CIRCULAR RNA VACCINES FOR SEASONAL FLU AND METHODS OF USESRelated applications
The application claims priority to, and the benefit of PCT Application No. PCT/CN2023/106381, filed on July 7, 2023, the contents of which are incorporated herein by reference in their entirety.
Incorporation by reference of sequence listing
The contents of electronic sequence listing (TFH01060PCT-sequence listing. xml; Size: 1, 080, 415 bytes; and Date of Creation: July 8, 2024) are herein incorporated by reference in its entirety.
1.FieldThe present invention relates to the field of molecular biology and immunology, in particular to constructs and methods for the manufacture and therapeutic use of circular RNAs as vaccines for influenza viruses.
2.BackgroundCircular RNAs (circRNAs) are a category of RNA molecules formed by head-to-tail ligation, which were demonstrated to have multiple biological functions in recent years. (Yang et al., Cell Research, 27 (5) : 626-641 (2017) ; Abe et al., Scientific Reports, 5: 16435 (2015) ; Gao et al., Nature Cell Biology, 23 (3) : 278-291 (2021) ; Pamudurti et al., Molecular Cell, 66 (1) : 9-21 (2017) ) . Compared with linear RNAs, circRNAs have better stability and therefore provide a promising new platform for RNA drugs.
Influenza is an acute respiratory infection caused by influenza viruses -influenza A and influenza B viruses -that circulate the world. Vaccination plays a critical role in controlling annual influenza epidemics. Vaccines elicit immune responses that attack the viral glycoprotein hemagglutinin (HA) and the viral enzyme neuraminidase (NA) found on the surface of the influenza virus. Improved immunogenic compositions against influenza are in great need.
The compositions provided herein, which comprise circular RNAs as vaccines for influenza viruses, as well as related methods and systems address this need and provide related advantages.
3.SummaryProvided herein are circular ribonucleotide acid (circRNA) scomprising, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a hemagglutinin sequence (HA sequence) encoding a hemagglutinin antigen (HA antigen) or a neuraminidase sequence (NA sequence) encoding a neuraminidase antigen (NA antigen) .
In some embodiments, the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus that is type A or B.
In some embodiments, the Z sequence further comprises a second HA (HA2) sequence encoding an HA antigen from a second influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, HA1, L, and HA2, wherein L is a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence further comprises a third HA (HA3) sequence encoding an HA antigen from a third influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, HA1, L1, HA2, L2, and HA3, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first, second and third viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence further comprises a fourth HA (HA4) sequence encoding an HA antigen from a fourth influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, HA1, L1, HA2, L2, HA3, L3, and HA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus that is type A or B.
In some embodiments, the Z sequence further comprises a second NA (NA2) sequence encoding an NA antigen from a second influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, NA1, L, and NA2; wherein L is a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence further comprises a third NA (NA3) sequence encoding an NA antigen from a third influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, NA1, L1, NA2, L2, and NA3; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first, second, and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence further comprises a fourth NA (NA4) sequence encoding an NA antigen from a fourth influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI, NA1, L1, NA2, L2, NA3, L3, and NA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen, and a first NA (NA1) sequence encoding an NA antigen, and the HA and NA antigens are from a first influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: (1) TI, HA1, L, and NA1; or (2) TI, NA1, L, and HA1; wherein L is a linker sequence or absent.
In some embodiments, the Z sequence further comprises a second HA (HA2) sequence encoding an HA antigen and a second NA (NA2) sequence encoding an NA antigen, wherein the HA and NA antigens are from a second influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, and NA2; (2) TI, NA1, L1, HA1, L2, NA2, L3, and HA2; (3) TI, HA1, L1, HA2, L2, NA1, L3, and NA2; or (4) TI, NA1, L1, NA2, L2, HA1, L3, and HA2; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z sequence further comprises a third HA (HA3) sequence encoding an HA antigen and a third NA (NA3) sequence encoding an NA antigen, wherein the HA and NA antigens are from a third influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3; (2) TI, HA1, L1, HA2, L2, HA3, L3, NA1, L4, NA2, L5, and NA3; (3) TI, NA1, L1, NA2, L2, NA3, L3, HA1, L4, HA2, L5, and HA3; or (4) TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3; wherein L1, L2, L3, L4, and L5 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the circRNAs comprise in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises an HA sequence encoding an HA antigen, or an NA sequence encoding an NA antigen.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and the Z2 sequence comprises a second HA (HA2) sequence, encoding HA antigens from a first and a second influenza viruses, each independently is type A or B. In some embodiments, the circRNAs comprise in the following order: TI1, HA1, L1, TI2, HA2, and L2, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first NA (NA1) sequence and the Z2 sequence encodes a second NA (NA2) sequence, encoding NA antigens from a first and a second influenza viruses, each independently is type A or B. In some embodiments, the circRNAs comprise in the following order: TI1, NA1, L1, TI2, NA2, and L2; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, and the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence; wherein the HA1 and NA1 sequences encode HA and NA antigens from a first influenza virus, and the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus, each independently is type A or B. In some embodiments, the circRNAs comprise in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2, or (2) TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; or (3) A and B; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence encoding an HA antigen and the Z2 sequence comprises a first NA (NA1) sequence encoding an NA antigen, wherein the HA and NA antigens are from a first influenza virus that is type A or B.
In some embodiments, the Z1 sequence further comprise a second HA (HA2) sequence, and the Z2 sequence further comprises a second NA (NA2) sequence; wherein the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus that is type A or B.
In some embodiments, the circRNAs comprise in the following order: TI1, HA1, L1, HA2, TI2, NA1, L2, NA2, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence further comprises a third HA sequence (HA3) and the Z2 sequence further comprises a third NA sequence (NA3) , wherein the HA3 and NA3 sequences encode HA and NA antigens from a third influenza virus that is type A or B. In some embodiments, the circRNAs comprise in the following order: TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the circRNAs comprise in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, and a third protein-coding (Z3) sequence, wherein the Z1, Z2 and Z3 sequences each independently comprises an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, and the Z3 comprises encodes a third HA (HA3) sequence; wherein the HA1, HA2 and HA3 sequences encode HA antigens from a first, a second and a third influenza viruses, respectively. In some embodiments, the circRNAs comprise in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, and the Z3 sequence comprises a third NA (NA3) sequence; wherein the NA1, NA2 and NA3 sequences encode NA antigens from a first, a second and a third influenza viruses, respectively. In some embodiments, the circRNAs comprise in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, and the Z3 comprises encodes a third HA (HA3) sequence and a third NA (NA3) sequence; wherein the HA1 and NA1 sequences encode antigens from a first influenza virus, the HA2 and NA2 encode antigens from a second influenza virus, and the HA3 and NA3 encode antigens from a third influenza virus. In some embodiments, the circRNAs comprise in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3; or (2) TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the circRNAs comprise in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, a third protein-coding (Z3) sequence, a fourth translation initiation (TI4) sequence, a fourth protein-coding (Z4) sequence, wherein the Z1, Z2, Z3 and Z4 sequences each independently comprises an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, the Z3 comprises encodes a third HA (HA3) sequence, and the Z4 comprises encodes a fourth HA (HA4) sequence; wherein the HA1, HA2, HA3, and HA4 sequences encode HA antigens from a first, a second, a third and a fourth influenza viruses, respectively. In some embodiments, the circRNAs comprise in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, the Z3 sequence comprises a third NA (NA3) sequence, and the Z4 sequence comprises a fourth NA (NA4) sequence; wherein the NA1, NA2, NA3 and NA4 sequences encode NA antigens from a first, a second, a third and a fourth influenza viruses, respectively. In some embodiments, the circRNAs comprise in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments, the HA antigen and/or NA antigen is from a seasonal influenza virus. In some embodiments, the HA antigen and/or NA antigen is selected according to standardized criteria used by World Health Organization’s Global Influenza Surveillance and Response System (GISRS) . In some embodiments, the HA antigen and/or NA antigen is selected using a hemagglutinin inhibition (HAI) assay and/or neuraminidase-inhibition (NAI) assay to identify circulating influenza viruses that are antigenically similar to influenza viruses from a previous season’s vaccine. In some embodiments, the HA antigen and/or NA antigen is from an influenza A virus listed in Table 1 or an influenza B virus listed in Table 2.
In some embodiments, the HA antigen and/or NA antigen is from a H1N1 virus, a H3N2 virus, a virus of B/Victoria lineage or a virus of B/Yamagata lineage. In some embodiments, the HA antigen and/or NA antigen is from an influenza A virus strain that is A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022, A/Darwin/6/2021 or A/Darwin/6/2021. In some embodiments, the HA antigen and/or NA antigen is from an influenza B virus strain that is B/Austria/1359417/2021 or B/Phuket/3073/2013. In some embodiments, the HA antigen is at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 3, 5A, 7A, or 8A, and/or wherein the NA antigen is at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 4, 5B, 7B, or 8B.
In some embodiments, the HA sequence has a nucleotide sequence listed in Table 6A, 9A, 10A, or 14, and/or wherein the NA sequence has a nucleotide sequence listed in Table 6B, 9B, or 10B.
Provided herein are circRNAs comprising a TI sequence and a Z sequence comprising a HA sequence encoding a HA antigen.
In some embodiments, the HA antigen is from a H1N1 virus, a H3N2 virus, a virus of B/Victoria lineage, or a virus of B/Yamagata lineage.
In some embodiments, the HA antigen is Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182, 184-188, and 209-210. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 210.
In some embodiments, the HA antigen is Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 177. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 211 or 189.
In some embodiments, the HA antigen is Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 179. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 214 or 196.
In some embodiments, the HA antigen is Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 180. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 221 or 199.
In some embodiments, the HA antigen is Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 235. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 281-285 and 520-521. In some embodiments, the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 520 or 521.
In some embodiments, the circRNA comprises a linker sequence that encodes a 2A self-cleaving peptide.
In some embodiments, the TI sequence comprises: an IRES, an IRES-like nucleotide sequence, or a combination thereof. In some embodiments, the TI sequence comprises an optimized IRES sequence. In some embodiments, the IRES is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 11.
In some embodiments, the circRNAs provided herein have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identical to a nucleotide sequence listed in Table 13. In some embodiments, the circRNAs provided herein have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 499-519.
In some embodiments, the circRNAs provided herein are scarless or near scarless.
In some embodiments, the circRNAs provided herein induce a reduced innate immune response relative to a mRNA encoding the HA antigen and/or NA antigen when administered to a subject.
In some embodiments, the circRNAs provided herein comprises a modified RNA nucleotide and/or modified nucleoside. In some embodiments, the circRNAs provided herein comprise at least 10%modified RNA nucleotides and/or modified nucleosides. In some embodiments, at least one of the modified RNA nucleotide and/or modified nucleoside is 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2'fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6, 2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2, 7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
In some embodiments, the circRNAs provided herein do not comprise 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2' fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6, 2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2, 7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
In some embodiments, at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
Provided herein are also immunogenic compositions comprising the circRNAs described herein.
In some embodiments, the immunogenic compositions provided herein comprise at least two, at least three, at least four, at least five, or at least six of the circRNA described herein.
In some embodiments, the immunogenic compositions provided herein comprise two circRNAs. In some embodiments, the Z sequences of the two circRNAs comprise HA sequences encoding HA antigens from two influenza viruses. In some embodiments, the two influenza viruses comprise two influenza A viruses, two influenza B viruses, or one influenza A virus and one influenza B virus.
In some embodiments, the immunogenic compositions provided herein comprise four circRNAs. In some embodiments, the Z sequences of the four circRNA comprise HA sequences encoding HA antigens from four influenza viruses. In some embodiments, the four influenza viruses comprise two influenza A viruses and two influenza B viruses.
In some embodiments, the four influenza viruses are Influenza Type A/Wisconsin/588/2019 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) . In some embodiments, the four HA antigens are Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein, Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein, Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 175, 177, 179, and 180, respectively. In some embodiments, the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 210, 211, 214, and 221, respectively. In some embodiments, the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 501, 519, 514, and 508, respectively.
In some embodiments, the four influenza viruses are Influenza Type A/Wisconsin/67/2022 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the four HA antigens are Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein, Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein, Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 235, 177, 179, and 180, respectively. In some embodiments, the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 521, 189, 196, and 221, respectively. In some embodiments, the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 518, 519, 508, and 515, respectively.
In some embodiments, the immunogenic compositions provided herein, the circRNA (s) are formulated in a lipid nanoparticle (LNP) . In some embodiments, each circRNA is separately formulated in an LNP.
In some embodiments, the immunogenic compositions provided herein comprise an adjuvant.
In some embodiments, the immunogenic compositions provided herein are for use in inducing an anti-influenza immune response in a subject.
In some embodiments, provided herein are methods for inducing an anti-influenza immune response in a subject comprising administering to the subject a therapeutically effective amount of the immunogenic composition described herein.
In some embodiments, methods provided herein induce cross protective immunity against a heterologous influenza virus. In some embodiments, methods provided herein induce protective immunity against an influenza virus of the same subtype.
In some embodiments, the immune response is measured by anti-hemagglutinin (HA) antibody titer, hemagglutination inhibition (HAI) titer, or a microneutralization assay. In some embodiments, the immune response is measured by HAI titer. In some embodiments, the methods produce an HAI titer of at least 40, at least 80, at least 120, at least 160, at least 320, at least 640, or at least 1280 in the subject. In some embodiments, the methods produce an HAI titer that is at least 3, at least 4, at least 5, or at least 6 times relative to a baseline HAI level in the subject. In some embodiments, the methods produce an HAI titer of at least 40 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years.
In some embodiments, the immune response is measured by anti-HA antibody titer about 7 days, about 14 days, about 21 days, about 28 days, or about 35 days following vaccination. In some embodiments, the methods induce at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-HA IgG titer in the subject as compared to that before the administration. In some embodiments, the methods produce at least a 50-fold increase in anti-HA IgG titer for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years.
In some embodiments, the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, or intraperitoneally.
In some embodiments, the methods include a single administration, optionally followed with a boost administration.
In some embodiments, the subject is a human. In some embodiments, the subject is 60 years of age or older. In some embodiments, the subject is 18 years of age or younger.
4. Illustrative Embodiments
Embodiment 1: A circular ribonucleotide acid (circRNA) , wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises a hemagglutinin sequence (HA sequence) encoding a hemagglutinin antigen (HA antigen) or a neuraminidase sequence (NA sequence) encoding a neuraminidase antigen (NA antigen) .
Embodiment 2: The circRNA of Embodiment 1, wherein the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus that is type A or B.
Embodiment 3: The circRNA of Embodiment 2, wherein the Z sequence further comprises a second HA (HA2) sequence encoding an HA antigen from a second influenza virus that is type A or B.
Embodiment 4: The circRNA of Embodiment 3 comprising in the following order: TI, HA1, L, and HA2, wherein L is a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 5: The circRNA of Embodiment 3, wherein the Z sequence further comprises a third HA (HA3) sequence encoding an HA antigen from a third influenza virus that is type A or B.
Embodiment 6: The circRNA of Embodiment 5 comprising in the following order: TI, HA1, L1, HA2, L2, and HA3, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first, second and third viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 7: The circRNA of Embodiment 5, wherein the Z sequence further comprises a fourth HA (HA4) sequence encoding an HA antigen from a fourth influenza virus that is type A or B.
Embodiment 8: The circRNA of Embodiment 7 comprising in the following order: TI, HA1, L1, HA2, L2, HA3, L3, and HA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 9: The circRNA of Embodiment 1, wherein the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus that is type A or B.
Embodiment 10: The circRNA of Embodiment 9, wherein the Z sequence further comprises a second NA (NA2) sequence encoding an NA antigen from a second influenza virus that is type A or B.
Embodiment 12: The circRNA of Embodiment 10 comprising in the following order: TI, NA1, L, and NA2; wherein L is a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 12: The circRNA of Embodiment 10, wherein the Z sequence further comprises a third NA (NA3) sequence encoding an NA antigen from a third influenza virus that is type A or B.
Embodiment 13: The circRNA of Embodiment 12 comprising in the following order: TI, NA1, L1, NA2, L2, and NA3; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first, second, and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 14: The circRNA of Embodiment 12, wherein the Z sequence further comprises a fourth NA (NA4) sequence encoding an NA antigen from a fourth influenza virus that is type A or B.
Embodiment 15: The circRNA of Embodiment 14 comprising in the following order: TI, NA1, L1, NA2, L2, NA3, L3, and NA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 16: The circRNA of Embodiment 1, wherein the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen, and a first NA (NA1) sequence encoding an NA antigen, and the HA and NA antigens are from a first influenza virus that is type A or B.
Embodiment 17: The circRNA of Embodiment 16 comprising in the following order: (1) TI, HA1, L, and NA1; or (2) TI, NA1, L, and HA1; wherein L is a linker sequence or absent.
Embodiment 18: The circRNA of Embodiment 16, wherein the Z sequence further comprises a second HA (HA2) sequence encoding an HA antigen and a second NA (NA2) sequence encoding an NA antigen, wherein the HA and NA antigens are from a second influenza virus that is type A or B.
Embodiment 19: The circRNA of Embodiment 18 comprising in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, and NA2; (2) TI, NA1, L1, HA1, L2, NA2, L3, and HA2; (3) TI, HA1, L1, HA2, L2, NA1, L3, and NA2; or (4) TI, NA1, L1, NA2, L2, HA1, L3, and HA2; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 20: The circRNA of Embodiment 18, wherein the Z sequence further comprises a third HA (HA3) sequence encoding an HA antigen and a third NA (NA3) sequence encoding an NA antigen, wherein the HA and NA antigens are from a third influenza virus that is type A or B.
Embodiment 21: The circRNA of Embodiment 20 comprising in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3; (2) TI, HA1, L1, HA2, L2, HA3, L3, NA1, L4, NA2, L5, and NA3; (3) TI, NA1, L1, NA2, L2, NA3, L3, HA1, L4, HA2, L5, and HA3; or (4) TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3; wherein L1, L2, L3, L4, and L5 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 22: The circRNA of Embodiment 1 comprising in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises an HA sequence encoding an HA antigen, or an NA sequence encoding an NA antigen.
Embodiment 23: The circRNA of Embodiment 22, wherein the Z1 sequence comprises a first HA (HA1) sequence and the Z2 sequence comprises a second HA (HA2) sequence, encoding HA antigens from a first and a second influenza viruses, each independently is type A or B.
Embodiment 24: The circRNA of Embodiment 23 comprising in the following order: TI1, HA1, L1, TI2, HA2, and L2, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 25: The circRNA of Embodiment 22, wherein the Z1 sequence comprises a first NA (NA1) sequence and the Z2 sequence encodes a second NA (NA2) sequence, encoding NA antigens from a first and a second influenza viruses, each independently is type A or B.
Embodiment 26: The circRNA of Embodiment 25 comprising in the following order: TI1, NA1, L1, TI2, NA2, and L2; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 27: The circRNA of Embodiment 22, wherein the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, and the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence; wherein the HA1 and NA1 sequences encode HA and NA antigens from a first influenza virus, and the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus, each independently is type A or B.
Embodiment 28: The circRNA of Embodiment 27 comprising in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2, or (2) TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2; wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; or (3) A and B; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 29: The circRNA of Embodiment 22, wherein the Z1 sequence comprises a first HA (HA1) sequence encoding an HA antigen and the Z2 sequence comprises a first NA (NA1) sequence encoding an NA antigen, wherein the HA and NA antigens are from a first influenza virus that is type A or B.
Embodiment 30: The circRNA of Embodiment 29, wherein the Z1 sequence further comprise a second HA (HA2) sequence, and the Z2 sequence further comprises a second NA (NA2) sequence; wherein the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus that is type A or B.
Embodiment 32: The circRNA of Embodiment 30 comprising in the following order: TI1, HA1, L1, HA2, TI2, NA1, L2, NA2, wherein L1 and L2 each is independently a linker sequence or absent; and wherein the first and second influenza viruses are (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 32: The circRNA of Embodiment 30, wherein the Z1 sequence further comprises a third HA sequence (HA3) and the Z2 sequence further comprises a third NA sequence (NA3) , wherein the HA3 and NA3 sequences encode HA and NA antigens from a third influenza virus that is type A or B.
Embodiment 33: The circRNA of Embodiment 32 comprising in the following order: TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 34: The circRNA of Embodiment 1 comprising in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, and a third protein-coding (Z3) sequence, wherein the Z1, Z2 and Z3 sequences each independently comprises an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.
Embodiment 35: The circRNA of Embodiment 34, wherein the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, and the Z3 comprises encodes a third HA (HA3) sequence; wherein the HA1, HA2 and HA3 sequences encode HA antigens from a first, a second and a third influenza viruses, respectively.
Embodiment 36: The circRNA of Embodiment 35 comprising in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 37: The circRNA of Embodiment 34, wherein the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, and the Z3 sequence comprises a third NA (NA3) sequence; wherein the NA1, NA2 and NA3 sequences encode NA antigens from a first, a second and a third influenza viruses, respectively.
Embodiment 38: The circRNA of Embodiment 37 comprising in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 39: The circRNA of Embodiment 34, wherein the Z1 sequence comprises a first HA (HA1) sequence and first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, and the Z3 comprises encodes a third HA (HA3) sequence and a third NA (NA3) sequence; wherein the HA1 and NA1 sequences encode antigens from a first influenza virus, the HA2 and NA2 encode antigens from a second influenza virus, and the HA3 and NA3 encode antigens from a third influenza virus.
Embodiment 40: The circRNA of Embodiment 39 comprising in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3; or (2) TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3; wherein L1, L2, and L3 each is independently a linker sequence or absent; and wherein the first, second and third influenza virus are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 41: The circRNA of Embodiment 1 comprising in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, a third protein-coding (Z3) sequence, a fourth translation initiation (TI4) sequence, a fourth protein-coding (Z4) sequence, wherein the Z1, Z2, Z3 and Z4 sequences each independently comprises an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.
Embodiment 42: The circRNA of Embodiment 41, wherein the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, the Z3 comprises encodes a third HA (HA3) sequence, and the Z4 comprises encodes a fourth HA (HA4) sequence; wherein the HA1, HA2, HA3, and HA4 sequences encode HA antigens from a first, a second, a third and a fourth influenza viruses, respectively.
Embodiment 43: The circRNA of Embodiment 42, comprising in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 44: The circRNA of Embodiment 41, wherein the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, the Z3 sequence comprises a third NA (NA3) sequence, and the Z4 sequence comprises a fourth NA (NA4) sequence; wherein the NA1, NA2, NA3 and NA4 sequences encode NA antigens from a first, a second, a third and a fourth influenza viruses, respectively.
Embodiment 45: The circRNA of Embodiment 44 comprising in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
Embodiment 46: The circRNA of any one of Embodiments 1 to 45, wherein the HA antigen and/or NA antigen is from a seasonal influenza virus.
Embodiment 47: The circRNA of Embodiment 46, wherein the HA antigen and/or NA antigen is selected according to standardized criteria used by World Health Organization’s Global Influenza Surveillance and Response System (GISRS) .
Embodiment 48: The circRNA of Embodiment 46, wherein the HA antigen and/or NA antigen is selected using a hemagglutinin inhibition (HAI) assay and/or neuraminidase-inhibition (NAI) assay to identify circulating influenza viruses that are antigenically similar to influenza viruses from a previous season’s vaccine.
Embodiment 49: The circRNA of any one of Embodiments 1 to 40, wherein the HA antigen and/or NA antigen is from an influenza A virus listed in Table 1 or an influenza B virus listed in Table 2.
Embodiment 50: The circRNA of any one of Embodiments 1 to 40, wherein the HA antigen and/or NA antigen is from a H1N1 virus, a H3N2 virus, a virus of B/Victoria lineage or a virus of B/Yamagata lineage.
Embodiment 51: The circRNA of any one of Embodiments 1 to 40, wherein the HA antigen and/or NA antigen is from an influenza A virus strain that is A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022, A/Darwin/6/2021 or A/Darwin/6/2021.
Embodiment 52: The circRNA of any one of Embodiments 1 to 40, wherein the HA antigen and/or NA antigen is from an influenza B virus strain that is B/Austria/1359417/2021 or B/Phuket/3073/2013.
Embodiment 53: The circRNA of any one of Embodiments 1 to 40, wherein the HA antigen is at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 3, 5A, 7A, or 8A, and/or wherein the NA antigen is at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence listed in Table 4, 5B, 7B, or 8B.
Embodiment 54: The circRNA of Embodiment 53, wherein the HA sequence has a nucleotide sequence listed in Table 6A, 9A, 10A, or 14, and/or wherein the NA sequence has a nucleotide sequence listed in Table 6B, 9B, or 10B.
Embodiment 55: The circRNA of Embodiment 1, wherein the Z sequence comprises a HA sequence encoding a HA antigen.
Embodiment 56: The circRNA of Embodiment 55, wherein the HA antigen is from a H1N1 virus, a H3N2 virus, a virus of B/Victoria lineage, or a virus of B/Yamagata lineage.
Embodiment 57: The circRNA of Embodiment 56, wherein the HA antigen is Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein.
Embodiment 58: The circRNA of Embodiment 57, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 175.
Embodiment 59: The circRNA of Embodiment 58, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182, 184-188, and 209-210.
Embodiment 60: The circRNA of Embodiment 58, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 210.
Embodiment 61: The circRNA of Embodiment 56 wherein the HA antigen is Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein.
Embodiment 62: The circRNA of Embodiment 61, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 177.
Embodiment 63: The circRNA of Embodiment 62, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212.
Embodiment 64: The circRNA of Embodiment 62, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 211 or 189.
Embodiment 65: The circRNA of Embodiment 56, wherein the HA antigen is Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein.
Embodiment 66: The circRNA of Embodiment 65, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 179.
Embodiment 67: The circRNA of Embodiment 66, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219.
Embodiment 68: The circRNA of Embodiment 66, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 214 or 196.
Embodiment 69: The circRNA of Embodiment 56, wherein the HA antigen is Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein.
Embodiment 70: The circRNA of Embodiment 69, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 180.
Embodiment 71: The circRNA of Embodiment 70, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221.
Embodiment 72: The circRNA of Embodiment 70, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 221 or 199.
Embodiment 73: The circRNA of Embodiment 56 wherein the HA antigen is Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein.
Embodiment 74: The circRNA of Embodiment 73, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 235.
Embodiment 75: The circRNA of Embodiment 74, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 281-285 and 520-521.
Embodiment 76: The circRNA of Embodiment 74, wherein the HA sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 520 or 521.
Embodiment 77: The circRNA any one of Embodiments 1 to 76, wherein the circRNA comprises a linker sequence that encodes a 2A self-cleaving peptide.
Embodiment 78: The circRNA any one of Embodiments 1 to 77, wherein the TI sequence comprises: an IRES, an IRES-like nucleotide sequence, or a combination thereof.
Embodiment 79: The circRNA of Embodiment 77, wherein the TI sequence comprises an optimized IRES sequence.
Embodiment 80: The circRNA of Embodiment 77, wherein the IRES is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence listed in Table 11.
Embodiment 81: The circRNA of Embodiment 1, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identical to a nucleotide sequence listed in Table 13.
Embodiment 82: The circRNA of Embodiment 1 having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 499-519.
Embodiment 83: The circRNA of any one of Embodiments 1 to 82, wherein the circRNA is scarless or near scarless.
Embodiment 84: The circRNA of any one of Embodiments 1 to 83, wherein circRNA induces a reduced innate immune response relative to a mRNA encoding the HA antigen and/or NA antigen when administered to a subject.
Embodiment 85: The circRNA of any one of Embodiments 1 to 84, wherein the circRNA comprises a modified RNA nucleotide and/or modified nucleoside.
Embodiment 86: The circRNA of Embodiment 85, wherein the circRNA comprises at least 10%modified RNA nucleotides and/or modified nucleosides.
Embodiment 87: The circRNA of Embodiment 85, wherein at least one of the modified RNA nucleotide and/or modified nucleoside is 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2'fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6, 2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2,7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
Embodiment 88: The circRNA of any one of Embodiments 1 to 83 wherein the circRNA does not comprise 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2'fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6,2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2, 7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
Embodiment 89: The circRNA of any one of Embodiments 85 to 88, wherein at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
Embodiment 90: An immunogenic composition comprising the circRNA of any one of Embodiments 1 to 89.
Embodiment 91: An immunogenic composition comprising at least two, at least three, at least four, at least five, or at least six of the circRNA of any one of Embodiments 1 to 89.
Embodiment 92: The immunogenic composition of Embodiment 91 comprising two circRNAs.
Embodiment 93: The immunogenic composition of Embodiment 92, wherein the Z sequences of the two circRNAs comprise HA sequences encoding HA antigens from two influenza viruses.
Embodiment 94: The immunogenic composition of Embodiment 93, wherein the two influenza viruses comprise two influenza A viruses, two influenza B viruses, or one influenza A virus and one influenza B virus.
Embodiment 95: The immunogenic composition of Embodiment 91 comprising four circRNAs.
Embodiment 96: The immunogenic composition of Embodiment 95, wherein the Z sequences of the four circRNA comprise HA sequences encoding HA antigens from four influenza viruses.
Embodiment 97: The immunogenic composition of Embodiment 96, wherein the four influenza viruses comprise two influenza A viruses and two influenza B viruses.
Embodiment 98: The immunogenic composition of Embodiment 97, wherein the four influenza viruses are Influenza Type A/Wisconsin/588/2019 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) .
Embodiment 99: The immunogenic composition of Embodiment 98, wherein the four HA antigens are Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein, Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein, Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein.
Embodiment 100: The immunogenic composition of Embodiment 99, wherein the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 175, 177, 179, and 180, respectively.
Embodiment 101: The immunogenic composition of Embodiment 100, wherein the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 210, 211, 214, and 221, respectively.
Embodiment 102: The immunogenic composition of Embodiment 101, wherein the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 501, 519, 514, and 508, respectively.
Embodiment 103: The immunogenic composition of Embodiment 97, wherein the four influenza viruses are Influenza Type A/Wisconsin/67/2022 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein.
Embodiment 104: The immunogenic composition of Embodiment 103, wherein the four HA antigens are Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein, Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein, Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein, and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein.
Embodiment 105: The immunogenic composition of Embodiment 104, wherein the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 235, 177, 179, and 180, respectively.
Embodiment 106: The immunogenic composition of Embodiment 105, wherein the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 521, 189, 196, and 221, respectively.
Embodiment 107: The immunogenic composition of Embodiment 106, wherein the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 518, 519, 508, and 515, respectively.
Embodiment 108: The immunogenic composition of any one of Embodiments 90 to 107, wherein the circRNA (s) are formulated in a lipid nanoparticle (LNP) .
Embodiment 109: The immunogenic composition of Embodiment 108, wherein each circRNA is separately formulated in an LNP.
Embodiment 110: The immunogenic composition of any one of Embodiments 90 to 109, comprising an adjuvant.
Embodiment 111: The immunogenic composition of any one of Embodiments 90 to 110, for use in inducing an anti-influenza immune response in a subject.
Embodiment 112: A method for inducing an anti-influenza immune response in a subject comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of Embodiments 90 to 110.
Embodiment 113: The method of Embodiment 112, wherein the method induces cross protective immunity against a heterologous influenza virus.
Embodiment 114: The method of Embodiment 112, wherein the method induces protective immunity against an influenza virus of the same subtype.
Embodiment 115: The method of any one of Embodiments 112 to 114, wherein the immune response is measured by anti-hemagglutinin (HA) antibody titer, hemagglutination inhibition (HAI) titer, or a microneutralization assay.
Embodiment 116: The method of Embodiment 115, wherein the immune response is measured by HAI titer.
Embodiment 117: The method of Embodiment 116, wherein the method produces an HAI titer of at least 40, at least 80, at least 120, at least 160, at least 320, at least 640, or at least 1280 in the subject.
Embodiment 118: The method of Embodiment 116, wherein the method produces an HAI titer that is at least 3, at least 4, at least 5, or at least 6 times relative to a
baseline HAI level in the subject.
Embodiment 119: The method of any one of Embodiments 116 to 118, wherein method produces an HAI titer of at least 40 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years.
Embodiment 120: The method of Embodiment 115, wherein the immune response is measured by anti-HA antibody titer about 7 days, about 14 days, about 21 days, about 28 days, or about 35 days following vaccination.
Embodiment 121: The method of Embodiment 120, wherein the method induces at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-HA IgG titer in the subject as compared to that before the administration.
Embodiment 122: The method of Embodiment 120 or 121, wherein method produces at least a 50-fold increase in anti-HA IgG titer for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years.
Embodiment 123: The method of any one of Embodiments 112 to 122, wherein the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, or intraperitoneally.
Embodiment 124: The method of any one of Embodiments 112 to 123, wherein the method includes a single administration, optionally followed with a boost administration.
Embodiment 125: The method of any one of Embodiments 112 to 124 wherein the subject is a human.
Embodiment 126: The method of Embodiment 125, wherein the subject is 60 years of age or older.
Embodiment 127: The method of Embodiment 125, wherein the subject is 18 years of age or younger.
5.Brief Description of DrawingsFIG. 1 provides a circRNA vaccine which comprises, in the following order: TI, HA1, L1, HA2, L2, HA3, L3, and HA4.
FIG. 2 provides a circRNA vaccine which comprises, in the following order: TI, NA1, L1, NA2, L2, NA3, L3, and NA4.
FIG. 3 provides a circRNA vaccine which comprises, in the following order: TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3.
FIG. 4 provides a circRNA vaccine which comprises, in the following order: TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3.
FIG. 5 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, TI2, and NA1.
FIG. 6 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3.
FIG. 7 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, TI2, HA2, TI3, HA3.
FIG. 8 provides a circRNA vaccine which comprises, in the following order: TI1, NA1, TI2, NA2, TI3, NA3.
FIG. 9 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3.
FIG. 10 provides a circRNA vaccine which comprises, in the following order: TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3
FIG. 11 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L.
FIG. 12 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L1, HA2, L2.
FIG. 13 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L1, HA3, L2., HA2.
FIG. 14 provides a circRNA vaccine which comprises, in the following order: TI1, HA1, L1, HA2, L2 HA3, L3, HA4.
FIG. 15 provides representative results showing binding IgG titer for CircRNA-PD001.
FIG. 16 provides representative results showing HAI titer (H1N1) for CircRNA-PD001.
FIG. 17 provides representative results showing binding IgG titer for CircRNA-PD002.
FIG. 18 provides representative results showing HAI titer (H3N2) for CircRNA-PD002.
FIG. 19 provides representative results showing binding IgG titer for CircRNA-PD003.
FIG. 20 provides representative results showing HAI titer (Yamagata) for CircRNA-PD003.
FIG. 21 provides representative results showing binding IgG titer for CircRNA-PD004.
FIG. 22 provides representative results showing HAI titer (Victoria) for CircRNA-PD004.
FIG. 23 provides representative results showing binding IgG titer (H1N1) for CircRNA-PD005.
FIG. 24 provides representative results showing binding IgG titer (H3N2) for CircRNA-PD005.
FIG. 25 provides representative results showing binding IgG titer (Yamagata) for CircRNA-PD005.
FIG. 26 provides representative results showing binding IgG titer (Victoria) for CircRNA-PD005.
FIG. 27 provides representative results showing binding IgG titer (H1N1) for CircRNA-PD006.
FIG. 28 provides representative results showing HAI titer (H1N1) for CircRNA-PD006.
FIG. 29 provides representative results showing HAI titer (Wisconsin) for CircRNA-PD007.
FIG. 30 provides representative results showing HAI titer (Darwin) for CircRNA-PD007.
FIG. 31 provides representative results showing HAI titer (Victoria) for CircRNA-PD007.
FIG. 32 provides representative results showing HAI titer (Yamagata) for CircRNA-PD007.
FIG. 33 provides representative results showing HAI titer (H1N1) for CircRNA-PD009.
FIG. 34 provides representative results showing HAI titer (H3N2) for CircRNA-PD009.
FIG. 35 provides representative results showing HAI titer (Victoria) for CircRNA-PD009.
FIG. 36 provides representative results showing HAI titer (Yamagata) for CircRNA-PD009.
As provided in detail below, in the description of drawings above, TI refers to a translation initiation sequence; TI1, TI2, and TI3 each independently is a translation initiation sequence. HA refers to a sequence encoding a hemagglutinin antigen; HA1, HA2, HA3, and HA4 each is independently a sequence encoding a hemagglutinin antigen. NA refers to a sequence encoding a neuraminidase antigen; NA1, NA2, NA3, and NA4 each is independently a sequence encoding a hemagglutinin antigen. L refers to a linker sequence; L1, L2, L3, L4, and L5, each is independent an linker sequence or absent.
6.Detailed DescriptionProvided herein are immunogenic compositions comprising a circular ribonucleotide acid (circRNA) encoding a vaccine for an influenza virus. In some embodiments, the immunogenic compositions provided herein comprise a circRNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises an HA sequence encoding a hemagglutinin antigen or an NA sequence encoding a neuraminidase antigen. Methods of manufacture and methods of uses thereof are also provided herein.
6.1 Definitions
Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim (s) , when used in conjunction with the word “comprising, ” the words “a” or “an” may mean one or more than one.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or. ” As used herein “another” or “additional” may mean at least a second or more.
As used herein, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. The term “about” encompasses the exact number recited. In some embodiments, “about” means within plus or minus 10%of a given value or range. In certain embodiments, “about” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers. In some embodiments, “about” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1%of the value to which “about” refers.
As used herein, “essentially free, ” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.1%, preferably below 0.05%, and more preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
The terms “peptide, ” “polypeptide” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids comprising at least two or more contiguous amino acids chemically or biochemically modified or derivatized amino acids. The term “peptide” as used herein refers to a class of short polypeptides. The term peptide may refer to a polymer of amino acids (natural or non-naturally occurring) having a length of up to about 100 amino acids. For example, peptides may be about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, about 75 to about 100 amino acid residues in length. In some embodiments, the peptides may be about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, are about 5000 amino acid residues in length.
The term “antigen, ” as used herein and understood in the art, are immunogenic proteins, namely, proteins that are capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens) . As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of antigens of interest (e.g., the HA antigens or NA antigens) . As such, the term “antigen” encompasses full-length proteins as well as immunogenic fragments and variants thereof. For example, an antigen can be any immunogenic fragment of a reference protein. In addition to variants that are identical to the reference protein but are truncated, in some embodiments, an antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein. Antigens/antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to full length proteins. Accordingly, as used herein, a “hemagglutinin antigen” or “HA antigen” can be a full-length hemagglutinin protein, an immunogenic fragment thereof, or an immunogenic variant thereof. Likewise, “a neuraminidase antigen” or “NA antigen” can be a full-length neuraminidase protein, an immunogenic fragment thereof, or an immunogenic variant thereof.
The terms “nucleic acid, ” “polynucleotide, ” and “oligonucleotide” are used interchangeably herein and refer to a polymer or oligomer of nucleotides of any length. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated, or glycosylated) , non-natural nucleotides, non-nucleotide building blocks that exhibit similar structure and/or function as natural nucleotides (i.e., “nucleotide analogs” ) , and/or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The nucleic acids or polynucleotides can be heterogenous or homogenous in composition, can be isolated from naturally occurring sources, or can be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and can exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. Nucleic acid structures also include, for instance, a DNA/RNA helix, peptide nucleic acid (PNA) , morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 4 (14) : 4503-4510 (2002) and U.S. Patent 5, 034, 506) , locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000) ) , cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122: 8595-8602 (2000) ) , and/or a ribozyme.
As is understood in the art, a nucleic acid strand is inherently directional, as the carbon atoms in the sugar ring are numbered from 1’ to 5’ and the “5’ -end” has a free hydroxyl (or phosphate) on a 5’ carbon and the “3’ prime end” has a free hydroxyl (or phosphate) on a 3’ carbon. As used herein and understood in the art, a nucleic acid having certain sequence elements “from 5’ to 3’ ” means that these sequence elements are arranged linearly from the 5’ end to the 3’ end of the nucleic acid.
When referring to a nucleotide sequence or protein sequence, the term “identity” is used to denote similarity between two sequences. Sequence similarity or identity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2, 482 (1981) , by the sequence identity alignment algorithm of Needleman &Wunsch, J Mol. Biol. 48, 443 (1970) , by the search for similarity method of Pearson &Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988) , by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI) , the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984) , or by inspection. Another algorithm is the BLAST algorithm, described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993) . A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996) ;
blast. wustl/edu/blast/README. html. WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Further, an additional useful algorithm is gapped BLAST as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise indicated, percent identity is determined herein using the algorithm available at the internet address: blast. ncbi. nlm. nih. gov/Blast. cgi.
As used herein, terms “complementary” and “complementarity” refers to the relationship between two nucleic acid molecules having the capacity to form hydrogen bond (s) with one another by either traditional Watson-Crick base-paring or other non-traditional types of pairing. The two DNA/RNA strands with complementary sequences bind to form a duplex that follows the Watson–Crick base-pairing rules: A binds to T (U) with two hydrogen bonds; G binds to C with three hydrogen bonds. The degree of complementarity between two nucleotide sequences can be indicated by the percentage of nucleotides in a nucleotide sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleotide sequence (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, and 100%complementary) . Two nucleotide sequences are “perfectly complementary” or “100%complementary” if all the contiguous nucleotides of a nucleotide sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleotide sequence. Two nucleotide sequences are “substantially complementary” if the degree of complementarity between the two nucleotide sequences is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) over a region of at least 8 nucleotides (e.g., at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more nucleotides) , or if the two nucleotide sequences hybridize under at least moderate, or, in some embodiments high, stringency conditions. Exemplary moderate stringency conditions include overnight incubation at 37℃ in a solution comprising 20%formamide, 5%SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 mM sodium phosphate (pH 7.6) , 5x Denhardt’s solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1*SSC at about 37-50℃, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012) . High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1%sodium dodecyl sulfate (SDS) at 50℃, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1%bovine serum albumin (BSA) /0.1%Ficoll/0.1%polyvinylpyrrolidone (PVP) /50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42℃, or (3) employ 50%formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate) , 50 mM sodium phosphate (pH 6.8) , 0.1%sodium pyrophosphate, 5x Denhardt’s solution, sonicated salmon sperm DNA (50 pg/ml) , 0.1%SDS, and 10%dextran sulfate at 42℃, with washes at (i) 42℃ in 0.2*SSC, (ii) 55℃ in 50%formamide, and (iii) 55℃ in 0.1*SSC (optionally in combination with EDTA) . Additional details and an explanation of stringency of hybridization reactions are provided in, e.g., Sambrook, supra, and Ausubel et al., eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 5th ed., John Wiley &Sons, Inc., Hoboken, N.J. (2002) .
The term “operably linked” as used herein and understood in the art with reference to sequence elements in nucleic acid molecules means that these sequence elements (e.g., an intron fragment, a target sequence, a promoter, and a coding sequence) are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
The term “hybridization” or “hybridized” when referring to nucleotide sequences is the association formed between and/or among sequences having complementarity.
The term “in vitro transcription, ” or “IVT, ” refers to versatile method to produce RNA in vitro that uses an RNA polymerase, ribonucleotides, and appropriate buffer conditions to synthesize RNA from a DNA template.
The term “expression construct” or “expression cassette, ” as used herein, means a nucleotide sequence that directs translation.
The terms “coding sequence, ” “coding sequence region, ” “coding region, ” and “CDS, ” as used interchangeably here refer to the portion of a nucleic acid (e.g., a DNA or an RNA) that is or can be translated to protein.
The terms “reading frame, ” “open reading frame, ” and “ORF” as used interchangeably herein refer to a nucleotide sequence that begins with an initiation codon (e.g., ATG) and, in some embodiments, ends with a termination codon (e.g., TAA, TAG, or TGA) .
The term “control elements” as used herein refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES) , enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell.
The term “promoter” as used herein refers to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding to an RNA polymerase and allowing for the initiation of transcription of a downstream (3'direction) coding sequence. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription of a nucleic acid sequence. A promoter that is “operatively positioned, ” “operatively linked” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence, which is “under control” and “under transcriptional control” of the promoter.
The term “enhancer” as used herein means a nucleic acid sequence that, when positioned proximate to a promoter, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain.
The terms “internal ribosome entry site, ” “internal ribosome entry site sequence, ” “IRES” and “IRES sequence region” as used interchangeably herein refer to cis elements of viral or human cellular RNAs (e.g., messenger RNA (mRNA) and/or circRNAs) that bypass the steps of canonical eukaryotic cap-dependent translation initiation.
The term “IRES-like sequence” and “Internal Ribosome Entry Site-like sequence, ” as used interchangeably herein refer to non-naturally occurring nucleotide sequences that display a function of a naturally occurring IRES.
The term “vector” or “construct” (sometimes referred to as a gene delivery system or gene transfer “vehicle” ) refers to a vehicle that is used to carry genetic material (e.g., a nucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed.
The term “treat” as used herein refers to executing a protocol or plan, which can include administering one or more drugs or active agents to a patient, in an effort to alleviate signs or symptoms of the disease or the recurrence of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission, increased survival, improved quality of life or improved prognosis. Alleviation or prevention can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. As used herein, a “treatment” does not require complete alleviation of signs or symptoms, and does not require a cure.
As used herein, the term “therapeutic beneficial” or “therapeutically effective” when used in connection with a therapeutic refers to the property of the therapeutic that promotes or enhances the well-being of the subject. This includes, but is not limited to, a reduction in the frequency, severity, or rate of progression of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or a reduction in the rate of metastasis or recurrence. Treatment of cancer can also refer to prolonging survival of a subject with cancer.
As used herein, the term “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. For animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required, e.g., by the FDA Office of Biological Standards.
As used herein, the term “pharmaceutically acceptable carrier” includes any and all aqueous biocompatible solvents (e.g., saline solutions, phosphate buffered saline, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc. ) , antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases) , isotonic agents, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.
The terms “transfection, ” “transformation, ” and “transduction” are used interchangeably herein and refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.
As used herein, the term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.
Nomenclature for nucleotides, nucleic acids, nucleosides, and amino acids used herein is consistent with International Union of Pure and Applied Chemistry (IUPAC) standards (see, e.g., bioinformatics. org/smsylupac. html) . Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to Uniprot (https: //www. uniprot. org/) , GenBank
(ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
For nucleotide sequences in the sequence listings, the nucleotides uracil (U) and thymine (T) are considered equivalent and used interchangeably depending on whether the sequence is derived from RNA (U) or DNA (T) .
Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.
DSPC: The term “DSPC” used herein is a cylindrical-shaped lipid. DSPC is used to synthesize liposomes, and is the lipid component in the lipid nanoparticle (LNP) system. The detail was described on Cat. No. HY-W040193, MedChemexpress.
ALC-0315: The term “ALC-0315” used herein is a responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. ALC-0315 can be used to form lipid nanoparticle (LNP) delivery vehicles. The detail was described on Cat. No. HY-138170, MedChemexpress.
E-1-0635: The term “E-1-0635” used herein refers to the same compound designated as E-1-0635 in Example II-123 of PCT/CN2023/129346.
PEG-2000: The term “PEG-2000” used herein is a solvent for a large number of substances. PEG2000 can be used as a carrier material and modifying agent. PEG2000 is widely used in a variety of pharmaceutical formulations. The detail was described on Cat. No. HY-Y0873B, MedChemexpress.
ALC-0159: The term “ALC-0159” used herein is a polyethylene glycol (PEG) lipid conjugate, could be used as vaccine excipient. The detail was described on Cat. No. : HY-138300, MedChemexpress.
DOPE: The term “DOPE” used herein is a neutral helper lipid for cationic liposome and combines with cationic phospholipids to improve transfection efficiency of naked siRNA. The detail was described on Cat. No. HY-112005, MedChemexpress.
LNPs: The term “LNPs” used herein are nanoparticles composed of lipids. They are a novel pharmaceutical drug delivery system (and part of nanoparticle drug delivery) , and a novel pharmaceutical formulation.
PBI: The term “PBI” used herein is Polybenzimidazole (PBI, short for poly [2, 2’ - (m-phenylen) -5, 5’ -bisbenzimidazole] ) which is a synthetic fiber with a very high decomposition temperature. It does not exhibit a melting point, it has exceptional thermal and chemical stability, and it does not readily ignite.
PBS: The term “PBS” used herein is Phosphate-buffered saline (PBS) which is a buffer solution (pH ～ 7.4) commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solutions match those of the human body (isotonic) .
6.2 Influenza viruses
Influenza can cause mild to severe respiratory illness, which can result in hospitalization or death. Older adults and young children are at an increased risk of serious flu complications. The disease burden remains high, as the annual effectiveness of licensed vaccines varies from approximately 30-50%.
Influenza viruses belong to the Orthomyxoviridae family and are divided into types A, B, C and D. Influenza types A and B are responsible for epidemics of respiratory illness that are often associated with increased rates of hospitalization and death. All influenza viruses are negatives-strand RNA viruses with a segmented genome. Influenza type A and B viruses have 8 genes that code for 10 proteins, including the surface proteins hemagglutinin (HA) and neuraminidase (NA) . Influenza viruses comprise HA and NA proteins on the surface of the viral envelope. HA allows the virus's recognizing and binding to target cells, and also to infect the cell with viral RNA. NA is critical for the subsequent release of the daughter virus particles created within the infected cell so they can spread to other cells.
In the case of influenza type A viruses, further subdivision can be made into different subtypes according to differences in the two surface proteins. To date, 18 HA subtypes and 11 NA subtypes have been identified. All known subtypes of influenza type A viruses have been isolated from birds and can affect a range of mammal species. While more than 130 influenza A subtype combinations have been identified in nature, primarily from wild birds, there are potentially many more influenza A subtype combinations given the propensity for virus “reassortment. ” Reassortment is a process by which influenza viruses swap gene segments. Reassortment can occur when two influenza viruses infect a host at the same time and swap genetic information. Almost all influenza A pandemics have been caused by descendants of the 1918 virus, including “drifted” H1N1 viruses and reassorted H2N2 and H3N2 viruses.
Influenza type B viruses almost exclusively infect humans. Influenza B viruses are not classified into subtypes but can be broken down into lineages. Currently circulating influenza type B viruses belong to either B/Yamagata (B/Yamagata/16/88-like) or B/Victoria (B/Victoria/2/87-like) lineage. In recent years, flu B/Yamagata viruses have circulated much less frequently in comparison to flu B/Victoria viruses globally. Influenza virus B mutates at a rate 2 to 3 times slower than type A; however, it significantly impacts children and young adults annually. The influenza B virus capsid is enveloped while its virion consists of an envelope, a matrix protein, a nucleoprotein complex, a nucleocapsid, and a polymerase complex. It can be spherical or filamentous. Its 500 or so surface projections are made of HA and NA. The influenza B virus genome is 14, 548 nucleotides long and consists of eight segments of linear negative-sense, single-stranded RNA. The multipartite genome is encapsidated, each segment in a separate nucleocapsid, and the nucleocapsids are surrounded by one envelope.
Different subtypes of influenza A and different lineages of influenzas can be further divided into clades and subclades. As used herein and as is known in the art, a clade is a taxonomic subdivision of influenza viruses based on the similarity of their HA gene sequences. Viruses within a clade have similar genetic changes (e.g., nucleotide or amino acid mutations) and are therefore, genetically related, but they do not share the exact same viral genome. Viruses within a clade can be further subdivided in sub-clades. While clades and sub-clades may be genetically different from others, they are not necessarily antigenically different. Viruses from a specific clade or sub-clade may not have a mutation that impacts host immunity in comparison to other clades or sub-clades.
The hallmark of human influenza viruses is their ability to undergo antigenic change, which occurs in the following two ways: antigenic drift or antigenic shift.
Antigenic drift is a process of gradual and relatively continuous change in the viral HA and NA proteins. It results from the accumulation of point mutations in the HA and NA genes during viral replication. Both influenza type A and B viruses undergo antigenic drift, leading to new virus strains. The emergence of these new strains necessitates the frequent updating of influenza vaccine virus strains. Because antibodies to previous influenza infections may not provide full protection against the new strains resulting from antigenic drift, subjects can have many influenza infections over a lifetime.
In addition to antigenic drift, influenza type A viruses can also undergo a more dramatic and abrupt type of change called antigenic shift. By definition, a shift has occurred when an influenza type A virus emerges among humans bearing either an HA protein or a combination of HA and NA proteins that have not been circulating among humans in recent years. There are at least three possible mechanisms by which antigenic shift can occur: (a) a virus bearing new HA and NA proteins can arise through the genetic reassortment of non-human and human influenza viruses; (b) an influenza virus from other animals (e.g. birds or pigs) can infect a human directly without undergoing genetic reassortment; or (c) a non-human virus may be passed from one type of animal (e.g. birds) through an intermediate animal host (such as a pig) to humans.
Whereas antigenic drift occurs continuously, antigenic shift occurs infrequently and unpredictably. Since antigenic shift results in the emergence of a new influenza virus, a large proportion (or even all) of the world’s population will have no antibodies against it. If the new strain is capable of causing illness in humans and sustained chains of human-to-human transmission leading to community-wide outbreaks then such a virus has the potential to spread worldwide, causing a pandemic.
The most effective way to prevent the influenza virus infection is vaccination. Immunity from influenza virus vaccination, however, wanes over time, so annual vaccination is recommended to protect against the virus. Vaccination is most effective when circulating viruses are well-matched with viruses used to develop the vaccines. Due to the constant evolving nature of influenza viruses, the WHO Global Influenza Surveillance and Response System (GISRS) -a system of National Influenza Centers and WHO Collaborating Centers around the world -continuously monitors the influenza viruses circulating in humans and updates the recommended composition of influenza vaccines twice a year. Surveillance is the foundation underpinning all efforts to understand, prevent, and control influenza, and global influenza surveillance. Such surveillance activities also provide the vital information needed to establish the degree of seasonality of influenza in various parts of the world, and to estimate its impact and burden.
Traditionally, WHO provides a recommendation on the composition of the vaccine that targets the three (3) most representative virus types in circulation (two subtypes of influenza A viruses and one influenza B virus) (atrivalent vaccine) . Starting with the 2013-2014 northern hemisphere influenza season, a fourth component was recommended, supporting quadrivalent vaccine development. Quadrivalent vaccines include a second influenza B virus in addition to the viruses in trivalent vaccines and are thought to provide wider protection against influenza B virus infections.
The WHO, through the WHO GISRS system, in collaboration with other partners, monitors influenza activity globally and recommends seasonal influenza vaccine compositions twice a year for the Northern and Southern hemisphere influenza seasons. The WHO also guides countries in tropical and subtropical areas to choose vaccine formulations (Northern hemisphere vs.Southern hemisphere) and supports decisions for timing of vaccination campaigns. The organization also supports Member States to develop prevention and control strategies. The GISRS provides laboratory diagnosis and virological surveillance of circulating influenza viruses -both of which are key elements in influenza vaccine virus selection and the early detection of emerging viruses with pandemic potential.
The recommendations by WHO are updated twice a year (in February or March for the Northern hemisphere and in September for the Southern hemisphere) ; however, it is at least six or seven months before the vaccines are administered to the population. The recommendations are made so far in advance so that sufficient numbers of traditional vaccines (e.g., attenuated virus vaccines) can be designed and produced in advance of the flu season. During the six-or seven-month period; however, the influenza viruses may mutate, or other influenza strains may become more prevalent, such that the traditional vaccines become less effective. The traditional vaccines cannot adapt because they are already in production, and it would take an addition six to seven months to design and manufacture a new vaccine. In contrast, the RNA vaccines described herein are able to overcome these challenges. They can be produced in a matter of weeks, so that they can be designed against the influenza viruses circulating closer to the influenza season. As prediction of the circulating influenza viruses closer to the influenza season will be more accurate than predictions from six or seven months before the season, and the RNA vaccines described herein will also be more effective because they are designed to target circulating viruses closer to the influenza season.
In some embodiments, the circRNA vaccines described herein can be formulated as a supplemental booster vaccine. For example, if the traditional vaccines are made and further predictions indicate that a different influenza virus is more prevalent or infectious, a supplemental booster circRNA vaccine may be designed and administered, as described herein.
Additionally, as circRNAs lack the free ends necessary for exonuclease-mediated degradation, causing them to be resistant to several mechanisms of RNA degradation and granting extended half-lives when compared to an equivalent linear RNA. Circularization may allow for the stabilization of RNA polynucleotides that generally suffer from short half-lives and can improve the overall efficacy.
6.3 Antigens
In some embodiments, the immunogenic compositions provided herein comprise a circRNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises an HA sequence encoding a hemagglutinin antigen or an NA sequence encoding a neuraminidase antigen. As used herein, an “HA sequence” refers to a nucleotide sequence that encodes a hemagglutinin antigen; and an “NA sequence” refers to a nucleotide sequence that encodes a neuraminidase antigen.
Hemagglutinin is the target of current influenza virus vaccines, while neuraminidase has not been as widely used. The additional targeting of neuraminidase can provide additional protection during well-matched years and “fallback” protection during HA-drift years. In some embodiments, the circRNAs disclosed herein comprise an HA sequence. In some embodiments, the circRNAs disclosed comprise an NA sequence. In some embodiments, the circRNAs comprise both an HA sequence and an NA sequence. In some embodiments, the hemagglutinin antigen is from an influenza A virus. In some embodiments, the hemagglutinin antigen is from an influenza B virus. In some embodiments, the neuraminidase antigen is from an influenza A virus. In some embodiments, the neuraminidase antigen is from an influenza B virus.
Influenza A viruses are divided into subtypes based on HA and NA. There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 through H18 and N1 through N11, respectively) . Current subtypes of influenza A viruses that routinely circulate in people include A (H1N1) and A (H3N2) .
In some embodiments, circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H1-H18 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H1 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H3 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H2 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H5 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H7 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H9 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an HA sequence encoding an influenza A HA antigen of the H1 subtype and an HA sequence encoding an influenza A HA antigen of the H3 subtype.
In some embodiments, circRNA vaccines disclosed herein comprise an NA sequence encoding an influenza A NA antigen of the N1-N11 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an NA sequence encoding an influenza A NA antigen of the N1 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an NA sequence encoding an influenza A NA antigen of the N2 subtype. n some embodiments, the circRNA vaccines disclosed herein comprise an NA sequence encoding an influenza A NA antigen of the N8 subtype. In some embodiments, the circRNA vaccines disclosed herein comprise an NA sequence encoding an influenza A NA antigen of the N1 subtype and an influenza A NA antigen of the N2 subtype.
In some embodiments, circRNA vaccines provided herein encode an antigen from influenza A. The influenza A antigens can be from any strain known in the art. Exemplary influenza Type A viruses are listed in Table 1 below:
Table 1: Influenza A Viruses
In some embodiments, circRNA vaccines provided herein encode an antigen from influenza B antigen. The influenza B antigens can be from any strain known in the art. Examples of influenza Type B viruses are listed in Table 2 below.
Table 2: Influenza B Viruses
The sequences of the HA and NA antigens are known in the art and are available from GenBank. Exemplary amino acid sequences of the hemagglutinin antigens are provided in Table 3. Exemplary amino acid sequences of the neuraminidase antigens are provided in Table 4. In some embodiments, the circRNAs provided herein comprise at least an HA sequence encoding an HA antigen listed in Table 3, or an immunogenic fragment or variant thereof. In some embodiments, the circRNAs provided herein comprise at least an NA sequence encoding an NA antigen listed in Table 4, or an immunogenic fragment or variant thereof. In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to an amino acid selected from the group consisting of SEQ ID NOs: 1-84, or an immunogenic fragment thereof. In some embodiments, the circRNA vaccines provided herein comprise an NA sequence encoding an NA antigen that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-174, or an immunogenic fragment thereof.
In some embodiments, circRNA vaccines provided herein comprise an HA sequence that encodes an HA antigen of an influenza A virus strain of the A (H1N1) subtype. In some embodiments, circRNA vaccines provided herein comprise an HA sequence that encodes an HA antigen of an influenza A virus strain of the A (H3N2) subtype. In some embodiments, circRNA vaccines provided herein comprise an HA sequence that encodes an HA antigen of an influenza B virus strain of the B/Victoria lineage. In some embodiments, circRNA vaccines provided herein comprise an HA sequence that encodes an HA antigen of an influenza B virus strain of the B/Yamagata lineage.
In some embodiments, circRNA vaccines provided herein comprise an NA sequence that encodes an NA antigen of an influenza A virus strain of the A (H1N1) subtype. In some embodiments, circRNA vaccines provided herein comprise an NA sequence that encodes an NA antigen of an influenza A virus strain of the A (H3N2) subtype. In some embodiments, circRNA vaccines provided herein comprise an NA sequence that encodes an NA antigen of an influenza B virus strain of the B/Victoria lineage. In some embodiments, circRNA vaccines provided herein comprise an NA sequence that encodes an NA antigen of an influenza B virus strain of the B/Yamagata lineage.
In some embodiments, the HA antigen encoded by the circRNAs disclosed herein is a full-length HA. In some embodiments, the HA antigen encoded by the circRNAs disclosed herein is an immunogenic fragment of the full-length HA. In some embodiments, the immunogenic fragment comprises the stem region of the full-length HA. In some embodiments, the HA antigen encoded by the circRNAs disclosed herein is a wild-type HA. In some embodiments, the HA antigen is a modified HA. In some embodiments, the HA antigen comprises at least one mutation. In some embodiments, at least one amino acid is mutated relative to an influenza A or B hemagglutination wild type sequence. In some embodiments, the mutation is T2191, H371Y, I494M, H504P, M362L, HA0, APB, TB, or VASP. In some embodiments, more than one amino acid is mutated. In some embodiments, the mutation is selected from the group consisting of creation of a disulfide in the HA stem to link neighboring protomers, deletion of a cleavage site, and replacement of polybasic cleavage site (HPAI) by an LPAI sequence. In some embodiments, the mutation is a disulfide in the HA stem to link neighboring protomers. In some embodiments, the mutation is the deletion of a cleavage site. In some embodiments, the mutation is replacement of polybasic cleavage site (HPAI) by an LPAI sequence. As understood in the art, HPAI refers to highly pathogenic avian influenza (HPAI) viruses, while LPAI refers to low-pathogenic avian influenza viruses.
In some embodiments, the NA antigen encoded by the circRNAs disclosed herein is a full-length NA. In some embodiments, the NA antigen encoded by the circRNAs disclosed herein is an immunogenic fragment of the full-length NA. In some embodiments, the NA antigen is a wild-type NA. In some embodiments, the NA antigen is enzymatically active. In some embodiments, the NA antigen is a modified NA, such as an enzymatically inactive NA. As used herein, “enzymatically inactive NA” refers to a NA that has been mutated such that it possesses no or minimal catalytic activity (see, e.g., Richard et al., J Clin Virol., 2008, 41 (1) : 20-24; Yen et al., J Virol., 2006, 80 (17) : 8787-8795) . For example, in some embodiments, the enzymatically inactive NA possesses less than 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%of the catalytic activity of the wild-type NA (e.g., in an enzymatic activity assay, as is known in the art) . In some embodiments, at least one of Argll8, Aspl51, Argl52, Arg224, Glu276, Arg292, Arg371 and Tyr406 is mutated relative to an influenza A or B neuraminidase wild type sequence. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or all 8 amino acids are mutated. In some embodiments, at least one of Glull9, Argl56, Trpl78, Serl79, Aspl98, he222, Glu227, His274, Glu277, Asn294, and Glu425 is mutated relative to an influenza A or B neuraminidase wild type sequence. In some embodiments, the mutation is R118K, D151G, or E227D. In some embodiments, the mutation is a deletion of the cytoplasmic tail (dcytT) . In some embodiments, the mutation is a deletion of amino acids of the stalk region. In some embodiments, the mutation is a deletion of 15 amino acids of the stalk region (stalk_dl5) . In some embodiments, the mutation is a deletion of 30 amino acids of the stalk region (stalk_d30) . In some embodiments, the mutation is an insertion of amino acids of the stalk region. In some embodiments, the mutation is an insertion of 15 amino acids in the stalk region (stalk_insl5) .
Exemplary HA and NA antigens are known in the art and are publicly available, for example, on the websites of NCBI’s Influenza Virus Resource, Influenza Research Database on Bacterial and Viral Bioinformatics Resource Center (BV-BRC) , and GISRS.
The circRNA vaccines provided herein are amenable to inclusion of multiple antigen-encoding sequences and, as such, can include multiple HA sequences and/or NA sequences. In some embodiments, the circRNA vaccines provided herein comprise one or more HA sequences encoding one or more HA antigens, one or more NA sequences encoding one or more NA antigens, or a combination of both. In some embodiments, the circRNA vaccines provided herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA sequences that encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA antigens. In some embodiments, the circRNA vaccines provided herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NA sequences that encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA sequences that encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA antigens, and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NA sequences that encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NA antigens. For example, in some embodiments, the circRNA vaccines provided herein comprise 4 HA sequences that encode 4 HA antigens. In some embodiments, the circRNA vaccines provided herein comprise 4 NA sequences that encode 4 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 4 HA sequences that encode 4 HA antigens, and 4 NA sequences that encode 4 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 2 HA sequences that encode 2 HA antigens. In some embodiments, the circRNA vaccines provided herein comprise 2 NA sequences that encode 2 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 2 HA sequences that encode 2 HA antigens, and 2 NA sequences that encode 2 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 3 HA sequences that encode 3 HA antigens. In some embodiments, the circRNA vaccines provided herein comprise 3 NA sequences that encode 3 NA antigens. In some embodiments, the circRNA vaccines provided herein comprise 3 HA sequences that encode 3 HA antigens, and 3 NA sequences that encode 3 NA antigens.
In some embodiments, the circRNA vaccines provided herein comprises HA and NA sequences that collectively encode eight antigens: an H1 antigen, and H3 antigen, an N1 antigen, an N2 antigen, an HA B/Yamagata antigen, an HA B/Victoria antigen, an NA B/Yamagata antigen, and an NA B/Victoria antigen.
In some embodiments, the vaccines of the disclosure are designed to combat seasonal influenza, and as such are vaccines for use in an upcoming or forthcoming Northern hemisphere season or Southern hemisphere season. Based on an understanding of circulating influenza viruses (e.g., virus lineages, strains and/or subtypes) at a given point in time, the vaccines are designed to combat such viruses as they are predicted to be those that will be circulating or prevalent in the upcoming or forthcoming influenza season (e.g., the upcoming/forthcoming season in a particular geographic location, for example, northern or southern hemisphere) . As such, the circRNA vaccines of the instant invention comprise circRNA encoding hemagglutinin and/or neuraminidase antigens of the influenza viruses circulating at the time of design of the vaccines. Exemplary circRNA vaccines disclosed herein encode HA antigens and/or NA antigens of the circulating H1N1 viruses and H3N2 viruses. The circRNA vaccines disclosed herein can comprise HA sequences encoding the HA antigens of each circulating influenza A subtype or of each predominant influenza A subtype in combination with HA sequences encoding the HA antigens of each circulating influenza B lineage (or of each predominant influenza lineage) . In exemplary embodiments, the circRNA vaccines disclosed herein also comprise NA sequences encoding the NA antigens corresponding to the selected HA antigens. Predominant viruses, or those predominant in circulation, are those detected in the human population at an endemic frequency or at a frequency above a certain threshold understood by the skilled artisan is requisite to evidence that those strain (s) are in circulation within a population, e.g., within populations representative of the Northern or Southern hemisphere.
In some embodiments, the circRNAs provided herein comprise Z sequences containing HA sequences and/or NA sequences encoding HA antigens and/or NA antigens that are selected according to standardized criteria used GISRS. In some embodiments, the circRNA vaccines provided herein comprise a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to a sequence selected according to standardized criteria used GISRS.
Methods to characterize target antigens for circulating influenza viruses are known in the art, including, for example, hemagglutination inhibition assay (HAI) , neuraminidase inhibition assay (NAI) , microneutralization assay, virus identification by immunofluorescence antibody Staining, and genetic characterization.
The HAI test is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses and further determining the antigenic characteristics of influenza viral isolates provided that the reference antisera used contain antibodies to currently circulating viruses (see, e.g., Pedersen JC Methods Mol Biol. 2014; 1161: 11-25) . The antisera used are based on antigen preparations derived from either the wildtype strain or a high-growth reassortant made using the wild-type strain or an antigenically equivalent strain.
To perform the assay, a serial dilution of virus is prepared across the rows in a U or V-bottom shaped 96-well microtiter plate. As an example, the most concentrated sample in the first well may be diluted to be l/5x of the stock, and subsequent wells may be two-fold dilutions (1/10, 1/20, 1/40, etc. ) . The final well serves as a negative control with no virus. Each row of the plate typically has a different virus and the same pattern of dilutions. After serial dilutions, a standardized concentration of red blood cells (RBCs) is added to each well and mixed gently. The plate is incubated at room temperature. Following the incubation period, the assay can be analyzed to distinguish between agglutinated and non-agglutinated wells. The relative concentration, or titer, of the virus sample is based on the well with the last agglutinated appearance, immediately before a pellet is observed.
Serological methods such as the HAI test are essential for many epidemiological and immunological studies and for evaluation of the antibody response following vaccination. Serological methods are also very useful in situations where identification of the virus is not feasible (e.g., after viral shedding has stopped) . The HAI test is used to identify circulating influenza viruses that are antigenically similar to influenza viruses from a previous season’s vaccine. As used herein “antigenically similar” refers to a virus having an HAI titer that differs by two dilutions or less.
The neuraminidase-inhibition (NAI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus (see, e.g., Pedersen JC, Methods Mol Biol. 2014; 1161: 27-36) . A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses.
There are two basic forms of assay for influenza virus NA based on the use of different substrate molecules, a long-standing assay based on the use of a large substrate such as fetuin (e.g., the enzyme-linked lectin assay (ELLA) ) and newer assays which utilize small substrate molecules. The fetuin-based method is used to determine the potency of the viral NA and thus the standardized NA dose for use in the NA inhibition (NAI) assay. Once determined, the standardized dose is added to serial dilutions of test antisera, negative control serum and reference anti-NA serum. Any inhibitory effect of the sera on NA activity can then be determined and the NAI titer calculated. The small substrate based method can be a fluorescence assay that uses the substrate 2- (4-methylumbelliferyl) -a-D-N-acetylneuraminic acid (MUNANA) . The substrate is added to serially diluted test antisera and cleavage of the MUNANA substrate by NA releases the fluorescent product methylumbelliferone. The inhibitory effect of the sera on the influenza virus NA is determined based on the concentration of the sera that is required to reduce 50%of the NA activity, given as an IC50 value. The small substrate based method can, alternatively, be a chemiluminescence-based (CL) assay that uses a sialic acid 1, 2-dioxetane derivative (NA-Star) substrate or a modified NA-XTD substrate. The CL assays provide an extended-glow chemiluminescent light signal and neuraminidase inhibitor IC50 values are achieved over a range of virus dilutions.
In some embodiments, the HA antigens and/or NA antigens are selected using a HAI assay or NAI assay to identify circulating influenza viruses that are antigenically similar to influenza viruses from a previous season’s vaccine. In some embodiments, the influenza viruses are considered to be antigenically similar if their HAI titers differ by two dilutions or less.
Serological methods such as the HAI test rarely yield an early diagnosis of acute influenza virus infection. Although conventional neutralization tests for influenza viruses (based on the inhibition of cytopathogenic effect formation in MDCK cell culture) are productive, a microneutralization assay using microtiter plates in combination with an ELISA to detect virus-infected cells can yield results within two days. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses in human and animal sera, and in some embodiments, includes the detection of human antibodies to avian subtypes. Testing can be carried out quickly once a novel virus is identified and often before purified viral proteins become available for use in other assays.
Immunofluorescence antibody (IFA) staining of virus-infected cells in original clinical specimens and field isolates is a rapid and sensitive method for diagnosing respiratory and other viral infections. In some embodiments, IFA staining is performed on isolates rather than original clinical specimens, as this allows any virus that is present to first be amplified, and if required used in other studies. As commercially available rapid tests for diagnosing influenza infection differ with regard to the type of specimen required as well as their complexity, specificity and sensitivity, it is recommended that such assays should be used in conjunction with other laboratory tests.
The direct molecular identification of influenza isolates is a rapid and powerful technique. The reverse-transcription polymerase chain reaction (RT-PCR) allows template viral RNA to be reverse transcribed producing complementary DNA (cDNA) which can then be amplified and detected. This method can be used directly on clinical samples and the rapid nature of the results can greatly facilitate investigation of outbreaks of respiratory illness (e.g., influenza) . For example, genetic analysis of influenza virus genes (especially the HA and NA genes) can be used to identify an unknown influenza virus when the antigenic characteristics cannot be defined. Genetic analyses also can be used to monitor the evolution of influenza viruses and to determine the degree of relatedness between viruses from different geographical areas and those collected at different times of the year.
For illustrative purposes, circulating influenza A viruses include, for example, influenza A(H1N1) pdm09, A (H3N2) , and influenza type B viruses (B/Victoria/2/87 and B/Yamagata/16/88) . In some embodiments, the influenza A (H1N1) pdm09 viruses comprise HA genes that belong to phylogenetic clade 5a. 2a. n some embodiments, the influenza A(H1N1) pdm09 viruses comprise HA genes that belong to phylogenetic clade 5a. 2a. 1. n some embodiments, the influenza A (H1N1) pdm09 viruses comprise HA genes that belong to phylogenetic clade 5a. 1. In some embodiments, the influenza A (H3N2) viruses comprise HA genes that belong to phylogenetic clade 1 (e.g., 3C. 2a1b. 2a. 1) . In some embodiments, the influenza A (H3N2) viruses comprise HA genes that belong to phylogenetic clade 2 (e.g., 3C. 2alb. 2a. 2) . In some embodiments, the influenza B virus of the B/Victoria lineage belongs to genetic clade 1A. 3.
In some embodiments, the circulating influenza A (H1N1) pdm09 virus is A/Wisconsin/67/2022, A/Wisconsin/588/2019, A/Victoria/2570/2019, A/Victoria/4897/2022, or A/Sydney/5/2021. In some embodiments, the circulating influenza A (H3N2) virus is A/Darwin/09/2021 or A/Darwin/6/2021. In some embodiments, the circulating influenza B/Victoria lineage virus is B/Austria/1359417/2021 or B/Washington/02/2019. In some embodiments, the influenza B/Yamagata lineage virus is B/Phuket/3073/2013.
In some embodiments, the circRNAs provided herein encode an influenza virus antigen variant. Antigen variants or other polypeptide variants refers to molecules that differ in their amino acid sequence from a wild-type, native, or reference sequence. The antigen/polypeptide variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50%identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90%identity with a wild-type, native, or reference sequence.
Variant antigens/polypeptides encoded by nucleic acids of the disclosure can contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, enhance their expression, and/or improve their stability or PK/PD properties in a subject. Variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art and exemplary such assays are set forth in the Examples section. Similarly, PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response. The stability of protein (s) encoded by a variant nucleic acid can be measured by assaying thermal stability or stability upon urea denaturation or can be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art.
As such, circRNAs encoding polypeptide (e.g., antigen) containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends) . Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) can alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like can be substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In some embodiments, buried hydrogen bond networks can be replaced with hydrophobic resides to improve stability. In some embodiments, glycosylation sites can be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that can be deleted, for example, prior to use in the preparation of an circRNA vaccine.
Exemplary amino acid sequences of the hemagglutinin antigens and neuraminidase antigens of circulating virus are provided in Tables 5A, 7A, or 8A (hemagglutinin antigens) and 5B, 7B, or 8B (neuraminidase antigens) . In some embodiments, the circRNAs provided herein comprise at least an HA sequence encoding an HA antigen listed in Table 5A, 7A, or 8A, or an immunogenic fragment or variant thereof. In some embodiments, the circRNAs provided herein comprise at least an NA sequence encoding an NA antigen listed in Table 5B, 7B, or 8B, or an immunogenic fragment or variant thereof.
In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to an amino acid selected from the group consisting of SEQ ID NOs: 175, 177, 179-181, 234-236, and 240-257, or an immunogenic fragment thereof.
In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 175, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 177, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 179, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 180, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 181, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 234, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 235, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 236, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 240, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 241, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 242, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 243, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 244, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 245, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 246, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 247, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 248, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 249, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 250, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 251, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 252, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 253, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 254, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 255, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 256, or an immunogenic fragment thereof. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 257, or an immunogenic fragment thereof.
In some embodiments, the circRNA vaccines provided herein comprise an NA sequence encoding an NA antigen that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 176, 178, 237-239, and 258-275, or an immunogenic fragment thereof. In some embodiments, the NA sequence encodes an NA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 176, or an immunogenic fragment thereof. In some embodiments, the NA sequence encodes an NA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 178, or an immunogenic fragment thereof. In some embodiments, the NA sequence encodes an NA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 237, or an immunogenic fragment thereof. In some embodiments, the NA sequence encodes an NA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 238, or an immunogenic fragment thereof. In some embodiments, the NA sequence encodes an NA antigen having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 239, or an immunogenic fragment thereof.
In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen. In some embodiments, the HA antigen is from a H1N1 virus, a H3N2 virus, a virus of B/Victoria lineage, or a virus of B/Yamagata lineage. In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain A/Wisconsin/588/2019 (H1N1) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain A/Wisconsin/67/2022 (H1N1) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain A/Darwin/6/2021 (H3N2) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain A/Sydney/5/2021 (H1N1) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain B/Austria/1359417/2021. In some embodiments, the circRNA vaccines provided herein comprise an HA sequence encoding an HA antigen from strain B/Phuket/3073/2013.
In some embodiments, the HA antigen is Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the HA antigen is Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 177. In some embodiments, the HA antigen is Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 179. In some embodiments, the HA antigen is Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 180. In some embodiments, the HA antigen is Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 235.
The circRNA vaccines provided herein can encode HA antigens and/or NA antigens of circulating strains/lineages that represent multiple, distinct influenza clades and sub-clades, producing vaccines more efficacious at combatting an upcoming or forthcoming influenza season. In some embodiments, the circRNA vaccines provided herein comprise multiple HA sequences encoding the HA antigens from the most prevalent influenza strains. For example, in some embodiments, the circRNA vaccines provided herein comprise multiple HA sequences encoding HA antigens from the most prevalent A/H1N1 strain (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and A/H3N2 strain (e.g., A/Darwin/9/2021, A/Darwin/6/2021) . The circRNA vaccines provided herein can further comprise an HA sequence encoding an HA antigen from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) . The circRNA vaccines provided herein can further comprise an HA sequence encoding an HA antigen from the most prevalent B/Yamagata lineage (e.g., B/Phuket/3073/2013) . In some embodiments, the circRNA vaccines provided herein comprise multiple HA sequences encoding HA antigens from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
In some embodiments, the circRNA vaccines provided herein comprise multiple NA sequences encoding the NA antigens from the most prevalent influenza strains. In some embodiments, the circRNA vaccines provided herein comprise multiple NA sequences encoding NA antigens from the most prevalent A/H1N1 strain (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and A/H3N2 strain (e.g., A/Darwin/9/2021, A/Darwin/6/2021) . The circRNA vaccines provided herein can further comprise an NA sequence encoding an NA antigen from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) . The circRNA vaccines provided herein can further comprise an NA sequence encoding an NA antigen from the most prevalent B/Yamagata lineage (e.g., B/Phuket/3073/2013) . In some embodiments, the circRNA vaccines provided herein comprise multiple NA sequences encoding NA antigens from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
In some embodiments, the circRNA vaccines provided herein comprise multiple HA and NA sequences encoding the HA and NA antigens from the most prevalent influenza strains. For example, in some embodiments, the circRNA vaccines provided herein comprise multiple HA and NA sequences encoding the HA and NA antigens from the most prevalent A/H1N1 strain (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and A/H3N2 strain (e.g., A/Darwin/9/2021, A/Darwin/6/2021) . The circRNA vaccines provided herein can further comprise HA and NA sequences encoding HA and NA antigens from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) . The circRNA vaccines provided herein can further comprise HA and NA sequences encoding HA and NA antigens from the most prevalent B/Yamagata lineage (e.g., B/Phuket/3073/2013) . In some embodiments, the circRNA vaccines provided herein comprise multiple HA and NA sequences encoding HA and NA antigens from the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
In some embodiments, the circRNA vaccines provided herein can further include HA sequences and/or NA sequences encoding the HAs and NAs of a second prevalent A/H1N1 strain, A/H3N2 strain, B/Victoria lineage and/or B/Yamagata lineage. For example, in some embodiments, the circRNA vaccines provided herein comprise multiple HA and/or NA sequences encoding the HA and/or NA antigens from the most prevalent A/H1N1 strain (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and A/H3N2 strain (e.g., A/Darwin/9/2021, A/Darwin/6/2021) , the most prevalent B/Victoria lineage (e.g., B/Austria/1359417/2021) , optionally also the most prevalent B/Yamagata lineage (e.g., B/Phuket/3073/2013) , and further include HA and/or NA sequences encoding the HAs and NAs of a second prevalent A/H1N1 strain, A/H3N2 strain, B/Victoria lineage and/or B/Yamagata lineage B/Yamagata lineage.
6.4 Circular RNAs (circRNAs)
The circRNA vaccines disclosed herein provide a unique advantage over traditional protein-based vaccination approaches in which protein antigens are purified or produced in vitro, e.g., recombinant protein production technologies. The vaccines of the present disclosure feature circRNAs encoding the desired antigens, which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigens. Upon delivery and uptake by cells of the body, the circRNAs are translated in the cytosol and protein antigens are generated by the host cell machinery. The protein antigens are presented and elicit an adaptive humoral and cellular immune response. Neutralizing antibodies are directed against the expressed protein antigens and hence the protein antigens are considered relevant target antigens for vaccine development. The circRNAs provided herein induce a reduced innate immune response relative to their mRNA counterparts when administered to a subject.
In some embodiments, provided herein are immunogenic compositions comprising a circRNA, wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence, and wherein the Z sequence comprises an HA sequence encoding a hemagglutinin antigen or an NA sequence encoding a neuraminidase antigen. As described in the section above, the circRNAs disclosed herein can have at least one HA sequence and/or at least one NA sequence. The circRNAs disclosed herein can have one HA sequence or one NA sequence. The circRNAs disclosed herein can have one HA sequence and one NA sequence. The circRNAs disclosed herein can have multiple HA sequences or NA sequences. Further, the circRNAs disclosed herein can have multiple HA sequences and multiple NA sequences. Additionally, in some embodiments, the circRNA can have one TI sequence. In some embodiments, the circRNA can have two or more TI sequences.
6.4.1 Structures with one TI sequence
In some embodiments, circRNAs provided herein comprise a translation initiation (TI) sequence and a protein-coding (Z) sequence, wherein the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus. The first influenza virus can be type A. The first influenza virus can be type B.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus, and further comprise a second HA (HA2) sequence encoding an HA antigen from a second influenza virus. The second influenza virus can be type A. The second influenza virus can be type B. Accordingly, in some embodiments, the first and second influenza viruses can be two subtypes of type A (i.e., A1 and A2) . In some embodiments, the first and second influenza viruses can be two subtypes of type B viruses (i.e., B1 and B2) . In some embodiments, one of the two influenza viruses can be type A and the other type B. The HA1 sequence and the HA2 sequence can be linked by a linker (L) sequence. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: TI, HA1, L, and HA2, wherein L is a linker sequence or absent, and wherein the HA1 and HA2 encode HA antigens of the first and second influenza viruses that are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B. The first and second influenza viruses can be types B and A.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus, a second HA (HA2) sequence encoding an HA antigen from a second influenza virus, and further comprises a third HA (HA3) sequence encoding an HA antigen from a third influenza virus. The third influenza virus can be type A. The third influenza virus can be type B. Accordingly, in some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. The HA sequences can be arranged in any order. The HA sequences can be linked by linker sequences. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: TI, HA1, L1, HA2, L2, and HA3, wherein L1 and L2 each is independently a linker sequence or absent, wherein the HA1, HA2 and HA3 encode HA antigens of the first, second and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types A1, B, and A2. The first, second and third influenza viruses can be types B, A1, and A2. The first, second and third influenza viruses can be types A, B1, and B2. The first, second and third influenza viruses can be types B1, A, and B2. The first, second and third influenza viruses can be types B1, B2, and A.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen from a first influenza virus, a second HA (HA2) sequence encoding an HA antigen from a second influenza virus, a third HA (HA3) sequence encoding an HA antigen from a third influenza virus, and further comprises a fourth HA (HA4) sequence. The fourth influenza virus can be type A. The fourth influenza virus can be type B. An exemplary diagram is provided in FIG. 1. Accordingly, in some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . In some embodiments, three of the four influenza viruses can be three subtypes of type A (i.e., A1, A2, and A3) and the fourth one can be type B. In some embodiments, three of the four influenza viruses can be three subtypes of type B (i.e., B1, B2 and B3) and the fourth one can be type A. In some embodiments, all four influenza viruses can be type A. In some embodiments, all four influenza viruses can be type B. The HA sequences can be arranged in any order. In some embodiments, the circRNA comprises, in the following order: TI, HA1, L1, HA2, L2, HA3, L3, and HA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) A1, B1, A2, and B2; (3) B1, A1, B1, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second, third and fourth influenza viruses can be types A1, A2, B1 and B2. The first, second, third and fourth influenza viruses can be types A1, B1, A2, and B2. The first, second, third and fourth influenza viruses can be types B1, A1, B2, and A2. The first, second, third and fourth influenza viruses can be types B1, B2, A1, and A2.
In some embodiments, circRNAs provided herein comprise a translation initiation (TI) sequence and a protein-coding (Z) sequence, wherein the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus. The first influenza virus can be type A. The first influenza virus can be type B.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus, and further comprise a second NA (NA2) sequence encoding an NA antigen from a second influenza virus. The second influenza virus can be type A. The second influenza virus can be type B. Accordingly, in some embodiments, the first and second influenza viruses can be two subtypes of type A (i.e., A1 and A2) . In some embodiments, the first and second influenza viruses can be two subtypes of type B viruses (i.e., B1 and B2) . In some embodiments, one of the two influenza viruses can be type A and the other type B. The NA1 sequence and the NA2 sequence can be linked by a linker (L) sequence. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: TI, NA1, L, and NA2, wherein L is a linker sequence or absent, and wherein the NA1 and NA2 encode NA antigens of the first and second influenza viruses that are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A, wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B. The first and second influenza viruses can be types B and A.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus, a second NA (NA2) sequence encoding an NA antigen from a second influenza virus, and further comprises a third NA (NA3) sequence encoding an antigen from a third influenza virus. The third influenza virus can be type A. The third influenza virus can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. The NA sequences can be arranged in any order. The NA sequences can be linked by linker sequences. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: TI, NA1, L1, NA2, L2, and NA3, wherein L1 and L2 each is independently a linker sequence or absent, wherein the NA1, NA2 and NA3 encode NA antigens of the first, second and third influenza viruses that are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types A1, B, and A2. The first, second and third influenza viruses can be types B, A1, and A2. The first, second and third influenza viruses can be types A, B1, and B2. The first, second and third influenza viruses can be types B1, A, and B2. The first, second and third influenza viruses can be types B1, B2, and A.
In some embodiments of the circRNAs disclosed herein, the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen from a first influenza virus, a second NA (NA2) sequence encoding an NA antigen from a second influenza virus, a third NA (NA3) sequence encoding an antigen from a third influenza virus, and further comprises a fourth NA (NA4) sequence. The fourth influenza virus can be type A. The fourth influenza virus can be type B. An exemplary diagram is provided in FIG. 2. Accordingly, in some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . In some embodiments, three of the four influenza viruses can be three subtypes of type A (i.e., A1, A2, and A3) and the fourth one can be type B. In some embodiments, three of the four influenza viruses can be three subtypes of type B (i.e., B1, B2 and B3) and the fourth one can be type A. In some embodiments, all four influenza viruses can be type A. In some embodiments, all four influenza viruses can be type B. The NA sequences can be arranged in any order. In some embodiments, the circRNA comprises, in the following order: TI, NA1, L1, NA2, L2, NA3, L3, and NA4; wherein L1, L2 and L3 each is independently a linker sequence or absent; and wherein the NA1, NA2, NA3 and NA4 encode NA antigens of the first, second, third, and fourth influenza viruses that are types (1) A1, A2, B1, and B2; (2) A1, B1, A2, and B2; (3) B1, A1, B1, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second, third and fourth influenza viruses can be types A1, A2, B1 and B2. The first, second, third and fourth influenza viruses can be types A1, B1, A2, and B2. The first, second, third and fourth influenza viruses can be types B1, A1, B2, and A2. The first, second, third and fourth influenza viruses can be types B1, B2, A1, and A2.
In some embodiments of the circRNAs disclosed herein, the Z sequence can comprise a first HA (HA1) sequence encoding an HA antigen, and a first NA (NA1) sequence encoding an NA antigen. The HA and NA antigens are from a first influenza virus. The first influenza virus can be type A. The first influenza virus can be type B. The HA1 and NA1 sequences can be connected by a linker sequence. The HA1 and NA1 sequences can be arranged in any order. In some embodiments, the circRNA comprises, in the following order: TI, HA1, L, and NA1; wherein L is a linker sequence or absent. In some embodiments, the circRNA comprises, in the following order, TI, NA1, L, and HA1.
In some embodiments of the circRNAs disclosed herein, the Z sequence can comprise a first HA (HA1) sequence and a first NA (NA1) sequence encoding HA and NA antigens from a first influenza virus, and further comprise a second HA (HA2) sequence and a second NA (NA2) sequence encoding HA and NA antigens from a second influenza virus. The second influenza virus can be type A. The second influenza virus can be type B. Accordingly, in some embodiments, the first and second influenza viruses can be two subtypes of type A (i.e., A1 and A2) . In some embodiments, the first and second influenza viruses can be two subtypes of type B viruses (i.e., B1 and B2) . In some embodiments, one of the two influenza viruses can be type A and the other type B. Adjacent HA sequences and the NA sequences can be linked by a linker (L) sequence. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, and NA2; (2) TI, NA1, L1, HA1, L2, NA2, L3, and HA2; (3) TI, HA1, L1, HA2, L2, NA1, L3, and NA2; or (4) TI, NA1, L1, NA2, L2, HA1, L3, and HA2; wherein L1, L2 and L3 each is independently a linker sequence or absent. In some embodiments, the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B. The first and second influenza viruses can be types B and A.
In some embodiments of the circRNAs disclosed herein, the Z sequence can comprise a first HA (HA1) sequence and a first NA (NA1) sequence encoding HA and NA antigens from a first influenza virus, a second HA (HA2) sequence and a second NA (NA2) sequence encoding HA and NA antigens from a second influenza virus, and further comprise a third HA (HA3) sequence and a third NA (NA3) sequence encoding HA and NA antigens from a third influenza virus. The third influenza virus can be type A. The third influenza virus can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. The HA and NA sequences can be arranged in any order. Adjacent HA and NA sequences can be linked by linker sequences. As such, in some embodiments, the circRNAs provided herein comprise, in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3; (2) TI, HA1, L1, HA2, L2, HA3, L3, NA1, L4, NA2, L5, and NA3; (3) TI, NA1, L1, NA2, L2, NA3, L3, HA1, L4, HA2, L5, and HA3; or (4) TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3; wherein L1, L2, L3, L4, and L5 each is independently a linker sequence or absent. The circRNA can comprise, in the following order: TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3. An exemplary diagram is provided in FIG. 3. The circRNA can comprise, in the following order: TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3. An exemplary diagram is provided in FIG. 4. The circRNA can comprise, in the following order: TI, HA1, L1, HA2, L2, HA3, L3, NA1, L4, NA2, L5, and NA3. The circRNA can comprise, in the following order: TI, NA1, L1, NA2, L2, NA3, L3, HA1, L4, HA2, L5, and HA3. In some embodiments, the first, second and third influenza virus are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types A1, B, and A2. The first, second and third influenza viruses can be types B, A1, and A2. The first, second and third influenza viruses can be types A, B1, and B2. The first, second and third influenza viruses can be types B1, A, and B2. The first, second and third influenza viruses can be types B1, B2, and A.
6.4.2 Structures with two TI sequences
In some embodiments, the circRNAs disclosed herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, wherein the Z1 and the Z2 sequences each independently comprises an HA sequence encoding an HA antigen, or an NA sequence encoding an NA antigen.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and the Z2 sequence comprises a second HA (HA2) sequence, wherein the HA1 sequences encodes an HA antigen from a first influenza virus and the HA2 sequence encodes an HA antigen from second influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, TI2, HA2. The first influenza virus can be type A or B, and the second influenza virus can be type A or B. In some embodiments, the circRNAs provided herein can comprise in the following order: TI1, HA1, L1, TI2, HA2, and L2, wherein L1 and L2 each is independently a linker sequence or absent. In some embodiments, the HA1 and HA2 encode HA antigens of the first and second influenza viruses that are types (1) A1 and A2; (2) B1 and B2; or (3) A and B; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B.
In some embodiments, the Z1 sequence comprises a first NA (NA1) sequence and the Z2 sequence encodes a second NA (NA2) sequence, wherein the NA1 sequences encodes an NA antigen from a first influenza virus and the NA2 sequence encodes an NA antigen from second influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, NA1, TI2, NA2. In some embodiments, the circRNAs provided herein can comprise in the following order: TI1, NA1, L1, TI2, NA2, and L2, wherein L1 and L2 each is independently a linker sequence or absent. The first influenza virus can be type A or B, and the second influenza virus can be type A or B. In some embodiments, the NA1 and NA2 encode NA antigens of the first and second influenza viruses that are types (1) A1 and A2; (2) B1 and B2; or (3) A and B; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B.
In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, and the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence; wherein the HA1 and NA1 sequences encode HA and NA antigens from a first influenza virus, and the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus. Adjacent HA sequences and NA sequences can be linked by a linker (L) sequence. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2; wherein L1 and L2 each is independently a linker sequence or absent. In some embodiments, the circRNA comprises, in the following order: TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2. The first influenza virus can be type A or B, and the second influenza virus can be type A or B. In some embodiments, the first and second influenza viruses that are types (1) A1 and A2; (2) B1 and B2; or (3) A and B; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence encoding an HA antigen and the Z2 sequence comprises a first NA (NA1) sequence encoding an NA antigen, wherein the HA and NA antigens are from a first influenza virus. The first influenza virus can be type A. The first influenza virus can be type B. As such, the circRNA comprises, in the following order: TI1, HA1, TI2, and NA1. An exemplary diagram is provided in FIG. 5.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence and further comprise a second HA (HA2) sequence, and the Z2 sequence comprises a first NA (NA1) sequence and further comprises a second NA (NA2) sequence; wherein the HA1 and NA1 sequences encode HA and NA antigens from a first influenza virus, and the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus. Adjacent HA sequences and NA sequences can be linked by a linker (L) sequence. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, HA2, TI2, NA1, L2, NA2, wherein L1, and L2 each is independently a linker sequence or absent. The first influenza virus can be type A or B, and the second influenza virus can be type A or B. In some embodiments, the first and second influenza viruses are (1) A1 and A2; (2) B1 and B2; (3) A and B;or (4) B and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first and second influenza viruses can be types A1 and A2. The first and second influenza viruses can be types B1 and B2. The first and second influenza viruses can be types A and B. The first and second influenza viruses can be types B and A.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence, a second HA (HA2) sequence, and further comprise a third HA (HA3) sequence, and the Z2 sequence comprises a first NA (NA1) sequence, a second NA (NA2) sequence, and further comprises a third NA (NA3) sequence; wherein the HA1 and NA1 sequences encode HA and NA antigens from a first influenza virus, the HA2 and NA2 sequences encode HA and NA antigens from a second influenza virus, and the HA3 and NA3 sequences encode HA and NA antigens from a third influenza virus. The first influenza virus can be type A or B, the second influenza virus can be type A or B, and the third influenza virus can be type A or B.In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. Adjacent HA sequences and NA sequences can be linked by a linker (L) sequence. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3; wherein L1, L2, L3 and L4 each is independently a linker sequence or absent. An exemplary diagram is provided in FIG. 6. In some embodiments, the first, second, and third influenza viruses are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types A1, B, and A2.The first, second and third influenza viruses can be types B, A1, and A2. The first, second and third influenza viruses can be types A, B1, and B2. The first, second and third influenza viruses can be types B1, A, and B2. The first, second and third influenza viruses can be types B1, B2, and A.
6.4.3 Structures with three TI sequences
In some embodiments, the circRNAs provided herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, and a third protein-coding (Z3) sequence, wherein the Z1, Z2 and Z3 sequences each independently comprises an HA sequence or an NA sequence.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, and the Z3 sequence comprises a third HA (HA3) sequence, wherein the HA1 sequences encodes an HA antigen from a first influenza virus, the HA2 sequence encodes an HA antigen from second influenza virus, and the HA3 sequence encodes an HA antigen from third influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, TI2, HA2, TI3, HA3. An exemplary diagram is provided in FIG. 7. In some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent. The first influenza virus can be type A or B, the second influenza virus can be type A or B, and the third influenza virus can be type A or B. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. In some embodiments, the first, second, and third influenza viruses are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types B1, B2, and A.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, and the Z3 sequence comprises a third NA (NA3) sequence, wherein the NA1 sequences encodes an NA antigen from a first influenza virus, the NA2 sequence encodes an NA antigen from second influenza virus, and the NA3 sequence encodes an NA antigen from third influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, NA1, TI2, NA2, TI3, NA3. An exemplary diagram is provided in FIG. 8. In some embodiments, the circRNA comprises, in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3; wherein L1, L2, and L3 each is independently a linker sequence or absent. The first influenza virus can be type A or B, the second influenza virus can be type A or B, and the third influenza virus can be type A or B. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. In some embodiments, the first, second, and third influenza viruses are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types B1, B2, and A.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence and first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, and the Z3 comprises encodes a third HA (HA3) sequence and a third NA (NA3) sequence; wherein the HA1 and NA1 sequences encode antigens from a first influenza virus, the HA2 and NA2 encode antigens from a second influenza virus, and the HA3 and NA3 encode antigens from a third influenza virus. Adjacent HA sequences and NA sequences can be linked by a linker (L) sequence. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3. An exemplary diagram is provided in FIG. 9. In some embodiments, the circRNA comprises, in the following order: TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3. An exemplary diagram is provided in FIG. 10. L1, L2, and L3 each independently is a linker sequence. The first influenza virus can be type A or B, the second influenza virus can be type A or B, and the third influenza virus can be type A or B. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. In some embodiments, the first, second, and third influenza viruses are types (1) A1, A2 and B; or (2) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types B1, B2, and A.
6.4.4 Structures with four TI sequences
In some embodiments, the circRNAs provided herein comprise, in the following order: a first translation initiation (TI1) sequence, a first protein-coding (Z1) sequence, a second translation initiation (TI2) sequence, and a second protein-coding (Z2) sequence, a third translation initiation (TI3) sequence, a third protein-coding (Z3) sequence, a four translation initiation (TI4) sequence, and a fourth protein-coding (Z4) sequence, wherein the Z1, Z2, Z3 and Z4 sequences each independently comprises an HA sequence or an NA sequence.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, the Z3 sequence comprises a third HA (HA3) sequence, and the Z4 sequence comprises a fourth HA (HA4) sequence, wherein the HA1 sequences encodes an HA antigen from a first influenza virus, the HA2 sequence encodes an HA antigen from second influenza virus, the HA3 sequence encodes an HA antigen from third influenza virus, and the HA4 sequence encodes an HA antigen from a fourth influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, HA1, TI2, HA2, TI3, HA3, TI3, and HA4. An exemplary diagram is provided in FIG. 14. In some embodiments, the circRNA comprises, in the following order: TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4; wherein L1, L2, L3, and L4 each is independently a linker sequence or absent. The first influenza virus can be type A or B, the second influenza virus can be type A or B, the third influenza virus can be type A or B, and the fourth influenza virus can be type A or B. In some embodiments, three of the four influenza viruses can be three subtypes of type A (i.e., A1, A2, and A3) and the fourth one can be type B. In some embodiments, three of the four influenza viruses can be three subtypes of type B (i.e., B1 B2, and B3) and the fourth one can be type A. In some embodiments, all four influenza viruses can be type A. In some embodiments, all four influenza viruses can be type B. In some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . In some embodiments, the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
In some embodiments of the circRNAs provided herein, the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, the Z3 sequence comprises a third NA (NA3) sequence, and the Z4 sequence comprises a fourth NA (NA4) sequence, wherein the NA1 sequences encodes an NA antigen from a first influenza virus, the NA2 sequence encodes an NA antigen from second influenza virus, the NA3 sequence encodes an NA antigen from third influenza virus, and the NA4 sequence encodes an NA antigen from a fourth influenza virus. As such, in some embodiments, the circRNA comprises, in the following order: TI1, NA1, TI2, NA2, TI3, NA3, TI3, and NA4. An exemplary diagram is provided in FIG. 14. In some embodiments, the circRNA comprises, in the following order: TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4; wherein L1, L2, L3, and L4 each is independently a linker sequence or absent. The first influenza virus can be type A or B, the second influenza virus can be type A or B, the third influenza virus can be type A or B, and the fourth influenza virus can be type A or B. In some embodiments, three of the four influenza viruses can be three subtypes of type A (i.e., A1, A2, and A3) and the fourth one can be type B. In some embodiments, three of the four influenza viruses can be three subtypes of type B (i.e., B1 B2, and B3) and the fourth one can be type A. In some embodiments, all four influenza viruses can be type A. In some embodiments, all four influenza viruses can be type B. In some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . In some embodiments, the first, second, third and fourth influenza virus are types (1) A1, A2, B1, and B2; (2) , A1, B1, A2, and B2; (3) B1, A1, B2, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses.
6.4.5 Targeted viruses
In some embodiments, the circRNAs disclosed herein encode HA/NA antigens from one, two, three, or four influenza viruses. The first, second, third and fourth influenza viruses each independently can be any influenza A virus or influenza B virus described herein or known in the art. In some embodiments, the first, second, third and fourth influenza viruses each independently can be a circulating virus. In some embodiments, circRNAs provided herein encode HA and/or NA antigens from a first influenza virus. The first influenza virus can be any influenza A virus or influenza B virus described herein or known in the art. The first influenza virus can be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, the influenza A virus is an H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) . In some embodiments, the influenza A virus is an H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) . In some embodiments, the influenza B virus is B/Victoria lineage (e.g., B/Austria/1359417/2021) . In some embodiments, the influenza B virus is B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
In some embodiments, circRNAs provided herein encode HA and/or NA antigens from a first influenza virus and a second influenza virus. In some embodiments, the second influenza virus can be any influenza A virus or influenza B virus described herein or known in the art. The second influenza virus can be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, the first and second influenza viruses are types (1) A1 and A2; (2) B1 and B2; (3) A and B; or (4) B and A, wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. In some embodiments, the first and second influenza viruses can be two subtypes of influenza A virus. In some embodiments, the first and second influenza virus are H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) , or vice versa. In some embodiments, the first and second influenza viruses can be two subtypes of influenza B virus. In some embodiments, the first and second influenza virus is from B/Victoria lineage (e.g., B/Austria/1359417/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) , or vice versa. In some embodiments, the first and second influenza viruses can be types A and B. For example, the first and second influenza viruses can be H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and B/Victoria lineage (e.g., B/Austria/1359417/2021) , or vice versa. In some embodiments, the first and second influenza viruses can be H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) , or vice versa. In some embodiments, the first and second influenza viruses can be H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) and B/Victoria lineage (e.g., B/Austria/1359417/2021) , or vice versa. In some embodiments, the first and second influenza viruses can be H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
In some embodiments, circRNAs provided herein encode HA and/or NA antigens from a first influenza virus, a second influenza virus and a third influenza virus. In some embodiments, the third influenza virus can be any influenza A virus or influenza B virus described herein or known in the art. The third influenza virus can be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, two of the three influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the third one can be type B. In some embodiments, two of the three influenza viruses can be two subtypes of type B (i.e., B1 and B2) and the third one can be type A. In some embodiments, all three influenza viruses can be type A. In some embodiments, all three influenza viruses can be type B. In some embodiments, the first, second and third influenza virus are types (1) A1, A2 and B; (2) A1, B, and A2; or (3) B, A1, and A2; (4) A, B1, and B2; (5) B1, A, and B2; or (6) B1, B2, and A; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second and third influenza viruses can be types A1, A2, and B. The first, second and third influenza viruses can be types A1, B, and A2. The first, second and third influenza viruses can be types B, A1, and A2. The first, second and third influenza viruses can be types A, B1, and B2. The first, second and third influenza viruses can be types B1, A, and B2. The first, second and third influenza viruses can be types B1, B2, and A. In some embodiments, two of the three influenza viruses can be two subtypes of influenza A viruses and the third one can be an influenza B virus. The two subtypes of influenza A viruses can be, for example, an H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and an H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) ; and the influenza B virus can be B/Victoria lineage (e.g., B/Austria/1359417/2021) or B/Yamagata lineage (e.g., B/Phuket/3073/2013) . In some embodiments, two of the three influenza viruses can be two subtypes of influenza B virus and the third one can be influenza A virus. The two subtypes of influenza B viruses can be, for example, a B/Victoria lineage (e.g., B/Austria/1359417/2021) and a B/Yamagata lineage (e.g., B/Phuket/3073/2013) , and the influenza A virus can be an H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) or an H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) ; and the influenza B virus can be B/Victoria lineage (e.g., B/Austria/1359417/2021) . In some embodiments, circRNAs provided herein encode HA and/or NA antigens from a first influenza virus, a second influenza virus, a third influenza virus and a fourth influenza virus. In some embodiments, the fourth influenza virus can be any influenza A virus or influenza B virus described herein or known in the art. The fourth influenza virus can be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . In some embodiments, three of the four influenza viruses can be three subtypes of type A (i.e., A1, A2, and A3) and the fourth one can be type B. In some embodiments, three of the four influenza viruses can be three subtypes of type B (i.e., B1, B2 and B3) and the fourth one can be type A. In some embodiments, all four influenza viruses can be type A. In some embodiments, all four influenza viruses can be type B. In some embodiments, the first, second, third and fourth influenza viruses are types (1) A1, A2, B1, and B2; (2) A1, B1, A2, and B2; (3) B1, A1, B1, and A2, or (4) B1, B2, A1, and A2; wherein A1 and A2 are the first and second subtypes of influenza A viruses; and B1 and B2 are the first and second subtypes of influenza B viruses. The first, second, third and fourth influenza viruses can be types A1, A2, B1 and B2. The first, second, third and fourth influenza viruses can be types A1, B1, A2, and B2. The first, second, third and fourth influenza viruses can be types B1, A1, B2, and A2. The first, second, third and fourth influenza viruses can be types B1, B2, A1, and A2. In some embodiments, two of the four influenza viruses can be two subtypes of type A (i.e., A1 and A2) and the other two can be two subtypes of type B (i.e., B1 and B2) . For example, in some embodiments, two subtypes of the influenza A viruses are an H1N1 virus (e.g., A/Wisconsin/588/2019, A/Sydney/5/2021, A/Victoria/4897/2022, A/Wisconsin/67/2022) and an H3N2 virus (e.g., A/Darwin/6/2021 or A/Darwin/6/2021) . In some embodiments, two subtypes of the influenza B virus can be B/Victoria lineage (e.g., B/Austria/1359417/2021) and B/Yamagata lineage (e.g., B/Phuket/3073/2013) .
6.4.6 Fusion Proteins
CircRNAs provided herein comprise a protein-coding (Z) sequence. In some embodiments, the Z sequence encodes an antigenic fusion protein. In other words, the encoded proteins can include two or more proteins (e.g., HA antigens and/or NA antigens) joined together. Alternatively, the protein to which an influenza antigen is fused does not promote a strong immune response to itself, but rather to the influenza virus antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein.
The circRNAs vaccines as provided herein, in some embodiments, encode fusion proteins that comprise HA and/or NA antigens linked to scaffold moieties. In some embodiments, such scaffold moieties impart desired properties to an antigen encoded by a nucleic acid of the disclosure. For example, scaffold proteins can improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner.
In some embodiments, the scaffold moiety is protein that can self-assemble into protein nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10-150 nm, a highly suitable size range for optimal interactions with various cells of the immune system. In some embodiments, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold moiety is a hepatitis B surface antigen (HBsAg) . HBsAg forms spherical particles with an average diameter of about 22 nm and which lacked nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58-68) . In some embodiments, the scaffold moiety is a hepatitis B core antigen (HBcAg) self-assembles into particles of 24-31 nm diameter, which resembled the viral cores obtained from HBV-infected human liver. HBcAg produced in self-assembles into two classes of differently sized nanoparticles of 300 A and 360 A diameter, corresponding to 180 or 240 protomers. In some embodiments, the HA and/or NA antigen disclosed herein is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the influenza virus antigen.
In some embodiments, bacterial protein platforms can be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.
Ferritin is a protein whose main function is intracellular iron storage. Ferritin is made of 24 subunits, each composed of a four-alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol. 2009; 390: 83-98) . Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003; 8: 105-111; Fawson D. M. et al. Nature. 1991; 349: 541-544) . Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Thus, the ferritin nanoparticle is well-suited to carry and expose antigens.
Fumazine synthase (FS) is also well-suited as a nanoparticle platform for antigen display. FS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S. E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014) . The FS monomer is 150 amino acids long and consists of beta-sheets along with tandem alpha-helices flanking its sides. A number of different quaternary structures have been reported for FS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 A diameter. Even FS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol. 2006; 362: 753-770) .
Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, may also be used as a platform to present antigens on the surface of self-assembling nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947) . Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated as a cellular compartment that encapsulates proteins such as DyP (Dye decolorizing peroxidase) and Flp (Ferritin like protein) , which are involved in oxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104) .
In some embodiments, circRNAs of the present disclosure encode an influenza virus antigen (e.g., HA and/or NA antigen) fused to a foldon domain. The foldon domain may be, for example, obtained from bacteriophage T4 fibritin (see, e.g., Tao Y, et al. Structure. 1997 Jun 15; 5 (6) : 789-98) .
CircRNAs provided herein comprise a protein-coding (Z) sequence. In some embodiments, the Z sequence also encodes a signal peptide fused to the HA and/or NA antigen. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide can also facilitate the targeting of the protein to the cell membrane.
A signal peptide can have a length of 15-60 amino acids. For example, a signal peptide may have a length of 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.
Signal peptides from heterologous genes (which regulate expression of genes other than influenza virus antigens in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure.
6.4.7 Sequence optimization
CircRNAs provided herein comprise a protein-coding (Z) sequence. In some embodiments, the Z sequence can be codon optimized. In some embodiments, the HA sequence and/or NA sequence is codon optimized. Codon optimization methods are known in the art. Codon optimization, in some embodiments, can be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase stability or reduce secondary structures; minimize tandem repeat codons or base runs that can impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites) ; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art -non-limiting examples include services from GeneArt (Life Technologies) , DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the Z sequence is optimized using optimization algorithms. In some embodiments, the HA sequence and/or NA sequence is optimized using optimization algorithms.
In some embodiments, an HA or NA sequence shares less than 95%sequence identity to a naturally-occurring or wild-type counterpart (e.g., a naturally-occurring or wild-type mRNA sequence encoding an HA antigen or an NA antigen) . The HA or NA sequence can share less than 90%sequence identity to a naturally-occurring or wild-type counterpart. The HA or NA sequence can share less than 85%sequence identity to a naturally-occurring or wild-type counterpart. The HA or NA sequence can share less than 80%sequence identity to a naturally-occurring or wild-type counterpart. The HA or NA sequence can share less than 75%sequence identity to a naturally-occurring or wild-type counterpart.
In some embodiments, a codon-optimized sequence encodes an HA or NA antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200%more) , than an HA or NA encoded by a non-codon-optimized sequence.
Exemplary optimized HA and NA sequences encoding HA and NA antigens disclosed herein are provided in Tables 6A, 9A and 10A (HA sequences) and 6B, 9B, and 10B (NA sequences) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182-223, 276-290 and 306-397.
In some embodiments, the circRNA has an HA sequence encoding Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 175) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182, 184-188, and 209-210. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 182. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 184. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 185. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 186. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 187. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 188. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 209. The HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 210.
In some embodiments, the circRNA has an HA sequence encoding Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein (e.g., SEQ ID NO: 177) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 183. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 189. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 190. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 191. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 192. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 211. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 212.
In some embodiments, the circRNA has an HA sequence encoding Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein (e.g., SEQ ID NO: 179) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 194. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 195. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 196. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 197. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 198. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 213. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 214. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 215. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 216. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 217. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 218. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 219.
In some embodiments, the circRNA has an HA sequence encoding Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein (e.g., SEQ ID NO: 180) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 199. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 200. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 201. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 202. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 203. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 220. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 221.
In some embodiments, the circRNA has an HA sequence encoding Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 235) . In some embodiments, the circRNA vaccines provided herein comprise an HA sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 281-285 and 520-521. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 281. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 282. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 283. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 284. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 285. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 520. In some embodiments, the HA sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 521.
6.4.8 Translation initiation (TI) sequences
Provided herein are immunogenic compositions comprising a circRNA, wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence.
In some embodiments, the translation initiation element (or TI) is an IRES, or an IRES-like sequence. As used interchangeably herein and understood in the art, the terms “internal ribosome entry site, ” “internal ribosome entry site sequence, ” “IRES” and “IRES sequence region” refer to cis elements of viral or human cellular RNAs (e.g., messenger RNA (mRNA) and/or circRNAs) that bypass the steps of canonical eukaryotic cap-dependent translation initiation. The canonical cap-dependent mechanism used by the vast majority of eukaryotic mRNAs requires an m7G cap at the 5’ end of the mRNA, initiator Met-tRNAmet, more than a dozen initiation factor proteins, directional scanning, and GTP hydrolysis to place a translationally competent ribosome at the start codon. IRESs attracts a ribosomal (e.g., eukaryotic ribosomal) to form translation initiation complex and promotes translation initiation. IRESs typically comprise a long and highly structured 5-UTR which mediates the translation initiation complex binding and catalyzes the formation of a functional ribosome.
A multitude of naturally occurring IRES sequences are available and include sequences derived from or isolated from a wide variety of viruses, such as from leader sequences of piconaviruses such as the encephalomyocarditis virus (EMCV) UTR, the polio leader sequence, the hepatitis A virus leader sequence, the hepatitis C virus IRES, human rhinovirus type 2 IRES, an IRES element from the foot and mouth disease virus, a giardiavirus IRES, and the like.
In some embodiments, the naturally occurring IRES sequence is isolated or derived from an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis A virus HA 16, hepatitis GB virus, foot and mouth disease virus, human enterovirus 71, equine rhinitis virus, ectrapis obliqua picoma-like virus, encephalomyocarditis virus, drosophila C virus, human coxsackievirus B3, crucifer tobamovirus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, Hibiscus chlorotic ringspot virus, classical swine fever virus, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobimavirus, HCV QC64, human cosavirus E/D, human cosavirus F, human cosavirus JMY, rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, salivirus A SHI, salivirus FHB, salivirus NG-J1, human parechovirus 1, crohivirus B, Yc-3, rosavirus M-7, shanbavirus A, pasivirus A, pasivirus A 2, echovirus E14, human parechovirus 5, Aichi virus, phopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, pegivirus A 1220, pasivirus A 3, sapelovirus, rosavirus B, bakunsa virus, tremovirus A, swine pasivirus 1, PLV-CHN, pasivirus A, sicinivirus, hepacivirus K, hepacivirus A, BVDV1, border disease virus, BVDV2, CSFV-PK15C, SF573 dicistravirus, Hubei picoma-like virus, CRPV, salivirus A BN5, salivirus A BN2, salivirus A 02394, salivirus A GUT, salivirus A CH, salivirus A SZ1, salivirus FHB, coxsackievirus (e.g., CVA3, CVA12, CVB1, CVB3, CVB5) , echovirus 7, enterovirus A71, and/or EV24.
In some embodiments, a natural IRES sequence is isolated or derived from a eukaryotic IRES element selected from human FGF2, human SFTPA1, human AML1/RUNX1, drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFl alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, drosophila reaper, canine Scamper, drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, drosophila hairless, S. cerevisiae TFIID, and S. cerevisiae YAP1.
In some embodiments, a naturally occurring sequence is an endogenous IRES sequence, which is derived from or isolated from homo sapiens. In some embodiments, a natural IRES sequence is an endogenous IRES sequence, which is derived from or isolated from human tissue or human sample.
In some embodiments, an IRES sequence is isolated or derived from a cellular IRES element selected from AML1/RUNX1, Antp-D, Antp-DE, Antp-CDE, ATlR varl, ATlR_var2, ATlR_var3, ATlR_var4, BAGl_p36delta236nt, BAGl_p36, BiP_-222_-3, C-IAP1 285-1399, c IAP1 13 13-1462, c-jun, Cat-l_224, CCND1, eIF4GI-ext, eIF4GII, eIF4GII-long, FGF1A, FMR1, Gtx-l33-l4l, Gtx-l-l66, Gtx-l-l20, Gtx-l-l96, HAP4, HIFla, hSNMl, HsplOl, hsp70, hsp70, Hsp90, IGF2_leader2, L-myc, MNT 75-267, MNT 36-160, MTG8a, MYB, MYT2 997-1 152, NRF_-653_-l7, NtHSFl, ODC1, p27kipl, p53_l28-269, PDGF2/c-sis, PITSLRE_p58, Rbm3 , reaper, Scamper, TFIID, TIF4631, Ubx_l-966, Ubx_373-96l, UNR, Ure2, XIAP 5-464, XIAP 305-466, YAP1, (GAAA) l6, (PPTl9) 4 and XI.
In some embodiments, an IRES sequence is isolated or derived from a viral IRES element selected from ABPV IGRpred, AEV, ALPV IGRpred, BQCV IGRpred, BVDV1 1-385, BVDV1 29-391, CrPV 5NCR, CrPV IGR, crTMV_IRESmp228, CSFV, DCV IGR, EoPV_5NTR, ERBV_l62-920, EV7l_l-748, FMDV type C, GBV-A, GBV-C, HAV HM175, HiPVJGRpred, HIV-l, HoCVlJGRpred, IAPVJGRpred, idefix, KBV IGRpred, PSIV IGR, PV typel Mahoney, PV_type3_Leon, REV-A, RhPV 5NCR, RhPV IGR , SINV l IGRpred, SV40 661-830, TMEV, TMV_UI_IRESmp228, TRV 5NTR, TrV IGR, TSV, and IGR.
In some embodiments, the IRES sequence comprises a sequence isolated or derived from a natural IRES sequence. The term “IRES-like sequence” or “Internal Ribosome Entry Site-like sequence” refers to non-naturally occurring nucleotide sequences that display a function of a naturally occurring IRES. In some embodiments, the IRES-like sequence can recruit ribosomal components to mediate cap-independent translation. An IRES-like sequence may be identified by methods known in the art, such as in PCT application No. PCT/CN2022/095949.
In some embodiments, the IRES-like sequence is greater than or equal to 3 nucleic acid residues in length. In some embodiments, the IRES-like sequence is 3-300 nucleic acid residues in length. In some embodiments, the IRES-like sequence is 3-200, 4-200, 5-200, 6-200, 7-200, 3-100, 4-100, 5-100, 6-100, 7-100, 3-50, 4-50, 5-50, 6-50, 7-50, 3-40, 4-40, 5-40, 6-40, 7-40, 3-30, 4-30, 5-30, 6-30, 7-30, 3-20, 4-20, 5-20, 6-20, 7-20 nucleic acid residues in length. In some embodiments, IRES-like sequence is 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleic acid residues in length. In some embodiments, the IRES-like sequence is 5-12 nucleic acid residues in length.
In some embodiments, the IRES-like sequence described herein or a fragment thereof can be combined with a naturally occurring IRES sequence described herein or a fragment thereof, to form additional IRES-like sequences. In some embodiments, a complete natural IRES sequence is combined with a complete IRES-like sequence. In some embodiments, a fragment of a natural IRES sequence is combined with a complete IRES-like sequence. In some embodiments, a complete natural IRES sequence is combined with a fragment of an IRES-like sequence. In some embodiments, a fragment of a natural IRES sequence is combined with a fragment of an IRES-like sequence.
Provided herein are immunogenic compositions comprising a circRNA, wherein the circRNA comprises, a translation initiation (TI) sequence and a protein-coding (Z) sequence. A person of ordinary skill in the art would understand that the circRNA vaccines disclosed herein are not limited to specific TI sequences, and any other TI sequences known in the art can be incorporated so long as they allow sufficient expression of the HA antigen and/or NA antigen.
In some embodiments, the TI sequence comprises an optimized IRES sequence. In some embodiments, the IRES sequence is optimized for enhanced efficiency in initiation translation. In some embodiments, a TI sequence drives the translation of more than antigen, and the IRES sequence is optimized for the efficient translation of all encoding sequences. For example, in some embodiments, the circRNA comprises an TI sequence and a Z1 sequence, wherein the Z1 sequence comprises two, three, or four HA sequences, each encoding an HA antigen, and the TI sequence comprises an IRES sequence that is optimized for efficient translation of all HA sequences, such that administration of the circRNA to a subject can induce sufficient immune response against all HA antigens.
In some embodiments, the TI sequence comprises an IRES listed in Table 11. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 490-497. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 490. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 491. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 492. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 493. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 494. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 495. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 496. In some embodiments, the IRES sequence can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to SEQ ID NO: 497.
6.4.9 linkers
In some embodiments, the circRNAs provided herein encode more than one functional domain or peptide linked together as a fusion protein. In some embodiments, the circRNAs provided herein further comprise a linker (L) sequence that encodes a linker (L) located between at least two or each domain of the fusion protein. In some embodiments, circRNAs provided herein comprise more than one antigen-encoding sequence (e.g., HA sequences and/or NA sequences) . In some embodiments, adjacent antigen-encoding sequences are linked by a linker sequence (L) .
The linker can be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE 6: el8556) . In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain.
Cleavable linkers known in the art can be used in connection with the disclosure. Exemplary linkers include: F2A linkers, T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750) . The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure) . The skilled artisan can likewise appreciate that other polycistronic constructs can be suitable for use as provided herein.
In some embodiments, the linker sequence comprises a 5’ UTR, 3’ UTR, poly-Asequence, polyA-C sequence, poly-C sequence, poly-U sequence, poly-G sequence, ribosome binding site, aptamer, riboswitch, ribozyme, small RNA binding site, translation regulation elements (e.g., a Kozak sequence) , a protein binding site (e.g., PTBP1 or HUR) a non-natural nucleotide, or a non-nucleotide chemical-linker sequence.
In some embodiments, the linker sequence comprises a 3’ UTR. In some embodiments, the 3’ UTR can be the 3’ UTR from human beta globin, human alpha globin xenopus beta globin, xenopus alpha globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFa, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic linsgspot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.
In some embodiments, the linker sequence comprises a 5’ UTR. In some embodiments, the 5’ UTR can be the 5’ UTR from human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or the adenovirus tripartite leader.
In some embodiments, the linker sequence comprises a polyA region. In some embodiments the polyA region is at least 30 nucleotides or at least 60 nucleotides in length.
6.4.10 Exemplary circRNAs
For exemplary purposes, Table 13 provides exemplary circRNA sequences. In some embodiments, the circRNA can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 499-519.
In some embodiments, the circRNAs provided herein encode Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 499-501. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 499. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 500. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 501.
In some embodiments, the circRNAs provided herein encode Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 502-505 and 519. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 502. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 503. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 504. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 505. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 519.
In some embodiments, the circRNAs provided herein encode Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 512-516. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 512. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 513. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 514. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 515. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 516.
In some embodiments, the circRNAs provided herein encode Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 506-511. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 506. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 507. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 508. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 509. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 510. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 511.
In some embodiments, the circRNAs provided herein encode Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein. In some embodiments, the circRNAs provided herein can have s at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequence of SEQ ID NO: 517. In some embodiments, the circRNAs provided herein can have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 518.
6.4.11 Modified nucleosides
In some embodiments, the circRNAs provided herein are not chemically modified and comprise the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, the circRNAs provided herein comprise nucleotides and/or nucleosides that are modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB 2017/051367 all of which are incorporated by reference herein.
As such, circRNAs disclosed herein can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. In some embodiments, circRNAs disclosed herein comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a circRNA contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
In some embodiments, modified circRNAs disclosed herein, introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified circRNAs comprising standard nucleosides.
In some embodiments, modified circRNAs disclosed herein, introduced into a cell or organism, exhibit reduced immunogenicity in the cell or organism (e.g., a reduced innate response) relative to an unmodified circRNA comprising standard nucleosides.
In some embodiments, circRNAs disclosed herein comprise non-natural modified nucleosides that are introduced during synthesis or post-synthesis to achieve desired functions or properties. The modifications can be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification can be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain.
In some embodiments, the circRNAs provided herein provided herein have modified RNA nucleotides and/or modified nucleosides. In some embodiments, the modified nucleoside is m5C (5-methylcytidine) . In another embodiment, the modified nucleoside is m5U (5-methyluridine) . In another embodiment, the modified nucleoside is m6A (N6-methyladenosine) . In another embodiment, the modified nucleoside is s2U (2-thiouridine) . In another embodiment, the modified nucleoside is Y (pseudouridine) . In another embodiment, the modified nucleoside is Um (2'-O-methyluridine) . In other embodiments, the modified nucleoside is m! A (1-methyladenosine) ; m2A (2-methyladenosine) ; Am (2’ -0-methyladenosine) ; ms2 m6A (2-methylthio-N6-methyladenosine) ; i6A (N6-isopentenyladenosine) ; ms2i6A (2-methylthio-N6 isopentenyladenosine) ; io6A (N6- (cis-hydroxyisopentenyl) adenosine) ; ms2io6A (2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine) ; g6A (N6-glycinylcarbamoyladenosine) ; t6A (N6-threonylcarbamoyladeno sine) ; ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine) ; m6t6A (N6-methyl-N6-threonylcarbamoyladenosine) ; hn6A (N6-hydroxynorvalylcarbamoyladenosine) ; ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine) ; Ar (p) (2’ -0-ribosyladenosine (phosphate) ) ; I (inosine) ; m1I (1-methylinosine) ; m1hn (1, 2’ -O-dimethylinosine) ; m3C (3-methylcytidine) ; Cm (2’ -0-methylcytidine) ; s2C (2-thiocytidine) ; ac4C (N4-acetylcytidine) ; (5-formylcytidine) ; m5Cm (5 , 2'-O-dimethylcytidine) ; ac4Cm (N4-acetyl-2’ -O-methylcytidine) ; k2C (lysidine) ; m! G (1-methylguanosine) ; m2G (N2-methylguanosine) ; m7G (7-methylguanosine) ; Gm (2'-0-methylguanosine) ; m2 2G (N2, N2-dimethylguanosine) ; m2Gm (N2, 2’ -O-dimethylguanosine) ; m2 aGm (N2, N2, 2’ -O-trimethylguanosine) ; Gr (p) (2’ -0-ribosylguanosine (phosphate) ) ; yW (wybutosine) ; oayW (peroxywybutosine) ; OHyW (hydroxy wybutosine) ; OHyW* (undermodified hydroxywybutosine) ; imG (wyosine) ; mimG (methylwyosine) ; Q (queuosine) ; oQ (epoxyqueuosine) ; galQ (galactosyl-queuosine) ; manQ (mannosyl-queuosine) ; preQo (7-cyano-7-deazaguanosine) ; preQi (7-aminomethyl-7-deazaguanosine) ; G+ (archaeosine) ; D (dihydrouridine) ; m5Um (5, 2’ -0-dimethyluridine) ; s4U (4-thiouridine) ; m5s2U (5-methyl-2-thiouridine) ; s2Um (2-thio-2’ -0-methyluridine) ; acp3U (3- (3-amino-3-carboxypropyl) uridine) ; ho5U (5-hydroxyuridine) ; mo5U (5-methoxyuridine) ; cmo5U (uridine 5-oxy acetic acid) ; mcmo5U (uridine 5-oxy acetic acid methyl ester) ; chm5U (5- (carboxyhydroxymethyl) uridine) ) ; mchm5U (5- (carboxyhydroxymethyl) uridine methyl ester) ; mcm5U (5-methoxycarbonylmethyluridine) ; mcm5Um (5-methoxycarbonylmethyl-2’ -0-methyluridine) ; mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine) ; nm5S2U (5-aminomethyl-2-thiouridine) ; mnm5U (5-methylaminomethyluridine) ; mnm5s2U (5-methylaminomethyl-2-thiouridine) ; mnm5se2U (5-methylaminomethyl-2-selenouridine) ; ncm5U (5-carbamoylmethyluridine) ; ncm5Um (5-carbamoylmethyl-2'-O-methyluridine) ; cmnm5U (5-carboxymethylaminomethyluridine) ; cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyluridine) ; cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine) ; m6 2A (N6, N6-dimethyladenosine) ; Im (2’ -0-methylinosine) ; m4C (N4-methylcytidine) ; m4Cm (N4, 2’ -0-dimethylcytidine) ; hm5C (5-hydraxymethylcytidine) ; m3U (3-methyluridine) ; cm5U (5-carboxymethyluridine) ; m6Am (N6, 2’ -O-dimethyladenosine) ; m6 2Am (N6, N6, 0-2’ -trimethyladenosine) ; m2, 7G (N2, 7-dimethylguanosine) ; m2, 2, 7G (N2, N2, 7-trimethylguanosine) ; m3Um (3, 2’ -0-dimethyluridine) ; m5D (5-methyldihydrouridine) ; f5Cm (5-formyl-2’ -0-methylcytidine) ; m'Gm (l, 2’ -0-dimethylguanosine) ; m'Am (l, 2’ -0-dimethyladenosine) ; rm 5U (5-taurinomethyluridine) ; τm5s2U (5-taurinomethyl-2-thiouridine) ) ; imG-14 (4-demethylwyosine) ; imG2 (isowyosine) ; or ac6A (N6-acetyladenosine) .
In some embodiments, the modified nucleoside can include a compound selected from the group of: pyridin-4-one ribonucleoside, 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-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-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, 4-m ethoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-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, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 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-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, 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. In another embodiment, the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine and 1-methylpseudouridine.
In some embodiments of the circ RNAs disclosed herein, at least one of the modified RNA nucleotide and/or modified nucleoside is 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2'fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6, 2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2,7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
The circRNAs of the present disclosure can be partially or fully modified along the entire length of the molecule. The circRNAs of the present disclosure can contain from about 1%to about 100%modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1%to 20%, from 1%to 25%, from 1%to 50%, from 1%to 60%, from 1%to 70%, from 1%to 80%, from 1%to 90%, from 1%to 95%, from 10%to 20%, from 10%to 25%, from 10%to 50%, from 10%to 60%, from 10%to 70%, from 10%to 80%, from 10%to 90%, from 10%to 95%, from 10%to 100%, from 20%to 25%, from 20%to 50%, from 20%to 60%, from 20%to 70%, from 20%to 80%, from 20%to 90%, from 20%to 95%, from 20%to 100%, from 50%to 60%, from 50%to 70%, from 50%to 80%, from 50%to 90%, from 50%to 95%, from 50%to 100%, from 70%to 80%, from 70%to 90%, from 70%to 95%, from 70%to 100%, from 80%to 90%, from 80%to 95%, from 80%to 100%, from 90%to 95%, from 90%to 100%, and from 95%to 100%) . In some embodiments, the circRNAs comprise at least 10%modified RNA nucleotides and/or modified nucleosides. It is understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.
In some embodiments, at least one of the modified RNA nucleotide and/or modified nucleoside is introduced at in vitro transcription (IVT) .
In some embodiments, vaccines with the modified circRNA require at least 10-fold less RNA polynucleotide than is required for an unmodified circRNA vaccine to produce an equivalent antibody titer.
Both the chemically modified and unmodified circRNA vaccines of the disclosure produce better immune responses than counterpart mRNA vaccines. In some embodiments, the circRNAs provided herein do not comprise a modified RNA nucleotide and/or modified nucleoside. In some embodiments, the circRNAs provided herein do not comprise 5-methylcytidine (m5C) , N6-methyladenosine (m6A) , 3, 2'-O-dimethyluridine (m4U) , 2-thiouridine (s2U) , 2'fluorouridine, pseudouridine, 2'-O-methyluridine (Um) , 2'deoxy uridine (2'dU) , 4-thiouridine (s4U) , 5-methyluridine (m5U) , 2'-O-methyladenosine (m6A) , N6, 2'-O-dimethyladenosine (m6Am) , N6, N6, 2'-O-trimethyladenosine (m62Am) , 2'-O-methylcytidine (Cm) , 7-methylguanosine (m7G) , 2'-O-methylguanosine (Gm) , N2, 7-dimethylguanosine (m2, 7G) , N2, N2, 7-trimethylguanosine (m2, 2, 7G) , and inosine (I) , Y (pseudouridine) , or m1A (1-methyladenosine) .
6.4.12 Preparation of circRNAs
Provided herein are immunogenic compositions comprising circRNA vaccines. CircRNAs are single-stranded RNAs that are joined head to tail, which can be prepared using methods known in the art. In some embodiments, methods of circRNA synthesis in vitro comprise ligating the ends of linear RNA precursor to produce a covalently closed circle. Linear RNA can be produced by chemical synthesis or enzymatic strategies (Müller S. (2017) , RNA Biol. 14, 1018-1027; Obi P. (2021) . Methods S1046-2023, 00065-00067) . The advantage of chemical synthesis is that the 5’ monophosphate can be directly introduced during the synthesis process for future cyclization. However, limited by the high cost of purification and low yield, chemical synthesis can only produce RNA of less than 50 to 70 nucleotides in length. Therefore, enzymatic strategy is the primary linear RNA synthesis method at present. Enzymatic strategy is usually realized through an in vitro transcription (IVT) reaction (Beckert B. (2011) . Methods Mol. Biol. 703, 29–41) , which includes DNA template, reaction buffer, and phage RNA polymerase. The phage RNA polymerase usually derives from the T7, SP6, or T3 bacteriophages, and T7 RNA polymerase is the most common phage RNA polymerase. IVT reaction allows for longer RNA synthesis at a lower cost. In some embodiments, mutated phage polymerases with improved transcription quality and reduced side reactions are used.
Circularization is achieved when the linear RNA precursor is ligated in vitro. The ligation methods include chemical ligation, enzymatic ligation, and ribozyme method. In some embodiments, the linear RNA precursors are circularized by chemical ligation using, for example cyanogen bromide (BrCN) or 1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide to link DNA-RNA hybrids (Sokolova et al., (1988) . FEBS Lett. 232, 153–155) .
In some embodiments, the linear RNA precursors are circularized by enzymatic ligations by catalytic reactions of several enzymes from the bacteriophage T4, including T4 DNA ligase (T4 Dnl) , T4 RNA ligase 1 (T4 Rnl 1) , and T4 RNA ligase 2 (T4 Rnl 2) . As a linear RNA precursor needs a 3’ -OH on the acceptor substrate and a 5’ monophosphate on the donor substrate for enzymatic ligation, if the linear RNA precursor is produced by chemical synthesis, a 5’ monophosphate can be incorporated during the synthesis or added after the synthesis using ATP and T4 polynucleotide kinase. If the linear RNA precursor is synthesized by IVT reaction, it usually starts with 5’ -pppG, and the 5’ -terminus needs to be dephosphorylated prior using calf intestinal (CIP) enzyme or other phosphatases. Then, 5’ monophosphate can be added using ATP and T4 polynucleotide kinase. In some embodiments, GMP is added to the IVT reaction mixture for phosphorylating the transcript at its 5’ end. In some embodiments, the linear RNA precursors are circularized by T4 Dnl ligation. T4 Dnl can help ligate double-stranded duplexes, such as DNA/RNA hybrids, and therefore requires a complementary DNA (cDNA) template or bridge to achieve RNA ligation. In some embodiments, the linear RNA precursors are circularized by T4 Rnl 1 ligation. T4 Rnl 1 catalyzes the nucleophilic attack of the 3’ -OH terminus onto the activated 5’ -terminus to form a covalent 5’ , 3’ -phosphodiester bond and produces circRNAs. In some embodiments, the linear RNA precursors are circularized by T4 Rnl 2 ligation. Similar to T4 Rnl 1, T4 Rnl 2 also catalyzes the nucleophilic attack of the 3’ -OH terminus onto the activated 5’-terminus to form a covalent 5’ , 3’ -phosphodiester bond. However, T4 Rnl 2 is much more active at joining nicks in double-stranded RNA (dsRNA) than at ligating the ends of ssRNA. T4 Rnl 2 is more suitable for linear RNA precursor with the ligation junction in a double-stranded region.
In some embodiments, the linear RNA precursors are circularized by enzymatic ligations by ribozyme methods. Modified group I intron self-splicing system can be used, which is also called permuted introns and exons (PIE) method (Wesselhoeft et al. (2018) . Nat. Commun. 9, 2629; Rausch et al. (2021) Nucleic Acids Res. 49, E35) . PIE method requires only the addition of GTP and Mg2+ as cofactors and shows great potential for protein synthesis. This method realized RNA ligation through a regular group I intron self-splicing reaction, including two transesterifications at defined splice sites. PIE method can be used for RNA cyclization in vitro and in vivo. Compared to chemical ligation and enzymatic ligation, PIE method could be applied for the cyclization of larger linear RNA precursor, and the reaction condition and purification method of PIE method is simpler.
Group II introns can also be used for circRNA synthesis, which involves an inverse splicing reaction (Mikheeva S. et al., (1997) . Nucleic Acids Res. 25, 5085–94) . This splicing reaction involves the joining of the 5’s plice site at the end of an exon to the 3’s plice site at the beginning of the same exon. Compared to the group I introns, all exon sequences are dispensable for group II intron-catalyzed inverse splicing. Therefore, this method can enable more accurate linear RNA precursor ligation. Methods of preparing circRNAs using Group II introns are described in PCT Application Nos. PCT/CN2022/095749, PCT/CN2022/095949, PCT/CN2022/104527, PCT/CN2022/129435, PCT/CN2022/134803, PCT/CN2022/135585, all hereby incorporated by reference in their entireties.
Hairpin ribozyme (HPR) can produce circRNAs through rolling circle reaction and the self-splicing reaction from circular single-strand DNA template (Dallas et al. (2008) . Nucleic Acids Res. 36, 6752-66; Petkovic and Müller (2013) FEBS Lett. 587, 2435-40) . The linear RNA precursor with HPR will fold into two alternative cleavage-active conformations to remove the 3’-end and the 5’ -end. As a result, the intermediate will contain a 5’ -OH and a 2’ , 3’ -cyclic phosphate to produce the target circRNA. In this method, small circRNAs can be produced from long repeating RNAs transcribed by RNA polymerase through a rolling circle mechanism in vitro.
For illustrative purposes, in some embodiments, circRNAs provided herein can be prepared using Group II intron self-splicing as follows. First, vectors containing nucleotide sequences encoding the precursor RNA are designed and cloned, which comprise the following operably linked elements from 5’ to 3’ : (1) a 3’ intron fragment; (2) a target sequence consisting of (i) a 3’ target sequence fragment and (ii) a 5’ target sequence fragment, from 5’ to 3’ ; and (3) a 5’ intron fragment; wherein the RNA has group II intron activity and, upon self-splicing, can form a circRNA that comprises both the 5’and 3’ target sequence fragments with the 3’ -end of the 5’ target sequence fragment linked to the 5’ -end of the 3’ target sequence fragment. In some embodiments, the vectors are DNA vectors.
CircRNAs prepared using Group II intron self-splicing can be scarless or near scarless. The terms “scar, ” as used herein, refers to the non-target sequence region in the circRNA splicing product. The term “scarless splicing” as used herein means that the construct self-splices to produce circRNA that do not contain additional sequence elements beyond the target sequence. As such, a “scarless” circRNA contains no scar, meaning that it solely consists of the target sequence. The term “near-scarless splicing” as used herein means that the construct self-splices to produce circRNA that include no more than 20 nucleotides besides the target sequence. A “near-scarless circRNA” is a circRNA resulting from “near-scarless” self-splicing, which include no more than 20 nucleotides besides the target sequence.
As such, the self-splicing of these RNAs produce circRNAs that do not contain additional sequence elements beyond the target sequence and is therefore referred to herein as “scarless” splicing. A circRNAs that solely consists of the target sequence is referred to herein as a “scarless” circRNA. The terms “scar, ” as used herein, refers to the non-target sequence region in the circRNA splicing product. As such, a “scarless” circRNA contains no scar. In some embodiments, the circRNAs provided herein are scarless. In some embodiments, the circRNAs provided herein are near scarless.
Many vectors can be used, including, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus (AAV) , herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5-DESTTM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. Exemplary AAV serotypes include AAV1, AAV2, AAV4, AAV5, AAV6, AAV9 AAV8, and AAV9. In some embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) . Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus.
Additionally, vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. “Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5'and 3'untranslated regions-which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters can be used.
Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) , ferritin H (FerH) , ferritin L (FerL) , Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70kDa protein 5 (HSPA5) , heat shock protein 90kDa beta, member 1 (HSP90B1) , heat shock protein 70kDa (HSP70) , β-kinesin (β-KIN) , the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 -1482 (2007) ) , a Ubiquitin C promoter (UBC) , a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.
Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.
The vectors provided herein can be made using standard techniques of molecular biology. For example, the various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving the polynucleotides from a vector known to include the same. The various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, e.g., Edge, Nature (1981) 292: 756; Nambair et al., Science (1984) 223 : 1299; and Jay et al., J. Biol. Chem. (1984) 259: 631 1.
Thus, particular nucleotide sequences can be obtained from vectors harboring the desired sequences or synthesized completely, or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. One method of obtaining nucleotide sequences encoding the desired vector elements is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88: 4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54: 75-82) , oligonucleotide directed mutagenesis of preexisting nucleotide regions (Riechmann et al., Nature (1988) 332: 323-327 and Verhoeyen et al., Science (1988) 239: 1534-1536) , and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used.
Second, the linear RNA precursors can be generated by incubating a vector provided herein under conditions permissive of transcription of the RNAs encoded by the vector. For example, in some embodiments, the linear RNA precursors provided herein can be synthesized by incubating a vector provided herein that comprises an RNA polymerase promoter upstream of its 5’ duplex forming region and/or expression sequence with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the vector is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase P. In some embodiments, the linear RNA precursors can be generated by performing in vitro transcription using a vector provided herein as a template (e.g., a vector provided herein with an RNA polymerase promoter positioned upstream of the 5’ homology region) .
Third, the linear RNA precursors, which have group II intron activity, can be used to generate circular RNA by self-splicing. In some embodiments, the linear RNA precursors are incubated under conditions suitable for circularization (self-splicing) . Self-splicing of group II introns needs to be accomplished under high-salinity conditions, and does not require the introduction of GTP. As such, in some embodiments, the buffer used in the self-splicing reaction comprises 10 mM to 100 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM divalent magnesium ions, such as MgCl2. The self-splicing buffer can also comprise 10 mM to 100 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM NaCl.
In some embodiments, the self-splicing reaction is performed in vitro for about 5 min to about 1 h, such as about 5 min, about 10 min, about 15 min, about 20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45 min, about 50 min, about 55 min, and about 1 h.
In some embodiments, the self-splicing reaction is performed at a temperature between 20 and 60 ℃, between 20 and 50 ℃, between 20 and 40 ℃, between 20 and 30 ℃, between 30 and 40 ℃, between 40 and 50 ℃, or between 50 and 60 ℃.
In some embodiments, the precursor RNAs are capable of achieving a circularization rate of at least 30%, such as a circularization rate of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%.
Fourth, the circRNAs prepared by self-splicing the linear RNA precursors are purified. Purification includes, but is not limited to, the removal of non-circularized linear RNAs, dsRNAs, and other unwanted components. In some embodiments, the circRNAs disclosed herein are purified before being transfected into cells. The phosphate groups at both ends of a linear RNA and some dsRNAs might activate the RIG-1 signaling pathway, and the immune response resulted from RIG-1 signaling can lead to the degradation of exogenous RNAs, thus affecting the function of circular RNAs.
For illustrative purposes, the methods of purification can include any of the following: enzymatic treatment; chromatography, including but not limited to affinity column chromatography, reversed-phase silica gel column liquid chromatography, gel filtration chromatography, and gel exclusion liquid chromatography; and electrophoresis, including but not limited to gel electrophoresis such as agarose gel electrophoresis, and capillary electrophoresis; and any combination thereof. Methods for removing linear RNAs, for example, include enzymatic treatment, such as treatment with RNase R; and chromatography, such as high performance liquid chromatography (HPLC) . Methods for removing terminal phosphate groups, for example, include treatment with alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIP) .
In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, a purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and/or nicked RNA than unpurified RNA.
In some embodiments, the linear RNA precursors provided herein comprise a purification tag that is removed during self-splicing, which can be used for negative selection of the circRNAs. Specifically, a sample containing the circRNAs, such as the product of splicing reaction starting from the tagged linear precursors, can be mixed with a probe that is immobilized on a solid surface, wherein the tag-containing precursors or any other tag-containing impurities can bind to the probe and be removed from the solution, resulting in a circRNA solution substantially free from the precursor, intron and any other tag-containing impurities. In some embodiments, the purification tag is a 15-40 nt polynucleotides. A purification matrix includes, for example, magnetic resin or beads, silicone resin, Sephadex resin, affinity resin, nanoparticles, and nanomaterial surface or coated surfaces.
As such, in some embodiments, the circRNAs prepared by methods disclosed herein can have a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circRNAs provided herein provided herein have a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circRNAs provided herein provided herein have a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNAs encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis.
In some embodiments, the circRNAs provided herein comprise one or more expression sequences and are configured for persistent expression in a cell of a subject in vivo. In some embodiments, the circRNAs is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point. In such embodiments, the expression of the one or more expression sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.
In some embodiments, the circRNAs provided herein provided herein have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circRNAs provided herein provided herein have a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail. In some embodiments, the circRNAs provided herein provided herein have higher stability than an equivalent linear mRNA. In some embodiments, this can be shown by measuring receptor presence and density in vitro or in vivoi post electroporation, with time points measured over 1 week. In some embodiments, this can be shown by measuring RNA presence via qPCR or ISH.
In some embodiments, the circRNAs disclosed herein can be of any length or size. In some embodiments the circRNA is between 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and/or 1500 and 5000 nucleotides in length.
In some embodiments, the circRNAs disclosed herein can be at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt in length. In some embodiments, the circRNA is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.
In some embodiments, circRNAs disclosed herein can be about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.
In some embodiments, circRNAs disclosed herein can be at least 500 nucleotides in length, at least 1000 nucleotides in length, or at least 1500 nucleotides in length.
The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley &Sons (1987 and annual updates) Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed. ) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds. ) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed. ) (1995) ; each of which is incorporated herein by reference in its entirety.
6.5 Immunogenic compositions
Provided herein are immunologic compositions containing the circRNAs disclosed herein. In some embodiments, the immunological compositions provided herein are for use in inducing an anti-influenza immune response in a subject. In some embodiments, the immunological compositions provided herein are for prevention or treatment of influenza virus in humans and other mammals. The term “immunogenic composition” as used herein and consistently understood in the art refers to a composition with a potential to induce an immune response to a substance. An “immune response” as used herein and consistently understood in the art refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble molecules produced by any of these cells or the liver (including for example, Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. This composition can include, but is not limited to, purified antigens, recombinant proteins, inactivated or attenuated pathogens, synthetic peptides, or nucleic acid sequences encoding antigenic proteins. In some embodiments, the immunogenic compositions provided herein comprise circRNAs encoding antigenic proteins, such as HA antigens and/or NA antigens. In some embodiments, an immunogenic composition can contain adjuvants, stabilizers, preservatives, and other excipients that enhance the immunogenicity of the antigen and improve the stability and delivery of the active substance with immunogenic potential. The primary purpose of an immunogenic composition is to stimulate the host’s immune system to recognize, respond to, and remember the pathogen or its components, thereby conferring protection against future infections.
In some embodiments, immunogenic compositions provided herein comprise at least two, at least three, at least four, at least five, or at least six of the circRNA described herein. In some embodiments, the immunogenic compositions provided herein can comprise at least two circRNAs disclosed herein. In some embodiments, the immunogenic compositions provided herein can comprise at least three circRNAs disclosed herein. In some embodiments, the immunogenic compositions provided herein can comprise at least four circRNAs disclosed herein. In some embodiments, the immunogenic compositions provided herein can comprise at least five circRNAs disclosed herein. In some embodiments, the immunogenic compositions provided herein can comprise at least six circRNAs disclosed herein.
In some embodiments, immunogenic compositions disclosed herein comprise at least two circRNAs, wherein the first circRNA comprises an HA sequence encoding an HA antigen, and the second circRNA comprises a NA sequence encoding a NA antigen. In some embodiments, the HA antigen and NA antigen are from the same influenza virus. In some embodiments, the HA antigen and NA antigen are from two influenza viruses. In some embodiments, the HA antigen and NA antigen are from two Type A influenza viruses. In some embodiments, the HA antigen and NA antigen are from two Type B influenza viruses. In some embodiments, the HA antigen and NA antigen are from a Type A influenza virus and a Type B influenza virus. In some embodiments, the two circRNAs each comprise an HA sequence encoding an HA antigen. In some embodiments, the two HA antigens are from two influenza viruses. In some embodiments, the two HA antigens are from two Type A influenza viruses. In some embodiments, the two HA antigens are from two Type B influenza viruses. In some embodiments, the two HA antigens are from a Type A influenza virus and a Type B influenza virus. In some embodiments, the two circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the two NA antigens are from two influenza viruses. In some embodiments, the two NA antigens are from two Type A influenza viruses. In some embodiments, the two NA antigens are from two Type B influenza viruses. In some embodiments, the two NA antigens are from a Type A influenza virus and a Type B influenza virus. The HA antigens and/or NA antigens can be any HA antigens and/or NA antigens disclosed herein or otherwise known in the art.
In some embodiments, immunogenic compositions disclosed herein comprise at least three circRNAs, wherein the first circRNA comprises an HA sequence encoding an HA antigen, and the other two circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the first circRNA comprises a NA sequence encoding a NA antigen, and the other two circRNAs each comprise an HA sequence encoding an HA antigen. In some embodiments, the three circRNAs each comprise an HA sequence encoding an HA antigen. In some embodiments, the three circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the three HA/NA antigens are from one influenza virus. In some embodiments, the three HA/NA antigens are from two influenza viruses. In some embodiments, the three HA/NA antigens are from three influenza viruses. In some embodiments, the three HA/NA antigens are from three Type A influenza viruses. In some embodiments, the three HA/NA antigens are from three Type B influenza viruses. In some embodiments, the three HA/NA antigens are from two Type A influenza viruses and one Type B influenza virus. In some embodiments, the three HA/NA antigens are from two Type B influenza viruses and one Type A influenza viruses.
In some embodiments, immunogenic compositions disclosed herein comprise at least four circRNAs, wherein the first circRNA comprises an HA sequence encoding an HA antigen, and the other three circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the first circRNA comprises a NA sequence encoding a NA antigen, and the other three circRNAs each comprise an HA sequence encoding an HA antigen. In some embodiments, two of the four circRNAs each comprise an HA sequence encoding an HA antigen, and the other two circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the four circRNAs each comprise an HA sequence encoding an HA antigen. In some embodiments, the four circRNAs each comprise a NA sequence encoding a NA antigen. In some embodiments, the four HA/NA antigens are from one influenza virus. In some embodiments, the four HA/NA antigens are from two influenza viruses. In some embodiments, the four HA/NA antigens are from three influenza viruses. In some embodiments, the four HA/NA antigens are from four influenza viruses. In some embodiments, the four HA/NA antigens are from four Type A influenza viruses. In some embodiments, the four HA/NA antigens are from four Type B influenza viruses. In some embodiments, the four HA/NA antigens are from three Type A influenza viruses and one Type B influenza virus. In some embodiments, the four HA/NA antigens are from three Type B influenza viruses and one Type A influenza viruses. In some embodiments, the four HA/NA antigens are from two Type B influenza viruses and two Type A influenza viruses.
The HA antigens and/or NA antigens can be any HA antigens and/or NA antigens described herein or otherwise known in the art.
In some embodiments, immunogenic compositions disclosed herein comprise at least four circRNAs, each comprise an HA sequence encoding an HA antigen. In some embodiments, the four circRNA comprise HA sequences encoding HA antigens from four influenza viruses. In some embodiments, the four influenza viruses comprise two influenza A viruses and two influenza B viruses.
In some embodiments, the viruses are seasonal viruses. In some embodiments, the HA antigens are selected based on GISRS standard.
For exemplary purposes, in some embodiments, the four influenza viruses are Influenza Type A/Wisconsin/588/2019 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) . In some embodiments, the four HA antigens are Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 175) , Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein (e.g., SEQ ID NO: 177) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein (e.g., SEQ ID NO: 179) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein (e.g., SEQ ID NO: 180) . In some embodiments, the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 175, 177, 179, and 180, respectively.
In some embodiments, the HA sequences that encode the Influenza Type A/Wisconsin/588/2019 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 175) . In some embodiments, the HA sequences that encode as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182, 184-188, and 209-210. In some embodiments, the HA sequences that encode Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein (e.g., SEQ ID NO: 177) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212. In some embodiments, the HA sequences that encode Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein (e.g., SEQ ID NO: 179) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219. In some embodiments, the HA sequences that encode Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein (e.g., SEQ ID NO: 180) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221. In some embodiments, the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 210, 211, 214, and 221, respectively. In some embodiments, the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 501, 519, 514, and 508, respectively.
In some embodiments, the four influenza viruses are Influenza Type A/Wisconsin/67/2022 (H1N1) , Influenza Type A/Darwin/6/2021 (H3N2) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) . In some embodiments, the four HA antigens are Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 235) , Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein (e.g., SEQ ID NO: 177) , Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein (e.g., SEQ ID NO: 179) , and Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein (e.g., SEQ ID NO: 180) . In some embodiments, the four HA antigens have amino acid sequences that have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs: 235, 177, 179, and 180, respectively.
In some embodiments, the HA sequences that encode the Influenza Type A/Wisconsin/67/2022 (H1N1) pdm09-like virus HA protein (e.g., SEQ ID NO: 235) . In some embodiments, the HA sequences that encode as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 281-285 and 520-521. In some embodiments, the HA sequences that encode Influenza Type A/Darwin/6/2021 (H3N2) -like virus HA protein (e.g., SEQ ID NO: 177) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212. In some embodiments, the HA sequences that encode Influenza Type B/Austria/1359417/2021 (B/Victoria lineage) -like virus HA protein (e.g., SEQ ID NO: 179) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219. In some embodiments, the HA sequences that encode Influenza Type B/Phuket/3073/2013 (B/Yamagata lineage) -like virus HA protein (e.g., SEQ ID NO: 180) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221. In some embodiments, the four HA sequences have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 521, 189, 196, and 221, respectively. In some embodiments, the four circRNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the nucleotide sequences of SEQ ID NOs: 521, 189, 196, and 221, respectively.
In some embodiments, the immunogenic compositions containing circRNAs as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject) . In some embodiments, the provided herein are pharmaceutical compositions that can be used as therapeutic or prophylactic agents. The term “pharmaceutical composition” as used herein refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier, ” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in REMINGTON'S PHARMACEUTICAL SCIENCES. Vaccine compositions can be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, can be found, for example, in Remington: THE SCIENCE AND PRACTICE OF PHARMACY 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety) .
The immunogenic compositions provided herein can further include other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound can be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) can be given after an earlier administration of the prophylactic composition.
The circRNA vaccines described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous) .
Provided herein are pharmaceutical compositions including circRNA vaccines described herein in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions provided herein can further comprise other components including, but not limited to, adjuvants. In some embodiments, the pharmaceutical compositions provided herein do not include an adjuvant (they are adjuvant free) .
In some embodiments, circRNA vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both.
Formulations of the vaccine compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., circRNAs) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single-or multi-dose unit. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition can comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
In some embodiments, a circRNA vaccine is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation) ; (4) alter the biodistribution (e.g., target to specific tissues or cell types) ; (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject) , hyaluronidase, nanoparticle mimics and combinations thereof.
A range of carrier systems have been described which encapsulate or complex RNA in order to facilitate RNA delivery and consequent expression of encoded antigens as compared to RNA which is not encapsulated or complexed. The present compositions can utilize any suitable carrier system. Particular carrier systems of note are further described below.
In embodiments, the circRNAs used herein are complexed, encapsulated, partially encapsulated, or associated with one or more lipids (e.g., cationic lipids and/or neutral lipids) , thereby forming lipid-based carriers such as liposomes, LNPs, lipoplexes, and/or nanoliposomes, suitably lipid nanoparticles.
In some embodiments, the circRNAs used herein are formulated in a LNP, either separately or together.
In some embodiments, the circRNAs used herein are formulated separately (in any formulation or complexation agent defined herein) , suitably wherein circRNAs used herein are formulated in separate liposomes, LNPs, lipoplexes, and/or nanoliposomes.
In some embodiments, the circRNAs used herein are co-formulated, i.e., formulated together.
The term “lipid nanoparticle” , also referred to as “LNP” , is not restricted to any particular morphology, and include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of a nucleic acid, e.g. an RNA. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP) .
LNPs are non-virion liposome particles in which RNA can be encapsulated. The incorporation of a nucleic acid into LNPs is also referred to herein as “encapsulation” wherein the nucleic acid, e.g. the RNA is contained within the interior space of the liposomes, LNPs, lipoplexes, and/or nanoliposomes. LNP delivery systems and methods for their preparation are known in the art.
In some embodiments, the circRNAs disclosed herein are complexed with one or more lipids thereby forming LNPs, liposomes, nanoliposomes, lipoplexes, suitably LNPs. In some embodiments, LNPs are suitable for intramuscular and/or intradermal administration.
In embodiments, at least about 80%, 85%, 90%, 95%of lipid-based carriers, suitably the LNPs, have a spherical morphology, suitably comprising a solid core or partially solid core.
LNPs typically comprise a cationic lipid and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g. PEGylated lipid) . The circRNAs can be encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or the entire lipid portion of the LNP. The circRNAs or a portion thereof can also be associated and complexed with the LNP. An LNP can comprise any lipid capable of forming a particle to which the nucleic acids are attached, or in which the one or more nucleic acids are encapsulated. In some embodiments, the LNP comprising circRNAs comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.
In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, and a cationic lipid.
LNP can, for example, be formed of a mixture of (i) a PEG-modified lipid (ii) a noncationic lipid (iii) a sterol (iv) an ionisable cationic lipid. Alternatively, LNP can for example be formed of a mixture of (i) a PEG-modified lipid (ii) a non-cationic lipid (iii) a sterol (iv) a non-ionisable cationic lipid.
In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the cationic lipid is ionizable.
In vivo characteristics and behavior of LNPs can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG) , to the LNP surface to confer steric stabilization. Furthermore, LNPs (or liposomes, nanoliposomes, lipoplexes) can be used for specific targeting by attaching ligands (e.g. antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (e.g. via PEGylated lipids or PEGylated cholesterol) .
In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” or “PEG-modified lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-s-DMG) and the like. The terms “PEGylated lipid” and “PEG-modified lipid” are used interchangeably herein.
In certain embodiments, the LNP comprises a stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated lipid) . Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20) , PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N- [ (methoxy poly (ethylene glycol) 2000) carbamyl] -1 , 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA) . In some embodiments, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG) . In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a PEGylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’ , 3’ -di (tetradecanoyloxy) propyl-1-O- (w-methoxy (polyethoxy) ethyl) butanedioate (PEG-5-DMG) , a PEGylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as w-methoxy (polyethoxy) ethyl-N- (2, 3di (tetradecanoxy) propyl) carbamate or 2, 3-di (tetradecanoxy) propyl-N- (w-methoxy (polyethoxy) ethyl) carbamate.
In some embodiments, the PEG-modified lipid comprises PEG-DMG or PEG-cDMA. In embodiments, the PEGylated lipid is suitably derived from formula (IV) of published PCT patent application W02018078053A1. Accordingly, PEGylated lipids derived from formula (IV) of published PCT patent application W02018078053A1, and the respective disclosure relating thereto, are herewith incorporated by reference.
Further examples of PEG-lipids suitable in that context are provided in WO2024068545A1, US20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.
In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based on the total moles of lipid in the LNP.
In embodiments, the LNP comprises one or more additional lipids, which stabilize the formation of particles during their formulation or during the manufacturing process (e.g. neutral lipid and/or one or more steroid or steroid analogue) .
In embodiments, the circRNA is complexed with one or more lipids thereby forming lipid nanoparticles, wherein the LNP comprises one or more neutral lipid and/or one or more steroid or steroid analogue. Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.
In some embodiments, the non-cationic lipid is a neutral lipid, such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1 , 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1 , 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or sphingomyelin (SM) , preferably the neutral lipid is DSPC.
In embodiments, the LNP (or liposome, nanoliposome, lipoplex) comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC) , dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) , dioleoyl-phosphatidylethanolamine (DOPE) , palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal) , dipalmitoyl phosphatidyl ethanolamine (DPPE) , dimyristoylphosphoethanolamine (DM PE) , distearoyl-phosphatidylethanolamine (DSPE) , 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE) , and 1 , 2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE) , or mixtures thereof.
The cationic lipid of an LNP can be ionizable, i.e., it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid but is progressively 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 assumes a positive charge on pH decrease.
Such cationic lipids (for liposomes, LNPs, lipoplexes, and/or nanoliposomes) include, but are not limited to, DSDMA, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC) , N, N-distearyl-N, N-dimethylammonium bromide (DDAB) , 1 , 2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride and 1 , 2-Dioleyloxy-3-trimethylaminopropane chloride salt) , N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA) , N, N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA) , ckk-E12, ckk, 1 , 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA) , 1 , 2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA) , 1 , 2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-DLenDMA) , 98N12-5, 1 , 2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP) , 1 , 2-Dilinoleyoxy-3- (dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2-Dilinoleyoxy-3-morpholinopropane (DLin-MA) , 1 , 2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP) , 1 , 2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA) , 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP) , 1 , 2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI) , ICE (Imidazol-based) , HGT5000, HGT5001 , 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. CI) , 1 , 2-Dilinoleyloxy-3- (N-methylpiperazino) propane (DLin-MPZ) , or 3- (N, N-Dilinoleylamino) -1 , 2-propanediol (DLinAP) , 3- (N, N-Dioleylamino) -1 , 2-propanedio (DOAP) , 1 , 2-Dilinoleyloxo-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DM A) , 2, 2-Dilinoleyl-4-dimethylaminomethyl- [1 , 3] -dioxolane (DLin-K-DMA) or analogs thereof, (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-di ( (9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3aH-cyclopenta [d] [1 , 3] dioxol-5-amine, (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yl-4- (dimethylamino) butanoate (MC3) , ALNY-100 ( (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-di ( (9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3aH-cyclopenta [d] [1 , 3] dioxol-5-amine) ) , 1 , 1’ - (2- (4- (2- ( (2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazanediyl) didodecan-2-ol (C12-200) , 2, 2-dili noleyl-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) -N , N 16-diundecyl-4, 7, 10, 13-tetraazahexadecane-l, 16-diamide) , (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-M-C3-DMA) , 3- ( (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yloxy) -N, N-dimethylpropan-1 -amine (MC3 Ether) , 4- ( (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6, 9, 28, 31-tetraen-19-yloxy) -N, N-dimethylbutan-1 -amine (MC4 Ether) , (commercially available cationic liposomes comprising DOTMA and 1 , 2-dioleoyl-sn-3phosphoethanolamine (DOPE) , from GIBCO/BRL, Grand Island, N. Y. ) ; (commercially available cationic liposomes comprising N- (1- (2, 3dioleyloxy) propyl) -N- (2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) , from GIBCO/BRL) ; and (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis. ) or any combination of any of the foregoing. Further suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO2010053572 (and particularly, Cl 2-200 described at paragraph [00225] ) and WO2012170930, both of which are incorporated herein by reference, HGT4003, HGT5000, HGTS001 , HGT5001 , HGT5002 (see US20150140070A1) .
In embodiments, the cationic lipid of the liposomes, LNPs, lipoplexes, and/or nanoliposomes can be an amino lipid.
Representative amino lipids include, but are not limited to, 1 , 2-dilinoleyoxy-3- (dimethylamino) acetoxypropane (DLin-DAC) , 1 , 2-dilinoleyoxy-3morpholinopropane (DLin-MA) , 1 , 2-dilinoleoyl-3-dimethylaminopropane (DLinDAP) , 1 , 2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA) , 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP) , 1 , 2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. CI) , 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. CI) , 1 , 2-dilinoleyloxy-3- (N-methylpiperazino) propane (DLin-MPZ) , 3- (N, Ndilinoleylamino) -1 , 2-propanediol (DLinAP) , 3-(N,N-dioleylamino) -1 , 2-propanediol (DOAP) , 1 , 2-dilinoleyloxo-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) .
In embodiments, the cationic lipid of the liposomes, LNPs, lipoplexes, and/or nanoliposomes can be an aminoalcohol lipidoid. Aminoalcohol lipidoids may be prepared by the methods described in U.S. Patent No. 8, 450, 298, herein incorporated by reference in its entirety. Suitable (ionizable) lipids can also be the compounds as disclosed in Tables 1, 2 and 3 and as defined in WO2017075531 A1, hereby incorporated by reference.
Other suitable (cationic or ionizable) lipids are disclosed in W02009086558, W02009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601 , WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871 , US20130064894, US20130129785, US20130150625, US20130178541 , US20130225836, US20140039032 and WO2017112865. In that context, the disclosures of W02009086558, W02009127060, W02010048536, W02010054406, W02010088537, W02010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601 , WO2016118724, WO2016118725, W02017070613, W02017070620, WO2017099823, W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No. US20100036115, US20120202871 , US20130064894, US20130129785, US20130150625, US20130178541 , US20130225836 and US20140039032 and WO2017112865 specifically relating to (cationic) lipids suitable for LNPs (or liposomes, nanoliposomes, lipoplexes) are incorporated herewith by reference.
In embodiments, amino or cationic lipids as defined herein have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4) , and neutral at a second pH, suitably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention. In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7. LNPs (or liposomes, nanoliposomes, lipoplexes) can comprise two or more (different) cationic lipids as defined herein. Cationic lipids may be selected to contribute to different advantageous properties. For example, cationic lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the LNP (or liposomes, nanoliposomes, lipoplexes) . In particular, the cationic lipids can be chosen so that the properties of the mixed-LNP are more desirable than the properties of a single-LNP of individual lipids.
In some embodiments, the LNP compositions can comprise an ionizable cationic lipid, a neutral lipid, a cholesterol, and a PEG. In some embodiments, the ionizable cationic lipid can be MC3, SM-102, ALC-0315, or E-1-0635. In some embodiments, the neutral lipid can be DSPC or DOPE. In some embodiments, the PEG can be DMG-PEG2000 or ALC-0159. The different components can be mixed at different ratios. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of about 50: about 10: about 40: about 2. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 50±10: 10±2: 40±5: 2±1. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 50: 10: 38.5: 1.5. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 46.3: 9.4: 42.7: 1.6. In some embodiments, the ionizable cationic lipid, neutral lipid, cholesterol, and PEG are mixed at molar ratios of 46.3: 12: 39.2: 2.5.
In some embodiments, the following LNP compositions with specific components and ratios are used for preparation of the immunogenic compositions containing circRNAs provided herein. The ratios are molar ratios.
WO2017/070620 provides general information on LNP compositions and is incorporated herein by reference. Other useful LNPs are described in the following references: WO2012/006376; WO2012/030901; WO2012/031046; W02012/031043; WO2012/006378; WO2011/076807; WO2013/033563; WO2013/006825; WO2014/136086; WO2015/095340; WO2015/095346; WO2016/037053, which are also incorporated herein by reference.
6.6 Administration
Provided herein are immunizing compositions (e.g., circRNA vaccines) , methods, kits and reagents for prevention and/or treatment of influenza virus infection in humans and other mammals. Immunizing compositions can be used as therapeutic or prophylactic agents. In some embodiments, immunizing compositions are used to provide prophylactic protection from influenza virus infection. In some embodiments, immunizing compositions are used to treat an influenza virus infection. In some embodiments, embodiments, immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.
In some embodiments, provided herein are methods for inducing an anti-influenza immune response in a subject comprising administering to the subject a therapeutically effective amount of the immunogenic composition disclosed herein. In some embodiments, the methods provided herein induce protective immunity against an influenza virus of the same subtype. In some embodiments, the methods provided herein induce cross protective immunity against a heterologous influenza virus.
A subject can be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject. In some embodiments, the subject is 60 years of age or older (e.g., 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 years of age or older) . In some embodiments, the subject is under 18 years of age (e.g., under 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years of age) . In some embodiments, the subject is 60 years of age or older. In some embodiments, the subject is under 18 years of age. In some embodiments, the subject is between the ages of about 20 years and about 60 years (e.g., about 20, 25, 30, 35, 40, 45, 50, 55 or 60 years old) .
As used herein, an “effective amount” or “therapeutically effective amount” of a circRNA vaccine refers to the amount of vaccine that can induce an antigen-specific immune response in subject (e.g., a mammalian subject, such as a human subject) when administered to the subject. As used herein, an immune response to a vaccine of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more) influenza virus protein (s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (CTLs) . CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+and CD8+ T-cells.
Thus, an “effective amount” or “therapeutically effective amount” of an immunogenic composition is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the circRNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides) , other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. Increased antigen production can be demonstrated by increased cell transfection (the percentage of cells transfected with the circRNA vaccine) , increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide) , or altered antigen specific immune response of the host cell.
The circRNA encoding the influenza virus antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject. A composition can be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of circRNA vaccines provided to a cell, a tissue or a subject can be an amount effective for immune prophylaxis.
Prophylactic protection from an influenza virus can be achieved following administration of an immunizing composition (e.g., a circRNA vaccine) of the present disclosure. Immunizing compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster) . It is possible, although less desirable, to administer an immunizing composition to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.
A method of eliciting an immune response in a subject against an influenza virus antigen (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject an immunogenic composition comprising a circRNA encoding an influenza virus antigen, thereby inducing in the subject an immune response specific to the influenza virus antigen, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.
A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than RNA vaccines. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) . For example, the traditional vaccines include Fluzone, FluMist, Fluvirin, Fluarix, Afluria, Flublok, Flucelvax, FluLaval, Agriflu, and Fluad.
In some embodiments, the immune response in the subject induced by the methods disclosed herein is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure.
In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the influenza virus or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the influenza virus or an unvaccinated subject.
In other embodiments, the ability to promote a robust T cell response (s) is measured using art recognized techniques.
In some embodiments of the methods disclosed herein, the immune response is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the influenza virus. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments of the methods disclosed herein, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
An immunogenic composition (e.g., a circRNA vaccine) disclosed herein can be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous, intravenous, or intraperitoneal administration. In some embodiments, the immunogenic compositions disclosed herein can be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art. In some embodiments, the immunogenic compositions disclosed herein can be administered intramuscularly. The immunogenic compositions disclosed herein can be administered intranasally. The immunogenic compositions disclosed herein can be administered intradermally.
The present disclosure provides methods comprising administering circRNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The circRNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the circRNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
In some embodiments, methods provided herein comprise a single administration. The effective amount of the circRNA, as provided herein, can be as low as 50 pg (total RNA) , administered for example as a single dose or as two 25 pg doses. A “dose” as used herein, represents the sum total of circRNA in the composition (e.g., including all of the NA antigens and/or HA antigens in the formulation) . In some embodiments, the effective amount is a total dose of 50 pg-300 pg, 100 pg -300 pg, 150 pg -300 pg, 200 pg -300 pg, 250 pg -300 pg, 150 pg -200 pg, 150 pg -250 pg, 150 pg -300 pg, 200 pg -250 pg, or 250 pg -300 pg. For example, the effective amount may be a total dose of 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 210 pg, 220 pg, 230 pg, 240 pg, 250 pg, 260 pg, 270 pg, 280 pg, 290 pg, or 300 pg. In some embodiments, the effective amount is a total dose of 66 pg. In some embodiments, the effective amount is a total dose of 67 pg. In some embodiments, the effective amount is a total dose of 68 pg. In some embodiments, the effective amount is a total dose of 132 pg. In some embodiments, the effective amount is a total dose of 133 pg. In some embodiments, the effective amount is a total dose of 134 pg. In some embodiments, the effective amount is a total dose of 266 pg. In some embodiments, the effective amount is a total dose of 267 pg. In some embodiments, the effective amount is a total dose of 268 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a total dose of 200 pg. In some embodiments, the effective amount is a total dose of 300 pg.
6.7 Efficacy
The efficacy of the circRNA vaccines disclosed herein can be measured by various methods known in the art (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ; 201 (11) : 1607-10) .
Sterilizing Immunity. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control. For example, the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.
Detectable Antigen. In some embodiments, the effective amount of an immunogenic composition of the present disclosure is sufficient to produce detectable levels of influenza virus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the immunogenic compositions disclosed herein produce detectable levels of influenza virus antigen as measured in serum of the subject for a sustained period of time. In some embodiments, detectable levels of influenza virus antigen can be measured in serum of the subject at least 1 week post administration. In some embodiments, detectable levels of influenza virus antigen can be measured in serum of the subject at least two weeks, at least three weeks or at least one month post administration. In some embodiments, the effective amount of an immunogenic composition of the present disclosure is sufficient to translate a biologically effective amount of the antigen from the circular polyribonucleotide in vivo in a cell of the subject over a period of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, the effective amount of an immunogenic composition of the present disclosure is sufficient to express a biologically effective amount of the antigen from the circular polyribonucleotide in vivo in a cell of the subject over a period of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, the effective amount of an immunogenic composition of the present disclosure is sufficient to translate a biologically effective amount of the antigen from the circular polyribonucleotide in vivo in the subject over a period of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, the effective amount of an immunogenic composition of the present disclosure is sufficient to express a biologically effective amount of the antigen from the circular polyribonucleotide in vivo in the subject over a period of at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, the “biologically effective amount” means the “effective amount” that is sufficient to produce or sustain the immune response against the antigen in the subject.
Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-influenza virus antigen) . Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.
In some embodiments, the antigen-specific immune response is characterized by measuring an anti-influenza virus antigen antibody titer produced in a subject administered an immunizing composition as provided herein.
In some embodiments, circRNAs of the disclosure produce prophylactically-and/or therapeutically efficacious levels, concentrations and/or titers of antigen-specific antibodies in the blood or serum of a vaccinated subject. As defined herein, the term “antibody titer” refers to the amount of antigen-specific antibody produced in a subject, e.g., a human subject. Accordingly, for example, the term “IgG titer” refers to the amount of antigen-specific IgG produced in a subject, e.g., a human subject. In some embodiments, antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA) . In exemplary embodiments, antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1: 40, 1: 100, etc.
In some embodiments, an efficacious circRNA vaccine produces an antibody titer of greater than 1: 40, greater that 1: 100, greater than 1: 400, greater than 1: 1000, greater than 1: 2000, greater than 1: 3000, greater than 1: 4000, greater than 1: 500, greater than 1: 6000, greater than 1: 7500, greater than 1: 10000. In some embodiments, the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In some embodiments, the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose. ) In some embodiments, antigen-specific antibodies are measured in units of μg/ml or are measured in units of IU/L (International Units per liter) or mlU/ml (milli International Units per ml) . In some embodiments, an efficacious vaccine produces >0.5 μg/ml, >0.1 μg/ml, >0.2 μg/ml, >0.35 μg/ml, >0.5 μg/ml, >1 μg/ml, >2 μg/ml, >5 μg/ml or >10 μg/ml. In some embodiments, an efficacious vaccine produces >10 mlU/ml, >20 mlU/ml, >50 mlU/ml, >100 mlU/ml, >200 mlU/ml, >500 mlU/ml or >1000 mlU/ml. In some embodiments, the antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination. In some embodiments, the level or concentration is produced or reached following a single dose of vaccine administered to the subject. In some embodiments, the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose) . In some embodiments, antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA) . In exemplary embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay.
In some embodiments, an HA ELISA assay is performed to examine the HA antibody titers resulting from administration of a candidate vaccine (e.g., IgG antibody titers) . In some embodiments, the circRNA vaccines provided herein induces at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-HA IgG titer in the subject as compared to that before the administration. In some embodiments, the circRNA vaccines provided herein induces at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-HA IgG titer in the subject as compared to control (e.g., PBS) . In some embodiments, the circRNA vaccines provided herein induce an HA IgG antibody titer that is 1-log, 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, or 10-log increased relative to pre-administration. In some embodiments, the circRNA vaccines provided herein induce an HA IgG antibody titer that is 1-log, 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, or 10-log increased relative to a control (e.g., PBS) . In some embodiments, the control comprises the HA-reactive IgG antibody titer in a subject prior to administration of the composition (e.g., vaccine) . In some embodiments, a candidate vaccine has an HA IgG antibody titer that is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold increased relative to a control vaccine. In some embodiments, the antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination.
In some embodiments, an NA ELISA assay is performed to examine the NA antibody titers resulting from administration of a candidate vaccine (e.g., IgG antibody titers) . In some embodiments, the circRNA vaccines provided herein induces at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-NA IgG titer in the subject as compared to that before the administration. In some embodiments, the circRNA vaccines provided herein induces at least a 50-fold, at least a 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold increase in anti-NA IgG titer in the subject as compared to control (e.g., PBS) . In some embodiments, the circRNA vaccines provided herein induce an NA IgG antibody titer that is 1-log, 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, or 10-log increased relative to pre-administration. In some embodiments, the circRNA vaccines provided herein induce an NA IgG antibody titer that is 1-log, 2-log, 3-log, 4-log, 5-log, 6-log, 7-log, 8-log, 9-log, or 10-log increased relative to a control (e.g., PBS) . In some embodiments, the control comprises the NA-reactive IgG antibody titer in a subject prior to administration of the composition (e.g., vaccine) . In some embodiments, a candidate vaccine has an NA IgG antibody titer that is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold increased relative to a control vaccine. In some embodiments, the antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination.
In some embodiments, an anti-influenza virus antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-influenza virus antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-influenza virus antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-influenza virus antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-influenza virus antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control. In some embodiments, the antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination.
In some embodiments, a geometric mean, which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.
In some embodiments, the immunogenic compositions disclosed herein induce a lasting immunologic response. In some embodiments, the increased antibody titer can be maintained for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, the antibody titer can be maintained for at least 2 months. In some embodiments, the antibody titer can be maintained for at least 3 months. In some embodiments, the antibody titer can be maintained for at least 5 months. In some embodiments, the antibody titer can be maintained for at least 1 year.
HAI/NAI assays: The HAI assay can also be used to measure the effectiveness of a candidate vaccine, such as those provided herein. The hemagglutination inhibition titer (HAI titer) was defined as the reciprocal of the highest serum dilution that inhibited hemagglutination. For example, if hemagglutination is not observed (complete inhibition) in the well with a 1: 80 dilution of serum, but hemagglutination is observed in the well with a 1: 160 dilution of serum (no inhibition) , the HAI titer of the serum against the tested virus is 80.
In some embodiments, the circRNA vaccines provided herein have an HAI titer that is 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold increased relative to a control (e.g., HAI titer from a subject administered a traditional seasonal flu vaccine, such as ) . The NAI assay can also be used to measure the effectiveness of a candidate vaccine, such as those provided herein. In some embodiments, the circRNA vaccines provided herein have an NAI titer that is 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold increased relative to a control.
In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 40, at least 80, at least 120, at least 160, at least 320, at least 640, or at least 1280 in the subject. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 40. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 80. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 120. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 160. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 320. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 640. In some embodiments, the immunogenic compositions described herein can produce an HAI titer of at least 1280. In some embodiments, the immunogenic compositions described herein can produce an HAI titer that is at least 3, at least 4, at least 5, or at least 6 times relative to a baseline HAI level in the subject.
In some embodiments, the immunogenic compositions disclosed herein induce a lasting immunologic response. In some embodiments, the increased HAI titer can be maintained for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, administration of immunogenic compositions disclosed herein produces an HAI titer of at least 40 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, administration of immunogenic compositions disclosed herein produces an HAI titer of at least 80 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, the HAI titer can be maintained for at least 2 months. In some embodiments, the HAI titer can be maintained for at least 3 months. In some embodiments, the HAI titer can be maintained for at least 5 months. In some embodiments, the HAI titer can be maintained for at least 1 year.
In some embodiments, the immunogenic compositions disclosed herein induce a lasting immunologic response. In some embodiments, the increased NAI titer can be maintained for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, administration of immunogenic compositions disclosed herein produces an NAI titer of at least 40 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, administration of immunogenic compositions disclosed herein produces an NAI titer of at least 80 for at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, or at least 2 years. In some embodiments, the NAI titer can be maintained for at least 2 months. In some embodiments, the NAI titer can be maintained for at least 3 months. In some embodiments, the NAI titer can be maintained for at least 5 months. In some embodiments, the NAI titer can be maintained for at least 1 year.
Neutralizing antibody:
In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the influenza virus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the influenza virus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the influenza virus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the neutralizing antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination.
In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50. In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL) . For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL. In some embodiments, the neutralizing antibody level or concentration is produced or reached by 7 days following vaccination, by 14 days following vaccination, by 21 days following vaccination, by 28 days following vaccination, by 35 days following vaccination, or by 42 or more days following vaccination.
In some embodiments, an antigen-specific immune response is measured as a ratio of geometric mean titer (GMT) , referred to as a geometric mean ratio (GMR) , of serum neutralizing antibody titers to influenza virus. A geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data.
A control, in some embodiments, is an anti-influenza virus antigen antibody titer produced in a subject who has not been administered an immunizing composition (e.g., circRNA vaccine) . In some embodiments, a control is an anti-influenza virus antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine. Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism.
In some embodiments, the ability of an immunizing composition (e.g., circRNA vaccine) to be effective is measured in a murine model. For example, an immunizing composition may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies can also be used to assess the efficacy of a vaccine of the present disclosure. For example, an immunizing composition may be administered to a murine model, the murine model challenged with virus, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response) ) .
A “standard of care, ” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose, ” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent influenza virus infection or a related condition, while following the standard of care guideline for treating or preventing influenza virus infection or a related condition.
In some embodiments, the anti-influenza virus antigen antibody titer produced in a subject administered an effective amount of an immunizing composition is equivalent to an anti influenza virus antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine.
A control may be, for example, an unvaccinated subject, or a subject administered a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit vaccine.
In some embodiments, the efficacy of the circRNA vaccines provided herein is significantly higher than a traditional vaccine or an mRNA vaccine. For example, in some embodiments, the circRNA vaccines provided herein can produce significantly higher antibody titer against the antigen (e.g., HA) than a traditional vaccine or an mRNA vaccine. In some embodiments, the circRNA vaccines provided herein can produce significantly higher HAI titer than a traditional vaccine or an mRNA vaccine. In some embodiments, the circRNA vaccines provided herein can produce significantly higher titer of neutralizing antibodies against the antigen (e.g., HA) than a traditional vaccine or an mRNA vaccine. In some embodiments, the circRNA vaccines provided herein can result in an immune response that lasts significantly longer than a traditional vaccine or an mRNA vaccine. For example, the traditional vaccines include Fluzone, FluMist, Fluvirin, Fluarix, Afluria, Flublok, Flucelvax, FluLaval, Agriflu, and Fluad.
In some embodiments, vaccine efficacy can also be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy can be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas: Efficacy = (ARU -ARV) /ARU x 100; and Efficacy = (1-RR) x 100.
Vaccine effectiveness can also be assessed by how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness = (1 -OR) x 100.
Statistical analyses are adopted to measure differences in efficacy between different treatment groups. When the data satisfies the conditions of a normal distribution, a T-test is employed to compare the means of two groups. For comparisons involving more than two groups, an analysis of variance (ANOVA) is used. When the data does not meet the criteria for normal distribution, non-parametric tests, such as the Wilcoxon rank sum test, are applied. As used herein to describe the extent of a difference, the term “significantly” or its grammatical equivalent means the difference is statistically significant.
6.8 Experimental
The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
6.8.1 Example 1. IRES-like sequence-based vectors
Coding sequences of an expression protein with IRES-like sequence are designed and used to construct circRNA vectors. RNAs are in vitro transcribed ( “IVT” ) from the XbaI digested linearized plasmid DNA template using T7 RiboMAXTM Large-Scale RNA Production System (PromegaTM, P1320) in the presence of unmodified NTPs. After DNase I treatment, the RNA products are column purified with RNA Clean and Concentrator Kit (ZYMO research, R1013) to remove excess NTP and other salt in IVT buffer, as well as the possible small RNA fragments generated during IVT. In some experiments, the purified RNA is further circularized in a new circularization buffer. The RNAs are first heated to 75℃ for 5 min and quickly cooled down to 45℃, after which a buffer including indicated magnesium and sodium is added to a final concentration: 50mM Tris-HCl at pH 7.5, 50/100mM NaCl, 0-40mM MgCl2, and is then heated at 53℃ for indicated time for circularization. The best optimized reaction condition including concentration of magnesium and sodium, and incubation time at 53℃, is selected for further experiments.
6.8.2 Example 2. Expression from the circRNA of the present disclosure
The expression of circular RNA products of different target sequences after transfection of cells is further tested. To minimize immunodegradation caused by linear RNAs, the RNA products are treated in the following three steps prior to transfection.
(1) RNase R treatment is performed to digest linear RNAs. The reaction conditions are at 37℃ for 30 min.
(2) CIP treatment is performed to remove phosphate groups at both ends of the linear RNAs. The reaction conditions are to add quick CIP (NEB) , and react at 37℃ for 30 min.
(3) HPLC purification is performed to remove small linear RNAs. HPLC conditions: gel exclusion chromatographic column: WatersBEH450A, column temperature: 40℃, flow rate: 1 min/ml, and elution conditions: 0 to 30 min, 100%buffer A (prepared with 10 mM Tris, 0.5 mM EDTA, and DEPC-treated water) .
The target RNAs are transfected using lipo RNAmaxunder the transfection conditions according to the supplier’s instructions, for 24 hours.
Using the construction methods described in the Examples above, different target sequences are tested. To facilitate detection of protein expression, constructs comprising a fluorescent protein coding sequence together with the IRES sequences disclosed herein are constructed. The addition of IRES may initiate cap-independent non-canonical translation, enabling the translation of coding sequences in circular RNAs into proteins.
For different target sequences, different methods are used to detect protein expression.
In the case that the translation product is GFP, the fluorescence is observed under a microscope. In addition, the cells is lysed with RIPA lysis solution (Beyotime) , and then the protein expression is detected by Western blotting. In the case that the translation product is luciferase, the cells are first lysed with Passive lysis solution (PromegaTM) and then the protein expression is detected by a microplate reader using a luciferase detection kit (PromegaTM) . It is confirmed that the expression of Gluc protein is obtained by the method of the present disclosure.
6.8.3 Example 3. Administration of circRNAs into mice
The circular RNA is encapsulated in a lipid nanoparticle via the NanoAssemblr Ignite system as previously described (Corbett K. S. et al., Nature 586, 567 (2020) , Polack F. P., et al., N. Engl. J. Med. 383, 2063 (2020) . In brief, an aqueous solution of circRNA at pH 4.0 is rapidly mixed with a lipid mixture dissolved in ethanol, which contains different ionizable cationic lipid, distearoylphosphatidylcholine (DSPC) , DMG-PEG2000, and cholesterol. The ratios for the lipid mixture are MC3: DSPC: Cholesterol: PEG-2000=50: 10: 38.5: 1.5 for formulation 1, SM-102: DSPC: Cholesterol: PEG-2000 = 50: 10: 38.5: 1.5 for formulation 2, and ALC-0315: DSPC: Cholesterol: ALC-0159 = 46.3: 9.4: 42.7: 1.6 for formulation 3. The resulting LNP mixture is then dialyzed against PBS and stored at -80 ℃ at a concentration of 0.5 μg/μl for further application.
Female BALB/C mice aged 8 weeks are purchased from Shanghai Model Organisms Center. 20 μg of CircRNA-LNPs in PBS are administrated into mice intramuscularly with 3/10 insulin syringes (BD biosciences) . The serum is collected 24 hours after the administration of the LNP, and 50 μl serum is used for Luciferase activity assay in vitro. Bioluminescence imaging is performed with an IVIS Spectrum (Roper Scientific) . 24 hours after Gluc-LNP injection, 2 mg/kg of Coelenterazine (MedChemExpress, MCE) is administrated to mice intraperitoneally. Mice are then anesthetized after receiving the substrate in a chamber with 2.5%isoflurane (RWD Life Science Co. ) and placed on the imaging platform while being maintained on 2%isoflurane via a nose cone. Mice are imaged 5 minutes post substrate injection with 30 seconds exposure time to ensure the signal are effectively and sufficiently acquired.
For immunogenicity studies, 8-week-old female BALB/c mice (Shanghai Model Organisms Center, lnc) are used. 10 μg of CircRNA-HA-LNP are diluted in 50 μl 1XPBS and intramuscularly administrated into the mice in the same hind leg for both prime and boost shots. Mice in the control groups receive PBS or empty LNPs. The blood samples are collected 2 weeks after prime and boost shots. Mice spleens are also harvested at the end point (2 weeks after boost) for immunostaining and flow cytometry.
6.8.4 Example 4. circRNAs mediate prolonged protein production
A major advantage of circular mRNAs is their superb stability because of lacking the free ends, therefore the circRNA should have good shelf life for protein expression compared to linear counterparts. To directly test this, both linear and circular mRNA encoding the Gaussia luciferase (Gluc) are synthesized and stored parallelly in pure water at room temperature for different days before being transfecting into 293 T cells. The activity of the circRNA to direct protein translation is tested in comparison to that of the linear mRNA. The prolonged protein translation is expected for the circRNA.
6.8.5 Example 5. Purified circRNAs direct robust translation of target proteins
The mRNA purity was found to be a key factor for the protein production and induction of innate immunity, as the removal of dsRNA by HPLC can eliminate immune activation and improves translation of linear nucleoside-modified mRNA (Kariko, K. et al., (2011) . To examine if the circRNAs produced as disclosed herein can induce innate immune response and cell toxicity, the circRNAs are purified with gel purification or HPLC and measured if the circRNAs can induce cellular immune response upon transfection of circRNAs by testing cell survival and production of RIG-I and IFN-B1 (interferon-β1) .
6.8.6 Example 6. In vitro evaluation of a linear mRNA or circular RNA for protein expression
An RNA encodes a reporter protein (e.g., a luciferase) is employed to determine the in vitro protein expression efficiency of the RNA encapsulated in an LNP. Exemplary expression RNAs selected from, but are not limited to, Tables 6A-6B or a nucleic acid sequence encodes an amino acid sequence selected from Tables 3, 4, 5A-5B. Exemplary circular RNAs include those described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; and PCT/CN2022/095949, filed May 30, 2022; the disclosure of each of which is incorporated herein by reference in its entirety.
Briefly, a luciferase-coding linear mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example 2. Cells are seeded into a 24-well plate and incubated with the LNP-encapsulated luciferase-coding mRNA or circular RNA at 37 ℃ for 24 h. The cell lysis and supernatant are separately collected and analyzed in a luminescence assay using theReporter Assay System.
6.8.7 Example 7. In vivo evaluation of a linear mRNA or circular RNA for protein expression
An RNA encodes a reporter protein (e.g., a luciferase) is employed to determine the in vivo protein expression efficiency of the RNA encapsulated in an LNP, according to the procedures as described in WO 2017/049245 A2 or WO 2018/081480 A1, the disclosure of each of which is incorporated herein by reference in its entirety. Exemplary expression RNAs selected from, but are not limited to, Tables 6A-6B or a nucleic acid sequence encodes an amino acid sequence selected from Tables 3, 4, 5A-5B. Exemplary circular RNAs include those described in PCT Appl. Nos. PCT/CN2020/077026, filed February 27, 2020; PCT/CN2022/095749, filed May 27, 2022; and PCT/CN2022/095949, filed May 30, 2022; the disclosure of each of which is incorporated herein by reference in its entirety.
Briefly, a luciferase-coding mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example 2. The LNP-encapsulated luciferase-coding mRNA or circular RNA is systemically administered to 6-8 week old female C57BL/6 mice by tail vein injection at predetermined doses. The mice are euthanized at predetermined time points (e.g., 4 h, 6 h, 12 h, …14 days) post-administration. The liver, spleen, brain, lung, heart, kidney, pancreas, and muscle tissues of the mice are collected, immediately snap frozen in liquid nitrogen, and stored at -80 ℃ for analysis. The collected tissues of each type are homogenized. The homogenate is analyzed using the STEADY-GLO luciferase assay system according to the manufacturer’s protocol. The amount of proteins assayed is determined using a BCA protein assay kit. Relative luminescence units (RLU) can be normalized to total μg protein assayed and compared with a standard curve.
Briefly, a luciferase-coding mRNA or circular RNA is encapsulated in an LNP comprising a compound provided herein as described in Example B-1. The LNP-encapsulated luciferase-coding mRNA or circular RNA is systemically administered to 6-8 week old female C57BL/6 mice by tail vein injection at predetermined doses. The mice are euthanized at predetermined time points (e.g., 4h, 6h, 12h, …14 days) post-administration and placed on an imaging platform while being maintained on isoflurane via a nose cone. Bioluminescence imaging is performed with an IVIS Spectrum (Roper Scientific) . Mice are imaged 5 minutes post injection with 30 seconds exposure time to ensure the signal is effectively and sufficiently acquired.
6.8.8 Example 8. Immunogenicity evaluation of a linear mRNA or circular RNA
Immunogenicity is assessed in six-week-old female BALB/cJ, C57BL/6J, and/or B6C3F1/J mice by two intramuscular immunizations with a linear mRNA or circular RNA LNP at predetermined doses (e.g., 0.01, 0.1, or 1 μg) , separated by 2-week interval or 3-week interval. Sera are collected 2 weeks post-prime and 2 weeks post-boost and assessed for a target-specific IgG by an enzyme-linked immunosorbent assay (ELISA) . Mouse spleens are also harvested at the end point (2 weeks after boost or 3 weeks after boost) for immunostaining and flow cytometry. Time points are compared within each dose level by statistical method (e.g., two-sided Wilcoxon signed-rank test) , and doses are compared post-boost (via e.g., Kruskal–Wallis ANOVA with Dunn’s multiple comparisons test) .
6.8.9 Example 9. Immunization with a linear mRNA or circular RNA
Protective immunity is assessed in young adult BALB/cJ mice challenged with a linear mRNA or circular RNA LNP at predetermined doses. Mononuclear single-cell suspensions from each immunized mouse are generated using RWD tissue dissociator, followed by 70 μM filtration and density gradient centrifugation using the FICO/LITE-LM medium. Spleenocytes are cryopreserved for further analysis. Before a T cell flow cytometry analysis, spleenocytes are cultured in R10 media and incubated for 6 h at 37 ℃ with an mRNA or circular RNA LNP. The cells are then washed before staining with a ZOMBIE NIRTM solution. The cells are washed and permeabilized using a BD CYTOFIX/CYTOPERM fixation/permeabilization solution according to the manufacturer’s instructions. The cells are washed in a permeabilization/wash solution and resuspended in FC BLOCK before staining with an antibody cocktail containing one or more antibodies selected from the following: I-A/I-E PE, CD4 BV421, CD8a BUV510, CD44 PE-Cy7, CD62L BB515, CD3e AF700, IFN-γ PE/Dazzle, TNF BB700, IL-2 BV605, IL-4 BV650, and IL-5 APC and in 1 × permeabilization/wash solution diluted with a brilliant stain buffer. The cells are washed in permeabilization/wash solution and resuspended in the FC buffer (PBS supplemented with 2%heat-inactivated FBS and 0.05%NaN3) before running on a full spectrum flow cytometry system. Analysis is performed using FLOWJO software.
6.8.10 Example 10. Preclinical Demonstration of Immunogenicity by CircRNA Vaccines against A (H1N1) , A (H3N2) , B/Yamagata, and B/Victoria Influenza Viruses
Table 15. The sequence list of constructs
0A: ALC-0315: DSPC: Cholesterol: ALC-0159 = 46.3: 9.4: 42.7: 1.6 for formulation 3
1A: E-1-0635: DSPC: CHOL: PEG-2000=46.3: 12: 39.2: 2.5 for formulation 4
1A-O: E-1-0635: DOPE: CHOL: PEG-2000=46.3: 12: 39.2: 2.5 for formulation 5 (1A-O)
1A-S: E-1-0635: DSPC: CHOL: PEG-2000=46.3: 12: 39.2: 2.5 for formulation 4
0A-S: ALC-0315: DSPC: Cholesterol: ALC-0159 = 46.3: 9.4: 42.7: 1.6 for formulation 3
The circular RNA was encapsulated in a lipid nanoparticle via the NanoAssemblr Ignite system as previously described (Corbett K. S. et al., Nature 586, 567 (2020) , Polack F. P., et al., N. Engl. J. Med. 383, 2063 (2020) . In brief, an aqueous solution of circRNA at pH 4.0 was rapidly mixed with a lipid mixture dissolved in ethanol, which contains different ionizable cationic lipid, distearoylphosphatidylcholine (DSPC) , DMG-PEG2000, and cholesterol. The ratios for the lipid mixture were MC3: DSPC: Cholesterol: PEG-2000=50: 10: 38.5: 1.5 for formulation 1, SM-102: DSPC: Cholesterol: PEG-2000 = 50: 10: 38.5: 1.5 for formulation 2, ALC-0315: DSPC: Cholesterol: ALC-0159 = 46.3: 9.4: 42.7: 1.6 for formulation 3, E-1-0635: DSPC: CHOL: PEG-2000=46.3: 12: 39.2: 2.5 for formulation 4 and E-1-0635: DOPE: CHOL: PEG-2000=46.3: 12: 39.2: 2.5 for formulation 5.
The resulting LNP mixture with different formulations were dialyzed against PBS (pH 7.4) in dialysis cassettes for at least 18 hr. The resulting LNP mixture with different formulations were concentrated using Amicon ultra centrifugal filters (EMD Millipore) , passed through a 0ul. 22-μm filter, and stored at 4℃ until use. The resulting LNP mixture with different formulations were tested for CircRNA concentration, particle size, PDI, RNA encapsulation, endotoxin, and amount of trace ethanol and they were found to be between 400ng/μl-600ng/μl in RNA concentration, between 80 and 100 nm in size, with 0.3in PDI, with ≥ 75%encapsulation, with ≤0.5%trace amount of ethanol, and ≤20 EU/mL endotoxin (results not shown) .
All formulations were further tested for pH, osmolarity, CircRNA dimer and dsRNA. The pH was between 6.5-8.5. The osmolarity was ≤600 mOsmol/kg. The amount of cirRNA dimer was ≤1.0 %and the amount of dsRNA was ≤0.5%. The resulting LNP mixture was then stored at -80 ℃ at a concentration of 0.5 μg/μl for further application.
Female BALB/C mice aged 8 weeks were purchased from Shanghai Model Organisms Center. CircRNA Vaccines containing 1, 5 and 25μg of CircRNA in PBS were administrated into mice intramuscularly with 3/10 insulin syringes (BD biosciences) . See details in Table 15 and Table 16 and FIGs. 15-22.
Mice in the negative control groups received PBS or empty LNPs. Mice in the positive control groups received a commercially available influenza vaccine, with 5μg HA per strain.
The commercially available vaccine was selected from the following:
Flulaval Qualdrivalent (Flulaval) from ID Biomedical Corporation of Qucbec, 0.5 ml/vial, 60mcg HA total, 15 mcg HA H1N1, 15 mcg HA H3N2, 15 mcg HA B/ (Victoria lineage) , 15 mcg HA B/ (Yamagata lineage)
Fluarix Qualdrivalent (Fluarix) from GSK biologicals, 0.5 ml/vial, 60mcg HA total, 15 mcg HA H1N1, 15 mcg HA H3N2, 15 mcg HA B/ (Victoria lineage) , 15 mcg HA B/ (Yamagata lineage)
Fluzone Qualdrivalent (Fluzone) from Sanofi Pasteur Inc, 0.5 ml/vial, 60mcg HA total, 15 mcg HA H1N1, 15 mcg HA H3N2, 15 mcg HA B/ (Victoria lineage) , 15 mcg HA B/ (Yamagata lineage)
Afluria Qualdrivalent (Afluria) from Seqirus Pty Ltd, 0.5 ml/vial, 60mcg HA total, 15 mcg HA H1N1, 15 mcg HA H3N2, 15 mcg HA B/ (Victoria lineage) , 15 mcg HA B/ (Yamagata lineage)
Fluad Qualdrivalent (Fluad) from Seqirus Inc, 0.5 ml/vial, 60mcg HA total, 15 mcg HA H1N1, 15 mcg HA H3N2, 15 mcg HA B/ (Victoria lineage) , 15 mcg HA B/ (Yamagata lineage)
Fluzone-high Qualdrivalent (Fluzone-high) from Sanofi-Pasteur Inc, 0.7 ml/vial, 240 mcg HA total, 60 mcg HA H1N1, 60 mcg HA H3N2, 60 mcg HA B/ (Victoria lineage) , 60 mcg HA B/ (Yamagata lineage)
Four groups of strain A (H1N1) , A (H3N2) , B/Yamagata and B/Victoria were tested in the experiments. See detailed experiments and results in Section 6.8.11-6.8.14.
HAI assay: For the serological testing, detection, and identification of antibodies against influenza/avian influenza, hemagglutination inhibition (HI) tests were adopted and the following procedures were followed:
1) The serum samples were mixed with receptor-destroying enzyme (RDE) , incubated at 37℃ for 16-18 hours and then inactivated at 56℃ for 30 minutes. 1 volume of concentrated red blood cells was added to 20 volumes of the treated serum, mixed well, and placed at 4℃ for 1 hour. The mixture was then centrifuged at 1200 x g for 10 minutes, and the supernatant was collected. (2) 4 HA units of antigen for the hemagglutination inhibition (HI) test were prepared. “One HA unit” referred to the amount of virus that could cause hemagglutination of an equal volume of standardized red blood cells. The total amount of virus antigen required for the HI test was first calculated, and then the antigen was prepared. The freshly prepared 4 HA unit antigen was also re-titrated to ensure consistent and accurate antigen use in the HI test. 3) 25 μL of saline was added to each well in all but the first row on a horizontally placed microplate. 4) 25 μL serum was taken from each well of the first row, and a twofold serial dilution was performed from the first to the last row. 25 μL liquid was discarded from each well of the last row. 5) 25 μL of freshly prepared 4 HA units of antigen was added to each well of the HI test plate. 6) 25 μL of saline was added to each well of the serum control plate. 7) The microplate was gently tapped to mix the antigen and antibody thoroughly. The mixture was incubated at room temperature for 60 minutes. 8) 25 μL of 1%red blood cell solution was added to each well of the HI test plate. The microplate was gently tapped to mix thoroughly. 9) The mixture was incubated at room temperature for 30-60 minutes, and the results were observed.
The hemagglutination inhibition (HI) titer was defined as the reciprocal of the highest serum dilution that inhibited hemagglutination. For example, if hemagglutination was not observed (complete inhibition) in the well with a 1: 80 dilution of serum, but hemagglutination was observed in the well with a 1: 160 dilution of serum (no inhibition) , the HI titer of the serum against the tested virus was 80.
Binding IgG Titer: The following procedures were followed to determine the titer.
1. Coating: the HA antigen was prepared into a 2 μg/mL coating solution using coating buffer. 100 μL/well of the coating solution was added to a CORNING high-binding polystyrene 96-well plate. The plate was incubated at 2-8℃ for 16-18 hours. 2. Blocking: The coated plate was washed and dried with absorbent paper. 200 μL/well 5%skim milk was added. The plate was incubated at room temperature for 2 hours. 3. Sample treatment: The samples were diluted 200, 600, 1800, 5400, 48600, 145800, and 437400 times using 5%skim milk to prepare the corresponding pre-treated samples. 100 μL/well of the pre-treated samples were added. The plate was incubated at room temperature for 1.5 hours. 4. Secondary antibody incubation: The plate was again washed and dried on absorbent paper. 100 μL/well of the secondary antibody solution (dilution ratio 1: 5000) was added, and the plate was incubated at 37℃ in the dark for 0.5 hours. 5. Color development and detection: The plate was again washed and dried on absorbent paper 100 μL/well of TMB substrate solution was added, and the plate was incubated at room temperature in the dark for 6 minutes. 100 μL/well of stop solution was added to terminate the reaction. The absorbance of the 96-well plate was read at 450 nm using a microplate reader.
6.8.11 Example 11. Preclinical Demonstration of Immunogenicity by CircRNA-PD001
In terms of H1N1, a single dose of CircRNA vaccine containing 1, 5 or 25 μg of Construct A26B-0A was administered intramuscularly with 3/10 insulin syringes (BD biosciences) to the Balb/c mice (8 in each group) separately, which were labelled as G2-G4, and a negative control group G1 was also set up for comparison. Similarly, a single dose of CircRNA vaccine containing of 1, 5 or 25 μg A26A-0A, A26B-0A or A26C-0A was administered intramuscularly to the Balb/c mice, see Tables 16. Mice in control groups received either PBS buffer or empty LNPs.
Table 16: CircRNA-PD001
Note: LNP*control group received the amount of LNP equal to 25μg group
Binding IgG for CircRNA-PD001
The procedures described in Example 11 were followed to measure the binding IgG titer for CircRNA-PD001. The results were presented in FIG. 15.
HAI assay (H1N1) for CircRNA-PD001
The procedures described in Example 11 were followed to measure the HAI titer for CircRNA-PD001 (H1N1) . The results of the HAI assay were presented in FIG. 16 and compared to the IgG titer.
6.8.12 Example 12. Preclinical Demonstration of Immunogenicity by CircRNA-PD002
Table 17.
CircRNA-PD002 experiment design
Binding IgG for CircRNA-PD002 (H3N2)
The procedures described in Example 12 were followed to measure the binding IgG titer for CircRNA-PD002. The results of this test were presented in FIG. 17.
HAI assay (H3N2) for CircRNA-PD002 (H3N2)
The procedures described in Example 12 were followed to measure the HAI titer for CircRNA-PD002 (H3N2) . The results of the HAI assay were presented in FIG. 18 and compared to the IgG titer.
6.8.13 Example 13. Preclinical Demonstration of Immunogenicity by CircRNA-PD003
Table 18 CircRNA-PD003 experiment design
Binding IgG for CircRNA-PD003 (Yamagata)
The procedures described in Example 13 were followed to measure the binding IgG titer for CircRNA-PD003. The results of this test were presented in FIG. 19.
HAI assay (Yamagata) for CircRNA-PD003
The procedures described in Example 13 were followed to measure the HAI titer for CircRNA-PD003 (Yamagata) . The results of the HAI assay were presented in FIG. 20 and compared to the IgG titer.
6.8.14 Example 14. Preclinical Demonstration of Immunogenicity by CircRNA-PD004
Table 19 CircRNA-PD004 experiment design
Binding IgG for CircRNA-PD004 (Victoria)
The procedures described in Example 14 were followed to measure the binding IgG titer for CircRNA-PD004. The results of this test were presented in FIG. 21.
HAI assay (Victoria) for CircRNA-PD004
The procedures described in Example 14 were followed to measure the HAI titer for CircRNA-PD004 (Victoria) . The results of the HAI assay were presented in FIG. 22 and compared to the IgG titer.
6.8.15 Example 15. Preclinical Demonstration of Immunogenicity by CircRNA-PD005
Table 20: CircRNA-PD005 CircRNA Vaccines constructs against A (H1N1) , A (H3N2) , B/Yamagata, and B/Victoria Influenza Viruses
Table 21 CircRNA-PD005 experiment design
*Note: There were not enough animals provided for Group G9 so only 5 were administered in actual.
Binding IgG for CircRNA-PD005 (H1N1, H3N2, Yamagata, and Victoria)
The procedures described in Example 15 were followed to measure the binding IgG titer for CircRNA-PD005. The results of this test were presented in FIGs. 23 (H1N1) , 24 (H3N2) , 25 (Yamagata) and 26 (Victoria) .
HAI assay (Victoria) for CircRNA-PD005
The procedures described in Example 15 were followed to measure the HAI titer for CircRNA-PD005 (H1N1, H3N2, Yamagata, and Victoria) .
6.8.16 Example 16. Preclinical Demonstration of Immunogenicity by CircRNA-PD006
Table 22 CircRNA-PD006 experiment design
Table 23 CircRNA-PD006 experiment design-H1N1
注:*LNP equals to RNA 5μg
Binding IgG for CircRNA-PD006 (H1N1)
The procedures described in Example 16 were followed to measure the binding IgG titerfor CircRNA-PD006 (H1N1) . The results of this test were presented in FIG.27.
HAI assay (H1N1) for CircRNA-PD006
The procedures described in Example 16 were followed to measure the HAI titer forCircRNA-PD006 (H1N1) . The results of the HAI assay were presented in FIG.28 and comparedto the IgG titer.
Table 25 CircRNA-PD006 experiment design-Yamagata
Binding IgG for CircRNA-PD006 (Yamagata)
The procedures described in Example 16 were followed to measure the binding IgG titerfor CircRNA-PD006 (Yamagata) .
HAI assay (Yamagata) for CircRNA-PD006
The procedures described in Example 16 were followed to measure the HAI titer for CircRNA-PD006 (Yamagata) .
6.8.17 Example 17. Preclinical Demonstration of Immunogenicity by CircRNA-PD007
Table 26 CircRNA-PD007 experiment design
Table 27 CircRNA-PD007 experiment design
Balb/c mice, half male and half female, were injected IM with 50 μL per mouse. The D364 group was subjected to gross anatomical examination (provisional) , while the remaining groups were subjected to gross anatomical examination using D56.
Evaluation indicators: clinical observation, body weight, gross anatomical observation, HAI titer, and cellular immunity (determined by HAI titer)
Table 28 CircRNA-PD007 experiment design
HAI assay (Wisconsin) for CircRNA-PD007
The procedures described in Example 17 were followed to measure the HAI titer for CircRNA-PD007 (Wisconsin) . The results of the HAI assay were presented in FIG. 29.
Table 29 HAI for CircRNA-PD007
HAI assay (Darwin) for CircRNA-PD007
The procedures described in Example 17 were followed to measure the HAI titer for CircRNA-PD007 (Darwin) . The results of the HAI assay were presented in FIG. 30.
Table 30 HAI for CircRNA-PD007
Table 31 HAI (A27E-0A and A27E-1A) for CircRNA-PD007
HAI assay (Victoria B) for CircRNA-PD007
The procedures described in Example 10 were followed to measure the HAI titer for CircRNA-PD007 (Victoria B) . The results of the HAI assay were presented in FIG. 31.
Table 32 HAI for CircRNA-PD007
HAI assay (Yamagata B) for CircRNA-PD007
The procedures described in Example 10 were followed to measure the HAI titer for CircRNA-PD007 (Yamagata) . The results of the HAI assay were presented in FIG. 32.
Table 33 HAI for CircRNA-PD007
6.8.18 Example 18. Preclinical Demonstration of Immunogenicity of Flulaval, Fluarix. Fluzone, Afluria, Fluad and Fluzone-High by CirRNA-PD008
Table 34 CirRNA-PD008 experiment design
Table 35 CirRNA-PD008 experiment design
See below for detail information of each vaccine:
● Flulaval Qualdrivalent (Flulaval) Company: ID Biomedical Corporation of Qucbec, details: 0.5 ml/vial;
● Fluarix Qualdrivalent (Fluarix) from GSK biologicals, details: 0.5 ml/vial;
● Fluzone Qualdrivalent (Fluzone) from Sanofi Pasteur Inc, details: 0.5 ml/vial;
● Afluria Qualdrivalent (Afluria) from Seqirus Pty Ltd, details: 0.5 ml/vial;
● Fluad Qualdrivalent (Fluad) from Seqirus Inc, details: 0.5 ml/vial; Name: Fluzone-high Qualdrivalent (Fluzone-high) from Sanofi-Pasteur Inc, details: 0.7 ml/vial
6.8.19 Example 19. Preclinical Demonstration of Immunogenicity by CicRNA-PD009
Table 36 CicRNA-PD009 experiment design
Table 37 CicRNA-PD009 experiment design
H1N1 HAI assay
The procedures described in Example 19 were followed to measure the HAI titer for CircRNA-PD009 (H1N1) . The results of the HAI assay were presented in FIG. 33.
Table 38 HAI CircRNA-PD009
Table 39 HAI CircRNA-PD009
H3N2 HAI assay
The procedures described in Example 19 were followed to measure the HAI titer for CircRNA-PD009 (H3N2) . The results of the HAI assay were presented in FIG. 34.
Table 40 HAI CircRNA-PD009
Table 41 HAI CircRNA-PD009
Victoria HAI assay
The procedures described in Example 19 were followed to measure the HAI titer for CircRNA-PD009 (Victoria) . The results of the HAI assay were presented in FIG. 35.
Table 42 HAI CircRNA-PD009
Table 43 HAI CircRNA-PD009
Yamagata HAI assay
The procedures described in Example 19 were followed to measure the HAI titer for CircRNA-PD009 (Yamagata) . The results of the HAI assay were presented in FIG. 36.
Table 44 HAI CircRNA-PD009
Table 61 HAI CircRNA-PD009
6.8.20 Example 20. Long term evaluation of efficacy of the circRNAs provided herein
The data described above in Example 11-19 have demonstrated the efficacy of circRNA vaccines targeting the HA antigen by measuring serum IgG titers and utilizing the HAI assay, with observations recorded up to the indicated time post-administration. To further evaluate the durability of the immune response elicited by the circRNA vaccines, the immune responses induced by these vaccines will continue to be monitored over a longer period. Specifically, the same serum IgG titer measurements and HAI assays as described in Example 10 have been, are currently being, or will be carried out at about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 months, about 15 months, about 18 months, or about 24 months post-administration. We expect that these extended time points will provide comprehensive insights into the long-lasting effects of the circRNA vaccines, revealing sustained immune responses that contribute to long-term protection against the HA antigen. Parallel experiments are performed to compare the efficacy of the circRNA vaccines with that of mRNA vaccines and traditional vaccines. We expect to find that circRNA vaccines induce longer lasting immune responses against the HA antigens compared to mRNA vaccines and traditional vaccines,
6.8.21 Example 21. Evaluation of innate immune response and cell toxicity induced by circRNAs provided herein
The circRNA constructs listed in Table 15 of Example 10 are further evaluated for associated innate immune response and cell toxicity by testing cell survival and production of RIG-I and IFN-B1 (interferon-β1) . Parallel experiments are performed to compare the innate immune activation of circRNA vaccines with that of mRNA vaccines and traditional vaccines. We expect to find that circRNA vaccines induce a lower innate immune response compared to mRNA vaccines and traditional vaccines, as to be evidenced by reduced activation of RIG-I and lower levels of IFN-β production in cells treated with circRNA vaccines. The attenuated innate immune response of circRNA vaccines could be advantageous in reducing reactogenicity while maintaining efficacy, potentially offering a more favorable safety profile for widespread use.
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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Unless the context indicates otherwise, it is specifically intended that the various features described herein can be used in any combination. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Sequence Listing and Tables
Table 3: Exemplary HA Amino Sequence from NCBI database
NOTE: Exemplary “gi|221324|gb|BAA02775|” is index used in NCBI database. Download the influenza. faa. gz and influenza_aa. dat. gz files from the FTP site of the NCBI Influenza Virus Database (https: //ftp. ncbi. nih. gov/genomes/INFLUENZA/) . Extract the sequences of these two genes according to the keywords “hemagglutinin” and “neuraminidase” based on the descriptions in influenza_aa. dat. gz, keeping only the sequences with the host specified as “Human” . To eliminate incomplete or uncertain amino acid fragments, follow these two steps: 1. Retain the amino acid sequences of the gene hemagglutinin (HA) within the range of 1650-1750 amino acids in length, and retain the amino acid sequences of the gene neuraminidase (NA) within the range of 1300-1450 amino acids in length. 2. Remove sequences containing the letter 'X' in the amino acid sequence. Afterwards, remove redundant sequences that are completely identical, and then use cd-hit for clustering (version 4.6.1, parameter -c 0.95) . Select the central sequence as the representative sequence for each cluster and output it.
Table 4: Exemplary NA Amino Sequence from NCBI database
Tables 5A-5B: Exemplary HA (Table 5A) and NA (Table 5B) Amino sequence from WHO file named “Recommended composition of influenza virus vaccines for use in the 2022-2023 northern hemisphere influenza season”
Table 5A
Table 5B
Tables 6A-6B: Exemplary RNA sequences
Table 6A: Exemplary HA sequences (RNA)
Table 6B: Exemplary NA sequences (RNA)
Tables 7A-7B: Exemplary HA (Table 6A) and NA (Table 6B) Amino sequence from WHO file named “Recommended composition of influenza virus vaccines for use in the 2024-2025 northern and southern hemisphere influenza season”
Table 7A
Tables 8A-8B: Exemplary HA (Table 8A) and NA (Table 8B) Amino sequence from WHO file named “Recommended composition of influenza virus vaccines for use in the 2010-2023 northern and southern hemisphere influenza season”
Table 8A
Table 8B
Table 9A-9B: Exemplary RNA sequences (corresponding to Table 7A-7B)
Table 9A Exemplary HA sequences
Table 9B Exemplary NA sequences
Table 10A-10B: Exemplary RNA sequences (corresponding to Table 8A-8B)
Table 10A Exemplary HA RNA sequences
Table 10B. Exemplary NA sequences (RNA)
Table 11: Exemplary IRES sequence
Table 12: Exemplary vector
Table 13: Exemplary circRNA sequence (Upper case: Linker and CDS sequences; Lower case: IRES sequences)
Table 14: A36A and A36B RNA sequence list