技术领域Technical Field
本发明属于mRNA疫苗制备技术领域，具体涉及一种以包膜蛋白为靶标的基孔肯雅病毒的mRNA疫苗及其制备方法。The present invention belongs to the technical field of mRNA vaccine preparation, and specifically relates to an mRNA vaccine of Chikungunya virus with envelope protein as a target and a preparation method thereof.
背景技术Background technique
基孔肯雅病毒（Chikungunya Virus, CHIKV）是一种原发于非洲的蚊媒病毒，属甲病毒属，具有广泛的细胞和组织嗜性，可在除B和T细胞外的大多数细胞类型中复制，通过严重的病毒血症传播至全身，主要引发关节炎症，虽然基孔肯雅热是一种自限性疾病，但是会留下严重后遗症，对老人和小孩的危害较大。Chikungunya Virus (CHIKV) is a mosquito-borne virus originating in Africa. It belongs to the genus Alphavirus and has broad cell and tissue tropism. It can replicate in most cell types except B and T cells. It spreads throughout the body through severe viremia and mainly causes joint inflammation. Although Chikungunya fever is a self-limiting disease, it can leave serious sequelae and is more harmful to the elderly and children.
起初CHIKV在非洲东部爆发，随后在非洲各国流行，后在东南亚爆发，后来又在南亚次大陆爆发 CHIKV有四个主要谱系：（1）西非;（2） 东/中/南非 （ECSA）;（3）亚洲谱系；（4）印度洋谱系。起初CHIKV仅由埃及伊蚊传播，因此仅在热带地区发生局部爆发，但随着印度洋谱系的诞生，CHIKV可由分布在温带的白纹伊蚊传播，逐步向温带地区扩散，有全球爆发的趋势。而目前几乎没有上市的安全且效果好的疫苗可用，现有技术下，疫苗制备方法主要有：Initially, CHIKV broke out in East Africa, then became prevalent in African countries, then broke out in Southeast Asia, and later broke out in the South Asian subcontinent. CHIKV has four main lineages: (1) West Africa; (2) East/Central/South Africa (ECSA); (3) Asian lineage; (4) Indian Ocean lineage. Initially, CHIKV was only transmitted by Aedes aegypti, so only local outbreaks occurred in tropical regions. However, with the emergence of the Indian Ocean lineage, CHIKV can be transmitted by Aedes albopictus distributed in temperate zones, gradually spreading to temperate regions, and there is a trend of global outbreaks. However, there are almost no safe and effective vaccines available on the market. Under the existing technology, the main methods for preparing vaccines are:
（1）减毒活疫苗，通过对病毒进行改造使其毒力降低后制备成活疫苗，但病毒进入体内有返祖的趋势，存在安全隐患，且有副作用；(1) Live attenuated vaccines are prepared by modifying the virus to reduce its virulence. However, the virus has a tendency to revert to its original form once it enters the body, which poses a safety hazard and has side effects.
（2）灭活疫苗，通过将CHIKV病毒灭活后制成疫苗，但活毒制备，需重复进行灭活验证，更新代次慢，缺乏良好的T细胞免疫应答，中和抗体存在时间短；(2) Inactivated vaccines are made by inactivating the CHIKV virus. However, the live virus needs to be repeatedly verified for inactivation, has a slow generation rate, lacks a good T cell immune response, and has a short neutralizing antibody life.
（3）重组蛋白疫苗，利用表达系统直接表达出病毒蛋白，甚至可设计为多价或多聚体疫苗，可同时预防多个毒株，但多聚体蛋白折叠后可能不能完全展现抗原全貌，并且其免疫原性相对差，需要合适的佐剂，且免疫周期长；(3) Recombinant protein vaccines use expression systems to directly express viral proteins, and can even be designed as multivalent or polymeric vaccines to prevent multiple strains at the same time. However, after folding, the polymeric protein may not be able to fully display the full picture of the antigen, and its immunogenicity is relatively poor, requiring appropriate adjuvants, and the immunization cycle is long;
mRNA疫苗是一种疫苗形式，其基本原理是通过特定的递送系统将表达抗原靶标的mRNA导入体内，在体内表达出蛋白并刺激机体产生特异性免疫学反应，从而使机体获得免疫保护，以mRNA为基础的药物，特别是mRNA疫苗，已被广泛证明是一种很有前途的免疫治疗策略。mRNA疫苗的独特优势，包括其高效、相对较低的副作用和低成本，目前，针对基孔肯雅病毒相关的mRNA疫苗未见报道。mRNA vaccine is a form of vaccine. Its basic principle is to introduce mRNA expressing antigenic targets into the body through a specific delivery system, express proteins in the body and stimulate the body to produce specific immunological responses, so that the body can obtain immune protection. mRNA-based drugs, especially mRNA vaccines, have been widely proven to be a promising immunotherapy strategy. The unique advantages of mRNA vaccines include their high efficiency, relatively low side effects and low cost. At present, there are no reports of mRNA vaccines related to Chikungunya virus.
基于此，本发明采用寻找保守序列的方法设计疫苗，设计了一条以包膜蛋白为靶点的保守氨基酸序列，为基孔肯雅病毒的防治提供一种更安全和高效的疫苗。Based on this, the present invention adopts the method of finding conservative sequences to design a vaccine, designs a conservative amino acid sequence with envelope protein as the target, and provides a safer and more efficient vaccine for the prevention and treatment of Chikungunya virus.
发明内容Summary of the invention
本发明的主要目的是提供一种以包膜蛋白为靶标的基孔肯雅病毒的mRNA疫苗及其制备方法，为基孔肯雅病毒的防治提供新方法，具体的，本发明提供以下技术方案：The main purpose of the present invention is to provide an mRNA vaccine of Chikungunya virus targeting envelope protein and a preparation method thereof, so as to provide a new method for the prevention and treatment of Chikungunya virus. Specifically, the present invention provides the following technical solutions:
本发明提供了如SEQ ID NO：1所示的核苷酸序列在制备基孔肯雅病毒的mRNA疫苗中的应用。The present invention provides the use of a nucleotide sequence as shown in SEQ ID NO: 1 in preparing an mRNA vaccine for Chikungunya virus.
进一步的，本发明提供如SEQ ID NO：2所示的蛋白在制备基孔肯雅病毒的mRNA疫苗中的应用，如SEQ ID NO：2所示的蛋白由SEQ ID NO：1所示的序列转录、翻译获得。Furthermore, the present invention provides the use of a protein as shown in SEQ ID NO: 2 in the preparation of an mRNA vaccine for Chikungunya virus, wherein the protein as shown in SEQ ID NO: 2 is obtained by transcription and translation of a sequence as shown in SEQ ID NO: 1.
作为一种实施方式，本发明提供一种重组质粒，所述重组质粒的结构为：PUC57-T7-5’UTR-E3-E2-6k-E1-3’UTR-PolyA，编码区E3-E2-6k-E1的核苷酸序列如SEQ ID NO：1所示，5’UTR核苷酸序列如SEQ ID NO：3所示，3’UTR核苷酸序列如SEQ ID NO：4所示。As an embodiment, the present invention provides a recombinant plasmid, the structure of the recombinant plasmid is: PUC57-T7-5’UTR-E3-E2-6k-E1-3’UTR-PolyA, the nucleotide sequence of the coding region E3-E2-6k-E1 is shown in SEQ ID NO: 1, the 5’UTR nucleotide sequence is shown in SEQ ID NO: 3, and the 3’UTR nucleotide sequence is shown in SEQ ID NO: 4.
进一步的，本发明提供一种基孔肯雅病毒的mRNA疫苗，所述疫苗包括疫苗本体和疫苗载体，其特征在于，所述疫苗本体以基孔肯雅病毒包膜蛋白为靶标，所述包膜蛋白的序列如SEQ ID NO：2所示。Furthermore, the present invention provides an mRNA vaccine for Chikungunya virus, comprising a vaccine body and a vaccine vector, characterized in that the vaccine body targets the Chikungunya virus envelope protein, and the sequence of the envelope protein is shown in SEQ ID NO: 2.
进一步的，所述疫苗本体由所述的重组质粒在体外进行酶切线性化，并转录获得。Furthermore, the vaccine body is obtained by linearizing and transcribing the recombinant plasmid in vitro.
进一步的，所述疫苗本体的序列如SEQ ID NO：5所示。Furthermore, the sequence of the vaccine entity is shown in SEQ ID NO:5.
进一步的，所述疫苗载体为脂质纳米粒。Furthermore, the vaccine carrier is a lipid nanoparticle.
优选的，所述脂质纳米粒为： SM102、DMG-PEG2000、DSPC和胆固醇依次按照摩尔百分比50%，1.5%，10%，38.5%进行混合后溶解到无水乙醇溶液中获得。Preferably, the lipid nanoparticles are obtained by mixing SM102, DMG-PEG2000, DSPC and cholesterol in molar percentages of 50%, 1.5%, 10% and 38.5% in sequence and then dissolving them in anhydrous ethanol solution.
作为一种实施方式，本发明提供一种试剂盒，所述试剂盒含有所述的重组质粒或任一所述的疫苗。As an embodiment, the present invention provides a kit, which contains the recombinant plasmid or any of the vaccines.
作为一种实施方式，一种基孔肯雅病毒的mRNA疫苗的制备方法，包括以下步骤：As an embodiment, a method for preparing an mRNA vaccine of Chikungunya virus comprises the following steps:
S1：构建含SEQ ID NO：1所示的核苷酸序列的重组质粒；S1: construct a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO: 1;
S2：将重组质粒进行酶切线性化，对线性化模板进行体外转录，得到mRNA疫苗本体；S2: The recombinant plasmid is linearized by enzyme digestion, and the linearized template is transcribed in vitro to obtain the mRNA vaccine body;
S3：制备疫苗载体：脂质纳米粒溶液；S3: Preparation of vaccine carrier: lipid nanoparticle solution;
S4：将mRNA疫苗本体溶解在柠檬酸缓冲液，并与脂质纳米粒溶液混合，然后超滤获得所述mRNA疫苗。S4: Dissolve the mRNA vaccine in citric acid buffer, mix it with the lipid nanoparticle solution, and then ultrafilter to obtain the mRNA vaccine.
本发明达到的技术效果：The technical effects achieved by the present invention are:
本发明采用寻找保守序列的方法设计疫苗，设计了一条以包膜蛋白为靶点的保守氨基酸序列，并鉴于mRNA疫苗在SRAS-CoV-2爆发期间的出色表现，我们将其设计成mRNA疫苗mRNA-CHIKV-E，通过微流控的方法将其制备成脂质纳米颗粒（LNP），并免疫Balb/c小鼠，采用真病毒中和实验检测该mRNA疫苗免疫小鼠后产生中和抗体水平及用亚洲谱系毒株的灭活病毒及印度洋谱系的E2（Sino Biological 40440-V08B）蛋白进行Elispot实验检测细胞免疫。发现，小鼠在经过两次免疫后对马来西亚株产生了中和抗体，并对亚洲谱系及印度洋谱系的毒株均产生了高水平T细胞免疫应答，并且免疫后的小鼠完全控制了病毒血症的出现，这说明所设计的疫苗具有一定的免疫原性且可以引发良好的T细胞免疫应答，具有良好的保护效力。The present invention adopts the method of finding conservative sequences to design vaccines, designs a conservative amino acid sequence targeting envelope protein, and in view of the excellent performance of mRNA vaccines during the SRAS-CoV-2 outbreak, we design it into mRNA vaccine mRNA-CHIKV-E, prepare it into lipid nanoparticles (LNP) by microfluidics, and immunize Balb/c mice. The level of neutralizing antibodies produced by the mRNA vaccine after immunizing mice with mice was detected by real virus neutralization experiments, and the inactivated virus of the Asian lineage strain and the E2 (Sino Biological 40440-V08B) protein of the Indian Ocean lineage were used to perform Elispot experiments to detect cellular immunity. It was found that after two immunizations, the mice produced neutralizing antibodies to the Malaysian strain, and produced high levels of T cell immune responses to the strains of the Asian lineage and the Indian Ocean lineage, and the immunized mice completely controlled the occurrence of viremia, which shows that the designed vaccine has certain immunogenicity and can induce a good T cell immune response, and has good protective efficacy.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1 mRNA-CHIKV-E示意图。Fig. 1 Schematic diagram of mRNA-CHIKV-E.
图2 mRNA-CHIKV-E制备过程检测结果图；其中：a）体外转录RNA电泳，泳道2 ，3:mRNA-CHIKV-E；b）包封验证，泳道1：mRNA-CHIKV-E-LNP，泳道2：用tritonx-100处理充分释放mRNA后的mRNA-CHIKV-E-LNP；c）制备的mRNA-CHIKV-E-LNP粒径分布。Figure 2 Detection results of the mRNA-CHIKV-E preparation process; wherein: a) in vitro transcribed RNA electrophoresis, lanes 2, 3: mRNA-CHIKV-E; b) encapsulation verification, lane 1: mRNA-CHIKV-E-LNP, lane 2: mRNA-CHIKV-E-LNP after treatment with tritonx-100 to fully release mRNA; c) particle size distribution of prepared mRNA-CHIKV-E-LNP.
图3 mRNA-CHIKV-E转染细胞后蛋白表达量的WB实验结果图；其中： a）mRNA-CHIKV-E-LNP（图中简略标注为 “CV-E-LNP”）转染293T/17细胞，与商用试剂盒Mirus（MIR2225）转染裸RNA表达量对比；b）mRNA-CHIKV-E-LNP（图中简略标注为 “CV-E-LNP”）转染Vero，与商用试剂盒Mirus表达量对比。其中（-）为阴性对照，Mirus为采用商用试剂盒转染，CV-E-LNP是用制备的疫苗进行转染，β-actin为内参蛋白。Figure 3 WB experimental results of protein expression after mRNA-CHIKV-E transfected cells; a) mRNA-CHIKV-E-LNP (abbreviated as "CV-E-LNP" in the figure) was transfected into 293T/17 cells, and the expression was compared with that of naked RNA transfected with the commercial kit Mirus (MIR2225); b) mRNA-CHIKV-E-LNP (abbreviated as "CV-E-LNP" in the figure) was transfected into Vero, and the expression was compared with that of the commercial kit Mirus. (-) is the negative control, Mirus is transfected with a commercial kit, CV-E-LNP is transfected with the prepared vaccine, and β-actin is the internal reference protein.
图4 Vero细胞免疫荧光图。Fig. 4 Immunofluorescence images of Vero cells.
图5 Balb/c小鼠分别免疫不同剂量后五天内体重变化图。Figure 5 Body weight changes of Balb/c mice within five days after immunization with different doses.
图6 mRNA-CHIKV-E免疫小鼠后第7、14、21和28天采集血清检测E2蛋白（SinoBiological 40440-V08B）IgG结合抗体滴度图。Fig. 6. Serum was collected on days 7, 14, 21, and 28 after mice were immunized with mRNA-CHIKV-E to detect IgG binding antibody titers of E2 protein (SinoBiological 40440-V08B).
图7 Elispot实验，用印度洋谱系的E2蛋白和亚洲谱系的灭活病毒作为刺激物进行Elispot实验代表性结果图；其中： a）IFN-γ；b）IL-2。Figure 7 Elispot experiment, representative results of Elispot experiment using E2 protein of Indian Ocean lineage and inactivated virus of Asian lineage as stimuli; wherein: a) IFN-γ; b) IL-2.
图8 Elispot实验分析图；其中： a）用印度洋谱系的E2蛋白作为刺激物检测IFN-γ，产生的斑点统计；b）用亚洲谱系的灭活病毒作为刺激物检测针对IFN-γ产生斑点计数结果； c) 用印度洋谱系的E2蛋白作为刺激物检测针对IL-2产生的斑点统计；d）用亚洲谱系的灭活病毒作为刺激物检测针对IL-2产生斑点计数结果。Figure 8 Elispot experiment analysis diagram; wherein: a) IFN-γ is detected using the E2 protein of the Indian Ocean lineage as a stimulus, and the spot counts are generated; b) IFN-γ is detected using the inactivated virus of the Asian lineage as a stimulus, and the spot counts are generated; c) IL-2 is detected using the E2 protein of the Indian Ocean lineage as a stimulus, and the spot counts are generated; d) IL-2 is detected using the inactivated virus of the Asian lineage as a stimulus, and the spot counts are generated.
图9 免疫后第7、14、21、28天时Balb/c小鼠对马来西亚株（a）及其突变株（b）产生中和抗体滴度图。Fig. 9 Neutralizing antibody titers of Balb/c mice against the Malaysian strain (a) and its mutant strain (b) on days 7, 14, 21, and 28 after immunization.
图10 小鼠攻毒后五天内四肢关节肿胀情况图；其中： a）左前肢；b）右前肢；c）左后肢；d) 右后肢。Figure 10: Swelling of the limb joints of mice within five days after infection; a) left forelimb; b) right forelimb; c) left hindlimb; d) right hindlimb.
图11小鼠攻毒后各剂量组小鼠血液中病毒载量检测图。Figure 11 shows the detection of viral load in the blood of mice in each dose group after infection.
图12 攻毒7天后全身性组织器官的病毒载量检测结果图。Figure 12 shows the results of viral load detection of systemic tissues and organs 7 days after infection.
图13 肺组织的病理损伤分析结果图。Fig. 13 Results of pathological damage analysis of lung tissue.
图14 肝组织的病理损伤分析结果图。Fig. 14 Pathological damage analysis results of liver tissue.
图15 后腿肌肉的病理损伤分析结果图。Fig. 15 Pathological damage analysis results of hind leg muscles.
图16 脑组织的病理分析结果图。Fig. 16 Pathological analysis results of brain tissue.
图17 脾组织的病理分析结果图。Fig. 17 Pathological analysis results of spleen tissue.
图18 肾脏组织的病理分析结果图。Fig. 18 Pathological analysis results of kidney tissue.
图19 心脏组织的病理分析结果图。Fig. 19 Pathological analysis results of heart tissue.
图 20 肺组织、肝组织、后腿肌肉组织、脑组织、脾脏组织、肾脏组织及心脏组织病理评分结果分析统计图。Figure 20 Statistical analysis of pathological scoring results of lung tissue, liver tissue, hind leg muscle tissue, brain tissue, spleen tissue, kidney tissue and heart tissue.
具体实施方式Detailed ways
为了使本发明的目的、技术方案和优势更加清楚，以下结合实施例对本发明进行进一步说明。应当理解，此处所描述的实施例仅用于解释本发明，并不用于限制本发明。本发明中部分材料释义如下表1所示，本发明中所有材料均可由市场购买。In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with embodiments. It should be understood that the embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The interpretation of some materials in the present invention is shown in Table 1 below, and all materials in the present invention can be purchased from the market.
本发明中序列信息如下表2所示The sequence information in the present invention is shown in Table 2 below
实施例1Example 1
1、疫苗制备1. Vaccine preparation
在NCBI Virus数据库中检索CHIKV的全长结构蛋白，共得到来自于不同谱系毒株的788个全长结构蛋白的氨基酸序列，并将它们进行氨基酸序列比对，共1248个氨基酸，取每个氨基酸位点出现最多的氨基酸，将这1248个保守氨基酸重新按顺序组装成一条新的保守氨基酸序列。并按照人的密码子偏好性进行密码子优化，得到DNA序列，取包膜蛋白E3-E2-6k-E1序列构建到含有：The full-length structural proteins of CHIKV were searched in the NCBI Virus database, and the amino acid sequences of 788 full-length structural proteins from different lineages were obtained. The amino acid sequences were aligned, with a total of 1248 amino acids. The most common amino acids at each amino acid site were selected, and the 1248 conservative amino acids were reassembled in order into a new conservative amino acid sequence. Codon optimization was performed according to human codon preference to obtain a DNA sequence, and the envelope protein E3-E2-6k-E1 sequence was constructed to contain:
T7启动子、5’UTR、3’UTR及100个PolyA的质粒PUC57上（PUC57-T7-5’UTR-E3-E2-6k-E1-3’UTR-PolyA）（图1），E3-E2-6k-E1核苷酸序列如SEQ ID NO：1所示，E3-E2-6k-E1蛋白序列如SEQ ID NO：2所示，5’UTR核苷酸序列如SEQ ID NO：3所示，3’UTR核苷酸序列如SEQID NO：4所示。The plasmid PUC57 containing T7 promoter, 5’UTR, 3’UTR and 100 PolyA (PUC57-T7-5’UTR-E3-E2-6k-E1-3’UTR-PolyA) ( Figure 1 ), the E3-E2-6k-E1 nucleotide sequence is shown in SEQ ID NO: 1, the E3-E2-6k-E1 protein sequence is shown in SEQ ID NO: 2, the 5’UTR nucleotide sequence is shown in SEQ ID NO: 3, and the 3’UTR nucleotide sequence is shown in SEQ ID NO: 4.
使用Top10菌种进行质粒扩增，提取质粒后采用BsaI进行酶切线性化，使用70%体积的异丙醇沉淀线性化模板，并用70%乙醇洗涤。采用商用试剂盒（NEB）对线性化模板进行体外转录，转录前，将转录的原料UTP完全替换为N1-甲基假尿苷三磷酸（N1-Me-pUTP），转录过程检测结果如图2，转录后得到mRNA-CHIKV-E（简写为“mRNA-CV-E”），即疫苗本体，核苷酸序列如SEQ ID NO：5所示。Top10 strains were used for plasmid amplification. After plasmid extraction, BsaI was used for restriction enzyme linearization. The linearized template was precipitated with 70% volume of isopropanol and washed with 70% ethanol. The linearized template was transcribed in vitro using a commercial kit (NEB). Before transcription, the raw material UTP of transcription was completely replaced with N1-methyl pseudouridine triphosphate (N1-Me-pUTP). The detection results of the transcription process are shown in Figure 2. After transcription, mRNA-CHIKV-E (abbreviated as "mRNA-CV-E") was obtained, i.e., the vaccine body, and the nucleotide sequence is shown in SEQ ID NO: 5.
2、疫苗载体脂质纳米粒(LNP)包封2. Vaccine carrier lipid nanoparticle (LNP) encapsulation
将mRNA溶解到PH=4, 浓度50mM的柠檬酸缓冲液中，使其终浓度为108 ng/μL。接着配制脂质纳米粒混合溶液：将SM102，DMG-PEG2000，DSPC，胆固醇按照摩尔比为50%，1.5%，10%，38.5%的比例进行混合溶解到无水乙醇溶液中，并将脂质-乙醇溶液与mRNA-柠檬酸溶液分别通过0.22μm微孔滤膜过滤，按照mRNA-CHIKV-E中的磷元素含量与SM102的N元素含量按1:8的摩尔比例，使用微流控仪器进行混合，流速比为缓冲液相:乙醇相为15mL/min：5mL/min，将得到的mRNA-CHIKV-E-LNP立即用15ml上述柠檬酸缓冲液稀释，并用100KD超滤管进行超滤，离心力为3000g，超滤至1/4体积后加50mM的Tris-HCl缓冲液（PH=7.5）至15mL，反复两次后测浓度与包封率，确保包封率达到95%以上（图2），得到包封后的基孔肯雅病毒的mRNA疫苗mRNA-CHIKV-E-LNP。Dissolve the mRNA in a citric acid buffer at pH = 4, concentration 50mM, to a final concentration of 108 ng/μL. Then prepare a lipid nanoparticle mixed solution: SM102, DMG-PEG2000, DSPC, and cholesterol were mixed and dissolved in anhydrous ethanol solution at a molar ratio of 50%, 1.5%, 10%, and 38.5%, and the lipid-ethanol solution and the mRNA-citric acid solution were filtered through a 0.22μm microporous membrane, respectively, and mixed using a microfluidic instrument according to the molar ratio of 1:8 between the phosphorus content in mRNA-CHIKV-E and the N content in SM102, with a flow rate ratio of buffer phase:ethanol phase. The centrifugal force was 3000 g, and after ultrafiltration to 1/4 volume, 50 mM Tris-HCl buffer (PH = 7.5) was added to 15 mL. After repeated two times, the concentration and encapsulation rate were measured to ensure that the encapsulation rate reached more than 95% (Figure 2), and the encapsulated Chikungunya virus mRNA vaccine mRNA-CHIKV-E-LNP was obtained.
mRNA-CHIKV-E-LNP制备好后在293T/17细胞与Vero细胞以相应的mRNA为对照分别将mRNA-CHIKV-E-LNP转染不同的细胞，16h后收细胞总蛋白，分别用CHIKV的E1抗体（GeneTex GTX135187）和E2抗体（Alpha Diagnostic international #CHIKE21-A）WB检测是否有目标抗原的表达，确保mRNA-CHIKV-E-LNP组的细胞高表达E1和E2蛋白（图3）。为了明确疫苗的抗原在细胞上的表达定位，用mRNA-CHIKV-E-LNP转染Vero细胞后用E1抗体（GeneTex GTX135187）通过免疫应该检测定位抗原靶标的表达定位，确保抗原定位到细胞膜上以便于完成后续的抗原分泌和抗原呈递（图4）。After mRNA-CHIKV-E-LNP was prepared, mRNA-CHIKV-E-LNP was transfected into different cells in 293T/17 cells and Vero cells with the corresponding mRNA as the control. After 16 hours, the total cell protein was collected and the expression of the target antigen was detected by WB using CHIKV E1 antibody (GeneTex GTX135187) and E2 antibody (Alpha Diagnostic international #CHIKE21-A) to ensure that the cells in the mRNA-CHIKV-E-LNP group highly expressed E1 and E2 proteins (Figure 3). In order to clarify the expression location of the vaccine antigen on the cell, after Vero cells were transfected with mRNA-CHIKV-E-LNP, the expression location of the antigen target was detected by immunostaining using E1 antibody (GeneTex GTX135187) to ensure that the antigen was localized on the cell membrane to facilitate subsequent antigen secretion and antigen presentation (Figure 4).
验证其表达效果后，用以E蛋白为靶标的mRNA-CHIKV-E-LNP疫苗以高剂量15μg/只、中剂量8μg/只、低剂量4μg/只和LNP为对照分别在0d，14d共两次免疫。监测疫苗免疫后的体重变化，确保疫苗的基本安全（图5）。通过血清对E2蛋白的特异性IgG结合抗体检测，评估疫苗的免疫原性，以确保疫苗的免疫原性良好（图6）。After verifying its expression effect, the mRNA-CHIKV-E-LNP vaccine targeting E protein was used for two immunizations at 0d and 14d with a high dose of 15μg/mouse, a medium dose of 8μg/mouse, a low dose of 4μg/mouse and LNP as a control. The weight changes after vaccination were monitored to ensure the basic safety of the vaccine (Figure 5). The immunogenicity of the vaccine was evaluated by detecting the specific IgG binding antibody to the E2 protein in the serum to ensure that the immunogenicity of the vaccine was good (Figure 6).
为了检测该疫苗免疫后是否产生记忆性细胞免疫效果，运用 ElisPot方法检测了抗原刺激后的脾脏免疫细胞的IL-2和INF-γ的阳性细胞，以评估细胞免疫水平（图7-8）。In order to detect whether the vaccine produces memory cellular immunity after immunization, the ElisPot method was used to detect the positive cells of IL-2 and INF-γ in the spleen immune cells after antigen stimulation to evaluate the level of cellular immunity (Figures 7-8).
为了明确血清中和抗体水平和是否具备交叉保护作用，用CPE方法在Vero细胞中使用两个不同的CHIKV毒株检测免疫后不同时间点的中和抗体水平（图9）。In order to clarify the level of serum neutralizing antibodies and whether there is cross-protection, the neutralizing antibody levels at different time points after immunization were detected in Vero cells using two different CHIKV strains using the CPE method ( Figure 9 ).
最后通过攻毒保护实验等证明了该疫苗的有效性。由于CHIKV通常会引起关节肿胀和变形、攻毒的部位是小鼠的后退肌肉，监测了小鼠的四肢关节变化情况，使用游标卡尺测量关节的纵横两个数值（图10）。攻毒后不同时间点取血用qPCR方法检测血液中的病毒载量，监测病毒血症的动态变化情况（图11）。攻毒后7天解剖各组小鼠，用qPCR方法检测各组织器官的病毒载量，明确疫苗是否有效抑制病毒复制（图12）；同时用HE染色法检测各组织器官的病理损伤情况，并用相应的病理评分进行相对定量统计分析病理损伤程度（图13-图19）。Finally, the effectiveness of the vaccine was demonstrated through virus attack protection experiments. Since CHIKV usually causes joint swelling and deformation, and the site of virus attack is the posterior muscles of mice, the changes in the joints of the limbs of mice were monitored, and the vertical and horizontal values of the joints were measured using a vernier caliper (Figure 10). Blood was collected at different time points after virus attack, and the viral load in the blood was detected by qPCR to monitor the dynamic changes of viremia (Figure 11). Seven days after virus attack, mice in each group were dissected, and the viral load of each tissue and organ was detected by qPCR to determine whether the vaccine effectively inhibited viral replication (Figure 12); at the same time, the pathological damage of each tissue and organ was detected by HE staining, and the corresponding pathological scores were used for relative quantitative statistical analysis of the degree of pathological damage (Figures 13-19).
疫苗验证效果如下：The vaccine verification results are as follows:
体外转录的RNA电泳，及mRNA-CHIKV-E-LNP制备成功验证Electrophoresis of in vitro transcribed RNA and verification of successful preparation of mRNA-CHIKV-E-LNP
由图2中可知，转录得到的RNA未有明显降解及杂带，并且mRNA-CHIKV-E-LNP成功制备，且粒径小于100nm。As can be seen from Figure 2, the transcribed RNA showed no obvious degradation or mixed bands, and mRNA-CHIKV-E-LNP was successfully prepared with a particle size of less than 100 nm.
mRNA-CHIKV-E-LNP细胞水平表达验证Verification of mRNA-CHIKV-E-LNP expression at the cell level
如图3可见，mRNA-CHIKV-E转染后在293T/17细胞与Vero细胞中均得到表达，未转染的阴性对照（-）并没有目的条带。As shown in Figure 3, mRNA-CHIKV-E was expressed in both 293T/17 cells and Vero cells after transfection, and the untransfected negative control (-) did not have the target band.
图4中免疫荧光实验可知，mRNA-CHIKV-E在Vero中成功表达，并主要在细胞内与细胞膜上表达，阴性对照组并未发现目的蛋白。As shown in the immunofluorescence experiment in Figure 4, mRNA-CHIKV-E was successfully expressed in Vero and was mainly expressed in the cells and on the cell membrane. No target protein was found in the negative control group.
BALB/c小鼠注射疫苗后体重变化Body weight changes of BALB/c mice after vaccination
图5中可见，免疫后小鼠与注射空LNP的对照组体重略微下降后整体继续升高，并且体温正常，表明疫苗没有明显的毒性。As can be seen in Figure 5, the body weight of the immunized mice and the control group injected with empty LNP decreased slightly and then continued to increase overall, and the body temperature was normal, indicating that the vaccine had no obvious toxicity.
mRNA-CHIKV-E免疫BALB/c小鼠后血清对E2蛋白抗原结合抗体滴度明显升高：分别在0d，14d共两次免疫After BALB/c mice were immunized with mRNA-CHIKV-E, the titer of serum antibodies binding to E2 protein antigen increased significantly: two immunizations were performed at 0d and 14d, respectively.
图6可见一免后7d就可以产生血清结合抗体，二免后一周结合抗体显著升高，滴度可达105。Figure 6 shows that serum binding antibodies can be produced 7 days after the first immunization, and the binding antibodies increase significantly one week after the second immunization, with the titer reaching 105 .
mRNA-CHIKV-E引发Balb/c小鼠强烈的T细胞免疫，并对亚洲谱系毒株及印度洋谱系毒株均有效mRNA-CHIKV-E induces strong T cell immunity in Balb/c mice and is effective against both Asian and Indian Ocean lineage strains
如图7、8所示，Elispot结果显示与空LNP组相比，各剂量组均能产生强烈的T细胞免疫应答，并且对亚洲谱系与印度洋谱系均有作用。As shown in Figures 7 and 8, the Elispot results showed that compared with the empty LNP group, each dose group was able to produce a strong T cell immune response and had effects on both the Asian lineage and the Indian Ocean lineage.
mRNA-CHIKV-E免疫后对马来西亚株及其突变株产生中和抗体Immunization with mRNA-CHIKV-E produces neutralizing antibodies against the Malaysian strain and its mutants
由图9可知，在一免后21天中和抗体显著升高，并对两个毒株均产生了一定的中和抗体。As shown in Figure 9, the neutralizing antibodies increased significantly 21 days after the first immunization, and certain neutralizing antibodies were produced against both strains.
免疫后Balb/c小鼠攻毒实验Virus challenge experiment in Balb/c mice after immunization
由图10中可知，攻毒后，对照组与空LNP组的右前肢肿胀程度明显大于疫苗组。As can be seen from Figure 10, after the virus attack, the swelling degree of the right forelimbs in the control group and the empty LNP group was significantly greater than that in the vaccine group.
mRNA-CHIKV-E完全控制CHIKV感染小鼠后病毒血症的出现mRNA-CHIKV-E completely controls the emergence of viremia in CHIKV-infected mice
由图11中可知，高、中、低剂量组均能良好的控制病毒血症的出现，相比之下，空LNP组与对照组在感染后第一天就出现较为严重的病毒血症。As shown in Figure 11, the high, medium and low dose groups can all effectively control the occurrence of viremia. In contrast, the empty LNP group and the control group developed more severe viremia on the first day after infection.
鉴于CHIKV病毒感染后可造成全身性组织器官的侵染和组织损伤，因此检测了全身性组织器官的病毒载量，mRNA-CHIKV-E可以有效清除或显著降低全身性组织器官的病毒载量Given that CHIKV infection can cause systemic infection and tissue damage, the viral load of systemic tissues and organs was tested. mRNA-CHIKV-E can effectively eliminate or significantly reduce the viral load of systemic tissues and organs.
由上图12可知，CHIKV攻毒后mRNA-CHIKV-E免疫的小鼠可清除大部分组织器官的病毒载量，脾脏和攻毒注射原位后足肌肉与后足病毒载量显著下降，说明该疫苗可有效清除或显著降低全身性组织器官的病毒载量。As can be seen from Figure 12 above, mice immunized with mRNA-CHIKV-E can clear the viral load of most tissues and organs after CHIKV challenge, and the viral load of the spleen and the hind paw muscles and hind paws injected in situ was significantly reduced, indicating that the vaccine can effectively clear or significantly reduce the viral load of systemic tissues and organs.
mRNA-CHIKV-E免疫小鼠后可显著降低CHIKV攻毒对全身性组织器官的病理损伤程度Immunization of mice with mRNA-CHIKV-E can significantly reduce the pathological damage of systemic tissues and organs caused by CHIKV challenge
对肺组织的出血或淤血、炎性细胞浸润、肺泡结构的完整和支气管的堵塞等病理特点有效减轻，中剂量和低剂量具有显著性差异（P<0.05），见图13及图20的分析结果。It effectively alleviated the pathological features of lung tissue bleeding or congestion, inflammatory cell infiltration, integrity of alveolar structure and bronchial obstruction, with significant differences between the medium dose and the low dose (P < 0.05), as shown in the analysis results of Figures 13 and 20.
对肝脏组织的出血、血管内血栓、炎性细胞浸润和肝细胞肿胀融合等病理特点有效减轻，高剂量和中剂量具有显著性差异（P<0.05，P<0.01），见图14及图20的分析结果。It effectively alleviated the pathological features of liver tissue such as bleeding, intravascular thrombosis, inflammatory cell infiltration and hepatocyte swelling and fusion, with significant differences between high and medium doses (P < 0.05,P < 0.01), as shown in the analysis results of Figures 14 and 20.
对肌肉注射攻毒部位的后腿肌肉的炎性细胞浸润、出血和肌纤维化等病理特点有效减轻，三个剂量组均具有显著性差异（P<0.001），见图15及图20的分析结果。The pathological characteristics of inflammatory cell infiltration, hemorrhage and muscle fibrosis in the hind leg muscles at the site of intramuscular injection of the poison were effectively alleviated, and there were significant differences among the three dosage groups (P <0.001), as shown in the analysis results of Figures 15 and 20.
对脑组织的小胶质细胞增多、轻度出血、部分神经元死亡等病理特点有效减轻，三个剂量组均有显著性差异（P<0.0001,P<0.01），见图16及图20分析结果。It effectively alleviated the pathological features of brain tissue such as microgliosis, mild hemorrhage, and partial neuronal death, with significant differences among the three dosage groups (P <0.0001,P <0.01), as shown in the analysis results of Figures 16 and 20.
对脾脏组织的出血、炎性细胞增多、生发中心活跃或消失等病理特点有效减轻，三个剂量组均有显著性差异（P<0.0001），见图17及图20分析结果。The pathological features of spleen tissue such as bleeding, increased inflammatory cells, active or disappeared germinal centers were effectively alleviated, and there were significant differences among the three dosage groups (P <0.0001), as shown in the analysis results of Figures 17 and 20.
对肾脏组织的出血或淤血、炎性细胞浸润和渗出液等病理特点有效减轻，高剂量和中剂量组有显著性差异（P<0.01,P<0.001），见图18及图20分析结果。It effectively alleviated the pathological features of renal tissue such as bleeding or congestion, inflammatory cell infiltration and exudate, with significant differences between the high-dose and medium-dose groups (P <0.01,P <0.001), as shown in the analysis results in Figures 18 and 20.
对心脏组织的炎性细胞浸润、肌纤维化、出血或淤血等病理特点有效减轻，三个剂量组均具有显著性差异（P<0.0001）见图19及图20分析结果。The pathological characteristics of cardiac tissue, such as inflammatory cell infiltration, muscle fibrosis, hemorrhage or congestion, were effectively alleviated, and there were significant differences among the three dosage groups (P <0.0001). See the analysis results in Figures 19 and 20.
综上，本发明设计出针对CHIKV不同谱系具有交叉保护性的疫苗，降低生产成本与免疫周期，可有效的应对CHIKV的爆发。以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。In summary, the present invention designs a vaccine with cross-protection against different lineages of CHIKV, reduces production costs and immunization cycles, and can effectively respond to the outbreak of CHIKV. The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by equivalent replacement or equivalent conversion are within the scope of protection of the present invention.
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