以通过使用电穿孔装置的电穿孔来施用，例如，用于皮内或肌内递送的电穿孔装置。合适地，可以使用US7245963B2中描述的装置，特别是由US7245963B2的权利要求1至68限定的装置。In embodiments where plasmid DNA is used and included in a composition or vaccine or combination, the composition/vaccine/combination can be administered by electroporation using an electroporation device, e.g., an electroporation device for intradermal or intramuscular delivery. Suitably, the device described in US7245963B2, in particular the device defined by claims 1 to 68 of US7245963B2, can be used.
在其中使用腺病毒DNA并包含在组合物或疫苗或组合中的实施方式中，组合物/疫苗/组合可以通过鼻内施用来施用。In embodiments where adenoviral DNA is used and contained in a composition or vaccine or combination, the composition/vaccine/combination may be administered by intranasal administration.
在实施方式中，包含在本文限定的组合物或疫苗或组合中的核酸以约100ng至约500ug的量，约1ug至约200ug的量，约1ug至约100ug的量，约5ug至约100ug的量，优选约10ug至约50ug的量来提供，具体地，以约1ug、2ug、3ug、4ug、5ug、8ug、9ug、10ug、11ug、12ug、13ug、14ug、15ug、16ug、20ug、25ug、30ug、35ug、40ug、45ug、50ug、55ug、60ug、65ug、70ug、75ug、80ug、85ug、90ug、95ug或100ug的量来提供。In an embodiment, the nucleic acid contained in the composition, vaccine or combination defined herein is provided in an amount of about 100ng to about 500ug, about 1ug to about 200ug, about 1ug to about 100ug, about 5ug to about 100ug, preferably about 10ug to about 50ug, specifically, about 1ug, 2ug, 3ug, 4ug, 5ug, 8ug, 9ug, 10ug, 11ug, 12ug, 13ug, 14ug, 15ug, 16ug, 20ug, 25ug, 30ug, 35ug, 40ug, 45ug, 50ug, 55ug, 60ug, 65ug, 70ug, 75ug, 80ug, 85ug, 90ug, 95ug or 100ug.
在一些实施方式中，包含核酸的疫苗，或包含核酸的组合物以有效量配制以在受试者中产生抗原特异性免疫反应。在一些实施方式中，核酸的有效量为1ug至200ug，1ug至100ug或5ug至100ug的总剂量。In some embodiments, a vaccine comprising a nucleic acid, or a composition comprising a nucleic acid, is formulated in an effective amount to produce an antigen-specific immune response in a subject. In some embodiments, the effective amount of the nucleic acid is a total dose of 1 ug to 200 ug, 1 ug to 100 ug, or 5 ug to 100 ug.
在其中核酸在基于脂质的载体(例如，LNP)中提供的实施方式中，在一个剂量中包含的本文限定的PEG-脂质的量低于约50μg PEG脂质，优选低于约45μg PEG脂质，更优选低于约40μg PEG脂质。In embodiments where the nucleic acid is provided in a lipid-based carrier (e.g., LNP), the amount of PEG-lipid defined herein contained in one dose is less than about 50 μg PEG lipid, preferably less than about 45 μg PEG lipid, more preferably less than about 40 μg PEG lipid.
在一个剂量中具有低的PEG脂质量可以降低副作用(例如，过敏)的风险。Having a low amount of PEG lipid in a dose can reduce the risk of side effects (eg, allergies).
在特别优选的实施方式中，一个剂量中包含的PEG-脂质的量在约3.5μg PEG脂质至约35μg PEG脂质的范围内。In particularly preferred embodiments, the amount of PEG-lipid included in one dose is in the range of about 3.5 μg PEG lipid to about 35 μg PEG lipid.
在其中核酸在基于脂质的载体(例如，LNP)提供的实施方式中，在一个剂量中包含的本文限定的阳离子脂质的量低于约400μg阳离子脂质，优选低于约350μg阳离子脂质，更优选低于约300μg阳离子脂质。In embodiments where the nucleic acid is provided in a lipid-based carrier (e.g., LNP), the amount of cationic lipid defined herein contained in one dose is less than about 400 μg cationic lipid, preferably less than about 350 μg cationic lipid, more preferably less than about 300 μg cationic lipid.
在一个剂量中具有低的阳离子脂质量可以降低副作用(例如，发烧)的风险。Having a low amount of cationic lipid in a dose can reduce the risk of side effects (eg, fever).
在特别优选的实施方式中，在一个剂量中包含的阳离子脂质的量在约30μg PEG脂质至约300μg PEG脂质的范围内。In particularly preferred embodiments, the amount of cationic lipid included in one dose is in the range of about 30 μg PEG lipid to about 300 μg PEG lipid.
在一个实施方式中，针对冠状病毒(优选SARS-CoV-2冠状病毒)用于受试者治疗或预防的免疫方案包括一个单一剂量的组合物或疫苗。In one embodiment, a regimen for treating or preventing a subject against a coronavirus, preferably a SARS-CoV-2 coronavirus, comprises a single dose of a composition or vaccine.
在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的1ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的2ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的3ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的4ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的5ug的剂量，在一次疫苗接种中施用于受试者的6ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的7ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的8ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的9ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的10ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的11ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的12ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的13ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的14ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的16ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的20ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的25ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的30ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的40ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的50ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的100ug的剂量。在一些实施方式中，有效量为在一次疫苗接种中施用于受试者的200ug的剂量。在该情况中“剂量”涉及本文定义的核酸，优选mRNA的有效量。In some embodiments, the effective amount is a dose of 1 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 2 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 3 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 4 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 5 ug administered to a subject in one vaccination, and a dose of 6 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 7 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 8 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 9 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 10 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 11 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 12 ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 13ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 14ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 16ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 20ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 25ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 30ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 40ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 50ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 100ug administered to a subject in one vaccination. In some embodiments, the effective amount is a dose of 200ug administered to a subject in one vaccination. In this case, "dose" relates to a nucleic acid as defined herein, preferably an effective amount of mRNA.
在优选的实施方式中，用于冠状病毒(优选SARS-CoV-2冠状病毒)感染的治疗或预防的免疫方案包括一系列单剂量(dose或dosage)的组合物或疫苗。本文使用的单剂量分别指原始/第一剂量、第二剂量或任何更多剂量，其优选施用以“加强”免疫反应。In a preferred embodiment, the immunization regimen for the treatment or prevention of coronavirus (preferably SARS-CoV-2 coronavirus) infection includes a series of single doses (dose or dosage) of the composition or vaccine. Single doses used herein refer to the original/first dose, the second dose or any more doses, respectively, which are preferably administered to "boost" the immune response.
在一些实施方式中，有效量是总共两次施用于受试者的1ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的2ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的3ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的4ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的5ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的6ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的7ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的8ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的9ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的10ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的11ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的12ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的13ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的14ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的16ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的20ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的25ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的30ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的40ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的50ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的100ug剂量。在一些实施方式中，有效量是总共两次施用于受试者的200ug剂量。在该情况中“剂量”涉及本文定义的核酸，优选mRNA的有效量。In some embodiments, the effective amount is a total of two 1ug doses administered to the subject. In some embodiments, the effective amount is a total of two 2ug doses administered to the subject. In some embodiments, the effective amount is a total of two 3ug doses administered to the subject. In some embodiments, the effective amount is a total of two 4ug doses administered to the subject. In some embodiments, the effective amount is a total of two 5ug doses administered to the subject. In some embodiments, the effective amount is a total of two 6ug doses administered to the subject. In some embodiments, the effective amount is a total of two 7ug doses administered to the subject. In some embodiments, the effective amount is a total of two 8ug doses administered to the subject. In some embodiments, the effective amount is a total of two 9ug doses administered to the subject. In some embodiments, the effective amount is a total of two 10ug doses administered to the subject. In some embodiments, the effective amount is a total of two 11ug doses administered to the subject. In some embodiments, the effective amount is a total of two 12ug doses administered to the subject. In some embodiments, the effective amount is a total of two 13ug doses administered to the subject. In some embodiments, the effective amount is a total of two 14ug doses administered to the subject. In some embodiments, the effective amount is a total of 16ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 20ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 25ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 30ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 40ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 50ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 100ug doses applied to a subject twice. In some embodiments, the effective amount is a total of 200ug doses applied to a subject twice. In this case, "dose" relates to nucleic acid defined herein, preferably an effective amount of mRNA.
在优选的实施方式中，疫苗/组合物/组合针对冠状病毒(优选抗SARS-CoV-2冠状病毒)感染对受试者免疫(本文限定的施用时)至少1年，优选至少2年。在优选的实施方式中，疫苗/组合物/组合针对冠状病毒(优选抗SARS-CoV-2冠状病毒)对受试者免疫超过2年，更优选超过3年，甚至更优选超过4年，甚至更优选超过5-10年。In a preferred embodiment, the vaccine/composition/combination immunizes the subject against coronavirus (preferably anti-SARS-CoV-2 coronavirus) infection (when administered as defined herein) for at least 1 year, preferably at least 2 years. In a preferred embodiment, the vaccine/composition/combination immunizes the subject against coronavirus (preferably anti-SARS-CoV-2 coronavirus) for more than 2 years, more preferably more than 3 years, even more preferably more than 4 years, even more preferably more than 5-10 years.
治疗和使用的方法、诊断方法和用途：Methods of treatment and use, diagnostic methods and uses :
在另一方面中，本发明涉及治疗或预防障碍的方法。In another aspect, the invention relates to methods of treating or preventing a disorder.
值得注意的是，涉及第一方面的核酸、第二方面的组合物、第三方面的多肽和第四方面的疫苗、第五方面的试剂盒或部件套装、第六方面的组合物，或医疗用途的实施方式同样可以作为本文提供的治疗方法的合适的实施方式来阅读和理解。此外，涉及本文提供的治疗方法的特定特征和实施方式也可以应用于本发明的医疗用途。It is noteworthy that the embodiments of the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, the kit or component set of the fifth aspect, the composition of the sixth aspect, or the medical use can also be read and understood as suitable embodiments of the treatment methods provided herein. In addition, the specific features and embodiments of the treatment methods provided herein can also be applied to the medical use of the present invention.
预防(抑制)或治疗疾病，特别是冠状病毒感染，涉及抑制疾病或病症的完全发展，例如，在处于疾病如冠状病毒感染的风险中的受试者中。“治疗”涉及在疾病开始发展后改善疾病或病理性病症的体征或症状的治疗性干预。涉及疾病或病理性病症的术语“改善”是指任何可观察到的治疗的有效效果。抑制疾病可以包括预防或降低疾病的风险，如预防或降低病毒感染的风险。有益效果例如可以通过怀疑的受试者中疾病的临床症状的延迟发作、疾病的一些或全部临床症状的严重程度的减轻、疾病的减缓进展、病毒载量的减少、受试者整体健康或身体状况的改善或者通过特定疾病特征生的其他参数来证明。“预防性”处理是施用于未呈现疾病体征或仅呈现早期体征的受试者的处理，用于降低产生病理状况的风险的目的。Prevention (inhibition) or treatment of disease, particularly coronavirus infection, involves inhibiting the full development of the disease or condition, for example, in a subject at risk of a disease such as coronavirus infection."Treatment" involves therapeutic intervention to improve the signs or symptoms of a disease or pathological condition after the disease begins to develop. The term "improvement" related to a disease or pathological condition refers to any observable effective effect of treatment. Inhibiting a disease may include preventing or reducing the risk of a disease, such as preventing or reducing the risk of a viral infection. Beneficial effects may be demonstrated, for example, by delayed onset of clinical symptoms of the disease in a suspected subject, alleviation of the severity of some or all of the clinical symptoms of the disease, slowed progression of the disease, reduction in viral load, improvement in the overall health or physical condition of the subject, or by other parameters characterized by a specific disease. "Preventive" treatment is a treatment applied to a subject who does not present signs of the disease or only presents early signs, for the purpose of reducing the risk of developing a pathological condition.
在优选的实施方式中，本发明涉及治疗或预防障碍的方法，其中所述方法包括将至少一种第一方面的核酸、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合应用于或施用于需要的受试者。In a preferred embodiment, the present invention relates to a method for treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof at least one of the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, or the kit or set of parts of the fifth aspect, or the combination of the sixth aspect.
在优选的实施方式中，所述障碍是冠状病毒感染，或涉及所述感染的障碍，特别是SARS-CoV-2冠状病毒感染，或与这种感染相关的障碍，例如，COVID-19。In a preferred embodiment, the disorder is a coronavirus infection, or a disorder related to such an infection, particularly a SARS-CoV-2 coronavirus infection, or a disorder associated with such an infection, e.g., COVID-19.
在优选的实施方式中，本发明涉及治疗或预防如上限定的障碍的方法，其中所述方法包括将至少一种第一方面的核酸、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合应用于或施用于需要的受试者，其中需要的受试者优选是哺乳动物受试者。In a preferred embodiment, the present invention relates to a method for treating or preventing a disorder as defined above, wherein the method comprises applying or administering at least one of the nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect or the kit or set of parts of the fifth aspect, or the combination of the sixth aspect to a subject in need thereof, wherein the subject in need thereof is preferably a mammalian subject.
在某些实施方式中，通过将至少一种第一方面的核酸、第二方面的组合物、第三方面的多肽、第四方面的疫苗或第五方面的试剂盒或部件套装，或者第六方面的组合应用于或施用于需要的受试者来治疗或预防疾病的方法进一步限定为降低受试者中的疾病负荷的方法。例如，所述方法优选降低COVID-19疾病的一种或多种症状的严重程度和/或持续时间。在一些方面中，所述方法降低了受试者需要住院、入住ICU、辅助输氧治疗和/或呼吸机治疗的可能性。在进一步的方面中，所述方法降低了受试者出现发烧、呼吸困难；嗅觉丧失和/或味觉丧失的可能性。在优选的方面中，所述方法降低了受试者患严重或中度COVID-19疾病的可能性。在某些方面中，该实施方式的方法在受试者施用该实施方式的组合物后约2周至1个月、2个月、3个月、4个月、5个月、6个月、1年或2年防止受试者的严重或中度COVID-19疾病。在优选的方面中，该实施方式的方法防止有症状的COVID-19疾病。在进一步的方面中，该实施方式的方法在受试者施用该实施方式的组合物后约2周至1个月、2个月、3个月、4个月、5个月、6个月、1年或2年防止受试者中可检测的SARS-CoV-2核酸水平。在进一步的方面中，该实施方式的方法限定为用于在受试者中提供对冠状病毒感染(例如，SARS-CoV-2感染)的保护性免疫的方法。在再进一步的方面中，该实施方式的方法在至少80％、85％、90％或95％的治疗受试者中防止中度和重度COVID-19疾病。还在进一步的方面中，该实施方式的方法在施用第二或后续免疫原性组合物(例如，增强剂施用)后约2周到约1年，在至少80％、85％、90％或95％的治疗受试者中预防中度和重度COVID-19疾病。还在进一步的方面中，该实施方式的方法在施用第二或后续组合物后约2周至约3个月、6个月、9个月、1年、1.5年、2年或3年在至少80％、85％、90％或95％的治疗受试者防止中度和重度COVID-19疾病。In certain embodiments, the method for treating or preventing a disease by applying or administering to a subject in need at least one nucleic acid of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the vaccine of the fourth aspect, or the kit or component set of the fifth aspect, or the combination of the sixth aspect to a subject in need is further defined as a method for reducing the disease load in the subject. For example, the method preferably reduces the severity and/or duration of one or more symptoms of the COVID-19 disease. In some aspects, the method reduces the likelihood that the subject will require hospitalization, admission to the ICU, assisted oxygen therapy, and/or ventilator therapy. In a further aspect, the method reduces the likelihood that the subject will have a fever, dyspnea; loss of smell and/or loss of taste. In a preferred aspect, the method reduces the likelihood that the subject will suffer from severe or moderate COVID-19 disease. In certain aspects, the method of this embodiment prevents severe or moderate COVID-19 disease in the subject from about 2 weeks to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 2 years after the subject administers the composition of this embodiment. In a preferred aspect, the method of this embodiment prevents symptomatic COVID-19 disease. In a further aspect, the method of the embodiment prevents detectable SARS-CoV-2 nucleic acid levels in the subject from about 2 weeks to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or 2 years after the subject applies the composition of the embodiment. In a further aspect, the method of the embodiment is defined as a method for providing protective immunity to coronavirus infection (e.g., SARS-CoV-2 infection) in a subject. In a further aspect, the method of the embodiment prevents moderate and severe COVID-19 disease in at least 80%, 85%, 90% or 95% of the treated subjects. In a further aspect, the method of the embodiment prevents moderate and severe COVID-19 disease in at least 80%, 85%, 90% or 95% of the treated subjects from about 2 weeks to about 1 year after the second or subsequent immunogenic composition (e.g., enhancer administration). In still a further aspect, the methods of this embodiment prevent moderate and severe COVID-19 disease in at least 80%, 85%, 90% or 95% of treated subjects from about 2 weeks to about 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years or 3 years after administration of the second or subsequent composition.
在进一步的方面中，该实施方式的方法包括(i)获得该实施方式的组合物(例如，疫苗组合物)，其中所述组合物是冻干的；(ii)将冻干组合物溶解在药学上可接受的液体载体中以产生液体组合物；和(iii)向受试者施用有效量的液体组合物。在一些方面中，冻干组合物包含小于约10％的水含量。例如，冻干组合物可优选包含约0.1％至约10％、0.5％至7.5％或0.5％至5.0％的水。In a further aspect, the method of this embodiment comprises (i) obtaining a composition (e.g., a vaccine composition) of this embodiment, wherein the composition is lyophilized; (ii) dissolving the lyophilized composition in a pharmaceutically acceptable liquid carrier to produce a liquid composition; and (iii) administering an effective amount of the liquid composition to a subject. In some aspects, the lyophilized composition comprises less than about 10% water content. For example, the lyophilized composition may preferably comprise about 0.1% to about 10%, 0.5% to 7.5%, or 0.5% to 5.0% water.
在再进一步的方面中，该实施方式的方法包括向受试者施用包含至少两种不同mRNA的疫苗组合物，每种mRNA编码不同的SARS-CoV-2刺突多肽，其各自与SEQ ID NO:10至少约95％相同(例如，与LNP复合)。在进一步的方面中，这种方法在受试者中提供足够的免疫反应，以保护受试者免受严重的COVID-19疾病至少约6个月。例如，在一些方面中，受试者可以被保护在约6个月到约1年、1.5年、2年、2.5年、3年、4年或5年免受严重的COVID-19疾病。因此，在一些方面中，该实施方式的方法提供了可以为受试者提供长期(例如，大于6个月)的免于严重疾病的保护的单剂量疫苗组合物。In a further aspect, the method of this embodiment comprises administering to a subject a vaccine composition comprising at least two different mRNAs, each encoding a different SARS-CoV-2 spike polypeptide, each of which is at least about 95% identical to SEQ ID NO: 10 (e.g., complexed with LNP). In a further aspect, this method provides a sufficient immune response in the subject to protect the subject from severe COVID-19 disease for at least about 6 months. For example, in some aspects, the subject can be protected from severe COVID-19 disease for about 6 months to about 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, or 5 years. Therefore, in some aspects, the method of this embodiment provides a single-dose vaccine composition that can provide a subject with long-term (e.g., greater than 6 months) protection from severe disease.
如本文所用的，严重COVID-19疾病定义为经历以下一种或多种的受试者：As used herein, severe COVID-19 disease is defined as a subject experiencing one or more of the following:
·休息时表明严重全身性病态的临床体征(呼吸率≥30次/分钟，心率≥125次/分钟、海平面室内空气SpO2≤93％或PaO2/FIO2<300mm Hg(根据海拔高度调整))Clinical signs of severe systemic illness at rest (respiratory rate ≥30 breaths/minute, heart rate ≥125 beats/minute, SpO2 ≤93% on room air at sea level or PaO2/FIO2 <300 mm Hg (adjusted for altitude))
·呼吸衰竭(定义为需要高流量氧气、无创通气、机械通气或ECMO)Respiratory failure (defined as requiring high-flow oxygen, noninvasive ventilation, mechanical ventilation, or ECMO)
·休克的证据(SBP＜90mm Hg，DBP＜60mm Hg，或需要血管升压药)Evidence of shock (SBP <90 mm Hg, DBP <60 mm Hg, or need for vasopressors)
·严重的肾、肝或神经功能障碍Severe renal, hepatic or neurological dysfunction
·收治至ICUAdmitted to ICU
·死亡·die
如本文所用的，中度COVID-19疾病定义为经历以下一种或多种的受试者：As used herein, moderate COVID-19 illness is defined as a subject experiencing one or more of the following:
·呼吸急促或呼吸困难Shortness of breath or difficulty breathing
·呼吸率≥20次呼吸/分钟Respiratory rate ≥ 20 breaths/minute
·SpO2异常，但在海平面室内空气中仍>93％(根据海拔高度调整)SpO2 abnormal but still >93% on room air at sea level (adjusted for altitude)
·下呼吸道疾病的临床或影像学证据Clinical or radiographic evidence of lower respiratory tract disease
·深静脉血栓形成(DVT)的放射学证据Radiographic evidence of deep vein thrombosis (DVT)
如本文所用，轻度COVID-19疾病定义为经历以下所有的受试者：As used herein, mild COVID-19 illness is defined as a subject experiencing all of the following:
·有症状，和Have symptoms, and
·没有呼吸急促或呼吸困难，和No shortness of breath or difficulty breathing, and
·无低血氧症(根据海拔高度调整)，和No hypoxemia (adjusted for altitude), and
·不符合中度或重度COVID-19疾病的病例定义Do not meet the case definition for moderate or severe COVID-19 illness
在特别优选的实施方式中，需要的受试者是哺乳动物受试者，优选人类受试者(例如新生儿、怀孕者、免疫受损者和/或老年人)。在一些实施方式中，受试者年龄在6个月至100岁、6个月至80岁、1岁至80岁、1岁至70岁、2岁至80年或2岁至60岁。在其他实施方式中，受试者是年龄不超过3岁、不超过2岁、不超过1.5岁、不超过1岁(12个月)、不超过9个月、6个月或3个月的新生儿或婴儿。在某些实施方式中，人类受试者是老年人受试者。在一些其他实施方式中，受试者是年龄至少为50、60、65或70岁的老年受试者。在进一步的方面中，根据实施方式进行治疗的受试者为61岁或以上。在更进一步的方面中，受试者为18岁至60岁。In a particularly preferred embodiment, the subject in need is a mammalian subject, preferably a human subject (e.g., a newborn, a pregnant person, an immunocompromised person, and/or an elderly person). In some embodiments, the subject is aged between 6 months and 100 years old, 6 months and 80 years old, 1 year old and 80 years old, 1 year old and 70 years old, 2 years old and 80 years old, or 2 years old and 60 years old. In other embodiments, the subject is a newborn or infant no older than 3 years old, no older than 2 years old, no older than 1.5 years old, no older than 1 year old (12 months), no older than 9 months, 6 months, or 3 months. In certain embodiments, the human subject is an elderly subject. In some other embodiments, the subject is an elderly subject at least 50, 60, 65, or 70 years old. In a further aspect, the subject treated according to the embodiment is 61 years old or more. In a further aspect, the subject is 18 to 60 years old.
在进一步的实施方式中，哺乳动物受试者是60岁或以下的人类受试者。在某些实施方式中，人类受试者是55岁、50岁、45岁或40岁或以下的人类受试者。因此，在一些实施方式中，是约12至60；12至55；12至50；12至45；或12至40岁的人类受试者。在进一步的实施方式中，人受试者为约18至60；18至55；18至50；18至45；或18至40岁。在一些实施方式中，人类受试者是18至50岁或18至40岁。In further embodiments, the mammalian subject is a human subject 60 years of age or younger. In certain embodiments, the human subject is a human subject 55 years of age, 50 years of age, 45 years of age, or 40 years of age or younger. Thus, in some embodiments, the human subject is about 12 to 60; 12 to 55; 12 to 50; 12 to 45; or 12 to 40 years of age. In further embodiments, the human subject is about 18 to 60; 18 to 55; 18 to 50; 18 to 45; or 18 to 40 years of age. In some embodiments, the human subject is 18 to 50 years of age or 18 to 40 years of age.
在某些实施方式中，根据实施方式进行治疗的受试者是怀孕的受试者，如怀孕的人类。在一些方面中，受试者已经怀孕超过约一个月、两个月、三个月、四个月、五个月、六个月、七个月或八个月。In certain embodiments, the subject treated according to the embodiments is a pregnant subject, such as a pregnant human. In some aspects, the subject has been pregnant for more than about one month, two months, three months, four months, five months, six months, seven months or eight months.
在某些方面中，根据实施方式进行治疗的受试者具有美洲、非洲、亚洲或欧洲原住民血统。在一些方面中，受试者具有至少约10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％或90％的美洲、非洲、亚洲或欧洲原住民血统。在某些方面中，受试者具有美洲原住民血统，例如至少约10％、25％或50％的美洲原住民血统。在进一步的方面中，受试者是具有美洲原住民血统的老年受试者，例如，年龄至少为55、60、65或70岁的受试者。In some aspects, the subject treated according to the embodiment has the Americas, Africa, Asia or European aboriginal ancestry. In some aspects, the subject has at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the Americas, Africa, Asia or European aboriginal ancestry. In some aspects, the subject has the Native American ancestry, such as at least about 10%, 25% or 50% of the Native American ancestry. In further aspects, the subject is an elderly subject with Native American ancestry, for example, a subject of at least 55, 60, 65 or 70 years old.
在进一步的方面中，根据实施方式进行治疗的受试者患有疾病或是免疫受损的。在一些方面中，受试者患有肝病、肾病、糖尿病、高血压、心脏病或肺病。在进一步的方面中，根据实施方式进行治疗的受试者是具有过敏反应史的受试者，这样的受试者具有食物过敏。在一些方面中，受试者以前对疫苗有过过敏反应，如过敏性反应。在再进一步的方面中，根据所述方法进行治疗的受试者是在血清中具有可检测的抗-PEG抗体的受试者，如可检测的抗-PEG IgE。In further aspects, the subject treated according to the embodiments suffers from a disease or is immunocompromised. In some aspects, the subject suffers from liver disease, kidney disease, diabetes, hypertension, heart disease or lung disease. In further aspects, the subject treated according to the embodiments is a subject with a history of allergic reactions, such that the subject has food allergies. In some aspects, the subject has previously had an allergic reaction to a vaccine, such as anaphylaxis. In yet further aspects, the subject treated according to the method is a subject with detectable anti-PEG antibodies in serum, such as detectable anti-PEG IgE.
在进一步的方面中，根据实施方式进行治疗的受试者具有选自以下的至少一种共病：In a further aspect, the subject treated according to the embodiments has at least one comorbidity selected from:
(i)慢性肾病：肾功能从过去3-6个月内的血清肌酐测量确定，使用慢性肾病流行病学协作组(CKD-EPI)方程转换为估计的肾小球滤过率(eGFR)，肾功能受损定义为eGFR<60mL/min/173m2。(i) Chronic kidney disease: Renal function was determined from serum creatinine measurements within the past 3-6 months and converted to estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, with impaired renal function defined as eGFR < 60 mL/min/173 m2.
-轻度慢性肾病定义为eGFR在60-89mL/min/1.73m2之间。- Mild chronic kidney disease is defined as an eGFR between 60-89 mL/min/1.73m2 .
-中度慢性肾病定义为eGFR在31-59mL/min/1.73m2之间，稳定治疗和在至少6个月内良好维持(修改自慢性肾病临床实践临床指南：Am J kidney Dis，2002)。- Moderate CKD is defined as an eGFR between 31-59 mL/min/1.73m2 , stable on treatment and well maintained for at least 6 months (modified from Clinical Practice Guidelines for Chronic Kidney Disease: Am J Kidney Dis, 2002).
(ii)COPD(包括肺气肿和慢性支气管炎)。(ii) COPD (including emphysema and chronic bronchitis).
-轻度COPD伴有或不伴有咳嗽或生痰定义为1秒用力呼气量/用力肺活量(FEV1/FVC)<0.7，并预测FEV1≥80％。- Mild COPD with or without cough or sputum production defined as forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) < 0.7 and FEV1 ≥ 80% predicted.
-中度COPD伴有或不伴有咳嗽或生痰定义为FEV1/FVC＜0.7和FEV1≥50％，但在稳定治疗的情况下预测为＜80％(COPD严重程度的GOLD标准)。- Moderate COPD with or without cough or sputum production defined as FEV1/FVC <0.7 and FEV1 ≥50%, but predicted to be <80% in the setting of stable treatment (GOLD criteria for COPD severity).
(iii)体重指数(BMI)>32kg/m2的肥胖-任何极端病态肥胖也包括在内。(iii) Obesity with a body mass index (BMI) > 32 kg/m2 - any extreme morbid obesity is included.
(iv)慢性心血管病症(心力衰竭、冠状动脉疾病、心肌病、动脉高血压)，包括以下：(iv) Chronic cardiovascular disorders (heart failure, coronary artery disease, cardiomyopathy, arterial hypertension), including the following:
-I类心力衰竭，未来发生心力衰竭的潜在高风险，无功能性或结构性心脏障碍。- Class I heart failure, potentially high risk of future heart failure, without functional or structural heart disorders.
-II级心力衰竭：患有心脏病导致身体活动轻微受限的受试者。休息时舒适。- Class II Heart Failure: Subjects with heart disease resulting in slight limitation of physical activity. Comfortable at rest.
-日常体力活动导致疲劳、心悸、呼吸困难或心绞痛。- Fatigue, palpitations, dyspnea, or angina resulting from daily physical activity.
-III级心力衰竭，体力活动明显受限，但休息时感到舒适，而低于日常活动导致症状。- Class III heart failure, with marked limitation of physical activity but comfort at rest and less than normal daily activity causing symptoms.
-在任何阶段都没有症状的结构性心脏障碍。- Asymptomatic structural heart disorder at any stage.
-轻度左心室收缩或舒张功能障碍，通常没有太多产生的临床症状。- Mild left ventricular systolic or diastolic dysfunction, usually without many clinical symptoms.
-根据纽约心脏协会(NYHA)，中度左心室衰竭伴劳力性呼吸困难或正呼吸或阵发性夜间呼吸困难，用药稳定(II-III类)。- Moderate left ventricular failure with exertional dyspnea or orthostatic or paroxysmal nocturnal dyspnea according to the New York Heart Association (NYHA), stable on medication (Class II-III).
-冠心病，2级及以上代谢当量阈值(MET)达中度，用药稳定。(MET定义为坐着休息时消耗的氧气量，且等于每公斤体重3.5ml O2×min；4正常，可以爬楼梯或爬山，且可以参加其他剧烈活动；1可以照顾自己，可能无法维持自己的生活，并且锻炼受到限制。)- Coronary heart disease, 2 or above metabolic equivalent threshold (MET) reaching moderate, stable medication. (MET is defined as the amount of oxygen consumed while sitting at rest, and is equal to 3.5 ml O2 × min per kg body weight; 4 is normal, can climb stairs or mountains, and can participate in other strenuous activities; 1 can take care of themselves, but may not be able to maintain their own life, and exercise is limited.)
-2-3MET的非感染性和代谢性来源的心肌病，伴随用药。- Cardiomyopathy of non-infectious and metabolic origin of 2-3 METs, concomitant medication.
-1期高血压或2期高血压，通过药物稳定并控制。- Stage 1 hypertension or stage 2 hypertension, stabilized and controlled with medication.
(v)如通过入组前12个月内采集的血液样本证明的，慢性HIV感染伴稳定的病毒血症(<50拷贝/mL)和CD4计数>350/mL。(允许病毒载量<50拷贝/mL，瞬时变化为50-350拷贝/mL。)(v) Chronic HIV infection with stable viremia (<50 copies/mL) and CD4 count >350/mL as demonstrated by blood specimens collected within 12 months prior to enrollment. (Viral load <50 copies/mL with transient variation of 50-350 copies/mL was allowed.)
(vi)2型糖尿病，药物控制[血红蛋白A1c(HbA1c)<58mmol/mol(7.45％)]或未控制，近期HbA1c>58mmol/mol(7.45％)；[(以％计HbA1c：-2.15)×10.929＝以mmol/mmol计的HbA1c]；在未控制的DM中，HbA1c应在<10％的变化化化内，并且在前3个月内不应有任何糖尿病酮症酸中毒史或严重症状性低血糖发作。(vi) Type 2 diabetes, medically controlled [hemoglobin A1c (HbA1c) <58mmol/mol (7.45%)] or uncontrolled, recent HbA1c >58mmol/mol (7.45%); [(HbA1c in %: -2.15) × 10.929 = HbA1c in mmol/mmol]; in uncontrolled DM, HbA1c should be within a variance of <10%, and there should not be any history of diabetic ketoacidosis or severe symptomatic hypoglycemia in the previous 3 months.
(vii)至少一年前在稳定条件下接受肾移植并服用药物至少6个月的受试者，被归类为低排斥风险。(vii) Subjects who received a kidney transplant at least one year ago in stable conditions and took medication for at least 6 months were classified as low risk of rejection.
在再进一步的方面中，根据实施方式进行治疗的受试者在过去6个月内没有用免疫抑制剂药物治疗超过14天。在一些方面中，根据实施方式进行治疗的受试者在施用前至少28天没有接受活疫苗和/或在施用前至少14天没有接受灭活疫苗。在进一步的方面中，根据实施方式进行治疗的受试者没有：In yet further aspects, the subject treated according to the embodiments has not been treated with an immunosuppressant drug for more than 14 days in the past 6 months. In some aspects, the subject treated according to the embodiments has not received a live vaccine for at least 28 days prior to administration and/or an inactivated vaccine for at least 14 days prior to administration. In further aspects, the subject treated according to the embodiments has not:
-病毒学证实的COVID-19病；- Virologically confirmed COVID-19 disease;
-对于女性：在施用该实施方式的组合物前一个月内经历妊娠或哺乳；- For females: experiencing pregnancy or breastfeeding within one month before administration of the composition of this embodiment;
-在施用实施方式的组合物前28天内用研究或未注册的产品(例如疫苗或药物)进行治疗；- Treatment with an investigational or unregistered product (e.g., vaccine or drug) within 28 days prior to administration of a composition of an embodiment;
-在施用实施方式的组合物前28天(对于活疫苗)或14天(对于灭活疫苗)内接受许可的疫苗；- Receipt of a licensed vaccine within 28 days (for live vaccines) or 14 days (for inactivated vaccines) prior to administration of a composition of the embodiments;
-先前或同时使用任何研究性SARS-CoV-2疫苗或另一种冠状病毒(SARS-CoV、MERS-CoV)疫苗进行治疗；- Prior or concomitant treatment with any investigational SARS-CoV-2 vaccine or another coronavirus (SARS-CoV, MERS-CoV) vaccine;
-在施用实施方式的组合物前的6个月内，用免疫抑制剂或其他免疫改进药物(例如皮质类固醇、生物制品和甲氨蝶呤)治疗总计>14天；- Treatment with immunosuppressants or other immune-modifying drugs (e.g., corticosteroids, biologics, and methotrexate) for a total of >14 days within 6 months prior to administration of the composition of the embodiments;
-根据病史和体检，有任何医学诊断的或怀疑的免疫抑制或免疫缺陷病症，包括已知的人类免疫缺陷病毒(HIV)、乙型肝炎病毒(HBV)或丙型肝炎病毒(HCV)感染；癌症的当前诊断或治疗，包括白血病、淋巴瘤、霍奇金病、多发性骨髓瘤或全身恶性肿瘤；慢性肾衰竭或肾病综合征；以及接受器官或骨髓移植。- Any medically diagnosed or suspected immunosuppression or immunodeficiency condition based on medical history and physical examination, including known infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV); current diagnosis or treatment for cancer, including leukemia, lymphoma, Hodgkin's disease, multiple myeloma, or systemic malignancies; chronic renal failure or nephrotic syndrome; and receipt of an organ or bone marrow transplant.
-有血管性水肿史(遗传性或特发性)或任何过敏反应或pIMD史。- History of angioedema (hereditary or idiopathic) or any history of allergic reaction or pIMD.
-对CVnCoV疫苗的任何成分的过敏史。- History of allergy to any component of the CVnCoV vaccine.
-在施用实施方式的组合物之前的3个月内施用免疫球蛋白或任何血液制品；- Administration of immune globulin or any blood product within 3 months prior to administration of the composition of the embodiment;
-经历了显著的急性或慢性医疗或精神疾病；和/或- Experiencing significant acute or chronic medical or psychiatric illness; and/or
-经历了严重和/或不受控制的心血管疾病、胃肠道疾病、肝病、肾病、呼吸系统疾病、内分泌紊乱以及神经和精神疾病。- Experiencing severe and/or uncontrolled cardiovascular disease, gastrointestinal disease, liver disease, kidney disease, respiratory disease, endocrine disorders, and neurological and psychiatric disorders.
在某些方面中，根据实施方式的方法进行治疗的受试者不患有任何潜在的免疫介导的疾病(pIMD)。在进一步的方面中，实施方式的治疗方法不诱导治疗的受试者中的任何pIMD。如本文使用的，将pIMD限定为乳糜泻；克罗恩病；溃疡性结肠炎；溃疡性直肠炎；自身免疫性胆管炎；自身免疫性肝炎；原发性胆汁性肝硬化；原发性硬化性胆管炎；艾迪生病；自身免疫性甲状腺炎(包括桥本甲状腺炎；I型糖尿病；Grave病或Basedow病；抗合成酶综合征；皮肌炎；青少年慢性关节炎(包括Still病)；混合性结缔组织病；风湿性多肌痛；多发性肌炎；银屑病关节病；复发性多软骨炎；类风湿性关节炎；硬皮病(例如，包括弥漫性全身型和CREST综合征)；脊椎关节炎(例如，包括强直性脊柱炎、反应性关节炎(Reiter综合征)和未分化脊椎关节炎)；系统性红斑狼疮；系统性硬化症；急性播散性脑脊髓炎(包括位点特异性变型(如非传染性脑炎、脑脊髓炎、脊髓炎、骨髓根性脊髓炎)；颅神经障碍(例如，包括瘫痪/轻瘫(例如，贝尔氏麻痹))；格林-巴利综合征(例如，包括米勒-费雪综合征和其他变型)；免疫介导的外周神经病变、帕森斯-特纳综合征和神经丛病(例如，包括慢性炎性脱髓鞘多发性神经病、多灶性运动神经病以及与单克隆丙种球蛋白病相关的多发性神经病)；多发性硬化症；嗜睡症；视神经炎；横贯性脊髓炎；斑秃；自身免疫性大疱性皮肤病，包括天疱疮、类天疱疮和疱疹样皮炎；皮肤性红斑狼疮；结节性红斑；硬斑病；扁平苔藓；银屑病；斯威特综合征；白癜风；大血管血管炎(例如，包括：巨细胞动脉炎，如大动脉炎和颞动脉炎)；中型和/或小血管血管炎(例如，包括：结节性多动脉炎、川崎病、微观多血管炎、韦格纳肉芽肿病、Churg-Strauss综合征(过敏性肉芽肿性血管炎)、Buerger病血栓闭塞性血管炎、坏死性血管炎和抗中性粒细胞胞质抗体(ANCA)阳性血管炎(类型不详)、过敏性紫癜、白塞综合征、白细胞破裂性血管炎)；抗磷脂综合征；自身免疫性溶血性贫血；自身免疫性肾小球肾炎(包括IgA肾病、肾小球快速进展性、膜性肾小球肾炎、膜增殖性肾小球肾炎和系膜增生性肾小球肾炎)；自身免疫性心肌炎/心肌病；自身免疫性血小板减少症；Goodpasture综合征；特发性肺纤维化；恶性贫血；雷诺现象；结节病；综合征；Stevens-Johnson综合征；葡萄膜炎)。In certain aspects, the subject treated according to the method of the embodiment does not suffer from any potential immune-mediated disease (pIMD). In a further aspect, the treatment method of the embodiment does not induce any pIMD in the subject treated. As used herein, pIMD is limited to celiac disease; Crohn's disease; ulcerative colitis; ulcerative proctitis; autoimmune cholangitis; autoimmune hepatitis; primary biliary cirrhosis; primary sclerosing cholangitis; Addison's disease; autoimmune thyroiditis (including Hashimoto's thyroiditis; type I diabetes; Grave's disease or Basedow's disease; anti-synthetase syndrome; dermatomyositis; juvenile chronic arthritis (including Still's disease); mixed connective tissue disease; polymyalgia rheumatica; polymyositis; psoriatic arthropathy; relapsing polychondritis; rheumatoid arthritis; scleroderma (e.g., including diffuse systemic and CREST syndrome syndrome); spondyloarthritis (e.g., including ankylosing spondylitis, reactive arthritis (Reiter syndrome), and undifferentiated spondyloarthritis); systemic lupus erythematosus; systemic sclerosis; acute disseminated encephalomyelitis (including site-specific variants (such as noninfectious encephalitis, encephalomyelitis, myelitis, myeloid radiculitis); cranial nerve disorders (e.g., including paralysis/paresis (e.g., Bell's palsy)); Guillain-Barré syndrome (e.g., including Miller-Fisher syndrome and other variants); immune-mediated peripheral neuropathies, Parsons-Turner syndrome, and plexopathies (e.g., including chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, and polyneuropathy associated with monoclonal gammopathy); multiple sclerosis; narcolepsy; optic neuritis; transverse myelitis; alopecia areata; autoimmune bullous skin diseases, including pemphigus, pemphigoid, and dermatitis herpetiformis; cutaneous lupus erythematosus; erythema nodosum; morphea; lichen planus; psoriasis; Sweet's syndrome; vitiligo; large vessel vasculitis (e.g., including giant cell arteritis such as Takayasu's arteritis and temporal arteritis); medium and/or small vessel vasculitis (e.g., including polyarteritis nodosa, Kawasaki disease, microscopic polyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome (allergic granulomatosis vasculitis), Buerger's disease, thromboangiitis obliterans, necrotizing vasculitis, and antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis (type unknown), Henoch-Schönlein purpura, Behcet's syndrome, leukocytoclastic vasculitis); antiphospholipid syndrome; autoimmune hemolytic anemia; autoimmune glomerulonephritis (including IgA nephropathy, rapidly progressive glomerular disease, membranous glomerulonephritis, membranoproliferative glomerulonephritis, and mesangial proliferative glomerulonephritis); autoimmune myocarditis/cardiomyopathy; autoimmune thrombocytopenia; Goodpasture syndrome; idiopathic pulmonary fibrosis; pernicious anemia; Raynaud's phenomenon; sarcoidosis; syndrome; Stevens-Johnson syndrome; uveitis).
在某些方面中，实施方式的疫苗接种方法不导致受试者经历任何特别感兴趣的不良事件(AESI)。如本文所用，AESI被定义为上面列出的pIMD；过敏性反应；血管炎；加强的免疫接种后的疾病；儿童多系统炎症综合征；急性呼吸窘迫综合征；COVID-19病；急性心脏损伤；微血管病；心力衰竭和心源性休克；应激性心肌病；冠状动脉疾病；心律失常；心肌炎、心包炎；血小板减少症；深静脉血栓形成；肺栓塞；脑血管中风；肢体缺血；出血性疾病；急性肾损伤；肝损伤；全身性惊厥；Guillain-Barré综合征；急性播散性脑脊髓炎；嗅觉缺失症、味觉丧失；脑膜脑炎；冻疮样病变；单器官皮肤血管炎；多形性红斑；免疫后严重的局部/全身AR。In certain aspects, the vaccination method of the embodiment does not cause the subject to experience any adverse events of particular interest (AESI). As used herein, AESI is defined as the pIMD listed above; allergic reaction; vasculitis; disease after booster vaccination; multisystem inflammatory syndrome in children; acute respiratory distress syndrome; COVID-19 disease; acute cardiac injury; microangiopathy; heart failure and cardiogenic shock; stress cardiomyopathy; coronary artery disease; arrhythmia; myocarditis, pericarditis; thrombocytopenia; deep vein thrombosis; pulmonary embolism; cerebrovascular stroke; limb ischemia; hemorrhagic disease; acute kidney injury; liver injury; systemic convulsions; Guillain-Barré syndrome; acute disseminated encephalomyelitis; anosmia, loss of taste; meningoencephalitis; pernio-like lesions; single organ cutaneous vasculitis; erythema multiforme; severe local/systemic AR after immunization.
特别地，这样的治疗方法可以包括以下步骤：In particular, such treatment methods may include the following steps:
a)提供第一方面的至少一种核酸(例如DNA或RNA)，优选至少一种RNA，至少一种第二方面的组合物，至少一种第三方面的多肽，至少一种第四方面的疫苗，或第五方面的试剂盒或部件套装；a) providing at least one nucleic acid (e.g. DNA or RNA), preferably at least one RNA, of the first aspect, at least one composition of the second aspect, at least one polypeptide of the third aspect, at least one vaccine of the fourth aspect, or a kit or set of parts of the fifth aspect;
b)将所述核酸、组合物、多肽、疫苗或试剂盒或部件套装作为第一剂量应用或施用于受试者b) applying or administering the nucleic acid, composition, polypeptide, vaccine or kit or set of parts as a first dose to a subject
c)任选地，将所述核酸、组合物、多肽、疫苗或试剂盒或部件套装作为第二剂量或进一步的剂量应用或施用于受试者，优选在第一剂后至少3、4、5、6、7、8、9、10、11、12个月。c) Optionally, the nucleic acid, composition, polypeptide, vaccine or kit or set of parts is applied or administered to the subject as a second dose or further dose, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months after the first dose.
本文所用的第一剂量分别是指初始/第一剂量、第二剂量或任何进一步的剂量，这些剂量优选是为了“增强”免疫反应而施用的。在某些方面中，对受试者施用疫苗/组合物一次、二次、三次、四次或更多次。在一些方面中，疫苗/组合物至少第一次和第二次施用于受试者(例如，初免和加强)。在一些方面中，在第一次施用后至少10天、14天、21天、28天、35天、42天、49天或56天，进行第二次施用。在一些方面中，第一次施用和第二次施用之间的时间为约7天至约56天；约14天至约56天；约21天至约56天；或约28天至约56天。在进一步的方面中，对受试者施用疫苗/组合物三次或更多次。在某些方面中，每次疫苗/组合物施用之间至少隔10天、14天、21天、28天、35天、42天、49天或56天。The first dose used herein refers to the initial/first dose, the second dose or any further dose, respectively, and these doses are preferably administered in order to "enhance" immune response. In some aspects, the vaccine/composition is administered to the subject once, twice, three times, four times or more. In some aspects, the vaccine/composition is administered to the subject (for example, primary immunization and reinforcement) at least for the first and second time. In some aspects, at least 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days or 56 days after the first administration, the second administration is performed. In some aspects, the time between the first administration and the second administration is about 7 days to about 56 days; about 14 days to about 56 days; about 21 days to about 56 days; or about 28 days to about 56 days. In further aspects, the vaccine/composition is administered to the subject three times or more. In some aspects, each vaccine/composition is administered at least 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days or 56 days apart.
在一些方面中，根据实施方式进行治疗的受试者之前感染了SARS-CoV-2或之前用至少第一SARS-CoV-2疫苗组合物进行了治疗。在一些方面中，受试者用一、二、三个或更多个剂量的第一SARS-CoV-2疫苗组合物进行治疗。在一些方面中，用于治疗受试者的实施方式的组合物与之前用于治疗受试者的组合物是不同类型的疫苗组合物。在一些方面中，受试者之前用mRNA疫苗进行了治疗，如BNT162或mRNA-1273。在进一步的方面中，受试者之前用蛋白质亚基疫苗进行了治疗，如基于刺突蛋白的疫苗，例如，NVX-CoV2373或COVAX。在某些优选的方面中，蛋白质亚基疫苗组合物包含佐剂。在进一步的方面中，受试者之前用病毒载体疫苗进行了治疗，如基于腺病毒载体的疫苗，例如，ADZ1222或Ad26.COV-2.S。在更进一步的方面中，受试者之前用SARS-CoV-2的灭活病毒疫苗进行了治疗，如CoronaVac、BBIBP-CorV或BBV152。在进一步的方面中，之前用疫苗组合物治疗的受试者具有可检测的SARS-CoV-2结合抗体，如SARS-CoV-2 S蛋白结合抗体或SARS-CoV-2N蛋白结合抗体。在进一步的方面中，在至少约3个月、6个月、9个月、1年、1.5年、2年或3年前，根据实施方式进行治疗的受试者用第一SARS-CoV-2疫苗组合物进行治疗。在更进一步的方面中，在约3个月至2年前或在约6个月或2年前，根据实施方式进行治疗的受试者用第一SARS-CoV-2疫苗组合物进行治疗。在一些方面中，用实施方式的进一步疫苗组合物治疗的受试者在至少80％、85％、90％或95％的治疗受试者中保护免于中度和重度COVID-19疾病。例如，治疗的受试者在施用进一步的组合物后在至少80％、85％、90％或95％的治疗受试者中保护免受中度和重度COVID-19疾病约2周至约1年。在更进一步的方面中，施用实施方式的进一步疫苗组合物在所述施用后在至少80％、85％、90％或95％的治疗受试者中防止中度和重度COVID-19疾病约2周至约3个月、6个月、9个月、1年、1.5年、2年或3年。以下显示了这些组合疫苗接种策略的实例：In some aspects, the subject treated according to the embodiments has previously been infected with SARS-CoV-2 or has previously been treated with at least the first SARS-CoV-2 vaccine composition. In some aspects, the subject is treated with one, two, three or more doses of the first SARS-CoV-2 vaccine composition. In some aspects, the composition of the embodiments used to treat the subject is a different type of vaccine composition than the composition previously used to treat the subject. In some aspects, the subject has previously been treated with an mRNA vaccine, such as BNT162 or mRNA-1273. In further aspects, the subject has previously been treated with a protein subunit vaccine, such as a vaccine based on a spike protein, for example, NVX-CoV2373 or COVAX. In certain preferred aspects, the protein subunit vaccine composition comprises an adjuvant. In further aspects, the subject has previously been treated with a viral vector vaccine, such as an adenovirus vector-based vaccine, for example, ADZ1222 or Ad26.COV-2.S. In a further aspect, the subject was previously treated with an inactivated virus vaccine for SARS-CoV-2, such as CoronaVac, BBIBP-CorV, or BBV152. In a further aspect, the subject previously treated with the vaccine composition has detectable SARS-CoV-2 binding antibodies, such as SARS-CoV-2 S protein binding antibodies or SARS-CoV-2N protein binding antibodies. In a further aspect, the subject treated according to the embodiments was treated with a first SARS-CoV-2 vaccine composition at least about 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, or 3 years ago. In a further aspect, the subject treated according to the embodiments was treated with a first SARS-CoV-2 vaccine composition about 3 months to 2 years ago or about 6 months or 2 years ago. In some aspects, subjects treated with a further vaccine composition of the embodiments are protected from moderate and severe COVID-19 disease in at least 80%, 85%, 90%, or 95% of the treated subjects. For example, treated subjects are protected from moderate and severe COVID-19 disease in at least 80%, 85%, 90% or 95% of treated subjects for about 2 weeks to about 1 year after administration of the further composition. In a further aspect, administration of a further vaccine composition of an embodiment protects against moderate and severe COVID-19 disease in at least 80%, 85%, 90% or 95% of treated subjects for about 2 weeks to about 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years or 3 years after said administration. Examples of these combined vaccination strategies are shown below:
第1剂mRNA疫苗-T1-第2剂mRNA疫苗-T2-第3剂mRNA疫苗1st dose of mRNA vaccine - T1 - 2nd dose of mRNA vaccine - T2 - 3rd dose of mRNA vaccine
第1剂mRNA疫苗-T1-第2剂mRNA疫苗-T2-第3剂蛋白质亚基疫苗1st dose of mRNA vaccine - T1 - 2nd dose of mRNA vaccine - T2 - 3rd dose of protein subunit vaccine
第1剂mRNA疫苗-T1-第2剂mRNA疫苗-T2-第3剂病毒载体疫苗1st dose of mRNA vaccine - T1 - 2nd dose of mRNA vaccine - T2 - 3rd dose of viral vector vaccine
第1剂mRNA疫苗-T1-第2剂mRNA疫苗-T2-第3剂灭活病毒疫苗1st dose of mRNA vaccine - T1 - 2nd dose of mRNA vaccine - T2 - 3rd dose of inactivated virus vaccine
第1剂蛋白质亚基疫苗-T1-第2剂蛋白质亚基疫苗-T2-第3剂mRNA疫苗1st dose of protein subunit vaccine - T1 - 2nd dose of protein subunit vaccine - T2 - 3rd dose of mRNA vaccine
第1剂灭活病毒疫苗-T1-第2剂灭活病毒疫苗-T2-第3剂mRNA疫苗1st dose of inactivated virus vaccine - T1 - 2nd dose of inactivated virus vaccine - T2 - 3rd dose of mRNA vaccine
第1剂病毒载体疫苗-T1-第2剂病毒载体疫苗-T2-第3剂mRNA疫苗1st dose of viral vector vaccine - T1 - 2nd dose of viral vector vaccine - T2 - 3rd dose of mRNA vaccine
第1剂病毒载体疫苗-T2-第2剂mRNA疫苗First dose of viral vector vaccine - T2 - Second dose of mRNA vaccine
第1剂蛋白质亚基疫苗-T2-第2剂mRNA疫苗1st dose of protein subunit vaccine - T2 - 2nd dose of mRNA vaccine
第1剂灭活病毒疫苗-T2-第2剂mRNA疫苗1st dose of inactivated virus vaccine - T2 - 2nd dose of mRNA vaccine
第1剂mRNA疫苗-T2-第2剂mRNA疫苗1st dose of mRNA vaccine - T2 - 2nd dose of mRNA vaccine
在所述实例中，以上时间段1(T1)通常是2至6周，优选3至4周。时间段(T2)在一些情况中为约3个月、6个月、9个月、1年、1.5年、2年或三年。In the example, the above time period 1 (T1) is generally 2 to 6 weeks, preferably 3 to 4 weeks. The time period (T2) is about 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years or three years in some cases.
在一些方面中，该实施方式的方法包括将多个剂量的疫苗组合物施用于受试者。在进一步的方面中，提供了一种降低SARS-CoV-2加强疫苗组合物的反应原性的方法。在一些方面中，在初始接种后，呈现高水平的反应原性的受试者施用不同于初始疫苗组合物的加强疫苗。例如，在一些实施方式中，初始疫苗是BNT162或mRNA-1273，而加强疫苗是实施方式的mRNA疫苗组合物。在一些方面中，基于与之前施用的疫苗组合物相比具有较低浓度的PEG或PEG缀合物浓度来选择用于具有高反应原性的受试者的加强疫苗组合物。在一些方面中，基于与之前施用的疫苗组合物相比具有较低浓度的mRNA或LNP来选择用于具有高反应原性的受试者的加强疫苗组合物。In some aspects, the method of this embodiment includes administering multiple doses of a vaccine composition to a subject. In a further aspect, a method for reducing the reactogenicity of a SARS-CoV-2 booster vaccine composition is provided. In some aspects, after the initial vaccination, a subject presenting a high level of reactogenicity is administered a booster vaccine different from the initial vaccine composition. For example, in some embodiments, the initial vaccine is BNT162 or mRNA-1273, and the booster vaccine is an mRNA vaccine composition of an embodiment. In some aspects, a booster vaccine composition for a subject with high reactogenicity is selected based on a lower concentration of PEG or PEG conjugate concentration compared to a previously administered vaccine composition. In some aspects, a booster vaccine composition for a subject with high reactogenicity is selected based on a lower concentration of mRNA or LNP compared to a previously administered vaccine composition.
在某些方面中，根据实施方式进行治疗的受试者施用作为加强疫苗的疫苗组合物并且之前用一次或多次的冠状病毒疫苗组合物施用进行了治疗。在某些方面中，用加强疫苗治疗的受试者之前用包括刺突蛋白抗原或编码刺突蛋白抗原的核酸分子的疫苗组合物进行了治疗。在一些实施方式中，选择用加强疫苗治疗的受试者之前施用了包含或编码具有与加强疫苗的刺突蛋白不同的氨基酸序列的刺突蛋白的疫苗组合物。在某些方面中，之前施用的疫苗组合物包含或编码相对于加强疫苗组合物具有至少1、2、3、4、5、6、7、8、9或10个氨基酸差异的刺突(例如，SARS-CoV-2刺突)蛋白。在某些方面中，加强疫苗组合物包含编码相对于之前施用的疫苗组合物具有约1至50；约3至30；约5至30或约10至25个氨基酸差异的刺突蛋白的RNA。在再进一步的方面中，加强疫苗组合物包含编码2、3、4或更多种具有不同氨基酸序列的不同刺突蛋白的RNA。In certain aspects, the subject treated according to the embodiments is administered a vaccine composition as a booster vaccine and has been previously treated with one or more administrations of a coronavirus vaccine composition. In certain aspects, the subject treated with the booster vaccine was previously treated with a vaccine composition comprising a spike protein antigen or a nucleic acid molecule encoding a spike protein antigen. In some embodiments, the subject selected for treatment with the booster vaccine was previously administered a vaccine composition comprising or encoding a spike protein having an amino acid sequence different from the spike protein of the booster vaccine. In certain aspects, the previously administered vaccine composition comprises or encodes a spike (e.g., SARS-CoV-2 spike) protein having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences relative to the booster vaccine composition. In certain aspects, the booster vaccine composition comprises RNA encoding a spike protein having about 1 to 50; about 3 to 30; about 5 to 30 or about 10 to 25 amino acid differences relative to the previously administered vaccine composition. In a further aspect, the booster vaccine composition comprises RNA encoding 2, 3, 4 or more different spike proteins having different amino acid sequences.
在进一步的方面中，实施方式的方法包括向受试者施用2种或更多种加强疫苗组合物，其中每种加强疫苗组合物包含编码具有不同氨基酸序列的不同刺突蛋白的RNA。在一些方面中，这些不同的加强疫苗组合物基本上同时施用或间隔小于约10分钟、20分钟、30分钟、1小时或2小时施用。在一些方面中，将不同的加强疫苗组合物施用于相同部位，如肌内注射至受试者的相同手臂。在进一步方面中，将不同的加强疫苗组合物施用于不同的部位，如肌内注射至不同的手臂或者一个或两个手臂和一个或多个腿部肌肉。In a further aspect, the method of the embodiment comprises administering 2 or more booster vaccine compositions to the subject, wherein each booster vaccine composition comprises RNA encoding different spike proteins having different amino acid sequences. In some aspects, these different booster vaccine compositions are administered substantially simultaneously or at intervals of less than about 10 minutes, 20 minutes, 30 minutes, 1 hour, or 2 hours. In some aspects, different booster vaccine compositions are administered to the same site, such as intramuscularly to the same arm of the subject. In a further aspect, different booster vaccine compositions are administered to different sites, such as intramuscularly to different arms or one or both arms and one or more leg muscles.
在某些方面中，实施方式的方法进一步限定为刺激受试者中的抗体或CD8+T细胞反应的方法。在一些方面中，将方法限定为刺激受试者中的中和抗体反应的方法。在进一步方面中，将方法限定为刺激受试者中的保护性免疫反应的方法。还在进一步方面中，将方法限定为刺激受试者中的TH2定向免疫反应的方法。In certain aspects, the methods of the embodiments are further defined as methods for stimulating an antibody or CD8+T cell response in a subject. In some aspects, the methods are defined as methods for stimulating a neutralizing antibody response in a subject. In further aspects, the methods are defined as methods for stimulating a protective immune response in a subject. Still in further aspects, the methods are defined as methods for stimulating a TH2 directed immune response in a subject.
在进一步方面中，实施方式的疫苗/组合物/组合的施用刺激了抗体反应，其在受试者中针对每个冠状病毒中和抗体产生约10至约500个冠状病毒刺突蛋白结合抗体。例如，施用可以刺激针对每个冠状病毒中和抗体产生不超过约200个刺突蛋白结合抗体的抗体反应。在进一步方面中，施用刺激针对每个冠状病毒中和抗体产生约10至约300；约20至约300；约20至约200；约30至约100；或约30至约80个冠状病毒刺突蛋白结合抗体的抗体反应。在更进一步的方面中，实施方式的组合物的施用在受试者中刺激包括刺突蛋白结合抗体与冠状病毒中和抗体的比率的抗体反应，其具有平均康复期患者血清(来自已经从冠状病毒感染恢复的受试者)中发现的20％、15％、10％或5％的刺突蛋白结合抗体与冠状病毒中和抗体的比率。In a further aspect, administration of the vaccine/composition/combination of the embodiments stimulates an antibody response that produces about 10 to about 500 coronavirus spike protein binding antibodies for each coronavirus neutralizing antibody in the subject. For example, administration can stimulate an antibody response that produces no more than about 200 spike protein binding antibodies for each coronavirus neutralizing antibody. In a further aspect, administration stimulates an antibody response that produces about 10 to about 300; about 20 to about 300; about 20 to about 200; about 30 to about 100; or about 30 to about 80 coronavirus spike protein binding antibodies for each coronavirus neutralizing antibody. In a further aspect, administration of the composition of the embodiments stimulates an antibody response in a subject that includes a ratio of spike protein binding antibodies to coronavirus neutralizing antibodies, which has a ratio of 20%, 15%, 10% or 5% of the spike protein binding antibodies to coronavirus neutralizing antibodies found in average convalescent patient sera (from subjects who have recovered from coronavirus infection).
在更进一步的方面中，实施方式的疫苗/组合物/组合的施用在受试者中刺激针对每个冠状病毒中和抗体产生约1至约500个冠状病毒刺突蛋白受体结合结构域(RBD)-结合抗体的抗体反应。在进一步的方面中，施用刺激针对每个冠状病毒中和抗体产生不超过约50个刺突蛋白RBD结合抗体的抗体反应。在更进一步的方面中，施用刺激针对每个冠状病毒中和抗体产生约1至约200；约2至约100；约3至约200；约5至约100；约5至约50；或约5至约20个刺突蛋白RBD结合抗体的抗体反应。在更进一步的方面中，实施方式的组合物的施用刺激受试者中包括刺突蛋白RBD结合抗体与冠状病毒中和抗体的比率的抗体反应，其具有平均康复期患者血清(来自已经从冠状病毒感染恢复的受试者)中发现的20％、15％、10％或5％的刺突蛋白RBD结合抗体与冠状病毒中和抗体的比率。In a further aspect, administration of the vaccine/composition/combination of the embodiment stimulates an antibody response in a subject to produce about 1 to about 500 coronavirus spike protein receptor binding domain (RBD)-binding antibodies for each coronavirus neutralizing antibody. In a further aspect, administration stimulates an antibody response to produce no more than about 50 spike protein RBD binding antibodies for each coronavirus neutralizing antibody. In a further aspect, administration stimulates an antibody response to produce about 1 to about 200; about 2 to about 100; about 3 to about 200; about 5 to about 100; about 5 to about 50; or about 5 to about 20 spike protein RBD binding antibodies for each coronavirus neutralizing antibody. In a further aspect, administration of the composition of the embodiment stimulates an antibody response in a subject including a ratio of spike protein RBD binding antibodies to coronavirus neutralizing antibodies, which has a ratio of 20%, 15%, 10% or 5% of the spike protein RBD binding antibodies to coronavirus neutralizing antibodies found in the average convalescent patient serum (from subjects who have recovered from coronavirus infection).
在更进一步的方面中，实施方式的疫苗/组合物/组合的施用基本上不诱导受试者中IL-4、IL-13、TNF和/或IL-1β的增加。在进一步的方面中，实施方式的疫苗/组合物的施用基本上不诱导受试者中血清IL-4、IL-13、TNF和/或IL-1β的增加。在一些方面中，实施方式的疫苗/组合物的施用在受试者中基本上不诱导注射部位(例如，肌内注射部位)的IL-4、IL-13、TNF和/或IL-1β的增加。在更进一步的方面中，实施方式的方法包括将实施方式的疫苗/组合物施用于患病的人类受试者。在某些方面中，受试者患有心血管疾病、肾病、肺病或自身免疫疾病。在一些方面中，将实施方式的疫苗/组合物施用于正接受抗凝治疗的受试者。In a further aspect, the administration of the vaccine/composition/combination of the embodiment does not substantially induce an increase in IL-4, IL-13, TNF and/or IL-1β in the subject. In a further aspect, the administration of the vaccine/composition of the embodiment does not substantially induce an increase in serum IL-4, IL-13, TNF and/or IL-1β in the subject. In some aspects, the administration of the vaccine/composition of the embodiment does not substantially induce an increase in IL-4, IL-13, TNF and/or IL-1β at the injection site (e.g., intramuscular injection site) in the subject. In a further aspect, the method of the embodiment includes administering the vaccine/composition of the embodiment to a human subject who is ill. In certain aspects, the subject suffers from cardiovascular disease, kidney disease, lung disease or autoimmune disease. In some aspects, the vaccine/composition of the embodiment is administered to a subject who is receiving anticoagulant therapy.
在更进一步方面中，将实施方式的疫苗/组合物/组合施用于人类受试者导致不超过20％、15％、10％、7.5％或5％的受试者经历3级局部不良事件(参见以下的表3a)。例如，在一些方面中，在第一或第二剂量的组合物后不超过10％的受试者经历3级局部不良事件。在优选方面中，将实施方式的组合物施用于人类受试者导致不超过40％、30％、25％、20％、15％、10％、7.5％或5％的受试者经历2级或更高级别的局部不良事件。例如，在一些方面中，在第一或第二剂量的组合物后不超过30％的受试者经历2级或更高等级的局部不良事件。在一些方面中，将实施方式的组合物施用于人类受试者导致不超过10％的受试者在注射部位经历3级疼痛、发红、肿胀和/或瘙痒。In further aspects, the vaccine/composition/combination of the embodiment is applied to human subjects causing no more than 20%, 15%, 10%, 7.5% or 5% of the subject to experience 3 grade local adverse events (see Table 3a below). For example, in some aspects, no more than 10% of the subject experience 3 grade local adverse events after the composition of the first or second dose. In preferred aspects, the composition of the embodiment is applied to human subjects causing no more than 40%, 30%, 25%, 20%, 15%, 10%, 7.5% or 5% of the subject to experience 2 grade or higher grade local adverse events. For example, in some aspects, no more than 30% of the subject experience 2 grade or higher grade local adverse events after the composition of the first or second dose. In some aspects, the composition of the embodiment is applied to human subjects causing no more than 10% of the subject to experience 3 grade pain, redness, swelling and/or itching at the injection site.
在进一步的方面中，将实施方式的疫苗/组合物/组合施用于人类受试者导致不超过30％、25％、20％、15％、10％或5％的受试者经历3级全身性不良事件(参见以下的表B)。例如，在一些方面中，在第一剂量的组合物后不超过25％的受试者经历3级全身性不良事件。在一些方面中，在第二剂量的组合物后不超过40％的受试者经历3级全身性不良事件。在一些方面中，将实施方式的组合物施用于人类受试者导致不超过30％、25％、20％、15％、10％或5％的受试者经历3级发烧、头疼、疲劳、寒颤、肌痛、关节痛、恶心和/或腹泻。In further aspects, the vaccine/composition/combination of the embodiment is applied to human subjects causing no more than 30%, 25%, 20%, 15%, 10% or 5% of the subjects to experience grade 3 systemic adverse events (see Table B below). For example, in some aspects, no more than 25% of the subjects experience grade 3 systemic adverse events after the composition of the first dose. In some aspects, no more than 40% of the subjects experience grade 3 systemic adverse events after the composition of the second dose. In some aspects, the composition of the embodiment is applied to human subjects causing no more than 30%, 25%, 20%, 15%, 10% or 5% of the subjects to experience grade 3 fever, headache, fatigue, chills, myalgia, arthralgia, nausea and/or diarrhea.
表3a：针对引起的局部不良事件的强度分级*Table 3a: Grading of intensity of local adverse events*
表3b：针对引起的全身性不良事件的强度分级*Table 3b: Grading of Intensity of Elicited Systemic Adverse Events*
*FDA毒性分级表(美国卫生与公众服务部，美国食品药品管理局(FDA))。行业指导。参加预防性疫苗临床试验的健康成人和青少年志愿者的毒性分级量表。2007年，在万维网上网址为fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf；登录日期：2019年3月，通过引用并入本文)；IV＝静脉内。*FDA Toxicity Grading Scale (U.S. Department of Health and Human Services, U.S. Food and Drug Administration (FDA)). Guidance for Industry. Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Participating in Preventive Vaccine Clinical Trials. 2007, available on the World Wide Web at fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf; accession date: March 2019, incorporated herein by reference); IV = intravenous.
根据更多方面，本发明还提供了用于表达至少一个多肽的方法，包括至少一个源自冠状病毒的肽或蛋白质，或其片段或变体，其中该方法优选包括以下步骤：According to further aspects, the present invention also provides a method for expressing at least one polypeptide, comprising at least one peptide or protein derived from a coronavirus, or a fragment or variant thereof, wherein the method preferably comprises the following steps:
a)提供至少一种第一方面的核酸或至少一种第二方面的组合物；和a) providing at least one nucleic acid of the first aspect or at least one composition of the second aspect; and
b)将所述核酸或组合物应用或施用于表达系统(细胞)、组织、生物体。合适的用于表达多肽(由本发明的核酸编码的)的细胞可以是果蝇S2昆虫细胞系。b) applying or administering the nucleic acid or composition to an expression system (cell), tissue, organism. Suitable cells for expressing the polypeptide (encoded by the nucleic acid of the invention) may be the Drosophila S2 insect cell line.
用于表达的方法可以适用于实验室、研究、诊断、肽或蛋白质的商业生产和/或治疗目的。该方法可以进一步在特定疾病的治疗的情形中进行，特别是在传染病的治疗中，特别是冠状病毒感染，优选SARS-CoV-2冠状病毒感染和疾病COVID-19。The method for expression can be suitable for laboratory, research, diagnostic, commercial production of peptides or proteins and/or therapeutic purposes. The method can further be carried out in the context of the treatment of specific diseases, in particular in the treatment of infectious diseases, in particular coronavirus infections, preferably SARS-CoV-2 coronavirus infections and the disease COVID-19.
同样，根据另一方面，本发明还提供了核酸、组合物、多肽、疫苗或试剂盒或部件套装的用途，优选用于诊断或治疗目的，例如，用于编码的冠状病毒抗原肽或蛋白质的表达。Likewise, according to another aspect, the present invention also provides the use of a nucleic acid, a composition, a polypeptide, a vaccine or a kit or a component set, preferably for diagnostic or therapeutic purposes, for example, for the expression of an encoded coronavirus antigenic peptide or protein.
在特定实施方式中，将所述核酸、多肽、组合物、疫苗、组合应用或施用于组织或生物体后例如可以是获得诱导的冠状病毒抗体的步骤，例如，SARS-CoV-2冠状病毒特异性(单克隆)抗体，或获得所产生的SARS-CoV-2冠状病毒蛋白构建体(S蛋白)的步骤。In a specific embodiment, the application or administration of the nucleic acid, polypeptide, composition, vaccine, combination to a tissue or organism can be followed by, for example, a step of obtaining induced coronavirus antibodies, for example, SARS-CoV-2 coronavirus-specific (monoclonal) antibodies, or a step of obtaining the produced SARS-CoV-2 coronavirus protein construct (S protein).
所述用途可应用于(诊断)实验室、用于研究、用于诊断、用于肽、蛋白质或SARS-CoV-2冠状病毒抗体的商业生产和/或用于治疗目的。所述用途可以在体外、体内或离体进行。所述用途可以进一步在治疗特定疾病的情形中进行，特别是在冠状病毒感染(例如，COVID-19)或相关障碍的治疗中。The use can be applied in (diagnostic) laboratories, for research, for diagnosis, for commercial production of peptides, proteins or SARS-CoV-2 coronavirus antibodies and/or for therapeutic purposes. The use can be carried out in vitro, in vivo or ex vivo. The use can further be carried out in the context of treating a specific disease, particularly in the treatment of coronavirus infection (e.g., COVID-19) or related disorders.
根据进一步的方面，本发明还提供了制造组合物或疫苗的方法，包括以下步骤：According to a further aspect, the present invention also provides a method for producing a composition or a vaccine, comprising the following steps:
a)在帽类似物的存在下使用DNA模板的RNA体外转录步骤，以获得加帽的mRNA，优选具有表2中提供的核酸序列；a) an RNA in vitro transcription step using a DNA template in the presence of a cap analog to obtain a capped mRNA, preferably having a nucleic acid sequence as provided in Table 2;
b)使用RP-HPLC，和/或TFF，和/或寡(dT)纯化和/或AEX，优选使用RP-HPLC，纯化步骤a)所获得的加帽RNA；b) purifying the capped RNA obtained in step a) using RP-HPLC, and/or TFF, and/or oligo(dT) purification and/or AEX, preferably using RP-HPLC;
c)提供包含步骤b)纯化的加帽RNA的第一液体组合物；c) providing a first liquid composition comprising the capped RNA purified in step b);
d)提供包含至少一种本文限定的阳离子脂质、本文限定的中性脂质、本文限定的类固醇或类固醇类似物和本文限定的PEG-脂质的第二液体组合物；d) providing a second liquid composition comprising at least one cationic lipid as defined herein, a neutral lipid as defined herein, a steroid or steroid analogue as defined herein and a PEG-lipid as defined herein;
e)将第一液体组合物和第二液体组合物引入至少一个混合装置中，以使得形成包含加帽RNA的LNP；e) introducing the first liquid composition and the second liquid composition into at least one mixing device such that LNPs comprising the capped RNA are formed;
f)纯化所获得的包含加帽RNA的LNP；f) purifying the obtained LNPs containing the capped RNA;
g)任选地，将包含加帽RNA的纯化的LNP冻干。g) Optionally, lyophilizing the purified LNPs comprising the capped RNA.
优选地，步骤e)的混合装置是T-形件连接器或微流体混合装置。优选地，混合步骤f)包括至少一个选自沉淀步骤、渗析步骤、过滤步骤、TFF步骤的步骤。任选地，任何酶促聚腺苷酸化步骤可以在步骤a)或b)后进行。任选地，可以进行进一步的纯化步骤以例如除去残余DNA、缓冲液、小的RNA副产物等。任选地，在不存在帽类似物的情况下进行RNA体外转录，并且在RNA体外转录后进行酶促加帽步骤。任选地，在至少一种本文限定的修饰的核苷酸的存在下进行RNA体外转录。Preferably, the mixing device of step e) is a T-piece connector or a microfluidic mixing device. Preferably, the mixing step f) comprises at least one step selected from a precipitation step, a dialysis step, a filtration step, a TFF step. Optionally, any enzymatic polyadenylation step can be performed after step a) or b). Optionally, further purification steps can be performed to, for example, remove residual DNA, buffers, small RNA byproducts, etc. Optionally, RNA in vitro transcription is performed in the absence of a cap analog, and an enzymatic capping step is performed after RNA in vitro transcription. Optionally, RNA in vitro transcription is performed in the presence of at least one modified nucleotide as defined herein.
在实施方式中，步骤a，优选步骤a-c，更优选以上(a-g)列出的所有步骤，在用于RNA体外转录的自动化装置中进行。这样的装置也可以用于产生组合物或疫苗(参见方面2和3)。优选地，可以合适地使用如WO2020/002598中所述的装置，特别是，如WO2020/002598的权利要求1至59和/或68至76中所述的装置。In an embodiment, step a, preferably steps a-c, more preferably all steps listed in (a-g) above, are performed in an automated device for RNA in vitro transcription. Such a device can also be used to produce a composition or vaccine (see aspects 2 and 3). Preferably, a device as described in WO2020/002598, in particular, a device as described in claims 1 to 59 and/or 68 to 76 of WO2020/002598 can be suitably used.
列表和表格的简述A brief description of lists and tables
列表1a：用于置换、缺失和/或插入的氨基酸位置Table 1a: Amino acid positions for substitutions, deletions and/or insertions
列表1b：氨基酸置换、缺失或插入List 1b: Amino acid substitutions, deletions or insertions
表1：优选的冠状病毒构建体(氨基酸序列和核酸编码序列)Table 1: Preferred coronavirus constructs (amino acid sequences and nucleic acid coding sequences)
表2a：适用于冠状病毒疫苗的RNA构建体Table 2a: RNA constructs suitable for coronavirus vaccines
表2b：适用于冠状病毒疫苗的RNA构建体Table 2b: RNA constructs suitable for coronavirus vaccines
表3a：针对引起的局部不良事件的强度分级Table 3a: Grading of intensity of local adverse events
表3b：针对引起的全身性不良事件的强度分级Table 3b: Grading of severity of systemic adverse events
表4：编码不同的SARS-CoV-2 S抗原涉及的RNA构建体(实施例中使用的)Table 4: RNA constructs encoding different SARS-CoV-2 S antigens (used in the examples)
表5：实施例的基于脂质的载体组合物Table 5: Lipid-based carrier compositions of the examples
表6：疫苗接种方案Table 6: Vaccination schedule
表7：中值VNT(第42天)Table 7: Median VNT (Day 42)
表8：疫苗接种方案(实施例3)Table 8: Vaccination schedule (Example 3)
表9：疫苗接种方案(实施例4)Table 9: Vaccination schedule (Example 4)
表10：疫苗接种方案(实施例5)Table 10: Vaccination schedule (Example 5)
表11：疫苗接种方案(实施例6)Table 11: Vaccination schedule (Example 6)
表12：疫苗接种方案(实施例7)Table 12: Vaccination schedule (Example 7)
表13：疫苗接种方案(实施例8)Table 13: Vaccination schedule (Example 8)
表14A：疫苗接种方案(实施例9A)Table 14A: Vaccination schedule (Example 9A)
表14B：疫苗接种方案(实施例9B)Table 14B: Vaccination schedule (Example 9B)
表15：疫苗接种方案(实施例10)Table 15: Vaccination schedule (Example 10)
表16：疫苗接种方案(实施例11)Table 16: Vaccination schedule (Example 11)
表17：疫苗接种方案(实施例12)Table 17: Vaccination schedule (Example 12)
表18：疫苗接种方案(实施例13)Table 18: Vaccination schedule (Example 13)
表19：疫苗接种方案(实施例14)Table 19: Vaccination schedule (Example 14)
实施例Example
以下实施例说明了本发明的各种实施方式和方面。本发明并不旨在以任何方式通过本文描述的特定实施方式来限制范围。给出以下制备和实施例以使得本领域技术人员能够更清楚地理解和实施本发明。然而，本发明的范围不受限于具体说明的实施方式(其只是作为本发明的单个方面的说明)和方法(其在本发明的范围内在功能上是等同的)。实际上，除了本文描述的那些，本发明的各种改变从之前的描述、附图和以下的实施例将对本领域技术人员变得显而易见。所有这样的改变在所附权利要求的范围内。The following examples illustrate various embodiments and aspects of the present invention. The present invention is not intended to be limited in scope by the specific embodiments described herein in any way. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and implement the present invention. However, the scope of the present invention is not limited to the specifically described embodiments (which are only descriptions of a single aspect of the present invention) and methods (which are functionally equivalent within the scope of the present invention). In fact, in addition to those described herein, various changes of the present invention will become apparent to those skilled in the art from the previous description, drawings and the following examples. All such changes are within the scope of the appended claims.
实施例1：DNA和RNA构建体、组合物和疫苗的制备Example 1: Preparation of DNA and RNA constructs, compositions and vaccines
本实施例提供了获得本发明的RNA的方法以及产生本发明的组合物或疫苗的方法。This example provides methods for obtaining the RNA of the present invention and methods for producing the composition or vaccine of the present invention.
1.1.DNA和RNA构建体的制备：1.1. Preparation of DNA and RNA constructs :
制备编码不同SARS-CoV-2 S蛋白设计的DNA序列并用于随后的RNA体外转录反应。通过引入用于稳定和表达优化的G/C优化或修饰的编码序列(例如，“cds opt1”)而改变野生型或参考编码DNA序列来制备DNA序列。将序列引入pUC衍生的DNA载体中以产生稳定3’-UTR序列和5’-UTR序列，另外具有腺苷(例如，A64或A100)链，和任选地组蛋白-茎-环(hSL)结构和任选地30个胞嘧啶链(例如，C30)(参见表4，对于冠状病毒抗原设计的概述，参见列表1或表1)。DNA sequences encoding different SARS-CoV-2 S protein designs were prepared and used for subsequent RNA in vitro transcription reactions. The DNA sequences were prepared by changing the wild-type or reference coding DNA sequence by introducing a G/C optimized or modified coding sequence (e.g., "cds opt1") for stability and expression optimization. The sequences were introduced into a pUC-derived DNA vector to generate stable 3'-UTR sequences and 5'-UTR sequences, additionally having adenosine (e.g., A64 or A100) chains, and optionally a histone-stem-loop (hSL) structure and optionally a 30 cytosine chain (e.g., C30) (see Table 4, for an overview of coronavirus antigen design, see List 1 or Table 1).
使用本领域已知的常用方案在细菌中转化和扩增所获得的质粒DNA构建体。将质粒DNA构建体提取、纯化和用于随后的RNA体外转录(参见1.2.节)。The obtained plasmid DNA constructs were transformed and amplified in bacteria using common protocols known in the art. The plasmid DNA constructs were extracted, purified and used for subsequent RNA in vitro transcription (see section 1.2.).
或者，可以将DNA质粒用作用于PCR扩增的模板(参见1.3.节)。Alternatively, the DNA plasmid can be used as a template for PCR amplification (see Section 1.3.).
1.2.从质粒DNA模板的RNA体外转录：1.2. In vitro transcription of RNA from plasmid DNA template :
使用限制性酶将根据1.1节制备的DNA质粒酶促线性化并在合适的缓冲条件下在核苷酸混合物(ATP/GTP/CTP/UTP)和帽类似物(例如，m7GpppG、m7G(5’)ppp(5’)(2’OMeA)pG、m7G(5’)ppp(5’)(2’OMeG)pG)或3’OMe-m7G(5’)ppp(5’)(2’OMeA)pG)的存在下使用T7RNA聚合酶用于DNA依赖性RNA体外转录。使用RT-HPLC(CureVac AG，Tübingen，德国；WO2008/077592)纯化所获得的RNA构建体并用于体外和体内实验。DNA模板也可以使用PCR生成。这样的PCR模板可以使用本文列出的RNA聚合酶用于DNA依赖性RNA体外转录。The DNA plasmid prepared according to section 1.1 was enzymatically linearized using restriction enzymes and used for DNA-dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and a cap analog (e.g., m7GpppG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG) or 3'OMe-m7G(5')ppp(5')(2'OMeA)pG) under appropriate buffer conditions. RT-HPLC ( The RNA constructs obtained were purified by CureVac AG, Tübingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. DNA templates can also be generated using PCR. Such PCR templates can be used for DNA-dependent RNA in vitro transcription using the RNA polymerases listed herein.
为了获得化学修饰的mRNA，在修饰的核苷酸混合物的存在下进行RNA体外转录，所述混合物包含N(1)-甲基假尿苷(m1ψ)或假尿苷(ψ)替代尿嘧啶。使用RP-HPLC(CureVac AG，Tübingen，德国；WO2008/077592)纯化所获得的m1ψ或ψ修饰的RNA并用于进一步的实验。To obtain chemically modified mRNA, RNA in vitro transcription was performed in the presence of a modified nucleotide mixture containing N(1)-methylpseudouridine (m1ψ) or pseudouridine (ψ) instead of uracil. RP-HPLC ( The obtained m1ψ- or ψ-modified RNA was purified by CureVac AG, Tübingen, Germany; WO 2008/077592) and used for further experiments.
使用酶促加帽生成加帽的RNA(预测性的)：Generation of capped RNA using enzymatic capping (predictive):
一些RNA构建体在帽类似物不存在的情况下在体外转录。然后使用本领域通常已知的加帽酶通过酶促添加帽结构(帽0或帽1)。使用加帽试剂盒将体外转录的RNA加帽以获得帽0-RNA。使用帽特异性2’-O-甲基转移酶另外修饰帽0-RNA，以获得帽1-RNA。例如如上解释的，帽1-RNA进行纯化并用于进一步的实验。Some RNA constructs are transcribed in vitro in the absence of a cap analog. The cap structure (cap 0 or cap 1) is then enzymatically added using a capping enzyme commonly known in the art. The in vitro transcribed RNA is capped using a capping kit to obtain cap 0-RNA. Cap 0-RNA is additionally modified using a cap-specific 2'-O-methyltransferase to obtain cap 1-RNA. Cap 1-RNA is purified and used for further experiments, for example as explained above.
例如，根据WO2016/180430，在目前良好生产实践下生产用于临床研发的RNA，在DNA和RNA水平上实施各种质量控制步骤。For example, according to WO2016/180430, RNA for clinical development is produced under current good manufacturing practice, with various quality control steps being performed at the DNA and RNA levels.
实施例的RNA构建体：RNA constructs of the examples :
所生成的RNA序列/构建体提供于表4中，其中显示了编码的抗原性蛋白和相应的UTR元件。如果没有另外指出，在m7GpppG、m7G(5’)ppp(5’)(2’OMeA)pG的存在下使用RNA体外转录来产生表4的RNA序列/构建体；因此，RNA序列/构建体包含5’帽1结构。如果没有另外指出，表4的RNA序列/构建体在不存在化学修饰的核苷酸(例如，假尿苷(ψ)或N(1)-甲基假尿苷(m1ψ))的情况下产生。The RNA sequences/constructs generated are provided in Table 4, where the encoded antigenic proteins and corresponding UTR elements are shown. If not otherwise indicated, RNA sequences/constructs of Table 4 were generated using RNA in vitro transcription in the presence of m7GpppG, m7G(5')ppp(5')(2'OMeA)pG; therefore, the RNA sequences/constructs comprise a 5' cap 1 structure. If not otherwise indicated, the RNA sequences/constructs of Table 4 were generated in the absence of chemically modified nucleotides (e.g., pseudouridine (ψ) or N(1)-methyl pseudouridine (m1ψ)).
1.3.从PCR扩增的DNA模板的RNA体外转录(预测性的)：1.3. In vitro transcription of RNA from PCR-amplified DNA template (predictive):
在合适的缓冲条件下在核苷酸混合物(ATP/GTP/CTP/UTP)和帽类似物(m7GpppG或3’-O-Me-m7G(5’)ppp(5’)G)的存在下使用DNA依赖性T7 RNA聚合酶将根据段落1.1制备的纯化的PCR扩增的DNA模板在体外转录。或者，在合适的缓冲条件下在修饰的核苷酸混合物(ATP、GTP、CTP、N1-甲基假尿苷(m1ψ)或假尿苷(ψ)和帽类似物(m7GpppG、m7G(5’)ppp(5’)(2’OMeA)pG或m7G(5’)ppp(5’)(2’OMeG)pG))的存在下使用DNA依赖性T7 RNA聚合酶在体外转录PCR扩增的DNA。一些RNA构建体在不存在帽类似物的情况下在体外转录并且使用本领域通常已知的加帽酶通过酶促添加帽结构(帽0或帽1)。使用RP-HPLC(CureVac AG，Tübingen，德国；WO2008/077592)纯化所获得的mRNA并用于体外和体内实验。The purified PCR-amplified DNA template prepared according to paragraph 1.1 is transcribed in vitro using a DNA-dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and a cap analog (m7GpppG or 3'-O-Me-m7G(5')ppp(5')G) under suitable buffer conditions. Alternatively, the PCR-amplified DNA is transcribed in vitro using a DNA-dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N1-methylpseudouridine (m1ψ) or pseudouridine (ψ) and a cap analog (m7GpppG, m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG)) under suitable buffer conditions. Some RNA constructs are transcribed in vitro in the absence of a cap analog and a cap structure (cap 0 or cap 1) is enzymatically added using a capping enzyme commonly known in the art. RP-HPLC ( The obtained mRNA was purified by CureVac AG, Tübingen, Germany; WO 2008/077592) and used for in vitro and in vivo experiments.
表4：编码不同SARS-CoV-2 S抗原设计的RNA构建体Table 4: RNA constructs encoding different SARS-CoV-2 S antigen designs
**mRNA R10159、R10162、R10157、R10712、R10813、R10815、R10821、R10823、R10826、R10828、R10831、R10833、R11116、R11120、R11161、R11178用N(1)-甲基假尿苷(m1ψ)产生；R10727、R10728、R10812、R10814、R10820、R10822、R10825、R10827、R10830、R10832、R11115、R11119、R11160、R11177用假尿苷(ψ)产生**mRNA R10159, R10162, R10157, R10712, R10813, R10815, R10821, R10823, R10826, R10828, R10831, R10833, R11116, R11120, R11161, R11178 were generated using N(1)-methylpseudouridine (m1ψ); R10727, R10728, R10812, R10814, R10820, R10822, R10825, R10827, R10830, R10832, R11115, R11119, R11160, R11177 were generated using pseudouridine (ψ)
刺突融合前稳定的蛋白(＝S_stab)，刺突蛋白(＝S)Spike protein (=S_stab), spike protein (=S)
1.4.LNP配制的mRNA组合物的制备：1.4. Preparation of LNP-formulated mRNA compositions :
使用阳离子脂质、结构脂质、PEG-脂质和胆固醇制备LNP。使用微流体混合装置将脂质溶液(乙醇中)与RNA溶液(水性缓冲液)混合。将获得的LNP通过渗析在碳水化合物缓冲液中重新缓冲，并使用超速离心管浓缩至目标浓度。在用于体外或体内实验前，将LNP配置的mRNA储存在-80℃下。LNPs were prepared using cationic lipids, structural lipids, PEG-lipids, and cholesterol. A microfluidic mixing device was used to mix the lipid solution (in ethanol) with the RNA solution (in aqueous buffer). The obtained LNPs were rebuffered in a carbohydrate buffer by dialysis and concentrated to the target concentration using an ultracentrifuge tube. The LNP-configured mRNA was stored at -80°C before use in in vitro or in vivo experiments.
根据PCT公开No.WO2015/199952、WO2017/004143和WO2017/075531(将其全部公开内容在此按引用并入)中所述的一般程序，制备和测试脂质纳米颗粒。使用可离子化氨基脂质(阳离子脂质)、磷脂、胆固醇和PEG化的脂质制备了脂质纳米颗粒(LNP)-配制的mRNA。如下制备LNP。将根据式III-3的阳离子脂质(ALC-0315)、DSPC、胆固醇和根据式IVa的PEG-脂质(ALC-0159)以大约47.5:10:40.8:1.7的摩尔比溶解于乙醇中(参见表5)。以0.03-0.04w/w的mRNA(序列参见表4)与总脂质的比率来制备包含化合物III-3的脂质纳米颗粒(LNP)。简言之，将mRNA在10至50mM柠檬酸盐缓冲液中稀释至0.05至0.2mg/mL。使用注射泵来混合约1:5至1:3(vol/vol)比率的乙醇脂质溶液与mRNA水溶液，使用高于15ml/min的总流速。然后除去乙醇并通过渗析用PBS替代外部缓冲液。最后，将脂质纳米颗粒通过0.2μm孔无菌过滤器过滤。脂质纳米颗粒直径大小为60-90nm，如使用Malvern Zetasizer Nano(Malvern，UK)通过quasi-elastic光散射来测定的。According to the general procedures described in PCT Publication No. WO2015/199952, WO2017/004143 and WO2017/075531 (the entire disclosure of which is hereby incorporated by reference), lipid nanoparticles were prepared and tested. Lipid nanoparticles (LNP)-formulated mRNA were prepared using ionizable amino lipids (cationic lipids), phospholipids, cholesterol and PEGylated lipids. LNPs were prepared as follows. Cationic lipids (ALC-0315), DSPC, cholesterol and PEG-lipids (ALC-0159) according to formula III-3 were dissolved in ethanol at a molar ratio of about 47.5:10:40.8:1.7 (see Table 5). Lipid nanoparticles (LNP) containing compound III-3 were prepared with a ratio of 0.03-0.04w/w mRNA (see Table 4 for sequence) to total lipids. In brief, mRNA is diluted to 0.05 to 0.2 mg/mL in 10 to 50 mM citrate buffer. Use a syringe pump to mix an ethanol lipid solution of about 1:5 to 1:3 (vol/vol) ratio with an mRNA aqueous solution, using a total flow rate higher than 15 ml/min. Then remove ethanol and replace the external buffer with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. The lipid nanoparticle size is 60-90 nm in diameter, as measured by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).
表5：实施例的基于脂质的载体组成Table 5: Lipid-based carrier compositions of the examples
1.5.包含抗原组合的组合mRNA疫苗(二价或多价疫苗组合物)的制备：1.5. Preparation of combined mRNA vaccines (bivalent or multivalent vaccine compositions) containing antigen combinations :
以单独或共同配制的方式，用LNP配制组合mRNA疫苗。对于分开混合或配制的mRNA疫苗，制备每种mRNA组分并单独LNP配制，如实施例1.4中所述的，接着混合不同的LNP配制的组分。对于共同配制的mRNA疫苗，首先将不同的mRNA组分混合在一起，接着在LNP中共同配制，如实施例1.4中所述的。Combination mRNA vaccines are formulated with LNPs in a separate or co-formulated manner. For separately mixed or formulated mRNA vaccines, each mRNA component is prepared and LNP formulated separately, as described in Example 1.4, followed by mixing the different LNP formulated components. For co-formulated mRNA vaccines, the different mRNA components are first mixed together and then co-formulated in LNPs, as described in Example 1.4.
实施例2：大鼠中的多价研究：Wistar大鼠中i.m.施用时二价CV2CoV(R9709)和Example 2: Multivalent study in rats: Bivalent CV2CoV (R9709) andCV2CoV.351(R10384)疫苗的免疫原性Immunogenicity of the CV2CoV.351 (R10384) vaccine
在这个研究中，在Wistar大鼠中评价了由LNP配制的二价mRNA疫苗CV2CoV/CV2CoV.351(R9709/R10384)诱导的体液免疫原性，与CV2CoV(R9709)或CV2CoV.351(R10384)进行比较。In this study, the humoral immunogenicity induced by the bivalent mRNA vaccine CV2CoV/CV2CoV.351(R9709/R10384) formulated with LNP was evaluated in Wistar rats and compared with CV2CoV(R9709) or CV2CoV.351(R10384).
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2中所述的(RNA体外转录)制备了SARS-CoV-2 mRNA构建体(编码全长的、融合前稳定的祖先SARS-CoV-2 S的基于CV2CoV-mRNA的SARS-CoV-2疫苗和编码全长的、融合前稳定的SARS-CoV-2 B.1.351 S的基于CV2CoV.351 mRNA的SARS-CoV-2疫苗)。在用于体内疫苗接种实验前，根据实施例1.4和实施例1.5，HPLC纯化的mRNA用LNP配制(单独混合或配制的“混合2LNP”或共同配制的“混合1LNP”，用于二价mRNA疫苗(F、G、H组)。SARS-CoV-2 mRNA constructs (SARS-CoV-2 vaccines based on CV2CoV-mRNA encoding full-length, pre-fusion stable ancestral SARS-CoV-2 S and SARS-CoV-2 vaccines based on CV2CoV.351 mRNA encoding full-length, pre-fusion stable SARS-CoV-2 B.1.351 S) were prepared as described in Example 1.2 (RNA in vitro transcription). Before use in in vivo vaccination experiments, HPLC-purified mRNA was formulated with LNPs (separately mixed or formulated "mixed 2LNPs" or co-formulated "mixed 1LNPs" for bivalent mRNA vaccines (Groups F, G, H) according to Examples 1.4 and 1.5.
免疫：immunity :
大鼠肌内(i.m.)注射mRNA疫苗组合物并且剂量如表6中所示。接种缓冲液的动物作为阴性对照(组A)。所有动物在第0天和第21天接种。在第14天、第21天和第42天收集血样，用于确定体液免疫反应。Rats were injected intramuscularly (i.m.) with mRNA vaccine compositions and the doses were as shown in Table 6. Animals vaccinated with buffer were used as negative controls (Group A). All animals were vaccinated on days 0 and 21. Blood samples were collected on days 14, 21, and 42 for determination of humoral immune responses.
表6：疫苗接种方案(实施例2)Table 6: Vaccination regimen (Example 2)
CV2CoV在表4中显示为R9709，而CV2CoV.351在表4中显示为R10384。CV2CoV is shown as R9709 in Table 4, and CV2CoV.351 is shown as R10384 in Table 4.
使用ELISA测定IgG1和IgG2刺突结合抗体滴度：IgG1 and IgG2 spike-binding antibody titers were measured using ELISA :
在第14天和第21天测定了分离的血清中的抗SARS-CoV-2刺突RBD特异性结合抗体，显示为IgG1和IgG2a的终点滴度。将重组SARS-CoV-2刺突RBD蛋白或重组SARS-CoV-2B.1.351刺突蛋白RBD(K417N、E484K、N501Y)用于包被。包被的平板使用相应的血清稀释度孵育，并且用生物素化的抗体检测特异性抗体与RBD的结合。Anti-SARS-CoV-2 spike RBD-specific binding antibodies were measured in isolated sera on days 14 and 21, shown as endpoint titers of IgG1 and IgG2a. Recombinant SARS-CoV-2 spike RBD protein or recombinant SARS-CoV-2B.1.351 spike protein RBD (K417N, E484K, N501Y) was used for coating. The coated plates were incubated with the corresponding serum dilutions, and the binding of specific antibodies to RBD was detected with biotinylated antibodies.
VNT的测定Determination of VNT
为了分析大鼠血清的VNT，用100TCID50的SARS-CoV-2孵育一式两份使用1:10的起始稀释，随后1:2连续稀释测试的热灭活血清(56℃，30min)的系列稀释。为此，使用了不同的病毒：To analyze the VNT of rat sera, serial dilutions of heat-inactivated sera (56°C, 30 min) tested in duplicate were incubated with 100 TCID50 of SARS-CoV-2 using a 1:10 starting dilution followed by 1:2 serial dilutions. For this purpose, different viruses were used:
·祖先SARS-CoV-2：源自EVAg的2019-nCov/Italy-INMI株Ancestral SARS-CoV-2: 2019-nCov/Italy-INMI strain derived from EVAg
·B.1.351变体SARS-CoV-2：hCoV-19/Netherlands/NoordHolland_10159/2021株，南非变体，下一株进化枝20H，谱系B.1.351，由EVAg提供B.1.351 variant SARS-CoV-2: hCoV-19/Netherlands/NoordHolland_10159/2021 strain, South African variant, next evolutionary branch 20H, lineage B.1.351, provided by EVAg
·B.1.1.7变体SARS-CoV-2：由VisMederi Research分离的株14484人拭子，其与祖先病毒相比含有以下突变：N501Y、A570D、T572I、D614G、P681H、T716I、S735L、S982A、D1118H。值得注意的是，这些突变不同于变体4的共有序列，即，失去了缺失dH69/V70和dY144，且T572I和S735L表示另外的突变。B.1.1.7 variant SARS-CoV-2: strain 14484 human swab isolated by VisMederi Research, which contains the following mutations compared to the ancestral virus: N501Y, A570D, T572I, D614G, P681H, T716I, S735L, S982A, D1118H. Notably, these mutations differ from the consensus sequence of variant 4, i.e., deletions dH69/V70 and dY144 are lost, and T572I and S735L represent additional mutations.
·P.1变体SARS-CoV-2株：由University of Siena分离的PG_253，含有以下突变：野生型SARS-CoV-2的L18F T20N P26S D138Y R190S K417T E484K N501Y D614G H655YT1027I和V1176F(源自EVAg的2019-nCov/Italy-INMI株)或B.1.351变体SARS-CoV-2(hCoV-19/Netherlands/NoordHolland_10159/2021株，南非变体，下一株进化枝20H，谱系B.1.351，由EVAg提供)P.1 variant SARS-CoV-2 strain: PG_253 isolated by the University of Siena, containing the following mutations: L18F T20N P26S D138Y R190S K417T E484K N501Y D614G H655YT1027I and V1176F of wild-type SARS-CoV-2 (2019-nCov/Italy-INMI strain from EVAg) or B.1.351 variant SARS-CoV-2 (hCoV-19/Netherlands/NoordHolland_10159/2021 strain, South African variant, next evolutionary branch 20H, lineage B.1.351, provided by EVAg)
将病毒在37℃下孵育1小时。每个平板含有所示用于细胞对照的行(8孔)，其仅含有细胞和培养基，以及所示的病毒对照行，其仅含有细胞和病毒。将100μl病毒-血清混合物与Vero E6细胞(ATCC，Cat.1586)的汇合层一起孵育，然后在37℃下孵育3天(祖先SARS-CoV-2)或4天(SARS-CoV-2 B.1.351、B.1.1.7和P.1)，并对于CPE形成进行显微评分来对感染病毒进行定量。对每次运行进行反滴定以验证工作病毒溶液的TCID50的正确范围。根据Reed&Muench描述的方法计算VN滴度。如果没有观察到中和(MNt<10)，报告为5的任意值。在VisMederi srl(Siena，Italy)进行分析。The virus was incubated at 37 ° C for 1 hour. Each plate contained the rows (8 wells) for cell control shown, which contained only cells and culture medium, and the virus control rows shown, which contained only cells and viruses. 100 μl of virus-serum mixture was incubated with a confluent layer of Vero E6 cells (ATCC, Cat.1586), then incubated at 37 ° C for 3 days (ancestor SARS-CoV-2) or 4 days (SARS-CoV-2 B.1.351, B.1.1.7 and P.1), and microscopic scoring was performed for CPE formation to quantify the infected virus. Each run was back titrated to verify the correct range of the TCID50 of the working virus solution. VN titer was calculated according to the method described by Reed & Muench. If neutralization (MNt <10) is not observed, an arbitrary value of 5 is reported. Analyzed at VisMederi srl (Siena, Italy).
结果：result :
如图1所示，对于接种CV2CoV和CV2CoV.351的组，在第14天(图1A-D)和第21天(图1E-H)检测到对祖先SARS-CoV-2的受体结合结构域(RBD)和B.351变体的RBD的显著IgG1和IgG2a结合抗体反应。在第14天，如图1A所示，检测到对于所有组(祖先SARS-CoV-2受体结合结构域(RBD)蛋白包被)的相当的IgG1反应和对于所有组(祖先SARS-CoV-2受体结合结构域(RBD)蛋白包被)的相当的IgG2a滴度(图1B)。在第14天，如图1C所示，检测到对于所有疫苗接种设计(RBD B.1.351变体K417N、E484K、N501Y蛋白包被)相当的IgG1反应和对于所有疫苗接种设计(RBD B.1.351变体K417N、E484K、N501Y蛋白包被)相当的IgG2a滴度(图1D)。在第21天，如图1E所示，检测到对于所有疫苗接种设计(祖先SARS-CoV-2受体结合结构域(RBD)蛋白包被)相当的IgG1反应和对于所有疫苗接种设计(祖先SARS-CoV-2受体结合结构域(RBD)蛋白包被)的相当的IgG2a滴度(图1F)。在第21天，如图1G所示，显示了对于所有疫苗接种设计(RBD B.1.351变体K417N、E484K、N501Y蛋白包被)相当的IgG1反应和对于所有疫苗接种设计(RBD B.1.351变体K417N、E484K、N501Y蛋白包被)相当的IgG2a滴度(图1H)。总体而言，单独(组B-C)、按序组合或(组D-E)和作为两种疫苗变体(二价疫苗CV2CoV/CV2CoV.351)在一种和两种LNP中的共同递送给予的CV2CoV和CV2CoV.351，在同一条腿或不同条腿中注射(F-H组)诱导了与祖先SARS-CoV-2RBD和B.1.351变体的RBD相当的结合抗体水平。As shown in Figure 1, for the groups vaccinated with CV2CoV and CV2CoV.351, significant IgG1 and IgG2a binding antibody responses to the receptor binding domain (RBD) of the ancestral SARS-CoV-2 and the RBD of the B.351 variant were detected on days 14 (Figures 1A-D) and 21 (Figures 1E-H). On day 14, as shown in Figure 1A, comparable IgG1 responses for all groups (ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating) and comparable IgG2a titers for all groups (ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating) were detected (Figure 1B). On day 14, as shown in FIG1C , comparable IgG1 responses were detected for all vaccination designs (RBD B.1.351 variants K417N, E484K, N501Y protein coating) and comparable IgG2a titers were detected for all vaccination designs (RBD B.1.351 variants K417N, E484K, N501Y protein coating) ( FIG1D ). On day 21, as shown in FIG1E , comparable IgG1 responses were detected for all vaccination designs (ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating) and comparable IgG2a titers were detected for all vaccination designs (ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating) ( FIG1F ). On day 21, as shown in Figure 1G, comparable IgG1 responses for all vaccination designs (RBD B.1.351 variants K417N, E484K, N501Y protein coating) and comparable IgG2a titers for all vaccination designs (RBD B.1.351 variants K417N, E484K, N501Y protein coating) were shown (Figure 1H). Overall, CV2CoV and CV2CoV.351, given alone (groups B-C), in sequential combination or (groups D-E), and as co-delivery of two vaccine variants (bivalent vaccine CV2CoV/CV2CoV.351) in one and two LNPs, injected in the same leg or different legs (groups F-H), induced comparable binding antibody levels to the ancestral SARS-CoV-2 RBD and the RBD of the B.1.351 variant.
如图2A(第14天)和图2B(第21天)所示，用CV2CoV和CV2CoV.351的疫苗接种在大鼠中诱导了显著水平的针对祖先SARS-CoV-2的VNT(B-H组)。总体而言，用单独或组合(B-H组)的疫苗接种产生了高VNT水平。特别是在B组中用CV2CoV的两个接种，D组中用CV2CoV和CV2CoV.351的连续接种以及将两种疫苗变体(二价疫苗CV2CoV/CV2CoV.351)共同递送到不同的腿中(H组)在这些早期时间点显示出增加的VNT。在42天，对于所有组(组B-H)检测到增加的VNT水平(图2C)。尽管使用较低剂量的每种疫苗，但在第42天二价疫苗(F-H组)诱导的反应与单价疫苗(B-E组)相当。As shown in Figure 2A (the 14th day) and Figure 2B (the 21st day), vaccination with CV2CoV and CV2CoV.351 induced significant levels of VNT against ancestral SARS-CoV-2 in rats (groups B-H). In general, vaccination with either alone or in combination (groups B-H) produced high VNT levels. In particular, two vaccinations with CV2CoV in group B, continuous vaccination with CV2CoV and CV2CoV.351 in group D, and co-delivery of two vaccine variants (bivalent vaccine CV2CoV/CV2CoV.351) to different legs (group H) showed increased VNT at these early time points. At 42 days, increased VNT levels (Figure 2C) were detected for all groups (groups B-H). Despite the use of lower doses of each vaccine, the reaction induced by the bivalent vaccine (groups F-H) at day 42 was comparable to that of the monovalent vaccine (groups B-E).
如图3A(第14天)和3B(第21天)所示，用CV2CoV和CV2CoV.351的疫苗接种诱导了显著水平的针对B.1.351变体SARS-CoV-2的VNT(B-H组)。总体而言，用单独或组合的疫苗接种(B-H组)产生了足够的VNT水平。C组中用CV2CoV.351的两个接种，E组中用CV2CoV.351和CV2CoV的连续接种以及将两种疫苗变体(二价疫苗CV2CoV/CV2CoV.351)共同递送到不同的腿中(H组)在早期时间点显示出增加的VNT。在第42天，对于所有组(组B-H)检测到增加的VNT水平(图3C)。尽管在第42天使用较低剂量的每种疫苗，但二价疫苗(F-H组)诱导的反应与单价疫苗(B-E组)相当。As shown in Figures 3A (Day 14) and 3B (Day 21), vaccination with CV2CoV and CV2CoV.351 induced significant levels of VNT (Groups B-H) against B.1.351 variant SARS-CoV-2. In general, sufficient VNT levels were produced with vaccination alone or in combination (Groups B-H). Two vaccinations with CV2CoV.351 in Group C, continuous vaccination with CV2CoV.351 and CV2CoV in Group E, and co-delivery of two vaccine variants (bivalent vaccine CV2CoV/CV2CoV.351) to different legs (Group H) showed increased VNT at early time points. At Day 42, increased VNT levels (Figure 3C) were detected for all groups (Groups B-H). Despite the use of lower doses of each vaccine at Day 42, the response induced by the bivalent vaccine (Groups F-H) was comparable to that of the monovalent vaccine (Groups B-E).
如图4所示，在第42天检测到使用B.1.1.7变体SARS-CoV-2(图4A)或B.1.1.28P.1(图4B)的所有组(B-H组)的基于CPE的测定中评估的VNT的显著诱导。在第42天，将两种疫苗变体共同递送到相同的腿(F和G组)或不同的腿(H组)中可以产生针对两种变体的反应。表7总结了在第42天针对祖先、B.1.1.7、B.1.351和P1SARS-CoV-2变体的中值VNT。As shown in Figure 4, significant induction of VNTs evaluated in CPE-based assays was detected for all groups (Groups B-H) using B.1.1.7 variant SARS-CoV-2 (Figure 4A) or B.1.1.28P.1 (Figure 4B) on Day 42. At Day 42, co-delivery of the two vaccine variants to the same leg (Groups F and G) or to different legs (Group H) could generate responses against both variants. Table 7 summarizes the median VNTs for ancestral, B.1.1.7, B.1.351, and P1 SARS-CoV-2 variants on Day 42.
表7：中值VNT(第42天)Table 7: Median VNT (Day 42)
总体而言，二价疫苗引发稳健水平的RBD结合和病毒中和抗体，这些抗体能够中和祖先和B.1.351 SARS-CoV-2以及变体B.1.1.7和B.1.1.28P.1。Overall, the bivalent vaccine elicited robust levels of RBD-binding and virus-neutralizing antibodies that were able to neutralize ancestral and B.1.351 SARS-CoV-2 as well as variants B.1.1.7 and B.1.1.28P.1.
实施例3：剂量反应研究：在Wistar大鼠中i.m.施用时与CV2CoV相比CV2CoV.351的Example 3: Dose response study: CV2CoV.351 compared to CV2CoV when administered i.m. in Wistar rats免疫原性Immunogenicity
这个研究的目的是在剂量反应研究中评估大鼠中CV2CoV.351疫苗的免疫原性和早期先天性刺激。The aim of this study was to evaluate the immunogenicity and early innate stimulation of the CV2CoV.351 vaccine in rats in a dose-response study.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(CV2CoV-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长、融合前稳定的祖先SARS-CoV-2 S，和CV2CoV.351-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长、融合前稳定的SARS-CoV-2B.1.351S)。在用于体内疫苗接种实验前，根据实施例1.4将HPLC纯化的Mrna用LNP配制。SARS-CoV-2 mRNA constructs (mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV-LNP, encoding full-length, pre-fusion-stable ancestral SARS-CoV-2 S, and mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV.351-LNP, encoding full-length, pre-fusion-stable SARS-CoV-2 B.1.351S) were prepared as described in Example 1.2 (in vitro transcription of RNA). HPLC-purified Mra were formulated with LNPs according to Example 1.4 before use in in vivo vaccination experiments.
免疫：immunity :
给大鼠肌内(i.m.)注射表8所示的mRNA疫苗组合物和剂量。用缓冲液接种的动物作为阴性对照(A组)。所有动物在第0天和第21天接种。在第14天、第21天和第42天收集血样用于测定抗体滴度。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 8. Animals inoculated with buffer served as negative controls (Group A). All animals were inoculated on days 0 and 21. Blood samples were collected on days 14, 21, and 42 for determination of antibody titers.
表8：接种方案(实施例3)Table 8: Vaccination scheme (Example 3)
CV2CoV在表4中显示为R9709，而CV2CoV.351在表4中显示为R10384CV2CoV is shown as R9709 in Table 4, while CV2CoV.351 is shown as R10384 in Table 4
按照实施例2中所述的进行使用ELISA的IgG1和IgG2刺突结合抗体滴度的测定和进行VNT的测定。Determination of IgG1 and IgG2 spike binding antibody titers using ELISA and determination of VNT were performed as described in Example 2.
结果：result :
针对祖先和B.1.351 SARS-CoV-2的抗原特异性结合抗体滴度(通过ELISA分析)和VNT在用这两种疫苗接种的动物中以剂量依赖性方式可检测到。结合以及中和抗体随时间和剂量的增加而增加。Antigen-specific binding antibody titers (by ELISA analysis) and VNT against ancestral and B.1.351 SARS-CoV-2 were detectable in a dose-dependent manner in animals vaccinated with both vaccines. Binding as well as neutralizing antibodies increased with time and dose.
如图5A(IgG1)和5B(IgG2a)(祖先SARS-CoV-2RBD包被)和5C(IgG1)和5D(IgG2a)(B.1.351变体RBD K417N、E484K、N501Y包被)所示，在第14天，使用0.5μg、2μg、8μg和40μg的剂量，用CV2CoV(B-E组)和CV2CoV.351(F-I组)接种在大鼠中诱导刺突结合抗体滴度。As shown in Figures 5A (IgG1) and 5B (IgG2a) (ancestral SARS-CoV-2 RBD coated) and 5C (IgG1) and 5D (IgG2a) (B.1.351 variant RBD K417N, E484K, N501Y coated), on day 14, vaccination with CV2CoV (groups B-E) and CV2CoV.351 (groups F-I) induced spike-binding antibody titers in rats using doses of 0.5μg, 2μg, 8μg, and 40μg.
如图5E(IgG1)、图5F(IgG2a)(祖先SARS-CoV-2RBD包被)和图5G(IgG1)和图5H(IgG2a)(B.1.351变体RBD K417N、E484K、N501Y包被)所示，在第21天，使用0.5μg、2μg和8μg以及40μg的剂量，用CV2CoV(B-E组)和CV2CoV.351(F-I组)接种在大鼠中诱导刺突结合抗体滴度。As shown in Figure 5E (IgG1), Figure 5F (IgG2a) (ancestral SARS-CoV-2 RBD coated) and Figure 5G (IgG1) and Figure 5H (IgG2a) (B.1.351 variant RBD K417N, E484K, N501Y coated), on day 21, vaccination with CV2CoV (groups B-E) and CV2CoV.351 (groups F-I) induced spike-binding antibody titers in rats using doses of 0.5μg, 2μg, 8μg and 40μg.
如图6A所示，B.1.351变体疫苗CV2CoV.351(B-E组)在所有剂量组中在第14天诱导针对祖先SARS-CoV-2的剂量依赖性VNT(异源反应)。与接种CV2CoV时的反应(同源反应)进行比较，CV2CoV.351接种组中的VNT在第14天减少约2倍。图6B显示了CV2CoV.351(B-E组)在所有剂量组中在第14天诱导针对B.1.351SARS-CoV-2的剂量依赖性VNT(同源反应)。CV2CoV.351接种引发高水平的针对同源病毒的VNT，与针对祖先病毒的异源VNT相比，其在第14天增加45倍(所有剂量组的平均差异)。与接种CV2CoV(F-I组)相比，由CV2CoV.351诱导的VNT在第14天增加41倍(所有剂量组的平均差异)。As shown in Figure 6A, the B.1.351 variant vaccine CV2CoV.351 (groups B-E) induced dose-dependent VNTs (heterologous responses) against the ancestral SARS-CoV-2 on day 14 in all dose groups. Compared with the response when CV2CoV was vaccinated (homologous response), the VNTs in the CV2CoV.351-vaccinated group decreased by about 2 times on day 14. Figure 6B shows that CV2CoV.351 (groups B-E) induced dose-dependent VNTs (homologous responses) against B.1.351 SARS-CoV-2 on day 14 in all dose groups. CV2CoV.351 vaccination triggered high levels of VNTs against homologous viruses, which increased 45-fold on day 14 compared to heterologous VNTs against the ancestral virus (average difference across all dose groups). Compared with vaccination with CV2CoV (groups F-I), the VNTs induced by CV2CoV.351 increased 41-fold on day 14 (average difference across all dose groups).
如图6C所示，B.1.351变体疫苗CV2CoV.351(B-E组)在所有剂量组中在第21天诱导针对祖先SARS-CoV-2的剂量依赖性VNT(异源反应)。与接种CV2CoV时的反应(同源反应)相比，接种CV2CoV.351的组中的VNT在第21天降低大约2倍。如图6D所示，在所有剂量组中，CV2CoV.351在第21天诱导针对B.1.351SARS-CoV-2(同源反应)的轻微剂量依赖性VNT。与针对祖先病毒的异源VNT(所有剂量组的平均差异)相比，CV2CoV.351疫苗接种引发高水平的针对同源病毒的VNT，其在第21天增加35倍。与接种CV2CoV相比，CV2CoV.351诱导的VNT在第21天增加了42倍(所有剂量组的平均差异)。如图6E所示，B.1.351变体疫苗CV2CoV.351在第41天在所有剂量组中诱导了针对祖先SARS-CoV-2(异源反应)的VNT。除了0.5μg剂量组(F组)之外，在接种CV2CoV后显示出略高的反应(同源反应)。如图6F所示，CV2CoV.351在第42天在所有剂量组中诱导针对B.1.351 SARS-CoV-2的VNT(同源反应)。与接种CV2CoV相比，由CV2CoV.351诱导的VNT在第42天增加。如图6G所示，在所有剂量组中，B.1.351变体疫苗CV2CoV.351在第42天诱导针对B.1.1.7变体SARS-CoV-2的VNT(异源反应)。在用CV2CoV接种后显示出类似的反应(异源反应)。如图6H所示，在所有剂量组中，CV2CoV.351在第42天诱导针对B.1.1.28P.1SARS-CoV-2的VNT(同源反应)。在用CV2CoV接种后检测到较低的反应(异源反应)。As shown in Figure 6C, the B.1.351 variant vaccine CV2CoV.351 (groups B-E) induced dose-dependent VNTs against the ancestral SARS-CoV-2 on day 21 in all dose groups (heterologous response). The VNTs in the group vaccinated with CV2CoV.351 were reduced by approximately 2-fold on day 21 compared to the response when vaccinated with CV2CoV (homologous response). As shown in Figure 6D, in all dose groups, CV2CoV.351 induced slightly dose-dependent VNTs against B.1.351 SARS-CoV-2 (homologous response) on day 21. Compared with heterologous VNTs against the ancestral virus (average difference across all dose groups), CV2CoV.351 vaccination triggered high levels of VNTs against the homologous virus, which increased 35-fold on day 21. Compared with vaccination with CV2CoV, CV2CoV.351-induced VNTs increased 42-fold on day 21 (average difference across all dose groups). As shown in Figure 6E, the B.1.351 variant vaccine CV2CoV.351 induced VNTs against the ancestral SARS-CoV-2 (heterologous response) in all dose groups on day 41. Except for the 0.5μg dose group (Group F), a slightly higher response was shown after vaccination with CV2CoV (homologous response). As shown in Figure 6F, CV2CoV.351 induced VNTs against B.1.351 SARS-CoV-2 in all dose groups on day 42 (homologous response). Compared with vaccination with CV2CoV, the VNT induced by CV2CoV.351 increased on day 42. As shown in Figure 6G, in all dose groups, the B.1.351 variant vaccine CV2CoV.351 induced VNTs against the B.1.1.7 variant SARS-CoV-2 on day 42 (heterologous response). A similar response was shown after vaccination with CV2CoV (heterologous response). As shown in Figure 6H, in all dose groups, CV2CoV.351 induced VNT against B.1.1.28P.1SARS-CoV-2 (homologous response) on day 42. Lower responses were detected after vaccination with CV2CoV (heterologous response).
总体而言，SARS-CoV-2 B.1.351变体mRNA疫苗候选物CV2CoV.351在大鼠中诱导强的体液免疫反应，如通过结合和病毒中和抗体滴度所确定的。与接种CV2CoV相比，接种CV2CoV.351后，针对B.1.351的病毒中和滴度显著增加。Overall, the SARS-CoV-2 B.1.351 variant mRNA vaccine candidate CV2CoV.351 induced a strong humoral immune response in rats, as determined by binding and virus neutralizing antibody titers. Virus neutralization titers against B.1.351 were significantly increased after vaccination with CV2CoV.351 compared to vaccination with CV2CoV.
实施例4：延长的多价疫苗接种研究：在Wistar大鼠中i.m.施用时二价CV2CoV和Example 4: Extended multivalent vaccination study: Bivalent CV2CoV andCV2CoV.351疫苗的免疫原性(预测性的)Immunogenicity of CV2CoV.351 vaccine (predictive)
这个研究的目的是评估二价CV2CoV/CV2CoV.351疫苗在大鼠中第三次疫苗接种的免疫原性和早期先天性刺激。The aim of this study was to evaluate the immunogenicity and early innate stimulation of the third vaccination of the bivalent CV2CoV/CV2CoV.351 vaccine in rats.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(CV2CoV-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先SARS-CoV-2 S，和CV2CoV.351-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的SARS-CoV-2 B.1.351 S)。在用于体内疫苗接种实验前，HPLC纯化的mRNA根据实施例1.4和实施例1.5用LNP配制(对于二价Mrna疫苗单独混合或配制)。SARS-CoV-2 mRNA constructs (mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV-LNP, encoding full-length, pre-fusion stable ancestral SARS-CoV-2 S, and mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV.351-LNP, encoding full-length, pre-fusion stable SARS-CoV-2 B.1.351 S) were prepared as described in Example 1.2 (RNA in vitro transcription). Before being used in in vivo vaccination experiments, HPLC purified mRNA was formulated with LNP according to Example 1.4 and Example 1.5 (mixed or formulated separately for bivalent mRNA vaccines).
免疫：immunity :
大鼠肌内(i.m.)注射表9所示的mRNA疫苗组合物和剂量。用缓冲液接种的动物作为阴性对照(A组)。所有动物在第0天、第3周(第21天)接种，并且在第15周(第105天)给B组另外接种。在第0天、第14天、第21天、第42天、第77天、第105天、第119天和第133天收集血样用于测定抗体滴度。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and dosages shown in Table 9. Animals inoculated with buffer were used as negative controls (Group A). All animals were inoculated at Day 0, Week 3 (Day 21), and Group B was additionally inoculated at Week 15 (Day 105). Blood samples were collected at Day 0, Day 14, Day 21, Day 42, Day 77, Day 105, Day 119, and Day 133 for determination of antibody titers.
表9：疫苗接种方案(实施例4)Table 9: Vaccination schedule (Example 4)
CV2CoV在表4中显示为R9709，而CV2CoV.351在表4中显示为R10384CV2CoV is shown as R9709 in Table 4, while CV2CoV.351 is shown as R10384 in Table 4
按照实施例2中所述的进行使用ELISA的IgG1和IgG2刺突结合抗体滴度的测定和进行VNT的测定。Determination of IgG1 and IgG2 spike binding antibody titers using ELISA and determination of VNT were performed as described in Example 2.
实施例5：疫苗的共同递送：大鼠用编码祖先SARS-CoV2抗原(CV2CoV)和变体Example 5: Co-delivery of vaccines: Rats were treated with vaccines encoding ancestral SARS-CoV2 antigens (CV2CoV) and variantsB.1.351的SARS-CoV2-抗原(CV2CoV.351)疫苗接种Vaccination with SARS-CoV2-antigen B.1.351 (CV2CoV.351)
在这个研究中，测量了针对祖先和B.1.351 SARS-CoV-2的抗体反应。In this study, antibody responses to ancestral and B.1.351 SARS-CoV-2 were measured.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(基于CV2CoV-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先SARS-CoV-2 S，和基于CV2CoV.351-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的SARS-CoV-2 B.1.351S)。在体内使用前，HPLC纯化的mRNA根据实施例1.4和实施例1.5用LNP配制(对于二价mRNA疫苗单独混合或配制)。SARS-CoV-2 mRNA constructs (SARS-CoV-2 vaccines based on CV2CoV-mRNA encoding full-length, pre-fusion stable ancestral SARS-CoV-2 S, and SARS-CoV-2 vaccines based on CV2CoV.351-mRNA encoding full-length, pre-fusion stable SARS-CoV-2 B.1.351S) were prepared as described in Example 1.2 (RNA in vitro transcription). Before in vivo use, HPLC purified mRNA was formulated with LNP according to Example 1.4 and Example 1.5 (mixed or formulated separately for bivalent mRNA vaccines).
免疫：immunity :
大鼠肌内(i.m.)注射表10所示的mRNA疫苗组合物和剂量。作为阴性对照，一组大鼠接种缓冲液(A组)。所有动物在第0周和第3周(第21天)接种。在第0天、第14天、第21天和第42天收集血样用于测定抗体滴度。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 10. As a negative control, one group of rats was vaccinated with buffer (Group A). All animals were vaccinated at week 0 and week 3 (day 21). Blood samples were collected at day 0, day 14, day 21, and day 42 for determination of antibody titers.
表10：接种方案(实施例5)Table 10: Vaccination scheme (Example 5)
在表4中，CV2CoV显示为R9709，CV2CoV.351显示为R10384In Table 4, CV2CoV is shown as R9709 and CV2CoV.351 is shown as R10384
按照实施例2中所述的进行使用ELISA的IgG1和IgG2刺突结合抗体滴度的测定和进行VNT的测定。Determination of IgG1 and IgG2 spike binding antibody titers using ELISA and determination of VNT were performed as described in Example 2.
结果：result :
总之，二价疫苗组合物CV2CoV+CV2CoV.351在第14天诱导了相当水平的针对祖先和B.1.351变体RBD的刺突结合抗体(图7A-7D)。在注射后第14天，在用2μg或8μg二价疫苗组合物CV2CoV+CV2CoV.351接种的所有动物中可检测到显著水平的刺突结合抗体。在注射0.5μg、2μg或8μg二价疫苗组合物CV2CoV+CV2CoV.351的所有组中，均诱导了剂量依赖性水平的IgG1和IgG2a刺突结合抗体滴度。对于2μg剂量，IgG1和IgG2a抗体之间的比率显示与IgG1相比，IgG2a抗体的诱导略低。图7A和7B显示针对祖先SARS-CoV-2RBD的结合抗体，而图7C和7D显示针对B.1.351变体RBD的结合抗体。对于2和8μg组，随时间以剂量依赖性方式诱导针对祖先SARS-CoV-2(图7E(第14天)、7F(第21天)和7I(第42天))和针对B.1.351变体SARS-CoV-2(图7G(第14天)、7H(第21天)和7J(第42天))的稳健VNT。图7K和7L显示了对于2μg和8μg剂量组，分别针对变体B.1.1.7和P.1的VNT的剂量依赖性诱导。In summary, the bivalent vaccine composition CV2CoV+CV2CoV.351 induced comparable levels of spike-binding antibodies against the ancestral and B.1.351 variant RBDs on day 14 (Figures 7A-7D). On day 14 after injection, significant levels of spike-binding antibodies were detected in all animals vaccinated with 2μg or 8μg of the bivalent vaccine composition CV2CoV+CV2CoV.351. Dose-dependent levels of IgG1 and IgG2a spike-binding antibody titers were induced in all groups injected with 0.5μg, 2μg or 8μg of the bivalent vaccine composition CV2CoV+CV2CoV.351. For the 2μg dose, the ratio between IgG1 and IgG2a antibodies showed that the induction of IgG2a antibodies was slightly lower than that of IgG1. Figures 7A and 7B show binding antibodies to the ancestral SARS-CoV-2RBD, while Figures 7C and 7D show binding antibodies to the B.1.351 variant RBD. For the 2 and 8 μg groups, robust VNTs were induced against ancestral SARS-CoV-2 (Figures 7E (day 14), 7F (day 21), and 7I (day 42)) and against B.1.351 variant SARS-CoV-2 (Figures 7G (day 14), 7H (day 21), and 7J (day 42)) in a dose-dependent manner over time. Figures 7K and 7L show the dose-dependent induction of VNTs against variants B.1.1.7 and P.1, respectively, for the 2 μg and 8 μg dose groups.
实施例6：加强研究：Wistar大鼠中i.m.施用时用二价CVnCoV和CvnCoV.351疫苗接Example 6: Booster study: Vaccination with bivalent CVnCoV and CvnCoV.351 vaccines when administered i.m. in Wistar rats种大鼠Rat
设计这个研究来确定同源加强是否能够引发针对异源变体(B.1.351)的免疫反应的显著增加。This study was designed to determine whether homologous boosting could elicit a significant increase in the immune response against the heterologous variant (B.1.351).
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(CV2CoV-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先SARS-CoV-2 S，和CV2CoV.351-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的SARS-CoV-2 B.1.351 S)。如表4和11中所示的，在一些构建体中，尿苷被1-甲基假尿苷替代。在用于体内前，HPLC纯化的mRNA根据实施例1.4用LNP配制。SARS-CoV-2 mRNA constructs (mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV-LNP, encoding full-length, pre-fusion stable ancestral SARS-CoV-2 S, and mRNA-based SARS-CoV-2 vaccines formulated with CV2CoV.351-LNP, encoding full-length, pre-fusion stable SARS-CoV-2 B.1.351 S) were prepared as described in Example 1.2 (RNA in vitro transcription). As shown in Tables 4 and 11, in some constructs, uridine was replaced by 1-methyl pseudouridine. Before use in vivo, HPLC purified mRNA was formulated with LNP according to Example 1.4.
免疫：immunity :
大鼠肌内(i.m.)注射表11所示的mRNA疫苗组合物和剂量。作为阴性对照，一组大鼠接种缓冲液(A组)。所有动物在第0周(第1天)和第3周(第21天)接种相同的疫苗组合物。在第0天、第15天、第21天、第42天、第77天、第105天、第119天和第133天收集血样用于测定VNT。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 11. As a negative control, one group of rats was vaccinated with buffer (Group A). All animals were vaccinated with the same vaccine composition at week 0 (day 1) and week 3 (day 21). Blood samples were collected on days 0, 15, 21, 42, 77, 105, 119, and 133 for determination of VNT.
表11：疫苗接种方案(实施例6)Table 11: Vaccination schedule (Example 6)
CV2CoV在表4中显示为R9709，而CV2CoV.351在表4中显示为R10384CV2CoV is shown as R9709 in Table 4, while CV2CoV.351 is shown as R10384 in Table 4
按照实施例2中所述的进行VNT的测定。VNT determination was performed as described in Example 2.
结果：result :
总之，在用CVnCoV、CV2CoV或“m1ψ祖先S”(R10162)进行两次初免疫苗接种后三个月，用CV2CoV或CV2CoV.351进行加强免疫诱导了针对祖先和B.1.351 SARS-CoV-2的病毒中和抗体的显著增加(分别为图8A和8B)。用CV2CoV和CV2CoV.351进行加强免疫诱导了能够中和祖先SARS-CoV-2和SARS-CoV-2 B.1.351、B.1.1.7和P.1变体的VNT(图8C-8F)。In summary, three months after two primary vaccinations with CVnCoV, CV2CoV, or "m1ψ ancestral S" (R10162), booster immunization with CV2CoV or CV2CoV.351 induced a significant increase in virus-neutralizing antibodies against ancestral and B.1.351 SARS-CoV-2 (Figures 8A and 8B, respectively). Booster immunization with CV2CoV and CV2CoV.351 induced VNTs that were able to neutralize ancestral SARS-CoV-2 and SARS-CoV-2 B.1.351, B.1.1.7, and P.1 variants (Figures 8C-8F).
针对祖先SARS-CoV-2的VNT(图8A)：VNTs targeting ancestral SARS-CoV-2 (Figure 8A):
CVnCoV在大鼠中两次接种后(G和F组)诱导了针对祖先SARS-CoV-2的强VNT。滴度保持容易检测直到在d105的加强，随时间测量的滴度略有降低。CVnCoV induced strong VNTs against ancestral SARS-CoV-2 after two vaccinations in rats (groups G and F). Titers remained readily detectable until boosting at d105, with a slight decrease in titers measured over time.
CV2CoV(同源疫苗)在CVnCoV初免疫苗接种(F组)的背景下显示出高的加强能力：在CV2CoV加强后，针对祖先SARS-CoV-2的VNT显著增加109倍(d105 vs d119)。CV2CoV (homologous vaccine) showed high boosting capacity in the context of CVnCoV primary vaccination (group F): after CV2CoV boosting, VNT against ancestral SARS-CoV-2 increased significantly by 109-fold (d105 vs d119).
与用CV2CoV加强免疫的反应相比，用CV2CoV.351(异源疫苗)加强免疫诱导的针对祖先SARS-CoV-2的滴度较低(G组)。在d105和d119检测到的滴度之间观察到的差异增加了6倍。然而，差异在统计学上不显著。Compared to the response to booster immunization with CV2CoV, booster immunization with CV2CoV.351 (heterologous vaccine) induced lower titers against ancestral SARS-CoV-2 (group G). A six-fold increase in the difference observed between titers detected on d105 and d119 was observed. However, the difference was not statistically significant.
用CV2CoV或“m1ψ祖先S”(R10162)初免疫苗接种可以实现类似的结果，其中与CVnCoV诱导的VNT相比，在第105天第三次(“加强”)疫苗接种之前的滴度显著增加。Similar results could be achieved with priming vaccination with CV2CoV or “m1ψ ancestor S” (R10162), where titers increased significantly before the third (“boost”) vaccination on day 105 compared to CVnCoV-induced VNTs.
针对SARS-CoV-2 B.1.351的VNT(图8B)：VNT against SARS-CoV-2 B.1.351 (Figure 8B):
CVnCoV在大鼠中两次接种后诱导了针对SARS-CoV-2 B.1.351的强VNT。然而，滴度总体上低于针对祖先SARS-CoV-2的滴度(将图8B与图8A进行比较)。与在d105相对于d119(G组)检测的VNT相比，用CV2CoV.351(同源疫苗)加强接种诱导了109倍的显著增加。CVnCoV induced strong VNTs against SARS-CoV-2 B.1.351 after two vaccinations in rats. However, titers were generally lower than those against ancestral SARS-CoV-2 (compare Figure 8B with Figure 8A). Booster vaccination with CV2CoV.351 (homologous vaccine) induced a significant 109-fold increase compared to the VNTs detected at d105 relative to d119 (Group G).
CV2CoV(异源疫苗)在CVNCoV初免疫苗接种(F组)的背景下显示出高的加强能力：在CV2CoV加强后，针对SARS-CoV-2B.1.351的VNT显著增加了256倍(d105 vs d119)。CV2CoV (heterologous vaccine) showed high boosting capacity in the context of CVNCoV primary vaccination (Group F): after CV2CoV boosting, VNT against SARS-CoV-2B.1.351 increased significantly by 256 times (d105 vs d119).
用CV2CoV或“m1ψ祖先S”(R10162)初免接种可以实现类似的结果，其中与CVNCoV诱导的VNT相比，第三次(“加强”)接种之前的滴度显著增加。Similar results could be achieved with priming with CV2CoV or “m1ψ ancestor S” (R10162), where titers before the third (“boost”) vaccination were significantly increased compared to VNTs induced by CVNCoV.
在加强免疫后14天测试针对祖先SARS-CoV-2以及针对SARS-CoV-2 B.1.1.7(α)、B.1.351(β)和P.1(γ)变体的病毒中和反应(图8C-8F)。Virus neutralization responses against ancestral SARS-CoV-2 and against SARS-CoV-2 B.1.1.7(α), B.1.351(β), and P.1(γ) variants were tested 14 days after booster immunization (Figures 8C-8F).
用CV2CoV.351(同源疫苗，C、E和G组)加强不仅在d119诱导了针对SARS-CoV-2B.1.351的强VNT(图8D)，而且还诱导了针对祖先SARS-CoV-2(图8C)、SARS-CoV-2 B.1.1.7(图8E)和P.1(图8F)(异源疫苗)的强VNT。Boosting with CV2CoV.351 (homologous vaccine, groups C, E, and G) induced strong VNTs not only against SARS-CoV-2B.1.351 at d119 (Figure 8D), but also against ancestral SARS-CoV-2 (Figure 8C), SARS-CoV-2 B.1.1.7 (Figure 8E), and P.1 (Figure 8F) (heterologous vaccines).
实施例7：用编码SARS-CoV-2变体的mRNA疫苗接种小鼠Example 7: Vaccination of mice with mRNA encoding SARS-CoV-2 variants
设计这个研究来确定接种编码SARS-CoV-2变体的mRNA疫苗是否诱导具有交叉中和能力的免疫原性。This study was designed to determine whether vaccination with mRNA vaccines encoding SARS-CoV-2 variants induces immunogenicity with cross-neutralizing capacity.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体，基于CV2CoV-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先或变体SARS-CoV-2。在用于体内前，根据实施例1.4用LNP配制HPLC纯化的mRNA。SARS-CoV-2 mRNA constructs were prepared as described in Example 1.2 (RNA in vitro transcription), SARS-CoV-2 vaccines based on CV2CoV-mRNA encoding full-length, pre-fusion stable ancestral or variant SARS-CoV-2. Before use in vivo, HPLC purified mRNA was formulated with LNP according to Example 1.4.
免疫：immunity :
小鼠肌内(i.m.)注射表12所示的mRNA疫苗组合物和剂量。作为阴性对照，一组小鼠接种缓冲液(第1组)。在第0天和第21天给所有动物接种。在第0天、第14天、第21天和第42天收集血样用于测定抗体滴度。Mice were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 12. As a negative control, one group of mice was vaccinated with buffer (Group 1). All animals were vaccinated on days 0 and 21. Blood samples were collected on days 0, 14, 21, and 42 for determination of antibody titers.
表12：疫苗接种方案(实施例7)Table 12: Vaccination schedule (Example 7)
使用ELISA测定IgG1抗体滴度：Determination of IgG1 antibody titer using ELISA :
使用重组SARS-CoV-2 S蛋白(祖先SARS-CoV-2 RBD或B.1.351 RBD变体(K427N、E484K、N501Y))用于包被来进行ELISA，使用相应的血清稀释液孵育包被的板，并使用生物素化的同种型特异性抗小鼠抗体，然后使用链霉亲和素-HRP(辣根过氧化物酶)以Amplex作为底物检测特异性抗体与SARS-CoV-2 RBD或RBD变体的结合。在初免接种后第14天通过ELISA测量抗体的终点滴度。ELISA was performed using recombinant SARS-CoV-2 S protein (ancestral SARS-CoV-2 RBD or B.1.351 RBD variant (K427N, E484K, N501Y)) for coating, the coated plates were incubated with the corresponding serum dilutions, and the binding of specific antibodies to SARS-CoV-2 RBD or RBD variants was detected using biotinylated isotype-specific anti-mouse antibodies, followed by streptavidin-HRP (horseradish peroxidase) with Amplex as substrate. The endpoint titer of antibodies was measured by ELISA on day 14 after the primary vaccination.
VNT的测定：Determination of VNT :
在基于CPE的分析中评估的VNT测定如实施例2中所述进行。为了检测针对SARS-CoV-2δ变体的VNT，使用包含以下突变的病毒株The VNT assay evaluated in the CPE-based assay was performed as described in Example 2. To detect VNT against the SARS-CoV-2 delta variant, a virus strain containing the following mutations was used
hCoV-19/France/IDF-APHP-hEGP-20-23-2131905084/2021|EPI_ISL_20 29113|2021-04-27：T19R E156G d157F d158R L452R T478K D614G P681R D950N。hCoV-19/France/IDF-APHP-hEGP-20-23-2131905084/2021|EPI_ISL_20 29113|2021-04-27: T19R E156G d157F d158R L452R T478K D614G P681R D950N.
胞内细胞因子染色：Intracellular cytokine staining :
根据本领域已知的标准方案，在第42天分离来自接种疫苗的小鼠的脾细胞。简言之，将分离的脾通过细胞过滤器研磨并在PBS/1％FBS中洗涤，然后裂解红细胞。在用PBS/1％ FBS彻底洗涤步骤后，将脾细胞接种到96孔板中(每孔2×106个细胞)。在存在2.5μg/ml抗CD28抗体(BD Biosciences)的情况下，在37℃下，在蛋白质转运抑制剂存在下用SARS-CoV-2祖先S蛋白特异性肽(1μg/ml)的混合物刺激细胞6小时。重复相同的程序以用SARS-CoV-2 B.1.351 S蛋白特异性肽的混合物刺激脾细胞。刺激后，洗涤细胞并根据制造商的说明书使用Cytofix/Cytoperm试剂(BD Biosciences)对于胞内细胞因子进行染色。使用以下抗体进行染色：Thy1.2-FITC(1:200)、CD8-APC-Cy7(1:200)、TNF-PE(1:100)、IFNγ-APC(1:100)(eBioscience)、CD4-BD Horizon V450(1:200)(BD Biosciences)，并与1:100稀释的Fcγ-封闭一起孵育。使用Aqua染料来区分活/死细胞(Invitrogen)。使用ZE5流式细胞仪(Bio-Rad)获得细胞。使用FlowJo软件包(Tree Star，Inc.)分析流式细胞术数据。According to standard protocols known in the art, splenocytes from vaccinated mice were isolated on the 42nd day. In brief, the separated spleen was ground through a cell strainer and washed in PBS/1% FBS, and then the red blood cells were lysed. After a thorough washing step with PBS/1% FBS, splenocytes were inoculated into 96-well plates (2×106 cells per well). In the presence of 2.5 μg/ml anti-CD28 antibodies (BD Biosciences), cells were stimulated with a mixture of SARS-CoV-2 ancestor S protein-specific peptides (1 μg/ml) at 37°C in the presence of protein transport inhibitors for 6 hours. The same procedure was repeated to stimulate splenocytes with a mixture of SARS-CoV-2 B.1.351 S protein-specific peptides. After stimulation, the cells were washed and stained for intracellular cytokines using Cytofix/Cytoperm reagents (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences), and incubated with Fcγ-block at a dilution of 1:100. Aqua dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a ZE5 flow cytometer (Bio-Rad). Flow cytometry data were analyzed using the FlowJo software package (Tree Star, Inc.).
结果：result :
第14天，所有测试的编码全长的、融合前稳定的祖先或变体SARS-CoV-2的SARS-CoV-2 mRNA疫苗构建体诱导针对祖先SARS-CoV-2 RBD(图9A)和B.1.351变体RBD(K417N、E484K、N501Y)的高IgG抗体反应，图9B。On day 14, all tested SARS-CoV-2 mRNA vaccine constructs encoding full-length, prefusion-stabilized ancestral or variant SARS-CoV-2 induced high IgG antibody responses against the ancestral SARS-CoV-2 RBD ( Figure 9A ) and the B.1.351 variant RBD (K417N, E484K, N501Y), Figure 9B .
所有测试的mRNA疫苗构建体在第42天显示了强的VNT诱导，这是最显著的，并且对于同源中和在较早时间点(d14，d21)也是可检测的(图9C：第2组，祖先；图9D：第3组，B.1.1.7；图9E：第4组和第7组，B.1.351；图F：第6组，P1)。All tested mRNA vaccine constructs showed strong VNT induction, which was most prominent at day 42, and homologous neutralization was also detectable at earlier time points (d14, d21) (Figure 9C: Group 2, ancestors; Figure 9D: Group 3, B.1.1.7; Figure 9E: Groups 4 and 7, B.1.351; Figure F: Group 6, P1).
如图9G-J所示，接种编码不同的变体全长S稳定的蛋白质的mRNA在第42天在两次接种后诱导了高水平的抗原特异性CD4+和CD8+IFNα/TNF双阳性T细胞，与用祖先(图G和H)或B.1.351(图I和J)肽文库刺激脾细胞时的程度相似。As shown in Figures 9G-J, vaccination with mRNA encoding different variant full-length S-stabilized proteins induced high levels of antigen-specific CD4+ and CD8+ IFNα/TNF double-positive T cells on day 42 after two vaccinations, to a similar extent when splenocytes were stimulated with the ancestral (Figures G and H) or B.1.351 (Figures I and J) peptide libraries.
实施例8：小鼠接种编码SARS-CoV-2变体的mRNA疫苗Example 8: Vaccination of mice with mRNA vaccines encoding SARS-CoV-2 variants
设计这个研究来确定接种编码SARS-CoV-2变体的mRNA疫苗是否诱导具有交叉中和能力的免疫原性。This study was designed to determine whether vaccination with mRNA vaccines encoding SARS-CoV-2 variants induces immunogenicity with cross-neutralizing capacity.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(CV2CoV-LNP配制的基于mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先或变体SARS-CoV-2 S(S_stab)。在用于体内前，根据实施例1.4用LNP配制HPLC纯化的mRNA。SARS-CoV-2 mRNA constructs (Cv2CoV-LNP-formulated mRNA-based SARS-CoV-2 vaccines encoding full-length, prefusion-stabilized ancestral or variant SARS-CoV-2 S (S_stab) were prepared as described in Example 1.2 (RNA in vitro transcription). HPLC-purified mRNA was formulated with LNPs according to Example 1.4 before use in vivo.
免疫：immunity :
小鼠肌内(i.m.)注射表13所示的mRNA疫苗组合物和剂量。作为阴性对照，一组小鼠接种缓冲液(第13组)。在第0天和第21天给所有动物接种。在第0天、第14天、第21天和第42天收集血样用于测定抗体滴度。Mice were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 13. As a negative control, one group of mice was vaccinated with buffer (Group 13). All animals were vaccinated on days 0 and 21. Blood samples were collected on days 0, 14, 21, and 42 for determination of antibody titers.
表13：疫苗接种方案(实施例8)Table 13: Vaccination schedule (Example 8)
可以按照实施例2所述的进行使用同源和异源变体的VNT测定。对于T细胞分析，在第42天分离脾细胞。VNT assays using homologous and heterologous variants can be performed as described in Example 2. For T cell analysis, splenocytes were isolated at day 42.
通过细胞内细胞因子染色(ICS)的T细胞分析：T cell analysis by intracellular cytokine staining (ICS) :
根据本领域已知的标准方案分离来自接种疫苗的小鼠的脾细胞。简言之，将分离的脾通过细胞过滤器研磨并在PBS/1％ FBS中洗涤，然后裂解红细胞。在用PBS/1％ FBS彻底洗涤步骤后，将脾细胞接种到96孔板中(每孔2×106个细胞)。在存在2.5μg/ml抗CD28抗体(BD Biosciences)的情况下，在37℃下，在蛋白质转运抑制剂存在下用SARS-CoV-2祖先S蛋白特异性肽(1μg/ml)的混合物刺激细胞6小时。刺激后，洗涤细胞并根据制造商的说明书使用Cytofix/Cytoperm试剂(BD Biosciences)对强胞内细胞因子进行染色。使用以下抗体进行染色：Thy1.2-FITC(1:200)、CD8-APC-Cy7(1:200)、TNF-PE(1:100)、IFNγ-APC(1:100)(eBioscience)、CD4-BD Horizon V450(1:200)(BD Biosciences)，并与1:100稀释的Fcγ-封闭一起孵育。使用Aqua染料来区分活/死细胞(Invitrogen)。使用ZE5流式细胞仪(Bio-Rad)获得细胞。使用FlowJo软件包(Tree Star，Inc.)分析流式细胞术数据。Splenocytes from vaccinated mice were isolated according to standard protocols known in the art. In brief, the separated spleen was ground through a cell strainer and washed in PBS/1% FBS, and then the red blood cells were lysed. After a thorough washing step with PBS/1% FBS, the splenocytes were inoculated into 96-well plates (2×106 cells per well). In the presence of 2.5 μg/ml anti-CD28 antibodies (BD Biosciences), cells were stimulated with a mixture of SARS-CoV-2 ancestor S protein-specific peptides (1 μg/ml) in the presence of protein transport inhibitors at 37°C for 6 hours. After stimulation, the cells were washed and strong intracellular cytokines were stained using Cytofix/Cytoperm reagents (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences), and incubated with Fcγ-block at a dilution of 1:100. Aqua dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a ZE5 flow cytometer (Bio-Rad). Flow cytometry data were analyzed using the FlowJo software package (Tree Star, Inc.).
结果：result :
如图10所示，用祖先肽文库刺激脾细胞时，在第42天两次接种后接种编码不同的变体全长S稳定的蛋白质的mRNA诱导了高水平的抗原特异性CD4+和CD8+ IFNα/TNF双阳性T细胞(分别为图10A和B)。看起来可能是体液应答以实施例7中所示的相似方式进行(ELISA或VNT)(对于表13的疫苗构建体尚未测试)。As shown in Figure 10, when spleen cells were stimulated with the ancestral peptide library, vaccination with mRNA encoding different variant full-length S stable proteins induced high levels of antigen-specific CD4+ and CD8+ IFNα/TNF double positive T cells after two vaccinations on day 42 (Figure 10A and B, respectively). It seems likely that humoral responses were performed in a similar manner as shown in Example 7 (ELISA or VNT) (not yet tested for the vaccine constructs of Table 13).
实施例9A：小鼠接种编码SARS-CoV-2变体的mRNA疫苗(预测性的)Example 9A: Vaccination of mice with mRNA vaccines encoding SARS-CoV-2 variants (predictive)
在这个研究内，可以确定接种编码SARS-CoV-2变体的mRNA疫苗是否诱导具有交叉中和能力的免疫原性。Within this study, it will be determined whether vaccination with mRNA vaccines encoding SARS-CoV-2 variants induces immunogenicity with cross-neutralizing capacity.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(基于CV2CoV-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先或变体SARS-CoV-2 S(S_stab))。在用于体内前，根据实施例1.4用LNP配制HPLC纯化的mRNA。SARS-CoV-2 mRNA constructs (SARS-CoV-2 vaccines based on CV2CoV-mRNA encoding full-length, prefusion-stabilized ancestral or variant SARS-CoV-2 S (S_stab)) were prepared as described in Example 1.2 (RNA in vitro transcription). HPLC-purified mRNA was formulated with LNP according to Example 1.4 before use in vivo.
免疫：immunity :
小鼠肌内(i.m.)注射表14A所示的mRNA疫苗组合物和剂量。作为阴性对照，一组小鼠接种缓冲液(第1组)。在第0天和第21天给所有动物接种。在第0天和第21天给所有动物接种。在第0天、第14天、第21天和第42天收集血样用于测定抗体滴度。Mice were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 14A. As a negative control, one group of mice was vaccinated with buffer (Group 1). All animals were vaccinated on Day 0 and Day 21. All animals were vaccinated on Day 0 and Day 21. Blood samples were collected on Day 0, Day 14, Day 21, and Day 42 for determination of antibody titers.
表14A：疫苗接种方案Table 14A: Vaccination schedule
按照实施例2和实施例7所述的进行使用同源和异源变体的VNT测定。对于T细胞分析，在第42天分离脾细胞。按照实施例8中所述的进行细胞内细胞因子染色(ICS)的T细胞分析。编码新出现的变体的进一步构建体可以以相似的方式来测试。VNT assays using homologous and heterologous variants were performed as described in Examples 2 and 7. For T cell analysis, splenocytes were isolated at day 42. T cell analysis by intracellular cytokine staining (ICS) was performed as described in Example 8. Further constructs encoding the newly emerged variants can be tested in a similar manner.
实施例9B：大鼠接种编码SARS-CoV-2变体的mRNA疫苗(预测性的)Example 9B: Rats vaccinated with mRNA vaccines encoding SARS-CoV-2 variants (predictive)
在这个研究内，可以确定接种编码SARS-CoV-2变体的mRNA疫苗是否诱导具有交叉中和能力的免疫原性。Within this study, it will be determined whether vaccination with mRNA vaccines encoding SARS-CoV-2 variants induces immunogenicity with cross-neutralizing capacity.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(基于CV2CoV-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的omicron变体SARS-CoV-2 S(S_stab))。在用于体内前，根据实施例1.4用LNP配制HPLC纯化的mRNA。The SARS-CoV-2 mRNA construct (SARS-CoV-2 vaccine based on CV2CoV-mRNA encoding the full-length, pre-fusion stabilized omicron variant SARS-CoV-2 S (S_stab)) was prepared as described in Example 1.2 (RNA in vitro transcription). Before use in vivo, HPLC purified mRNA was formulated with LNP according to Example 1.4.
免疫：immunity :
大鼠肌内(i.m.)注射表14B所示的mRNA疫苗组合物和剂量。作为阴性对照，一组大鼠接种缓冲液(第1组)。所有动物在第0天和第21天接种。在第0天、第14天、第21天和第42天收集血样用于测定抗体滴度。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 14B. As a negative control, one group of rats was vaccinated with buffer (Group 1). All animals were vaccinated on Day 0 and Day 21. Blood samples were collected on Day 0, Day 14, Day 21, and Day 42 for determination of antibody titers.
表14B：疫苗接种方案Table 14B: Vaccination schedule
CV2CoV.529在表4中例如显示为R11175、R11176、R11177**、R11178**CV2CoV.529 is shown in Table 4 as R11175, R11176, R11177**, R11178**
按照实施例2所述的进行使用同源和异源SARS-CoV-2变体的VNT测定。对于T细胞分析，在第42天分离脾细胞。按照实施例8中所述的进行细胞内细胞因子染色(ICS)的T细胞分析。编码新出现的变体的进一步构建体可以以相似的方式来测试。VNT assays using homologous and heterologous SARS-CoV-2 variants were performed as described in Example 2. For T cell analysis, splenocytes were isolated at day 42. T cell analysis by intracellular cytokine staining (ICS) was performed as described in Example 8. Further constructs encoding newly emerged variants can be tested in a similar manner.
实施例10：扩展的多价接种研究：Wsitar大鼠中i.m.施用时二价CV2CoV和Example 10: Extended multivalent vaccination study: bivalent CV2CoV andCV2CoV.351疫苗的免疫原性Immunogenicity of the CV2CoV.351 vaccine
这个研究的目的是评估第三次接种多价CV2CoV/CV2CoV.351时在CVnCoV接种后的加强反应。The aim of this study was to evaluate the booster response after CVnCoV vaccination with a third vaccination of polyvalent CV2CoV/CV2CoV.351.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(基于CV2CoV-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的祖先SARS-CoV-2 S，和基于CV2CoV.351-mRNA的SARS-CoV-2疫苗，其编码全长的、融合前稳定的SARS-CoV-2 B.1.351S)。在用于体内接种实验前，根据实施例1.4和实施例1.5用LNP配制HPLC纯化的mRNA(对于二价mRNA疫苗单独混合或配制)。SARS-CoV-2 mRNA constructs (SARS-CoV-2 vaccines based on CV2CoV-mRNA encoding full-length, pre-fusion stable ancestral SARS-CoV-2 S, and SARS-CoV-2 vaccines based on CV2CoV.351-mRNA encoding full-length, pre-fusion stable SARS-CoV-2 B.1.351S) were prepared as described in Example 1.2 (RNA in vitro transcription). HPLC purified mRNA was formulated with LNP according to Example 1.4 and Example 1.5 (mixed or formulated separately for bivalent mRNA vaccines) before use in in vivo vaccination experiments.
免疫：immunity :
大鼠肌内(i.m.)注射表14所示的mRNA疫苗组合物和剂量。接种缓冲液的动物用作阴性对照(C组)。在第0周、第3周(第21天)和第15周(第105天)给所有动物接种。在第0天、第14天、第21天、第42天、第77天、第105天、第119天和第133天收集血样用于测定抗体滴度。Rats were injected intramuscularly (i.m.) with the mRNA vaccine compositions and dosages shown in Table 14. Animals inoculated with buffer were used as negative controls (Group C). All animals were inoculated at week 0, week 3 (day 21), and week 15 (day 105). Blood samples were collected at day 0, day 14, day 21, day 42, day 77, day 105, day 119, and day 133 for determination of antibody titers.
表15：疫苗接种方案(实施例10)Table 15: Vaccination schedule (Example 10)
CVnCoV在表4中显示为R9515，CV2CoV显示为R9709和CV2CoV.351显示为R10384。CVnCoV is shown in Table 4 as R9515, CV2CoV as R9709 and CV2CoV.351 as R10384.
按照实施例2所述的进行VNT的测定。VNT was determined as described in Example 2.
结果：result :
如图11所示，在大鼠中接种两次后，CVnCoV和CV2CoV诱导了强的针对祖先SARS-CoV-2的VNT。对于CVnCoV，滴度保护容易检测到直到第105天增强，滴度随着时间的推移略有下降。As shown in Figure 11, CVnCoV and CV2CoV induced strong VNTs against ancestral SARS-CoV-2 after two vaccinations in rats. For CVnCoV, titer protection was easily detectable until day 105, and titers declined slightly over time.
在CVnCoV初免接种的背景下，二价CV2CoV+CV2CoV.351疫苗组合物显示出高的加强能力：在CV2CoV+CV2CoV.351加强后，针对祖先SARS-CoV-2的VNT显著增加了30倍(d105vs d119)和45倍(d105vs d133)(B组)。在CV2CoV初免接种的背景下，二价CV2CoV+CV2CoV.351疫苗组合物在第105天显示出已经很高的VNT的进一步增强能力。In the context of CVnCoV priming, the bivalent CV2CoV+CV2CoV.351 vaccine composition showed high boosting capacity: after CV2CoV+CV2CoV.351 boosting, VNT against ancestral SARS-CoV-2 increased significantly by 30-fold (d105 vs d119) and 45-fold (d105 vs d133) (Group B). In the context of CV2CoV priming, the bivalent CV2CoV+CV2CoV.351 vaccine composition showed further boosting capacity of the already high VNT on day 105.
如图11B所示，大鼠中接种两次后，CVnCoV诱导了强的针对SARS-CoV-2 B.1.351的VNT。针对B.1.351的滴度总体上低于针对祖先病毒的滴度。对于接种CVnCoV和CV2CoV的动物，滴度保持容易检测直到第105天增强，测量的VNT随着时间的推移有小的下降。在CVnCoV初免接种的背景下，二价CV2CoV+CV2CoV.351疫苗组合物显示出高的加强能力：在CV2CoV+CV2CoV.351加强后，针对SARS-CoV-2 B.1.351的VNT显著增加了19倍(d105 vs d119)和75倍(d105 vs d133)。在CV2CoV初免接种的背景下，二价CV2CoV+CV2CoV.351疫苗组合物在第105天显示出已经很高的针对SARS-CoV-2 B.1.351的VNT的进一步增强能力。As shown in Figure 11B, after two vaccinations in rats, CVnCoV induced a strong VNT against SARS-CoV-2 B.1.351. The titer for B.1.351 is generally lower than that for the ancestral virus. For animals inoculated with CVnCoV and CV2CoV, the titer remained easily detectable until the 105th day, and the measured VNT had a small decline over time. In the context of the initial vaccination of CVnCoV, the bivalent CV2CoV+CV2CoV.351 vaccine composition showed a high strengthening ability: after CV2CoV+CV2CoV.351 was strengthened, the VNT for SARS-CoV-2 B.1.351 increased significantly by 19 times (d105 vs d119) and 75 times (d105 vs d133). In the context of the initial vaccination of CV2CoV, the bivalent CV2CoV+CV2CoV.351 vaccine composition showed a further strengthening ability of the already high VNT for SARS-CoV-2 B.1.351 on the 105th day.
尽管检测到的针对B.1.351的中和滴度保持低于针对祖先SARS-CoV-2诱导的滴度直到实验的第105天，但在用CV2CoV+CV2CoV.351疫苗组合物加强时测得的针对两种病毒的VNT在第133天达到了相当的水平(图11A与图11B)。Although the neutralization titers detected against B.1.351 remained lower than those induced against the ancestral SARS-CoV-2 until day 105 of the experiment, the VNTs measured against both viruses reached comparable levels on day 133 when boosted with the CV2CoV+CV2CoV.351 vaccine composition (Figures 11A and 11B).
如图11C-11F所示，在第119天，B组和C组中不仅针对祖先和B.1.351 SARS-CoV-2，而且针对B.1.1.7和P.1 SARS-CoV-2变体，都诱导了稳定且高的VNT(图11C：祖先，图11D：B.1.351，图11E：B.17，图11F：P.1)。As shown in Figures 11C-11F, on day 119, stable and high VNTs were induced in groups B and C not only against the ancestral and B.1.351 SARS-CoV-2, but also against the B.1.1.7 and P.1 SARS-CoV-2 variants (Figure 11C: ancestral, Figure 11D: B.1.351, Figure 11E: B.17, Figure 11F: P.1).
总之，在用CVnCoV或CV2CoV两次初免后三个月，用二价CV2CoV+CV2CoV.351疫苗组合物加强诱导了针对祖先SARS-CoV-2以及SARS-CoV-2 B.1.351的VNT的显著增加，并引发了针对B.1.1.7和P.1变体的高水平VNT。In summary, three months after two primings with CVnCoV or CV2CoV, boosting with the bivalent CV2CoV+CV2CoV.351 vaccine composition induced a significant increase in VNTs against ancestral SARS-CoV-2 as well as SARS-CoV-2 B.1.351, and triggered high levels of VNTs against the B.1.1.7 and P.1 variants.
实施例11：大鼠接种编码SARS-CoV-2变体B1.617.2.S_stab抗原的mRNAExample 11: Rats vaccinated with mRNA encoding SARS-CoV-2 variant B1.617.2.S_stab antigen
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备编码δ变体(B1.617.2)的稳定刺突(S_stab)的mRNA构建体。如表4和11中所示，在一些构建体中，尿苷被假尿苷(ψ)或1-甲基假尿苷(m1ψ)替代。在用于体内接种实验前，根据实施例1.4用LNP配制HPLC纯化的mRNA。The mRNA construct encoding the stable spike (S_stab) of the delta variant (B1.617.2) was prepared as described in Example 1.2 (RNA in vitro transcription). As shown in Tables 4 and 11, in some constructs, uridine was replaced by pseudouridine (ψ) or 1-methyl pseudouridine (m1ψ). Before being used in the in vivo inoculation experiment, HPLC purified mRNA was prepared with LNP according to Example 1.4.
免疫：immunity :
Wistar大鼠(n＝8)肌内(i.m.)注射表13所示的mRNA疫苗组合物和剂量。接种缓冲液的动物用作阴性对照(第1组，n＝6)。在第0天和第21天给所有动物接种。在第21天(初免后)和第42天(加强后)收集血样用于测定抗体滴度。Wistar rats (n = 8) were injected intramuscularly (i.m.) with the mRNA vaccine compositions and doses shown in Table 13. Animals inoculated with buffer were used as negative controls (Group 1, n = 6). All animals were inoculated on days 0 and 21. Blood samples were collected on days 21 (after primary immunization) and 42 (after booster) for determination of antibody titers.
表16：疫苗接种方案(实施例11)：Table 16: Vaccination schedule (Example 11) :
表4中CV2CoV.617.2显示为R10630，CVCoV.617.2w/o hsl显示为R10824，CV2CoV.617.2ψ显示为R10827，和CV2CoV.617.2m1ψ显示为R10828。对于R10827，尿苷被ψ(假尿苷)替代，而对于R10828，尿嘧啶被m1ψ(1-甲基假尿嘧啶)替代。CV2CoV.617.2 is shown as R10630, CVCoV.617.2w/o hsl is shown as R10824, CV2CoV.617.2ψ is shown as R10827, and CV2CoV.617.2m1ψ is shown as R10828 in Table 4. For R10827, uridine is replaced by ψ (pseudouridine), and for R10828, uracil is replaced by m1ψ (1-methylpseudouridine).
如实施例2所述，使用ELISA测定总IgG刺突结合抗体滴度并进行VNT测定。重组SARS CoV-2刺突B.1.617.2RBD蛋白(L452R，T478K，δ变体)用于ELISA IgG测定。(对于针对δB.1.617.2的VNT，使用以下株：谱系：δ-B.1.617.2，株：hCoV-19/France/IDF-APHP-HEGP-20-23-2131905084/2021|EPI_ISL_2029113|2021-04-27(T19R E156G d157F d158R L452RT478K D614G P681R D950N)。As described in Example 2, ELISA was used to determine total IgG spike binding antibody titers and VNT assays were performed. Recombinant SARS CoV-2 spike B.1.617.2 RBD protein (L452R, T478K, delta variant) was used for ELISA IgG assays. (For VNT against delta B.1.617.2, the following strains were used: Lineage: delta-B.1.617.2, strain: hCoV-19/France/IDF-APHP-HEGP-20-23-2131905084/2021|EPI_ISL_2029113|2021-04-27 (T19R E156G d157F d158R L452RT478K D614G P681R D950N).
结果：result :
如图12A和B所示，使用2μg、8μg和20μg的剂量，用配制在LNP中的编码全长S稳定蛋白(δ变体B1.617.2)的不同mRNA形式接种，在第14天和第42天在大鼠中诱导了显著水平的刺突结合抗体滴度。第二次接种导致抗体滴度的进一步增加。As shown in Figures 12A and B, vaccination with different mRNA forms encoding full-length S stable protein (delta variant B1.617.2) formulated in LNPs using doses of 2 μg, 8 μg, and 20 μg induced significant levels of spike-binding antibody titers in rats at days 14 and 42. A second vaccination resulted in a further increase in antibody titers.
2、8和20μg组以剂量依赖的方式诱导稳定的针对SARS CoV-2变体B1.617.2的VNT，其随着时间增加。对于包括2μg剂量(图12A，第14天)、图12B(第21天)和图12C(第42天)的所有组，早在第一次注射后第14天可检测到VNT。还诱导了针对祖先SARS-CoV-2(图12F)、针对SARS-CoV-2变体B.1.351(图12G)和针对SARS-CoV-2变体P.1(图12H)的稳定的异源VNT。2, 8 and 20 μg groups induced stable VNTs against SARS CoV-2 variant B1.617.2 in a dose-dependent manner, which increased over time. For all groups including 2 μg doses (Figure 12A, day 14), Figure 12B (day 21) and Figure 12C (day 42), VNTs were detectable as early as day 14 after the first injection. Stable heterologous VNTs against ancestral SARS-CoV-2 (Figure 12F), SARS-CoV-2 variant B.1.351 (Figure 12G) and SARS-CoV-2 variant P.1 (Figure 12H) were also induced.
结果证明将天然核苷酸(第2-7组)或化学修饰的核苷酸((ψ(假尿苷，第8-10组)或m1ψ(1-甲基假尿苷，第11-13组))引入mRNA构建体中，在第42天诱导了相当水平的针对不同变体的VNT，并有通过使用化学修饰的核苷酸提高VNT的趋势(第8-13组)。The results demonstrated that the introduction of natural nucleotides (groups 2-7) or chemically modified nucleotides ((ψ(pseudouridine, groups 8-10) or m1ψ(1-methylpseudouridine, groups 11-13)) into the mRNA constructs induced comparable levels of VNT against different variants at day 42, with a trend toward increased VNT by the use of chemically modified nucleotides (groups 8-13).
实施例12：大鼠中的多价研究：在Wistar大鼠中i.m.施用时二价疫苗组合物的免Example 12: Multivalent studies in rats: Immunization of a bivalent vaccine composition when administered i.m. in Wistar rats疫原性Immunogenicity
在这个研究中，在Wistar大鼠中评价了由不同LNP配制的二价mRNA疫苗组合物诱导的体液免疫原性。In this study, the humoral immunogenicity induced by bivalent mRNA vaccine compositions formulated with different LNPs was evaluated in Wistar rats.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备SARS-CoV-2 mRNA构建体(基于mRNA的SARS-CoV-2，编码全长的、融合前稳定的变体SARS-CoV-2 S)。在用于体内接种实验前，根据实施例1.4和实施例1.5用LNP配制HPLC纯化的mRNA(对于二价mRNA疫苗单独混合或配制)。SARS-CoV-2 mRNA constructs (mRNA-based SARS-CoV-2, encoding full-length, prefusion-stabilized variant SARS-CoV-2 S) were prepared as described in Example 1.2 (RNA in vitro transcription). HPLC-purified mRNA was formulated with LNPs according to Examples 1.4 and 1.5 (mixed or formulated separately for bivalent mRNA vaccines) before use in in vivo vaccination experiments.
免疫：immunity :
大鼠肌内(i.m.)注射表6所示的二价mRNA疫苗组合物和剂量。接种缓冲液的动物用作阴性对照(第1组)。在第0天和第21天给所有动物接种。在第14天、第21天和第42天收集血样用于测定体液免疫反应。Rats were injected intramuscularly (i.m.) with the bivalent mRNA vaccine compositions and doses shown in Table 6. Animals vaccinated with buffer were used as negative controls (Group 1). All animals were vaccinated on days 0 and 21. Blood samples were collected on days 14, 21, and 42 for determination of humoral immune responses.
表17：疫苗接种方案(实施例12)Table 17: Vaccination schedule (Example 12)
对于一些构建体，尿苷被m1ψ(1-甲基假尿苷)替代：R10828、R10159、R10813。For some constructs, uridine was replaced by mlψ (1-methylpseudouridine): R10828, R10159, R10813.
如实施例2所述，使用ELISA测定总IgG刺突结合抗体滴度。使用祖先SARS-CoV-2的重组刺突RBD蛋白、变体B.1.617.2(L452R，T478K:δ)的RBD或B.1.351(K417N，E484K，N501Y:beta)的RBD进行包被。Total IgG spike-binding antibody titers were determined using ELISA as described in Example 2. Coating was performed using recombinant spike RBD proteins of ancestral SARS-CoV-2, RBD of variant B.1.617.2 (L452R, T478K: δ), or RBD of B.1.351 (K417N, E484K, N501Y: beta).
结果：result :
用包含在LNP中配制的编码不同变体的全长稳定的刺突蛋白的不同mRNA形式的二价疫苗组合物接种，所述不同mRNA形式包含天然或化学修饰核苷酸(m1ψ(1-甲基假尿苷))(更多详细内容参见表17)，在第14天在大鼠中诱导了稳定且高水平的刺突结合抗体滴度。图13证明了同源以及异源反应。(图13A：祖先SARS-CoV-2 RBD；图13B：B.1.617.2 RBD(L452R，T478K，δ)；图13C：B.1.351 RBD(K417N，E484K，N501Y，β)。对于同源以及异源反应，具有修饰核苷酸的构建体比具有天然核苷酸的构建体诱导更高的总IgG滴度。Vaccination with a bivalent vaccine composition comprising different mRNA forms encoding different variants of the full-length stabilized spike protein formulated in LNPs, comprising natural or chemically modified nucleotides (m1ψ(1-methylpseudouridine)) (see Table 17 for more details), induced stable and high levels of spike-binding antibody titers in rats on day 14. Figure 13 demonstrates homologous as well as heterologous responses. (Figure 13A: ancestral SARS-CoV-2 RBD; Figure 13B: B.1.617.2 RBD (L452R, T478K, δ); Figure 13C: B.1.351 RBD (K417N, E484K, N501Y, β). For both homologous and heterologous responses, constructs with modified nucleotides induced higher total IgG titers than constructs with natural nucleotides.
实施例13：在k18-hACE2小鼠中用SARS-CoV-2 B.1.351和B.1.617.2的挑战研究Example 13: Challenge studies with SARS-CoV-2 B.1.351 and B.1.617.2 in k18-hACE2 mice
通常，小鼠不易感染SARS-CoV-2，但已经开发出一种遗传工程化小鼠模型，该模型表达人类受体ACE2(hACE2)，这是病毒在K18启动子下进入宿主细胞中所需的。该模型最初是为了研究SARS(SARS-CoV)的致病因子而开发的(MCCRAY，Paul B.等，Lethal infectionof K18-hACE2 mice infected with severe acute respiratory syndromecoronavirus.Journal of virology,2007,81.Jg.,Nr.2,S.813-821)，但现在也被用作用于COVID-19的合适的小动物模型。此前，hACE2小鼠已被证明对SARS-CoV-2易感，并表现出具有体重减轻、肺部病理和与人类中相似的症状的病程(例如，BAO，Linlin等，Thepathogenicity of SARS-CoV-2 in hACE2 transgenic mice.Nature,2020,583.Jg.,Nr.7818,S.830-833，或YINDA,Claude Kwe等，K18-hACE2 mice develop respiratorydisease resembling severe COVID-19.PLoS pathogens,2021,17.Jg.,Nr.1,S.e1009195；DE ALWIS,Ruklanthi M.等，A Single Dose of Self-Transcribing andReplicating RNA Based SARS-CoV-2 Vaccine Produces Protective AdaptiveImmunity In Mice.BioRxiv,2020.)。原则上，K18-hACE2小鼠适用于疫苗研究，以研究SARS-CoV-2或SARS-CoV-2变体感染的预防或病毒载量的减少，同时用公知的免疫学方法(其通常适用于小鼠模型)研究抗COVID-19的mRNA疫苗的保护作用的相关因素和原因。Normally, mice are not susceptible to SARS-CoV-2 infection, but a genetically engineered mouse model has been developed that expresses the human receptor ACE2 (hACE2), which is required for the virus to enter host cells under the K18 promoter. This model was originally developed to study the pathogenicity of SARS (SARS-CoV) (MCCRAY, Paul B., et al., Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. Journal of virology, 2007, 81. Jg., Nr. 2, S. 813-821), but is now also used as a suitable small animal model for COVID-19. Previously, hACE2 mice have been shown to be susceptible to SARS-CoV-2 and exhibit a disease course with weight loss, lung pathology, and symptoms similar to those in humans (e.g., BAO, Linlin et al., The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature, 2020, 583. Jg., Nr. 7818, S. 830-833, or YINDA, Claude Kwe et al., K18-hACE2 mice develop respiratory disease resembling severe COVID-19. PLoS pathogens, 2021, 17. Jg., Nr. 1, S. e1009195; DE ALWIS, Ruklanthi M. et al., A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice. BioRxiv, 2020.). In principle, K18-hACE2 mice are suitable for vaccine research to study the prevention of SARS-CoV-2 or SARS-CoV-2 variant infection or the reduction of viral load, while studying the factors and causes of the protective effect of mRNA vaccines against COVID-19 using well-known immunological methods (which are generally applicable to mouse models).
本实施例显示了SARS-CoV-2变体S mRNA疫苗能够保护K18-hACE2小鼠免受SARS-CoV-2病毒攻击，这可以通过例如测量受感染动物的病毒载量，通过监测伴随体重减轻、肺部病理学和其他症状的疾病进展，或通过组织病理学和存活率来显示。This example shows that the SARS-CoV-2 variant S mRNA vaccine is able to protect K18-hACE2 mice from SARS-CoV-2 virus challenge, which can be shown by, for example, measuring the viral load of infected animals, by monitoring disease progression accompanied by weight loss, lung pathology and other symptoms, or by histopathology and survival rate.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1.2所述(RNA体外转录)制备用于SARS-CoV-2疫苗的mRNA构建体。在用于体内接种实验前，根据实施例1.4和实施例1.5用LNP配制HPLC纯化的mRNA(对于二价mRNA疫苗单独混合或配制)。The mRNA constructs for the SARS-CoV-2 vaccine were prepared as described in Example 1.2 (RNA in vitro transcription). Before use in the in vivo vaccination experiment, HPLC purified mRNA was formulated with LNP according to Example 1.4 and Example 1.5 (mixed or formulated separately for the bivalent mRNA vaccine).
免疫和攻击：Immunity and Attack :
K18-hACE2转基因小鼠(雌性，n＝2×10)肌内(i.m.)注射如表18所示的mRNA疫苗组合物(第1-4组)。作为阴性对照，一组小鼠用缓冲液处理(第5组)。动物在第0天和第28天接种了20μl体积的指示剂量。在第0天、第28天(初免后)、第56天(加强后)和第66天(攻击后)采集血样用于测定抗体滴度。在第56天，用SARS-CoV-2病毒(每只小鼠104.375TCID50SARS-CoV-2 B.1.351和104.375TCID50 SARS-CoV-2 B.1.617.2，从原始材料的反向滴定计算)对动物进行i.n.攻击/感染，并监测10天的体重变化、总体健康和存活，这表明对攻击的保护作用。其他的保护参数包括减少的肺和其他器官中的病毒载量以及减少的肺的病理学。RNA提取和RT-qPCR和sgRNA RT-PCR按照Hoffman等2021(Hoffmann,D.，Corleis,B.,Rauch,S.等，CVnCoV and CV2CoV protect human ACE2 transgenic mice fromancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.Nat Commun 12,4048(2021))中所述的进行。K18-hACE2 transgenic mice (female, n = 2 × 10) were injected intramuscularly (im) with mRNA vaccine compositions as shown in Table 18 (Groups 1-4). As a negative control, one group of mice was treated with buffer (Group 5). Animals were vaccinated with an indicated dose of 20 μl volume on days 0 and 28. Blood samples were collected on days 0, 28 (after primary immunization), 56 (after boosting), and 66 (after attack) for antibody titer determination. On day 56, animals were challenged/infected with SARS-CoV-2 virus (104.375 TCID50 SARS-CoV-2 B.1.351 and 104.375 TCID50 SARS-CoV-2 B.1.617.2 per mouse, calculated from reverse titration of the original material), and weight changes, overall health, and survival were monitored for 10 days, indicating protection against attack. Other protective parameters include reduced viral load in the lungs and other organs and reduced lung pathology. RNA extraction and RT-qPCR and sgRNA RT-PCR were performed as described in Hoffman et al. 2021 (Hoffmann, D., Corleis, B., Rauch, S. et al., CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat Commun 12, 4048 (2021)).
表18：疫苗接种方案(实施例13)：Table 18: Vaccination schedule (Example 13):
RBD抗体酶联免疫吸附测定(ELISA)RBD antibody enzyme-linked immunosorbent assay (ELISA)
使用基于SARS-CoV-2的RBD(祖先)的间接多物种ELISA分析血清。为此，在0.1M碳酸盐缓冲液(1.59g Na2CO3和2.93g NaHCO3，约1L去离子水，pH 9.6)中，在4℃下用100ng/孔RBD包被ELISA板(Greiner Bio-One GmbH)过夜，或仅用包被缓冲液处理。然后，使用PBS中的5％脱脂乳在37℃下将板封闭1h。血清在TBS-Tween(TBST)中预稀释1/100，并在RT下在包被和未包被的孔上孵育1h。将多物种缀合物(SBVMILK；从IDSchmallenberg病毒乳间接ELISA获得；IDvet)稀释1/80，然后在RT下加入1h。加入四甲基联苯胺底物(IDEXX)后，在Tecan光谱微型仪器(Tecan Group Ltd.)上以450nm的波长采集ELISA读数。在每一步之间，用TBST洗涤平板三次。通过从相应样品的蛋白质包被孔获得的值中减去在未包被孔上测量的光密度来计算吸光度。值得注意的是，ELISA确定了抗RBD Ig水平的相对丰度，且因此不允许在不同研究之间进行直接比较。Sera were analyzed using an indirect multispecies ELISA based on the RBD (ancestor) of SARS-CoV-2. For this purpose, ELISA plates (Greiner Bio-One GmbH) were coated with 100 ng/well RBD in 0.1 M carbonate buffer (1.59 g Na2 CO3 and 2.93 g NaHCO3 , about 1 L deionized water, pH 9.6) at 4°C overnight or treated with coating buffer alone. The plates were then blocked with 5% skim milk in PBS for 1 h at 37°C. Sera were pre-diluted 1/100 in TBS-Tween (TBST) and incubated on coated and uncoated wells for 1 h at RT. A multispecies conjugate (SBVMILK; from ID Schmallenberg virus milk indirect ELISA was obtained; IDvet) was diluted 1/80 and then added at RT for 1h. After adding tetramethylbenzidine substrate (IDEXX), ELISA readings were collected at a wavelength of 450nm on a Tecan spectral micro instrument (Tecan Group Ltd.). Between each step, the plate was washed three times with TBST. The absorbance was calculated by subtracting the optical density measured on the uncoated wells from the value obtained from the protein-coated wells of the corresponding samples. It is worth noting that ELISA determines the relative abundance of anti-RBD Ig levels and therefore does not allow direct comparison between different studies.
病毒中和测试(VNT)Virus Neutralization Test (VNT)
血清用DMEM在96孔深孔母板中预稀释1/16或1/32。在三个重复研究中，将100μl这种预稀释样品转移到96孔板中。通过使50μl DMEM中的50μl血清稀释液进行log2稀释，在每个孔中留下50μl的血清稀释液。随后，向每个孔中加入50μl相应的SARS-CoV-2(B.1.351或B.1.617.2)病毒稀释液(100TCID50/孔)，并在37℃下孵育1小时。最后，向每个孔中加入100μl在补充1％青霉素/链霉素的DMEM中的胰蛋白酶化VeroE6细胞(每100ml一个汇合TC175烧瓶的细胞)。在37℃下孵育72小时后，通过光学显微镜对孔进行评估。在没有观察到特异性CPE的情况下，将血清稀释液计为中和。通过病毒滴定法确认病毒滴度，包括阳性和阴性血清样品。The serum was pre-diluted 1/16 or 1/32 with DMEM in a 96-well deep-well mother plate. In three replicate studies, 100 μl of this pre-diluted sample was transferred to a 96-well plate. By making a log2 dilution of 50 μl serum dilution in 50 μl DMEM, 50 μl of serum dilution was left in each well. Subsequently, 50 μl of the corresponding SARS-CoV-2 (B.1.351 or B.1.617.2) virus dilution (100 TCID50/well) was added to each well and incubated at 37 ° C for 1 hour. Finally, 100 μl of trypsinized VeroE6 cells (cells of a confluent TC175 flask per 100 ml) in DMEM supplemented with 1% penicillin/streptomycin were added to each well. After incubation at 37 ° C for 72 hours, the wells were evaluated by optical microscopy. In the absence of specific CPE, the serum dilution was counted as neutralization. Virus titers were confirmed by viral titration, including both positive and negative serum samples.
结果：result :
通过用SARS-CoV-2变体B.1.351或SARS-CoV-2变体B.1.627.2攻击小鼠来测试疫苗效力。如图14所示，所有接种组(第1-4组)的小鼠受益于用包含编码SARS-CoV-2祖先或变体刺突蛋白的mRNA的组合物进行的接种。图14A和B证明了受攻击小鼠在感染/攻击后几天的存活(图14A：用B.1.351攻击，图14B：用B.1.617.2攻击)。用所有测试的mRNA疫苗接种导致小鼠对两种测试的SARS-CoV-2变体的完全保护(100％存活)，而与编码的刺突变体无关(第1组：祖先，第2组：B.1.351，第3组B.1.617.2，第4组B.1.351+3B.1.617.2)。图14C和D证明了感染/攻击后几天的体重变化百分比(图14C：用B.1.351攻击，图14D：用B.1.617.2攻击，平均体重百分比)。所有接种组(第1-4组)的小鼠没有显示出显著的体重减轻。Vaccine efficacy was tested by attacking mice with SARS-CoV-2 variant B.1.351 or SARS-CoV-2 variant B.1.627.2. As shown in Figure 14, mice in all vaccination groups (Groups 1-4) benefited from vaccination with a composition containing mRNA encoding SARS-CoV-2 ancestor or variant spike protein. Figures 14A and B demonstrate the survival of attacked mice a few days after infection/attack (Figure 14A: attack with B.1.351, Figure 14B: attack with B.1.617.2). Vaccination with all tested mRNA vaccines resulted in complete protection (100% survival) of mice against two tested SARS-CoV-2 variants, regardless of the encoded spike mutants (Group 1: ancestor, Group 2: B.1.351, Group 3 B.1.617.2, Group 4 B.1.351+3B.1.617.2). Figures 14C and D demonstrate the percent body weight change several days after infection/challenge (Figure 14C: challenge with B.1.351, Figure 14D: challenge with B.1.617.2, mean percent body weight). Mice in all vaccinated groups (Groups 1-4) did not show significant weight loss.
为了研究接种疫苗是否阻止了生产性感染或复制性SAR-CoV-2的传播，在感染后第4天采集口腔拭子，以监测唾液中的病毒RNA载量。在假手术组中，8/9或6/9个样本在分别感染SAR-CoV-2变体B.1.351或SAR-CoV-2 B.1.617.2后对病毒基因组呈阳性(图14E：B.1.351攻击组，图14F：B.1.617.2攻击组)。相反，在mRNA接种后，无论疫苗组如何，在任一挑战组的口腔拭子中均未检测到病毒基因组(仅在CV2CoV接种组的一只小鼠中检测到病毒基因，图14E，第1组)。为了进一步探索攻击后病毒复制的预防，在感染后10天分析了上呼吸道(URT)(鼻甲)和下呼吸道(LRT)(肺)以及中枢神经系统(脑，小脑/大脑)的病毒载量。在用SAR-CoV-2 B.1.351攻击的小鼠中，在URT中与未接种的小鼠(第5组)相比，在所有接种组中观察到可检测病毒复制的减少(图14G)。这种作用对于用SAR-CoV-2变体B.1.617.2攻击的小鼠更为显著(图14H)。接种编码SAR-CoV-2刺突变体B.1.617.2的LNP mRNA的小鼠(第3组)在同源病毒攻击后在鼻甲中未显示复制(图14H)。在LRT中，没有动物在低水平上对SAR-CoV-2 RNA呈阳性，这表明在所有组中都能保护动物免受SAR-CoV-2变体B.1.351和SAR-CoV-2变体B.1.617.2的感染(分别为图14I和J)。对于脑，获得了类似的结果(图14K和L用于小脑，图14M和N用于大脑(对于攻击组B.1.351：图14K和M，对于B.1.617.2：图14L与N)。To investigate whether vaccination prevented productive infection or the spread of replicative SAR-CoV-2, oral swabs were collected on day 4 post-infection to monitor viral RNA load in saliva. In the sham group, 8/9 or 6/9 samples were positive for viral genomes after infection with SAR-CoV-2 variant B.1.351 or SAR-CoV-2 B.1.617.2, respectively (Figure 14E: B.1.351 challenge group, Figure 14F: B.1.617.2 challenge group). In contrast, after mRNA vaccination, no viral genomes were detected in oral swabs from either challenge group, regardless of vaccine group (viral genes were detected only in one mouse in the CV2CoV-vaccinated group, Figure 14E, Group 1). To further explore the prevention of viral replication after challenge, viral loads in the upper respiratory tract (URT) (nasal concha) and lower respiratory tract (LRT) (lungs) as well as the central nervous system (brain, cerebellum/cerebrum) were analyzed 10 days after infection. In mice challenged with SAR-CoV-2 B.1.351, a reduction in detectable viral replication was observed in all vaccinated groups compared to unvaccinated mice (Group 5) in the URT (Figure 14G). This effect was more pronounced for mice challenged with SAR-CoV-2 variant B.1.617.2 (Figure 14H). Mice vaccinated with LNP mRNA encoding SAR-CoV-2 spike mutant B.1.617.2 (Group 3) showed no replication in the nasal turbinates after homologous virus challenge (Figure 14H). No animals were positive for SAR-CoV-2 RNA at low levels in the LRT, indicating that animals were protected from infection with SAR-CoV-2 variant B.1.351 and SAR-CoV-2 variant B.1.617.2 in all groups (Figures 14I and J, respectively). Similar results were obtained for the brain (Figures 14K and L for the cerebellum and Figures 14M and N for the cerebrum (for challenge group B.1.351: Figures 14K and M, for B.1.617.2: Figures 14L and N).
二价疫苗(第4组)诱导的对两种病毒变体(B.1.351和B.1.617.2)的保护作用与单价疫苗(第1-3组)相当，尽管使用较低剂量的每种疫苗。The bivalent vaccine (Group 4) induced protection against two viral variants (B.1.351 and B.1.617.2) comparable to that of the monovalent vaccines (Groups 1-3), despite the use of lower doses of each vaccine.
在第28天(仅针对攻击组B.1.351进行测试)和第56天(针对两组)收集的来自所有接种小鼠的血清显示了强的抗RBD总免疫球蛋白(Ig)的诱导，无论mRNA编码哪种变体刺突(图14O：攻击组B.1.351，图14P：攻击组B.1617.2)。mRNA疫苗组中的抗RBD抗体的强诱导由高病毒中和滴度(VNT)反映(图14Q：攻击后组B.1.351，图14R：攻击前组B.1.617.2，图14S：攻击后组B.1.617.2)。总之，测试的mRNA疫苗在初免-加强方案中诱导了稳定的抗体反应，能够在体外有效中和SAR-CoV-2变体B.1.351和SAR-CoV-2 B.1.617.2。Sera from all vaccinated mice collected on day 28 (tested only for the attack group B.1.351) and day 56 (for both groups) showed strong induction of total immunoglobulins (Ig) against RBD, regardless of which variant spike was encoded by the mRNA (Figure 14O: attack group B.1.351, Figure 14P: attack group B.1617.2). The strong induction of anti-RBD antibodies in the mRNA vaccine group was reflected by high virus neutralization titers (VNT) (Figure 14Q: post-attack group B.1.351, Figure 14R: pre-attack group B.1.617.2, Figure 14S: post-attack group B.1.617.2). In summary, the tested mRNA vaccines induced a stable antibody response in the prime-boost regimen, and were able to effectively neutralize SAR-CoV-2 variants B.1.351 and SAR-CoV-2 B.1.617.2 in vitro.
实施例14：接种CV2CoV或CV2CoV.351的仓鼠的攻击研究研究了在LNP中配制的编码变体B.1.351 S_stab的LNP-mRNA(CV2CoV.351)对叙利亚仓鼠中的保护作用。该模型代表了轻度至中度人类肺部疾病病理学，并且是研究人类相关免疫原性和发病机制的公认和接受的模型之一(Fontela等，PMID 32967005)。仓鼠易受野生型SARS-CoV-2感染，导致高水平的病毒复制，以及病毒靶器官中的组织病理学变化。Example 14: Challenge study of hamsters vaccinated with CV2CoV or CV2CoV.351 The protective effect of LNP-mRNA (CV2CoV.351) encoding variant B.1.351 S_stab formulated in LNP was studied in Syrian hamsters. This model represents mild to moderate human lung disease pathology and is one of the recognized and accepted models for studying human-related immunogenicity and pathogenesis ( Fontela et al., PMID 32967005). Hamsters are susceptible to infection with wild-type SARS-CoV-2, resulting in high levels of viral replication and histopathological changes in viral target organs.
LNP配制的mRNA疫苗的制备：Preparation of LNP-formulated mRNA vaccines :
如实施例1所述(RNA体外转录)制备了SARS-CoV-2 S mRNA构建体。在用于体内接种实验前，根据实施例1.4用LNP配制HPLC纯化的mRNA。The SARS-CoV-2 S mRNA construct was prepared as described in Example 1 (RNA in vitro transcription). Before use in in vivo vaccination experiments, HPLC purified mRNA was formulated with LNP according to Example 1.4.
免疫和攻击：Immunity and Attack :
给叙利亚金色仓鼠(n＝9/组)肌内(i.m.)注射如表19所示的mRNA疫苗组合物和剂量。作为阴性对照，一组仓鼠未处理并模拟注射(缓冲液)(A组)，另一组注射了NaCl作为缓冲液对照。动物在第56天，使用每只仓鼠105.25TCID50(从原始材料的反向滴定计算)用70μl的SARS-CoV-2变体B.1.351在短期吸入麻醉下对动物进行鼻内攻击。在感染后的几天里，除了每天的身体检查和体重称量常规外，还监测病毒的脱落。为了评估病毒脱落，在短期异氟醚麻醉下，从每只仓鼠身上单独收集鼻腔冲洗液。在第28天(初免后)、第55天(加强后)和第60天(攻击感染后)采集血样用于测定抗体滴度。Syrian golden hamsters (n = 9/group) were injected intramuscularly (im) with mRNA vaccine compositions and doses as shown in Table 19. As a negative control, one group of hamsters was untreated and simulated injected (buffer) (Group A), and the other group was injected with NaCl as a buffer control. On the 56th day, animals were challenged intranasally with 70 μl of SARS-CoV-2 variant B.1.351 under short-term inhalation anesthesia using 105.25 TCID50 per hamster (calculated from the reverse titration of the original material). In the days after infection, in addition to daily physical examinations and body weight weighing routines, viral shedding was monitored. To assess viral shedding, nasal washes were collected separately from each hamster under short-term isoflurane anesthesia. Blood samples were collected on the 28th day (after primary immunization), the 55th day (after boosting), and the 60th day (after attack infection) for determination of antibody titers.
表19：疫苗接种方案(实施例14)：Table 19: Vaccination schedule (Example 14) :
抗体分析Antibody analysis
在第0、28、55和60天采集血样用于通过ELISA测定总IgG抗体。用1μg/ml的SARS-CoV-2 S祖先RBD在37℃下包被平板4-5h。将板在10％牛奶中封闭过夜，洗涤并用血清在室温下孵育2h。为了检测，仓鼠血清与生物素山羊抗仓鼠(叙利亚)IgG抗体(BioLegend，Cat：405601)孵育，然后与HRP-链霉亲和素(BD，Cat：554066)孵育。在BioTek SynergyHTX平板读取器中进行特定信号的检测，激发530/25，发射检测590/35和灵敏度45。Blood samples were collected on days 0, 28, 55, and 60 for determination of total IgG antibodies by ELISA. Plates were coated with 1 μg/ml of SARS-CoV-2 S ancestral RBD at 37°C for 4-5 h. The plates were blocked overnight in 10% milk, washed, and incubated with serum for 2 h at room temperature. For detection, hamster serum was incubated with biotin goat anti-hamster (Syrian) IgG antibody (BioLegend, Cat: 405601) and then incubated with HRP-streptavidin (BD, Cat: 554066). Detection of specific signals was performed in a BioTek SynergyHTX plate reader with excitation 530/25, emission detection 590/35, and sensitivity 45.
如实施例13中所述的，通过仅使用SARS-CoV-2病毒变体B.1.351，分析了仓鼠血清样品的病毒中和抗体滴度(VNT)。As described in Example 13, the virus neutralizing antibody titer (VNT) of hamster serum samples was analyzed using only the SARS-CoV-2 virus variant B.1.351.
呼吸道中的病毒载量Viral load in the respiratory tract
随着时间从鼻腔冲洗液以及攻击感染后4天的肺组织样品(头部、内侧、尾部)分离RNA。按照Hoggmann等2021(Hoffmann,D.,Corleis,B.,Rauch,S.et al.CVnCoV and CV2CoVprotect human ACE2 transgenic mice from ancestral B BavPat1 and emergingB.1.351 SARS-CoV-2.Nat Commun 12,4048(2021))中所述的，进行RNA提取及之后通过RT-qPCR检测亚基因组RNA(sgRNA)。RNA was isolated from nasal washes and lung tissue samples (head, medial, tail) 4 days after challenge infection over time. According to Hoggmann et al. 2021 (Hoffmann, D., Corleis, B., Rauch, S. et al. CVnCoV and CV2CoVprotect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2. Nat Commun 12, 4048 (2021)), RNA extraction and subsequent detection of subgenomic RNA (sgRNA) by RT-qPCR.
结果result
通过用105.25TCID50剂量/仓鼠的SARS-CoV-2变体B.1.351攻击仓鼠来测试疫苗效力。图15A证明了在攻击后几天中的体重变化百分比。所有接种组(第2-4组)的小鼠没有表现出显著的体重减轻。未处理的小鼠的体重随着时间下降至90％体重(第1组)。The vaccine efficacy was tested by challenging hamsters with 105.25 TCID50 doses/hamster of SARS-CoV-2 variant B.1.351. Figure 15A demonstrates the percentage of body weight change in a few days after the challenge. Mice in all vaccination groups (Groups 2-4) did not show significant weight loss. The weight of untreated mice dropped to 90% body weight over time (Group 1).
为了研究接种是否能预防SARS-CoV-2的生产性感染，在感染后第2、4、8和12天分析鼻腔冲洗液，以监测受感染动物的病毒RNA载量。在接种组(第2-4组)中，通过检测sgRNA确定的病毒载量与未处理的对照组(第1组)相比略有降低(图15B)。在mRNA接种后，在下呼吸道(LRT)(头部、内侧、尾部肺叶)中检测到更明显、显著减少的病毒基因组(通过检测sgRNA)。在12μg CV2CoV.351疫苗组(第4组)中，只有一只动物对SARS-CoV-2 B.1.351 RNA呈低水平阳性，这表明对SARS-CoV-2变体B.1.351的感染具有保护作用(图15C)。To investigate whether vaccination prevents productive infection with SARS-CoV-2, nasal washes were analyzed on days 2, 4, 8, and 12 post-infection to monitor viral RNA loads in infected animals. In the vaccinated groups (Groups 2-4), viral loads determined by detection of sgRNA were slightly reduced compared to the untreated control group (Group 1) (Figure 15B). After mRNA vaccination, a more pronounced and significantly reduced number of viral genomes (by detection of sgRNA) was detected in the lower respiratory tract (LRT) (head, medial, and caudal lobes). In the 12μg CV2CoV.351 vaccine group (Group 4), only one animal was positive for low levels of SARS-CoV-2 B.1.351 RNA, indicating protection against infection with the SARS-CoV-2 variant B.1.351 (Figure 15C).
在第28天、第55天和第60天收集的来自小鼠的血清显示出强烈的抗RBD总免疫球蛋白(Ig)诱导(图15D)。mRNA疫苗组中抗RBD抗体的这种诱导通过稳定的病毒中和滴度(VNT)反映(参见图15E(空心符号攻击前(第55天)，实心符号激发后第60天)。总体而言，测试的mRNA疫苗在初免-加强方案中诱导了稳定的抗体反应，能够有效中和SARS-CoV-2变体B.1.351。Sera from mice collected on days 28, 55, and 60 showed strong induction of total immunoglobulins (Ig) against RBD (Figure 15D). This induction of anti-RBD antibodies in the mRNA vaccine group was reflected by stable virus neutralization titers (VNT) (see Figure 15E (open symbols before attack (day 55), solid symbols after stimulation day 60). Overall, the tested mRNA vaccines induced a stable antibody response in the prime-boost regimen that was able to effectively neutralize the SARS-CoV-2 variant B.1.351.
实施例15(预测性的)：Example 15 (Prophetic) :
为了证明mRNA疫苗组合物的安全性和有效性，启动了临床试验(I期)。在临床试验中，用根据本发明的在LNP中配制的编码变体SARS-CoV-2刺突蛋白的mRNA对一组人类志愿者进行至少两次肌肉内注射(例如，第0天和第28天)。为了评估根据本发明的疫苗组合物的安全性特征，在施用后监测受试者(生命体征、接种部位耐受性评估、血液学分析)。通过测定接种受试者血清中的病毒中和滴度(VNT)来分析免疫的功效。在作为基线的第0天和完成接种后采集血样。对血清进行病毒中和抗体分析。In order to demonstrate the safety and effectiveness of the mRNA vaccine composition, a clinical trial (Phase I) was initiated. In the clinical trial, a group of human volunteers were injected intramuscularly at least twice (e.g., day 0 and day 28) with mRNA encoding variant SARS-CoV-2 spike protein formulated in LNP according to the present invention. In order to evaluate the safety characteristics of the vaccine composition according to the present invention, the subjects were monitored after administration (vital signs, vaccination site tolerance assessment, hematological analysis). The efficacy of immunity was analyzed by measuring the virus neutralization titer (VNT) in the serum of the vaccinated subjects. Blood samples were collected on day 0 as a baseline and after completion of the vaccination. Serum was analyzed for virus neutralizing antibodies.
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