Circular RNA vaccine for seasonal influenza and method of use thereof

Abstract

An immunogenic composition containing circular RNA is disclosed as a vaccine against influenza virus. Related preparation methods and their therapeutic applications are also disclosed.

Description

Detailed Description of the Invention

[0001] Related Applications This application claims the priority and benefit of PCT application PCT / CN2023 / 106381 filed on July 7, 2023, the entire content of which is incorporated herein by reference. Incorporated Sequence Listing The content of the electronic sequence listing (TFH01060PCT-sequence listing.xml, size: 1,080,415 bytes, creation date: July 8, 2024) is incorporated herein by reference. [1. Technical Field] The present invention relates to the fields of molecular biology and immunology, and particularly to constructs of circular RNAs as influenza virus vaccines, methods of production, and therapeutic uses. [2. Background Art]

[0002] Circular RNA (circRNA) is a type of RNA molecule formed by head-to-tail ligation and has been demonstrated to have diverse 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 RNA, circular RNA is superior in stability and thus becomes a promising new platform for RNA drug development.

[0003] Influenza is an acute respiratory infection caused by influenza viruses (influenza A and influenza B viruses) that spread worldwide. Vaccination plays a crucial role in controlling annual influenza outbreaks. Vaccines induce an immune response that attacks the viral glycoprotein hemagglutinin (HA) and viral enzyme neuraminidase (NA) present on the surface of the influenza virus. Improved immunogenic compositions against influenza are urgently needed.

[0004] The compositions (containing circular RNA) as influenza virus vaccines, as well as related methods and systems, provided herein address this need and offer relevant advantages. [3. Summary of the Invention]

[0005] This application provides a circular ribonucleic acid (circular RNA, circRNA) comprising a translation initiation (TI) sequence and a protein coding (Z) sequence, 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).

[0006] In some embodiments, the Z sequence includes a first HA(HA1) sequence encoding an HA antigen derived from a first influenza virus of type A or type B.

[0007] In some embodiments, the Z sequence further comprises a second HA(HA2) sequence encoding an HA antigen derived from a second influenza virus of type A or B. In some embodiments, the circular RNA comprises the TI, HA1, L, and HA2 sequences in this order, where L is a linker sequence or is absent, and the first and second influenza viruses These are (1) A1 and A2 types, (2) B1 and B2 types, (3) A and B types, or (4) B and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0008] In some embodiments, the Z sequence further comprises a third HA(HA3) sequence encoding an HA antigen derived from a third influenza virus of type A or B. In some embodiments, the circular RNA comprises the sequences TI, HA1, L1, HA2, L2, and HA3 in this order, where L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0009] In some embodiments, the Z sequence further comprises a fourth HA(HA4) sequence encoding an HA antigen derived from a fourth influenza virus of type A or B. In some embodiments, the circular RNA comprises the sequences TI, HA1, L1, HA2, L2, HA3, L3, and HA4 in this order, where L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0010] In some embodiments, the Z sequence includes a first NA(NA1) sequence encoding an NA antigen derived from a first influenza A or B virus.

[0011] In some embodiments, the Z sequence further comprises a second NA(NA2) sequence encoding an NA antigen derived from a second influenza virus of type A or B. In some embodiments, the circular RNA comprises the TI, NA1, L, and NA2 sequences in this order, where L is a linker sequence or is absent, and the first and second influenza viruses are (1) A1 and A2, (2) B1 and B2, (3) A and B, or (4) B and A, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0012] In some embodiments, the Z sequence further comprises a third NA(NA3) sequence encoding an NA antigen derived from a third influenza virus of type A or B. In some embodiments, the circular RNA comprises the sequences TI, NA1, L1, NA2, L2, and NA3 in this order, where L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0013] In some embodiments, the Z sequence further comprises a fourth NA(NA4) sequence encoding an NA antigen derived from a fourth influenza virus of type A or B. In some embodiments, the circular RNA comprises the sequences TI, NA1, L1, NA2, L2, NA3, L3, and NA4 in this order, where L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0014] 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, wherein the HA antigen and the NA antigen are derived from a first influenza virus of type A or B. In some embodiments, the circular RNA comprises the sequences in the following order: (1) TI, HA1, L, and NA1; or (2) TI, NA1, L, and HA1, where L is a linker sequence or is absent.

[0015] 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 antigen and NA antigen are derived from a second influenza virus of type A or B. In some embodiments, the circular RNA is sequenced 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, where L1, L2, and L3 are independently linker sequences or are absent, and the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; (3) A and B types; or (4) B and A types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0016] In some embodiments, the Z sequence further comprises a third HA(HA3) sequence encoding the HA antigen and a third NA(NA3) sequence encoding the NA antigen, wherein the HA antigen and the NA antigen are derived from a third influenza virus of type A or B. In some embodiments, the circular RNA is sequenced 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, with L1, L2, L3, L4, and Each L5 is either an independent linker sequence or absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0017] In some embodiments, the circular RNA comprises 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 in that order, wherein the Z1 and Z2 sequences independently interact with HA anti- It contains an HA sequence encoding the progenitor or an NA sequence encoding the NA antigen.

[0018] In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, encoding HA antigens derived from first and second influenza viruses, and the first and second influenza viruses are independently type A or type B. In some embodiments, the circular RNA comprises TI1, HA1, L1, TI2, HA2, and L2 in this order, where L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, where A1 and A2 are first and second subtypes of type A influenza virus, and B1 and B2 are first and second subtypes of type B influenza virus.

[0019] In some embodiments, the Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence includes a second NA(NA2) sequence, and encodes HA antigens derived from first and second influenza viruses, the first and second influenza viruses being independently type A or type B. In some embodiments, the circular RNA includes TI1, NA1, L1, TI2, NA2, and L2 in this order, where L1 and L2 are independently linker sequences or are absent, the first and second influenza viruses being (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, where A1 and A2 are first and second subtypes of type A influenza virus, and B1 and B2 are first and second subtypes of type B influenza virus.

[0020] In some embodiments, the Z1 sequence includes a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence includes a second HA (HA2) sequence and a second NA (NA2) sequence, the HA1 and NA1 sequences encode HA and NA antigens derived from a first influenza virus, the HA2 and NA2 sequences encode HA and NA antigens derived from a second influenza virus, and the first and second influenza viruses are independently type A or type B. In some embodiments, the circular RNA is sequenced 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, where L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; or (3) A and B types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0021] In some embodiments, the Z1 sequence includes a first HA(HA1) sequence encoding the HA antigen, and the Z2 sequence includes a first NA(NA1) sequence encoding the NA antigen, wherein the HA antigen and the NA antigen are derived from a first influenza virus of type A or type B.

[0022] In some embodiments, the Z1 sequence further comprises 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 derived from a second influenza virus of type A or B.

[0023] In some embodiments, the circular RNA contains the sequences TI1, HA1, L1, HA2, TI2, NA1, L2, and NA2 in this order, where L1 and L2 are independently phosphorus The Kerr sequence is either present or absent, and the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; (3) A and B types; or (4) B and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0024] In some embodiments, the Z1 sequence further comprises a third HA (HA3) sequence, and the Z2 sequence further comprises a third NA (NA3) sequence, wherein the HA3 and NA3 sequences encode HA and NA antigens derived from a third influenza virus of type A or B. In some embodiments, the circular RNA contains the sequences TI1, HA1, L1, HA2, L2, HA3, TI3, NA1, L3, NA2, L4, and NA3 in this order, where L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0025] In some embodiments, the circular RNA comprises, in this 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, where each of the Z1, Z2, and Z3 sequences independently comprises an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0026] In some embodiments, 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 HA1 sequence, HA2 sequence, and HA3 sequence each encode an HA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively. In some embodiments, the circular RNA comprises TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3 in this order, where L1, L2, and L3 are each independently a linker sequence or do not exist, and the first, second, and third influenza viruses are each (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0027] 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 NA1 sequence, NA2 sequence, and NA3 sequence each encode an NA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively. In some embodiments, the circular RNA comprises TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3 in this order, where L1, L2, and L3 are each independently a linker sequence or do not exist, and the first, second, and third influenza viruses are each (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0028] In some embodiments, the Z1 sequence comprises a first HA (HA1) sequence and a first NA (N A1) comprising an array, wherein the Z2 array comprises a second HA (HA2) array and a second NA (NA2) array, the Z3 array comprises a third HA (HA3) array and a third NA (NA3) array, the HA1 array and the NA1 array encode antigens derived from a first influenza virus, the HA2 array and the NA2 array encode antigens derived from a second influenza virus, and the HA3 array and the NA3 array encode antigens derived from a third influenza virus. In some embodiments, the circular RNA has sequences 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, where L1, L2, and L3 are each independently a linker sequence or do not exist, and the first, second, and third influenza viruses are each (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0029] In some embodiments, the circular RNA comprises, in this 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, and a fourth protein-coding (Z4) sequence, wherein the Z1, Z2, Z3, and Z4 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0030] In some embodiments, the Z1 sequence includes a first HA(HA1) sequence, the Z2 sequence includes a second HA(HA2) sequence, the Z3 sequence includes a third HA(HA3) sequence, and the Z4 sequence includes a fourth HA(HA4) sequence, wherein the HA1, HA2, HA3, and HA4 sequences encode HA antigens derived from a first influenza virus, a second influenza virus, a third influenza virus, and a fourth influenza virus, respectively. In some embodiments, the circular RNA comprises the sequences TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4 in this order, wherein L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2 types; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0031] In some embodiments, the Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence includes a second NA(NA2) sequence, the Z3 sequence includes a third NA(NA3) sequence, and the Z4 sequence includes a fourth NA(NA4) sequence, wherein the NA1, NA2, NA3, and NA4 sequences encode NA antigens derived from a first influenza virus, a second influenza virus, a third influenza virus, and a fourth influenza virus, respectively. In some embodiments, the circular RNA contains the sequences TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4 in this order, where L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2 types, respectively; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. It's that type.

[0032] In some embodiments, the HA antigen and / or NA antigen are derived from seasonal influenza virus. In some embodiments, the HA antigen and / or NA antigen are derived from the World Health Organization's Global Influenza Surveillance System (WHS). The selection is made according to standardized criteria adopted by the and Response System (GISRS). In some embodiments, the HA antigen and / or NA antigen are selected using a hemagglutinin inhibitory (HAI) assay and / or neuraminidase inhibitory (NAI) assay to identify currently circulating influenza viruses that are antigenically similar to the influenza virus of the previous season's vaccine. In some embodiments, the HA antigen and / or NA antigen are derived from influenza A viruses listed in Table 1 or influenza B viruses listed in Table 2.

[0033] In some embodiments, the HA antigen and / or NA antigen are derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus. In some embodiments, the HA antigen and / or NA antigen are derived 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 are derived from an influenza B virus strain that is B / Austria / 1359417 / 2021 or B / Phuket / 3073 / 2013. In some embodiments, the HA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 3, Table 5A, Table 7A, or Table 8A, and / or the NA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 4, Table 5B, Table 7B, or Table 8B.

[0034] In some embodiments, the HA sequence has a nucleotide sequence described in Table 6A, Table 9A, Table 10A, or Table 14, and / or the NA sequence has a nucleotide sequence described in Table 6B, Table 9B, or Table 10B.

[0035] This application provides a circular RNA comprising a TI sequence and a Z sequence containing an HA sequence encoding the HA antigen.

[0036] In some embodiments, the HA antigen is derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus.

[0037] In some embodiments, the HA antigen is the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 175. 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 with the nucleotide sequences of SEQ ID NOs: 182, 184-188, and 209-210. In some embodiments, the HA antigen has at least 90%, at least 95%, at least 97%, at least It has 98%, at least 99%, or 100% sequence identity.

[0038] In some embodiments, the HA antigen is the HA protein of the A / Darwin / 6 / 2021(H3N2)-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 177. 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 with the nucleotide sequences of SEQ ID NO: 183, 189-193, and 211-212. 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 with the nucleotide sequence of SEQ ID NO: 211 or 189.

[0039] In some embodiments, the HA antigen is the HA protein of influenza B / Austria / 1359417 / 2021 (Victoria lineage)-like virus. 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 with the amino acid sequence of SEQ ID NO: 179. 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 with the nucleotide sequences of SEQ ID NOs: 194-198 and 213-219. 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 with the nucleotide sequence of SEQ ID NO: 214 or 196.

[0040] In some embodiments, the HA antigen is the HA protein of the B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 180. 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 with the nucleotide sequences of SEQ ID NOs: 199-203 and 220-221. 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 with the nucleotide sequence of SEQ ID NO: 221 or 199.

[0041] In some embodiments, the HA antigen is the HA protein of the A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 235. 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 with the nucleotide sequences of SEQ ID NOs: 281-285 and 520-521. 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 with the nucleotide sequence of SEQ ID NO: 520 or 521.

[0042] In some embodiments, the circular RNA includes a linker sequence encoding a 2A self-cleaving peptide.

[0043] In some embodiments, the TI sequence is an IRES, an IRES-like nucleotide sequence, or a combination thereof. In some embodiments, the TI sequence includes an optimized IRES sequence. In some embodiments, the IRES sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences listed in Table 11.

[0044] In some embodiments, the circular RNAs provided in this application have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences listed in Table 13. In some embodiments, the circular RNAs provided in this application have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with nucleotide sequences selected from SEQ ID NOs. 499-519.

[0045] In some embodiments, the circular RNA provided in this application is scarless or nearly scarless.

[0046] In some embodiments, the circular RNA provided in this application induces a reduced innate immune response when administered to a subject, compared to mRNA encoding HA antigen and / or NA antigen.

[0047] In some embodiments, the circular RNA provided in this application comprises modified RNA nucleotides and / or modified nucleosides. In some embodiments, the circular RNA provided in this application comprises at least 10% modified RNA nucleotides and / or modified nucleosides. In some embodiments, at least one of the modified RNA nucleotides and / or modified nucleosides is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyluridine These are ruadenosine (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 (pseudolidine), or m1A (1-methyladenosine).

[0048] In some embodiments, the circular RNAs provided in this application include 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), It does not contain 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), or inosine (I), Y (pseuduridine), or m1A (1-methyladenosine).

[0049] In some embodiments, at least one of the modified RNA nucleotides and / or modified nucleosides is introduced by in vitro transcription (IVT).

[0050] In some embodiments, this application also includes immunogenicity, comprising the circular RNA described herein. A composition is provided.

[0051] In some embodiments, the immunogenic compositions provided in this application comprise at least two, at least three, at least four, at least five, or at least six of the circular RNAs described in this application.

[0052] In some embodiments, the immunogenic composition provided in this application comprises two circular RNAs. In some embodiments, the Z sequences of the two circular RNAs comprise HA sequences encoding HA antigens derived 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.

[0053] In some embodiments, the immunogenic composition provided in this application comprises four circular RNAs. In some embodiments, the Z sequences of the four circular RNAs comprise HA sequences encoding HA antigens derived from four influenza viruses. In some embodiments, the four influenza viruses comprise two influenza A viruses and two influenza B viruses.

[0054] In some embodiments, the four influenza viruses are the A / Wisconsin / 588 / 2019(H1N1) influenza virus, the A / Darwin / 6 / 2021(H3N2) influenza virus, the B / Austria / 1359417 / 2021(B / Victoria lineage) influenza virus, and the B / Phuket / 3073 / 2013(B / Yamagata lineage) influenza virus. In some embodiments, the four HA antigens are the HA proteins of influenza A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, influenza A / Darwin / 6 / 2021(H3N2)-like virus, influenza B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus, and influenza B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus. In some embodiments, the amino acid sequences of the four HA antigens have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences 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 with the nucleotide sequences of SEQ ID NOs. 210, 211, 214, and 221, respectively. In some embodiments, the four circular RNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 501, 519, 514, and 508, respectively.

[0055] In some embodiments, the four influenza viruses are the HA proteins of the A / Wisconsin / 67 / 2022(H1N1) influenza virus, the A / Darwin / 6 / 2021(H3N2) influenza virus, the B / Austria / 1359417 / 2021(B / Victoria lineage)-like influenza virus, and the B / Phuket / 3073 / 2013(B / Yamagata lineage)-like influenza virus. In some embodiments, the four HA antigens are the HA proteins of the A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus, the HA proteins of the A / Darwin / 6 / 2021(H3N2)-like influenza virus, the HA proteins of the B / Austria / 1359417 / 2021(B / Victoria lineage)-like influenza virus, and the B / Phuket / The HA protein is of influenza virus 3073 / 2013 (B / Yamagata strain). In some embodiments, the amino acid sequences of the four HA antigens have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences 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 with the nucleotide sequences of SEQ ID NOs. 521, 189, 196, and 221, respectively. In some embodiments, the four circular RNAs have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 518, 519, 508, and 515, respectively.

[0056] In some embodiments, the immunogenic compositions provided in this application contain the cyclic RNAs in lipid nanoparticles (LNPs). In some embodiments, each cyclic RNA is contained separately in the LNPs.

[0057] In some embodiments, the immunogenic compositions provided in this application include an adjuvant.

[0058] In some embodiments, the immunogenic compositions provided in this application are used to induce an anti-influenza immune response in a subject.

[0059] In some embodiments, the present application provides a method for inducing an anti-influenza immune response in a subject, comprising administering an effective amount of the immunogenic composition described herein to the subject.

[0060] In some embodiments, the methods provided in this application induce cross-protective immunity against heterologous influenza viruses. In some embodiments, the methods provided in this application induce protective immunity against the same subtype of influenza virus.

[0061] In some embodiments, the immune response is measured by anti-hemagglutinin (HA) antibody titer, hemagglutinin inhibitor (HAI) titer, or microneutralization assay. In some embodiments, the immune response is measured by HAI titer. In some embodiments, 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. In some embodiments, the HAI titer produced in the subject by the method is at least 3 times, at least 4 times, at least 5 times, or at least 6 times the baseline HAI level. In some embodiments, the HAI titer produced by the method is at least 40 and lasts 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.

[0062] In some embodiments, the immune response is measured by anti-HA antibody titer approximately 7, 14, 21, 28, or 35 days after vaccination. In some embodiments, the method induces an increase of at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, or at least 10,000-fold in the subject's anti-HA IgG antibody titer compared to baseline. In some embodiments, the method results in an increase of at least 50-fold in the anti-HA IgG antibody titer, which lasts 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. do.

[0063] In some embodiments, the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, or intraperitoneally.

[0064] In some embodiments, the method comprises a single dose, followed optionally by a boost dose.

[0065] In some embodiments, the subject is 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.

[0066] 4. Exemplary Embodiments Embodiment 1: A circular ribonucleic acid (circular RNA, circRNA) comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, 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).

[0067] Embodiment 2: The circular RNA according to Embodiment 1, wherein the Z sequence comprises a first HA(HA1) sequence encoding an HA antigen derived from a first influenza virus of type A or B.

[0068] Embodiment 3: The circular RNA according to Embodiment 2, wherein the Z sequence further comprises a second HA(HA2) sequence encoding an HA antigen derived from a second influenza virus of type A or B.

[0069] Embodiment 4: The circular RNA according to Embodiment 3, wherein the circular RNA contains the sequences TI, HA1, L, and HA2 in that order, where L is a linker sequence or is absent, and the first and second influenza viruses are (1) A1 and A2, (2) B1 and B2, (3) A and B, or (4) B and A, respectively, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0070] Embodiment 5: The circular RNA according to Embodiment 3, wherein the Z sequence further comprises a third HA(HA3) sequence encoding an HA antigen derived from a third influenza virus of type A or B.

[0071] Embodiment 6: The circular RNA according to Embodiment 5, wherein the circular RNA contains the sequences TI, HA1, L1, HA2, L2, and HA3 in that order, and L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, and A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0072] Embodiment 7: The circular RNA according to Embodiment 5, wherein the Z sequence further comprises a fourth HA (HA4) sequence encoding an HA antigen derived from a fourth influenza virus of type A or B.

[0073] Embodiment 8: The circular RNA is TI, HA1, L1, HA2, L2, HA3, L3, The circular RNA according to Embodiment 7, comprising the and HA4 sequences in this order, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2 types, respectively; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0074] Embodiment 9: The circular RNA according to Embodiment 1, wherein the Z sequence comprises a first NA(NA1) sequence encoding an NA antigen derived from a first influenza virus of type A or B.

[0075] Embodiment 10: The circular RNA according to Embodiment 9, wherein the Z sequence further comprises a second NA(NA2) sequence encoding an NA antigen derived from a second influenza virus of type A or B.

[0076] Embodiment 12: The circular RNA according to Embodiment 10, wherein the circular RNA contains the sequences TI, NA1, L, and NA2 in that order, where L is a linker sequence or is absent, and the first and second influenza viruses are (1) A1 and A2, (2) B1 and B2, (3) A and B, or (4) B and A, respectively, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0077] Embodiment 12: The circular RNA according to Embodiment 10, wherein the Z sequence further comprises a third NA(NA3) sequence encoding an NA antigen derived from a third influenza virus of type A or B.

[0078] Embodiment 13: The circular RNA according to Embodiment 12, wherein the circular RNA contains the sequences TI, NA1, L1, NA2, L2, and NA3 in that order, and L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0079] Embodiment 14: The circular RNA according to Embodiment 12, wherein the Z sequence further comprises a fourth NA(NA4) sequence encoding an NA antigen derived from a fourth influenza virus of type A or B.

[0080] Embodiment 15: The circular RNA according to Embodiment 14, wherein the circular RNA contains the sequences TI, NA1, L1, NA2, L2, NA3, L3, and NA4 in this order, and L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0081] Embodiment 16: The Z sequence includes a first HA (HA1) sequence encoding the HA antigen and a first NA (NA1) sequence encoding the NA antigen, wherein the HA antigen and the NA antigen The circular RNA according to Embodiment 1, wherein the first influenza virus is of type A or type B.

[0082] Embodiment 17: The circular RNA according to Embodiment 16, having the following sequence: (1) TI, HA1, L, and NA1; or (2) TI, NA1, L, and HA1, wherein L is a linker sequence or is absent.

[0083] Embodiment 18: The circular RNA according to 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, and the HA antigen and the NA antigen are derived from a second influenza virus of type A or B.

[0084] Embodiment 19: The circular RNA according to Embodiment 18, having the following sequence: (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 are independently linker sequences or absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, wherein A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0085] Embodiment 20: The circular RNA according to 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, and the HA antigen and the NA antigen are derived from a third influenza virus of type A or type B.

[0086] Embodiment 21: Arranged 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) including TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3, where L1, L2, L3, L4, and L5 are each independently linkers The circular RNA according to Embodiment 20, wherein the sequence is or is absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0087] Embodiment 22: The circular RNA according to Embodiment 1, comprising 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 in this order, wherein the Z1 and Z2 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0088] Embodiment 23: The circular RNA according to Embodiment 22, wherein the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, encodes HA antigens derived from first and second influenza viruses, and the first and second influenza viruses are independently type A or type B.

[0089] Embodiment 24: The circular RNA according to Embodiment 23, comprising TI1, HA1, L1, TI2, HA2, and L2 in this order, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0090] Embodiment 25: The circular RNA according to Embodiment 22, wherein the Z1 sequence comprises a first NA(NA1) sequence, the Z2 sequence comprises a second NA(NA2) sequence, encodes HA antigens derived from first and second influenza viruses, and the first and second influenza viruses are independently type A or type B.

[0091] Embodiment 26: The circular RNA according to Embodiment 25, comprising TI1, NA1, L1, TI2, NA2, and L2 in this order, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0092] Embodiment 27: The circular RNA according to Embodiment 22, wherein the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, the HA1 sequence and the NA1 sequence encode HA antigen and NA antigen derived from a first influenza virus, the HA2 sequence and the NA2 sequence encode HA antigen and NA antigen derived from a second influenza virus, and the first and second influenza viruses are independently type A or type B.

[0093] Embodiment 28: The circular RNA according to Embodiment 27, having the following sequence: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2; or (2) comprising TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; or (3) type A and type B, wherein A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0094] Embodiment 29: The circular RNA according to 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, and the HA antigen and the NA antigen are derived from a first influenza virus of type A or type B.

[0095] Embodiment 30: The circular RNA according to Embodiment 29, wherein the Z1 sequence further comprises a second HA (HA2) sequence, the Z2 sequence further comprises a second NA (NA2) sequence, and the HA2 sequence and the NA2 sequence encode an HA antigen and an NA antigen derived from a second influenza virus of type A or B.

[0096] Embodiment 32: The sequence TI1, HA1, L1, HA2, TI2, NA1, L2, and NA2 is included in this order, and L1 and L2 are independently linker sequences, or The circular RNA according to Embodiment 30, wherein the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0097] Embodiment 32: The circular RNA according to Embodiment 30, wherein the Z1 sequence further comprises a third HA (HA3) sequence, the Z2 sequence further comprises a third NA (NA3) sequence, and the HA3 sequence and the NA3 sequence encode an HA antigen and an NA antigen derived from a third influenza virus of type A or B.

[0098] Embodiment 33: A circular RNA according to Embodiment 32, comprising the sequences TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3 in this order, wherein L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0099] Embodiment 34: The circular RNA according to Embodiment 1, comprising 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 in this order, wherein the Z1, Z2, and Z3 sequences each independently contain an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0100] Embodiment 35: The circular RNA according to Embodiment 34, wherein 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 HA1 sequence, the HA2 sequence, and the HA3 sequence each encode an HA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively.

[0101] Embodiment 36: The circular RNA according to Embodiment 35, comprising TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3 in this order, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (1) A1, A2, and B types, or (2) B1, B2, and A types, respectively, wherein A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0102] Embodiment 37: The circular RNA according to Embodiment 34, 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 NA1 sequence, the NA2 sequence, and the NA3 sequence each encode an NA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively.

[0103] Embodiment 38: comprising TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3 in this order, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first, second, and third influenza viruses are respectively The circular RNA according to Embodiment 37, wherein (1) is of type A1, type A2, and type B, or (2) is of type B1, type B2, and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0104] Embodiment 39: The circular RNA according to Embodiment 34, wherein the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, the Z3 sequence comprises a third HA (HA3) sequence and a third NA (NA3) sequence, the HA1 sequence and NA1 sequence encode an antigen derived from a first influenza virus, the HA2 sequence and NA2 sequence encode an antigen derived from a second influenza virus, and the HA3 sequence and NA3 sequence encode an antigen derived from a third influenza virus.

[0105] Embodiment 40: The circular RNA according to Embodiment 39, with the sequence in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3; or (2) comprising TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3, wherein L1, L2, and L3 are each independently linker sequences or are absent, and the first, second, and third influenza viruses are each (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, wherein A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

[0106] Embodiment 41: The circular RNA according to Embodiment 1, comprising 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, and a fourth protein coding (Z4) sequence in this order, wherein the Z1, Z2, Z3, and Z4 sequences each independently contain an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0107] Embodiment 42: The circular RNA according to Embodiment 41, wherein the Z1 sequence includes a first HA (HA1) sequence, the Z2 sequence includes a second HA (HA2) sequence, the Z3 sequence includes a third HA (HA3) sequence, the Z4 sequence includes a fourth HA (HA4) sequence, and the HA1, HA2, HA3, and HA4 sequences each encode an HA antigen derived from a first influenza virus, a second influenza virus, a third influenza virus, and a fourth influenza virus, respectively.

[0108] Embodiment 43: The circular RNA according to Embodiment 42, comprising the sequences TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4 in this order, wherein L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2, respectively; (2) A1, B1, A2, and B2; (3) B1, A1, B2, and A2; or (4) B1, B2, A1, and A2, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0109] Embodiment 44: The Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence includes a second NA(NA2) sequence, the Z3 sequence includes a third NA(NA3) sequence, and the Z4 sequence includes a fourth NA(NA4) sequence, with the NA1 sequence, the NA2 sequence, the NA3 sequence, and the NA4 sequence each representing a first influenza virus and a second influenza virus. The circular RNA according to Embodiment 41, encoding NA antigens derived from influenza virus, a third influenza virus, and a fourth influenza virus.

[0110] Embodiment 45: The circular RNA according to Embodiment 44, comprising the sequences TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4 in this order, wherein L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2, respectively; (2) A1, B1, A2, and B2; (3) B1, A1, B2, and A2; or (4) B1, B2, A1, and A2, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0111] Embodiment 46: A circular RNA according to any one of Embodiments 1 to 45, wherein the HA antigen and / or the NA antigen are derived from a seasonal influenza virus.

[0112] Embodiment 47: The circular RNA according to Embodiment 46, wherein the HA antigen and / or the NA antigen are selected according to standardized criteria adopted by the World Health Organization's Global Influenza Surveillance and Response System (GISRS).

[0113] Embodiment 48: The circular RNA according to Embodiment 46, wherein the HA antigen and / or the NA antigen are selected using a hemagglutinin inhibitory (HAI) assay and / or neuraminidase inhibitory (NAI) assay to identify a currently circulating influenza virus that is antigenically similar to the influenza virus of the previous season's vaccine.

[0114] Embodiment 49: A circular RNA according to any one of Embodiments 1 to 40, wherein the HA antigen and / or the NA antigen are derived from influenza A virus listed in Table 1 or influenza B virus listed in Table 2.

[0115] Embodiment 50: A circular RNA according to any one of Embodiments 1 to 40, wherein the HA antigen and / or the NA antigen are derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus.

[0116] Embodiment 51: A circular RNA according to any one of Embodiments 1 to 40, wherein the HA antigen and / or the NA antigen is derived from an influenza A virus strain in which 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.

[0117] Embodiment 52: The circular RNA according to any one of Embodiments 1 to 40, wherein the HA antigen and / or the NA antigen are derived from an influenza B virus strain which is B / Austria / 1359417 / 2021 or B / Phuket / 3073 / 2013.

[0118] Embodiment 53: The HA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 3, Table 5A, Table 7A, or Table 8A, and / or the NA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 4, Table 5B, Table 7B, or Table 8B. A cyclic RNA having one property, as described in any one of Embodiments 1 to 40.

[0119] Embodiment 54: The circular RNA according to Embodiment 53, wherein the HA sequence has a nucleotide sequence listed in Table 6A, Table 9A, Table 10A, or Table 14, and / or the NA sequence has a nucleotide sequence listed in Table 6B, Table 9B, or Table 10B.

[0120] Embodiment 55: The circular RNA according to Embodiment 1, wherein the Z sequence includes an HA sequence encoding the HA antigen.

[0121] Embodiment 56: The circular RNA according to Embodiment 55, wherein the HA antigen is derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus.

[0122] Embodiment 57: The circular RNA according to Embodiment 56, wherein the HA antigen is the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus.

[0123] Embodiment 58: The circular RNA according to 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 with the amino acid sequence of SEQ ID NO: 175.

[0124] Embodiment 59: The circular RNA according to Embodiment 58, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 182, 184-188, and 209-210.

[0125] Embodiment 60: The circular RNA according to Embodiment 58, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 210.

[0126] Embodiment 61: The circular RNA according to Embodiment 56, wherein the HA antigen is the HA protein of the A / Darwin / 6 / 2021(H3N2)-like influenza virus.

[0127] Embodiment 62: The circular RNA according to 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 with the amino acid sequence of SEQ ID NO: 177.

[0128] Embodiment 63: The circular RNA according to Embodiment 62, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 183, 189-193, and 211-212.

[0129] Embodiment 64: The circular RNA according to Embodiment 62, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 211 or 189.

[0130] Embodiment 65: The circular RNA according to Embodiment 56, wherein the HA antigen is the HA protein of influenza B / Austria / 1359417 / 2021 (Victoria lineage)-like virus.

[0131] Embodiment 66: The circular RNA according to 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 with the amino acid sequence of SEQ ID NO: 179.

[0132] Embodiment 67: The circular RNA according to Embodiment 66, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 194-198 and 213-219.

[0133] Embodiment 68: The circular RNA according to Embodiment 66, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 214 or 196.

[0134] Embodiment 69: The circular RNA according to Embodiment 56, wherein the HA antigen is the HA protein of influenza virus B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0135] Embodiment 70: The circular RNA according to 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 with the amino acid sequence of SEQ ID NO: 180.

[0136] Embodiment 71: The circular RNA according to Embodiment 70, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 199-203 and 220-221.

[0137] Embodiment 72: The circular RNA according to Embodiment 70, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 221 or 199.

[0138] Embodiment 73: The circular RNA according to Embodiment 56, wherein the HA antigen is the HA protein of the A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus.

[0139] Embodiment 74: The circular RNA according to 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 with the amino acid sequence of SEQ ID NO: 235.

[0140] Embodiment 75: The circular RNA according to Embodiment 74, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 281-285 and 520-521.

[0141] Embodiment 76: The circular RNA according to Embodiment 74, wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 520 or 521.

[0142] Embodiment 77: The circular RNA according to any one of Embodiments 1 to 76, wherein the circular RNA comprises a linker sequence encoding a 2A self-cleaving peptide.

[0143] Embodiment 78: The circular RNA according to any one of Embodiments 1 to 77, wherein the TI sequence comprises an IRES, an IRES-like nucleotide sequence, or a combination thereof.

[0144] Embodiment 79: The circular RNA according to Embodiment 77, wherein the TI sequence comprises an optimized IRES sequence.

[0145] Embodiment 80: The circular RNA according to Embodiment 77, wherein the IRES sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences listed in Table 11.

[0146] Embodiment 81: The circular RNA according to Embodiment 1, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences described in Table 13.

[0147] Embodiment 82: The circular RNA according to Embodiment 1, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from SEQ ID NOs. 499-519.

[0148] Embodiment 83: The circular RNA according to any one of Embodiments 1 to 82, wherein the circular RNA is scar-free or nearly scar-free.

[0149] Embodiment 84: The circular RNA according to any one of Embodiments 1 to 83, wherein, when administered to a subject, the innate immune response induced by the circular RNA is reduced compared to mRNA encoding the HA antigen and / or NA antigen.

[0150] Embodiment 85: The circular RNA according to any one of Embodiments 1 to 84, wherein the circular RNA comprises a modified RNA nucleotide and / or a modified nucleoside.

[0151] Embodiment 86: The circular RNA according to Embodiment 85, wherein the circular RNA comprises at least 10% modified RNA nucleotides and / or modified nucleosides.

[0152] Embodiment 87: At least one of the modified RNA nucleotides and / or modified nucleosides is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A) The cyclic RNA according to Embodiment 85, wherein 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 (pseudolidine), or m1A (1-methyladenosine).

[0153] Embodiment 88: The cyclic RNA is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (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- A circular RNA according to any one of Embodiments 1 to 83, which does not contain methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G), and inosine (I), Y (pseuduridine), or m1A (1-methyladenosine).

[0154] Embodiment 89: The circular RNA according to any one of Embodiments 85 to 88, wherein at least one of the modified RNA nucleotides and / or modified nucleosides is introduced by in vitro transcription (IVT).

[0155] Embodiment 90: An immunogenic composition comprising the circular RNA described in any one of Embodiments 1 to 89.

[0156] Embodiment 91: An immunogenic composition comprising at least two, at least three, at least four, at least five, or at least six circular RNAs as described in any one of Embodiments 1 to 89.

[0157] Embodiment 92: The immunogenic composition according to Embodiment 91, comprising two circular RNAs.

[0158] Embodiment 93: The immunogenic composition according to Embodiment 92, wherein the Z sequences of the two circular RNAs contain HA sequences encoding HA antigens derived from two influenza viruses.

[0159] Embodiment 94: The immunogenic composition according to 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.

[0160] Embodiment 95: The immunogenic composition according to Embodiment 91, comprising four circular RNAs.

[0161] Embodiment 96: The immunogenic composition according to Embodiment 95, wherein the Z sequences of the four circular RNAs contain HA sequences encoding four influenza virus-derived HA antigens.

[0162] Embodiment 97: The immunogenic composition according to Embodiment 96, wherein the four influenza viruses comprise two influenza A viruses and two influenza B viruses.

[0163] Embodiment 98: The immunogenic composition according to Embodiment 97, wherein the four influenza viruses are A / Wisconsin / 588 / 2019(H1N1) influenza virus, A / Darwin / 6 / 2021(H3N2) influenza virus, B / Austria / 1359417 / 2021(B / Victoria lineage) influenza virus, and B / Phuket / 3073 / 2013(B / Yamagata lineage) influenza virus.

[0164] Embodiment 99: The immunogenic composition according to Embodiment 98, wherein the four HA antigens are the HA protein of influenza A / Wisconsin / 588 / 2019(H1N1)pdm09, the HA protein of influenza A / Darwin / 6 / 2021(H3N2), the HA protein of influenza B / Austria / 1359417 / 2021(B / Victoria lineage), and the HA protein of influenza B / Phuket / 3073 / 2013(B / Yamagata lineage).

[0165] Embodiment 100: The amino acid sequences of the four HA antigens are, respectively, SEQ ID NO: 175, 1 The immunogenic composition according to Embodiment 99, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences of 77, 179, and 180.

[0166] Embodiment 101: The immunogenic composition according to 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 with the nucleotide sequences of SEQ ID NOs. 210, 211, 214, and 221, respectively.

[0167] Embodiment 102: The immunogenic composition according to Embodiment 101, wherein the four circular RNAs each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 501, 519, 514, and 508, respectively.

[0168] Embodiment 103: The immunogenic composition according to Embodiment 97, wherein the four influenza viruses are the A / Wisconsin / 67 / 2022(H1N1) influenza virus, the A / Darwin / 6 / 2021(H3N2) influenza virus, the HA protein of the B / Austria / 1359417 / 2021(B / Victoria lineage) influenza virus, and the HA protein of the B / Phuket / 3073 / 2013(B / Yamagata lineage) influenza virus.

[0169] Embodiment 104: The immunogenic composition according to Embodiment 103, wherein the four HA antigens are the HA protein of influenza A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus, the HA protein of influenza A / Darwin / 6 / 2021(H3N2)-like virus, the HA protein of influenza B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus, and the HA protein of influenza B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus.

[0170] Embodiment 105: The immunogenic composition according to Embodiment 104, wherein the amino acid sequences of the four HA antigens have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences of SEQ ID NOs. 235, 177, 179, and 180, respectively.

[0171] Embodiment 106: The immunogenic composition according to 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 with the nucleotide sequences of SEQ ID NOs. 521, 189, 196, and 221, respectively.

[0172] Embodiment 107: The immunogenic composition according to Embodiment 106, wherein the four circular RNAs each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 518, 519, 508, and 515.

[0173] Embodiment 108: The immunogenic composition according to any one of Embodiments 90 to 107, wherein the cyclic RNA(s) are incorporated into lipid nanoparticles (LNPs).

[0174] Embodiment 109: The immunogenic composition according to Embodiment 108, wherein each of the circular RNAs is separately incorporated into the LNP.

[0175] Embodiment 110: An immunogenic composition according to any one of Embodiments 90 to 109, comprising an adjuvant.

[0176] Embodiment 111: An immunogenic composition according to any one of Embodiments 90 to 110, used to induce an anti-influenza immune response in a subject.

[0177] Embodiment 112: A method for inducing an anti-influenza immune response in a subject, comprising administering to the subject an effective amount of an immunogenic composition described in any one of Embodiments 90 to 110.

[0178] Embodiment 113: The method according to Embodiment 112, which induces cross-protective immunity against heterologous influenza viruses.

[0179] Embodiment 114: The method according to Embodiment 112, which induces protective immunity against the same subtype of influenza virus.

[0180] Embodiment 115: The method according to any one of Embodiments 112 to 114, wherein the immune response is measured by an anti-hemagglutinin (HA) antibody titer, a hemagglutinin inhibitor (HAI) titer, or a microneutralization assay.

[0181] Embodiment 116: The method according to Embodiment 115, wherein the immune response is measured by HAI titer.

[0182] Embodiment 117: The method according to Embodiment 116, which 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.

[0183] Embodiment 118: The method according to Embodiment 116, wherein the HAI titer produced in the subject is at least 3 times, at least 4 times, at least 5 times, or at least 6 times the baseline HAI level.

[0184] Embodiment 119: The method according to any one of Embodiments 116 to 118, wherein the HAI titer produced by the method is at least 40 and lasts 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.

[0185] Embodiment 120: The method according to Embodiment 115, wherein the immune response is measured by anti-HA antibody titer approximately 7, 14, 21, 28, or 35 days after vaccination.

[0186] Embodiment 121: The method according to Embodiment 120, which induces an increase of at least 50 times, at least 100 times, at least 500 times, at least 1,000 times, at least 5,000 times, or at least 10,000 times in the anti-HA IgG antibody titer of the subject compared to before administration.

[0187] Embodiment 122: The method according to Embodiment 120 or 121, wherein the anti-HA IgG antibody titer is increased by at least 50 times and lasts 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.

[0188] Embodiment 123: The method according to any one of Embodiments 112 to 122, wherein the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, or intraperitoneally.

[0189] Embodiment 124: The method according to any one of Embodiments 112 to 123, comprising a single dose, followed optionally by a boost dose.

[0190] Embodiment 125: The method according to any one of Embodiments 112 to 124, wherein the subject is a human.

[0191] Embodiment 126: The method according to Embodiment 125, wherein the subject is 60 years of age or older.

[0192] Embodiment 127: The method according to Embodiment 125, wherein the subject is 18 years of age or younger. [5. Brief description of the drawing] [Brief explanation of the drawing]

[0193] [Figure 1] Figure 1 shows a circular RNA vaccine containing the sequences TI, HA1, L1, HA2, L2, HA3, L3, and HA4 in that order.

[0194] [Figure 2]Figure 2 shows a circular RNA vaccine containing the sequences TI, NA1, L1, NA2, L2, NA3, L3, and NA4 in that order.

[0195] [Figure 3] Figure 3 shows a circular RNA vaccine containing the sequences TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3 in that order.

[0196] [Figure 4] Figure 4 shows a circular RNA vaccine containing the sequences TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3 in that order.

[0197] [Figure 5] Figure 5 shows a circular RNA vaccine containing the TI1, HA1, TI2, and NA1 sequences in that order.

[0198] [Figure 6] Figure 6 shows a circular RNA vaccine containing the sequences TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3 in that order.

[0199] [Figure 7] Figure 7 shows a circular RNA vaccine containing the sequences TI1, HA1, TI2, HA2, TI3, and HA3 in that order.

[0200] [Figure 8] Figure 8 shows a circular RNA vaccine containing the sequences TI1, NA1, TI2, NA2, TI3, and NA3 in that order.

[0201] [Figure 9] Figure 9 shows a circular RNA vaccine containing the sequences TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3 in that order.

[0202] [Figure 10] Figure 10 shows a circular RNA vaccine containing the sequences TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3 in that order.

[0203] [Figure 11] Figure 11 shows a circular RNA vaccine containing the TI1, HA1, and L sequences in that order.

[0204] [Figure 12] Figure 12 shows a circular RNA vaccine containing the sequences TI1, HA1, L1, HA2, and L2 in that order.

[0205] [Figure 13] Figure 13 shows a circular RNA vaccine containing the sequences TI1, HA1, L1, HA3, L2, and HA2 in that order.

[0206] [Figure 14] Figure 14 shows a circular RNA vaccine containing the sequences TI1, HA1, L1, HA2, L2, HA3, L3, and HA4 in that order.

[0207] [Figure 15] Figure 15 provides representative results showing the binding IgG titer of CircRNA-PD001.

[0208] [Figure 16] Figure 16 provides representative results showing the HAI titer (H1N1) of CircRNA-PD001.

[0209] [Figure 17] Figure 17 provides representative results showing the binding IgG titer of CircRNA-PD002.

[0210] [Figure 18] Figure 18 provides representative results showing the HAI titer (H3N2) of CircRNA-PD002.

[0211] [Figure 19] Figure 19 provides representative results showing the binding IgG titer of CircRNA-PD003.

[0212] [Figure 20] Figure 20 provides representative results showing the HAI titer (Yamagata) of CircRNA-PD003.

[0213] [Figure 21] Figure 21 provides representative results showing the binding IgG titer of CircRNA-PD004.

[0214] [Figure 22] Figure 22 provides representative results showing the HAI titer (Victoria) of CircRNA-PD004.

[0215] [Figure 23] Figure 23 provides representative results showing the binding IgG titer (H1N1) of CircRNA-PD005.

[0216] [Figure 24] Figure 24 provides representative results showing the binding IgG titer (H3N2) of CircRNA-PD005.

[0217] [Figure 25] Figure 25 provides representative results showing the binding IgG titer (Yamagata) of CircRNA-PD005.

[0218] [Figure 26] Figure 26 provides representative results showing the binding IgG titer (Victoria) of CircRNA-PD005.

[0219] [Figure 27] Figure 27 provides representative results showing the binding IgG titer (H1N1) of CircRNA-PD006.

[0220] [Figure 28] Figure 28 provides representative results showing the HAI titer (H1N1) of CircRNA-PD006.

[0221] [Figure 29] Figure 29 provides representative results showing the HAI titer (Wisconsin) of CircRNA-PD007.

[0222] [Figure 30] Figure 30 provides representative results showing the HAI titer (Darwin) of CircRNA-PD007.

[0223] [Figure 31] Figure 31 provides representative results showing the HAI titer (Victoria) of CircRNA-PD007.

[0224] [Figure 32] Figure 32 provides representative results showing the HAI titer (Yamagata) of CircRNA-PD007.

[0225] [Figure 33] Figure 33 provides representative results showing the HAI titer (H1N1) of CircRNA-PD009.

[0226] [Figure 34] Figure 34 provides representative results showing the HAI titer (H3N2) of CircRNA-PD009.

[0227] [Figure 35] Figure 35 provides representative results showing the HAI titer (Victoria) of CircRNA-PD009.

[0228] [Figure 36]Figure 36 provides representative results showing the HAI titer (Yamagata) of CircRNA-PD009. [Modes for carrying out the invention]

[0229] As detailed below, in the description of the above diagram, TI refers to the translation initiation sequence, and TI1, TI2, and TI3 ​​are each independently translation initiation sequences. HA refers to the sequence encoding the hemagglutinin antigen, and HA1, HA2, HA3, and HA4 are each independently sequences encoding the hemagglutinin antigen. NA refers to the sequence encoding the neuraminidase antigen, and NA1, NA2, NA3, and NA4 are each independently sequences encoding the hemagglutinin antigen. L refers to the linker sequence, and L1, L2, L3, L4, and L5 are each independently linker sequences or are absent. [6. Modes for carrying out the invention]

[0230] This application provides immunogenic compositions comprising circular ribonucleic acid (circular RNA, circRNA) encoding an influenza virus vaccine. In some embodiments, the immunogenic compositions provided herein comprise circular RNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, 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). This application also provides methods for producing and using the same.

[0231] 6.1 Definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. Generally, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0232] As used herein, “a” or “an” may mean one or more. As used in combination with the phrase “comprising” as used in the claims herein, “a” or “an” may mean one or more.

[0233] In this specification, the term “or” in a claim is used to mean “and / or” unless it is explicitly indicated to mean only alternatives, or unless the alternatives are mutually exclusive; however, this disclosure supports the definition of alternatives and “and / or” only. In this application, “another” or “additional” may mean at least two or more. In this specification, the term “approximately” is used to indicate that a value includes the inherent error variation of the device, the method used to determine the value, or the variation present between the subjects of study. The term “approximately” encompasses the exact numerical value stated. In some embodiments, “approximately” means within ±10% of a given value or range. In certain embodiments, “approximately” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to. In some embodiments, “approximately” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to.

[0234] In this specification, "substantially absent" with respect to a particular component means that the particular component is not intentionally included in the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from accidental contamination of the composition is far less than 0.1%, preferably less than 0.05%, and more preferably less than 0.01%. Most preferably, the amount of the particular component is undetectable by standard analytical methods.

[0235] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms comprising at least two consecutive amino acids, which are chemically or biochemically modified or derivatized amino acids. In this specification, the term “peptide” refers to a type of short polypeptide. The term peptide refers to a polymer of amino acids (natural or unnatural) having a length of up to about 100 amino acid residues. For example, peptides may have lengths of about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, and about 75 to about 100 amino acid residues. In some embodiments, the peptide may have a length of 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, or about 5000 amino acid residues.

[0236] As used herein and as understood in the art, the term “antigen” refers to an immunogenic protein, i.e., a protein capable of inducing an immune response (e.g., causing the immune system to produce antibodies against an antigen). As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to fall within the scope of the antigen of interest (e.g., the HA antigen or NA antigen). Thus, the term “antigen” encompasses not only full-length proteins but also their immunogenic fragments and variants. For example, an antigen may be any immunogenic fragment of a reference protein. In addition to variants that are identical to the reference protein but cleaved, in some embodiments, an antigen may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein. The length of the antigen / antigen polypeptide may range from about 4, 6, or 8 amino acids to the full-length protein. Therefore As used herein, “hemagglutinin antigen” or “HA antigen” may be full-length hemagglutinin protein, its immunogenic fragment, or its immunogenic variant. Similarly, “neuraminidase antigen” or “NA antigen” may be full-length neuraminidase protein, its immunogenic fragment, or its immunogenic variant.

[0237] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably herein and refer to polymers or oligomers of nucleotides of any length. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated, or glycosylated), non-natural nucleotides, non-nucleotide components exhibiting similar structure and / or function to natural nucleotides (i.e., “nucleotide analogs”), and / or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The composition of nucleic acids or polynucleotides may be heterogeneous or homogeneous and may be isolated from natural resources or produced artificially or synthetically. Furthermore, nucleic acids may be DNA, RNA, or mixtures thereof and may exist permanently or transiently in single-stranded or double-stranded forms, including homo-double-stranded, hetero-double-stranded, and hybrid states. Nucleic acid structures include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 4(14):4503-10 (2002) and U.S. Patent Nos. 5,034,506), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see, e.g., Wang, Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes.

[0238] As understood in the art, nucleic acid chains have an inherent orientation, and the carbon atoms in the sugar ring are numbered sequentially from 1' to 5', with the "5' end" having a free hydroxyl group (or phosphate group) on the 5' carbon atom and the "3' end" having a free hydroxyl group (or phosphate group) on the 3' carbon atom. As used herein and as understood in the art, a nucleic acid having a particular sequence element "from 5' end to 3' end" means that these sequence elements are arranged linearly from the 5' end to the 3' end of the nucleic acid.

[0239] When referring to nucleotide sequences or protein sequences, the term "identity" is used to indicate similarity between two sequences. Sequence similarity or identity can be determined using standard methods known in the art, including but not limited to the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48, 443 (1970), the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequencing program described in Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or visual inspection methods. Another algorithm is the BLAST algorithm, as described by Altschul et al., J Mol. Biol. 215, 403-410 (1990) and Karlin et al. al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993) describes a particularly useful BLAST program. et al.,Methods in Enzymology,266,460-480(1996);blast.wustl / edu / blast / README.htm This is the WU-BLAST-2 program obtained from l. WU-BLAST-2 uses several search parameters, which can be optionally set to default values. These parameters are dynamic values ​​and are set by the program itself depending on the composition of a particular sequence and the composition of a particular database compared to the target sequence being searched. However, it is also possible to adjust the values ​​to increase sensitivity. Furthermore, there is a useful algorithm called Gap BLAST reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise noted, identity percentages in this specification are calculated using the algorithm available at the internet address blast.ncbi.nlm.nih.gov / Blast.cgi.

[0240] As used herein, the terms “complementary” and “complementarity” refer to the relationship between two nucleic acid molecules that have the ability to form hydrogen bonds with each other by traditional Watson-Crick base pairing or other non-traditional types of pairing. Two DNA / RNA strands with complementary sequences join to form a double helix that follows the Watson-Crick base pairing rule; that is, A is bonded to T(U) by two hydrogen bonds, and G is bonded to C by three hydrogen bonds. The degree of complementarity between two nucleotide sequences can be indicated by the percentage of nucleotides in one nucleotide sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with the 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 consecutive nucleotides in one nucleotide sequence are hydrogen-bonded with the same number of consecutive nucleotides in the 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 stringency conditions (high stringency conditions in some embodiments).Exemplary moderate stringency conditions include incubation overnight at 37°C in a solution containing 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 shear salmon sperm DNA, followed by washing the filter in 1x SSC at approximately 37–50°C, or substantially similar conditions, e.g., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory. A moderate stringency condition is mentioned in the Press; 4th edition (June 15, 2012). High stringency conditions include, for example, (1) conditions using low ionic strength and high temperature for washing (e.g., 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50°C), (2) using a denaturing agent such as formamide during hybridization (e.g., 50% (v / v) formamide, 0.1% bovine serum albumin (BSA) / 0.1% Ficol / 0.1% polyvinylpyrrolidone (PVP) / 50 mM sodium phosphate buffer (pH 6.5, containing 750 mM sodium chloride and 75 mM sodium citrate) at 42°C), or (3) 50% formamide, 5xSSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium phosphate This refers to a process in which sodium iophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 pg / ml), 0.1% SDS, and 10% dextran sulfate are mixed at 42°C, and washed with (i) 0.2*SSC at 42°C, (ii) 50% formamide at 55°C, and (iii) 0.1*SSC at 55°C (optionally in combination with EDTA). For explanations and details regarding the stringency of the hybridization reaction, see, for example, Sambrook (cited above) and Ausubel et al., eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 5th ed., John Wiley & Sons, Inc., Hoboken, NJ (2002).

[0241] As used herein and as understood in the art with respect to sequence elements in nucleic acid molecules, the term “operatably linked” means that these sequence elements (e.g., intron fragments, target sequences, promoters, and coding sequences) are functionally related to one another. For example, a promoter is operatably linked to a coding sequence if it controls the transcription of that sequence. Similarly, a ribosome binding site is operatably linked to a coding sequence if it is positioned to enable translation.

[0242] In relation to nucleotide sequences, the terms "hybridization" or "hybridized" refer to the binding that occurs between complementary sequences.

[0243] The term "in vitro transcription" or "IVT" refers to a general method of producing RNA in vitro, which involves synthesizing RNA from a DNA template using RNA polymerase, ribonucleotides, and appropriate buffer conditions.

[0244] As used herein, the terms “expression construct” or “expression cassette” mean a nucleotide sequence that directs translation.

[0245] In this specification, the terms “coding sequence,” “coding sequence region,” “coding region,” and “CDS” are used interchangeably to refer to the portion of nucleic acid (e.g., DNA or RNA) that is translated into protein or can be translated into protein.

[0246] The terms “reading frame,” “open reading frame,” and “ORF,” as used interchangeably herein, refer to a nucleotide sequence that begins with a start codon (e.g., ATG) and, in some embodiments, ends with a stop codon (e.g., TAA, TAG, or TGA).

[0247] As used herein, the term “regulatory elements” collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRESs), enhancers, splice junctions, etc., which comprehensively provide replication, transcription, post-transcriptional processing, and translation of coding sequences in receptor cells.

[0248] As used herein, the term “promoter” refers to a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence originates from a gene that binds to RNA polymerase and enables the initiation of transcription of a downstream (3' direction) coding sequence. This region may include a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. A “operatably positioned” or “operatably linked” promoter means that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence and controls the transcription initiation and / or expression of that sequence, and that the sequence is “under the control” and “transcriptionally regulated” of the promoter.

[0249] As used herein, the term "enhancer" refers to a nucleic acid sequence that, when placed in close proximity to a promoter, increases transcriptional activity compared to the transcriptional activity resulting from a promoter that does not contain an enhancer domain.

[0250] The terms “internal ribosome entry site,” “internal ribosome entry site sequence,” “IRES,” and “IRES sequence region” are used interchangeably herein and refer to cis-elements of viral or human cellular RNA (e.g., messenger RNA (mRNA) and / or circular RNA) that bypass the standard eukaryotic cap-dependent translation initiation step.

[0251] The terms "IRES-like sequence" and "internal ribosome entry site-like sequence" are used interchangeably in this specification and refer to unnaturally occurring nucleotide sequences that exhibit the function of naturally occurring IRESs.

[0252] The term “vector” or “construct” (sometimes called a gene delivery system or gene transport “medium”) refers to a medium used to carry genetic material (e.g., a nucleotide sequence) that is introduced into a host cell where it can be replicated and / or expressed.

[0253] As used herein, the term “treat” means to implement a protocol or plan that may involve administering one or more drugs or activators to a patient in order to reduce the signs or symptoms of a disease or the recurrence of the disease. Desired effects of treatment include a slower rate of disease progression, improvement or relief of the condition, remission, extended survival, improved quality of life, or improved prognosis. Relief or prevention may occur not only before the signs or symptoms of the disease or condition appear, but also after they appear. In this specification, “treatment” does not require complete relief of the signs or symptoms, nor does it require a cure.

[0254] In this specification, the terms “therapeutically beneficial” or “therapeutably effective,” when used in relation to a treatment, refer to the characteristics of a treatment that promote or improve the health of a 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, cancer treatment may include a reduction in tumor volume, a decrease in tumor invasiveness, a decrease in the rate of cancer growth, or a decrease in the rate of metastasis or recurrence. Cancer treatment may also refer to an extension of the survival time of a cancer patient.

[0255] In this specification, the term “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that, when administered to animals such as humans, do not cause adverse reactions, allergic reactions, or other undesirable reactions. It will be understood that, for administration to animals (e.g., humans), the formulations must meet standards for sterility, pyrogenicity, general safety, and purity, such as those required by the FDA Office of Biological Standards.

[0256] In this specification, the term “pharmaceutically acceptable carrier” includes all aqueous biocompatible solvents well known to those skilled in the art (e.g., parenteral media such as physiological saline, phosphate-buffered saline, sodium chloride, and ringel dextrose), antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, and similar materials and combinations thereof. The pH and precise concentrations of the various components in the pharmaceutical composition may be adjusted according to well known parameters.

[0257] 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 physical or chemical means.

[0258] As used herein, the term "subject" refers to any animal (e.g., mammal) that is the subject of a particular treatment, including but not limited to humans, non-human primates, canids, felines, rodents, etc. A subject may be human. A subject may have a particular disease or condition.

[0259] The nomenclature for nucleotides, nucleic acids, nucleosides, and amino acids used herein conforms to the standards of the International Union of Pure and Applied Chemistry (IUPAC) (see, for example, bioinformatics.org / smsylupac.html). Exemplary genes and polypeptides are described herein with reference to their GenBank numbers, GI numbers, and / or sequence numbers. Those skilled in the art will understand that homologous sequences can be readily identified by referring to sequence sources, including but not limited to Uniprot (https: / / www.uniprot.org / ), GenBank (ncbi.nlm.nih.gov / genbank / ), and EMBL (embl.org / ).

[0260] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that range form is for convenience and conciseness only and should not be interpreted as a strict limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0261] For nucleotide sequences in sequence listings, uracil (U) and thymine (T) nucleotides are considered equivalent and interchangeable depending on whether the sequence is derived from RNA (U) or DNA (T).

[0262] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the specific embodiments described herein. It should also be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting.

[0263] DSPC: The term "DSPC" used herein is a cylindrical lipid. DSPC is used in the synthesis of liposomes and is a lipid component of the lipid nanoparticle (LNP) system. Details are described in catalog number HY-W040193 (MedChemexpress).

[0264] ALC-0315: The term "ALC-0315" used herein is involved in the condensation of mRNA and promotes the intracellular delivery of mRNA and its release into the cytoplasm through the destabilization of pseudosomes. ALC-0315 can be used in the formation of lipid nanoparticle (LNP) delivery media. Details are described in catalog number HY-138170 (MedChemexpress).

[0265] E-1-0635: The term "E-1-0635" used herein refers to the same compound as the compound shown as E-1-0635 in Example II-123 of PCT / CN2023 / 129346.

Chemical formula

[0266] PEG-2000: The term "PEG-2000" used herein is a solvent for many substances. PEG2000 can be used as a carrier material and a modifier. PEG2000 is widely used in various pharmaceutical formulations. Details are described in catalog number HY-Y0873B of MedChemexpress.

[0267] ALC-0159: As used herein, the term "ALC-0159" refers to a polyethylene glycol (PEG) lipid complex that can be used as a vaccine excipient. Further details are provided in MedChemexpress catalog number HY-138300.

[0268] DOPE: As used herein, the term “DOPE” refers to a neutral helper lipid for cationic liposomes that enhances the transfection efficiency of naked siRNA by binding to cationic phospholipids. Further details are provided in MedChemexpress catalog number HY-112005.

[0269] LNP: As used herein, the term "LNP" refers to lipid-based nanoparticles. LNPs are novel drug delivery systems (part of nanoparticle drug delivery systems) and novel drug formulations.

[0270] PBI: As used herein, the term "PBI" refers to polybenzimidazole (PBI, an abbreviation for poly[2,2'-(m-phenylene)-5,5'-bisbenzimidazole]), a synthetic fiber with a very high decomposition temperature. It has no melting point, possesses excellent thermal and chemical stability, and does not ignite easily.

[0271] PBS: As used herein, "PBS" refers to phosphate-buffered saline (PBS), a buffer solution (pH approximately 7.4) commonly used in biological research. It is an aqueous salt solution containing disodium hydrogen phosphate, sodium chloride, and in some formulations, potassium chloride and potassium dihydrogen phosphate. This buffer helps maintain a constant pH. The osmotic pressure and ion concentration of the solution are approximately the same as those of the human body (isotonic).

[0272] 6.2 Influenza virus Influenza can cause mild to severe respiratory illness, potentially leading to hospitalization and death. The elderly and young children are at higher risk of serious influenza complications. The burden of this disease remains high, as the annual effectiveness of approved vaccines varies widely, from approximately 30% to 50%.

[0273] Influenza viruses belong to the Orthomyxoviridae family and are classified into types A, B, C, and D. Influenza types A and B are the cause of respiratory illness outbreaks, often accompanied by increased hospitalization and mortality rates. All influenza viruses are negative-strand RNA viruses with segmented genomes. Influenza types A and B contain 10 proteins, including the surface proteins hemagglutinin (HA) and neuraminidase (NA). It has eight genes that encode [something]. The influenza virus contains HA and NA proteins on the surface of its viral envelope. HA allows the virus to recognize and bind to target cells and infect them with viral RNA. NA is essential for daughter virus particles produced within infected cells to be released and spread to other cells.

[0274] In the case of influenza A viruses, they can be further classified into different subtypes depending on the differences in two surface proteins. To date, 18 hemagglutinin (HA) subtypes and 11 neuraminidase (NA) subtypes have been identified. All known subtypes of influenza A viruses have been isolated from birds and can infect various mammalian species. In nature, more than 130 combinations of influenza A subtypes have been identified, mainly from wild birds, but considering the tendency of viruses to "reassort," there may be many more combinations of influenza A subtypes. Reassortment is the process by which influenza viruses exchange genetic fragments. Reassortment occurs when two influenza viruses infect a host simultaneously and exchange 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.

[0275] Influenza B viruses infect almost exclusively humans. While not classified into subtypes, influenza B viruses can be subdivided into lineages. Currently circulating influenza B viruses belong to either the B / Yamagata (B / Yamagata / 16 / 88-like) or B / Victoria (B / Victoria / 2 / 87-like) lineages. In recent years, the frequency of B / Yamagata viruses circulating worldwide has decreased significantly compared to B / Victoria viruses. Although the mutation rate of influenza B viruses is 2-3 times slower than that of influenza A viruses, they still have a significant impact on children and young adults each year. The capsid of influenza B viruses is covered by an envelope, and the viral particle consists of the envelope, matrix proteins, nucleoprotein complex, nucleocapsid, and polymerase complex. The viral particle is spherical or filamentous, and its approximately 500 surface protrusions are composed of HA and NA. The genome of influenza B virus is 14,548 nucleotides long and consists of eight linear minus-stranded single-stranded RNA segments. This multi-segmental genome is enclosed in capsids, with each segment being enclosed in an independent nucleocapsid, and each nucleocapsid being surrounded by a single envelope.

[0276] Different subtypes of influenza A and different lineages of influenza can be further classified into clades and subclades. As is known herein and in the art, a clade is a taxonomic division based on the similarity of the HA gene sequences of influenza viruses. Viruses belonging to the same clade group are genetically related because they have similar genetic changes (such as nucleotide or amino acid mutations), but they do not share the exact same viral genome. Viruses belonging to the same clade are further classified into subclades. Clades and subclades may be genetically distinct, but not necessarily antigenically distinct. Viruses belonging to a particular clade or subclade may not have mutations that affect host immunity compared to viruses in other clades or subclades.

[0277] A key characteristic of human influenza viruses is their ability to cause antigenic changes, which occur in two ways: antigenic drift and antigenic shift.

[0278] Antigen drift is a process in which the viral hemagglutinin (HA) and neuraminidase (NA) proteins change gradually and relatively continuously. This occurs when point mutations accumulate in the HA and NA genes during replication. Both influenza A and B viruses undergo antigenic drift, giving rise to new virus strains. The emergence of these new strains necessitates frequent updates of influenza vaccine virus strains. Because antibodies against past influenza infections may not provide complete protection against new strains resulting from antigenic drift, subjects may be infected with influenza multiple times throughout their lives.

[0279] In addition to antigenic drift, influenza A viruses can also undergo antigenic shifts, which are more dramatic and rapid changes. By definition, an antigenic shift is considered to have occurred when an influenza A virus appears in humans with an HA protein, or a combination of HA and NA proteins, that has not been circulating among humans in recent years. There are at least three possible mechanisms for antigenic shifts: (a) the emergence of a new virus with new HA and NA proteins through genetic reassortment between a non-human influenza virus and a human influenza virus; (b) the direct infection of humans by influenza viruses from other animals (such as birds or pigs) without undergoing genetic reassortment; and (c) the infection of humans by non-human viruses from certain types of animals (such as birds) via an intermediate host animal (such as a pig).

[0280] Antigen drift occurs continuously, whereas antigen shift is rare and unpredictable. Antigen shift can lead to the emergence of new influenza viruses, meaning that a large portion (or all) of the world's population will not have antibodies against that virus. If a new strain causes disease in humans and has the ability to spread throughout a community through a persistent chain of human-to-human transmission, such a virus could spread globally and cause a pandemic.

[0281] The most effective way to prevent influenza virus infection is vaccination. However, immunity from the influenza virus vaccine weakens over time, so annual vaccination is recommended to prevent viral infection. Vaccination is most effective when the circulating virus closely matches the virus used in vaccine development. Because influenza viruses are constantly evolving, the WHO Global Influenza Surveillance and Response System (GISRS), comprised of national influenza centers and WHO collaborating centers worldwide, continuously monitors influenza viruses circulating among humans and updates the recommended influenza vaccine composition twice a year. Surveillance is the foundation of all efforts toward understanding, preventing, controlling, and global influenza surveillance. Such surveillance activities also provide essential information needed to understand the degree of influenza seasonality around the world and to estimate its impact and burden.

[0282] Traditionally, WHO has provided recommendations on the composition of vaccines targeting the three most representative viruses currently in circulation (two subtypes of influenza A virus and one of influenza B virus) (trivalent vaccines). Since the 2013–2014 influenza season in the Northern Hemisphere, a fourth component has been recommended and the development of quadrivalent vaccines has been supported. Quadrivalent vaccines contain, in addition to the viruses of trivalent vaccines, a second influenza B virus, which is thought to provide broader protection against influenza B virus infections.

[0283] WHO collaborates with other partners through the WHO GISRS system to monitor influenza activities worldwide and recommends the composition of seasonal influenza vaccines twice a year, in line with the influenza seasons in the Northern and Southern Hemispheres. WHO also provides guidance on the selection of vaccine formulations (Northern Hemisphere versus Southern Hemisphere) for countries in tropical and subtropical regions and supports the determination of the timing of vaccination campaigns. This organization also supports Member States in formulating prevention and control strategies. GISRS provides laboratory diagnosis of circulating influenza viruses and virological surveillance. All of these are important elements in the selection of influenza vaccine viruses and the early detection of emerging viruses with pandemic potential.

[0284] WHO recommendations are updated twice a year (February or March in the Northern Hemisphere, and September in the Southern Hemisphere), but it takes at least 6-7 months for vaccines to be administered to the public. These early recommendations are issued to allow sufficient quantities of conventional vaccines (such as attenuated virus vaccines) to be designed and manufactured before the flu season begins. However, during these 6-7 months, influenza viruses may mutate or other strains may become more widespread, potentially reducing the effectiveness of conventional vaccines. Conventional vaccines cannot be adapted as they are already in production, and designing and manufacturing new vaccines takes another 6-7 months. In contrast, the RNA vaccines described herein can overcome these challenges. Because they can be manufactured in a few weeks, they can be designed for influenza viruses that circulate closer to the flu season. Predicting influenza viruses that circulate closer to the flu season is more accurate than predictions made 6-7 months prior to the season, and the RNA vaccines described herein are more effective because they are designed to target viruses that circulate closer to the flu season.

[0285] In some embodiments, the circular RNA vaccines described herein can be formulated as supplemental booster vaccines. For example, if a conventional vaccine has been developed and subsequent predictions indicate that another influenza virus is more prevalent or infectious, a supplemental booster circular RNA vaccine can be designed and administered as described herein.

[0286] Furthermore, because circular RNA lacks the free ends necessary for degradation by exonucleases, it exhibits resistance to several RNA degradation mechanisms and has a longer half-life compared to equivalent linear RNA. Circularization can stabilize RNA polynucleotides, which generally have short half-lives, and improve their overall efficacy.

[0287] 6.3 Antigens In some embodiments, the immunogenic compositions provided herein comprise a circular RNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, wherein the Z sequence comprises an HA sequence encoding a hemagglutinin antigen or an NA sequence encoding a neuraminidase antigen. As used herein, “HA sequence” refers to a nucleotide sequence encoding a hemagglutinin antigen, and “NA sequence” refers to a nucleotide sequence encoding a neuraminidase antigen.

[0288] While hemagglutinin is a target in current influenza virus vaccines, neuraminidase is not as widely used. Adding neuraminidase as an additional target can provide additional protection in fitted years and "fallback" protection in HA drift years. In some embodiments, the circular RNA disclosed herein includes an HA sequence. In some embodiments, the disclosed circular RNA includes an NA sequence. In some embodiments, the circular RNA includes both an HA sequence and an NA sequence. In some embodiments, the hemagglutinin antigen is derived from influenza A virus. In some embodiments, the hemagglutinin antigen is derived from influenza B virus. In some embodiments, the neuraminidase antigen is derived from influenza A virus. In some embodiments, the neuraminidase antigen is derived from influenza B virus.

[0289] Influenza A viruses are classified into subtypes based on HA and NA. There are 18 hemagglutinin subtypes and 11 neuraminidase subtypes (H1-H18 and N1-N11, respectively). Currently, the subtypes of influenza A viruses that routinely circulate among humans are A(H1N1) and A(H3N2).

[0290] In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H1-H18 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H1 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H3 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H2 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H5 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H7 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H9 subtype influenza A HA antigen. In some embodiments, the circular RNA vaccine disclosed herein includes an HA sequence encoding the H1 subtype influenza A HA antigen and an HA sequence encoding the H3 subtype influenza A HA antigen.

[0291] In some embodiments, the circular RNA vaccines disclosed herein include NA sequences encoding N1-N11 subtype influenza A NA antigens. In some embodiments, the circular RNA vaccines disclosed herein include NA sequences encoding N1 subtype influenza A NA antigens. In some embodiments, the circular RNA vaccines disclosed herein include NA sequences encoding N2 subtype influenza A NA antigens. In some embodiments, the circular RNA vaccines disclosed herein include NA sequences encoding N8 subtype influenza A NA antigens. In some embodiments, the circular RNA vaccines disclosed herein include NA sequences encoding N1 subtype influenza A NA antigens and N2 subtype influenza A NA antigens.

[0292] In some embodiments, the circular RNA vaccines provided herein encode antigens derived from influenza A. The influenza A antigen may be derived from any strain known in the art. Exemplary influenza A viruses are shown in Table 1 below. [Table 1] TIFF2026523117000003.tif232170TIFF2026523117000004.tif158169

[0293] In some embodiments, the circular RNA vaccines provided in this application encode antigens derived from influenza B antigens. Influenza B antigens may be derived from any strain known in the art. Examples of influenza B viruses are shown in Table 2 below. [Table 2]

[0294] The sequences of HA antigen and NA antigen are known in the art and are available from GenBank. An exemplary amino acid sequence of hemagglutinin antigen is shown in Table 3. An exemplary amino acid sequence of neuraminidase antigen is shown in Table 4. In some embodiments, the circular RNA provided in this application comprises at least an HA sequence encoding the HA antigen listed in Table 3, or an immunogenic fragment or variant thereof. In some embodiments, the circular RNA provided in this application comprises at least an NA sequence encoding the NA antigen, or an immunogenic fragment or variant thereof, listed in Table 4. In some embodiments, the circular RNA vaccine provided in this application comprises an HA sequence encoding 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%, or at least 99% identity with an amino acid selected from the group consisting of SEQ ID NOs: 1 to 84, or an immunogenic fragment thereof. In some embodiments, the circular RNA vaccines provided in this application include an NA sequence encoding 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%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.85 to 174, or an immunogenic fragment thereof.

[0295] In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding the HA antigen of an influenza A virus strain of the A(H1N1) subtype. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding the HA antigen of an influenza A virus strain of the A(H3N2) subtype. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding the HA antigen of an influenza B virus strain of the B / Victoria lineage. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding the HA antigen of an influenza B virus strain of the B / Yamaga lineage.

[0296] In some embodiments, the circular RNA vaccine provided in this application is A(H1N1) The circular RNA vaccine provided in this application includes an NA sequence encoding the NA antigen of a subtype influenza A virus strain. In some embodiments, the circular RNA vaccine provided in this application includes an NA sequence encoding the NA antigen of an influenza A virus strain of the A(H3N2) subtype. In some embodiments, the circular RNA vaccine provided in this application includes an NA sequence encoding the NA antigen of an influenza B virus strain of the B / Victoria lineage. In some embodiments, the circular RNA vaccine provided in this application includes an NA sequence encoding the NA antigen of an influenza B virus strain of the B / Yamagata lineage.

[0297] In some embodiments, the HA antigen encoded by the circular RNA disclosed herein is full-length HA. In some embodiments, the HA antigen encoded by the circular RNA disclosed herein is an immunogenic fragment of full-length HA. In some embodiments, the immunogenic fragment includes the stem region of full-length HA. In some embodiments, the HA antigen encoded by the circular RNA disclosed herein is wild-type HA. In some embodiments, the HA antigen is modified HA. In some embodiments, the HA antigen contains at least one mutation. In some embodiments, at least one amino acid is mutated compared to the hemagglutinin wild-type sequence of influenza A or B. In some embodiments, the mutation is T2191, H371Y, I494M, H504P, M362L, HA0, APB, TB, or VASP. In some embodiments, multiple amino acids are mutated. In some embodiments, the mutation is selected from the group consisting of the formation of a disulfide in the HA stem for linking adjacent protomers, deletion of a cleavage site, and substitution of a polynucleotide cleavage site of highly pathogenic avian influenza (HPAI) with an LPAI sequence. In some embodiments, the mutation is a disulfide in the HA stem for linking adjacent protomers. In some embodiments, the mutation is a deletion of a cleavage site. In some embodiments, the mutation is substitution of a polynucleotide cleavage site (HPAI) with an LPAI sequence. As understood in the art, HPAI refers to highly pathogenic avian influenza (HPAI) virus, and LPAI refers to low pathogenic avian influenza virus.

[0298] In some embodiments, the NA antigen encoded by the circular RNA disclosed herein is full-length NA. In some embodiments, the NA antigen encoded by the circular RNA disclosed herein is an immunogenic fragment of full-length NA. In some embodiments, the NA antigen is wild-type NA. In some embodiments, the NA antigen is enzymatically active. In some embodiments, the NA antigen is modified NA, such as enzymatically inactive NA. In this specification, “enzymatically inactive NA” refers to NA that has been mutated to have no catalytic activity or minimal catalytic activity (e.g., Richard et al., J (See Clin Virol., 2008, 41(1):20-24; Yen et al., J Virol., 2006, 80(17):8787-8795). For example, in some embodiments, enzymatically inactive NA has 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 wild-type NA (for example, in enzyme activity assays known in the art). In some embodiments, at least one of Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and Tyr406 is mutated relative to the wild-type sequence of influenza A or B neuraminidase. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or all 8 amino acids are mutated. In some embodiments, at least one of Glu119, Arg156, Trp178, Ser179, Asp198, he222, Glu227, His274, Glu277, Asn294, and Glu425 is mutated from the wild-type sequence of influenza A or B neuraminidase. In some embodiments, the mutation is R118K, D151G, or E227D. In some embodiments, the mutation is a deletion in the cytoplasmic tail (dcytT). In some embodiments, the mutation is a deletion of an amino acid in the stalk region. In some embodiments, The mutation is a deletion of 15 amino acids in the stalk region (stalk_dl5). In some embodiments, the mutation is a deletion of 30 amino acids in the stalk region (stalk_d30). In some embodiments, the mutation is an insertion of amino acids in the stalk region. In some embodiments, the mutation is an insertion of 15 amino acids into the stalk region (stalk_insl5).

[0299] Exemplary HA and NA antigens are publicly known in the art and are available, for example, in the NCBI influenza virus resource, the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) influenza research database, and on the GISRS website.

[0300] The circular RNA vaccine provided in this application may contain multiple antigen-coding sequences and therefore may contain multiple HA sequences and / or NA sequences. In some embodiments, the circular RNA vaccine provided in this application may contain 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 circular RNA vaccine provided in this application may contain one, two, three, four, five, six, seven, eight, nine, or ten HA sequences encoding one, two, three, four, five, six, seven, eight, nine, or ten HA antigens. In some embodiments, the circular RNA vaccine provided in this application may contain one, two, three, four, five, six, seven, eight, nine, or ten NA sequences encoding one, two, three, four, five, six, seven, eight, nine, or ten NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA sequences encoding 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 antigens. For example, in some embodiments, the circular RNA vaccine provided in this application comprises 4 HA sequences encoding 4 HA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises 4 NA sequences encoding 4 NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises 4 HA sequences encoding 4 HA antigens and 4 NA sequences encoding 4 NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises 2 HA sequences encoding 2 HA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises 2 NA sequences encoding 2 NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises two HA sequences encoding two HA antigens and two NA sequences encoding two NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises three HA sequences encoding three HA antigens.In some embodiments, the circular RNA vaccine provided in this application comprises three NA sequences encoding three NA antigens. In some embodiments, the circular RNA vaccine provided in this application comprises three HA sequences encoding three HA antigens and three NA sequences encoding three NA antigens.

[0301] In some embodiments, the circular RNA vaccine provided in this application comprises HA sequences and NA sequences that comprehensively encode eight antigens (H1 antigen, H3 antigen, N1 antigen, N2 antigen, HA B / Yamagata antigen, HA B / Victoria antigen, NA B / Yamagata antigen, and NA B / Victoria antigen).

[0302] In some embodiments, the vaccines of the present disclosure are designed to combat seasonal influenza and are therefore vaccines for use in the coming season in the Northern or Southern Hemisphere. Based on an understanding of the influenza virus circulating at a particular time (e.g., lineage, strain, and / or subtype of the virus), the vaccine is intended for use in the coming influenza season (e.g., in a specific geographical area, e.g., the coming season in the Northern or Southern Hemisphere). The circular RNA vaccines are designed to combat viruses that are expected to be circulating or spreading. Accordingly, the circular RNA vaccines of the present invention include circular RNA encoding the hemagglutinin antigen and / or neuraminidase antigen of an influenza virus circulating at the time of vaccine design. Exemplary circular RNA vaccines disclosed herein encode the HA antigen and / or NA antigen of circulating H1N1 and H3N2 viruses. The circular RNA vaccines disclosed herein may include a combination of HA sequences encoding the HA antigen of each subtype or major subtype of circulating influenza A and HA sequences encoding the HA antigen of each lineage (or major influenza lineage) of circulating influenza B. In exemplary embodiments, the circular RNA vaccines disclosed herein also include an NA sequence encoding the NA antigen corresponding to the selected HA antigen. A major virus, or currently circulating major virus, is a virus detected in a human population at a localized epidemic frequency or at a frequency exceeding a certain threshold, which will be understood by those skilled in the art to be an essential criterion for demonstrating the spread of a strain within a human population, for example, within a human population representing the Northern or Southern Hemisphere.

[0303] In some embodiments, the circular RNA provided in this application includes a Z sequence containing an HA sequence and / or an NA sequence encoding an HA antigen and / or an NA antigen selected according to standardized criteria used in GISRS. In some embodiments, the circular RNA vaccine provided in this application includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a sequence selected according to standardized criteria used in GISRS.

[0304] Methods for characterizing the target antigens of circulating influenza viruses are known in the art, and include, for example, hemagglutination inhibition tests (HAIs), neuraminidase inhibition tests (NAIs), microneutralization assays, viral identification by immunofluorescence antibody staining, and genetic characterization.

[0305] The HAI test is a classic experimental procedure for classifying or subtyping hemagglutinating viruses, and for determining the antigenic characteristics of influenza virus isolates, and requires that the reference antiserum used contains antibodies against currently circulating viruses (see, for example, Pedersen JC Methods Mol Biol. 2014;1161:11-25). The antiserum used is based on the wild-type strain, or an antigen preparation derived from a highly proliferative reassembly using the wild-type strain or an antigenically equivalent strain.

[0306] To perform the assay, serial dilutions of the virus are prepared across each row of a 96-well microtiter plate with a U-bottom or V-bottom. For example, the highest concentration sample in the first well is diluted 1 / 5 times the stock, and subsequent wells are diluted 2 times (1 / 10, 1 / 20, 1 / 40, etc.). The last well serves as a negative control without the virus. Each row of the plate typically contains the same dilution pattern for a different virus. After serial dilutions, standardized concentrations of red blood cells (RBCs) are added to each well and gently mixed. The plate is incubated at room temperature. After the incubation period, the assay can be analyzed to distinguish between agglutinated and non-agglutinated wells. The relative concentration, i.e., titer, of the virus sample is based on the well that shows the last agglutinated appearance just before the pellet is observed.

[0307] Serological methods such as HAI testing are essential for many epidemiological and immunological studies, as well as for evaluating antibody responses after vaccination. Serological methods are also very useful in situations where viral identification is impossible (e.g., after viral shedding has stopped). HAI testing is used to identify influenza viruses that are antigenically similar to influenza viruses derived from the previous season's vaccine. "Antigenically similar" here refers to viruses whose HAI titer difference is within two dilution factors.

[0308] Neuraminidase inhibition (NAI) assays are laboratory procedures for identifying neuraminidase (NA) glycoprotein subtypes in influenza viruses, or the NA subtype specificity of antibodies against influenza viruses (see, e.g., Pedersen JC, Methods Mol Biol. 2014;1161:27-36). Procedures for serologically determining NA glycoprotein subtypes are essential for the identification and classification of avian influenza (AI) viruses.

[0309] There are two basic forms of influenza virus NA assays that use different substrate molecules. One is a long-term assay (such as enzyme-binding lectin assay (ELLA)) that uses a large substrate such as fetwin, and the other is a newer assay that uses a small substrate molecule. Fetwin-based methods are used to measure the potency of viral NA and determine a standardized NA dose to be used in NA inhibition (NAI) assays. Once the standardized dose is determined, it is added to serial dilutions of test antiserum, negative control serum, and reference anti-NA serum. The inhibitory effect of the serum on NA activity can then be determined, and the NAI titer can be calculated. A small substrate-based method may be a fluorescence assay using the substrate 2-(4-methylumbelliferyl)-aDN-acetylneuraminic acid (MUNANA). In addition to the test antiserum with serially diluted substrate, the cleavage of the MUNANA substrate by NA releases the fluorescent substance methylumbelliferone. The inhibitory effect of the serum on influenza virus NA is determined based on the serum concentration required to reduce NA activity by 50% (IC50 value). Alternatively, a small substrate-based method may be a chemiluminescent (CL) assay using a 1,2-dioxetane sialytic acid derivative (NA-Star) substrate or a modified NA-XTD substrate. The CL assay provides a long-lasting chemiluminescent signal, and the IC50 values ​​of the neuraminidase inhibitor are achieved over a range of viral dilutions.

[0310] In some embodiments, the HA antigen and / or NA antigen are selected using an HAI assay or NAI assay to identify circulating influenza viruses antigenically similar to the influenza viruses contained in the previous season's vaccine. In some embodiments, influenza viruses are considered antigenically similar if the difference in HAI titers of the influenza viruses is within two dilution factors.

[0311] Serological methods such as the HAI test rarely provide an early diagnosis of acute influenza virus infection. While conventional influenza virus neutralization tests (based on inhibition of cytopathic effect formation in MDCK cell cultures) are effective, a method combining a micro-neutralization assay using microtiter plates with enzyme-linked immunosorbent assay (ELISA) can detect virus-infected cells within two days. The micro-neutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies against influenza virus in human and animal serum, and in some embodiments, it includes the detection of human antibodies against avian subtypes. If a novel virus is identified, detection can be performed immediately, usually before the preparation of purified viral proteins required for other assays is completed.

[0312] Immunofluorescence (IFA) staining of virus-infected cells in original clinical specimens and field isolates is a rapid and highly sensitive method for diagnosing respiratory and other viral infections. In some embodiments, IFA staining is performed on isolates rather than original clinical specimens. This allows the present virus to be amplified first and used in other studies as needed. Because commercially available rapid diagnostic tests for influenza differ in the types of specimens required, complexity of the tests, specificity, and sensitivity, these tests are recommended to be used in conjunction with other clinical tests.

[0313] Direct molecular identification of influenza isolates is a rapid and powerful technique. Reverse transcription polymerase chain reaction (RT-PCR) allows for the reverse transcription of template viral RNA to generate complementary DNA (cDNA), which can then be amplified and detected. This method can be used directly on clinical samples and provides rapid results, significantly accelerating outbreak investigations for respiratory diseases (such as influenza). For example, genetic analysis of influenza virus genes (particularly the HA and NA genes) can be used to identify unknown influenza viruses when antigenic characteristics cannot be determined. Genetic analysis can also be used to monitor the evolution of influenza viruses and determine the degree of correlation between viruses originating from different geographical regions and viruses collected at different times of the year.

[0314] Examples of circulating influenza A viruses include, for example, influenza A(H1N1)pdm09 virus, A(H3N2), and influenza B viruses (B / Victoria / 2 / 87 and B / Yamagata / 16 / 88). In some embodiments, the influenza A(H1N1)pdm09 virus contains an HA gene belonging to phylogenetic group 5a.2a. In some embodiments, the influenza A(H1N1)pdm09 virus contains an HA gene belonging to phylogenetic group 5a.2a.1. In some embodiments, the influenza A(H1N1)pdm09 virus contains an HA gene belonging to phylogenetic group 5a.1. In some embodiments, the influenza A(H3N2) virus contains an HA gene belonging to phylogenetic group 1 (e.g., 3C.2a1b.2a.1). In some embodiments, the influenza A(H3N2) virus contains an HA gene belonging to phylogenetic group 2 (e.g., 3C.2alb.2a.2). In some embodiments, the B / Victoria strain of influenza B virus belongs to gene clade 1A.3.

[0315] 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 / Yamaga lineage virus is B / Phuket / 3073 / 2013.

[0316] In some embodiments, the circular RNA provided in this application encodes an influenza virus antigen variant. An antigen variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from the wild-type, native-type, or reference sequence. The antigen / polypeptide variant may have substitutions, deletions, and / or insertions at specific positions in its amino acid sequence compared to the native-type or reference sequence. Typically, the variant has at least 50% identity with the wild-type, native-type, or reference sequence. In some embodiments, the variant has at least 80% or at least 90% homology with the wild-type, native-type, or reference sequence.

[0317] The mutant antigens / polypeptides encoded by the nucleic acids of this disclosure may include amino acid changes that confer any of several desirable properties, such as enhanced immunogenicity, enhanced expression, and / or improved stability or PK / PD properties in a subject. Mutant antigens / polypeptides can be prepared using conventional mutagenesis techniques and, if necessary, assays can be performed to determine whether they possess the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and examples of such assays are described in the Examples section. Similarly, the PK / PD properties of protein variants are also known in the art. Using field-proven techniques, this can be measured, for example, by measuring changes in antigen expression over time in vaccinated subjects and / or examining the persistence of the induced immune response. The stability of proteins encoded by mutant nucleic acids can be measured by assaying thermal stability or stability under urea denaturation, or by using predictions based on computer simulations. Such experimental methods and computer simulation techniques are known in the art.

[0318] Accordingly, the scope of this application encompasses polypeptides (e.g., antigens) encoded in circular RNA having substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, particularly polypeptide (e.g., antigen) sequences disclosed herein. For example, a sequence tag or amino acids (such as one or more lysines) can be added to a peptide sequence (e.g., N-terminus or C-terminus). Sequence tags can be used for the detection, purification, or localization of peptides. Lysines can be used to increase the solubility of peptides or to enable biotinylation. Alternatively, a shortened sequence can be provided by optionally deleting amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein. Alternatively, specific amino acids (e.g., C-terminal or N-terminal residues) can be deleted depending on the intended use of the sequence, such as soluble or as part of a larger sequence bound to a solid support. In some embodiments, sequences (or sequences encoding sequences) such as signal sequences, termination sequences, transmembrane domains, linkers, and multimerization domains (e.g., Foldon regions) can be substituted with alternative sequences that achieve the same or similar function. In some embodiments, stability can be improved by filling cavities within the protein core, for example, by introducing larger amino acids. In some embodiments, stability can be improved by substituting the embedded hydrogen bond network with hydrophobic residues. In some embodiments, glycosylation sites can be removed and replaced with appropriate residues. Such sequences are readily identifiable to those skilled in the art. It should also be understood that some of the sequences provided herein include sequence tags or terminal peptide sequences (e.g., N-terminus or C-terminus) that can be removed before use, for example, in the preparation of circular RNA vaccines.

[0319] Exemplary amino acid sequences of the hemagglutinin antigen and neuraminidase antigen of circulating viruses are shown in Tables 5A, 7A, or 8A (hemagglutinin antigen) and 5B, 7B, or 8B (neuraminidase antigen). In some embodiments, the circular RNA provided herein comprises at least an HA sequence encoding the HA antigen listed in Table 5A, 7A, or 8A, or an immunogenic fragment or variant thereof. In some embodiments, the circular RNA provided herein comprises at least an NA sequence encoding the NA antigen listed in Table 5B, 7B, or 8B, or an immunogenic fragment or variant thereof.

[0320] In some embodiments, the circular RNA vaccines provided in this application include 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 175, 177, 179-181, 234-236, and 240-257, or an immunogenic fragment thereof.

[0321] 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 with SEQ ID NO: 175 or its immunogenic fragment. In some embodiments, the HA sequence encodes an HA antigen having at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identity with SEQ ID NO: 177 or its immunogenic fragment. 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 with SEQ ID NO: 179 or its immunogenic fragment. 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 with SEQ ID NO: 180 or its immunogenic fragment. 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 with SEQ ID NO: 181 or its immunogenic fragment. 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 with SEQ ID NO: 234 or its immunogenic fragment. 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 with SEQ ID NO: 235 or its immunogenic fragment.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 with SEQ ID NO: 236 or its immunogenic fragment. 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 with SEQ ID NO: 240 or its immunogenic fragment. 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 with SEQ ID NO: 241 or its immunogenic fragment. 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 with SEQ ID NO: 242 or its immunogenic fragment. 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 with SEQ ID NO: 243 or its immunogenic fragment. 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 with SEQ ID NO: 244 or its immunogenic fragment.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 with SEQ ID NO: 245 or its immunogenic fragment. In some embodiments, the HA sequence has at least 90%, at least 9% identity with SEQ ID NO: 246 or its immunogenic fragment. The HA sequence encodes an HA antigen having 1%, 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. 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 with SEQ ID NO: 247 or its immunogenic fragment. 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 with SEQ ID NO: 248 or its immunogenic fragment. 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 with SEQ ID NO: 249 or its immunogenic fragment. 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 with SEQ ID NO: 250 or its immunogenic fragment. 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 with SEQ ID NO: 251 or its immunogenic fragment.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 with SEQ ID NO: 252 or its immunogenic fragment. 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 with SEQ ID NO: 253 or its immunogenic fragment. 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 with SEQ ID NO: 254 or its immunogenic fragment. 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 with SEQ ID NO: 255 or its immunogenic fragment. 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 with SEQ ID NO: 256 or its immunogenic fragment. 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 with SEQ ID NO: 257 or its immunogenic fragment.

[0322] In some embodiments, the circular RNA vaccine provided in this application comprises 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, comprising at least 90%, at least 91%, and at least 92% of the original amino acid sequence, at least 92% of the original amino acid sequence, at least 91% of the original amino acid sequence, The NA sequence comprises an NA sequence encoding an NA antigen having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. 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 with SEQ ID NO: 176 or its immunogenic fragment. 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 with SEQ ID NO: 178 or its immunogenic fragment. In some embodiments, the NA sequence encodes 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 with SEQ ID NO: 237 or its immunogenic fragment. In some embodiments, the NA sequence encodes 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 with SEQ ID NO: 238 or its immunogenic fragment. In some embodiments, the NA sequence encodes 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 with SEQ ID NO: 239 or its immunogenic fragment.

[0323] In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen. In some embodiments, the HA antigen is derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from the A / Wisconsin / 588 / 2019(H1N1) strain. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from the A / Wisconsin / 67 / 2022(H1N1) strain. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from the A / Darwin / 6 / 2021(H3N2) strain. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from the A / Sydney / 5 / 2021(H1N1) strain. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from strain B / Austria / 1359417 / 2021. In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence encoding an HA antigen derived from strain B / Phuket / 3073 / 2013.

[0324] In some embodiments, the HA antigen is the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 175. In some embodiments, the HA antigen is the HA protein of the A / Darwin / 6 / 2021(H3N2)-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 177. In some embodiments, the HA antigen is the HA protein of the B / Austria / 1359417 / 2021(B / Victoria lineage)-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 177. It has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the HA antigen is the HA protein of the B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 180. In some embodiments, the HA antigen is the HA protein of the A / Wisconsin / 67 / 2022 (H1N1)pdm09-like influenza virus. 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 with the amino acid sequence of SEQ ID NO: 235.

[0325] The circular RNA vaccines provided in this application can encode HA antigens and / or NA antigens of epidemic strains / lineages representing multiple different influenza clades and subclades, thereby preparing a more effective vaccine to combat the upcoming influenza season. In some embodiments, the circular RNA vaccines provided in this application include multiple HA sequences encoding HA antigens derived from the most prevalent influenza strains. For example, in some embodiments, the circular RNA vaccines provided in this application include multiple HA sequences encoding HA antigens derived from the most prevalent A / H1N1 strains (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and A / H3N2 strains (e.g., A / Darwin / 9 / 2021, A / Darwin / 6 / 2021). The circular RNA vaccine provided in this application may further include HA sequences encoding HA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021). The circular RNA vaccine provided in this application may further include HA sequences encoding HA antigens derived from the most prevalent B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013). In some embodiments, the circular RNA vaccine provided in this application includes multiple HA sequences encoding HA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013).

[0326] In some embodiments, the circular RNA vaccine provided in this application includes multiple NA sequences encoding NA antigens derived from the most prevalent influenza strains. In some embodiments, the circular RNA vaccine provided in this application includes multiple NA sequences encoding NA antigens derived from the most prevalent A / H1N1 strains (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and A / H3N2 strains (e.g., A / Darwin / 9 / 2021, A / Darwin / 6 / 2021). The circular RNA vaccine provided in this application may further include NA sequences encoding NA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021). The circular RNA vaccines provided in this application may include NA sequences encoding NA antigens derived from the most prevalent B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013). In some embodiments, the circular RNA vaccines provided in this application may include multiple NA sequences encoding NA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013).

[0327] In some embodiments, the circular RNA vaccine provided in this application is most prevalent The circular RNA vaccine provided in this application includes a plurality of HA sequences and NA sequences encoding HA antigens and NA antigens derived from influenza strains. For example, in some embodiments, the circular RNA vaccine provided in this application includes a plurality of HA sequences and NA sequences encoding HA antigens and NA antigens derived from the most prevalent A / H1N1 strains (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and A / H3N2 strains (e.g., A / Darwin / 9 / 2021, A / Darwin / 6 / 2021). The circular RNA vaccine provided in this application may further include HA sequences and NA sequences encoding HA antigens and NA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021). The circular RNA vaccine provided in this application may further include HA and NA sequences encoding HA and NA antigens derived from the most prevalent B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013). In some embodiments, the circular RNA vaccine provided in this application includes multiple HA and NA sequences encoding HA and NA antigens derived from the most prevalent B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013).

[0328] In some embodiments, the circular RNA vaccines provided in this application may further include HA sequences and / or NA sequences encoding HA and NA of a second circulating strain, such as A / H1N1, A / H3N2, B / Victoria lineage, and / or B / Yamagata lineage. For example, in some embodiments, the circular RNA vaccines provided in this application may include the most circulating A / H1N1 strains (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and A / H3N2 strains (e.g., A / Darwin / 9 / 2021, A / Darwin / 6 / 2021), and the most common B / Victoria lineage (e.g., The formulation comprises multiple HA sequences and / or NA sequences encoding HA antigens and / or NA antigens derived from the most prevalent B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013) of arbitrary selection, and further comprises HA sequences and / or NA sequences encoding HA and NA of the second-prevalent strains A / H1N1, A / H3N2, B / Victoria lineage and / or B / Yamagata lineage.

[0329] 6.4 Circular RNA (circRNA) The circular RNA (circRNA) vaccines disclosed in this application offer unique advantages over conventional protein-based vaccination approaches (e.g., recombinant protein production technologies) that purify or prepare protein antigens in vitro. The vaccines of this disclosure feature circular RNA encoding a desired antigen, which, when introduced into the body—i.e., administered in vivo to a mammalian subject (e.g., a human)—causes cells in the body to express the desired antigen. Upon delivery to and uptake by cells in the body, the circular RNA is translated in the cytoplasm, generating protein antigens through host cell mechanisms. These protein antigens are presented, inducing adaptive humoral and cellular immune responses. Since neutralizing antibodies target the expressed protein antigens, these protein antigens are considered relevant target antigens in vaccine development. The circular RNAs provided in this application, when administered to a subject, reduce the induced innate immune response compared to their mRNA counterparts.

[0330] In some embodiments, the application provides an immunogenic composition comprising a circular RNA, the circular RNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, wherein the Z sequence comprises an HA sequence encoding a hemagglutinin antigen or an NA sequence encoding a neuraminidase antigen. As described in the above section, the circular RNA disclosed in the application may have at least one HA sequence and / or at least one NA sequence. The circular RNA disclosed in this application may have one HA sequence or one NA sequence. The circular RNA disclosed in this application may have one HA sequence and one NA sequence. The circular RNA disclosed in this application may have multiple HA sequences or NA sequences. Furthermore, the circular RNA disclosed in this application may have multiple HA sequences and multiple NA sequences. Furthermore, in some embodiments, the circular RNA may have one TI sequence. In some embodiments, the circular RNA may have two or more TI sequences.

[0331] 6.4.1 Structure having one TI array In some embodiments, the circular RNA provided in this application comprises 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 derived from a first influenza virus. The first influenza virus may be of type A. The first influenza virus may be of type B.

[0332] In some embodiments of the circular RNA disclosed in this application, the Z sequence comprises a first HA(HA1) sequence encoding an HA antigen derived from a first influenza virus, and further comprises a second HA(HA2) sequence encoding an HA antigen derived from a second influenza virus. The second influenza virus may be of type A. The second influenza virus may be of type B. Thus, in some embodiments, the first and second influenza viruses may be two subtypes of type A (i.e., A1 and A2). In some embodiments, the first and second influenza viruses may be two subtypes of type B virus (i.e., B1 and B2). In some embodiments, one of the two influenza viruses may be of type A and the other may be of type B. The HA1 and HA2 sequences may be linked by a linker(L) sequence. Accordingly, in some embodiments, the circular RNA provided in this application comprises TI, HA1, L, and HA2 in this order, where L is a linker sequence or is absent, and HA1 and HA2 encode HA antigens of first and second influenza viruses which are (1) types A1 and A2, (2) types B1 and B2, (3) types A and B, or (4) types B and A, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first and second influenza viruses may be types A1 and A2, respectively. The first and second influenza viruses may be types B1 and B2, respectively. The first and second influenza viruses may be types A and B, respectively. The first and second influenza viruses may be types B and A, respectively.

[0333] In some embodiments of the circular RNA disclosed in this application, the Z sequence includes 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 includes a third HA(HA3) sequence encoding an HA antigen from a third influenza virus. The third influenza virus may be type A. The third influenza virus may be type B. Therefore, in some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B viruses (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. The HA sequences can be arranged in any order. The HA sequences can be linked by a linker sequence. Therefore, in some embodiments, the circular RNA provided in this application comprises the sequences TI, HA1, L1, HA2, L2, and HA3 in this order, where L1 and L2 are independently linker sequences or present. HA1, HA2, and HA3 each encode the HA antigens of the first, second, and third influenza viruses, which are (1) A1, A2, and B types, (2) A1, B, and A2 types, or (3) B, A1, and A2 types, (4) A, B1, and B2 types, (5) B1, A, and B2 types, or (6) B1, B2, and A types, respectively, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, and third influenza viruses may be A1, A2, and B types, respectively. The first, second, and third influenza viruses may be A1, B, and A2 types, respectively. The first, second, and third influenza viruses may be B, A1, and A2 types, respectively. The first, second, and third influenza viruses may be of type A, type B1, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type A, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type B2, and type A, respectively.

[0334] In some embodiments of the circular RNA disclosed in this application, the Z sequence includes 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 includes a fourth HA(HA4) sequence. The fourth influenza virus may be type A. The fourth influenza virus may be type B. An illustrative diagram is shown in Figure 1. Thus, in some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the other two may be two subtypes of type B virus (i.e., B1 and B2). In some embodiments, three of the four influenza viruses may be three subtypes of type A (i.e., A1, A2, and A3), and the fourth may be type B. In some embodiments, three of the four influenza viruses may be three subtypes of type B virus (i.e., B1, B2, and B3), and the fourth may be type A. In some embodiments, all four influenza viruses may be type A. In some embodiments, all four influenza viruses may be type B. The HA sequences may be arranged in any order. In some embodiments, the circular RNA contains the sequences TI, HA1, L1, HA2, L2, HA3, L3, and HA4 in this order, where L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2 types, respectively; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, third, and fourth influenza viruses may be A1, A2, B1, and B2 types, respectively. The first, second, third, and fourth influenza viruses may be of types B1, A1, B2, and A2, respectively.The first, second, third, and fourth influenza viruses may be of types B1, A1, B2, and A2, respectively.

[0335] In some embodiments, the circular RNA provided in this application comprises 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 derived from a first influenza virus. The first influenza virus may be of type A. The first influenza virus may be of type B.

[0336] In some embodiments of the circular RNA disclosed in this application, the Z sequence comprises a first NA(NA1) sequence encoding an NA antigen derived from a first influenza virus, and further The sequence includes a second NA(NA2) sequence encoding an NA antigen derived from a second influenza virus. The second influenza virus may be type A. The second influenza virus may be type B. Therefore, in some embodiments, the first and second influenza viruses may be two subtypes of type A (i.e., A1 and A2). In some embodiments, the first and second influenza viruses may be two subtypes of type B virus (i.e., B1 and B2). In some embodiments, one of the two influenza viruses may be type A and the other may be type B. The NA1 and NA2 sequences may be linked by a linker (L) sequence. Accordingly, in some embodiments, the circular RNA provided in this application comprises TI, NA1, L, and NA2 in this order, where L is a linker sequence or is absent, and NA1 and NA2 encode NA antigens of first and second influenza viruses which are (1) types A1 and A2, (2) types B1 and B2, (3) types A and B, or (4) types B and A, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first and second influenza viruses may be types A1 and A2, respectively. The first and second influenza viruses may be types B1 and B2, respectively. The first and second influenza viruses may be types A and B, respectively. The first and second influenza viruses may be types B and A, respectively.

[0337] In some embodiments of the circular RNA disclosed in this application, the Z sequence includes 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 includes a third NA(NA3) sequence encoding an NA antigen from a third influenza virus. The third influenza virus may be type A. The third influenza virus may be type B. Therefore, in some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B virus (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. The NA sequences can be arranged in any order. The NA sequences can be linked by a linker sequence. Accordingly, in some embodiments, the circular RNA provided in this application comprises the sequences TI, NA1, L1, NA2, L2, and NA3 in this order, where L1 and L2 are independently linker sequences or are absent, and NA1, NA2, and NA3 encode the NA antigens of first, second, and third influenza viruses, which are (1) A1, A2, and B types, (2) A1, B, and A2 types, or (3) B, A1, and A2 types, (4) A, B1, and B2 types, (5) B1, A, and B2 types, or (6) B1, B2, and A types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first, second, and third influenza viruses may be A1, A2, and B types, respectively. The first, second, and third influenza viruses may be of type A1, type B, and type A2, respectively.The first, second, and third influenza viruses may be of type A, type B1, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type A, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type B2, and type A, respectively.

[0338] In some embodiments of the circular RNA disclosed in this application, the Z sequence comprises a first NA(NA1) sequence encoding an NA antigen derived from a first influenza virus, and a second NA sequence. The sequence includes a second NA(NA2) sequence encoding an NA antigen derived from influenza virus, a third NA(NA3) sequence encoding an NA antigen derived from a third influenza virus, and further includes a fourth NA(NA4) sequence. The fourth influenza virus may be type A. The fourth influenza virus may be type B. An illustrative diagram is shown in Figure 2. Thus, in some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the other two may be two subtypes of type B viruses (i.e., B1 and B2). In some embodiments, three of the four influenza viruses may be three subtypes of type A (i.e., A1, A2, and A3), and the fourth may be type B. In some embodiments, three of the four influenza viruses may be three subtypes of type B viruses (i.e., B1, B2, and B3), and the fourth may be type A. In some embodiments, all four influenza viruses may be type A. In some embodiments, all four influenza viruses may be type B. The NA sequences can be arranged in any order. In some embodiments, the circular RNA contains the sequences TI, NA1, L1, NA2, L2, NA3, L3, and NA4 in this order, where L1, L2, and L3 are independently linker sequences or are absent, and NA1, NA2, NA3, and NA4 encode first, second, third, and fourth influenza viruses, respectively, which are (1) A1, A2, B1, and B2 types; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first, second, third, and fourth influenza viruses may be of types A1, A2, B1, and B2, respectively. The first, second, third, and fourth influenza viruses may be of types B1, A1, B2, and A2, respectively. The first, second, third, and fourth influenza viruses may be of types B1, A1, B2, and A2, respectively.The first, second, third, and fourth influenza viruses may be of types B1, B2, A1, and A2, respectively.

[0339] In some embodiments of the circular RNA disclosed in this application, the Z sequence may include 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 derived from a first influenza virus. The first influenza virus may be type A. The first influenza virus may be type B. The HA1 and NA1 sequences may be linked by a linker sequence. The HA1 and NA1 sequences may be arranged in any order. In some embodiments, the circular RNA includes the TI, HA1, L, and NA1 sequences in this order, where L is either a linker sequence or absent. In some embodiments, the circular cRNA includes the TI, NA1, L, and HA1 sequences in this order.

[0340] In some embodiments of the circular RNA disclosed in this application, the Z sequence includes a first HA(HA1) sequence and a first NA(NA1) sequence encoding HA and NA antigens derived from a first influenza virus, and further includes a second HA(HA2) sequence and a second NA(NA2) sequence encoding HA and NA antigens derived from a second influenza virus. The second influenza virus may be type A. The second influenza virus may be type B. Thus, in some embodiments, the first and second influenza viruses may be two subtypes of type A (i.e., A1 and A2). In some embodiments, the first and second influenza viruses may be two subtypes of type B virus (i.e., B1 and B2). In some embodiments, one of the two influenza viruses may be type A and the other may be type B. Adjacent HA and NA sequences may be linked by a linker (L) sequence. Therefore, in some embodiments, the circular RNAs provided herein are sequenced in the following order: (1) TI, HA1, L1, NA1, L2, HA2, L3, and NA2; (2) T (1) I, NA1, L1, HA1, L2, NA2, L3, and HA2; (2) TI, HA1, L1, HA2, L2, NA1, L3, and NA2; or (3) TI, NA1, L1, NA2, L2, HA1, L3, and HA2, where L1, L2, and L3 are independently linker sequences or are absent. In some embodiments, the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; (3) A and B types; or (4) B and A types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first and second influenza viruses may be A1 and A2 types, respectively. The first and second influenza viruses may be B1 and B2 types, respectively. The first and second influenza viruses may be of type A and type B, respectively.

[0341] In some embodiments of the circular RNA disclosed in this application, the Z sequence may include a first HA(HA1) sequence and a first NA(NA1) sequence encoding HA and NA antigens derived from a first influenza virus, a second HA(HA2) sequence and a second NA(NA2) sequence encoding HA and NA antigens derived from a second influenza virus, and further may include a third HA(HA3) sequence and a third NA(NA3) sequence encoding HA and NA antigens derived from a third influenza virus. The third influenza virus may be type A. The third influenza virus may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. The HA and NA sequences can be arranged in any order. Adjacent HA and NA sequences can be linked by a linker sequence. Therefore, in some embodiments, the circular RNA provided in this application comprises sequences 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, where L1, L2, L3, L4, and L5 are independently linker sequences or are absent. A circular RNA can contain the sequences TI, HA1, L1, NA1, L2, HA2, L3, NA2, L4, HA3, L5, and NA3 in this order. An example diagram is shown in Figure 3.A circular RNA can contain the TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3 sequences in this order. An exemplary diagram is shown in Figure 4. A circular RNA can contain the TI, HA1, L1, HA2, L2, HA3, L3, NA1, L4, NA2, L5, and NA3 sequences in this order. A circular RNA can contain the TI, NA1, L1, NA2, L2, NA3, L3, HA1, L4, HA2, L5, and HA3 sequences in this order. In some embodiments, the first, second, and third influenza viruses are (1) types A1, A2, and B, (2) types A1, B, and A2, or (3) types B, A1, and A2, (4) types A, B1, and B2, (5) types B1, A, and B2, or (6) types B1, B2, and A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, and third influenza viruses may be types A1, A2, and B, respectively. The first, second, and third influenza viruses may be types A1, B, and A2, respectively. The influenza viruses can be of types B, A1, and A2, respectively. The first, second, and third influenza viruses can be of types A, B1, and B2, respectively. The first, second, and third influenza viruses can be of types B1, A, and B2, respectively. The first, second, and third influenza viruses can be of types B1, B2, and A, respectively.

[0342] 6.4.2 Structure having two TI arrays In some embodiments, the circular RNA disclosed herein comprises, in this 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 Z2 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

[0343] In some embodiments, the Z1 sequence includes a first HA sequence (HA1), the Z2 sequence includes a second HA sequence (HA2), the HA1 sequence encodes an HA antigen derived from a first influenza virus, and the HA2 sequence encodes an HA antigen derived from a second influenza virus. Thus, in some embodiments, the circular RNA includes TI1, HA1, TI2, and HA2 in this order. The first influenza virus may be type A or type B, and the second influenza virus may be type A or type B. In some embodiments, the circular RNA provided in this application comprises TI1, HA1, L1, TI2, HA2, and L2 in this order, where L1 and L2 are independently linker sequences or are absent, and HA1 and HA2 encode HA antigens of first and second influenza viruses, which are (1) A1 and A2 types; (2) B1 and B2 types; or (3) A and B types, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first and second influenza viruses may be A1 and A2 types, respectively. The first and second influenza viruses may be B1 and B2 types, respectively. The first and second influenza viruses may be A and B types, respectively.

[0344] In some embodiments, the Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence encodes a second NA(NA2) sequence, the NA1 sequence encodes an NA antigen derived from a first influenza virus, and the NA2 sequence encodes an NA antigen derived from a second influenza virus. Thus, in some embodiments, the circular RNA comprises TI1, NA1, TI2, and NA2 in this order. In some embodiments, the circular RNA provided in this application comprises TI1, NA1, L1, TI2, NA2, and L2 in this order, where L1 and L2 are independently linker sequences or are absent. The first influenza virus is type A or B, and the second influenza virus is type A or B. In some embodiments, NA1 and NA2 encode NA antigens of first and second influenza viruses which are (1) A1 and A2 types, (2) B1 and B2 types, or (3) A and B types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus. The first and second influenza viruses may be A1 and A2 types, respectively. The first and second influenza viruses may be B1 and B2 types, respectively. The first and second influenza viruses may be A and B types, respectively.

[0345] In some embodiments, the Z1 sequence includes a first HA (HA1) sequence and a first NA (NA1) sequence, and the Z2 sequence includes a second HA (HA2) sequence and a second NA (NA2) sequence, where the HA1 and NA1 sequences encode the HA antigen and NA antigen derived from a first influenza virus, respectively, and the HA2 and NA2 sequences encode the HA antigen and NA antigen derived from a second influenza virus, respectively. Adjacent HA and NA sequences can be linked by a linker (L) sequence. Thus, in some embodiments, a circular RN is formed. A comprises TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2 in this order, where L1 and L2 are independently linker sequences or are absent. In some embodiments, the circular RNA comprises TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2 in this order. The first influenza virus may be type A or type B, and the second influenza virus may be type A or type B. In some embodiments, the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; or (3) type A and type B, where A1 and A2 are first and second subtypes of type A influenza virus, and B1 and B2 are first and second subtypes of type B influenza virus. The first and second influenza viruses may be type A1 and type A2. The first and second influenza viruses may be type B1 and type B2. The first and second influenza viruses may be of type A and type B, respectively.

[0346] In some embodiments of the circular RNA provided in this application, the Z1 sequence comprises a first HA(HA1) sequence encoding the HA antigen, and the Z2 sequence comprises a first NA(NA1) sequence encoding the NA antigen, wherein the HA and NA antigens are derived from a first influenza virus. The first influenza virus may be type A. The first influenza virus may be type B. Therefore, the circular RNA comprises TI1, HA1, TI2, and NA1 in this order. An illustrative diagram is shown in Figure 5.

[0347] In some embodiments of the circular RNA provided in this application, the Z1 sequence comprises a first HA(HA1) sequence and a second HA(HA2) sequence, the Z2 sequence comprises a first NA(NA1) sequence and a second NA(NA2) sequence, the HA1 and NA1 sequences encode HA and NA antigens derived from a first influenza virus, and the HA2 and NA2 sequences encode HA and NA antigens derived from a second influenza virus. Adjacent HA and NA sequences may be linked by a linker (L) sequence. Thus, in some embodiments, the circular RNA comprises the sequences TI1, HA1, L1, HA2, TI2, NA1, L2, and NA2 in this order, where L1 and L2 are independently linker sequences or are absent. The first influenza virus is type A or B, and the second influenza virus is type A or B. In some embodiments, the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; (3) A and B types; or (4) B and A types, respectively, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first and second influenza viruses may be A1 and A2 types, respectively. The first and second influenza viruses may be B1 and B2 types, respectively. The first and second influenza viruses may be A and B types, respectively. The first and second influenza viruses may be B and A types, respectively.

[0348] In some embodiments of the circular RNA provided in this application, the Z1 sequence comprises a first HA(HA1) sequence, a second HA(HA2) sequence, and a third HA(HA3) sequence; the Z2 sequence comprises a first NA(NA1) sequence, a second NA(NA2) sequence, and a third NA(NA3) sequence; the HA1 and NA1 sequences encode HA and NA antigens derived from a first influenza virus; the HA2 and NA2 sequences encode HA and NA antigens derived from a second influenza virus; and the HA3 and NA3 sequences encode HA and NA antigens derived from a third influenza virus. The first influenza virus may be type A or B, the second influenza virus may be type A or B, and the third influenza virus may be type A or B. In some embodiments, two of the three influenza viruses are two subtypes of type A (i.e., type A1 and type A2), and the third is type B. In this embodiment, two of the three influenza viruses are two subtypes of type B (i.e., types B1 and B2), and the third is type A. In some embodiments, all three influenza viruses are type A. In some embodiments, all three influenza viruses are type B. Adjacent HA and NA sequences can be linked by a linker (L) sequence. Thus, in some embodiments, the circular RNA contains the sequences TI1, HA1, L1, HA2, L2, HA3, TI3, NA1, L3, NA2, L4, and NA3 in this order, where L1, L2, L3, and L4 are either independently linker sequences or absent. An exemplary figure is shown in Figure 6. In some embodiments, the first, second, and third influenza viruses are (1) A1, A2, and B types, (2) A1, B, and A2 types, or (3) B, A1, and A2 types, (4) A, B1, and B2 types, (5) B1, A, and B2 types, or (6) B1, B2, and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, and third influenza viruses may be A1, A2, and B types, respectively. The first, second, and third influenza viruses may be A1, B, and A2 types, respectively. The first, second, and third influenza viruses may be B, A1, and A2 types, respectively. The first, second, and third influenza viruses may be of type A, type B1, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type A, and type B2, respectively. The first, second, and third influenza viruses may be of type B1, type B2, and type A, respectively.

[0349] 6.4.3 Structure having three TI sequences In some embodiments, the circular RNA provided in this application comprises, in this 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 each of the Z1, Z2, and Z3 sequences independently comprises either an HA sequence or an NA sequence.

[0350] In some embodiments of the circular RNA provided in this application, the Z1 sequence includes a first HA (HA1) sequence, the Z2 sequence includes a second HA (HA2) sequence, the Z3 sequence includes a third HA (HA3) sequence, the HA1 sequence encodes an HA antigen from a first influenza virus, the HA2 sequence encodes an HA antigen from a second influenza virus, and the HA3 sequence encodes an HA antigen from a third influenza virus. Thus, in some embodiments, the circular RNA includes TI1, HA1, TI2, HA2, TI3, and HA3 in this order. An illustrative figure is shown in Figure 7. In some embodiments, the circular RNA includes TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3 in this order, where L1, L2, and L3 are independently linker sequences or are absent. The first influenza virus may be type A or B, the second influenza virus may be type A or B, and the third influenza virus may be type A or B. In some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. In some embodiments, the first, second, and third influenza viruses are (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, respectively, where A1 and A2 are the first and second subtypes of type A influenza virus, and B1 and B2 are the first and second subtypes of type B influenza virus. The influenza viruses may be of type A1, A2, and B, respectively. The first, second, and third influenza viruses may be of type B1, B2, and A, respectively.

[0351] In some embodiments of the circular RNA provided in this application, the Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence includes a second NA(NA2) sequence, the Z3 sequence includes a third NA(NA3) sequence, the NA1 sequence encodes an NA antigen from a first influenza virus, the NA2 sequence encodes an NA antigen from a second influenza virus, and the NA3 sequence encodes an NA antigen from a third influenza virus. Thus, in some embodiments, the circular RNA includes TI1, NA1, TI2, NA2, TI3, and NA3 in this order. An illustrative figure is shown in Figure 8. In some embodiments, the circular RNA includes TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3 in this order, where L1, L2, and L3 are independently linker sequences or are absent. The first influenza virus may be type A or B, the second influenza virus may be type A or B, and the third influenza virus may be type A or B. In some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. In some embodiments, the first, second, and third influenza viruses are (1) type A1, type A2, and type B, or (2) type B1, type B2, and type A, where A1 and A2 are the first and second subtypes of type A influenza virus, and B1 and B2 are the first and second subtypes of type B influenza virus. The first, second, and third influenza viruses may be type A1, type A2, and type B, respectively. The first, second, and third influenza viruses may be type B1, type B2, and type A, respectively.

[0352] In some embodiments of the circular RNA provided in this application, the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, and the Z3 sequence comprises a third HA (HA3) sequence and a third NA (NA3) sequence, wherein the HA1 and NA1 sequences encode antigens derived from a first influenza virus, the HA2 and NA2 sequences encode antigens derived from a second influenza virus, and the HA3 and NA3 sequences encode antigens derived from a third influenza virus. Adjacent HA and NA sequences can be linked by a linker (L) sequence. Thus, in some embodiments, the circular RNA comprises the sequences TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3 in this order. An illustrative figure is shown in Figure 9. In some embodiments, the circular RNA contains the sequences TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3 in this order. An exemplary diagram is shown in Figure 10. L1, L2, and L3 are each independently linker sequences. The first influenza virus may be type A or B, the second influenza virus may be type A or B, and the third influenza virus may be type A or B. In some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B (i.e., B1 and B2), and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. In some embodiments, the first, second, and third influenza viruses are (1) A1, A2, and B types, respectively, or (2) B1, B2, and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are of type B virus. These are the first and second subtypes of the influenza virus. The first, second, and third influenza viruses may be types A1, A2, and B, respectively. The first, second, and third influenza viruses may be types B1, B2, and A, respectively.

[0353] 6.4.4 Structure having four TI sequences In some embodiments, the circular RNA provided in this application comprises, in this 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, and a fourth protein coding (Z4) sequence, wherein each of the Z1, Z2, Z3, and Z4 sequences independently comprises an HA sequence or an NA sequence.

[0354] In some embodiments of the circular RNA provided in this application, the Z1 sequence includes a first HA (HA1) sequence, the Z2 sequence includes a second HA (HA2) sequence, the Z3 sequence includes a third HA (HA3) sequence, the Z4 sequence includes a fourth HA (HA4) sequence, the HA1 sequence encodes an HA antigen derived from a first influenza virus, the HA2 sequence encodes an HA antigen derived from a second influenza virus, the HA3 sequence encodes an HA antigen derived from a third influenza virus, and the HA4 sequence encodes an HA antigen derived from a fourth influenza virus. Thus, in some embodiments, the circular RNA includes the sequences TI1, HA1, TI2, HA2, TI3, HA3, TI3, and HA4 in this order. An illustrative diagram is shown in Figure 14. In some embodiments, the circular RNA contains the sequences TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4 in this order, where L1, L2, L3, and L4 are independently linker sequences or are absent. The first influenza virus is type A or B, the second influenza virus is type A or B, the third influenza virus is type A or B, and the fourth influenza virus is type A or B. In some embodiments, three of the four influenza viruses may be type A subtypes (i.e., A1, A2, and A3), and the fourth virus may be type B. In some embodiments, three of the four influenza viruses may be type B subtypes (i.e., B1, B2, and B3), and the fourth virus may be type A. In some embodiments, all four influenza viruses may be type A. In some embodiments, all four influenza viruses may be type B. In some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the remaining two may be two subtypes of type B (i.e., B1 and B2).In some embodiments, the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2 types, respectively; (2) A1, B1, A2, and B2 types; (3) B1, A1, B2, and A2 types; or (4) B1, B2, A1, and A2 types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

[0355] In some embodiments of the circular RNA provided in this application, the Z1 sequence includes a first NA(NA1) sequence, the Z2 sequence includes a second NA(NA2) sequence, the Z3 sequence includes a third NA(NA3) sequence, the Z4 sequence includes a fourth NA(NA4) sequence, the NA1 sequence encodes an NA antigen derived from a first influenza virus, the NA2 sequence encodes an NA antigen derived from a second influenza virus, the NA3 sequence encodes an NA antigen derived from a third influenza virus, and the NA4 sequence encodes an NA antigen derived from a fourth influenza virus. Thus, in some embodiments, the circular RNA includes the sequences TI1, NA1, TI2, NA2, TI3, NA3, TI3, and NA4 in this order. An illustrative figure is shown in Figure 14. In some embodiments, the circular RNA includes TI1 The sequence NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4 is included in this order, where L1, L2, L3, and L4 are independently linker sequences or are absent. The first influenza virus is type A or B, the second influenza virus is type A or B, the third influenza virus is type A or B, and the fourth influenza virus is type A or B. In some embodiments, three of the four influenza viruses may be three subtypes of type A (i.e., A1, A2, and A3), and the fourth virus may be type B. In some embodiments, three of the four influenza viruses may be three subtypes of type B (i.e., B1, B2, and B3), and the fourth virus may be type A. In some embodiments, all four influenza viruses may be type A. In some embodiments, all four influenza viruses may be type B. In some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the other two may be two subtypes of type B (i.e., B1 and B2). In some embodiments, the first, second, third and fourth influenza viruses are (1) types A1, A2, B1, and B2; (2) types A1, B1, A2, and B2; (3) types B1, A1, B2, and A2; or (4) types B1, B2, A1, and A2, where A1 and A2 are the first and second subtypes of type A influenza virus, and B1 and B2 are the first and second subtypes of type B influenza virus.

[0356] 6.4.5 Targeted Viruses In some embodiments, the circular RNA disclosed herein encodes HA / NA antigens derived from one, two, three, or four influenza viruses. The first, second, third, and fourth influenza viruses may each independently be any influenza A or B virus described in this application or known in the art. In some embodiments, the first, second, third, and fourth influenza viruses may each independently be circulating viruses. In some embodiments, the circular RNA provided herein encodes HA and / or NA antigens derived from the first influenza virus. The first influenza virus may be any influenza A or B virus described in this application or known in the art. The first influenza virus may 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 a B / Victoria lineage (e.g., B / Austria / 1359417 / 2021). In some embodiments, the influenza B virus is a B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013).

[0357] In some embodiments, the circular RNA provided herein encodes HA antigens and / or NA antigens derived from a first influenza virus and a second influenza virus. In some embodiments, the second influenza virus may be any influenza A or B virus described herein or known in the art. The second influenza virus may be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, the first and second influenza viruses may be (1) A1 and A2 types, (2) B1 and B2 types, (3) A and B types, or (4) B and A types, respectively. A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. In some embodiments, the first and second influenza viruses may be two subtypes of influenza A virus. In some embodiments, the first and second influenza viruses may be H1N1 viruses (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and H3N2 viruses (e.g., A / Darwin / 6 / 2021 or A / Darwin / 6 / 2021), or vice versa. In some embodiments, the first and second influenza viruses may be two subtypes of influenza B virus. In some embodiments, the first and second influenza viruses are derived from the B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and the B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013), or vice versa. In some embodiments, the first and second influenza viruses may be type A and type B, respectively. For example, the first and second influenza viruses may be H1N1 viruses (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and the B / Victoria lineage (e.g., B / Austria / 1359417 / 2021), or vice versa. In some embodiments, the first and second influenza viruses may be H1N1 viruses (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and B / Yamagata lineages (e.g., B / Phuket / 3073 / 2013), or vice versa.In some embodiments, the first and second influenza viruses may be H3N2 viruses (e.g., A / Darwin / 6 / 2021 or A / Darwin / 6 / 2021) and B / Victoria lineages (e.g., B / Austria / 1359417 / 2021), or vice versa. In some embodiments, the first and second influenza viruses may be H3N2 viruses (e.g., A / Darwin / 6 / 2021 or A / Darwin / 6 / 2021) and B / Yamagata lineages (e.g., B / Phuket / 3073 / 2013).

[0358] In some embodiments, the circular RNA provided herein encodes HA antigens and / or NA antigens derived from a first influenza virus, a second influenza virus, and a third influenza virus. In some embodiments, the third influenza virus may be any influenza A or B virus described herein or known in the art. The third influenza virus may be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, two of the three influenza viruses may be two subtypes of type A (i.e., A1 and A2) and the third may be type B. In some embodiments, two of the three influenza viruses may be two subtypes of type B (i.e., B1 and B2) and the third may be type A. In some embodiments, all three influenza viruses may be type A. In some embodiments, all three influenza viruses may be type B. In some embodiments, the first, second, and third influenza viruses are (1) A1, A2, and B types, (2) A1, B, and A2 types, or (3) B, A1, and A2 types, (4) A, B1, and B2 types, (5) B1, A, and B2 types, or (6) B1, B2, and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, and third influenza viruses may be A1, A2, and B types, respectively. The viruses can be of type A1, type B, and type A2, respectively. The first, second, and third influenza viruses can be of type B, type A1, and type A2, respectively. The first, second, and third influenza viruses can be of type A, type B1, and type B2, respectively. The first, second, and third influenza viruses can be of type B1, type A, and type B2, respectively. The first, second, and third influenza viruses can be of type B1, type B2, and type A, respectively. In some embodiments, two of the three influenza viruses can be two subtypes of influenza A virus, and the third can be influenza B virus. For example, two subtypes of influenza A virus could be H1N1 viruses (e.g., A / Wisconsin / 588 / 2019, A / Sydney / 5 / 2021, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022) and H3N2 viruses (e.g., A / Darwin / 6 / 2021 or A / Darwin / 6 / 2021), and influenza B virus could be a B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) or a B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013). In some embodiments, two of the three influenza viruses could be two subtypes of influenza B virus, and the third could be an influenza A virus. For example, the two subtypes of influenza B virus are the B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and the B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013), 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 influenza B virus can be of the B / Victoria lineage (e.g., B / Austria / 1359417 / 2021). In some embodiments, the circular RNA provided herein encodes HA antigens and / or NA antigens derived 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 may be any influenza A or B virus described herein or known in the art. The fourth influenza virus may be a circulating influenza A virus or a circulating influenza B virus. In some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2) and the other two may be two subtypes of type B (i.e., B1 and B2). In some embodiments, three of the four influenza viruses may be three subtypes of type A (i.e., A1, A2, and A3) and the fourth may be type B. In some embodiments, three of the four influenza viruses may be three subtypes of type B (i.e., B1, B2, and B3) and the fourth may be type A. In some embodiments, all four influenza viruses may be type A. In some embodiments, all four influenza viruses may be of type B. In some embodiments, the first, second, third, and fourth influenza viruses are (1) types A1, A2, B1, and B2; (2) types A1, B1, A2, and B2; (3) types B1, A1, B2, and A2; or (4) types B1, B2, A1, and A2, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus. The first, second, third, and fourth influenza viruses may be of type A1, A2, B1, and B2. The first, second, third, and fourth influenza viruses may be of type A1, B1, A2, and B2, respectively. The first, second, third, and fourth influenza viruses may be of type B1, A1, B2, and A2, respectively. It may be type A2. In some embodiments, two of the four influenza viruses may be two subtypes of type A (i.e., A1 and A2), and the other two may be two subtypes of type B (i.e., B1 and B2). For example, in some embodiments, the two subtypes of influenza A 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). In some embodiments, the two subtypes of influenza B virus may be the B / Victoria lineage (e.g., B / Austria / 1359417 / 2021) and the B / Yamagata lineage (e.g., B / Phuket / 3073 / 2013).

[0359] 6.4.6 Fusion Proteins The circular RNA provided in this application comprises a protein-coding (Z) sequence. In some embodiments, the Z sequence encodes an antigen fusion protein. In other words, the encoded protein may include two or more proteins (e.g., HA antigen and / or NA antigen) bound together. Alternatively, a protein fused with an influenza antigen may promote a strong immune response not to itself, but to the influenza virus antigen. In some embodiments, the antigen fusion protein retains the functional properties of each of the original proteins.

[0360] In some embodiments, the circular RNA vaccines provided in this application encode a fusion protein comprising HA antigens and / or NA antigens linked to a scaffold portion. In some embodiments, such scaffold portions can confer desired properties to the antigen encoded by the nucleic acid of this application. For example, the scaffold protein can improve the immunogenicity of the antigen by altering the structure of the antigen, altering the uptake and processing of the antigen, and / or binding the antigen to a binding partner.

[0361] In some embodiments, the scaffold portion is a protein that can self-assemble into highly symmetrical, stable, and structurally organized protein nanoparticles with a diameter of 10–150 nm, a size range very suitable for optimal interaction with various cells of the immune system. In some embodiments, a stable nanoparticle structure can be formed using a viral protein or virus-like particle. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold portion is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of approximately 22 nm and is non-infectious because it lacks nucleic acid (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14(2016) 58-68). In some embodiments, the scaffold portion is hepatitis B core antigen (HBcAg) and self-assembles into particles with a diameter of 24–31 nm, similar to the viral core obtained from the liver of a human infected with HBV. The generated HBcAg self-assembles into two different sizes of nanoparticles, 300 Å and 360 Å in diameter, corresponding to 180 or 240 protomers. In some embodiments, the HA and / or NA antigens disclosed herein are fused to HBsAG or HBcAG to facilitate the self-assembly of nanoparticles presenting the influenza virus antigen.

[0362] In some embodiments, a bacterial protein platform can be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine, and encapsulin.

[0363] Ferritin is a protein whose primary function is intracellular iron storage. Ferritin is composed of 24 subunits, each subunit consisting of four α-helix bundles, and self-assembles into a quaternary structure with octahedral symmetry (Cho K. et al. J Mol Biol. 2009;390:83-98). Several high-resolution structures of ferritin have been determined, and it has been confirmed that ferritin in Helicobacter pylori is composed of 24 identical protomers. In animals, on the other hand, the ferritin light and heavy chains are assembled individually or combined in different ratios to form particles consisting of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003;8:105-111; Fawson DM et al. Nature. 1991;349:541-544). Through self-assembly, ferritin forms nanoparticles with high thermal and chemical stability. Therefore, ferritin nanoparticles are suitable for delivering and exposing antigens.

[0364] Fumazine synthase (FS) is also suitable as a nanoparticle platform for antigen presentation. FS, which plays the second-to-last catalytic role in riboflavin biosynthesis, is an enzyme found in a wide range of organisms, including archaea, bacteria, fungi, plants, and bacteria (Weber SEFlavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The FS monomer is 150 amino acids long and consists of a β-sheet and tandem α-helices on either side. Various quaternary structures have been reported for FS, demonstrating its morphological diversity. These range from homopentamers to structures where 12 pentamers symmetrically assemble to form a 150 Å diameter capsid. FS cages consisting of more than 100 subunits have also been reported (Zhang X. et al. J Mol Biol. 2006;362:753-770).

[0365] Encapsrin, a novel protein cage nanoparticle isolated from the thermophilic bacterium Thermotoga maritima, may also be usable as a platform for presenting antigens on the surface of self-assembling nanoparticles. Encapsrin consists of 60 identical 31kDa monomers, each possessing a thin icosahedral T=1 symmetric cage structure with an inner diameter of 20 nm and an outer diameter of 24 nm (Sutter M. et al. Nat Struct Mol Biol. 2008, 15:939-947). Although the precise function of encapsrin in T. maritima is not yet clear, its crystal structure has recently been elucidated, and its function is presumed to be a cellular compartment that encapsulates proteins such as DyP (dye decolorization peroxidase) and Flp (ferritin-like protein) involved in the oxidative stress response (Rahmanpour R. et al. FEBS J. 2013, 280:2097-2104).

[0366] In some embodiments, the circular RNA of this application encodes influenza virus antigens (e.g., HA antigen and / or NA antigen) fused to a Foldon domain. The Foldon domain can be obtained, for example, from bacteriophage T4 fibrin (see, e.g., Tao Y, et al. Structure. 1997 Jun 15;5(6):789-98).

[0367] The circular RNA provided in this application includes a protein-coding (Z) sequence. In some embodiments, the Z sequence also encodes a signal peptide fused to the HA antigen and / or NA antigen. The signal peptide, comprising the N-terminal 15-60 amino acids of the protein, is typically required for membrane translocation in the secretory pathway and therefore universally controls the entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. In eukaryotes, the signal peptide of the nascent precursor protein (preprotein) causes ribosomes to enter the rough endoplasmic reticulum. The signal peptide is guided to the ER membrane, allowing the growing peptide chain to permeate the ER membrane and initiate processing. Processing in the ER produces mature proteins, but the signal peptide is typically either cleaved from the precursor protein by the host cell's resident ER signal peptidases or remains intact and functions as a membrane anchor. Signal peptides can also promote the localization of proteins to the cell membrane.

[0368] The length of a signal peptide is between 15 and 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 the embodiment of the soil, the signal peptide is 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 They have a length of ~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.

[0369] Signal peptides derived from heterologous genes (which regulate the expression of genes other than influenza virus antigens in nature) are known in the art and can be incorporated into the nucleic acids of this application after being tested for desired properties.

[0370] 6.4.7 Array Optimization The circular RNA provided in this application comprises a protein-coding (Z) sequence. In some embodiments, the Z sequence may be codon-optimized. In some embodiments, the HA sequence and / or NA sequence are codon-optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to ensure proper protein folding by matching the codon frequencies of the target organism and the host organism, to increase stability or reduce secondary structures by biasing the GC content, to minimize tandem repeat codons or nucleotide sequences that may impair gene construction or expression, to customize transcription and translation regulatory regions, to insert or remove protein transport sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) within the encoded protein, to add, remove or shuffle protein domains, to insert or delete restriction enzyme sites, to modify ribosome binding sites and degradation sites, to adjust the translation rate so that various domains of the protein fold properly, or to reduce or remove problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and examples, to name a few, include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, the Z sequence is optimized using an optimization algorithm. In some embodiments, the HA sequence and / or NA sequence are optimized using an optimization algorithm.

[0371] In some embodiments, the HA or NA sequence has less than 95% sequence identity with its natural or wild-type counterpart (e.g., a natural or wild-type mRNA sequence encoding the HA or NA antigen). The HA or NA sequence may have less than 90% sequence identity with its natural or wild-type counterpart. The HA or NA sequence may have less than 85% sequence identity with its natural or wild-type counterpart. The HA or NA sequence has 80% sequence identity with its natural or wild-type counterpart. The sequence may have less than 75% sequence identity. The HA or NA sequence may have less than 75% sequence identity with its natural or wild-type counterpart.

[0372] In some embodiments, HA or NA antigens encoded by codon-optimized sequences have immunogenicity equal to or greater than that of HA or NA encoded by non-codon-optimized sequences (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% higher).

[0373] Exemplary optimized HA and NA sequences encoding the HA and NA antigens disclosed in this application are shown in Tables 6A, 9A, and 10A (HA sequences) and Tables 6B, 9B, and 10B (NA sequences). In some embodiments, the circular RNA vaccines provided in this application include an HA sequence 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 with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182-223, 276-290, and 306-397.

[0374] In some embodiments, the circular RNA has an HA sequence encoding the HA protein of the A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus (e.g., SEQ ID NO: 175). In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence 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 with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 182, 184-188, and 209-210. In some embodiments, the HA sequence may 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 with SEQ ID NO: 182. The HA sequence may 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 with sequence number 184. The HA sequence may 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 with sequence number 185. The HA sequence may 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 with sequence number 185. The HA sequence may 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 with sequence number 186.The HA sequence may 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 with sequence number 187. The HA sequence may 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 with sequence number 188. The HA sequence may 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 with sequence number 209. The HA sequence may have at least 90%, at least 91% identity with sequence number 210. It may have 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.

[0375] In some embodiments, the circular RNA has an HA sequence encoding the HA protein of the A / Darwin / 6 / 2021(H3N2)-like influenza virus (e.g., SEQ ID NO: 177). In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence 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%, or at least 99% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 183, 189-193, and 211-212. In some embodiments, the HA sequence may 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 with SEQ ID NO: 183. The HA sequence may 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 with sequence number 189. The HA sequence may 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 with sequence number 190. The HA sequence may 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 with sequence number 191. The HA sequence may 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 with sequence number 192.The HA sequence may 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 with sequence number 211. The HA sequence may 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 with sequence number 212.

[0376] In some embodiments, the circular RNA has an HA sequence encoding the HA protein of the B / Austria / 1359417 / 2021 (B / Victoria lineage)-like influenza virus (e.g., SEQ ID NO: 179). In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence 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%, or at least 99% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 194-198 and 213-219. In some embodiments, the HA sequence may 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 with SEQ ID NO: 194. The HA sequence may 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 with sequence number 195. The HA sequence may 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%, or at least 98% identity with sequence number 196. The HA sequence may have at least 99% or 100% identity with sequence number 197. The HA sequence may 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 with sequence number 198. The HA sequence may 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 with sequence number 213. The HA sequence may 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 with at least sequence number 214. The HA sequence may 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 with sequence number 215. The HA sequence may 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 with sequence number 216. The HA sequence may 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 with sequence number 217.The HA sequence may 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 with sequence number 218. The HA sequence may 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 with sequence number 219.

[0377] In some embodiments, the circular RNA has an HA sequence encoding the HA protein of a B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like influenza virus (e.g., SEQ ID NO: 180). In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence 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%, or at least 99% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 199-203 and 220-221. In some embodiments, the HA sequence may 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 with SEQ ID NO: 199. The HA sequence may 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 with sequence number 200. The HA sequence may 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 with sequence number 201. The HA sequence may 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 with sequence number 202. The HA sequence may 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 with sequence number 202. 3 may 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. The HA sequence may 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 with sequence number 220. The HA sequence may 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 with at least sequence number 221.

[0378] In some embodiments, the circular RNA has an HA sequence encoding the HA protein of an A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus (e.g., SEQ ID NO: 235). In some embodiments, the circular RNA vaccine provided in this application includes an HA sequence 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%, or at least 99% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 281-285 and 520-521. In some embodiments, the HA sequence may 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 with SEQ ID NO: 281. The HA sequence may 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 with sequence number 282. The HA sequence may 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 with sequence number 283. The HA sequence may 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 with sequence number 284. The HA sequence may 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 with sequence number 285.The HA sequence may 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 with sequence number 520. The HA sequence may 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 with sequence number 521.

[0379] 6.4.8 Translation Initiation (TI) Sequence This application provides an immunogenic composition comprising a circular RNA, the circular RNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence.

[0380] In some embodiments, the translation initiation element (or TI) is an IRES or IRES-like sequence. As used interchangeably herein and as understood in the art, the terms “internal ribosome entry site,” “internal ribosome entry site sequence,” “IRES,” and “IRES sequence region” refer to the cis-element of viral or human cellular RNA (e.g., messenger RNA (mRNA) and / or circular RNA) that bypasses the standard eukaryotic cap-dependent translation initiation step. In most eukaryotic organisms, the translation initiation element (TI) is an IRES or IRES-like sequence. In the standard cap-dependent mechanism used by RNA, an m7G cap at the 5' end of mRNA, the initiator Met-tRNAmet, more than 12 initiation factor proteins, directional scanning, and GTP hydrolysis are required to position a translatable ribosome at the start codon. IRESs attract ribosomes (e.g., eukaryotic ribosomes) to form a translation initiation complex and promote translation initiation. IRESs typically contain a long, highly structured 5' untranslated region (5-UTR) that mediates the binding of the translation initiation complex and catalyzes the formation of a functional ribosome.

[0381] Numerous naturally occurring IRES sequences are available, including piconavirus leader sequences such as encephalomyocarditis virus (EMCV) UTR, polio leader sequences, hepatitis A virus leader sequences, hepatitis C virus IRES, human rhinovirus type 2 IRES, foot-and-mouth disease virus IRES elements, and giardiavirus IRES, among others, and sequences derived from or isolated from various viruses.

[0382] In some embodiments, naturally occurring IRES sequences include Taura syndrome virus, assassin bug virus, Tyler's encephalomyelitis virus, monkey virus 40, Solenopsis invicta virus 1, Loparoshipum padi virus, reticuloendotheliosis virus, human poliovirus 1, Plautia enterovirus, Casimir bee virus, human rhinovirus 2, Homalodisca coagulatavirus 1, human immunodeficiency virus type 1, pygmy kite p virus, hepatitis C virus, hepatitis A virus, hepatitis A virus HA16, hepatitis GB virus, foot-and-mouth disease virus, and human rhinovirus 1. Terrorvirus 71, Equine Rhinitis Virus, Ectrapis obliqua picoma-like Virus, Encephalomyocarditis Virus, Drosophila C Virus, Human Sackievirus B3, Brassicaceae 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, Swine Fever Virus, Tobacco Etch Virus, Cactus Crinkle Virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, picobimavirus, HCV QC64, anthropocosavirus E / D, anthropocosavirus F, anthropocosavirus 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, shambavirus A, pacivirus A, pacivirus A2, echovirus E14, human parechovirus 5, aichivirus, fopivirus, CVA10, enterovirus C, enterovirus D, enterovirus J, human pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, pegivirus A 1220, pacivirus A3. Saperovirus, Rosavirus B, Bakunsavirus, Tremovirus A, Porcine Pacivirus 1, PLV-CHN, Pacivirus A, Sisinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Borderline Disease Virus, BVDV2, CSFV-PK15C, SF573 Disistoravirus, Hubei Picoma-like Virus, CRPV, Sarivirus A BN5, Sarivirus A BN2, Sarivirus A 02394, Sarivirus A The IRES sequences are derived from or isolated from GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, Coxsackievirus (e.g., CVA3, CVA12, CVB1, CVB3, CVB5), Echovirus 7, Enterovirus A71, and / or EV24.

[0383] In some embodiments, the natural IRES sequences are human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila Antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p5 3. Isolated from or derived from IRES elements of eukaryotes selected from human Pim-1, mouse Rbm3, Drosophila reaper, dog Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, and S. cerevisiae YAP1.

[0384] In some embodiments, the naturally occurring IRES sequence is an endogenous IRES sequence derived from or isolated from Homo sapiens. In some embodiments, the natural IRES sequence is an endogenous IRES sequence derived from or isolated from human tissue or a human sample.

[0385] In some embodiments, the IRES sequence is 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 These elements were isolated from or derived from cellular IRES elements selected from 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.

[0386] In some embodiments, the IRES sequence is 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 type l Mahoney, PV_type3_Leon, REV-A, RhPV 5NCR, RhPV IGR, SINV l IGRpred, SV40 Isolated from or derived from viral IRES elements selected from 661-830, TMEV, TMV_UI_IRESmp228, TRV 5NTR, TrV IGR, TSV, and IGR.

[0387] In some embodiments, the IRES sequence includes sequences isolated from or derived from native IRES sequences. The terms “IRES-like sequence” or “internal ribosome entry site-like sequence” refer to non-native nucleotide sequences that exhibit the function of native IRESs. In some embodiments, the IRES-like sequence can recruit ribosomal components to mediate cap-independent translation. The IRES-like sequence can be identified by methods known in the art, such as PCT application number PCT / CN2022 / 095949.

[0388] In some embodiments, the length of the IRES-like sequence is 3 nucleic acid residues or more. In some embodiments, the length of the IRES-like sequence is 3 to 300 nucleic acid residues. The lengths of IRES-like sequences are 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, and 7-20 nucleic acid residues. In some embodiments, the lengths of the IRES-like arrays are 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, 4 These are 5, 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 some embodiments, the length of the IRES-like sequence is 5 to 12 nucleic acid residues.

[0389] In some embodiments, the IRES-like sequence or fragment thereof described in this application can be combined with the naturally occurring IRES sequence or fragment thereof described in this application to form another IRES-like sequence. 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.

[0390] This application provides an immunogenic composition comprising a circular RNA, the circular RNA comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence. Those skilled in the art will understand that the circular RNA vaccine disclosed herein is not limited to a specific TI sequence and may incorporate any other TI sequence known in the art, as long as it allows for sufficient expression of the HA antigen and / or NA antigen.

[0391] In some embodiments, the TI sequence includes an optimized IRES sequence. In some embodiments, the IRES sequence is optimized to increase the efficiency of initiation translation. In some embodiments, the TI sequence is not optimized to facilitate the translation of antigens only, but the IRES sequence is optimized for the efficient translation of all coding sequences. For example, in some embodiments, the circular RNA includes a TI sequence and a Z1 sequence, the Z1 sequence includes two, three, or four HA sequences, each encoding an HA antigen, and the TI sequence includes an IRES sequence optimized for the efficient translation of all HA sequences, so that when the circular RNA is administered to a subject, a sufficient immune response to all HA antigens can be induced.

[0392] In some embodiments, the TI sequence includes the IRESs listed in Table 11. In some embodiments, the IRES sequence may 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs.490. In some embodiments, the IRES sequence may 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 with SEQ ID NOs.490. In some embodiments, the IRES sequence may 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 with sequence number 491. In some embodiments, the IRES sequence may have at least 90%, at least 91%, at least 92%, or less identity with sequence number 492. It may have at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. In some embodiments, the IRES sequence may 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 with sequence number 493. In some embodiments, the IRES sequence may 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 with sequence number 494. In some embodiments, the IRES sequence may 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 with sequence number 495. In some embodiments, the IRES sequence may 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 with sequence number 496. In some embodiments, the IRES sequence may 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 with sequence number 497.

[0393] 6.4.9 Linker In some embodiments, the circular RNA provided herein encodes multiple functional domains or peptides linked together as a fusion protein. In some embodiments, the circular RNA provided herein further comprises linker(L) sequences encoding at least two domains of the fusion protein or linkers(L) located between each domain. In some embodiments, the circular RNA provided herein comprises multiple antigen-coding sequences (e.g., HA sequences and / or NA sequences). In some embodiments, adjacent antigen-coding sequences are linked by linker(L) sequences.

[0394] The linker may be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linkers, P2A linkers, T2A linkers, E2A linkers, and combinations thereof. This family of self-cleaving peptide linkers, called 2A peptides, is known in the art (see, for example, Kim, J. Het 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 comprises three domains having an intervening linker and has a domain-linker-domain-linker-domain structure.

[0395] Severable linkers known in the art can be used in connection with this disclosure. Examples of linkers include the F2A linker, T2A linker, P2A linker, and E2A linker (see, for example, WO2017127750). Those skilled in the art will understand that other linkers known in the art may be suitable for use in the structures of this application (e.g., those encoded by the nucleic acids of this disclosure). Similarly, those skilled in the art will understand that other polycistronic structures may be suitable for use as described herein.

[0396] In some embodiments, the linker sequence may include a 5'UTR, 3'UTR, polyA sequence, polyAC sequence, polyC sequence, polyU sequence, polyG sequence, ribosome binding site, aptamer, riboswitch, ribozyme, small RNA binding site, translation regulatory element (e.g., Kozak sequence), protein binding site (e.g., PTBP1 or HUR), and non-natural nuclei. Contains a rheotide or a non-nucleotide chemical linker sequence.

[0397] In some embodiments, the linker sequence includes a 3'UTR. In some embodiments, the 3'UTR may be derived from human β-globin, human α-globin, African clawed frog β-globin, African clawed frog α-globin, human prolactin, human GAP-43, human eEF1a1, human tau, human TNFα, dengue virus, hantavirus small molecule mRNA, bunyavirus small molecule mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ring spot virus, woodchuck hepatitis virus posttranslational regulatory element, Sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

[0398] In some embodiments, the linker sequence includes a 5'UTR. In some embodiments, the 5'UTR may be the 5'UTR of human β-globin, African clawed frog β-globin, human α-globin, African clawed frog α-globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, clawed frog virus, or adenovirus ternary leader.

[0399] In some embodiments, the linker sequence includes a polyA region. In some embodiments, the polyA region is at least 30 nucleotides or at least 60 nucleotides long.

[0400] 6.4.10 Exemplary circRNA For illustrative purposes, Table 13 shows exemplary circular RNA sequences. In some embodiments, the circular RNA may 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 with a nucleotide sequence selected from the group consisting of SEQ ID NOs.

[0401] In some embodiments, the circular RNA provided in this application encodes the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 499 to 501. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 499. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 500. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 501.

[0402] In some embodiments, the circular RNA provided in this application encodes the HA protein of the A / Darwin / 6 / 2021(H3N2)-like influenza virus. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 502-505 and 519. In some embodiments, the circular RNA provided in this application encodes the HA protein of SEQ ID NOs. 50 Sequence ID 2 may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with Sequence ID 503. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with Sequence ID 504. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with Sequence ID 505. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 519.

[0403] In some embodiments, the circular RNA provided in this application encodes the HA protein of influenza virus B / Austria / 1359417 / 2021 (B / Victoria lineage). In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 512-516. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 512. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 513. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 514. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 515. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 516.

[0404] In some embodiments, the circular RNA provided in this application encodes the HA protein of influenza virus B / Phuket / 3073 / 2013 (B / Yamagata strain). In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 506 to 511. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs. 507 may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 508. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 509. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 510. In this state, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 511.

[0405] In some embodiments, the circular RNA provided in this application encodes the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 517. In some embodiments, the circular RNA provided in this application may have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 518.

[0406] 6.4.11 Modified Nucleosides In some embodiments, the circular RNA provided in this application is unmodified and contains a standard ribonucleotide consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the circular RNA provided in this application contains nucleotides and / or nucleosides that are modified as known in the art. In some embodiments, the nucleotides and nucleosides of this application contain modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or unnaturally occurring modified nucleotides and nucleosides. Such modifications may include modifications to the sugar, backbone, or nucleic acid base portion of nucleotides and / or nucleosides that are recognized in the art.

[0407] In some embodiments, the naturally occurring modified nucleotides or nucleotides described herein are those commonly known or recognized in the art. A non-limiting list of such naturally occurring modified nucleotides and nucleotides can be found, among other things, in the widely recognized MODOMICS database.

[0408] In some embodiments, the non-naturally occurring modified nucleotides or nucleosides of this application are those that are commonly known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides are described, among others, in published U.S. applications 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 / IB2017 / 051367, all of which are incorporated herein by reference.

[0409] Accordingly, the circular RNAs disclosed in this application may include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, unnaturally occurring nucleotides and nucleosides, or any combination thereof. In some embodiments, the circular RNAs disclosed in this application may include a variety (more than one) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of the circular RNA may include one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0410] In some embodiments, the modified circular RNAs disclosed herein, when introduced into cells or organisms, exhibit reduced degradation in those cells or organisms compared to unmodified circular RNAs containing standard nucleosides.

[0411] In some embodiments, the modified circular RNAs disclosed herein, when introduced into cells or organisms, exhibit reduced immunogenicity (e.g., reduced innate immune response) in those cells or organisms compared to unmodified circular RNAs containing standard nucleosides.

[0412] In some embodiments, the circular RNAs disclosed herein include unnaturally modified nucleosides introduced during or after synthesis to achieve a desired function or property. The modifications may be present in internucleotide bonds, purine or pyrimidine bases, or glycans. These modifications may be introduced by chemical synthesis or by polymerase enzymes at the ends of the chain or elsewhere within the chain.

[0413] In some embodiments, the circular RNA provided in this application has a modified RNA nucleotide and / or a modified nucleoside. 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 (pseudolidine). 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'-O-methyladenosine), ms2m6A (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-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6 -Hydroxynorvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyladenosine), Ar(p)(2'-O-ribosyladenosine (phosphate ester)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-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'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate ester)); yW(wybutosine), oayW(peroxywybutosine), OHyW(hydroxywybutosine), OHyW*(inadequately modified hydroxywybutosine), imG(wyosine), mimG(methylwyosine), Q(quosine), oQ(epoxyquosine), galQ(galactosylquosine), manQ(mannosylquosine), preQo(7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archeosin), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5, U(5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U(5-methoxycarbonylmethyluridine), mcm5Um(5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine), nm5S2U(5-aminomethyl-2-thiouridine), mnm5U(5-methylaminomethyluridine), mnm5s2U(5-methylaminomethyl-2-thiouridine) Uridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A(N6,N6-dimethyladenosine), Im(2'-O-methylinosine), m4C(N4-methylcytidine), m4Cm(N4,2'-O-dimethylcytidine), hm5C(5-hydroxymethylcytidine), m3U(3-methyluridine), cm5U(5-carboxymethyluridine), m6Am(N6,2'-O-dimethyladenosine), m6 These are 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 (1,2'-0-dimethylguanosine), m'Am (1,2'-0-dimethyladenosine), rm5U (5-taurinomethyluridine), τm5s2U (5-taurinomethyl-2-thiouridine), imG-14 (4-demethylyosine), imG2 (isoyosine), or ac6A (N6-acetyladenosine).

[0414] In some embodiments, the modified nucleoside is pyridine-4-onribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine Zin, 1-taurinomethyl-4-thiouridine, 5-methyluridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, 4-methoxy -2-thio-pseudridine, 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudridine, pyrrolocitidine, pyrroloceudridine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopseudridine, 4-thio-1-methylpseudridine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudo Socitidine, zebralin, 5-azazebralin, 5-methylzebralin, 5-aza-2-thiozebralin, 2-thiozebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methylpseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deazaadenine, 7-deaza-8-azaadenine, 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-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthioadenine, 2-Methoxyadenine, Inosine, 1-Methylinosine, Wyosin, Wyobutosin, 7-Deazaguanosine, 7-Deaza-8-Azaguanosine, 6-Thioguanosine, 6-Thio-7-Deazaguanosine, 6-Thio-7-Deaza-8-Azaguanosine The compounds may include those selected from the group consisting of nosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine. In another embodiment, the modification is independently selected from the group consisting of 5-methylcytosine, pseudouridine, and 1-methylpseudridine.

[0415] In some embodiments of the circular RNA disclosed in this application, at least one of the modified RNA nucleotides and / or modified nucleosides is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), These are 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 (pseudolidine), or m1A (1-methyladenosine).

[0416] The circular RNA of this application can be partially or completely modified along the entire length of the molecule. The circular RNA of this application may have approximately 1% to approximately 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., one or more of A, G, U, or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%). It may include 0% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). In some embodiments, the circular RNA contains at least 10% modified RNA nucleotides and / or modified nucleosides. It is understood that the remaining proportions are explained by the presence of unmodified A, G, U, or C.

[0417] In some embodiments, at least one modified RNA nucleotide and / or modified nucleoside is introduced in vitro (IVT).

[0418] In some embodiments, vaccines containing modified circular RNA require at least 10 times fewer RNA polynucleotides to produce equivalent antibody titers than unmodified circular RNA vaccines.

[0419] The present application relates to a chemically modified circular RNA vaccine and an unmodified circular RNA vaccine. Both cins produce a superior immune response compared to the corresponding mRNA vaccines. In some embodiments, the circular RNAs provided in this application do not contain modified RNA nucleotides and / or modified nucleosides. In some embodiments, the circular RNAs provided in this application include 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), It does not contain 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), or inosine (I), Y (pseuduridine), or m1A (1-methyladenosine).

[0420] 6.4.12 Preparation of circular RNA This application provides immunogenic compositions comprising a circular RNA vaccine. Circular RNA is a single-stranded RNA with a head and tail joined, and can be prepared using methods known in the art. In some embodiments, methods for synthesizing circular RNA in vitro involve ligating the ends of linear RNA precursors to produce a circular structure closed by covalent bonds. Linear RNA can be produced by chemical synthesis or enzymatic strategies (Muller S. (2017), RNA Biol. 14, 1018-1027; Obi P. (2021). Methods S1046-2023, 00065-00067). The advantage of chemical synthesis is that a 5' monophosphate group can be directly introduced during the synthesis process for future cyclization. However, it has limitations such as high purification costs and low yields, and chemical synthesis can only produce RNA of less than 50-70 nucleotides in length. Therefore, enzymatic strategies are currently the primary method for synthesizing linear RNA. Enzymatic strategies are typically implemented by in vitro transcription (IVT) reactions involving a DNA template, reaction buffer, and phage RNA polymerase (Beckert B. (2011). Methods Mol. Biol. 703, 29-41). Phage RNA polymerases are usually derived from T7, SP6, or T3 bacteriophages, with T7 RNA polymerase being the most common. IVT reactions allow for lower-cost and longer RNA synthesis. In some embodiments, mutant phage polymerases with improved transcription quality and reduced side reactions are used.

[0421] Cyclization is achieved by in vitro ligation of linear RNA precursors. Ligation methods include chemical ligation, enzymatic ligation, and ribozyme methods. In some embodiments, linear RNA precursors are cyclized by chemical ligation using, for example, cyanogen bromide (BrCN) or 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide to ligate the DNA-RNA hybrid (Sokolova et al., (1988). FEBS Lett. 232, 153-155).

[0422] In some embodiments, linear RNA precursors are cyclized by enzymatic ligation catalyzed by several enzymes derived from bacteriophage T4 (including T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2)). Since linear RNA precursors require a 3'-OH group on the receptor substrate and a 5'-monophosphate group on the donor substrate for enzymatic ligation, when linear RNA precursors are prepared chemically, the 5'-monophosphate group can be incorporated during synthesis or added after synthesis using ATP and T4 polynucleotide kinase. When linear RNA precursors are synthesized by in vitro transcription (IVT), the process usually starts with 5'-pppG and is performed using calf intestinal phosphatase (CIP) or other phosphatases beforehand. -The ends need to be dephosphorylated. Next, a 5' monophosphate group can be added using ATP and T4 polynucleotide kinase. In some embodiments, GMP is added to the IVT reaction mixture to phosphorylate the 5' end of the transcript. In some embodiments, the linear RNA precursor is cyclized by T4 Dnl ligation. T4 Dnl promotes the ligation of double-stranded double helixes such as DNA / RNA hybrids, so a complementary DNA (cDNA) template or bridge is required to achieve RNA ligation. In some embodiments, the linear RNA precursor is cyclized by T4 Rnl 1 ligation. T4 Rnl 1 catalyzes a nucleophilic attack of the 3'-OH end to the activated 5' end, forming a covalent 5',3'-phosphodiester bond and generating circular RNA. In some embodiments, the linear RNA precursor is cyclized by T4 Rnl 2 ligation. Similar to T4 Rnl 1, T4 Rnl 2 also catalyzes nucleophilic attack of the 3'-OH terminus to the activated 5' terminus, forming a covalent 5',3'-phosphodiester bond. However, T4 Rnl 2 is far more active in ligating cleavage sites of double-stranded RNA (dsRNA) than in ligating the ends of ssRNA. T4 Rnl 2 is more suitable for linear RNA precursors that have a ligation site in the double-stranded region.

[0423] In some embodiments, linear RNA precursors are cyclized by enzymatic ligation using ribozymes. A modified group I intronic auto-splicing system can be used, also known as the permutation intron and exon (PIE) method (Wesselhoeft et al. (2018). Nat.Commun. 9, 2629; Rausch et al. (2021) Nucleic Acids Res. 49, E35). The PIE method requires only the addition of GTP and Mg2+ as cofactors and shows great potential for protein synthesis. This method achieves RNA ligation via a standard group I intronic auto-splicing reaction involving two transesterifications at specific splice sites. The PIE method can be used for RNA cyclization in vitro and in vivo. Compared to chemical and enzymatic ligation, the PIE method can be applied to the cyclization of larger linear RNA precursors, and the reaction conditions and purification methods of the PIE method are simpler.

[0424] Group II introns can also be used in circular RNA synthesis involving reverse splicing (Mikheeva S. et al., (1997). Nucleic Acids Res. 25, 5085-94). In this splicing reaction, the 5' price site at the end of an exon is joined to the 3' price site at the beginning of the same exon. Compared to reverse splicing catalyzed by group I introns, reverse splicing catalyzed by group II introns does not require the entire exon sequence. Therefore, this method enables more accurate linear RNA precursor ligation. Methods for preparing circular RNA using group II introns are described in PCT applications PCT / CN2022 / 095749, PCT / CN2022 / 095949, PCT / CN2022 / 104527, PCT / CN2022 / 129435, PCT / CN2022 / 134803, and PCT / CN2022 / 135585, all of which are incorporated herein by reference.

[0425] Hairpin ribozymes (HPRs) can produce circular RNA via rolling circle reactions and self-splicing reactions from circular single-stranded DNA templates (Dallas et al. (2008). Nucleic Acids Res. 36, 6752-66; Petkovic and Muller (2013) FEBS Lett. 587, 2435-40). Linear RNA precursors containing HPRs fold into two selective cleavage-active structures to remove the 3' and 5' ends. As a result, the intermediate contains a 5'-OH group and a 2',3'-cyclic phosphate group, generating the target circular RNA. This method allows for the generation of small circular RNAs from long repeat RNAs transcribed in vitro by RNA polymerase via the rolling circle mechanism.

[0426] For illustrative purposes, in some embodiments, the circular RNA provided in this application can be prepared using group II intron self-splicing as follows: First, a vector containing a nucleotide sequence encoding a precursor RNA is designed and cloned. This vector contains the following operably ligated 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. Here, the RNA has group II intron activity and self-splicing can form a circular RNA containing both a 5' target sequence fragment and a 3' target sequence fragment, with the 3' end of the 5' target sequence fragment ligated to the 5' end of the 3' target sequence fragment. In some embodiments, the vector is a DNA vector.

[0427] Circular RNA prepared using Group II intronic self-splicing may be scarless or nearly scarless. As used herein, the term “scar” refers to a non-target sequence region in a circular RNA splicing product. As used herein, the term “scarless splicing” means that the construct self-splices to produce a circular RNA that does not contain any additional sequence elements other than the target sequence. Thus, “scarless” circular RNA means that there is no scar and it consists only of the target sequence. As used herein, the term “nearly scarless splicing” means that the construct self-splices to produce a circular RNA that contains 20 or fewer nucleotides in addition to the target sequence. “Nearly scarless circular RNA” is a circular RNA produced by “nearly scarless” self-splicing that contains 20 or fewer nucleotides in addition to the target sequence.

[0428] Therefore, since the self-splicing of these RNAs produces circular RNA that does not contain additional sequence elements other than the target sequence, it is referred to herein as “scarless” splicing. Circular RNA consisting only of the target sequence is referred herein as “scarless” circular RNA. As used herein, the term “scar” refers to the non-target sequence region in the circular RNA splicing product. Therefore, “scarless” circular RNA does not contain scars. In some embodiments, the circular RNA provided herein is scarless. In some embodiments, the circular RNA provided herein is substantially scarless.

[0429] For example, many vectors can be used that can contain operable selectable sequences or markers for stable integration into the chromosome of a host cell, such as expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAVs), herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, 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 maintained outside the chromosome. The vector is engineered to contain a sequence encoding a DNA origin or "ori" derived from lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a lymphotropic herpesvirus or gamma herpesvirus origin corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus is Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV). It could be thymiriherpesvirus (HS) or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses.

[0430] Furthermore, the vector can include one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes can, for example, confer resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply essential nutrients not present in the culture medium. “Expression regulatory sequences,” “regulatory elements,” or “regulatory sequences” in an expression vector are the untranslated regions of the vector (origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylated sequences, 5' and 3' untranslated regions) that interact with host cell proteins to perform transcription and translation. The intensity and specificity of these elements vary. Depending on the vector system and host used, any number of appropriate transcription and translation elements, such as ubiquitous promoters or inducible promoters, can be used.

[0431] Exemplary universal expression regulatory sequences usable in this disclosure include the cytomegalovirus (CMV) very early promoter, the viral simian virus 40 (SV40) promoter (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Roussarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the vaccinia virus-derived H5, P7.5, and P11 promoters, and the elongation factor 1-alpha (E This list includes, but is not limited to, the F1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde triphosphate 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), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0432] Examples of inductive promoters / systems include steroid-inducible promoters such as promoters of genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormones), metallothione promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), and the "GeneSwitch" mifepristone regulatory system (Sirin et al.). Examples include, but are not limited to, al., 2003, Gene, 323:67), Kumart-inducible gene switches (WO2002 / 088346), and tetracycline-dependent regulatory systems.

[0433] The vectors provided in this application can be prepared using standard molecular biology techniques. For example, various elements of the vectors provided in this application can be obtained by recombination methods such as screening cDNA and genomic libraries from cells, or by methods such as deriving polynucleotides from vectors known to contain the same elements. Various elements of the vectors provided in this application can also be prepared by synthesis rather than cloning, based on known sequences. Complete sequences can be assembled from duplicate oligonucleotides prepared by standard methods. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:631.

[0434] Therefore, a specific nucleotide sequence can be obtained from a vector containing the desired sequence, or, as needed, can be fully or partially synthesized using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR). One method for obtaining a nucleotide sequence encoding a desired vector element is to anneal a complementary set of duplicate synthetic oligonucleotides prepared in a conventional automated polynucleotide synthesizer, then ligate them with a suitable DNA ligase, and amplify the ligated nucleotide sequence by PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Furthermore, oligonucleotide-induced synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide-induced mutagenesis of existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239:1534-1536), and enzymatic filling of gap oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-10033) can be used.

[0435] Secondly, linear RNA precursors can be generated by incubating the vector provided in this application under conditions that allow transcription of the RNA encoded by the vector. For example, in some embodiments, linear RNA precursors provided herein can be synthesized by incubating the vector provided in this application, which includes an RNA polymerase promoter upstream of the 5' double-strand formation region and / or expression sequence, with a suitable RNA polymerase enzyme under conditions that allow in vitro transcription. In some embodiments, the vector is incubated in a cell by bacteriophage RNA polymerase or in the cell nucleus by host RNA polymerase P. In some embodiments, linear RNA precursors can be generated by in vitro transcription using the vector provided in this application (e.g., the vector provided in this application having an RNA polymerase promoter upstream of the 5' homologous region) as a template.

[0436] Thirdly, linear RNA precursors having group II intron activity can be used to generate circular RNA by auto-splicing. In some embodiments, the linear RNA precursor is incubated under conditions suitable for circularization (auto-splicing). Auto-splicing of group II introns must be carried out under high-salinity conditions, and the introduction of GTP is not required. Therefore, in some embodiments, the buffer used for the auto-splicing reaction contains 10 mM to 100 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM of divalent magnesium ions (such as MgCl2). The auto-splicing buffer may also contain 10 mM to 100 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM of NaCl.

[0437] In some embodiments, the self-splicing reaction is performed in vitro for approximately 5 minutes to 1 hour, for example, approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, and 1 hour.

[0438] In some embodiments, the self-splicing reaction is carried out at temperatures of 20-60°C, 20-50°C, 20-40°C, 20-30°C, 30-40°C, 40-50°C, or 50-60°C.

[0439] In some embodiments, the precursor RNA has a cyclization rate of at least 30%, for example, At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95% of the cyclization rate can be achieved.

[0440] Fourth, circular RNA prepared by self-splicing a linear RNA precursor is purified. Purification includes, but is not limited to, the removal of uncircularized linear RNA, dsRNA, and other unwanted components. In some embodiments, the circular RNA disclosed herein is purified before being introduced into cells. The phosphate groups at both ends of linear RNA and some double-stranded RNA may activate the RIG-1 signaling pathway, and the immune response resulting from RIG-1 signaling may lead to the degradation of exogenous RNA, potentially affecting the function of circular RNA.

[0441] For illustrative purposes, purification methods include enzymatic treatment; chromatography, including but not limited to affinity column chromatography, reverse-phase silica gel column liquid chromatography, gel filtration chromatography, and gel exclusion liquid chromatography; electrophoresis, including but not limited to gel electrophoresis such as agarose gel electrophoresis and capillary electrophoresis; and any combination thereof. Methods for removing linear RNA include, for example, enzymatic treatment such as RNase R treatment and chromatography such as high-performance liquid chromatography (HPLC). Methods for removing terminal phosphate groups include, for example, alkaline phosphatase treatment such as calf intestinal alkaline phosphatase (CIP).

[0442] In some embodiments, purification includes one or more steps of phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification includes the steps of RNase R digestion, phosphatase treatment, and HPLC size exclusion purification in this order. In some embodiments, purification includes reverse-phase HPLC. In some embodiments, the purified composition has lower content of double-stranded RNA, DNA sprints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes, and / or nic RNA than unpurified RNA.

[0443] In some embodiments, the linear RNA precursor provided in this application includes a purified tag that is removed during self-splicing, and this purified tag can be used for negative selection of circular RNA. Specifically, a sample containing circular RNA (e.g., the product of a splicing reaction starting from a tagged linear precursor) can be mixed with a probe immobilized on a solid surface, and the tag-containing precursor or other tag-containing impurities can be bound to the probe and removed from the solution, resulting in a circular RNA solution substantially free of precursors, introns, and other tag-containing impurities. In some embodiments, the purified tag is a polynucleotide of 15 to 40 nucleotides. Examples of purification matrices include magnetic resins or beads, silicone resins, Sephadex resins, affinity resins, nanoparticles, and nanomaterial surfaces or coated surfaces.

[0444] Accordingly, in some embodiments, circular RNA prepared by the methods disclosed herein may have a functional half-life of at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 30 hours, at least 40 hours, at least 50 hours, at least 60 hours, at least 70 hours, or at least 80 hours. In some embodiments, the circular RNA provided herein has a functional half-life of 5–80 hours, 10–70 hours, 15–60 hours, and / or 20–50 hours. In some embodiments, the circular RNA provided herein has a functional half-life that is longer (e.g., at least 1.5 times, at least 2 times longer) than an equivalent linear RNA encoding the same protein. In some embodiments, the functional half-life can be evaluated through the detection of functional protein synthesis.

[0445] In some embodiments, the circular RNA provided in this application comprises one or more expression sequences and is configured to be continuously expressed in vivo within the cells of a subject. In some embodiments, the circular RNA is configured such that the expression of one or more expression sequences in the cell at a later time point is equal to or greater than that at an earlier time point. In such embodiments, the expression of one or more expression sequences can be maintained at a relatively stable level or can increase over time. The expression of expression sequences can be relatively stable over long periods. For example, in some cases, the expression of one or more expression sequences in the cell does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over periods of at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 23 days, or longer. In some cases, the expression of one or more intracellular expression sequences is maintained at a level that does not change by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days, or longer.

[0446] In some embodiments, the circular RNA provided in this application has a higher expression level than equivalent linear mRNA, for example, a higher expression level 24 hours after RNA administration to cells. In some embodiments, the circular RNA provided in this application has a higher expression level than mRNA containing the same expression sequence, 5moU modification, optimized UTR, cap, and / or poly-A tail. In some embodiments, the circular RNA provided in this application has higher stability than equivalent linear mRNA. In some embodiments, this can be demonstrated by measuring the presence and density of the receptor in vitro or in vivo after electroporation over a period of one week. In some embodiments, this can be demonstrated by measuring the presence of RNA via qPCR or ISH.

[0447] In some embodiments, the circular RNA disclosed herein may be of any length or size. In some embodiments, the length of the circular RNA is 300-10000, 400-9000, 500-8000, 600-7000, 700-6000, 800-5000, 900-5000, 1000-5000, 1100-5000, 1200-5000, 1300-5000, 1400-5000, and / or 1500-5000 nucleotides.

[0448] In some embodiments, the length of the circular RNA disclosed herein may 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 some embodiments, the length of the circular RNA is 3000 nt or less, 3500 nt or less, 4000 nt or less, 4500 nt or less, 5000 nt or less, 6000 nt or less, 7000 nt or less, 8000 nt or less, 9000 nt or less, or 10000 nt or less.

[0449] In some embodiments, the lengths of the circular RNA disclosed in this application are approximately 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, and 5 nt. It could be 000nt, 6000nt, 7000nt, 8000nt, 9000nt, or 10000nt.

[0450] In some embodiments, the length of the circular RNA disclosed herein may be at least 500 nucleotides, at least 1000 nucleotides, or at least 1500 nucleotides.

[0451] Unless otherwise specified, the implementation of this invention utilizes prior art in molecular biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields in the art. These techniques are described in and are well explained in the references cited in this application. For example, 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 See 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). All of these references are incorporated herein by reference.

[0452] 6.5 Immunogenic compositions This application provides immunogenic compositions comprising circular RNA disclosed herein. In some embodiments, the immunogenic compositions provided herein are used to induce an anti-influenza immune response in a subject. In some embodiments, the immunogenic compositions provided herein are used for the prevention or treatment of influenza viruses in humans and other mammals. As used herein and as consistently understood in the art, the term “immunogenic composition” means a composition that is capable of inducing an immune response to a substance. As used herein and as consistently understood in the art, “immune response” refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble molecules (e.g., antibodies, cytokines, and complement) produced by these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of pathogens that have entered the body of a vertebrate, cells or tissues infected with pathogens, cancer cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. This composition includes, 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 in this application are described herein. This includes circular RNA encoding antigenic proteins such as HA antigen and / or NA antigen. In some embodiments, the immunogenic composition may include adjuvants, stabilizers, preservatives, and other excipients that enhance the immunogenicity of the antigen and improve the stability and delivery of the immunogenic active substance. The primary objective of the immunogenic composition is to stimulate the host's immune system to recognize, react to, and remember the pathogen or its components, thereby conferring protection against future infections.

[0453] In some embodiments, the immunogenic compositions provided in this application include at least two, at least three, at least four, at least five, or at least six circular RNAs described in this application. In some embodiments, the immunogenic compositions provided in this application may include at least two circular RNAs disclosed in this application. In some embodiments, the immunogenic compositions provided in this application may include at least three circular RNAs disclosed in this application. In some embodiments, the immunogenic compositions provided in this application may include at least four circular RNAs disclosed in this application. In some embodiments, the immunogenic compositions disclosed in this application may include at least five circular RNAs disclosed in this application. In some embodiments, the immunogenic compositions disclosed in this application may include at least six circular RNAs disclosed in this application.

[0454] In some embodiments, the immunogenic compositions disclosed herein comprise at least two circular RNAs, the first circular RNA comprising an HA sequence encoding an HA antigen, and the second circular RNA comprising an NA sequence encoding an NA antigen. In some embodiments, the HA antigen and NA antigen are derived from the same influenza virus. In some embodiments, the HA antigen and NA antigen are derived from two influenza viruses. In some embodiments, the HA antigen and NA antigen are derived from two influenza A viruses. In some embodiments, the HA antigen and NA antigen are derived from two influenza B viruses. In some embodiments, the HA antigen and NA antigen are derived from influenza A virus and influenza B virus. In some embodiments, each of the two circular RNAs comprises an HA sequence encoding an HA antigen. In some embodiments, the two HA antigens are derived from two influenza viruses. In some embodiments, the two HA antigens are derived from two influenza A viruses. In some embodiments, the two HA antigens are derived from two influenza B viruses. In some embodiments, the two HA antigens are derived from influenza A virus and influenza B virus. In some embodiments, each of the two circular RNAs comprises an NA sequence encoding an NA antigen. In some embodiments, the two NA antigens are derived from two influenza viruses. In some embodiments, the two NA antigens are derived from two influenza A viruses. In some embodiments, the two NA antigens are derived from two influenza B viruses. In some embodiments, the two NA antigens are derived from one influenza A virus and one influenza B virus. The HA antigen and / or NA antigen may be any HA antigen and / or NA antigen disclosed in this application or known in the art.

[0455] In some embodiments, the immunogenic compositions disclosed herein comprise at least three circular RNAs, the first circular RNA comprising an HA sequence encoding the HA antigen, and the other two circular RNAs each comprising an NA sequence encoding the NA antigen. In some embodiments, the first circular RNA comprises an NA sequence encoding the NA antigen, and the other two circular RNAs each comprising an HA sequence encoding the HA antigen. In some embodiments, the three circular RNAs each comprise an HA sequence encoding the HA antigen. In some embodiments, the three circular RNAs each comprise an NA sequence encoding the NA antigen. In some embodiments, the three HA / NA antigens are derived from one influenza virus. In some embodiments, the three HA / NA antigens are derived from two influenza viruses. In some embodiments, the three HA / NA antigens are derived from three influenza viruses. In some embodiments, the three HA / NA antigens are derived from three influenza A viruses. In some embodiments, the three HA / NA antigens are derived from three influenza B viruses. In some embodiments, the three HA / NA antigens are derived from two influenza A viruses and one influenza B virus. In some embodiments, the three HA / NA antigens are derived from two influenza B viruses and one influenza A virus.

[0456] In some embodiments, the immunogenic composition disclosed herein comprises at least four circular RNAs, the first circular RNA comprising an HA sequence encoding the HA antigen, and the other three circular RNAs each comprising an NA sequence encoding the NA antigen. In some embodiments, the first circular RNA comprises an NA sequence encoding the NA antigen, and the other three circular RNAs each comprising an HA sequence encoding the HA antigen. In some embodiments, two of the four circular RNAs each comprise an HA sequence encoding the HA antigen, and the other two each comprise an NA sequence encoding the NA antigen. In some embodiments, each of the four circular RNAs comprises an HA sequence encoding the HA antigen. In some embodiments, each of the four circular RNAs comprises an NA sequence encoding the NA antigen. In some embodiments, the four HA / NA antigens are derived from one influenza virus. In some embodiments, the four HA / NA antigens are derived from two influenza viruses. In some embodiments, the four HA / NA antigens are derived from three influenza viruses. In some embodiments, the four HA / NA antigens are derived from four influenza viruses. In some embodiments, the four HA / NA antigens are derived from four influenza A viruses. In some embodiments, the four HA / NA antigens are derived from four influenza B viruses. In some embodiments, the four HA / NA antigens are derived from three influenza A viruses and one influenza B virus. In some embodiments, the four HA / NA antigens are derived from three influenza B viruses and one influenza A virus. In some embodiments, the four HA / NA antigens are derived from two influenza B viruses and two influenza A viruses.

[0457] The HA antigen and / or NA antigen may be any HA antigen and / or NA antigen described in this application or known in the art.

[0458] In some embodiments, the immunogenic compositions disclosed herein comprise at least four circular RNAs, each containing an HA sequence encoding an HA antigen. In some embodiments, the four circular RNAs comprise HA sequences encoding HA antigens derived from four influenza viruses. In some embodiments, the four influenza viruses comprise two influenza A viruses and two influenza B viruses.

[0459] In some embodiments, the virus is a seasonal virus. In some embodiments, the HA antigen is selected based on GISRS criteria.

[0460] For illustrative purposes, in some embodiments, the four influenza viruses are A / Wisconsin / 588 / 2019(H1N1) influenza virus, A / Darwin / 6 / 2021(H3N2) influenza virus, B / Austria / 1359417 / 2021(B / Victoria lineage) influenza virus, and B / Phuket / 3073 / 2013(B / Yamagata lineage) influenza virus. In some embodiments, the four HA antigens are A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus HA protein (e.g., SEQ ID NO: 175), A / Darwin / 6 / 2021(H3N2) influenza virus HA protein (e.g., SEQ ID NO: 177), and B / Austria / 1359 These are the 417 / 2021 (B / Victoria lineage) influenza virus HA protein (e.g., SEQ ID NO: 179) and the B / Phuket / 3073 / 2013 (B / Yamagata lineage) influenza virus HA protein (e.g., SEQ ID NO: 180). In some embodiments, the four HA antigens each have an amino acid sequence that has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or ...

Claims

1. A circular ribonucleic acid (circular RNA) comprising a translation initiation (TI) sequence and a protein-coding (Z) sequence, 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).

2. The circular RNA according to claim 1, wherein the Z sequence comprises a first HA (HA1) sequence encoding an HA antigen derived from a first influenza virus of type A or type B.

3. The circular RNA according to claim 2, wherein the Z sequence further comprises a second HA (HA2) sequence encoding an HA antigen derived from a second influenza virus of type A or B.

4. The circular RNA according to claim [0068], wherein the circular RNA comprises the sequences TI, HA1, L, and HA2 in that order, where L is a linker sequence or is absent, and the first and second influenza viruses are (1) A1 and A2, (2) B1 and B2, (3) A and B, or (4) B and A, respectively, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

5. The circular RNA according to claim 3, wherein the Z sequence further comprises a third HA (HA3) sequence encoding an HA antigen derived from a third influenza virus of type A or B.

6. The circular RNA according to claim [0070], wherein the circular RNA contains the sequences TI, HA1, L1, HA2, L2, and HA3 in that order, and L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

7. The circular RNA according to claim [0070], wherein the Z sequence further comprises a fourth HA (HA4) sequence encoding an HA antigen derived from a fourth influenza virus of type A or B.

8. The circular RNA according to claim [0072], wherein the circular RNA comprises the sequences TI, HA1, L1, HA2, L2, HA3, L3, and HA4 in that order, and L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (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, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

9. The circular RNA according to claim 1, wherein the Z sequence comprises a first NA (NA1) sequence encoding an NA antigen derived from a first influenza virus of type A or type B.

10. The circular RNA according to claim [0074], wherein the Z sequence further comprises a second NA (NA2) sequence encoding an NA antigen derived from a second influenza virus of type A or B. 。

11. The circular RNA according to claim [0075], wherein the circular RNA comprises the sequences TI, NA1, L, and NA2 in that order, where L is a linker sequence or is absent, and the first and second influenza viruses are (1) type A1 and type A2, (2) type B1 and type B2, (3) type A and type B, or (4) type B and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

12. The circular RNA according to claim [0075], wherein the Z sequence further comprises a third NA (NA3) sequence encoding an NA antigen derived from a third influenza virus of type A or type B.

13. The circular RNA according to claim [0077], wherein the circular RNA comprises the sequences TI, NA1, L1, NA2, L2, and NA3 in that order, and L1 and L2 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

14. The circular RNA according to claim [0077], wherein the Z sequence further comprises a fourth NA (NA4) sequence encoding an NA antigen derived from a fourth influenza virus of type A or B.

15. The circular RNA according to claim [0079], wherein the circular RNA comprises the sequences TI, NA1, L1, NA2, L2, NA3, L3, and NA4 in that order, and L1, L2, and L3 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2, respectively; (2) A1, B1, A2, and B2; (3) B1, A1, B2, and A2; or (4) B1, B2, A1, and A2, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

16. The circular RNA according to claim 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 antigen and the NA antigen are derived from a first influenza virus of type A or type B.

17. The circular RNA according to claim [0081], having the sequence in the following order: (1) TI, HA1, L, and NA1; or (2) TI, NA1, L, and HA1, wherein L is a linker sequence or is absent.

18. The circular RNA according to claim [0081], 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, and the HA antigen and the NA antigen are derived from a second influenza virus of type A or B.

19. The arrangement is 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) T The circular RNA according to claim [0083], comprising I, HA1, L1, HA2, L2, NA1, L3, and NA2; or (4) TI, NA1, L1, NA2, L2, HA1, L3, and HA2, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first and second influenza viruses are (1) A1 and A2 types; (2) B1 and B2 types; (3) A and B types; or (4) B and A types, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

20. The circular RNA according to claim [0083], 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, the HA antigen and the NA antigen being derived from a third influenza virus of type A or B.

21. The arrangement is 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) including TI, NA1, L1, HA1, L2, NA2, L3, HA2, L4, NA3, L5, and HA3, where L1, L2, L3, L4, and L5 are each independently a linker arrangement. The circular RNA according to claim [0085], wherein the first, second, and third influenza viruses are (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, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

22. The circular RNA according to claim 1, comprising 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 in this order, wherein the Z1 and Z2 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

23. The circular RNA according to claim [0087], wherein the Z1 sequence comprises a first HA (HA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence, encodes HA antigens derived from first and second influenza viruses, and the first and second influenza viruses are independently type A or type B.

24. The circular RNA according to claim [0088], comprising TI1, HA1, L1, TI2, HA2, and L2 in this order, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

25. The circular RNA according to claim [0087], wherein the Z1 sequence comprises a first NA (NA1) sequence, the Z2 sequence comprises a second NA (NA2) sequence, encodes HA antigens derived from first and second influenza viruses, and the first and second influenza viruses are independently type A or type B.

26. The first and second inf are included in this order, and L1 and L2 are either independently in the linker array or absent, and the first and second inf The circular RNA according to claim [0090], wherein the influenza virus is (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

27. The circular RNA according to claim [0087], wherein the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, the HA1 sequence and the NA1 sequence encode an HA antigen and an NA antigen derived from a first influenza virus, the HA2 sequence and the NA2 sequence encode an HA antigen and an NA antigen derived from a second influenza virus, and the first and second influenza viruses are independently type A or type B.

28. The circular RNA according to claim [0092], having the sequence in the following order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, and NA2; or (2) comprising TI1, NA1, L1, HA1, TI2, NA2, L2, and HA2, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; or (3) type A and type B, wherein A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

29. The circular RNA according to claim [0087], 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, and the HA antigen and the NA antigen are derived from a first influenza virus of type A or type B.

30. The circular RNA according to claim [0094], wherein the Z1 sequence further comprises a second HA (HA2) sequence, and the Z2 sequence further comprises a second NA (NA2) sequence, and the HA2 sequence and the NA2 sequence encode an HA antigen and an NA antigen derived from a second influenza virus of type A or B.

31. The circular RNA according to claim [0095], comprising the sequences TI1, HA1, L1, HA2, TI2, NA1, L2, and NA2 in this order, wherein L1 and L2 are independently linker sequences or are absent, and the first and second influenza viruses are (1) type A1 and type A2; (2) type B1 and type B2; (3) type A and type B; or (4) type B and type A, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

32. The circular RNA according to claim [0095], wherein the Z1 sequence further comprises a third HA (HA3) sequence, and the Z2 sequence further comprises a third NA (NA3) sequence, and the HA3 sequence and the NA3 sequence encode an HA antigen and an NA antigen derived from a third influenza virus of type A or B.

33. The sequence TI1, HA1, L1, HA2, L2, HA3, TI2, NA1, L3, NA2, L4, and NA3 is included in this order, and L1, L2, L3, and L4 are each independently linker sequences or are absent, and the first, second, and third influenza viruses are (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, and A1 and A2 The circular RNA according to claim [0097], wherein B1 and B2 are first and second subtypes of influenza A virus.

34. The circular RNA according to claim 1, comprising 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 in this order, wherein the Z1, Z2, and Z3 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

35. The circular RNA according to claim [0099], wherein 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 HA1 sequence, the HA2 sequence, and the HA3 sequence each encode an HA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively.

36. The circular RNA according to claim [00100], comprising TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, and L3 in this order, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (1) A1, A2, and B types, or (2) B1, B2, and A types, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

37. The circular RNA according to claim [0099], 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 NA1 sequence, the NA2 sequence, and the NA3 sequence each encode an NA antigen derived from a first influenza virus, a second influenza virus, and a third influenza virus, respectively.

38. The circular RNA according to claim [00102], comprising TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, and L3 in this order, wherein L1, L2, and L3 are independently linker sequences or are absent, and the first, second, and third influenza viruses are (1) A1, A2, and B types, or (2) B1, B2, and A types, respectively, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

39. The circular RNA according to claim [0099], wherein the Z1 sequence comprises a first HA (HA1) sequence and a first NA (NA1) sequence, the Z2 sequence comprises a second HA (HA2) sequence and a second NA (NA2) sequence, the Z3 sequence comprises a third HA (HA3) sequence and a third NA (NA3) sequence, the HA1 sequence and the NA1 sequence encode an antigen derived from a first influenza virus, the HA2 sequence and the NA2 sequence encode an antigen derived from a second influenza virus, and the HA3 sequence and the NA3 sequence encode an antigen derived from a third influenza virus.

40. The following sequences are arranged in the order: (1) TI1, HA1, L1, NA1, TI2, HA2, L2, NA2, TI3, HA3, L3, and NA3; or (2) including TI1, NA1, L1, HA1, TI2, NA2, L2, HA2, TI3, NA3, L3, and HA3, where L1, L2, and L3 are each independently linker sequences or are absent, and the first, The circular RNA according to claim [00104], wherein the 2. and 3. influenza viruses are (1) A1, A2, and B types, or (2) B1, B2, and A types, where A1 and A2 are the first and second subtypes of influenza A virus, and B1 and B2 are the first and second subtypes of influenza B virus.

41. The circular RNA according to claim 1, comprising 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, and a fourth protein coding (Z4) sequence in this order, wherein the Z1, Z2, Z3, and Z4 sequences each independently comprise an HA sequence encoding an HA antigen or an NA sequence encoding an NA antigen.

42. The circular RNA according to claim [00106], wherein the Z1 sequence includes a first HA (HA1) sequence, the Z2 sequence includes a second HA (HA2) sequence, the Z3 sequence includes a third HA (HA3) sequence, the Z4 sequence includes a fourth HA (HA4) sequence, and the HA1 sequence, the HA2 sequence, the HA3 sequence, and the HA4 sequence each encode a first influenza virus, a second influenza virus, a third influenza virus, and an HA antigen derived from the third influenza virus, respectively.

43. The circular RNA according to claim [00107], comprising the sequences TI1, HA1, L1, TI2, HA2, L2, TI3, HA3, L3, TI4, HA4, and L4 in this order, wherein L1, L2, L3, and L4 are each independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are each (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, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

44. The circular RNA according to claim [00106], 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, the Z4 sequence comprises a fourth NA (NA4) sequence, and the NA1 sequence, the NA2 sequence, the NA3 sequence, and the NA4 sequence each encode an NA antigen derived from a first influenza virus, a second influenza virus, a third influenza virus, and a fourth influenza virus, respectively.

45. The circular RNA according to claim [00109], comprising the sequences TI1, NA1, L1, TI2, NA2, L2, TI3, NA3, L3, TI4, NA4, and L4 in this order, wherein L1, L2, L3, and L4 are independently linker sequences or are absent, and the first, second, third, and fourth influenza viruses are (1) A1, A2, B1, and B2, respectively; (2) A1, B1, A2, and B2; (3) B1, A1, B2, and A2; or (4) B1, B2, A1, and A2, where A1 and A2 are first and second subtypes of influenza A virus, and B1 and B2 are first and second subtypes of influenza B virus.

46. The circular RNA according to any one of claims 1 to [00110], wherein the HA antigen and / or the NA antigen are derived from a seasonal influenza virus.

47. The HA antigen and / or the NA antigen are used in the World Health Organization's Global Influenza Surveillance and Response System. The circular RNA according to claim [00111], selected according to standardized criteria adopted by the system (GISRS).

48. The circular RNA according to claim [00111], wherein the HA antigen and / or the NA antigen are selected using a hemagglutinin inhibitory (HAI) assay and / or a neuraminidase inhibitory (NAI) assay to identify a currently circulating influenza virus that is antigenically similar to the influenza virus of the previous season's vaccine.

49. The circular RNA according to any one of claims 1 to [00105], wherein the HA antigen and / or the NA antigen is derived from the influenza A virus listed in Table 1 or the influenza B virus listed in Table 2.

50. The circular RNA according to any one of claims 1 to [00105], wherein the HA antigen and / or the NA antigen are derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus.

51. The circular RNA according to any one of claims 1 to [00105], wherein the HA antigen and / or the NA antigen is derived from an influenza A virus strain which 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.

52. The circular RNA according to any one of claims 1 to [00105], wherein the HA antigen and / or the NA antigen is derived from an influenza B virus strain which is B / Austria / 1359417 / 2021 or B / Phuket / 3073 / 2013.

53. The circular RNA according to any one of claims 1 to [00105], wherein the HA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 3, Table 5A, Table 7A, or Table 8A, and / or the NA antigen has at least 85%, at least 90%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence described in Table 4, Table 5B, Table 7B, or Table 8B.

54. The circular RNA according to claim [00118], wherein the HA sequence has a nucleotide sequence listed in Table 6A, Table 9A, Table 10A, or Table 14, and / or the NA sequence has a nucleotide sequence listed in Table 6B, Table 9B, or Table 10B.

55. The circular RNA according to claim 1, wherein the Z sequence comprises an HA sequence encoding an HA antigen.

56. The circular RNA according to claim [00120], wherein the HA antigen is derived from an H1N1 virus, an H3N2 virus, a B / Victoria lineage virus, or a B / Yamagata lineage virus.

57. The circular RNA according to claim [00121], wherein the HA antigen is the HA protein of the A / Wisconsin / 588 / 2019(H1N1)pdm09-like influenza virus.

58. The HA antigen is identical in sequence to at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence of Sequence ID No.

175. A circular RNA having unisexuality, as described in claim [00324].

59. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 182, 184-188, and 209-210.

60. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of Sequence ID No.

210.

61. The circular RNA according to claim [00121], wherein the HA antigen is the HA protein of the A / Darwin / 6 / 2021 (H3N2)-like influenza virus.

62. The circular RNA according to claim [00126], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of Sequence ID No.

177.

63. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 183, 189-193, and 211-212.

64. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 211 or 189.

65. The circular RNA according to claim [00121], wherein the HA antigen is the HA protein of influenza virus B / Austria / 1359417 / 2021 (Victoria lineage).

66. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of Sequence ID No.

179.

67. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 194-198 and 213-219.

68. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 214 or 196.

69. The circular RNA according to claim [00121], wherein the HA antigen is the HA protein of influenza virus B / Phucket / 3073 / 2013 (B / Yamagata lineage).

70. The HA antigen is identical in sequence to at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequence of Sequence ID No.

180. A circular RNA having unisexuality, as described in claim [00134].

71. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 199-203 and 220-221.

72. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 221 or 199.

73. The circular RNA according to claim [00121], wherein the HA antigen is the HA protein of the A / Wisconsin / 67 / 2022(H1N1)pdm09-like influenza virus.

74. The circular RNA according to claim [00138], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of Sequence ID No.

235.

75. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 281-285 and 520-521.

76. The circular RNA according to claim [00324], wherein the HA antigen has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 520 or 521.

77. The circular RNA according to any one of claims 1 to [00401], wherein the circular RNA includes a linker sequence encoding a 2A self-cleaving peptide.

78. The circular RNA according to any one of claims 1 to [00142], wherein the TI sequence comprises IRES, an IRES-like nucleotide sequence, or a combination thereof.

79. The circular RNA according to claim [00142], wherein the TI sequence comprises an optimized IRES sequence.

80. The circular RNA according to claim [00142], wherein the IRES sequence has at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences listed in Table 11.

81. The circular RNA according to claim 1, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences listed in Table 13.

82. The circular RNA according to claim 1, having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a nucleotide sequence selected from sequence numbers 499 to 519.

83. Claims 1 to [00147], wherein the circular RNA is scar-free or nearly scar-free. A circular RNA described in any one of the items.

84. The circular RNA according to any one of claims 1 to [00148], wherein, when administered to a subject, the innate immune response induced by the circular RNA is reduced compared to mRNA encoding the HA antigen and / or NA antigen.

85. The circular RNA according to any one of claims 1 to [00149], wherein the circular RNA comprises a modified RNA nucleotide and / or a modified nucleoside.

86. The circular RNA according to claim [00150], wherein the circular RNA comprises at least 10% modified RNA nucleotides and / or modified nucleosides.

87. At least one of the modified RNA nucleotides and / or modified nucleosides is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2 The cyclic RNA according to claim [00150], wherein the RNA is '-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 (pseudolidine), or m1A (1-methyladenosine).

88. The aforementioned circular RNA is 5-methylcytidine (m5C), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), A circular RNA according to any one of claims 1 to [00148], which does not contain 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 (pseudolidine), or m1A (1-methyladenosine).

89. The circular RNA according to any one of claims [00150] to [00417], wherein at least one of the modified RNA nucleotides and / or modified nucleosides is introduced by in vitro transcription (IVT).

90. An immunogenic composition comprising the circular RNA described in any one of claims 1 to [00417].

91. An immunogenic composition comprising at least two, at least three, at least four, at least five, or at least six circular RNAs as described in any one of claims 1 to [00417].

92. The immunogenic composition according to claim [00453], comprising two circular RNAs.

93. The Z sequences of the two circular RNAs described above contain the HA antigens derived from the two influenza viruses. The immunogenic composition according to claim [00157], comprising an HA sequence.

94. The immunogenic composition according to claim [00454], wherein the two influenza viruses comprise two influenza A viruses, two influenza B viruses, or one influenza A virus and one influenza B virus.

95. The immunogenic composition according to claim [00453], comprising four circular RNAs.

96. The immunogenic composition according to claim [00160], wherein the Z sequences of the four circular RNAs include HA sequences encoding four influenza virus-derived HA antigens.

97. The immunogenic composition according to claim [00458], wherein the four influenza viruses comprise two influenza A viruses and two influenza B viruses.

98. The immunogenic composition according to claim [00458], wherein the four influenza viruses are A / Wisconsin / 588 / 2019 (H1N1) influenza virus, A / Darwin / 6 / 2021 (H3N2) influenza virus, B / Austria / 1359417 / 2021 (B / Victoria lineage) influenza virus, and B / Phuket / 3073 / 2013 (B / Yamagata lineage) influenza virus.

99. The immunogenic composition according to claim [00460], wherein the four HA antigens are the HA protein of influenza virus A / Wisconsin / 588 / 2019(H1N1)pdm09, the HA protein of influenza virus A / Darwin / 6 / 2021(H3N2), the HA protein of influenza virus B / Austria / 1359417 / 2021(B / Victoria lineage), and the HA protein of influenza virus B / Phuket / 3073 / 2013(B / Yamagata lineage).

100. The immunogenic composition according to claim [00460], wherein the amino acid sequences of the four HA antigens have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences of SEQ ID NOs: 175, 177, 179, and 180, respectively.

101. The immunogenic composition according to claim [00461], wherein the four HA sequences each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of sequence numbers 210, 211, 214, and 221.

102. The immunogenic composition according to claim [00166], wherein the four circular RNAs each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 501, 519, 514, and 508.

103. The immunogenic composition according to claim [00458], wherein the four influenza viruses are the A / Wisconsin / 67 / 2022 (H1N1) influenza virus, the A / Darwin / 6 / 2021 (H3N2) influenza virus, the HA protein of the B / Austria / 1359417 / 2021 (B / Victoria lineage) influenza virus, and the HA protein of the B / Phuket / 3073 / 2013 (B / Yamagata lineage) influenza virus.

104. The immunogenic composition according to claim [00462], wherein the four HA antigens are the HA protein of influenza virus A / Wisconsin / 67 / 2022(H1N1)pdm09, the HA protein of influenza virus A / Darwin / 6 / 2021(H3N2), the HA protein of influenza virus B / Austria / 1359417 / 2021(B / Victoria lineage), and the HA protein of influenza virus B / Phuket / 3073 / 2013(B / Yamagata lineage).

105. The immunogenic composition according to claim [00462], wherein the amino acid sequences of the four HA antigens have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequences of SEQ ID NOs. 235, 177, 179, and 180, respectively.

106. The immunogenic composition according to claim [00462], wherein the four HA sequences each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of sequence numbers 521, 189, 196, and 221, respectively.

107. The immunogenic composition according to claim [00463], wherein the four circular RNAs each have at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences of SEQ ID NOs. 518, 519, 508, and 515.

108. The immunogenic composition according to any one of claims [00453] to [00172], wherein the cyclic RNA is incorporated into lipid nanoparticles (LNPs).

109. The immunogenic composition according to claim [00173], wherein each of the aforementioned circular RNAs is separately incorporated into the LNP.

110. An immunogenic composition according to any one of claims [00453] to [00174], comprising an adjuvant.

111. An immunogenic composition according to any one of claims [00453] to [00175], used to induce an anti-influenza immune response in a subject.

112. A method for inducing an anti-influenza immune response in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to any one of claims [00453] to [00175].

113. The method according to claim [00177], which induces cross-protective immunity against heterologous influenza viruses.

114. The method according to claim [00177], which induces protective immunity against the same subtype of influenza virus.

115. The method according to any one of claims [00177] to [00179], wherein the immune response is measured by an anti-hemagglutinin (HA) antibody titer, a hemagglutinin inhibitor (HAI) titer, or a microneutralization assay.

116. The method according to claim [00180], wherein the immune response is measured by HAI titer.

117. The method according to claim [00181], wherein the subject 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.

118. The method according to claim [00181], wherein the HAI titer produced in the subject is at least three times, at least four times, at least five times, or at least six times the baseline HAI level.

119. The method according to any one of claims [00181] to [00534], wherein the HAI titer produced by the method is at least 40 and lasts for at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least one year, or at least two years.

120. The method according to claim [00180], wherein the immune response is measured by anti-HA antibody titer approximately 7, 14, 21, 28, or 35 days after vaccination.

121. The method according to claim [00185], which induces an increase of at least 50 times, at least 100 times, at least 500 times, at least 1,000 times, at least 5,000 times, or at least 10,000 times in the anti-HA IgG antibody titer of the subject compared to before administration.

122. The method according to claim [00185] or [00186], wherein the anti-HA IgG antibody titer is increased by at least 50 times and lasts 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.

123. The method according to any one of claims [00177] to [00187], wherein the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, or intraperitoneally.

124. The method according to any one of claims [00177] to [00188], comprising a single dose followed optionally by a boost dose.

125. The method according to any one of claims [00177] to [00189], wherein the subject is a human.

126. The method according to claim [00190], wherein the subject is 60 years of age or older.

127. The method according to claim [00190], wherein the subject is 18 years of age or younger.

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