Cyclic RNA vaccines against SARS-COV-2 variants and methods of use thereof

Abstract

The present application provides a circular RNA (circRNA) that encodes an antigen polypeptide of an SARS-CoV-2 variant. In some embodiments, the present application provides a circRNA vaccine against an SARS-CoV-2 variant, such as a Delta or Aumike variant. In some embodiments, a circRNA vaccine comprises a circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide comprising a spike (S) protein of the SARS-CoV-2 variant, or a fragment thereof. Also provided are methods of treating or preventing SARS-CoV-2 infection using the circRNA disclosed herein, or a composition thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Patent Application No. PCT / CN2022 / 071347 filed on January 11, 2022 and International Patent Application No. PCT / CN2022 / 074752 filed on January 28, 2022, the disclosures of which are incorporated herein by reference in their entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (165392001043seqlist.xml; size: 95,801 bytes; creation date: January 6, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to circular RNA (circRNA) vaccines against SARS-Co-V-2 variants, and methods of using the same. Background Art

[0006] Coronavirus disease 2019 (COVID-19) is a serious global public health emergency caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). 1,2 To date, COVID-19 has resulted in more than 200 million confirmed cases and more than 5 million confirmed deaths (World Health Organization). Therefore, there is an urgent need to develop safe and effective vaccines against SARS-CoV-2 and emerging variants.

[0007] SARS-CoV-2, along with severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV (two other highly pathogenic coronaviruses), belongs to the genus Betacoronavirus in the family Coronaviridae. 3 SARS-CoV-2 is a single-stranded, positive-sense enveloped virus, whose virion consists of an inner capsid formed by a 30kb RNA genome wrapped by nucleocapsid (N) protein and a lipid envelope of tegument (M), envelope (E), and trimeric spike (S) proteins. 4 The S protein of SARS-CoV-2 is composed of S1 and S2 subunits and is the main surface protein of the virion. The S protein binds to its receptor, angiotensin-converting enzyme 2 (ACE2), via the receptor binding domain (RBD) at the C-terminus of the S1 subunit, mediating the entry of the virus into host cells. This binding subsequently induces fusion between the SARS-CoV-2 envelope and the host cell membrane, mediated by the S2 subunit, leading to the release of the viral genome into the cytoplasm. 5-8.

[0008] The S protein, S1 subunit or RBD antigen of SARS-CoV-2 can induce B cell and T cell responses to produce highly potent neutralizing antibodies against SARS-CoV-2 9-11 Vaccination is the most promising approach to end the COVID-19 pandemic. Traditional vaccine platforms such as inactivated vaccines, virus-like particle vaccines, and viral vector-based vaccines have been adopted to develop SARS-CoV-2 vaccines. 12-20 Importantly, mRNA vaccines against SARS-CoV-2 have been developed at an extremely fast pace and have been approved for emergency use. 21-27 , although this strategy has never been used commercially before 28 mRNA vaccines contain linear single-stranded RNA, which consists of a 5' cap, untranslated region (UTR), antigen coding region and 3' polyA tail, and are delivered into the body via lipid nanoparticle (LNP) encapsulation 28 Clinical-scale mRNA vaccines can be produced rapidly after viral antigen sequences are released 21 However, current mRNA vaccines still have certain limitations due to their inherent instability and suboptimal thermal stability after LNP encapsulation for in vivo administration. 29-31 , and potential immunogenic side effects 32,33 .

[0009] Circular RNAs (circRNAs) are covalently closed single-stranded RNA transcripts that comprise a large class of noncoding RNAs generated by a non-canonical RNA splicing event in eukaryotic cells called backsplicing. 34-36 Some viral genomes happen to be circular RNA, such as hepatitis D virus and plant viruses 33 In recent years, thousands of circRNAs have been identified in eukaryotes, including fungi, plants, insects, fish, and mammals, through high-throughput RNA sequencing and circRNA-specific bioinformatics. 36 Unlike linear mRNA, circRNA is highly stable because its covalently closed ring structure protects it from exonuclease-mediated degradation. 36-38 So far, only a few endogenous circRNAs have been shown to function as templates for protein translation. 39-42 Although circRNA lacks essential elements for cap-dependent translation, it can be engineered to enable protein translation through internal ribosome entry sites (IRES) or by incorporating m6A modifications upstream of open reading frames (ORFs). 43,44 . SUMMARY OF THE INVENTION

[0011] The present application provides novel vaccines based on circular RNA (circRNA) against SARS-CoV-2 variants (e.g., Delta or Omicron). Also provided are methods for producing circRNA vaccines and methods for using circRNA vaccines to treat or prevent SARS-CoV-2 infection.

[0012] One aspect of the present application provides a circular RNA (circRNA) comprising a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant, wherein the SARS-CoV-2 variant is a delta variant or an omega variant. In some embodiments, the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant. In some embodiments, the antigenic polypeptide comprises a receptor binding domain (RBD) of the S protein. In some embodiments, the RBD comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 variant is a delta variant, and wherein the antigenic polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the SARS-CoV-2 variant is an omega variant, and wherein the antigenic polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0013] In some embodiments according to any of the above circRNAs, the antigenic polypeptide further comprises a multimerization domain. In some embodiments, the multimerization domain is a C-terminal foldon (Fd) domain of T4 fibrin, which mediates trimerization of T4 fibrin. In some embodiments, the Fd domain comprises the amino acid sequence of SEQ ID NO: 3.

[0014] In some embodiments of any of the above circRNAs, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigen polypeptide.

[0015] In some embodiments of the circRNA according to any one of the above, the circRNA further comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigen polypeptide.

[0016] In some embodiments according to any of the above circRNAs, the circRNA further comprises an internal ribosome entry site (IRES) sequence operably linked to a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the IRES sequence is an IRES sequence selected from the group consisting of: IRES sequences of CVB3 virus, EV71 virus, EMCV virus, PV virus, and CSFV virus. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, and a nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA further comprises a polyAC or polyA sequence located at the 5' end of the IRES sequence.

[0017] In some embodiments according to any of the above circRNAs, the circRNA further comprises an m6A modification motif sequence operably linked to a nucleic acid sequence encoding an antigen polypeptide. In some embodiments, the circRNA further comprises a nucleic acid sequence, which comprises, from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence, and a nucleic acid sequence encoding the antigen polypeptide.

[0018] In some embodiments of any of the above circRNAs, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide.

[0019] Another aspect of the present application provides a composition comprising a plurality of circRNAs according to any of the above circRNAs, wherein the antigenic polypeptides corresponding to the plurality of circRNAs are different from each other. In some embodiments, the plurality of circRNAs target a plurality of SARS-CoV-2 variants.

[0020] One aspect of the present application provides a circRNA vaccine comprising any of the above-mentioned circRNA or any of the above-mentioned compositions. In some embodiments, the circRNA vaccine further comprises a transfection agent. In some embodiments, the transfection agent is polyethyleneimine (PEI). In some embodiments, the transfection agent is a lipid nanoparticle (LNP). In some embodiments, LNP comprises ionizable lipids, PEG2000-DMG, DSPC and cholesterol. In some embodiments, circRNA is not formulated with a transfection agent.

[0021] Also provided is a pharmaceutical composition comprising any of the above-mentioned circRNAs and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a transfection agent. In some embodiments, the transfection agent is PEI. In some embodiments, the transfection agent is LNP. In some embodiments, LNP includes ionizable lipids, PEG2000-DMG, DSPC and cholesterol. In some embodiments, the circRNA is not formulated with a transfection agent.

[0022] Further provided is a method for treating or preventing an individual SARS-CoV-2 infection, comprising administering to the individual an effective amount of any of the above-mentioned circRNAs, any of the above-mentioned compositions, any of the above-mentioned pharmaceutical compositions, or any of the above-mentioned circRNA vaccines. In some embodiments, the circRNA is administered in the form of naked circRNA. In some embodiments, the circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, the circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about four weeks, such as at least any one of five, six, seven, or eight weeks. In some embodiments, the SARS-CoV-2 infection is caused by a SARS-CoV-2 variant. In some embodiments, the SARS-CoV-2 infection is caused by more than one SARS-CoV-2 variant. In some embodiments, the SARS-CoV-2 infection is caused by a delta variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 infection is caused by an Omicron variant of SARS-CoV-2. In some embodiments, the SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariant (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1). In some embodiments, the circRNA is translated by the ribosome in an individual in a rolling circle.

[0023] Other aspects of the present application provide linear RNAs capable of forming any circRNA provided herein. In some embodiments, the linear RNA can be cyclized by self-catalysis of a group I intron comprising a 5' catalytic group I intron fragment and a 3' catalytic group I intron fragment. In some embodiments, the linear RNA comprises: a 3' catalytic group I intron fragment flanked by the 5' end of a 3' exon sequence that can be recognized by a group I intron, and a 5' catalytic group I intron fragment flanked by the 3' end of a 5' exon sequence that can be recognized by a group I intron. In some embodiments, the 3' catalytic group I intron fragment comprises a sequence of SEQ ID NO: 28, and the 5' catalytic group I intron fragment comprises a sequence of SEQ ID NO: 29. In some embodiments, the linear RNA further comprises: a 5' homologous sequence flanked by the 5' end of the 3' catalytic group I intron fragment, and a 3' homologous sequence flanked by the 3' end of the 5' catalytic group I intron fragment. In some embodiments, the 5' homologous sequence comprises the nucleic acid sequence of SEQ ID NO:23 and the 3' homologous sequence comprises the nucleic acid sequence of SEQ ID NO:24.

[0024] In some embodiments, the linear RNA can be cyclized by a ligase (e.g., RNA ligase). In some embodiments, the ligase is selected from the group consisting of T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2). In some embodiments, the linear RNA comprises: a 5' linker sequence at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' linker sequence at the 3' end of the nucleic acid sequence encoding the circRNA, wherein the 5' linker sequence and the 3' linker sequence can be connected to each other by a ligase.

[0025] One aspect of the present application provides a nucleic acid construct comprising a nucleic acid sequence encoding any one of the above-mentioned linear RNAs. In some embodiments, the nucleic acid construct comprises a T7 promoter operably linked to the nucleic acid sequence encoding the linear RNA.

[0026] One aspect of the present application provides a method for producing circRNA, comprising: (a) subjecting any one of the above-mentioned linear RNAs to conditions that activate the autocatalysis of a 5' catalytic group I intron fragment and a 3' catalytic group I intron fragment to provide a circularized RNA product, wherein the linear RNA comprises: a 3' catalytic group I intron fragment flanked by the 5' end of a 3' exon sequence that can be recognized by a group I intron, and a 5' catalytic group I intron fragment flanked by the 3' end of a 5' exon sequence that can be recognized by a group I intron; and (b) isolating the circularized RNA product to provide a circRNA vaccine.

[0027] One aspect of the present application provides a method for producing circRNA, comprising: (a) contacting any one of the above-mentioned linear RNAs with a single-stranded adapter nucleic acid, wherein the linear RNA comprises: a 5' linker sequence located at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' linker sequence located at the 3' end of the nucleic acid sequence encoding the circRNA, wherein the single-stranded adapter nucleic acid comprises from the 5' end to the 3' end: a first sequence complementary to the 3' linker sequence, and a second sequence complementary to the 5' linker sequence, and wherein the 5' linker sequence and the 3' linker sequence hybridize with the single-stranded adapter nucleic acid to provide a double-stranded nucleic acid intermediate, wherein the double-stranded nucleic acid intermediate comprises a single-stranded break between the 3' end of the 5' linker sequence and the 5' end of the 3' linker sequence; (b) contacting the intermediate with an RNA ligase under conditions that allow the 5' linker sequence to be ligated to the 3' linker sequence to provide a circularized RNA product; and (c) isolating the circularized RNA product to provide a circRNA vaccine.

[0028] One aspect of the present application provides a method for producing circRNA, comprising: (a) contacting any one of the above-mentioned linear RNAs with an RNA ligase under conditions that allow the 5' linker sequence to be ligated to the 3' linker sequence to provide a circularized RNA product, wherein the linear RNA comprises: a 5' linker sequence located at the 5' end of the nucleic acid sequence encoding the circRNA, and a 3' linker sequence located at the 3' end of the nucleic acid sequence encoding the circRNA; and (b) isolating the circularized RNA product to provide a circular RNA.

[0029] In some embodiments of the method for producing a circRNA vaccine according to any of the above, the method further comprises: obtaining the linear RNA by in vitro transcription of a nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA. In some embodiments of the method for producing a circRNA vaccine according to any of the above, the method further comprises purifying the circularized RNA product.

[0030] Compositions, kits, and articles of manufacture for use in any of the methods described above are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A-1I The expression of SARS-CoV-2 RBD antigen using circular RNA in vitro is shown. Figure 1A Shown is the autocatalytic activity of circRNAs by group I ribozymes RBD Schematic diagram of cyclization. SP, signal peptide sequence of human tPA protein. Foldon, trimerization domain from bacteriophage T4 fiber protein. RBD, receptor binding domain of SARS-CoV-2 spike protein. Arrows indicate the design of primers used for PCR analysis. Figure 1BDisplay linear RNA RBD and circRNA RBD Agarose gel electrophoresis results of PCR products. Figure 1C Western Blot analysis showing the transfection of circRNAs autocatalyzed by group I ribozymes RBD Expression level of RBD antigen in HEK293T cell supernatant. EGFP and linear RNA RBD was set as control. Figure 1D A quantitative ELISA assay is shown for measuring the concentration of RBD antigen in the supernatant. Figure 1E Western Blot analysis shows transfection of circRNA RBD Expression level of RBD antigen in the supernatant of mouse NIH3T3 cells. EGFP was set as control. Figure 1F is a circRNA circularized by T4 RNA ligase RBD Schematic diagram of the spike protein. SP, signal peptide sequence of human tPA protein. Foldon, trimerization domain from bacteriophage T4 fiber protein. RBD, receptor binding domain of SARS-CoV-2 spike protein. Figure 1G Western Blot analysis showing transfection of circRNA circularized by T4 RNA ligase RBD Expression level of RBD antigen in the supernatant of HEK293T cells. EGFP and linear RNA RBD was set as control. Figure 1H A quantitative ELISA assay is shown for measuring the concentration of RBD antigen in the supernatant. Fig. 1I circRNA RBD - Quantification of competitive inhibition of SARS-CoV-2 pseudoviral infection (EGFP) by translated RBD antigen. circRNA EGFP and linear RNA RBD was set as the control. Figure 1E and Figure 1H Data are shown as mean ± SEM (n = 3). Fig. 1I Data are shown as mean ± SEM (n = 2).

[0032] Figure 2A-2O The immunogenicity and protective effect of SARS-CoV-2 circRNA vaccine in mice were shown. Figure 2A is a schematic diagram of the LNP-circRNA complex. Figure 2BShowing LNP-circRNA measured by dynamic light scattering RBD Concentration size diagram. Figure 2C LNP-circRNA RBD Schematic representation of the vaccination procedure in BALB / c mice and the serum collection schedule for specific antibody analysis. Figure 2D SARS-CoV-2 specific IgG antibody titers measured by ELISA are shown. Data are shown as mean ± SEM (n = 4 or 5). Figure 2E Shown using circRNA RBD Neutralization test of sera from vaccine-immunized mice against D614G pseudovirus based on VSV. Serum samples were collected 5 weeks after booster immunization. Data are shown as mean ± SEM (n = 5). Figure 2F Shown using circRNA RBD Neutralization test of sera from mice immunized with the vaccine against SARS-CoV-2 real virus. Serum samples were collected 5 weeks after booster immunization. Data are shown as mean ± SEM Figure 2G CircRNAs detected by ELISA are shown RBD-β SARS-CoV-2 (β)-specific IgG antibody titers in immunized mice. Data are shown as mean ± SEM (n = 4 or 5). Figure 2H and 2I Shown using circRNA RBD or circRNA RBD-β Neutralization test of sera from vaccinated mice against D614G, alpha or beta pseudoviruses based on VSV. Serum samples were collected 1 week after booster immunization. Data are shown as mean ± SEM (n = 5). Figure 2J Shown using circRNA RBD-β Neutralization test of vaccine-immunized mouse serum against SARS-CoV-2(β) real virus. Figure 2K Shown using circRNA RBD-β Neutralization test of vaccine-immunized mouse sera against SARS-CoV-2 (D614G) real virus. Data are shown as mean ± SEM (n = 4 or 5). Figure 2L circRNA RBD-β In vitro alternative neutralization test of sera from vaccine-immunized mice. Figure 2M The infectious viral load of SARS-CoV-2 (β) true virus in BALB / c mice is shown. Normalized with GAPDH. Data are shown as mean ± SEM (n=3). Figure 2N Shown are body weight changes of immunized or placebo mice after SARS-CoV-2(β) real virus challenge. Fig.2OVirus load in lung tissue of challenged mice is shown. Data are shown as mean ± SEM (n ≥ 5). NT50 was determined using infectious SARS-CoV-2 real virus. Each symbol represents an individual mouse. Statistical testing was performed by unpaired two-sided Student's t-test.

[0033] Figure 3A-3O SARS-CoV-2 circRNA is shown RBD-德尔塔 Humoral and T cell immune responses elicited by the vaccine in mice. Figure 3A The circRNA produced by group I ribozymes autocatalytically as determined by ELISA is shown. RBD-德尔塔 SARS-CoV-2 (Delta)-specific IgG antibody titers of the vaccine. Data are shown as mean ± SEM (n=5). Figure 3B The circRNA generated by T4 RNA ligase as determined by ELISA is shown RBD-德尔塔 SARS-CoV-2 (Delta)-specific IgG antibody titers of the vaccine. Data are shown as mean ± SEM (n=5). Figure 3C Shown using circRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against VSV-based SARS-CoV-2 (Delta) pseudovirus. Serum samples were collected 1 week after booster immunization. Data are shown as mean ± SEM (n = 5). Figure 3D and Figure 3E The results show that mice treated with placebo or different doses of circRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against SARS-CoV-2(D614G), SARS-CoV-2(α), SARS-CoV-2(β), and SARS-CoV-2(Delta) pseudoviruses. Serum samples were collected 5 weeks after booster immunization. Data are shown as mean ± SEM (n = 5). Figure 3F , 3G 3H and 3H are the results of FACS analysis, showing the expression of single and live CD44 + CD62L - CD8 + The percentages of IFN-γ, IL-2, and TNF-α positive cells assessed in T Fig. 3I , 3J 3K and 3L are the results of FACS analysis, showing that single and live CD44 + CD62L - CD4 + The percentage of IFN-γ, IL-2, TNF-α, and IL-4 positive cells was evaluated in T. Figure 3F-3LData are shown as mean ± SEM (n = 3 or 4). Each symbol represents an individual mouse.

[0034] Figures 4A-4N It was shown that circRNA vaccines stimulated a higher proportion of neutralizing antibodies and stronger Th1-biased T cell immune responses than mRNA vaccines. Figure 4A The antigen expression levels of circRNA, 1mΨmRNA and non-modified mRNA in HEK293T cells at different time points after liposome transfection were compared by ELISA. Data are shown as mean ± SEM (n = 3). Figure 4B The dynamic changes of RNA levels are shown, and the data are shown as mean ± SEM (n = 3). Figure 4C The antigen expression levels of circRNA, 1mΨmRNA and non-modified mRNA in HEK293T cells at different time points after LNP transfection were compared by ELISA. Data are shown as mean ± SEM (n = 3). Figure 4D Western Blot analysis shows transfection of circRNA RBD Expression level of RBD antigen in the supernatant of HEK293T cells. RBD Stored at room temperature, transfected on days 0, 3, 7, and 14. Untreated and linear precursors were set as controls. Figure 4E , 4F and 4G show quantitative ELISA to measure circRNA transfected with LNP RBD , Expression levels of 1 mΨ mRNA and non-modified mRNA. LNP-RNA was stored at different temperatures and transfected at different time points. Data are shown as mean ± SEM (n = 3). Figure 4H Display of circRNAs determined by ELISA RBD , SARS-CoV-2 (Delta)-specific IgG antibody titers of sera from mice immunized with 1 mΨ mRNA. Data are shown as mean ± SEM (n = 5). Fig. 4I CircRNAs detected by ELISA are shown RBD-德尔塔 , SARS-CoV-2 (delta)-specific IgG isotype titers of sera from mice immunized with 1 mΨmRNA RBD-delta. Data are shown as mean ± SEM (n = 5). Figure 4J circRNA RBD-德尔塔 , 1mΨmRNA RBD-德尔塔SARS-CoV-2 (delta)-specific IgG2a / IgG1, IgG2c / IgG1, and (IgG2a+IgG2c) / IgG1 ratios of sera from immunized mice. Data are shown as mean ± SEM (n = 5). Figure 4K , 4L and 4M show the expression of circRNAs at different doses RBD-德尔塔 or 1mΨmRNA RBD-德尔塔 Neutralization assay of SARS-CoV-2 (Delta) pseudovirus based on VSV by sera from vaccine-immunized mice. Serum samples were collected 1 week after booster immunization. Data are shown as mean ± SEM (n = 5). Figure 4N circRNA RBD-德尔塔 or 1mΨmRNA RBD-德尔塔 Neutralizing antibody / binding antibody ratio of the vaccine. The ratio was calculated by dividing the NT50 of each mouse by the end-point IgG titer. Data are shown as mean ± SEM (n = 5).

[0035] Figures 5A-5J Demonstrating circRNA vaccine-induced immunogenicity and protection against SARS-CoV-2 infection in rhesus macaques. Figure 5A The LNP-circRNA of rhesus monkey is shown RBD Schematic representation of the vaccination process and serum collection schedule for specific antibody analysis. Figure 5B CircRNAs detected by ELISA are shown RBD SARS-CoV-2 (D614G)-specific IgG antibody titers in immunized rhesus macaques. Data are shown as mean ± SEM (n = 4). Figure 5C CircRNAs detected with VSV-based SARS-CoV-2(D614G) pseudovirus are shown RBD NT50 of immunized rhesus monkeys. Data are shown as mean ± SEM (n = 4). Figure 5D Different doses of circRNA were shown RBD Neutralization test of vaccine-immunized mouse sera against VSV-based SARS-CoV-2(D614G), SARS-CoV-2(α), SARS-CoV-2(β), and SARS-CoV-2(Delta) pseudoviruses. Data are shown as mean ± SEM (n=4). Figure 5E Different doses of circRNA were shown RBDNeutralization test of vaccine-immunized mouse sera against SARS-CoV-2(D614G), SARS-CoV-2(α), SARS-CoV-2(β) and SARS-CoV-2(Delta) veraviruses. Data are shown as mean ± SEM (n = 4). Fig. 5F The expression of circRNA from ELISPOT assay is shown. RBD Specific SARS-CoV-2 (D614G) IFN-γ, IL-2 and IL-4 responses of PBMCs from vaccinated rhesus macaques. Each point represents an independent biological replicate. Figure 5G Virus load (N gene) and subgenomic RNA load (E gene) in lung tissue of challenged rhesus monkeys are shown. Data are shown as mean ± SEM (n=4). Figure 5H circRNA RBD Pathological sections and HE staining of lung lobes of vaccine-immunized rhesus monkeys or control group. Fig.5I circRNA RBD Pathological scores of vaccine-immunized rhesus monkeys or control group. Data are shown as mean ± SEM (n = 4). Figure 5J The B cells (B), T cells (T), and protection ranking of each rhesus monkey in different dose groups are shown.

[0036] Figures 6A-6F It was shown that the circRNA vaccine did not cause obvious clinical disease symptoms in rhesus macaques. Fig. 6A , 6B , 6C, 6D, and 6E show the levels of IL-6, MCP1, TNF-α, IL-1β, and IFNα in the plasma of immunized rhesus monkeys. Fig. 6F The body temperature of rhesus macaques is shown. The body temperature was monitored within three days after immunization.

[0037] Figure 7 The circRNA vaccine showed the neutralization effect on pseudovirus of Omicron variant. RBD or LNP-circRNA RBD-β or LNP-circRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against VSV-based SARS-CoV-2 (Omicron) pseudovirus and corresponding pseudoviruses. Serum samples were collected 2 weeks after booster immunization. Data are shown as mean ± SEM (n = 5 or 6).

[0038] Figures 8A-8B Display circRNA RBD-奥密克戎 Vaccine-induced SARS-CoV-2 Omicron variant-specific antibodies. Fig. 8AThe results show that the circRNAs from the rats were treated with two doses of 5 μg or 10 μg (1 week apart). RBD-奥密克戎 SARS-CoV-2 Omicron variant-specific IgG antibody titers in sera of vaccine-immunized mice measured by ELISA. Data are shown as mean ± SEM (n = 3). Figure 8B The results show that the circRNAs from the experiment were expressed as 1 dose of 30 μg. RBD-奥密克戎 Serum from mice immunized with the vaccine, SARS-CoV-2 Omicron variant-specific IgG antibody titers measured by ELISA. Data are shown as mean ± SEM (n = 3). Each symbol represents an individual mouse.

[0039] Figures 9A-9C circRNA RBD Agarose gel electrophoresis and HPLC purification. Fig. 9A Differently treated linear RNAs are shown RBD and circRNA RBD Agarose gel electrophoresis results. Fig. 9B The circRNA digested by RNase R for different times ranging from 5 minutes to 120 minutes is shown. RBD and linear RNA RBD Agarose gel electrophoresis results. Fig. 9C Display of circRNAs not processed by RNaseR RBD (Left) and circRNA treated with RNase R RBD (Right) HPLC chromatogram.

[0040] Figures 10A-10D Shown by circRNA 3 days before challenge RBD-β Binding and neutralizing antibodies elicited by the vaccine. Fig. 10A Schematic diagrams of the constructs are provided. Fig. 10B SARS-CoV-2 specific IgG antibody titers determined by ELISA are shown. Data are shown as mean ± SEM (n = 5). Each symbol represents an individual mouse. Fig. 10C Sigmoidal curves showing the inhibition rate of sera from immune mice in the alternative virus neutralization test. Figures 10B-10D In the experiment, circRNAs were collected 3 days before challenge with the real SARS-CoV-2β strain. RBD-β (50 μg) serum from immunized mice.

[0041] Fig.11 Flow plots and gating strategies used to quantify SARS-CoV-2-RBD-specific T cells in mice are shown. The gating strategy for single and live T cells from the spleen is shown. CD4+ or CD8 + TEM cells (CD44 + CD62 - ).

[0042] Figures 12A-12M Display circRNA RBD-德尔塔 Vaccines and circRNAs RBD-奥密克戎 Both stimulated high levels of neutralizing antibodies against the Omicron variant circRNA. Fig. 12A The results show that the expression of 10 μg of LNP-circRNA RBD 、LNP-circRNA RBD-β 、LNP-circRNA RBD-德尔塔 or LNP-1mΨ-mRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against VSV-based SARS-CoV-2 pseudovirus. Serum samples were collected 2 weeks after booster immunization. Data are shown as mean ± SEM (n = 5 or 6). Fig. 12B The results show that the expression of 10 μg LNP-circRNA RBD-德尔塔 or LNP-1mΨ-mRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against VSV-based SARS-CoV-2 pseudovirus. Serum samples were collected 7 weeks after booster immunization. Data are shown as mean ± SEM (n = 5). Fig. 12C and 12D LNP-circRNA RBD-德尔塔 or LNP-1mΨ-mRNA RBD -德尔塔 Neutralizing antibody / binding antibody ratio of vaccine against Omicron variant. The ratio was calculated by dividing the NT50 of each mouse by the endpoint IgG titer. The NT50 and endpoint IgG titer were calculated 2 weeks after booster immunization ( Fig. 12C ) or 7 weeks after booster immunization ( Fig.12D The data are shown as mean ± SEM (n = 5). Fig.12E LNP-circRNA assayed by ELISA is shown RBD -奥密克戎 Endpoint titers of Omicron-Spike-specific IgG antibodies in sera from immunized mice. Data are shown as mean ± SEM (n = 5). Fig.12F Display of LNP-circRNA using VSV-based Omicron pseudovirus RBD-奥密克戎 NT50 of sera from immunized mice. Serum samples were collected 1 week after booster doses at two-week intervals. Data are shown as mean ± SEM (n = 4 or 5). Figure 12GThe results show that the expression of LNP-circRNA in the presence of 5 μg or 10 μg of LNP-circRNA was RBD-奥密克戎 Neutralization test of sera from vaccine-immunized mice against VSV-based pseudovirus. Serum samples were collected 1 week after booster immunization at two-week intervals. Data are shown as mean ± SEM (n = 4 or 5). Fig.12H Schematic diagram of the LNP-circRNA vaccination process in BALB / c mice and the serum collection schedule for specific antibody analysis is shown. Fig.12I LNP-circRNA RBD-β 、LNP-circRNA RBD-德尔塔 or LNP-circRNA RBD-奥密克戎 The serum of mice boosted with immunization received two doses of LNP-circRNA RBD-德尔塔Delta Then, the NT50 values ​​of the VSV-based delta pseudovirus were used. Data are shown as mean ± SEM (n = 3). Fig.12J LNP-circRNA RBD-β 、LNP-circRNA RBD-德尔塔 or LNP-circRNA RBD-奥密克戎 The serum of mice boosted with immunization received two doses of LNP-circRNA RBD-德尔塔 Then, the NT50 values ​​of the VSV-based omixiron pseudovirus were used. Data are shown as mean ± SEM (n = 3). Figure 12K Schematic diagram of the LNP-circRNA vaccination process in BALB / c mice and the serum collection schedule for specific antibody analysis is shown. Figure 12L LNP-circRNA RBD or LNP-circRNA RBD-德尔塔 The serum of mice boosted with immunization received two doses of LNP-circRNA RBD The NT50 values ​​of the VSV-based delta pseudovirus were then used. Data are shown as mean ± SEM (n = 2 or 3). Figure 12M LNP-circRNA RBD or LNP-circRNA RBD-德尔塔 The serum of mice boosted with immunization received two doses of LNP-circRNA RBD Afterwards, NT50 values ​​of VSV-based omixiron pseudovirus were used. Data are shown as mean ± SEM (n = 2 or 3). Each symbol represents an individual mouse.

[0043] Figures 13A-13C circRNA RBD-德尔塔 The vaccine produced high levels of neutralizing antibodies against the Omicron variant 7 weeks after booster immunization. Fig.13AThe results show that the expression of 5 μg LNP-circRNA RBD-德尔塔 or LNP-1mΨ-mRNA RBD-德尔塔 Neutralization test of sera from vaccine-immunized mice against VSV-based SARS-CoV-2 pseudovirus. Serum samples were collected 7 weeks after booster immunization. Data are shown as mean ± SEM (n = 5). Fig. 13B and 13C LNP-circRNA RBD-德尔塔 or LNP-1mΨ-mRNA RBD-德尔塔 Neutralizing antibody / binding antibody ratio of the vaccine against the Omicron variant. The ratio was calculated by dividing the NT50 of each mouse by the endpoint IgG titer. The NT50 and endpoint IgG titer were calculated 2 weeks after the booster immunization ( Fig. 13B ) or 7 weeks after booster immunization ( Fig. 13C The data are shown as mean ± SEM (n = 5).

[0044] Figures 14A-14C , Mice imaged by the IVIS imaging system. Fig.14A , unprocessed. Fig. 14B , injected with PBS. Fig. 14C , injected with naked circRNA Luc .

[0045] Figures 15A-15B , containing naked circRNA RBD-β SARS-CoV-2 circRNA RBD-β Vaccine immunogenicity in mice. Fig.15A , circRNA RBD-β Schematic representation of the vaccination process in BALB / c mice and the serum collection schedule for specific antibody analysis. Fig. 15B , SARS-CoV-2-specific IgG antibody titers were measured by ELISA. Data are shown as geometric mean ± geometric SD.

[0046] Figures 16A-16B , containing naked circRNA RBD-德尔塔 SARS-CoV-2 circRNA RBD-德尔塔 Vaccine immunogenicity in mice. Fig.16A , circRNA RBD-德尔塔 Schematic representation of the vaccination process in BALB / c mice and the serum collection schedule for specific antibody analysis. Fig. 16B , SARS-CoV-2-specific IgG antibody titers were measured by ELISA. Data are shown as geometric mean ± geometric SD.

[0047] Figures 17A-17CDisplay naked RNA RBD-β Vaccine immunogenicity in mice. Fig.17A Display naked RNA RBD-β Schematic representation of the vaccination process in BALB / c mice and the serum collection schedule for specific antibody analysis. Fig. 17B Comparative results of SARS-CoV-2RBD-delta-specific antibody titers induced by naked circRNA vaccine or naked mRNA vaccine are shown. Fig. 17C Comparison of SARS-CoV-2RBD-delta-specific antibody titers induced by naked circRNA vaccines dissolved in PBS or Ringer's solution is shown. Data are shown as geometric mean ± geometric SD.

[0048] Figures 18A-18F Optimization of the circRNA vaccine encoding the Omicron BA.1 RBD antigen is shown. Fig.18A Shown using circRNA RBD-BA.1 Results of neutralization test of SARS-CoV-2 pseudovirus using sera from vaccine-immunized mice. Fig.18B Shown is the optimized circRNA RBD-BA.1-mut1 Results of the neutralization test of SARS-CoV-2 pseudovirus performed with sera from immunized mice. Fig. 18C Shown is the optimized circRNA RBD-BA.1-mut2 Results of neutralization test of immunized mouse sera against SARS-CoV-2 pseudovirus. Fig.18D Display optimized circRNA RBD-BA.1-mut3 Results of neutralization test of immunized mouse sera against SARS-CoV-2 pseudovirus. Fig.18E Shown is the optimized circRNA RBD-BA.1-mut2-Folden Results of neutralization test of immunized mouse sera against SARS-CoV-2 pseudovirus. Fig.18F Shown is the optimized circRNA RBD-BA.1-mut3-Folden Neutralization test results of immunized mouse sera against SARS-CoV-2 pseudovirus. Data are shown as geometric mean ± geometric SD.

[0049] Figures 19A-19B Show, CircRNA RBD-BA.4 / 5 The vaccine induced potent neutralizing antibodies against some variants of Omicron and its sublineages. Fig.19A The results show that 5 μg of circRNA RBD-BA.4 / 5 Results of SARS-CoV-2 pseudovirus neutralization test using sera from mice immunized twice with the vaccine (3 weeks apart each time). Fig.19B The results show that 10 μg of circRNA RBD-BA.4 / 5 Results of the neutralization test of SARS-CoV-2 pseudovirus by sera from mice immunized twice with the vaccine (3 weeks apart each time). Data are presented as geometric mean ± geometric SD.

[0050] Figures 20A-20D Proved the use of circRNA RBD-BA.4 / 5 Results of neutralization tests of sera from vaccine-immunized mice against SARS-CoV-2 pseudovirus showed that the circRNARBD-trimer vaccine induced potent neutralizing antibodies against some variants of Omicron and its sublineages. Fig. 20A The results show that 5 μg of circRNA RBD-三聚体1 Results of neutralization test against SARS-CoV-2 pseudovirus using sera from mice immunized twice with the vaccine (2 weeks apart each time). Fig. 20B The results show that 10 μg of circRNA RBD-三聚体1 Results of neutralization test against SARS-CoV-2 pseudovirus using sera from mice immunized twice with the vaccine (2 weeks apart each time). Fig. 20C The results show that 5 μg of circRNA RBD-三聚体1-折叠子 Results of neutralization test against SARS-CoV-2 pseudovirus using sera from mice immunized twice with the vaccine (2 weeks apart each time). Fig.20D The results show that 10 μg of circRNA RBD-三聚体1-折叠子 Results of the neutralization test of SARS-CoV-2 pseudovirus by sera from mice immunized twice with the vaccine (2 weeks apart). Data are presented as geometric mean ± geometric SD. DETAILED DESCRIPTION

[0051] The present application provides a circRNA vaccine, which encodes an antigenic polypeptide comprising a spike protein or a fragment thereof of a SARS-CoV-2 variant, such as a Delta or Omicron variant or a subvariant thereof. The circRNA vaccine described herein is effective against SARS-CoV-2 or its emerging variants, and it induces a high proportion of neutralizing antibodies and a strong Th1-biased cellular response in mice, and effectively protects mice and rhesus monkeys from infection with SARS-CoV-2 and variants.

[0052] Unlike other types of coronavirus vaccines, the circRNA vaccines described herein do not require the handling of large amounts of infectious particles during production. In addition, compared to linear RNA vaccines, the circRNA vaccines described herein can provide enhanced stability and efficacy. For example, given their circular nature, circRNAs are particularly stable compared to many linear RNAs because they are resistant to exonucleolytic degradation by cellular exosomal ribonuclease complexes. In some embodiments, the circRNA in the circRNA vaccine disclosed herein can be rolled circle translated by ribosomes in individuals to whom the vaccine has been administered, thereby producing a large amount of antigenic polypeptides. The production of such circRNA vaccines can be carried out using a variety of methods, such as chemical ligation, enzyme catalysis, or ribozyme autocatalysis. The circRNA vaccines described herein provide a platform for the rapid development of vaccines against emerging coronavirus strains. In addition, circular RNAs can be rapidly and massively generated in vitro, and they do not require any nucleotide modification, which is significantly different from classic mRNA vaccines. Our data show that exemplary circRNAs and encapsulated circRNA-LNP complexes are highly thermally stable for 7-14 days at 4°C or room temperature. Due to its specific properties, circRNAs have potential in biomedical applications.

[0053] I. Definitions

[0054] Unless otherwise defined below, the terms used herein are as commonly used in the art.

[0055] As used herein, a "variant" virus refers to an isolate of a virus whose genomic sequence is different from that of a reference virus, and the difference in the genomic sequence confers new phenotypic properties, such as increased adaptability compared to a reference virus. When referring to the virus species of the present application, such as SARS-CoV-2, it should be understood that the species includes variants as well as reference viruses that are isolated and identified for the first time. In some embodiments, the variant virus is a variant (VBM) being monitored by the Centers for Disease Control and Prevention ("CDC") or the World Health Organization ("WHO"). Variants designated as VBM include those that data show potential or clear effects on approved or authorized medical countermeasures, or variants that are associated with more severe disease or increased transmission but are no longer detected, or variants that circulate at very low levels in the United States. In some embodiments, the variant viruses described herein are "variants of interest", i.e., variants with specific genetic markers that are associated with changes in receptor binding, reduced neutralization of antibodies produced by previous infection or vaccination, reduced therapeutic efficacy, potential diagnostic effects, and / or predicted increased transmissibility and / or disease severity. In some embodiments, the variant viruses described herein are "variants of concern", i.e., variants for which there is evidence of increased transmissibility, more severe disease (e.g., increased hospitalizations and / or deaths), significantly reduced neutralization by antibodies produced during previous infection or vaccination, reduced effectiveness of treatment or vaccines, and / or failure of diagnostic tests. In some embodiments, the variant viruses described herein are "high consequence variants", i.e., high consequence variants have clear evidence that preventive measures or medical countermeasures (MCMs) are significantly less effective than previously circulated variants.

[0056] The terms "polynucleotide", "nucleic acid", "nucleotide sequence" and "nucleic acid sequence" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0057] The "naked circRNA" described herein refers to a circRNA without a delivery vehicle (eg, liposome, lipid nanoparticle, or colloidal party).

[0058] The term "vaccine" is understood to refer to an immunologically active pharmaceutical preparation. In certain embodiments, the vaccine induces adaptive immunity when administered to a host. The vaccine preparation may further contain a pharmaceutical carrier, which may be designed for a specific mode of administration of the vaccine.

[0059] The terms "group I intron" and "group I catalytic intron" are used interchangeably and refer to a self-splicing ribozyme that can catalyze its own excision from an RNA precursor. Group I introns contain two fragments, the 5' catalytic group I intron fragment and the 3' catalytic group I intron fragment, which retain their folding and catalytic functions (i.e., self-splicing activity). In its natural environment, the 5' catalytic group I intron fragment is flanked at its 5' end by a 5' exon, which contains a 5' exon sequence that is recognized by the 5' catalytic group I intron fragment; and the 3' catalytic group I intron fragment is flanked at its 3' end by a 3' exon, which contains a 3' exon sequence that is recognized by the 3' catalytic group I intron fragment. The terms "5' exon sequence" and "3' exon sequence" used herein are labeled according to the order of the exons relative to the group I introns in their natural environment, for example, Figure 1A shown.

[0060] The term "antigenic polypeptide" refers to a polypeptide that can be used to trigger the immune system of a mammal to produce antibodies specific to the polypeptide or a portion thereof. Antigenic polypeptides described herein include naturally occurring proteins, protein domains, and short peptide fragments derived from naturally occurring proteins. Antigenic polypeptides may contain one or more known epitopes of naturally occurring proteins. Antigenic polypeptides may include carrier proteins or multimeric proteins that enhance immunogenicity.

[0061] The term "functional variant" of a reference protein refers to a variant polypeptide derived from a reference protein or a portion thereof, and the variant has substantially the same activity (e.g., binding to a target or enzymatic activity) as the reference protein. "Substantially the same activity" refers to an activity level of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the reference protein.

[0062] As used herein, the term "introducing / introduction" refers to the delivery of one or more polynucleotides (such as circRNA) or one or more constructs including vectors described herein, or one or more transcripts thereof to a host cell. The method of the present application can use many delivery systems, including but not limited to: viruses, liposomes, electroporation, microinjection and conjugation, to achieve the introduction of circRNA or constructs described herein into host cells. Conventional viral-based and non-viral-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to apply nucleic acids encoding circRNA of the present application to cells or host organisms in culture medium. Non-viral vector delivery systems include: DNA plasmids, RNA (e.g., transcripts of constructs described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles (such as liposomes). Viral vector delivery systems include DNA viruses and RNA viruses, which have free genomes or integrated genomes for delivery to host cells.

[0063] As used herein, when referring to a first nucleic acid sequence that is operably linked to a second nucleic acid sequence, "operably linked" refers to the situation when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, the promoter is operably linked to the coding sequence. Similarly, if a signal peptide affects the extracellular secretion of a polypeptide, the coding sequence of the signal peptide is operably linked to the coding sequence of the polypeptide. In general, operably linked nucleic acid sequences are continuous, and when it is necessary to connect two protein coding regions, the open reading frames are aligned.

[0064] As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid by traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, 10 of 10, respectively about 50%, 60%, 70%, 80%, 90% and 100% complementary). "Complete complementarity" refers to that all continuous residues of a nucleic acid sequence form hydrogen bonds with the same number of continuous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, with at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% complementary degree, or refers to two nucleic acids that hybridize under stringent conditions.

[0065] As used herein, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease, preventing or delaying the occurrence or recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing disease relief (whether partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life, and / or prolonging the survival period. "Treatment" also includes reducing the pathological consequences of the disease. The method of the present application takes into account any one or more of these treatment aspects.

[0066] The terms "individual," "subject," and "patient" are used interchangeably herein to describe mammals, including humans. In some embodiments, the individual is a human. In some embodiments, the individual is a rodent, such as a mouse. In some embodiments, the individual has a genetic disease or condition. In some embodiments, the individual has a SARS-CoV-2 infection. In some embodiments, the individual is at risk of contracting a SARS-CoV-2 infection. In some embodiments, the individual is in need of treatment.

[0067] As understood in the art, an "effective amount" refers to an amount of a composition sufficient to produce a desired therapeutic outcome (e.g., stimulating the production of antibodies and improving immunity against one or more SARS-CoV-2 variants, reducing the severity or duration of SARS-CoV-2 infection, stabilizing its severity, or eliminating one or more symptoms thereof). For therapeutic uses, beneficial or desired outcomes include, for example, reducing one or more symptoms caused by the disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological phenotypes that occur during the course of disease development, improving the quality of life of those suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, delaying the progression of the disease, and / or prolonging the survival of the patient. In some embodiments, an effective amount of a therapeutic agent can prolong survival (including overall survival and progression-free survival); cause an objective response (including a complete response or a partial response); alleviate one or more signs or symptoms of a disease or condition to some extent; and / or improve the quality of life of the subject. In some embodiments, the effective amount is a prophylactically effective amount, which is an amount of the composition sufficient to prevent or reduce the severity of one or more future symptoms of SARS-CoV-2 infection when administered to an individual susceptible to and / or likely to develop SARS-CoV-2 infection. For prophylactic use, beneficial or desired results include, for example, results such as elimination or reduction of risk, lessening of severity of future disease, or delaying the onset of disease (e.g., delaying biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during the future development of the disease).

[0068] As used herein, the term "wild type" is a term of art understood by skilled artisans to refer to the typical form of an organism, strain, gene or trait as it occurs in nature, as distinct from mutant or variant forms.

[0069] The present disclosure provides several types of compositions based on polynucleotides or polypeptides, including variants and derivatives. These include, for example, substitutions, insertions, deletions and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that has been modified and / or changed in any way relative to a reference molecule or a starting molecule.

[0070] Therefore, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications relative to a reference sequence, particularly polypeptide sequences disclosed herein, are included within the scope of the present disclosure. For example, a sequence tag or amino acids such as one or more lysines can be added to a peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification or positioning. Lysine can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxyl and amino terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminal residues or N-terminal residues) may be deleted depending on the purpose of the sequence, for example, as a sequence expression of a larger sequence part that is soluble or connected to a solid support.

[0071] The term "identity" refers to the overall correlation between polymeric molecules, such as polynucleotide molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules. The calculation of the percentage identity of two polynucleotide sequences, for example, can be performed by comparing the two sequences for the best comparison purpose (for example, a gap can be introduced in one or both of the first nucleic acid sequence and the second nucleic acid sequence to obtain the best comparison, and different sequences can be ignored for the purpose of comparison). In certain embodiments, the length of the sequence compared for the purpose of comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. Then, the nucleotides of the corresponding nucleotide positions are compared. When the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules at this position are the same. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, and considering the number of gaps and the length of each gap, it is necessary to introduce the best comparison to achieve the two sequences. The comparison of sequences and the determination of the percentage identity between the two sequences can be completed using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference.For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), which uses a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the GAP program in the GCG software package can be used to determine the percent identity between two nucleic acid sequences using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity have been incorporated into publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Molec. Biol., 215, 403 (1990)).

[0072] "Percentage (%) of amino acid sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the compared polypeptide, after alignment of the sequences with any conservative substitutions considered as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, the values ​​of % amino acid sequence identity are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32 (5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5 (1): 113, 2004, each of which is incorporated herein by reference in its entirety for all purposes).

[0073] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate that human hand is involved. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated in nature and as found in nature.

[0074] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include mRNA splicing in eukaryotic cells.

[0075] As used herein, the term "polypeptide" or "peptide" encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0076] As used herein, the term "simultaneous administration" refers to the first therapy and the second therapy in the combination therapy, which are administered at a time interval of no more than about 15 min (e.g., no more than about 10, 5, or 1 min). When the first therapy and the second therapy are administered simultaneously, the first therapy and the second therapy can be included in the same composition (e.g., a composition comprising the first therapy and the second therapy) or in separate compositions (e.g., the first therapy is included in one composition, and the second therapy is included in another composition).

[0077] As used herein, the term "sequential administration" refers to the first therapy and the second therapy in the combination therapy, which are administered at a time interval of more than about 15 min (such as more than about 20, 30, 40, 50, 60 min or more minutes). The first therapy or the second therapy can be administered first. The first therapy and the second therapy are contained in separate compositions, which can be contained in the same or different packages or kits.

[0078] As used herein, the term "concurrent administration" means that the administration of a first therapy and the administration of a second therapy in a combination therapy overlap with each other.

[0079] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredients contained therein to be effective, and which contains no additional components which would be unacceptably toxic to a subject to which the preparation would be administered.

[0080] "Pharmaceutically acceptable carrier" refers to one or more ingredients in a pharmaceutical formulation that are non-toxic to a subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, cryoprotectants, tonicity agents, preservatives, and combinations thereof. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology and manufacturing testing and / or are included in the Inactive Ingredients Guide compiled by the U.S. Food and Drug Administration or other state / federal governments or are listed in the U.S. Pharmacopeia or other recognized pharmacopoeias for use in mammals, especially humans.

[0081] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0082] An "article of manufacture" is any article (e.g., a package or container) or kit that contains at least one agent (e.g., a drug for treating SARS-CoV-2 infection), or a probe that specifically detects a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0083] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0084] Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, description of "about X" includes description of "X".

[0085] As used herein, reference to "not" a value or parameter generally means and describes "except" that value or parameter. For example, the method is not used to treat a disease type X means that the method is used to treat a disease type other than type X.

[0086] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0087] As used herein and in the appended claims, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise.

[0088] As used herein, the term "and / or", phrases such as "A and / or B" are intended to include: both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or", phrases such as "A, B and / or C" are intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0089] II. Circular RNA Vaccine

[0090] The present application provides circular RNA (circRNA) encoding antigenic polypeptides of SARS-CoV-2 variants (such as any of the SARS-CoV-2 variants described in Section C-"SARS-CoV-2 variants" below). Exemplary antigenic polypeptides are described in Section A-"Antigenic polypeptides" below. The circRNA may include any of the circRNA expression and / or circularization elements described in Section B-"Additional circRNA expression and circularization elements" below.

[0091] In some embodiments, the circRNA is stable for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20 days when stored at 4°C or room temperature. In some embodiments, the circRNA is stable for at least 7 days when stored at 4°C or room temperature. In some embodiments, the circRNA is stable for at least 14 days when stored at 4°C or room temperature. In some embodiments, the circRNA is stable for at least 30 days when stored at 4°C. In some embodiments, the degradation rate of the circRNA is less than 40% after 14 days of storage at room temperature. In some embodiments, the degradation rate of the circRNA is less than 30% after 14 days of storage at room temperature. In some embodiments, the degradation rate of the circRNA is less than 20% after 14 days of storage at room temperature. In some embodiments, the degradation rate of the circRNA is less than 10% after 14 days of storage at room temperature. In some embodiments, the degradation rate of the circRNA is less than 40% after 7 days of storage at room temperature. In some embodiments, the degradation rate of the circRNA is less than 30% after 7 days of storage at room temperature. In some embodiments, after storage at room temperature for 7 days, the degradation rate of the circRNA is less than 20%. In some embodiments, after storage at room temperature for 7 days, the degradation rate of the circRNA is less than 10%. In some embodiments, after storage at room temperature for 7 days, the degradation rate of the circRNA is less than 5%.

[0092] In some embodiments, the present application provides a circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant, and (b) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgE SP) fused to the N-terminus of the antigenic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' linker sequence at the 5' end of the circRNA, and a 3' linker sequence at the 3' end of the circRNA, wherein the 5' linker sequence and the 3' linker sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0093] In some embodiments, the present application provides a circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant; (b) an IRES sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide; and (c) an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgE SP) fused to the N-terminus of the antigenic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, a nucleic acid sequence encoding an antigenic polypeptide, and an in-frame 2A peptide coding sequence. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' linker sequence at the 5' end of the circRNA, and a 3' linker sequence at the 3' end of the circRNA, wherein the 5' linker sequence and the 3' linker sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0094] In some embodiments, the present application provides a circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant; and (b) an m6A modified motif sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgESP) fused to the N-terminus of the antigenic polypeptide. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an m6A modified motif sequence, a Kozak sequence, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigenic polypeptide, and a 5' exon sequence that can be recognized by a 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA further comprises a 5' linker sequence at the 5' end of the circRNA, and a 3' linker sequence at the 3' end of the circRNA, wherein the 5' linker sequence and the 3' linker sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1) of Omicron Omicron subvariants.

[0095] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising a circRNA, wherein the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide containing the S protein or a fragment thereof of SARS-CoV-2.

[0096] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, wherein the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide of a spike (S) protein or a fragment thereof containing a SARS-CoV-2 variant. In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0097] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising: (a) an S protein or a fragment thereof of a SARS-CoV-2 variant; and (b) a multimerization domain. In some embodiments, the multimerization domain is a C-terminal folding sub (Fd) domain of T4 fiber protein, which mediates the trimerization of T4 fiber protein. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises an amino acid sequence as shown in SEQ ID NOs: 3-4. In some embodiments, the multimerization domain is fused to the RBD domain of the S protein via a peptide linker (e.g., a peptide linker comprising an amino acid sequence of SEQ ID NO: 5). In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0098] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising a circRNA, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising a receptor binding domain (RBD) of the S protein of a SARS-CoV2 variant. In some embodiments, the RBD comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the RBD comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0099] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising: (a) RBD of an S protein fragment of a SARS-CoV-2 variant, and (b) a multimerization domain. In some embodiments, the RBD comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the multimerization domain is a C-terminal folding sub (Fd) domain of T4 fiber protein, which mediates the trimerization of T4 fiber protein. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises an amino acid sequence as shown in SEQ ID NOs: 3-4. In some embodiments, the multimerization domain is fused to the RBD domain of the S protein via a peptide linker (e.g., a peptide linker comprising an amino acid sequence of SEQ ID NO: 5). In some embodiments, the SARS-CoV-2 variant is a delta variant, and the RBD comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the SARS-CoV-2 variant is an omega variant, and the RBD comprises the amino acid sequence of SEQ ID NO: 19.

[0100] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising a circRNA, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising the S2 region of the S protein of a SARS-CoV2 variant. In some embodiments, the S2 region comprises amino acid residues 686 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises an amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0101] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising a circRNA, the circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising amino acid residues 2 to 1273 of the full-length S protein of the SARS-CoV-2 variant, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (e.g., deletion of amino acid residues 681 to 684). In some embodiments, the antigenic polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 8-10 and 40. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0102] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising an S protein or a fragment thereof of a SARS-CoV-2 variant; and (b) an internal ribosome entry site (IRES) sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgE SP) fused to the N-terminus of the S protein or its fragment. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence, the nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, an SP, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' connection sequence at the 5' end of the circRNA, and a 3' connection sequence at the 3' end of the circRNA, wherein the 5' connection sequence and the 3' connection sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, the antigen polypeptide comprises the RBD of the S protein. In some embodiments, the antigen polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper domain based on GCN-4). In some embodiments, the antigen polypeptide comprises the S2 region of the S protein. In some embodiments, the antigen polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of the SARS-CoV-2 variant, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations (e.g., deletion of amino acid residues 681-684) that inhibit S protein cleavage. In some embodiments, the circRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 20-21. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant.In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0103] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising an S protein or a fragment thereof of a SARS-CoV-2 variant; (b) an IRES sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide; and (c) an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgE SP) fused to the N-terminus of the S protein or its fragment. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence, the nucleic acid sequence comprising from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, an SP, a nucleic acid sequence encoding an antigenic polypeptide, and an in-frame 2A peptide coding sequence. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by a 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' connection sequence at the 5' end of the circRNA, and a 3' connection sequence at the 3' end of the circRNA, wherein the 5' connection sequence and the 3' connection sequence are connected to each other by a ligase (e.g., T4RNA ligase). In some embodiments, the antigen polypeptide comprises the RBD of the S protein. In some embodiments, the antigen polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper domain based on GCN-4). In some embodiments, the antigen polypeptide comprises the S2 region of the S protein. In some embodiments, the antigen polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of the SARS-CoV-2 variant, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations (e.g., deletion of amino acid residues 681-684) that inhibit S protein cleavage. In some embodiments, the circRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 20-21. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant.In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1).

[0104] In some embodiments, the present application provides a circular RNA (circRNA) vaccine comprising circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising an S protein or a fragment thereof of a SARS-CoV-2 variant; and (b) an m6A modification motif sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgESP) fused to the N-terminus of the S protein or its fragment. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence, an SP, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by a 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' connection sequence at the 5' end of the circRNA, and a 3' connection sequence at the 3' end of the circRNA, wherein the 5' connection sequence and the 3' connection sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, the antigen polypeptide comprises the RBD of the S protein. In some embodiments, the antigen polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper domain based on GCN-4). In some embodiments, the antigen polypeptide comprises the S2 region of the S protein. In some embodiments, the antigen polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of the SARS-CoV-2 variant, wherein the numbering is based on SEQID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations that inhibit S protein cleavage (e.g., deletion of amino acid residues 681-684). In some embodiments, the circRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 20-21. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5).In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the Omicron subvariants (e.g., BA.2.75, BA.4.6, BF.7, BQ.1.1).

[0105] The present application further provides a cocktail composition comprising a plurality of circRNAs, each circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant. In some embodiments, the plurality of circRNAs encode different antigenic polypeptides from each other, such as different mutants of an antigenic polypeptide (e.g., an S protein or a fragment thereof), or antigenic polypeptides of different SARS-CoV-2 variants.

[0106] A. Antigenic Peptide

[0107] The circRNA vaccines described herein comprise circular RNA (circRNA) encoding an antigenic polypeptide. In some embodiments, the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant, such as any of the S proteins or fragments thereof described in the subsection “Spike Protein or Fragments thereof” below. In some embodiments, the antigenic polypeptide comprises a multimerization domain, such as a natural multimerization domain of the S protein, or an exogenous multimerization domain. Suitable multimerization domains are described in the subsection “Multimerization Domain” below. The S protein or its fragment can be fused to the multimerization domain via a peptide linker, such as any of the peptide linkers described in the subsection “Peptide Linker” below.

[0108] The antigen polypeptide comprises at least one epitope that can be recognized by a T cell receptor (TCR). In some embodiments, the antigen polypeptide is a full-length protein or a fragment thereof, or an antigen fusion protein that can trigger an immune response in a subject. In some embodiments, the antigen polypeptide is a short peptide of no more than 100 amino acids in length. The antigen polypeptide can be a naturally derived peptide fragment from a protein antigen containing one or more epitopes, or an artificially designed peptide with one or more natural epitope sequences, wherein a peptide linker can be optionally placed between adjacent epitope sequences. In some embodiments, the antigen polypeptide comprises a single epitope of an antigen protein. In some embodiments, the antigen polypeptide comprises any one of about 1, 2, 3, 4, 5, 10 or more epitopes from a single antigen protein. In some embodiments, the antigen polypeptide comprises epitopes from a variety of (e.g., 2, 3, 4, 5, 10 or more) different antigen proteins. In some embodiments, the antigen polypeptide comprises a major histocompatibility complex (MHC) class I restricted epitope. In some embodiments, the antigen polypeptide comprises an MHC class II restricted epitope. In some embodiments, the antigenic polypeptide comprises an MHC class I restricted epitope and an MHC class II restricted epitope.

[0109] In some embodiments, the antigenic polypeptide is an antigenic protein or a fragment or variant thereof of an autoantigen (such as an antigen related to a disease or condition). In some embodiments, the antigenic polypeptide is a tumor antigen peptide. Tumor antigen peptide sequences are known in the art and can be found in public databases, such as the Cancer Antigen Peptide Database (van der Bruggen P et al. (2013) "Peptide database: T cell-defined tumor antigens." Cancer Immunity. URL: caped.icp.ucl.ac.be). The coding RNA sequence in the linear RNA or circRNA described herein can encode any known tumor antigen peptide or a combination thereof. In some embodiments, the antigenic polypeptide comprises an epitope of a tumor-associated antigen (TAA). In some embodiments, the antigenic polypeptide comprises an epitope of a tumor-specific antigen. In some embodiments, the antigenic polypeptide comprises an epitope of a neoantigen, i.e., a newly acquired and expressed antigen present in an individual tumor cell.

[0110] In some embodiments, a bioinformatics tool for T cell epitope prediction is used to predict the amino acid sequence of one or more epitope peptides based on the sequence of the antigen protein (including the neoantigen). Exemplary bioinformatics tools for T cell epitope prediction are known in the art, for example, see Yang X. and Yu X. (2009) "An introduction to epitope prediction methods and software" Rev. Med. Virol. 19 (2): 77-96. In some embodiments, the sequence of the antigen protein is known in the art or can be obtained in a public database. In some embodiments, the sequence of the antigen protein (including the neoantigen) is determined by sequencing a sample (e.g., a tumor sample) of the treated individual.

[0111] In some embodiments, the antigenic polypeptide comprises a spike (S) protein of a SARS-CoV-2 variant or a fragment thereof. In some embodiments, the antigenic polypeptide is a full-length S protein. In some embodiments, the antigenic polypeptide is a fragment of a naturally occurring S protein.

[0112] In some embodiments, the antigenic polypeptide comprises a variant of the S protein or a fragment thereof of a SARS-CoV-2 variant. Variants of the SARS-CoV-2 genome have been described. See, for example, Forster et al. (2020). Phylogenetic network analysis of SARS-CoV-2 genomes. PNAS 117 (17) 9241-9243, which is incorporated herein by reference in its entirety. In some embodiments, the antigenic polypeptide comprises a variant of the S protein or a fragment thereof that confers a fitness advantage (such as enhanced infectivity) to the SARS-CoV-2 variant. In some embodiments, the antigenic polypeptide comprises an S protein or a fragment thereof of SARS-CoV-2 with a D614G mutation. In some embodiments, the antigenic polypeptide is capable of stimulating an immune response against different strains and variants of SARS-CoV-2 in an individual. In some embodiments, the antigenic polypeptide is capable of stimulating an immune response against a specific strain or variant of a SARS-CoV-2 variant in an individual.

[0113] In some embodiments, the antigenic polypeptide comprises a receptor binding domain (RBD) of the S protein of a SARS-CoV2 variant. In some embodiments, the RBD comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the RBD comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 18. In some embodiments, the RBD comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 19.

[0114] In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein of the SARS-CoV2 variant. In some embodiments, the S2 region comprises amino acid residues 686 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises K986P and V987P mutations. In some embodiments, the S2 region comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 7.

[0115] In some embodiments, the antigenic polypeptide comprises the RBD and S2 regions of the S protein of the SARS-CoV2 variant. In some embodiments, the antigenic polypeptide comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0116] In some embodiments, the antigenic polypeptide comprises a spike (S) protein fragment and a multimerization domain of a SARS-CoV2 variant, which can be operably connected to the S protein fragment. In some embodiments, the multimerization domain is a C-terminal folding sub (Fd) domain of T4 fiber protein that mediates T4 fiber protein trimerization. In some embodiments, the multimerization domain is an isoleucine zipper domain based on GCN-4. In some embodiments, the multimerization domain comprises an amino acid sequence as shown in SEQ ID NO: 3 or 4. In some embodiments, the multimerization domain is fused to the S protein fragment through a peptide linker. In some embodiments, the antigenic polypeptide comprises an RBD domain of an S protein fused to the multimerization domain through a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NO: 5.

[0117] In some embodiments, the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of SARS-CoV-2 fused to a multimerization domain. In some embodiments, the antigenic polypeptide comprises an S protein fragment fused to a C-terminal folding sub (Fd) domain (e.g., SEQ ID NO: 3) of a T4 fiber protein that mediates trimerization of T4 fiber protein (e.g., SEQ ID NO: 4). In some embodiments, the antigenic polypeptide comprises an S protein fragment fused to an isoleucine zipper domain based on GCN-4. In some embodiments, the antigenic polypeptide comprises a receptor binding domain (RBD) of the S protein of SARS-CoV-2 fused to a multimerization domain via a peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NO: 5.

[0118] The antigen polypeptide may include a signal peptide (SP). In some embodiments, the SP is fused to the N-terminus of the S protein or a fragment thereof. In a non-limiting example, the signal peptide is a signal sequence and propeptide from human tissue plasminogen activator (tPA), a signal sequence from human IgE immunoglobulin, or a signal peptide sequence of MHC I. In some embodiments, the signal peptide can promote the secretion of the antigen polypeptide encoded by the circRNA vaccine.

[0119] In some embodiments, the circRNA comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA does not contain a stop codon at the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the in-frame 2A peptide coding sequence replaces the stop codon. In some embodiments, the circRNA does not contain a stop codon and the number of nucleotides constituting the RNA is a multiple of three. In some embodiments, the circRNA, which does not have a stop codon and the number of nucleotides constituting the RNA is a multiple of three, allows rolling circle translation of the circRNA. In some embodiments, the 2A peptide coding sequence allows rolling circle translation of the circRNA. In some embodiments, the 2A peptide allows the polypeptide generated by rolling circle translation to be cut into monomeric polypeptide sequences. In a non-limiting example, the 2A peptide coding sequence encodes a P2A or T2A peptide, such as a sequence shown in SEQ ID NO: 30 or 31.

[0120] Also provided is a circRNA comprising a nucleic acid sequence encoding any one of the antigenic polypeptides described herein. The nucleic acid sequence encoding the antigenic polypeptide can be codon optimized. In some embodiments, the circRNA comprises a nucleic acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 98% or more, or 100%) sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 20-21.

[0121] Spike protein or its fragments

[0122] The circRNA vaccine described herein comprises a circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide containing a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant.

[0123] In some embodiments, the S protein or fragment thereof comprises amino acid residues 2 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S protein or fragment thereof comprises a deletion of amino acid residues 681 to 684. In some embodiments, the S protein or fragment thereof comprises at least one point mutation in the S2 region, such as K986P, V987P, F817P, A892P, A899P or A942P mutations or a combination thereof. In some embodiments, the S protein or its fragment comprises a mutation selected from the following Table A, such as A222V, E406W, K417N, K417T, N439K, L452R, L452Q, L455N, L478K, E484K, Q493F, F490S, N501Y, A570D, D614G, P681H, A701V, T716I, at least one mutation of S982A or a combination thereof. In some embodiments, the S protein or its fragment comprises a N501Y point mutation. In some embodiments, the S protein or its fragment comprises a K417N, E484K and / or N501Y point mutation. In some embodiments, the S protein or its fragment comprises an E484K point mutation. In some embodiments, the S protein or its fragment comprises a K417T, E484K and N501Y point mutation. In some embodiments, the S protein or fragment thereof of SARS-CoV-2 comprises K986P and V987P point mutations, alone or in combination with a deletion of amino acid residues 681 to 684. In some embodiments, the S protein or fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-2, SEQ ID NOs: 6-10, SEQ ID NOs: 18-19, and SEQ ID NOs: 41. In some embodiments, the S protein or fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 18. In some embodiments, the S protein or fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 19.

[0124] In some embodiments, the S protein or fragment thereof comprises the N-terminal domain (NTD) of the S protein of a SARS-CoV-2 variant.

[0125] In some embodiments, the S protein or a fragment thereof comprises an amino acid sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more sequence identity with a wild-type S protein of a SARS-CoV-2 variant or a fragment thereof, or has an amino acid sequence as shown in any one of SEQ ID NOs: 1-2, SEQ ID NOs: 6-10, SEQ ID NOs: 18-19, and SEQ ID NO: 41.

[0126] RBD domain

[0127] In some embodiments, the S protein or its fragment comprises a receptor binding domain (RBD) of the S protein. In some embodiments, the RBD comprises amino acid residues 319 to 542 of the full-length S protein of the SARS-CoV-2 variant, wherein the numbering is based on SEQID NO: 1. In some embodiments, the RBD comprises an amino acid sequence of SEQ ID NO: 18 or 19. In some embodiments, the RBD comprises a sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more sequence identity with the amino acid sequence of SEQ ID NO: 18. In some embodiments, the RBD comprises a sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the RBD is connected to the multimerization domain. In some embodiments, the RBD is fused to the multimerization domain via a flexible peptide linker.

[0128] S2 Zone

[0129] In some embodiments, the S protein or a fragment thereof comprises the S2 region of the S protein. In some embodiments, the S2 region comprises amino acid residues 686 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the S2 region comprises one or more mutations that stabilize the prefusion conformation of the S protein. In some embodiments, the S2 region comprises K986P and V987P mutations, for example, as in the sequence shown in SEQ ID NO: 7. In some embodiments, the S2 region comprises a single point mutation, such as a K986P, V987P, F817P, A892P, A899P, or A942P mutation. In some embodiments, the S2 region comprises a combination of point mutations, including K986P, V987P, F817P, A892P, A899P, or A942P. In some embodiments, the S2 region comprises a wild-type sequence of the S protein of a SARS-CoV-2 variant, such as the sequence of SEQ ID NO:6, or a sequence having about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98%, or more sequence identity to the amino acid sequence of SEQ ID NO:6.

[0130] Multimerization domain

[0131] In some embodiments, the antigen polypeptide further comprises a multimerization domain, such as a dimerization domain, a trimerization domain, or a domain that mediates the formation of higher-order multimers. In some embodiments, the multimerization domain is a trimerization domain. In a non-limiting example, the multimerization domain comprises a C-terminal foldon (Fd) domain of T4 fiber protein, wherein the C-terminal foldon domain is a domain that mediates the trimerization of T4 fiber protein, such as the amino acid sequence shown in SEQ ID NO: 3. In another example, the multimerization domain comprises a GCN4 isoleucine zipper (IZ) domain, which is based on the trimerization domain of the GCN4 transcription activator from Saccharomyces cerevisiae, such as the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the multimerization domain has a sequence identity of about 80%, at least 85%, at least about 90%, at least about 95%, at least about 98% or more with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the GCN4 IZ domain or the T4 fibrin Fd domain can be modified according to techniques known in the art to reduce their immunogenicity. For example, the GCN4 IZ domain can be modified with an N-linked glycosylation site to reduce its immunogenicity (Sliepen et al. Immunosilencing a Highly Immunogenic Protein Trimerization Domain. The Journal of Biol. Chem. Vol. 290, No. 12, pp. 7436–7442). In some embodiments, the multimerization domain is fused to the N-terminus of the S protein or its fragment. In some embodiments, the multimerization domain is fused to the C-terminus of the S protein or its fragment.

[0132] Peptide linker

[0133] In some embodiments, the various domains in the antigenic polypeptide (e.g., various domains of the spike protein or its fragment) can be fused to each other or contain domains (e.g., antigenic polypeptide domains and carrier protein or multimerization domain) fused to each other through peptide linkers. In some embodiments, the antigenic polypeptide is a domain of the S protein of SARS-CoV-2 fused to the multimerization domain through a peptide linker. Flexible peptide linkers, such as glycine linkers, glycine-serine linkers, and linkers containing other amino acids are known in the art (e.g., suitable peptide linkers are described by Chen et al. in Fusion Protein Linkers: Property, Design and Functionality. Adv. Drug Deli Rev. 2013 October 15; 65 (10): 1357–1369, which is incorporated herein by reference in its entirety). Peptide linkers can also be designed by computational methods. Peptide linkers can be any length of 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or greater than 50 amino acids. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO:5.

[0134] B. Additional circRNA Expression and Circularization Elements

[0135] The circRNA of the circRNA vaccine described herein comprises one or more additional expression elements that promote the expression and / or circularization of the circRNA.

[0136] In some embodiments, the circRNA comprises a Kozak sequence operably linked to a nucleic acid sequence encoding an antigenic polypeptide, wherein the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant. In some embodiments, the Kozak sequence functions as a protein translation initiation site.

[0137] In some embodiments, the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant, which is operably linked to an internal ribosome entry site (IRES). In a non-limiting example, the IRES sequence can be an IRES sequence of a CVB3 virus, an EV71 virus, an EMCV virus, a PV virus, or a CSFV virus. See, e.g., Searching for IRES.RNA.2006Oct; 12(10): 1755–1785, which is incorporated herein by reference in its entirety. In some embodiments, the IRES sequence is a cellular IRES sequence. In some embodiments, the IRES sequence is followed by a Kozak sequence operably linked to a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence.

[0138] In some embodiments, the polyA sequence or polyAC spacer is located at the 5' end of IRES. In some embodiments, the polyA or polyAC sequence is located between the 5' end of IRES and the exon-exon splicing junction. The length of the internal polyA sequence or polyAC spacer can be in the range of 1 to 500 nucleotides (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450 or 500 nucleotides). In some embodiments, the length of the polyA sequence or polyAC sequence can be in the range of 10-70, 20-60, or 30-60 nucleotides. In some embodiments, circRNA comprises a polyAC sequence as shown in SEQ ID NO: 23, which is located at the 5' end of the IRES sequence. In some embodiments, there is no polyA sequence or polyAC sequence at the 5' end of the IRES sequence. Without being bound by any theory or hypothesis, the internal polyA sequence or polyAC spacer added before the IRES sequence can help maintain the functional secondary structure of the IRES element to achieve efficient protein translation initiated by the IRES. In some embodiments, the polyA sequence or polyAC spacer increases the expression of the RNA construct.

[0139] In some embodiments, the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide comprising a spike (S) protein or a fragment thereof of a SARS-CoV-2 variant, wherein the nucleic acid sequence is identical to mA (N 6-methyladenosine) modification motif sequence is operably linked. The m6A modification sequence may include an m6A consensus sequence. The m6A consensus sequence is known in the art (e.g., Ke et al., 2017, m 6 AmRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes & Dev. 2017. 31: 990-1006 identified a consensus sequence, which is incorporated herein by reference in its entirety) and can be downloaded from GEO (GSE86336). In some embodiments, the m6A modification motif sequence comprises a sequence as shown in SEQ ID NO: 24. In some embodiments, the m6A modification motif sequence is followed by a Kozak sequence operably linked to a nucleic acid sequence encoding an antigenic polypeptide.

[0140] In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the 3' catalytic group I intron fragment comprises the nucleic acid sequence of SEQ ID NO: 32, and the 5' catalytic group I intron fragment sequence comprises the nucleic acid sequence of SEQ ID NO: 33.

[0141] In some embodiments, the group I catalytic intron of the T4 phage Td gene is split into two in such a way as to retain structural elements that are essential for ribozyme folding. Exon fragment 2 is then connected to the upstream of exon fragment 1, and a nucleic acid sequence encoding an antigenic polypeptide is inserted between the exon-exon junction, wherein the antigenic polypeptide comprises a spike (S) protein of a SARS-CoV-2 variant or a fragment thereof. In some embodiments, an antigenic polypeptide comprising an IRES or m6A sequence, a Kozak sequence, a signal peptide coding sequence, an S protein or a fragment thereof containing a SARS-CoV-2 variant, and a stop codon or an in-frame 2A peptide sequence are inserted between the exon-exon junction.

[0142] In some embodiments, the circRNA comprises a 5' linker sequence located at the 5' end of the circRNA, and a 3' linker sequence located at the 3' end of the circRNA, wherein the 5' linker sequence and the 3' linker sequence are ligated to each other by a ligase (eg, T4 RNA ligase).

[0143] C. SARS-CoV-2 variants

[0144] Exemplary SARS-CoV-2 variants and spike protein mutations associated with these variants are shown in Table A below. The circRNA vaccines and compositions described herein can be used to treat any one of the SARS-CoV-2 variants described herein or a combination thereof. The SARS-COV-2 variants described herein are named by the World Health Organization or named according to the phylogenetic assignment of the named global epidemic (PANGO) pedigree software. It should be understood that different naming systems and algorithms in the art can be used to refer to the same variant. The classification and definition of SARS-CoV-2 variants and a list of known SARS-CoV-2 variants can be found at world wide web.cdc.gov / coronavirus / 2019-ncov / variants / variant-classifications.html.

[0145] Table A. SARS-CoV-2 variants

[0146]

[0147]

[0148] The reference SARS-CoV-2 virus is βCoV / WIV04 / 2019 / (Accession No.: epi-isl-402124). In some embodiments, the SARS-CoV-2 variant is a variant being monitored, a variant of interest, a variant of concern, or a variant of high consequence. In some embodiments, the SARS-CoV-2 variant is selected from the group consisting of α (i.e., B.1.1.7 and Q) variants, β (i.e., B.1.351) variants, γ (i.e., P.1, also known as B.1.28.1) variants, Epsilon (i.e., B.1.427 or B.1.429) variants, Aita (i.e., B.1.525) variants, Yota (i.e., B.1.526) variants, Kappa (i.e., B.1.617.1) variants, B.1.617.3 variants, Zeta (i.e., P.2) variants, Miyou (i.e., B.1.621 or B.1.621.1) variants, Delta (i.e., B.1.617.2 or AY) variants, and Omicron (i.e., B.1.1.529 or BA) variants. In some embodiments, the SARS-CoV-2 variant is a Delta variant, such as a B.1.617.2 variant, or an AY variant. In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is an Omicron variant, such as a B.1.529 variant or a BA variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1). In some embodiments, the SARS-CoV-2 variant has one or more mutations (e.g., insertions, deletions, and / or substitutions) in the spike protein. In some embodiments, one or more mutations in the spike protein may affect the fitness of the virus, such as transmissibility, virulence, and / or drug resistance (e.g., resistance to neutralizing antibodies and / or resistance to vaccines). In some embodiments, one or more mutations in the spike protein do not substantially change the fitness of the virus. In some embodiments, the SARS-CoV-2 variant does not have a mutation in the spike protein.

[0149] III. Treatment Methods

[0150] circRNA and compositions described herein can be used to treat or prevent SARS-CoV-2 infection in individuals, including but not limited to infections caused by delta variants or Omicron variants of SARS-CoV-2. In some embodiments, circRNA is translated by ribosomes in individuals. In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, circRNA is administered in the form of lipid nanoparticle formulations. In some embodiments, circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about four weeks, such as at least any one of five, six, seven or eight weeks. In some embodiments, circRNA is administered with an initial dose, followed by a secondary dose in any one of about 4, 6, 8 or 10 weeks.

[0151] In some embodiments, a method for treating or preventing SARS-CoV-2 infection in an individual is provided, comprising administering to the individual an effective amount of circRNA comprising a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant. In some embodiments, the antigenic polypeptide is an S protein or a fragment thereof. In some embodiments, the method comprises administering an effective amount of a cocktail composition comprising a plurality of circRNAs encoding different antigenic polypeptides. In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of Omicron subvariants (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1). In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, the circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, the circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about 4 weeks, such as at least any one of 5, 6, 7 or 8 weeks.

[0152] In some embodiments, the present application provides the use of a circRNA vaccine comprising a circRNA in the manufacture of a vaccine for treating or preventing SARS-CoV-2 infection in an individual, wherein the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide of a spike (S) protein or a fragment thereof containing a SARS-CoV-2 variant. In some embodiments, the circRNA is administered in the form of naked circRNA. In some embodiments, the circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, the circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about 4 weeks, for example, at least any one of 5, 6, 7 or 8 weeks.

[0153] In some embodiments, the present application provides a method for preventing or reducing the risk of infection of an individual SARS-CoV-2, comprising administering to an individual an effective amount of a circRNA of any one of the above embodiments, wherein the circRNA encodes an antigenic polypeptide of a SARS-CoV-2 variant. In some embodiments, the method comprises administering a cocktail composition comprising a plurality of circRNAs encoding different antigenic polypeptides. In some embodiments, the circRNA is translated by a ribosome in an individual by a rolling circle. In some embodiments, the circRNA is administered in the form of a naked circRNA, or in the form of a pharmaceutical composition comprising a transfection agent. In some embodiments, SARS-CoV-2 infection is caused by one or more SARS-CoV-2 variants. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an Omicron variant. In some embodiments, SARS-CoV-2 infection is caused by one or more Omicron subvariants (e.g., BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5). In some embodiments, SARS-CoV-2 infection is caused by one or more sublineages of the subvariant of Omicron (e.g., BA.2.75.2, BA.4.6, BF.7, BQ.1.1). In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about 4 weeks, for example, at least any one of 5, 6, 7, or 8 weeks.

[0154] In some embodiments, the present application provides a method for treating or preventing an infection caused by multiple SARS-CoV-2 strains in an individual, comprising administering to an individual an effective amount of a circRNA vaccine in any one of the embodiments described herein. In some embodiments, the method comprises administering to an individual an effective amount of a plurality of different circRNA vaccines in any one of the embodiments described herein. In some embodiments, the method comprises administering to an individual a composition comprising multiple (e.g., two or more) circRNAs, wherein the first circRNA encodes the S protein or a fragment thereof of the first strain of SARS-CoV-2, and the second circRNA encodes the S protein or a fragment thereof of the second strain of SARS-CoV-2. In some embodiments, at least one of the multiple circRNAs encodes a mutated S protein or a fragment thereof found in a variant (such as a delta or an Omicron variant) of Table A. In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about 4 weeks, for example, at least any one of 5, 6, 7, or 8 weeks.

[0155] In some embodiments, the present application provides a method for treating or preventing SARS-CoV-2 infection in an individual, comprising administering to the individual an effective amount of a circRNA vaccine comprising a circRNA, wherein the circRNA comprises a nucleic acid sequence encoding an antigenic polypeptide of an S protein or a fragment thereof containing a SARS-CoV-2 variant. In some embodiments, the antigenic polypeptide comprises the RBD of the S protein. In some embodiments, the antigenic polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper domain based on GCN-4). In some embodiments, the antigenic polypeptide comprises the S2 region of the S protein. In some embodiments, the antigenic polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P), which stabilize the pre-fusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations (e.g., deletion of amino acid residues 681 to 684), which inhibit the cleavage of the S protein. In some embodiments, the antigenic polypeptide comprises an S protein or a fragment thereof of SARS-CoV-2 having a D614G mutation. In some embodiments, the circRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 20-21. In some embodiments, the circRNA is translated by a ribosome in an individual. In some embodiments, the circRNA is administered in the form of naked circRNA. In some embodiments, the circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, the circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about four weeks, for example, any one of at least five, six, seven or eight weeks.

[0156] In some embodiments, the present application provides a method for treating or preventing SARS-CoV-2 infection in an individual, comprising administering to the individual an effective amount of a circRNA vaccine comprising circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising an S protein or a fragment thereof of a SARS-CoV-2 variant, and (b) an IRES sequence, wherein the IRES sequence is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA further comprises an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (eg, human tPA or IgE SP) fused to the N-terminus of the S protein or a fragment thereof. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence comprising, from the 5' end to the 3' end: an IRES sequence, a Kozak sequence, an SP, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a polyA or polyAC sequence located at the 5' end of the IRES sequence. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' connection sequence at the 5' end of the circRNA, and a 3' connection sequence at the 3' end of the circRNA, wherein the 5' connection sequence and the 3' connection sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, the antigen polypeptide comprises the RBD of the S protein. In some embodiments, the antigen polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper domain based on GCN-4). In some embodiments, the antigen polypeptide comprises the S2 region of the S protein. In some embodiments, the antigen polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P) that stabilize the prefusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations (e.g., deletion of amino acid residues 681 to 684) that inhibit the cleavage of the S protein. In some embodiments, the antigenic polypeptide comprises an S protein or a fragment thereof of SARS-CoV-2 having a D614G mutation.In some embodiments, circRNA comprises a nucleic acid sequence of SEQ ID NO:20 or SEQ ID NO:21. In some embodiments, circRNA is translated by ribosomes in an individual. In some embodiments, circRNA vaccines are administered by intramuscular injection (im). In some embodiments, one or more doses of circRNA vaccines are administered. In some embodiments, the interval between each dose is about 2 weeks (e.g., 12, 13, 14, 15 or 16 days). In some embodiments, the method includes administering a first dose of circRNA vaccine and administering a second dose of circRNA vaccine 2 weeks or about 2 weeks later. In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about four weeks, for example, any one of at least five, six, seven or eight weeks.

[0157] In some embodiments, the present application provides a method for treating or preventing SARS-CoV-2 infection in an individual, comprising administering to the individual an effective amount of a circRNA vaccine comprising circRNA, the circRNA comprising: (a) a nucleic acid sequence encoding an antigenic polypeptide, the antigenic polypeptide comprising a fragment of an S protein or a variant thereof, and (b) an m6A modification motif sequence, which is operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the nucleic acid sequence further encodes an SP (e.g., human tPA or IgE SP) fused to the N-terminus of the S protein or its fragment. In some embodiments, the circRNA further comprises a Kozak sequence operably linked to the nucleic acid sequence encoding the antigenic polypeptide. In some embodiments, the circRNA comprises a nucleic acid sequence, the nucleic acid sequence comprising from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence, an SP, and a nucleic acid sequence encoding an antigenic polypeptide. In some embodiments, the circRNA further comprises a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide. In some embodiments, the circRNA further comprises a 5' connection sequence at the 5' end of the circRNA, and a 3' connection sequence at the 3' end of the circRNA, wherein the 5' connection sequence and the 3' connection sequence are connected to each other by a ligase (e.g., T4 RNA ligase). In some embodiments, the antigen polypeptide comprises the RBD of the S protein. In some embodiments, the antigen polypeptide further comprises a multimerization domain (e.g., a C-terminal Fd domain, or an isoleucine zipper structure based on GCN-4). In some embodiments, the antigen polypeptide comprises the S2 region of the S protein. In some embodiments, the antigen polypeptide comprises amino acid residues 2 to 1273 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO:1. In some embodiments, the S2 region of the S protein comprises one or more mutations (e.g., K986P and V987P), which stabilize the pre-fusion conformation of the S protein. In some embodiments, the antigenic polypeptide comprises one or more mutations (e.g., deletion of amino acid residues 681 to 684), which inhibit the cleavage of the S protein. In some embodiments, the antigenic polypeptide comprises an S protein or a fragment thereof of SARS-CoV-2 having a D614G mutation. In some embodiments, the circRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 20-21. In some embodiments, the circRNA is translated by rolling circles in an individual by ribosomes. In some embodiments, the circRNA vaccine is administered by intramuscular injection (im). In some embodiments, one or more doses of the circRNA vaccine are administered.In some embodiments, the interval between each dose is about 2 weeks (e.g., 12, 13, 14, 15 or 16 days). In some embodiments, the method includes administering a first dose of circRNA vaccine and administering a second dose of circRNA vaccine 2 weeks or about 2 weeks later. In some embodiments, circRNA is administered in the form of naked circRNA. In some embodiments, circRNA is administered in the form of a lipid nanoparticle formulation. In some embodiments, circRNA is administered twice or more. In some embodiments, the interval between each administration is at least about four weeks, such as at least any one of five, six, seven or eight weeks.

[0158] Preparation and administration

[0159] In some embodiments, the circRNA composition (eg, circRNA vaccine or pharmaceutical composition) for administration further comprises a transfection agent. In a non-limiting example, the transfection agent is polyethyleneimine (PEI) or lipid nanoparticles (LNP). Suitable lipid nanoparticles for administering circRNA have been described, for example, in Ickenstein, LM & Garidel, P. Lipid-based nanoparticle formulations for small molecules and RNA drugs. 890 Expert Opin Drug Deliv 16, 1205-1226, doi: 10.1080 / 17425247.2019.1669558 (2019), US Patent App. Pub. No. 20200121809, US Patent App. Pub. No. 20200163878, US Patent App. Pub. No. 20190022247, and International Patent App. Pub. No. WO2021 / 030701, the contents of which are incorporated herein by reference in their entirety. In some embodiments, LNP is formed by a lipid mixture of MC3-lipid: DSPC: cholesterol: PEG2000-DMG. In some embodiments, MC3-lipid:DSPC:cholesterol:PEG2000-DMG are mixed in a molar ratio of 50:10:38.5:1.5.

[0160] Other examples of liposomes that can be used to administer circRNA compositions (e.g., circRNA vaccines or pharmaceutical compositions) include protamine, cationic nanoemulsions, modified dendritic nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipid transfection agents sold under the trademark LIPOFECTAMINE, non-liposomal transfection agents sold under the trademark FUGENE, or any combination thereof can be used as a transfection agent.

[0161] In some embodiments, liposome preparations may be subject to, but are not limited to, the selection of cationic lipid components, the saturation of cationic lipids, the properties of PEGylation, the ratio of all components, and the influence of biophysical parameters (such as size). In some embodiments, liposome preparations include cationic lipids, cholesterol, and PEGylated lipids. For example, liposome preparations may include cationic lipids, dipalmitoylphosphatidylcholine, cholesterol, and PEG-c-DMA. See, for example, Semple et al. Nature Biotech. 2010 28: 172-176, which is incorporated herein by reference as a whole. In some embodiments, liposome preparations may include about 35% to about 45% of cationic lipids, about 40% to about 50% of cationic lipids, about 50% to about 60% of cationic lipids, and / or about 55% to about 65% of cationic lipids. In some embodiments, the ratio of lipid to RNA in liposomes may be about 5: 1 to about 20: 1, about 10: 1 to about 25: 1, about 15: 1 to about 30: 1, and / or at least 30: 1. Suitable liposome formulations have been described, for example, in WO2020237227, the contents of which are incorporated herein by reference in their entirety.

[0162] In some embodiments, circRNA is delivered in the form of naked RNA. In some embodiments, circRNA is delivered by gene gun or by electroporation. In some embodiments, circRNA vaccines are substantially free of transfection agents (including, for example, polyethyleneimine (PEI), liposomes and / or lipid nanoparticles (LNP)). In some embodiments, circRNA vaccines are substantially free of adjuvants. In some embodiments, circRNA vaccines include adjuvants, which can make the vaccine stimulate a higher immune response. In some embodiments, the adjuvant is not aluminum hydroxide. In some embodiments, circRNA vaccines are substantially free of aluminum hydroxide. In some embodiments, circRNA vaccines are substantially free of one or more (or all) of the following substances: protamine, cationic nanoemulsions, modified dendritic nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid-cholesterol nanoparticles, cationic lipid-cholesterol PEG nanoparticles, cationic lipid transfection reagents, non-liposomal transfection reagents, or any combination thereof.

[0163] The circRNA vaccine (e.g., circRNA vaccine or pharmaceutical composition) for administration can be administered to a subject by systemic injection into the vascular system, systemic injection into lymph nodes, subcutaneous injection or depot, or by local injection. In some embodiments, the circRNA is administered intranasally. In some embodiments, the circRNA is administered by intramuscular injection or intradermal injection.

[0164] In some embodiments, the circRNA vaccine herein (e.g., S protein or fragment thereof encoding SARS-CoV-2 variants) is administered by intramuscular injection (im). In some embodiments, one or more doses of circRNA vaccine are administered. In some embodiments, two or more doses of circRNA vaccine are administered. In some embodiments, the interval between each dose is about 2 weeks (e.g., 12, 13, 14, 15 or 16 days) or more. In some embodiments, the method includes administering a first dose of circRNA vaccine, and administering a second dose of circRNA vaccine 2 weeks or about 2 weeks later. In some embodiments, the second dose of circRNA is administered after more than 2 weeks, including, for example, administration after any one week of 3, 4, 5, 6, 7 or 8 weeks.

[0165] In some embodiments, the circRNA may be formulated in lipid nanoparticles, such as those described in International Publication No. WO2012170930, which is herein incorporated by reference in its entirety.

[0166] In some embodiments, synthetic nanocarriers can be formulated for controlled and / or sustained release of circRNA as described herein. As non-limiting examples, synthetic nanocarriers for sustained release can be formulated by methods known in the art, as described herein, and / or as described in International Publication No. WO2010138192 and U.S. Publication No. 20100303850, each of which is incorporated herein by reference in its entirety.

[0167] In some embodiments, the circRNA can be formulated for controlled and / or sustained release, wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer. CYSC polymers are described in U.S. Pat. No. 8,399,007, which is incorporated herein by reference in its entirety.

[0168] In some embodiments, synthetic nanocarriers can be formulated for use as vaccines. In some embodiments, synthetic nanocarriers can encapsulate at least one circRNA encoding at least one antigen. As a non-limiting example, synthetic nanocarriers can include at least one antigen and an excipient for a vaccine formulation (see International Publication No. WO201 1150264 and U.S. Publication No. US201 10293723, each of which is incorporated herein by reference in its entirety). As another non-limiting example, a vaccine formulation can include at least two synthetic nanocarriers and excipients with the same or different antigens (see International Publication No. WO2011150249 and U.S. Publication No. US20110293701, each of which is incorporated herein by reference in its entirety). Vaccine formulations can be selected by methods described herein, methods known in the art, and / or methods described in International Publication No. WO2011150258 and U.S. Publication No. US20120027806, each of which is incorporated herein by reference in its entirety).

[0169] In some embodiments, the synthetic nanocarrier may include at least one circRNA encoding at least one adjuvant. As a non-limiting example, the adjuvant may include dimethyldioctadecyl ammonium bromide, dimethyldioctadecyl ammonium chloride, dimethyldioctadecyl ammonium phosphate or dimethyldioctadecyl ammonium acetate (DDA) and a polar portion of a total lipid extract of mycobacteria or a portion of the polar portion (see, e.g., U.S. Patent No. 8,241,610; which is incorporated herein by reference in its entirety). In another embodiment, the synthetic nanocarrier may include at least one circRNA and an adjuvant. As a non-limiting example, the synthetic nanocarrier and adjuvant may be prepared by the methods described in International Publication No. WO2011150240 and U.S. Publication No. US20110293700, each of which is incorporated herein by reference in its entirety.

[0170] In some embodiments, circRNA acts as an adjuvant. As an example, RNA induction in the cytoplasm can trigger innate immunity, and innate immune signals are known to promote adaptive immunity through different pathways. Therefore, a circRNA containing an antigenic polypeptide or a second circRNA (e.g., a circRNA that does not encode a polypeptide) can be used as an adjuvant to enhance the adaptive immune response to the antigenic polypeptide.

[0171] In some embodiments, the circRNA composition (e.g., circRNA vaccine or pharmaceutical composition) for administration can be administered intranasally. For example, the circRNA vaccine can be administered intranasally, similar to the administration of live vaccines. In some embodiments, the circRNA can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in the art.

[0172] In some embodiments, the circRNA vaccine comprises an adjuvant, which can cause the vaccine to stimulate a higher immune response. As a non-limiting example, the adjuvant can be a submicron oil-in-water emulsion, which can stimulate a higher immune response in human pediatric populations (see, e.g., adjuvanted vaccines described in U.S. Patent Publication No. US20120027813 and U.S. Patent No. US8506966, the contents of which are incorporated herein by reference in their entirety).

[0173] In some embodiments, the circRNA composition of the present application can be administered with other preventive or therapeutic compounds. As a non-limiting example, the preventive or therapeutic compound can be an adjuvant or a booster. As used herein, when referring to a preventive composition, such as a vaccine, the term "booster" refers to the additional administration of a preventive composition. A booster (or booster vaccine) can be given after early administration of a preventive composition. The time between the initial administration and the booster of the prophylactic composition can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours ... 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years.

[0174] IV. Preparation Method

[0175] The present application further provides nucleic acid constructs (e.g., linear RNA and vectors, etc.) for preparing circRNA as described herein, and methods for preparing circRNA, such as by chemical ligation, enzymatic ligation, or ribozyme autocatalysis of linear RNA. In some embodiments, circRNA is prepared by in vitro cyclization of linear RNA.

[0176] Linear RNA and nucleic acid construct encoding the linear RNA

[0177] In some embodiments, the application provides a linear RNA capable of forming a circRNA of any of the above embodiments. In some embodiments, the linear RNA can be cyclized by a chemical cyclization method using cyanogen bromide or a similar condensing agent. In some embodiments, the linear RNA can be cyclized by the autocatalysis of the I group intron comprising a 5' catalytic I group intron fragment and a 3' catalytic I group intron fragment. In some embodiments, the linear RNA can be cyclized by a ligase. In some embodiments, the linear RNA can be cyclized by T4 RNA ligase. In some embodiments, the linear RNA can be cyclized by DNA ligase. Suitable ligases include but are not limited to T4 DNA ligase (T4Dnl), T4 RNA ligase 1 (T4 Rnl1) and T4 RNA ligase 2 (T4Rnl2).

[0178] In some embodiments, the present application provides a linear RNA capable of forming a circRNA of any of the above embodiments, wherein the linear RNA can be cyclized by autocatalysis of a group I intron. In some embodiments, the group I intron comprises a 5' catalytic group I intron fragment and a 3' catalytic group I intron fragment. In some embodiments, the linear RNA comprises a 3' catalytic group I intron fragment (such as a sequence shown in SEQ ID NO: 32), which is flanked by a 5' end of a 3' exon sequence that can be recognized by a 3' catalytic group I intron fragment (such as a sequence shown in SEQ ID NO.25), and a 5' catalytic group I intron fragment (such as a sequence shown in SEQ ID NO: 33), which is flanked by a 5' catalytic group I intron fragment (such as a sequence shown in SEQ ID NO: 26) The 3' end of the 5' exon sequence is recognized.

[0179] In some embodiments, the linear RNA comprises a 3' intron-IRES-Kozak-SP-spike-5' intron sequence from the 5' to the 3' end. In some embodiments, the spike sequence comprises one of the sequences shown in SEQ ID NOs: 11-15, 18-19 and 41.

[0180] In some embodiments, the linear RNA comprises a 3' intron-IRES-Kozak-SP-RBD-5' intron sequence from the 5' to the 3' end. In some embodiments, the RBD sequence comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO: 1.

[0181] In some embodiments, the linear RNA further comprises a 5' homologous sequence flanking the 5' end of the 3' catalytic group I intron fragment, and a 3' homologous sequence flanking the 3' end of the 5' catalytic group I intron fragment. In some embodiments, the linear RNA comprises a 5' homology arm-3' catalytic group I intron fragment-3' exon sequence-IRES-Kozak-SP-antigenic polypeptide (e.g., spike protein or fragment thereof)-5' exon sequence-5' catalytic group I intron fragment-3' homology arm sequence from 5' to 3' end. In some embodiments, the length of the homologous sequence can be 1 to 100, 5 to 80, 5 to 60, 10 to 50, or 12 to 50 nucleotides. In some embodiments, the length of the homologous sequence is about 20-30 nucleotides. In some embodiments, the 5' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 27, and the 3' homologous sequence comprises the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the homology arms increase the efficiency of RNA circularization by about 0 to 20%, more than 20%, more than 30%, more than 40%, or more than 50%.

[0182] In some embodiments, a nucleic acid construct is provided, comprising a nucleic acid sequence encoding a linear RNA. In some embodiments, a T7 promoter is operably linked to a nucleic acid sequence encoding a linear RNA. In some embodiments, the T7 promoter comprises a sequence as shown in SEQ ID NO: 29. In some embodiments, the T7 promoter is capable of driving in vitro transcription.

[0183] Plasmids

[0184] In some embodiments, the application provides a plasmid comprising a nucleotide sequence described herein. In some embodiments, the plasmid is obtained by cloning a sequence encoding a linearized RNA into a plasmid vector. The plasmid can be produced by techniques known in the art, such as Gibson cloning or cloning using a restriction enzyme. In some embodiments, the plasmid vector includes an antibiotic expression cassette, allowing antibiotic selection of bacteria expressing the plasmid. In some embodiments, the provided plasmid can be purified from bacteria and used to produce a linear circRNA construct. Any plasmid vector suitable for in vitro transcription of linear RNA can be used.

[0185] In some embodiments, the plasmid is linearized before in vitro transcription of the linear RNA. In some embodiments, the recombinant plasmid is linearized by restriction enzyme digestion. In some embodiments, the recombinant plasmid is linearized by PCR amplification. In some embodiments, the method further comprises in vitro transcription with the linearized plasmid template. In some embodiments, the in vitro transcription is driven by a T7 promoter.

[0186] Circularization of linear RNA by chemical ligation

[0187] In some embodiments, a method for preparing a circRNA described herein is provided, comprising: (a) chemically ligating the 5' end and the 3' end of a linear RNA comprising a nucleic acid sequence encoding the circRNA; and (b) isolating the circularized RNA product, thereby providing the circRNA.

[0188] In some embodiments, the step of circularizing the linear RNA comprises a chemical circularization method using cyanogen bromide or a similar condensing agent.

[0189] In some embodiments, linear RNA can be cyclized by chemical methods. In some chemical methods, the 5' and 3' ends of nucleic acids (e.g., linear cyclic polyribonucleotides) include chemically reactive groups that, when brought into close proximity, can form new covalent connections between the 5' and 3' ends of the molecule. The 5' end can contain an NHS ester reactive group, and the 3' end can contain a 3' amino-terminal nucleotide, so that in an organic solvent, the 3' amino-terminal nucleotide on the 3' end of the linear RNA molecule will undergo nucleophilic attack on the 5'-NHS-ester moiety to form a new 5'- / 3'-amide bond.

[0190] In some embodiments, the cyclization efficiency of the cyclization method provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or 100%. In some embodiments, the cyclization efficiency of the cyclization method provided herein is at least about 40%.

[0191] Circularization of linear RNA by ribozyme autocatalysis

[0192] In some embodiments, circRNA can be obtained by cyclizing linear RNA by ribozyme autocatalysis. In some embodiments, linear RNA is cyclized in vitro. In some embodiments, cyclization by ribozyme autocatalysis includes (a) placing the linear RNA under conditions that activate the autocatalysis of the group I intron (or its 5' and 3' catalytic group I intron fragments) to provide a cyclized RNA product; and (b) isolating the cyclized RNA product, thereby providing a circRNA.

[0193] In some embodiments, the method includes the step of obtaining linear RNA by first cloning a sequence encoding linearized RNA into a plasmid vector and then linearizing the recombinant plasmid. In some embodiments, the recombinant plasmid is linearized by restriction enzyme digestion. In some embodiments, the recombinant plasmid is linearized by PCR amplification. In some embodiments, the method further includes in vitro transcription with a linearized plasmid template. In some embodiments, in vitro transcription is driven by a T7 promoter. In some embodiments, the method further includes purifying linear RNA transcripts. In some embodiments, the linear RNA is purified by gel purification.

[0194] In some embodiments, the present application provides a method for cyclizing a linear RNA (e.g., purified linear RNA) by ribozyme autocatalysis of a group I intron. During the splicing process, the 3' hydroxyl group of the guanosine nucleotide undergoes an esterification reaction at the 5' splice site. Half of the 5' intron is removed, and the free hydroxyl group at the intermediate end undergoes a second esterification reaction at the 3' splice site, resulting in cyclization of the middle region and removal of the 3' intron. In some embodiments, the conditions for activating the autocatalysis of the group I intron or the 5' and 3' catalytic group I intron fragments are the addition of GTPs and Mg. 2+ In some embodiments, a method is provided in which GTPs and Mg are added at 55°C. 2+ In some embodiments, the method further comprises treating with RNase R to digest the linear RNA transcript. In some embodiments, the method further comprises isolating circular RNA (circRNA). In some embodiments, the step of isolating circRNA comprises gel purifying circRNA. In some embodiments, the purified circRNA can be stored at -80°C.

[0195] In some embodiments, the efficiency of cyclization is at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, or at least 50%. In some embodiments, the efficiency of cyclization is about 40% to about 50% or more.

[0196] Circularization of linear RNA by ligation

[0197] In some embodiments, circRNA can be obtained by cyclizing linear RNA using a ligase such as RNA ligase. In some embodiments, linear RNA is cyclized in vitro. In some embodiments, linear RNA can be cyclized by T4 RNA ligase. In certain embodiments, linear RNA comprises a 5' connection sequence at the 5' end of the nucleic acid sequence encoding circRNA, and a 3' connection sequence at the 3' end of the nucleic acid sequence encoding circRNA, wherein the 5' connection sequence and the 3' connection sequence can be connected to each other by RNA ligase. In a non-limiting example, linear RNA can be cyclized by a ligase such as T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1) and T4 RNA ligase 2 (T4 Rnl2). Linear RNA can be cyclized with or without single-stranded nucleic acid adapters, such as splint DNA.

[0198] In some embodiments, the present application provides a method for producing any of the above-mentioned circRNAs, comprising: (a) contacting any linear RNA comprising a 5' linker sequence located at the 5' end of a nucleic acid sequence encoding the circRNA, and a 3' linker sequence located at the 3' end of a nucleic acid sequence encoding the above-mentioned circRNA with a single-stranded adapter nucleic acid, wherein the adapter nucleic acid comprises, from the 5' end to the 3' end: a first sequence complementary to the 3' linker sequence and a second sequence complementary to the 5' linker sequence, wherein the 5' linker sequence and the 3' linker sequence hybridize with the single-stranded adapter nucleic acid to provide a double-stranded nucleic acid intermediate, which comprises a single-stranded break between the 3' end of the 5' linker sequence and the 5' end of the 3' linker sequence; (b) contacting the intermediate with an RNA ligase under conditions that allow the 5' linker sequence to be ligated to the 3' linker sequence to provide a circularized RNA product; and (c) isolating the circularized RNA product to provide a circRNA.

[0199] In some embodiments, the method described herein includes circularizing a linear RNA in vitro, comprising: (a) contacting any linear RNA comprising a 5' linker sequence located at the 5' end of a nucleic acid sequence encoding a circular RNA, and a 3' linker sequence located at the 3' end of a nucleic acid sequence encoding the circular RNA with an RNA ligase under conditions that allow ligation of the 5' linker sequence and the 3' linker sequence to provide a circularized RNA product; and (b) isolating the circularized RNA product to provide a circular RNA.

[0200] In some embodiments, the method further comprises treating with RNase R to digest the linear RNA transcript. In some embodiments, the method further comprises isolating circular RNA (circRNA). In some embodiments, the step of isolating circRNA comprises gel purifying circRNA. In some embodiments, the purified circRNA can be stored at -80°C.

[0201] In some embodiments, a DNA or RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., a linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) to form a new phosphodiester bond. In one reaction example, the linear circular RNA is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) at 37°C for 1 hour according to the manufacturer's protocol. The ligation reaction can occur in the presence of a linear nucleic acid capable of base pairing juxtaposed with the 5'- and 3'-regions to assist in the enzymatic ligation reaction. In some embodiments, the ligation is a splint ligation. For example, a splint ligase, such as Ligase can be used for splint ligation. For splint ligation, a single-stranded polynucleotide (splint), such as single-stranded RNA, can be designed to hybridize with the two ends of a linear polyribonucleotide so that the two ends can be juxtaposed when hybridizing with the single-stranded splint. Splint ligase can therefore catalyze the connection of the two juxtaposed ends of a linear polyribonucleotide to generate a circular polyribonucleotide.

[0202] In some embodiments, DNA or RNA ligase can be used for the synthesis of circular RNA. As a non-limiting example, the ligase can be a circ ligase or a circular ligase.

[0203] Purification of circRNA

[0204] In some embodiments, the method for producing circRNA provided herein further includes the step of purifying the cyclized RNA product. In a non-limiting example, the circRNA is purified by gel purification or by high performance liquid chromatography (HPLC). In some embodiments, agarose gel electrophoresis can simply and effectively separate the circular splicing product from the linear precursor molecule, the ring with a nick, the splicing intermediate, and the excised intron. In some embodiments, the method includes purifying the circular RNA by chromatography, such as HPLC. In some embodiments, the purified circular RNA can be stored at -80 ° C.

[0205] V. Pharmaceutical Compositions, Kits, and Articles of Manufacture

[0206] The application further provides a pharmaceutical composition comprising any circRNA described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by mixing the therapeutic agent described herein with the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), which is incorporated herein by reference in its entirety), in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dose and concentration used, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium pyrosulfite); preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA and / or nonionic surfactants.

[0207] In some embodiments, the pharmaceutical composition is contained in a disposable vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a container in bulk form. In some embodiments, the pharmaceutical composition is stored frozen.

[0208] The present application further provides kits and articles of manufacture for use in any embodiment of the treatment methods described herein. The kits and articles of manufacture may contain any one of the formulations and pharmaceutical compositions described herein.

[0209] In some embodiments, a kit is provided comprising any one of the circRNAs described herein and instructions for treating or preventing SARS-CoV-2 infection.

[0210] In some embodiments, a kit is provided comprising any plasmid or linear RNA described herein, and instructions for preparing any circRNA. In some embodiments, a kit is provided comprising any plasmid, linear RNA or circRNA described herein, and instructions for administering the circRNA.

[0211] The kit of the present application is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packages (e.g., sealed Mylar or plastic bags), etc. The kit may optionally provide additional ingredients, such as buffers and explanatory information. Therefore, the present application also provides articles, which include vials (e.g., sealed vials), bottles, cans, flexible packages, etc.

[0212] Instructions related to the use of the composition generally include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. For example, a kit containing a sufficient dose of the circRNA disclosed herein may be provided to provide effective treatment for an individual or many individuals. In addition, a kit containing a sufficient dose of circRNA may be provided so that multiple administrations are performed to an individual (e.g., in the case of a circRNA vaccine, initial vaccine administration and subsequent booster administration). The kit may also include multiple unit doses of the pharmaceutical composition and instructions for use, and may be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[0213] In some embodiments, the kit includes a delivery system. The delivery system can be a unit dose delivery system. The volume of the solution or suspension delivered by each dose can be about 5 to about 2000 microliters, about 10 to about 1000 microliters, or about 50 to about 500 microliters. The delivery system for these different dosage forms can be a syringe, a dropper, a plastic extrusion unit, a sprayer, atomizer or a pharmaceutical aerosol in a unit dose or multi-dose package. In some embodiments, a delivery system of any circRNA described herein is provided, which comprises circRNA and a device for delivering circRNA.

[0214] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless explicitly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0215] Example

[0216] By reference to the following examples, the application will be more fully understood. However, they should not be construed as limiting the scope of the application. It should be understood that the examples and embodiments described herein are only for illustrative purposes, and various modifications or changes based on this will be suggested to those skilled in the art, and are included in the spirit and scope of the application and the scope of the attached embodiments.

[0217] Example 1. circRNA RBD Generation of functional SARS-CoV-2 RBD antigen

[0218] Using a Group I ribozyme autocatalytic strategy 43 To generate the SARS-CoV-2 RBD antigen 23 Circular RNA, called circRNA RBD ( Figure 1A). In this construct, an IRES element was placed before the RBD coding sequence to initiate its translation. To enhance the immunogenicity of the RBD antigen, the signal peptide sequence of human tissue plasminogen activator (tPA) was fused to the N-terminus of the RBD to ensure secretion of the antigen. 17,45-47 In addition, recent studies have reported that RBD trimers are superior to monomeric RBD in binding hACE2 6,7,48 Therefore, the phage T4 fiber protein (foldon) 49 The trimerization motif of the IRES-SP-RBD-foldon was fused to its C-terminus to further improve the immunogenicity of the RBD antigen. The IRES-SP-RBD-foldon sequence was then cloned into a vector to construct a template for in vitro transcription (IVT) for the production of circRNA RBD ( Figure 1A ). CircRNA was verified by reverse transcription-PCR analysis using specific primers and Sanger sequencing RBD The precise cyclization ( Figure 1A-1B ).

[0219] Due to its covalently closed circular structure, circRNA RBD Migrate faster in electrophoresis ( Fig. 9A ) and appear to be more resistant to the exonuclease RNase R than linear precursor RNA ( Fig. 9B ). High-performance liquid chromatography (HPLC) showed that RNase R treatment removed a large amount of linear precursor RNA, which is essential for purifying circRNA RBD Important steps ( Fig. 9C ).

[0220] To test the RBD Secretory expression of the generated RBD antigen and purified circRNA RBD Transfected into HEK293T cells. Western blot results showed that a large amount of RBD antigen was produced in the supernatant ( Figure 1C ). Quantitative ELISA assay showed that circRNA RBD The resulting RBD antigen concentration reached ~1400 ng / mL, which is 600 times higher than that of the linear precursor RNA ( Figure 1D ). circRNA RBD RBD antigen can also be produced in mouse NIH3T3 cells, indicating the high compatibility of circRNA ( Figure 1E ). Taken together, these results suggest that the use of circRNA RBD Abundant secreted RBD antigen can be produced in both human and mouse cells.

[0221] In addition to the group I ribozyme autocatalytic strategy, T4 RNA ligase was used to generate circRNAsRBD ( Figure 1F ). Similarly, western blot and quantitative ELISA results showed that RBD antigen was effectively expressed in the supernatant, with the antigen concentration reaching ∼1000 ng / mL, which is ∼200-fold higher than its linear precursor RNA ( Figure 1G-1H ).

[0222] In order to verify the RBD To determine whether the secreted SARS-CoV-2 RBD antigen produced was functional, we used hACE2-overexpressing HEK293 cells (HEK293T-ACE2) and a SARS-CoV-2 pseudovirus carrying an EGFP reporter gene to express the circRNA RBD The supernatant of transfected cells was used for competition assay 50 Secretion of SARS-CoV-2 RBD antigen can effectively block infection by SARS-CoV-2 pseudovirus ( Fig. 1I ). In short, circRNA RBD It showed robust protein expression and high stability, suggesting that it has the potential to be engineered into a new vaccine platform.

[0223] Example 2: SARS-CoV-2 circRNA RBD Vaccine induces sustained humoral immune response with high levels of neutralizing antibodies

[0224] To explore whether circRNAs could be developed into novel vaccines, we then evaluated circRNAs encapsulated in lipid nanoparticles. RBD Immunogenicity in BALB / c mice ( Figure 2A ). circRNA RBD The encapsulation efficiency exceeds 93%, and its average diameter is 100nm ( Figure 2B ). Mice were injected intramuscularly with LNP-circRNA RBD Immunizations were performed twice, two weeks apart, with a dose of 10 μg or 50 μg per mouse, while empty LNP was used as a placebo control ( Figure 2C ). The amount of RBD-specific binding IgG and neutralizing antibodies was assessed two or five weeks after the booster dose. RBD High titers of RBD-specific IgG were elicited in a dose-dependent manner, with ~3 × 10 2 and 5 weeks after each dose and booster immunization. 4 and ~1×10 6 , indicating that circRNA RBD Can induce long-lasting antibodies against SARS-CoV-2 RBD ( Figure 2D ).

[0225] To test the neutralization capacity of serum samples from vaccinated mice, a neutralization assay was performed. RBD Serum from vaccinated mice can effectively neutralize SARS-CoV-2 pseudoviruses ( Figure 2E ) and the real SARS-CoV-2 virus ( Figure 2F ), mouse (50 μg circRNA RBD ) have NT50 values ​​of 5.6×10 3 ( Figure 2E ) and ~2.2×10 5 ( Figure 2F The large amount of RBD-specific IgG, potent RBD antigen neutralization, and sustained SARS-CoV-2 neutralization ability indicate that circRNA RBD The vaccine did induce a long-lasting humoral immune response in mice.

[0226] Example 3: SARS-CoV-2 circRNA RBD-β Vaccine-induced antibodies show preferential neutralizing activity against the beta variant

[0227] Next, a circRNA vaccine encoding the RBD / K417N-E484K-N501Y antigen derived from the SARS-CoV-2β variant (referred to as circRNA RBD-β BALB / c mice were injected intramuscularly with circRNA RBD-β The vaccine was used for immunization, followed by booster immunization every two weeks. The sera of the immunized mice were collected 1 and 2 weeks after the booster immunization. ELISA showed that the RBD-β-specific IgG endpoint titer could reach 7×10 at 2 weeks after the booster immunization. 4 ( Figure 2G ). Alternative neutralization assays showed that circRNA RBD-β The serum of immunized mice effectively neutralized RBD antigen ( Fig. 10C ). SARS-CoV-2 pseudovirus neutralization assay showed that circRNA RBD The stimulated antibodies can effectively neutralize all three virus strains, with the highest neutralizing activity against the D614G strain ( Figure 2H ). circRNA RBD-β The elicited antibodies were also able to neutralize all three pseudoviruses, with the highest neutralizing activity against their corresponding variant β strains ( Fig.2I ).

[0228] Consistent with the pseudovirus neutralization test, the serum can effectively neutralize the real SARS-CoV-2β strain with an NT50 of 7.1×10 4 ( Figure 2J), can neutralize the real SARS-CoV-2D164G strain, with an NT50 of 9.8×10 3 ( Figure 2K ), with poorer effects. Overall, antibodies elicited by circRNA vaccines showed the best neutralizing activity against their corresponding variant strains. Notably, both vaccines neutralized all three strains, albeit with varying potencies. Nevertheless, newer vaccines or multivalent vaccines based on mutations of emerging variants are likely to provide better and more effective protection against circulating SARS-CoV-2 variants, especially the recent Omicron variants with severe antibody escape capabilities.

[0229] Example 4: circRNA RBD-β Vaccine protects mice from infection with SARS-CoV-2 beta variant

[0230] To further evaluate the SARS-CoV-2 circRNA RBD-β The protective efficacy of the vaccine in vivo was tested by real virus challenge with the β strain. 59 Consistent, due to spike protein, such as K417N, E484Q and N501Y 59,60 SARS-CoV-2β variants can infect and replicate in the lungs of BALB / c mice due to mutations in Figure 2L Nearly two months after the booster dose, the titer of RBD-501Y.V2-specific IgG was approximately 2×10 4 ( Fig. 10B ), and the serum showed significant neutralization ability against RBD-β antigen ( Figure 2M Then, each immunized mouse received 5 × 10 4 PFU of authentic SARS-CoV-2 B.1.351 / 501Y.V2 strain, and lung tissue was collected 3 days after challenge for detection of viral RNA. It was found that mice in the placebo group lost more weight than mice vaccinated ( Figure 2N Consistently, the viral titers in the lungs of vaccinated mice were significantly reduced compared with those of mice that received a placebo ( Fig.2O These results indicate that circRNA RBD-β The vaccine can effectively protect mice from infection with the SARS-CoV-2β variant.

[0231] Example 5: circRNA RBD-德尔塔 Vaccine induces potent neutralizing antibodies against SARS-CoV-2 delta variant

[0232] Currently, the delta variant of SARS-CoV-2 is the dominant circulating strain worldwide. Similar to the beta variant, the delta variant can also partially escape antibodies produced by survivors or vaccinated individuals. Therefore, there is an urgent need to develop newer vaccines against the delta variant. We used a group I ribozyme autocatalysis and T4 RNA ligation strategy to generate circRNA encoding the RBD antigen of the delta variant. RBD-德尔塔 Then the circRNA RBD-德尔塔 Encapsulation with LNP (PRECISION NANOSYSTEMS) to generate LNP-circRNA RBD-德尔塔 Vaccine. Use 0.5μg, 2.5μg, 5μg or 10μg of LNP-circRNA RBD-德尔塔 The mice were immunized with the vaccine intramuscularly twice, 2 weeks apart, while empty LNPs were used as a placebo control. Two weeks after the booster dose, serum samples of immunized mice were collected to detect RBD-specific antibodies. Group I ribozyme autocatalytic strategy or circRNA based on T4 RNA ligation strategy RBD -德尔塔 The vaccines can induce high titers of IgG binding antibodies in mice ( Figure 3A-3B ). From circRNA RBD-德尔塔 The vaccinated mouse serum can effectively neutralize SARS-CoV-2 delta pseudovirus in a dose-dependent manner, and the NT50 of a dose of 10 μg can even reach ~2×10 5 ( Figure 3C ).

[0233] Importantly, circRNA RBD-德尔塔 Sera from immunized mice also effectively neutralized other variants, including local strains, α and β variants, but with different neutralizing abilities. RBD-德尔塔 The sera from immunized mice showed high neutralizing activity against the corresponding delta variants. The neutralizing activity against local strains and alpha variants was partially decreased ( Figure 3D-3E ), β variants in circRNA RBD-德尔塔 The worst escape was observed in the serum of immunized mice, especially at the 10 μg dose ( Figure 3E ). The neutralization capacity against these four SARS-CoV-2 variants increased as the vaccination dose increased from 0.5μg to 5μg. However, when the dose was further increased from 5μg to 10μg, there was no increase in neutralization activity against local strains, α or β variants ( Figure 3E ). However, the neutralizing activity against the corresponding delta variant continued to increase, almost 10-fold ( Figure 3E ). RBD-β The results are consistent with ( Figures 2H-2K), these results strongly suggest that developing newer vaccines based on corresponding mutations of emerging variants may provide more effective protection than the current strategy of using the original SARS-CoV-2 vaccine as a third booster.

[0234] Example 6: circRNA RBD-德尔塔 Vaccines stimulate T cell immune responses

[0235] B cells, CD4 + T cells and CD8 + T cells are one of the three pillars of adaptive immunity and they mediate effector functions implicated in the control of SARS-CoV-2 in both non-hospitalized and hospitalized cases of COVID-19 (Sette and Crotty, 2021).

[0236] In order to explore and compare circRNA RBD-德尔塔 1mΨ-modified mRNA RBD-德尔塔 (1mΨ-mRNA RBD-德尔塔 ) Vaccine-stimulated CD4 + and CD8 + T cell immune response, collection of circRNA RBD-德尔塔 Vaccine or 1mΨ-mRNA RBD-德尔塔 Splenocytes from mice immunized with the vaccine were stimulated with the SARS-CoV-2 RBD-Delta mixed peptide (Table B below), and the effector memory T cells (Tem, CD44 + CD62L - ) to quantify cytokine-producing T cells ( Fig.11 ). Under the stimulation of the RBD peptide pool, the expression of circRNA RBD-德尔塔 Vaccine or 1mΨU-mRNA RBD-德尔塔 CD8 + T cells produce interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), and interleukin-2 (IL-2) ( Figure 3F-3H ), indicating that RBD-specific CD8 + T cell responses mediated by circRNA RBD-德尔塔 and 1mΨU-mRNA RBD-德尔塔 Vaccine stimulation. Using circRNA RBD-德尔塔 Vaccine or 1mΨU-mRNA RBD-德尔塔 CD4 + T cells induced strong IFN-γ, TNF-α, and IL-2 responses, but minimal IL-4 responses ( Figure 3I-3L ), indicating that circRNA RBD-德尔塔 and 1mΨ-mRNARBD-德尔塔 The vaccines all induced a Th1-biased T cell immune response, while 1mΨ-mRNA RBD-德尔塔 Vaccines show better performance than circRNA RBD-德尔塔 Vaccine slightly stronger CD4 + T cell response ( Figures 3I-3K ).

[0237] Table B. Peptide sequences of RBD-delta antigens

[0238]

[0239]

[0240] Example 7: circRNA vaccines have higher antigen encoding efficiency than mRNA vaccines

[0241] It has been reported that circRNA is more stable than mRNA due to its covalently closed circular structure (Fischer and Leung, 2017). To test whether the stability of circRNA vaccines can confer higher antigen encoding efficiency, 1mΨ-modified mRNA, referred to as 1mΨ-mRNA, and unmodified mRNA were generated, both of which contained the same RBD coding sequence as circRNA. The purified circRNA, 1mΨ-mRNA, and unmodified mRNA were transfected into HEK239T cells, respectively. The cell supernatant was collected and the abundance of RBD antigen at different time points from 12 to 96 hours was measured. ELISA results showed that circRNA not only produced higher levels of RBD antigen at each time point, but also produced RBD antigen that was more stable than 1mΨ-mRNA and unmodified mRNA ( Figure 4A ). RT-qPCR results showed that circRNA was more stable than 1mΨ-mRNA and unmodified mRNA ( Figure 4B ). Similarly, LNP-encapsulated circRNA also produced more and more persistent RBD antigen than 1mΨ-mRNA and unmodified mRNA ( Figure 4C Interestingly, LNP encapsulation increased the antigen encoding efficiency of unmodified mRNA to a level comparable to that of 1mΨ-mRNA, but both were still much lower than those of circRNA ( Figure 4C ).

[0242] Thermostable RNA vaccines are highly desirable for efficient vaccine delivery. We found that purified circRNAs were readily expressed without detectable loss even after storage at room temperature (~25°C) for two weeks ( Figure 4D), highlighting its remarkable thermal stability. In addition, to compare the thermal stability of LNP-circRNA formulations with LNP-1mΨ-mRNA and LNP-unmodified-mRNA, LNP-encapsulated circRNA, 1mΨ-mRNA, or unmodified-mRNA were stored at 4°C, room temperature (~25°C), or 37°C for 1, 3, 11, 14, 21, and 28 days before transfection. ELISA results showed that the antigen encoding efficiency of circRNA was much higher than that of the other two groups of mRNA at 4°C, 25°C, or 37°C. At 4°C, there was almost no detectable reduction in RBD antigen produced by LNP-encapsulated circRNA within 1-28 days ( Figure 4E ), while the reduction of RBD antigen produced by LNP-encapsulated 1mΨ-mRNA or unmodified -mRNA started from day 21 and day 28, respectively ( Figure 4E ). When the storage temperature was increased to 25°C or 37°C, the stability of LNP-encapsulated circRNA, 1mΨ-mRNA, and unmodified -mRNA decreased, especially at 37°C ( Figure 4F-4G ). In summary, these results indicate that deterioration effects do occur with extended shelf life and elevated temperatures, but LNP-encapsulated circRNAs exhibit high stability at 4°C ( Figure 4E-4G ).

[0243] Example 8: circRNA vaccines stimulated a higher proportion of neutralizing antibodies and stronger Th1-biased T cell immune responses than mRNA vaccines

[0244] Currently, there are two widely used mRNAs, mRNA-1273 (Moderna) and BNT162b2 (Pfizer / BNT), both of which contain modified 1mΨ. RBD-德尔塔 The vaccine has higher stability and antigen encoding efficiency. It is still unclear whether the LNP-circRNA vaccine shows higher immunogenicity than the LNP-1mΨ-mRNARBD-Delta vaccine. RBD-德尔塔 Vaccines and 1mΨ-mRNA RBD-德尔塔 The balance of Th1 and Th2 immune responses between vaccines is important because Th2-biased immune responses may induce vaccine-associated enhanced respiratory disease (VAERD). Stimulating a strong Th1-biased immune response is a major safety consideration in the design of SARS-CoV-2 vaccines. Therefore, we compared circRNAs RBD -德尔塔 Vaccine or 1mΨ-mRNA RBD-德尔塔 The endpoint titers of IgG2a / c and IgG1 stimulated by the vaccine represent Th1 and Th2 immune responses. ELISA showed that although circRNARBD-德尔塔 Vaccine-induced total IgG levels and 1mΨ-mRNA RBD-德尔塔 Vaccine equivalent ( Figure 4H ), but circRNA RBD-德尔塔 The ratios of IgG2a / IgG1, IgG2c / IgG1 or (IgG2a+IgG2c) / IgG1 stimulated by the vaccine are much higher than those of 1mΨ-mRNA RBD-德尔塔 vaccine( Figures 4I-4J ), indicating that circRNA RBD-德尔塔 The vaccine tended to induce a stronger Th1-biased immune response, about ~7 times higher than the mRNA vaccine.

[0245] Antibody-mediated enhancement (ADE) of infection by virus-specific antibodies is another potential problem for vaccines, which has been reported in some viral infections, including Zika, dengue virus, and coronavirus. Previous studies have reported that virus-binding antibodies without neutralizing activity elicited by viral infection or vaccination may cause ADE effects, especially for those viruses with different serotypes. Therefore, circRNAs were studied. RBD-德尔塔 Vaccines and 1mΨ-mRNA RBD-德尔塔 The differences in the ratio of neutralizing antibodies to binding antibodies between vaccines. RBD-德尔塔 1mΨ-mRNA RBD-德尔塔 The vaccine showed the same neutralizing ability ( Figure 4K-4M ),but 德尔塔 At different doses (0.5 μg, 2.5 μg and 5 μg), circRNA RBD-德尔塔 Vaccine vs. 1mΨ-mRNA RBD-德尔塔 Vaccines can induce a higher proportion of neutralizing antibodies ( Figure 4N ), indicating that circRNA RBD-德尔塔 The proportion of virus-binding but non-neutralizing antibodies elicited by the vaccine was much lower. With this unique feature, the circRNA platform may have important advantages in meeting the potential ADE effect and virus mutation.

[0246] Together, these results suggest that 1mΨ-mRNA RBD-德尔塔 Compared with vaccines, circRNA RBD-德尔塔 It can stimulate a higher proportion of neutralizing antibodies and stronger Th1-biased T cell immune responses.

[0247] Example 9: circRNA RBD The vaccine elicited potent neutralizing antibodies and Th1-biased immune responses in rhesus macaques

[0248] To further evaluate the immunogenicity of the circRNA vaccine in nonhuman primates, animals were treated with 20 μg, 100 μg, or 500 μg of LNP-circRNA on days 0 and 21. RBD vaccine, or 100 μg of LNP-circRNA EV Control (empty circRNA vector without antigen encoding), or PBS control, 2.5-4 years old rhesus monkeys were immunized intramuscularly ( Figure 5A Total RBD-specific IgG binding antibodies and neutralizing antibodies were measured using plasma from rhesus macaques two weeks after the booster dose ( Figure 5A ). ELISA results showed that LNP-circRNA RBD The endpoint IgG titer of the vaccine reached ~5.8×10 4 (20 μg or 100 μg dose level) and ~5.5×10 4 (500 μg dose level), while from LNP-circRNA EV Plasma samples from rhesus macaques immunized with either PBS or immunized with WT or PBS had no detectable RBD-specific IgG binding antibodies ( Figure 5B ).

[0249] Next, the VSV-based SARS-CoV-2 pseudovirus and real SARS-CoV-2 neutralization assays were used to detect the expression of LNP-circRNA. RBD Virus entry inhibition ability of the plasma of vaccine-immunized rhesus monkeys. Pseudovirus neutralization assay showed that the average NT50 reached ~241 (20μg dose level), ~1656 (100μg dose level) and ~1532 (500μg dose level) ( Figure 5C ). The real SARS-CoV-2 neutralization test showed that the average NT50 values ​​reached ~156 (20μg dose level), ~244 (100μg dose level) and ~152 (500μg dose level) ( Figure 5D ). Both fake and real SARS-CoV-2 neutralization assays showed that the 100 μg dose level could elicit high levels of neutralizing antibodies, while a higher dose level (500 μg) did not further increase the levels of neutralizing antibodies ( Figure 5C-5D ).

[0250] Considering the circulating variants of SARS-CoV-2, cross-neutralization assays were performed using plasma samples from immunized rhesus macaques. Both sham and real SARS-CoV-2 neutralization assays showed that LNP-circRNA RBD Plasma from vaccinated rhesus macaques can effectively inhibit the corresponding local strains, while the α and delta variants can also be inhibited but have a slight escape compared with the local strains ( Figure 5D , 5E). The β variant can also be derived from LNP-circRNA RBD Plasma samples from vaccinated rhesus macaques neutralized the virus, but it partially escaped ( Figure 5D , 5E).

[0251] Detection of LNP-circRNA using peripheral blood mononuclear cells (PBMCs) collected one day before SARS-CoV-2 challenge RBD 、LNP-circRNA EV RBD-specific T cell responses elicited by LNP-circRNA or PBS in rhesus monkeys. RBD In vaccinated rhesus macaques, there was a clear IFN-γ and IL-2 response, but almost no IL-4 response ( Fig. 5F ), indicating that LNP-circRNA RBD The vaccine stimulated a Th1-biased T cell immune response in rhesus macaques.

[0252] Example 10: circRNA RBD Vaccine protects rhesus macaques from SARS-CoV-2 infection

[0253] As described above, 5 weeks after the booster dose, LNP-circRNA RBD Vaccine (20 μg, 100 μg or 500 μg), LNP-circRNA EV Rhesus monkeys immunized with 1×10 6 The challenged macaques were euthanized at 7 dpi in the days following the viral challenge, and lung tissues were collected and analyzed for viral load and histopathological analysis. Reverse transcription quantitative PCR (RT-qPCR) using primers targeting SARS-CoV-2 genomic RNA (N protein region) showed that the LNP-circRNA was significantly upregulated after treatment with 100 μg or 500 μg of LNP-circRNA. RBD Vaccinated rhesus macaques are highly protected against SARS-CoV-2 infection due to the interaction with LNP-circRNA EV Compared with the PBS control, the number of SARS-CoV-2 genomic RNA copies decreased by nearly 1000 times ( Figure 5G ). To detect the viral load of actively replicating SARS-CoV-2, qPCR was performed using primers targeting SARS-CoV-2 subgenomic RNA (E protein region). Quantitative PCR showed that all 20 μg, 100 μg, and 500 μg of LNP-circRNA RBD Immunized rhesus macaques had almost no detectable viral subgenomic RNA in lung tissue ( Figure 5G ).

[0254] In addition, further histopathological examination showed that LNP-circRNA RBD Rhesus macaques immunized with the (20 μg, 100 μg, or 500 μg) vaccine were well protected against pneumonia caused by SARS-CoV-2 infection because RBD Very mild pneumonia was observed in the immunized groups, especially in the 100 μg or 500 μg dose groups ( Figure 5H ). In contrast, in LNP-circRNA EV Severe pneumonia symptoms were observed in the lungs of the control group or PBS group, such as local septal thickening, moderate septal hemorrhage, a large number of scattered dust cells and a large number of inflammatory cell infiltration ( Figure 5H ). Pathological scores further showed that LNP-circRNA RBD Immunization can significantly protect rhesus macaques from pneumonia caused by SARA-CoV-2 infection ( Fig.5I ), which may be LNP-circRNA RBD Synergistic effect between humoral immune response and T cell immune response stimulated by vaccine ( Figure 5J ).

[0255] Example 11: circRNA vaccine did not cause obvious clinical disease symptoms in rhesus monkeys

[0256] In addition to the efficacy of circRNA vaccines in rhesus monkeys, the immunogenicity and safety of circRNA vaccines are also important for new types of vaccine platforms. To evaluate the immunogenicity and safety of circRNA vaccines in rhesus monkeys, some physiological and biochemical indicators were monitored, including adverse reactions, cytokines indicating innate immune activation, body weight, body temperature, and blood routine examination. No serious clinical adverse reactions were observed after primary and booster immunizations. ELISA results showed that circRNA RBD The vaccine induced high levels of IL-6 and MCP-1 ( Fig. 6A , 6B ), while TNF-α, IL-1β and IFN-α were almost undetectable ( Figure 6C-6E After primary and booster immunizations, the body temperature of immunized rhesus monkeys was continuously monitored for 3 days, which showed that circRNA RBD , circRNA EV The body temperatures of rhesus monkeys immunized with PBS were within the normal physiological range ( Fig. 6F In conclusion, our study provides preliminary evidence for the safety of the circRNA vaccine platform in non-human primates.

[0257] Example 12: circRNA vaccine induces reduced neutralizing antibodies against SARS-CoV-2 Omicron variant

[0258] Currently, the SARS-CoV-2 Omicron variant is spreading rapidly around the world and causing significant humoral immune escape in subjects vaccinated against local strains of SARS-CoV-2. Therefore, it is imperative to evaluate our vaccine, especially against the β and delta variants of the Omicron variant. As shown in the above results, group I ribozyme autocatalysis was used to generate circRNAs encoding the RBD antigens of local strains and 2 variants (β and delta) of SARS-CoV-2. RBD To evaluate the efficacy of these vaccines against the Omicron variant, 10 μg LNP-circRNA RBD / RBD-β / RBD-德尔塔 Serum samples from mice immunized with the vaccine were used to detect neutralizing antibodies against the Omicron variant by pseudovirus neutralization assay. RBD / RBD-β / RBD-德尔塔 - Serum from vaccinated mice can neutralize SARS-CoV-2 Omicron pseudoviruses, but both strongly escape ( Figure 7 ). For NT50 determined by pseudovirus neutralization assay, circRNA RBD The serum of the group had a ~37.0-fold decrease, and circRNA RBD-β The serum of the group had a ~23.8-fold decrease ( Figure 7 ). RBD-德尔塔 The neutralizing activity of serum was better preserved (~13.4-fold reduction) ( Figure 7 ).

[0259] Example 13: circRNA RBD-奥密克戎 Vaccine induces SARS-CoV-2 Omicron variant-specific antibodies

[0260] circRNA RBD-奥密克戎 Encapsulation with LNP to generate LNP-circRNA RBD-德尔塔 Vaccine. Use 5μg or 10μg of LNP-circRNA RBD-奥密克戎 The mice were immunized with the vaccine intramuscularly twice, 1 week apart, while empty LNP was used as a placebo control. Four days after the booster dose, serum samples from immunized mice were collected to detect RBD-specific antibodies. RBD-奥密克戎 The vaccine induced Omicron variant-specific antibodies with endpoint titers of 300 (5 μg dose) and 900 (10 μg dose) ( Fig. 8A ). In addition, 1 dose of 30 μg of LNP-circRNA was used RBD-奥密克戎 Mice were immunized intramuscularly with the vaccine ( Figure 8B), while empty LNP was used as a placebo control. Serum samples from immunized mice were collected 11 days after the booster dose for detection of RBD-specific antibodies. RBD-奥密克戎 The vaccine can induce Omicron variant-specific antibodies with an endpoint titer of 500 ( Figures 8A-8B ).

[0261] Example 14: circRNA RBD-德尔塔 Vaccine stimulates high levels of neutralizing antibodies against Omicron variant

[0262] Recently, the SARS-CoV-2 Omicron variant has spread rapidly, which contains more than 30 mutations in the spike region and escapes most previously reported neutralizing antibodies and sera from vaccine recipients who received two doses of the original SARS-CoV-2 vaccine. In response to the Omicron emergency, the above circRNAs were first tested. RBD , circRNA RBD-β and circRNA RBD-德尔塔 Neutralization activity of vaccines against Omicron variants. Pseudovirus neutralization assays showed that sera from mice immunized with the three circRNA vaccines could neutralize Omicron variants, especially circRNA RBD-德尔塔 Vaccine, NT50 more than 2×10 4 ( Fig. 12A ). Omicron variant escape circRNA RBD The serum of mice immunized with the vaccine was the most serious, with a 37-fold decrease in neutralization capacity, while circRNA RBD-德尔塔 The neutralizing activity of the vaccine was better preserved, decreasing only 13.4-fold ( Fig. 12A ), suggesting that ongoing SARS-CoV-2 vaccines targeting the Delta variant may still effectively protect against infection with the Omicron variant.

[0263] In addition, circRNAs were compared RBD-德尔塔 Vaccines and 1mΨ-mRNA RBD-德尔塔 Efficacy of vaccines against Omicron variants. Short-term (2 weeks after booster) and long-lasting (7 weeks after booster) 10 μg circRNA RBD-德尔塔 The vaccines all stimulated more effective neutralizing antibodies against the Omicron variant, with higher NT50 values ​​and better retention of neutralizing activity ( Figures 12A-12B ). circRNA RBD-德尔塔 The NT50 value of neutralizing antibodies stimulated by the vaccine reached 2×10 4 , or 4×10 7 weeks after booster immunization 3 , while 1mΨ-mRNA RBD-德尔塔 The NT50 value of neutralizing antibodies stimulated by the vaccine reached 4×103 , or 9×10 7 weeks after booster immunization 2 ( Figure 5A-5B ). Similar to the above results ( Figures 12H-12K ), discovered circRNA RBD-德尔塔 The vaccine elicited a higher proportion of neutralizing antibodies against the Omicron variant than 1mΨ-mRNA at 2 weeks after booster immunization (short-term) and 7 weeks after booster immunization (persistent) RBD-德尔塔 vaccine( Figures 12C-12D , Figures 13A-13C ), indicating that circRNA vaccines have potential advantages against variable variants due to the high proportion of neutralizing antibodies.

[0264] Example 15: circRNA RBD-奥密克戎 Vaccine stimulates high levels of neutralizing antibodies against Omicron variant

[0265] In addition, new circRNA vaccines have been developed, called circRNA RBD-奥密克戎 Vaccine encoding the RBD antigen of the SARS-CoV-2 Omicron variant. Mice were treated with 5 μg or 10 μg of LNP-circRNA RBD-奥密克戎 The vaccine was administered intramuscularly twice, 2 weeks apart, while empty LNP was used as a placebo control. One week after the second dose, serum samples from immunized mice were collected for detection of Omicron RBD / spike-specific antibodies. RBD-奥密克戎 The vaccine can induce Omicron spike-specific antibodies, with an endpoint titer of ~6×10 4 The endpoint titer of the 10 μg dose was ∼2×10 5 ( Fig.12E ). From circRNA RBD-奥密克戎 The vaccinated mouse sera can effectively neutralize SARS-CoV-2 Omicron pseudovirus in a dose-dependent manner, and the NT50 of 5 μg dose can reach 3×10 3 , a dose of 10 μg can reach ~1×10 4 ( Fig.12F ), while those from circRNA RBD-奥密克戎 Sera from vaccinated mice showed litter neutralizing activity against the SARS-CoV-2 delta variant and the original SARS-CoV-2 strain ( Figure 12G ).

[0266] Example 16: The third booster dose of circRNARBD-delta vaccine significantly increased neutralizing antibodies against the Omicron variant

[0267] In addition, to verify whether an additional third booster dose of circRNA vaccine after the previous two doses of immunization can stimulate effective neutralizing antibodies against the Omicron variant. RBD-德尔塔 Vaccinated mice received circRNA 7 weeks after the second dose RBD-β , circRNA RBD-德尔塔 or circRNA RBD-奥密克戎 The third booster dose of the vaccine ( Fig.12H ). One week after the third booster immunization, neutralizing antibodies against the Omicron variant pseudovirus were detected ( Fig.12H ). The results showed that after 2 doses of circRNA RBD-德尔塔 After vaccination, the same circRNA RBD-德尔塔 The third booster dose of the vaccine significantly increased the neutralization capacity against Delat and Omicron variants by almost 5-10 times ( Figures 12I-12J ). However, circRNA RBD-德尔塔 or circRNA RBD-奥密克戎 The third booster dose of the vaccine failed to improve the neutralization ability against the Omicron variant, circRNA RBD-奥密克戎 The third booster dose of the vaccine even sharply reduced its effectiveness against the Omicron variant ( Figures 12I-12J ).

[0268] In addition, 2 doses of circRNA RBD Vaccinated mice received circRNA 8 weeks after the second dose RBD or circRNA RBD-德尔塔 The third booster dose of the vaccine ( Figure 12K Neutralizing antibodies to the Omicron variant pseudovirus were also detected one week after the third booster ( Figure 12K ). The results showed that the same circRNA RBD Vaccine or circRNA RBD-德尔塔 The third booster dose of the vaccine greatly enhanced the neutralization ability. The results showed that the same circRNA RBD Vaccine or circRNA RBD-德尔塔 The third booster dose of the vaccine greatly improved the neutralization ability against both Delta and Omicron variants, especially circRNA RBD-德尔塔 The third booster dose of the vaccine has an NT50 value of 6×10 2 Increased to 3.8×10 4 ( Figure 12L-12M ).

[0269] In general, the above results indicate that not only two doses of the new circRNARBD-Omicron vaccine are a strategy to deal with the emerging SARS-CoV-2 Omicron variant, but also the circRNA after two doses of immunization RBD-德尔塔 A 3rd booster dose of the vaccine could be an effective solution to the current prevalence of the SARS-CoV-2 Delta and Omicron variants.

[0270] Example 17: circRNA vaccine

[0271] COVID-19 remains a rapidly growing global health crisis, with circulating SAS-CoV-2 variants escaping antibodies elicited by current vaccines 53,65,66 Examples 1-13 demonstrate the effectiveness of using circRNA to produce SARS-CoV-2 vaccines.

[0272] Several studies have reported that full-length spike proteins (mRNA-1273 and BNT162b2) 21,22,27 Or RBD-based mRNA vaccines stimulate neutralizing antibodies and cellular immune responses 23-26,67 As reported, most of the effective neutralizing antibodies recognize the RBD region of the S protein 62,63,68-71 Targeting RBD may induce a smaller number of non-neutralizing antibodies 23-26,67 Given that the RBD trimer is similar to the trimerized extracellular domain of the full-length spike protein 48 , and with the monomeric counterpart 48 Compared with , circRNA has more advantages in binding hACE2, so circRNA was selected as the immunogen expressing RBD trimer. RBD trimer has functional ( Fig. 1I ), and indeed induced potent and sustained neutralizing antibodies against the real SARS-CoV-2 virus ( Figure 2A-2O and Figures 5A-5J ).

[0273] CircRNA RBD-501Y.V2 The immunized mice produced high titers of neutralizing antibodies. Given that the K417N-E484K-N501Y mutation in the RBD reduced its interaction with certain neutralizing antibodies 66,72 , prove that circRNA RBD or circRNA RBD-β Neutralizing antibodies produced by immunized mice have preferential neutralizing ability against their corresponding virus strains. Recent studies have shown that β variants do not show higher infectivity but have immune evasion ability. 53-58 , and various vaccines are reported to be less effective against SARS-CoV-2 variants 65,73-77 There have also been reports of vaccine breakthroughs against SARS-CoV-2 variants78 Therefore, the development and implementation of vaccines against emerging variants is urgent, and cricRNA vaccines are such platforms that can be quickly customized to specific variants. For example, a vaccine containing E484K, N501Y, and L452R mutations in RBD can be quickly developed through a circRNA platform to respond to potential outbreaks caused by SARS-CoV-2 variants.

[0274] This generalizable strategy for designing immunogens is highlighted in this disclosure. The coding sequences of circular RNAs can be rapidly adapted to address any emerging SARS-CoV-2 variants of concern, including alpha, beta, gamma, delta, and omicron. 66,85,86 In addition, circular RNA can be rapidly produced in large quantities in vitro, and they do not require any nucleotide modification, which is significantly different from classic mRNA vaccines. Since circular RNA has a covalently closed ring structure, it has inherent stability. 61 Our data suggest that circRNA RBD and encapsulated circRNA RBD-LNP The complex is highly thermally stable at 4°C for approximately 4 weeks ( Figure 4E ).

[0275] Example 18. Luciferase expression of naked circRNALuc in mice

[0276] Using a group I ribozyme autocatalytic strategy to generate circRNA encoding firefly luciferase (Luc) Luc Then the circRNA Luc Diluted in PBS. Mice were injected intramuscularly with PBS or 20 μg of naked circRNA Luc , while the untreated group served as a mock control. 24 hours after injection, mice were intraperitoneally injected with luciferin and transferred to the IVIS imaging system. Naked circRNA Luc The injection site showed local expression of firefly luciferase ( Figures 14A-14C ).

[0277] Example 19. circRNA vaccines containing naked circRNA induce SARS-CoV-2 RBD-specific antibodies in mice

[0278] First, a group I ribozyme autocatalytic strategy was used to generate circRNA RBD-β A vaccine comprising a naked circRNA encoding a beta variant RBD antigen RBD-Beβ , without LNP encapsulation. Then the circRNA RBD-β Dilute in PBS. Mice received 2 doses of 100 μg circRNA RBD-βThe vaccine was administered intramuscularly twice, 2 weeks apart, and a third dose of 250 μg circRNA was administered 3 weeks after the booster immunization. RBD-β The vaccine was used for immunization, while PBS was used as a placebo control. Two weeks after the third booster immunization, serum samples of immunized mice were collected to detect RBD-specific antibodies ( Fig.15A ). CircRNA RBD-β The vaccine can induce RBD-specific IgG binding antibodies in mice ( Fig. 15B ).

[0279] Considering that RBD-Delta is more immunogenic and LNP-encapsulated circRNA RBD-德尔塔 Vaccines vs LNP-encapsulated circRNA RBD-β The vaccine induced higher IgG titers in mice. Using a group I ribozyme autocatalytic strategy to generate circRNA RBD-德尔塔 Vaccine comprising naked circRNA encoding delta variant RBD antigen RBD-德尔塔 , without LNP encapsulation. Then the circRNA RBD-德尔塔 Dilute in PBS. Use 10 or 50 μg of circRNA RBD-德尔塔 The mice were immunized with the vaccine intramuscularly twice, 4 weeks apart, and PBS was used as a placebo control. Serum samples of the immunized mice were collected 25 days after the booster immunization to detect RBD-delta-specific antibodies ( Fig.16A ). CircRNA RBD-德尔塔 The vaccine induced RBD-delta-specific IgG binding antibodies in mice, and the 50 μg group produced higher IgG levels than the 10 μg group ( Fig. 16B ).

[0280] Example 20. Naked circRNA vaccine induces higher antibodies in mice than mRNA vaccine

[0281] A group I ribozyme autocatalytic strategy was used to generate naked circRNA encoding the delta variant of the RBD antigen RBD-德尔塔 Vaccines, without LNP encapsulation. For naked mRNA RBD-德尔塔 Vaccines were prepared with or without 1mΨ-modified UTP to generate modified or unmodified mRNA. The RNA vaccine was then diluted in PBS. Mice were immunized twice with 50 μg of naked RNA vaccine, 3 weeks apart. Seven days after booster immunization, serum samples from immunized mice were collected for detection of RBD-delta-specific antibodies, including total IgG, IgG1, IgG2a, and IgG2c ( Fig.17A ). Naked circRNA RBD-德尔塔 Vaccines can induce potent antibodies in mice, while naked modified or unmodified mRNARBD-德尔塔 Vaccines cannot Fig. 17B ). In addition, given that Ringer's solution had been used previously for naked mRNA vaccines, the differences between PBS and Ringer's solution as buffers for naked circRNA were compared. Using Ringer's solution, circRNA vaccines could induce higher IgG2a and IgG2c, indicating that the quality of the antibodies was higher ( Fig. 17C ).

[0282] Example 21. Optimization of circRNA vaccine encoding Omicron BA.1 RBD antigen

[0283] Previously, a group I ribozyme autocatalytic strategy was used to generate circRNA encoding the RBD antigen of the omikonen variant. RBD -奥密克戎 vaccine. It showed low efficacy against native or delta variants but still has some potential against the emerging Omicron and its sublineage variants. Here, some mutations were rationally introduced and the length of the RBD antigen was modified in an attempt to improve the performance of the Omicron BA.1 vaccine. In addition, the benefit of the trimerization domain foldon was tested. All antigens were expressed from circRNAs generated by a group I intron autocatalytic strategy.

[0284] Mice were immunized twice with 5 μg of LNP-circRNA vaccine, 2 weeks apart. Serum samples from immunized mice were collected 7 days after booster immunization for detection of neutralizing antibodies against multiple SARS-CoV-2 variants by pseudovirus neutralization assay. For the original Omicron BA.1 vaccine, it showed low efficacy against Omicron and some Omicron sublineages ( Fig.18A After optimization, all new vaccines showed good performance but were not able to effectively protect against the BA.4 / 5 variant ( Fig.18B -F).

[0285] Detailed antigen information for each vaccine is shown below:

[0286] RBD-BA.1: RBD(319-541)-Omicron BA.1

[0287] RBD-BA.1-mut1: RBD(319-541)-OmicronBA.1+S375A.

[0288] RBD-BA.1-mut2: RBD(319-537)-OmicronBA.1+S375A.

[0289] RBD-BA.1-mut3: RBD(319-537)-Omicron BA.1+S375A+L452R

[0290] Example 22. CircRNA RBD-BA.4 / 5 Vaccine induces potent neutralizing antibodies against Omicron and some variants of its sublineage

[0291] A group I ribozyme autocatalytic strategy was used to generate circRNA encoding the RBD antigen of Omicron BA.4 / 5 RBD -BA.4 / 5 Vaccine. Mice were immunized twice with 5 μg or 10 μg of LNP-circRNA vaccine, 3 weeks apart. Seven days after booster immunization, serum samples from immunized mice were collected for detection of neutralizing antibodies against multiple SARS-CoV-2 variants by pseudovirus neutralization assay. Sera from vaccinated mice effectively neutralized pseudoviruses in a dose-dependent manner and showed preferential neutralizing activity against BA.4 / 5 variants ( Fig.19A -B).

[0292] Example 23. CircRNA RBD-三聚体 Vaccine induces potent neutralizing antibodies against Omicron and some variants of its sublineage

[0293] To generate a broad-spectrum SARS-CoV-2 vaccine, a strategy of tandemly expressing multiple domains of different variants was used. For proof of concept, two trimeric vaccines encoding three domains of three variants were generated. A group I ribozyme autocatalytic strategy was used to generate circRNAs RBD-三聚体 Vaccine. Mice were immunized twice with 5 μg or 10 μg of LNP-circRNA vaccine, 2 weeks apart. Serum samples from immunized mice were collected 7 days after booster immunization for detection of neutralizing antibodies against multiple SARS-CoV-2 variants by pseudovirus neutralization assay. Serum from vaccinated mice effectively neutralized pseudoviruses and showed some broad-spectrum neutralizing activity ( Fig. 20A -D).

[0294] Detailed antigen information for each vaccine is shown below:

[0295] Trimer 1: RBD 德尔塔 -RBD BA.1 -RBD BA.4 / 5

[0296] Trimer 2: NTD BA.4 / 5 -RBD BA.4 / 5 -RBD 德尔塔

[0297] The above examples are provided to allow those of ordinary skill in the art to fully disclose and describe how to make and use the embodiments of the circRNA, vaccines, compositions and methods of the present application, and are not intended to limit the scope of what the inventors consider to be their inventions. Modifications to the above modes for practicing the present application that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification indicate the technical level of the artisans involved in the present application.

[0298] Many modifications and variations may be made thereto without departing from the spirit and scope of the present application, as will be apparent to those skilled in the art.The specific embodiments and examples described herein are provided by way of example only.

[0299] All headings and section names are used for clarity and reference purposes and are not to be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of appropriately combining various aspects from different headings and sections according to the spirit and scope of the present application as described herein.

[0300] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

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[0391] Exemplary sequence

[0392] SEQ ID NO:1 Full-length S protein sequence of SARS-CoV-2

[0393]

[0394] SEQ ID NO:2 RBD amino acid residues 319 to 542 of S protein

[0395] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0396] SEQ ID NO:3 C-terminal foldon domain of T4 fibrin domain

[0397] GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRS

[0398] SEQ ID NO:4 Leucine zipper domain based on GCN4

[0399] RMKQIEDKIEEILSKIYHIENEIARIKKLIGER

[0400] SEQ ID NO: 5 Exemplary peptide linker

[0401] GGGGSGGGGS

[0402] SEQ ID NO:6 Wild-type S2 region of SARS-CoV-2 S protein

[0403] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0404] SEQ ID NO:7 SARS-CoV-2 S protein K986P / V987P S2 region sequence

[0405] SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0406] SEQ ID NO:8 Wild-type amino acid residue 2 to 1273 sequence of the S protein of SARS-CoV-2

[0407]

[0408] SEQ ID NO:9 Amino acid residues 2 to 1273 of the S protein of SARS-CoV-2, Δ681-684

[0409]

[0410] SEQ ID NO:10 Amino acid residues 2 to 1273 of the S protein of SARS-CoV-2, K986P V987P Δ681-684 sequence

[0411]

[0412] SEQ ID NO: 11 Nucleic acid sequence of wild-type S2 region sequence

[0413]

[0414] SEQ ID NO: 12K986P / V987P S2 region sequence nucleic acid sequence

[0415]

[0416] SEQ ID NO: 13 corresponds to wild-type 2 to 1273 nucleic acid sequence

[0417]

[0418] SEQ ID NO:14 is a nucleotide sequence of the Δ681-684 sequence

[0419]

[0420] SEQ ID NO: 15 K986P / V987P Δ681-684 nucleic acid sequence

[0421]

[0422] SEQ ID NO: 16 Tissue plasminogen activator SP

[0423] DAMKRGLCCVLLLCGAVFVSPSQEIHARFRR

[0424] SEQ ID NO: 17 Human IgE immunoglobulin SP

[0425] DWTWILFLVAAATRVHS

[0426] SEQ ID NO:18 Amino acid sequence of SARS-CoV-2 delta variant RBD

[0427] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0428] SEQ ID NO:19 Amino acid sequence of SARS-CoV-2 Omicron variant RBD

[0429] RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDF TGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0430] SEQ ID NO:20 Nucleic acid sequence of SARS-CoV-2 delta variant RBD

[0431] CGCGTCCAGCCAACCGAGAGCATCGTCAGATTTCCCAACATTACAAATCTGTGTCCCTTCGGCGAGGTGTTCAACGCCACACGCTTCGCTTCAGTGTACGCATGGAACCGCAAGCGCATATCTAACTGCGTCGCGGATTATTCTGTCCTCTACAACTCCGCCTCTTTCTCCACCTTCAAGTGCTACGGAGTGTCACCGACTAAGCTGAACGATCTCTGCTTTACCAACGTCTACGCGGACTCCTTCGTGATAAGAGGTGATGAAGTGAGACAAATAGCCCCAGGTCAGACTGGTAAGATCGCAGATTACAACTACAAATTGCCTGATGATTTCACTGGTTGCGTTATCGCGTGGAACTCTAATAACCTCGATTCTAAGGTCGGTGGTAACTACAATTACaGGTACCGCTTGTTTAGGAAGTCAAACCTGAAGCCTTTCGAGAGGGATATTTCAACCGAAATCTATCAAGCGGGTTCAAagCCGTGTAACGGTGTGgAAGGATTTAACTGCTACTTCCCCCTGCAGTCTTACGGATTCCAGCCAACCAATGGCGTGGGTTACCAACCTTATCGCGTGGTGGTTCTGAGTTTCGAACTGTTGCACGCTCCCGCCACGGTATGCGGTCCCAAGAAGAGCACTAACTTGGTGAAGAATAAGTGCGTGAATTTC

[0432] SEQ ID NO:21 Nucleic acid sequence of the RBD of the SARS-CoV-2 Omicron variant

[0433] CGCGTCCAGCCAACCGAGAGCATCGTCAGATTTCCCAACATTACAAATCTGTGTCCCTTCGaCGAGGTGTTCAACGCCACACGCTTCGCTTCAGTGTACGCATGGAACCGCAAGCGCATATCTAACTGCGTCGCGGATTATTCTGTCCTCTACAACctCGCCcCTTTCTtCACCTTCAAGTGCTACGGAGTGTCACCGACTAAGCTGAACGATCTCTGCTTTACCAACGTCTACGCGGACTCCTTCGTGATAAGAGGTGATGAAGTGAGACAAATAGCCCCAGGTCAGACTGGTAAcATCGCAGATTACAACTACAAATTGCCTGATGATTTCACTGGTTGCGTTATCGCGTGGAACTCTAATAAgCTCGATTCTAAGGTCaGTGGTAACTACAATTACCTGTACCGCTTGTTTAGGAAGTCAAACCTGAAGCCTTTCGAGAGGGATATTTCAACCGAAATCTATCAAGCGGGTaacAagCCGTGTAACGGTGTGgcAGGATTTAACTGCTACTTCCCCCTGCgGTCTTACaGcTTCCgGCCAACCtATGGCGTGGGTcACCAACCTTATCGCGTGGTGGTTCTGAGTTTCGAACTGTTGCACGCTCCCGCCACGGTATGCGGTCCCAAGAAGAGCACTAACTTGGTGAAGAATAAGTGCGTGAATTTC

[0434] SEQ ID NO:22 Kozak nucleic acid sequence

[0435] GCCACCAUG

[0436] SEQ ID NO:23 polyAC sequence

[0437] GAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACA

[0438] SEQ ID NO:24 m6A modification sequence

[0439] ACGAGTCCTGGACTGAAACGGACTTGT

[0440] SEQ ID NO:25 3' exon sequence recognized by 3' catalytic group I intron fragment

[0441] AAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCAC

[0442] SEQ ID NO:26 5' exon sequence recognized by 5' catalytic group I intron fragment

[0443] GAGACGCUACGGACUU

[0444] SEQ ID NO: 27 Exemplary 5' homologous sequence

[0445] GGGAGACCCUCGACCGUCGAUUGUCCACUGGUC

[0446] SEQ ID NO:28 Exemplary 3' homologous sequence

[0447] ACCAGUGGACAAUCGACGGAUAACAGCAUAUCUAG

[0448] SEQ ID NO: 29T7 promoter

[0449] UAAUACGACUCACUAUAGG

[0450] SEQ ID NO:30 T2A peptide coding sequence

[0451] GAGGGCAGAGGAAGUCUUCUAACAUGCGGUGACGUGGAGGAGAAUCCCGGCCCU

[0452] SEQ ID NO:31P2A peptide coding sequence

[0453] GCUACUAACUUCAGCCUGCUGAAGCAGGCUGGAGACGUGGAGGAGAACCCUGGACCU

[0454] SEQ ID NO:32 Catalytic group I intron fragment

[0455] AACAAUAGAUGACUUACAACUAAUCGGAAGGUGCAGAGACUCGACGGGAGCUACCCUAACGUCAAGACGAGGGUAAAGAGAGAGUCCAAUUCUCAAAGCCAAUAGGCAGUAGCGAAAGCUGCAAGAGAAUG

[0456] SEQ ID NO:33 5' catalytic group I intron fragment

[0457] AAAUAAUUGAGCCUUAAAGAAGAAAUUCUUUAAGUGGAUGCUCUCAAACUCAGGGAAACCUAAAUCUAGUUAUAGACAAGGCAAUCCUGAGCCAAGCCGAAGUAGUAAUUAGUAAG

[0458] SEQ ID NO:34 Nucleotide sequence of the full-length S protein sequence of SARS-CoV-2

[0459]

[0460] SEQ ID NO:35 Nucleotide sequence of RBD amino acid residues 319 to 542 of S protein

[0461] CGCGTCCAGCCAACCGAGAGCATCGTCAGATTTCCCAACATTACAAATCTGTGTCCCTTCGGCGAGGTGTTCAACGCCACACGCTTCGCTTCAGTGTACGCATGGAACCGCAAGCGCATATCTAACTGCGTCGCGGATTATTCTGTCCTCTACAACTCCGCCTCTTT CTCCACCTTCAAGTGCTACGGAGTGTCACCGACTAAGCTGAACGATCTCTGCTTTACCAACGTCTACGCGGACTCCTTCGTGATAAGAGGTGATGAAGTGAGACAAATAGCCCCAGGTCAGACTGGTAAGATCGCAGATTACAACTACAAATTGCCTGATGATTTCA CTGGTTGCGTTATCGCGTGGAACTCTAATAACCTCGATTCTAAGGTCGGTGGTAACTACAATTACCTGTACCGCTTGTTTAGGAAGTCAAACCTGAAGCCTTTCGAGAGGGATATTTCAACCGAAATCTATCAAGCGGGTTCAACACCGTGTAACGGTGTGGAAGGA TTTAACTGCTACTTCCCCCTGCAGTCTTACGGATTCCAGCCAACCAATGGCGTGGGTTACCAACCTTATCGCGTGGTGGTTCTGAGTTTCGAACTGTTGCACGCTCCCGCCACGGTATGCGGTCCCAAGAAGAGCACTAACTTGGTGAAGAATAAGTGCGTGAATTTC

[0462] SEQ ID NO:36 Nucleic acid sequence of the C-terminal foldon domain of T4 fibrin

[0463] GGAAGCGGCTACATCCCAGAAGCCCCTAGAGACGGACAGGCTTACGTGCGAAAAGACGGCGAGTGGGTGCTGCTGAGCACATTCCTGGGAAGGAGC

[0464] SEQ ID NO:37 Nucleic acid sequence based on the isoleucine zipper domain of GCN4

[0465] CGAATGAAGCAGATTGAGGATAAAATTGAGGAGATTCTCAGCAAAATTTACCACATAGAAATGAGATCGCTCGGATTAAAAACTGATCGGAGAAAGA

[0466] SEQ ID NO:38 Nucleic acid sequence of GS peptide linker

[0467] GGCGGAGGAGGCAGCGGCGGAGGAGGCAGC

[0468] SEQ ID NO:39CVB3 virus IRES

[0469] TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAA

[0470] SEQ ID NO:40 Amino acid residue sequence of the S protein of SARS-CoV-2 from 2 to 1273, K986P V987P

[0471]

[0472] SEQ ID NO:41SARS-CoV-2 strain B.1.351 Amino acid sequence of RBD RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKL PDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

[0473] SEQ ID NO:42 Nucleic acid sequence encoding SARS-CoV-2 strain B.1.351 RBD

[0474] CGCGTCCAGCCAACCGAGAGCATCGTCAGATTTCCCAACATTACAAATCTGTGTCCCTTCGGCGAGGTGTTCAACGCCACACGCTTCGCTTCAGTGTACGCATGGAACCGCAAGCGCATATCTAACTGCGTCGCGGATTATTCTGTCCTCTACAACTCCGCCTCTTTCTCCACCTTCAAGTGCTACGGAGTGTCACCGACTAAGCTGAACGATCTCTGCTTTACCAACGTCTACGCGGACTCCTTCGTGATAAGAGGTGATGAAGTGAGACAAATAGCCCCAGGTCAGACTGGTAACATCGCAGATTACAACTACAAATTGCCTGATGATTTCACTGGTTGCGTTATCGCGTGGAACTCTAATAACCTCGATTCTAAGGTCGGTGGTAACTACAATTACCTGTACCGCTTGTTTAGGAAGTCAAACCTGAAGCCTTTCGAGAGGGATATTTCAACCGAAATCTATCAAGCGGGTTCAACACCGTGTAACGGTGTGAAAGGATTTAACTGCTACTTCCCCCTGCAGTCTTACGGATTCCAGCCAACCTATGGCGTGGGTTACCAACCTTATCGCGTGGTGGTTCTGAGTTTCGAACTGTTGCACGCTCCCGCCACGGTATGCGGTCCCAAGAAGAGCACTAACTTGGTGAAGAATAAGTGCGTGAATTTC

Claims

1. A circular RNA (circRNA) comprising a nucleic acid sequence encoding an antigenic polypeptide of a SARS-CoV-2 variant, wherein the SARS-CoV-2 variant is a delta variant or an omicron variant.

2. The circRNA of claim 1, wherein the antigenic polypeptide comprises a spike (S) protein or a fragment thereof of the SARS-CoV-2 variant.

3. The circRNA of claim 2, wherein the antigen polypeptide comprises the receptor binding domain (RBD) of the S protein.

4. A circRNA according to claim 3, wherein the RBD comprises amino acid residues 319 to 542 of the full-length S protein of SARS-CoV-2, wherein the numbering is based on SEQ ID NO:

1.

5. The circRNA of claim 3 or 4, wherein the SARS-CoV-2 variant is a delta variant, and wherein the antigenic polypeptide comprises the amino acid sequence of SEQ ID NO:

18.

6. The circRNA of claim 3 or 4, wherein the SARS-CoV-2 variant is an Omicron variant, and wherein the antigenic polypeptide comprises the amino acid sequence of SEQ ID NO:

19.

7. The circRNA according to any one of claims 1-6, wherein the antigen polypeptide further comprises a multimerization domain.

8. The circRNA of claim 7, wherein the multimerization domain is the C-terminal foldon (Fd) domain of T4 fiber protein, which mediates the trimerization of T4 fiber protein.

9. The circRNA according to claim 8, wherein the Fd domain comprises the amino acid sequence of SEQ ID NO:

3.

10. The circRNA according to any one of claims 1 to 9, further comprising a Kozak sequence operably linked to the nucleic acid sequence encoding the antigen polypeptide.

11. The circRNA according to any one of claims 1 to 10, further comprising an in-frame 2A peptide coding sequence operably linked to the 3' end of the nucleic acid sequence encoding the antigen polypeptide.

12. The circRNA according to any one of claims 1-11, further comprising an internal ribosome entry site (IRES) sequence operably linked to the nucleic acid sequence encoding the antigen polypeptide.

13. The circRNA according to claim 12, wherein the IRES sequence is an IRES sequence selected from the group consisting of IRES sequences of CVB3 virus, EV71 virus, EMCV virus, PV virus, and CSFV virus. 14 . The circRNA according to claim 13 , comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises, from the 5′ end to the 3′ end: an IRES sequence, a Kozak sequence and a nucleic acid sequence encoding the antigen polypeptide.

15. The circRNA according to any one of claims 12 to 14, further comprising a polyAC or polyA sequence located at the 5' end of the IRES sequence.

16. The circRNA according to any one of claims 1-11, further comprising an m6A modification motif sequence operably linked to the nucleic acid sequence encoding the antigen polypeptide.

17. The circRNA according to claim 16, comprising a nucleic acid sequence, wherein the nucleic acid sequence comprises, from the 5' end to the 3' end: an m6A modification motif sequence, a Kozak sequence and a nucleic acid sequence encoding the antigen polypeptide.

18. The circRNA according to any one of claims 1 to 17, further comprising a 3' exon sequence that can be recognized by the 3' catalytic group I intron fragment flanking the 5' end of the nucleic acid sequence encoding the antigen polypeptide, and a 5' exon sequence that can be recognized by the 5' catalytic group I intron fragment flanking the 3' end of the nucleic acid sequence encoding the antigen polypeptide.

19. A composition comprising a plurality of circRNAs according to any one of claims 1 to 18, wherein the antigenic polypeptides corresponding to the plurality of circRNAs are different from each other.

20. The composition of claim 19, wherein the plurality of circRNAs target a plurality of SARS-CoV-2 variants.

21. A circRNA vaccine comprising the circRNA of any one of claims 1-18 or the composition of claim 19 or 20.

22. A pharmaceutical composition comprising the circRNA according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

23. The circRNA vaccine according to claim 21 or the pharmaceutical composition according to claim 22, further comprising a transfection agent.

24. The circRNA vaccine or pharmaceutical composition according to claim 23, wherein the transfection agent is polyethyleneimine (PEI) or lipid nanoparticles (LNP).

25. The circRNA vaccine of claim 21 or the pharmaceutical composition of claim 22, wherein the circRNA is not formulated with a transfection agent.

26. A method for treating or preventing SARS-CoV-2 infection in an individual, comprising administering to the individual an effective amount of the circRNA of any one of claims 1-18, the composition of any one of claims 19-20, the pharmaceutical composition of any one of claims 22-25, or the circRNA vaccine of any one of claims 21 and 23-25.

27. The method of claim 26, wherein the SARS-CoV-2 infection is caused by a SARS-CoV-2 variant.

28. The method of claim 27, wherein the SARS-CoV-2 infection is caused by the Delta or Omicron variants of SARS-CoV-2.

29. The method according to any one of claims 26-28, wherein the circRNA undergoes rolling circle translation by ribosomes in the individual.

30. The method according to any one of claims 26-29, wherein the circRNA is administered in the form of naked circRNA.

31. The method according to any one of claims 26-30, wherein the circRNA is administered two or more times, and the interval between each administration is more than two weeks.

32. A linear RNA capable of forming a circRNA according to any one of claims 1-18.

33. A nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA of claim 32, optionally comprising a T7 promoter operably linked to the nucleic acid sequence.

Images