Messenger RNA vaccines against wide spectrum of coronavirus variants

Abstract

To provide better vaccines as well as better products and methods to prevent and treat coronavirus infections.SOLUTION: The present invention relates to the mRNA vaccine of coronavirus spike protein with deletion of glycosites in the receptor binding domain (RBD), the subunit 1 (S1) domain, or the subunit 2 (S2) domain, or a combination thereof. The vaccine elicits broadly protective immune responses to coronavirus and variants thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on April 12, 2022 is titled "G4590-15000PCT_SeqListing_20220412" and is 111 kilobytes in size.

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 173,752, filed April 12, 2021, and U.S. Provisional Patent Application No. 63 / 264,737, filed December 1, 2021, the contents of which are incorporated by reference in their entireties herein.

[0003] Field The present disclosure relates generally to the field of treatment and / or prevention of coronavirus infections. In particular, the present disclosure relates to messenger RNA (mRNA) vaccines against a wide range of coronavirus (CoV) variants. [Background technology]

[0004] In 1796, Edward Jenner created the world's first vaccine (cowpox) to protect against smallpox, successfully saving millions of people. Since then, vaccination has been recognized as the best way to protect against pathogens. Since the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019, which caused coronavirus-induced disease 2019 (COVID-19), the virus has spread worldwide, causing more than 200 million infections and more than 4 million deaths in 20 months. This pandemic has become a major threat to public health.

[0005] Many efforts have been directed towards the development of effective tools and vaccines to combat this pandemic. The trimeric spike (S) protein on the viral surface is the key immunogen and has been the target for the development of prophylactic vaccines and therapeutic antibodies. As of December 2020, the US FDA has authorized the mRNA vaccine candidates from Pfizer / BioNTech and Moderna, as well as Regeneron's antibody, for emergency use; however, several other vaccine candidates and human antibodies are in clinical trials, and a few of them are on the verge of approval, including those from Oxford / Astrazeneca and J&J. Among the various vaccines developed to control the spread of SARS-CoV-2 and its variants, the mRNA vaccines developed by Moderna and BioNTech / Pfizer have become the main breakthrough due to their rapidity and convenience. These vaccines are stabilized using novel mRNA technology and lipid nanoparticle (LNP) formulations for in vivo delivery and translation into the spike (S) protein to induce an immune response (Ewen Callaway. COVID vaccine excitement builds as Moderna reports third positive result. Nature. 587(7834):337-338 (2020); Polack P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 383(27):2603-2615 (2020)).

[0006] However, infections with emerging SARS-CoV-2 variants may continue to occur or become more frequent, due to the large number of variants that have circulated widely around the world and the potential increased ability of more infectious variants, such as the beta, delta and omicron variants, to reinfect previously vaccinated humans and / or those who have recovered from infection with earlier versions of the coronavirus. The S protein of this RNA virus is highly glycosylated and frequently mutates, with over 9 million sequences and over 1,000 mutation sites in its 1,273 amino acid sequence reported in GISAID (www.gisaid.org), including the highly infectious delta and omicron variants, thus posing a major challenge to developing broadly effective antibodies and vaccines. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ewen Callaway, “COVID vaccine excitement builds as Moderna reports third positive result.”, Nature., 2020, 587(7834), p.337~338 [Non-Patent Document 2] Polack P. et al., “Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine.”, N Engl J Med., 2020, 383(27), p. 2603~2615 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is an urgent need for better vaccines, as well as better products and methods for preventing and treating coronavirus infections. [Means for solving the problem]

[0009] Summary of the Invention The present disclosure provides a novel coronavirus mRNA vaccine, its preparation method and use. The novel vaccine is designed based on mRNA technology, which removes the glycan shield of the coronavirus (e.g., SARS-CoV-2) spike protein to more fully expose the conserved regions of the spike protein. The coronavirus spike protein mRNA vaccine has a deletion of glycosites in the receptor binding domain (RBD) or subunit 2 (S2) domain to expose highly conserved epitopes and elicit antibody and CD8 T cell responses with broader protection against alpha, beta, gamma, delta, omicron and various variants compared to unmodified mRNA. The mRNA vaccine provided herein is effective in inducing protective immunity against SARS-CoV-2 and variants (e.g., alpha, beta, gamma, delta, omicron). When used individually or in combination as an immunogenic composition or vaccine, the mRNA vaccine of the present disclosure can protect people from infection and / or protect them to reduce symptoms if infected.

[0010] In one aspect, the disclosure provides at least one immunogenic peptide, wherein the immunogenic peptide comprises an amino acid sequence selected from the group consisting of TESIVRFPNITNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), GPKKSTNLVKNKC (SEQ ID NO: 47), CDVVIGIVNNTVY (SEQ ID NO: 48), PELDSFKEELDKYFK[N]HTS (SEQ ID NO: 49), VNIQKEIDRLNEVA (SEQ ID NO: 50), NLNESLIDLQ (SEQ ID NO: 51), and LGKYEQYIKWP (SEQ ID NO: 52), or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, or 90% identity to any of SEQ ID NOs: 41-52.

[0011] In some embodiments, the immunogenic peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 41-43 and 45-51. In some embodiments, the immunogenic peptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids of SEQ ID NOs: 41-52. In some embodiments, the immunogenic peptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NOs: 41-43 and 45-51.

[0012] In one aspect, the disclosure provides modified nucleic acid molecules encoding modified spike proteins comprising one or more amino acid substitutions in an N-linked glycosylation sequon (NXS / T), where X is any amino acid residue except proline and S / T represents a serine or threonine residue.

[0013] In some embodiments, the modified spike proteins described herein comprise an asparagine (N) to glutamine (Q) substitution at the N-linked glycosylation sequon (NXS / T) to remove the N-linked glycan sequon.

[0014] In some embodiments, the modified spike proteins described herein comprise one or more amino acid substitutions in the N-linked glycosylation sequon (NXS / T) to remove the N-linked glycan sequon.

[0015] In some embodiments, the modified spike proteins described herein comprise one or more amino acid substitutions of S / T at the O-linked glycosylation site to eliminate the O-linked glycosylation site, an example being a substitution of S / T with alanine (A).

[0016] In one embodiment, the modified nucleic acid molecule is mRNA or is double- or single-stranded DNA.

[0017] In one embodiment, the modified spike protein is derived from a SARS-CoV-2 spike protein. The SARS-CoV-2 spike protein described herein comprises an amino acid sequence of SEQ ID NO:2, 16, 18 or 20, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to an amino acid sequence of SEQ ID NO:2, 16, 18 or 20.

[0018] In some embodiments, the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, 16, 18 or 20 is an mRNA comprising a nucleotide sequence of SEQ ID NO:1, 15, 17 or 19, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:1, 15, 17 or 19, respectively.

[0019] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 4, 22, 24 or 26, wherein the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site. The modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 4, 22, 24 or 26 comprises the nucleotide sequence of SEQ ID NO: 3, 21, 23 or 25, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 3, 21, 23 or 25, respectively.

[0020] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 6, 28, 30 or 32, wherein the modified spike protein comprises an S2 subunit lacking a glycosylation site. The modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 6, 28, 30 or 32 comprises the nucleotide sequence of SEQ ID NO: 5, 27, 29 or 31, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 5, 27, 29 or 31, respectively.

[0021] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 8 or 34, wherein the modified spike protein comprises an S2 subunit that consists of a single glycosylation site. In some embodiments, the single glycosylation site is at position N1194. In some embodiments, the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 8 or 34 comprises the nucleotide sequence of SEQ ID NO: 7 or 33, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 33, respectively.

[0022] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 10 or 36, wherein the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site and an amino acid substitution of N801 to Q801. The modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 10 or 36 comprises the nucleotide sequence of SEQ ID NO: 9 or 35, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 9 or 35, respectively.

[0023] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 12 or 38, wherein the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site and an amino acid substitution of N1194 with Q1194. The modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 12 or 38 comprises the nucleotide sequence of SEQ ID NO: 11 or 37, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 37, respectively.

[0024] In some embodiments, the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 14 or 40, wherein the modified spike protein comprises a modified receptor binding domain (RBD) lacking a glycosylation site and amino acid substitutions of N122 to Q122, N165 to Q165 and N234 to Q234. The modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 14 or 40 comprises the nucleotide sequence of SEQ ID NO: 13 or 39, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 13 or 39, respectively.

[0025] In some embodiments, the modified spike proteins described herein comprise an S1 subunit that lacks a glycosylation site.

[0026] In some embodiments, the modified spike proteins described herein comprise both an S1 subunit and an S2 subunit that lack glycosylation sites.

[0027] The present invention relates to an mRNA vaccine of coronavirus spike protein with deletions of glycosites in the receptor binding domain (RBD) or subunit 2 (S2) domain to expose highly conserved epitopes and elicit antibody and CD8 T cell responses with broader spectrum protection against alpha, beta, gamma, delta, omicron and various variants compared to unmodified mRNA.

[0028] In some embodiments, the coronavirus vaccine comprises a coronavirus spike protein mRNA with one or more mutations in a glycosite in the RBD domain or S2 domain or other domain with one or more exchanges or combinations of N to Q or S / T to A. In further embodiments, the mutation in the N-glycosite is a change of the putative sequon NXS / T to QXS / T and / or a change of S / T to A in the O-glycosite.

[0029] In some embodiments, mRNAs described herein having glycosites with N to Q exchanges include S-(deg-RBD) (S protein with two N-glycosites in RBD mutated N to Q and two O-glycosites mutated S / T to A, all), S-(deg-S2) (S protein with nine glycosites in S2 mutated N to Q, all), S-(deg-S2-1194) (S protein with eight glycosites in S2 mutated N to Q, except glycosite 1194), S-(deg-RBD-801) (S protein with two N-glycosites in RBD mutated N to Q and two O-glycosites mutated S / T to A ...S2-1194) (S protein with two N-glycosites in RBD mutated N to Q and two O-glycosites mutated S / T to A, all), S-(deg-S2-1194) (S protein with two N-glycosites in S2 mutated N to Q, all), S-(deg These include S-(deg-RBD-1194) (S protein with two N-glycosites in the RBD mutated N to Q and two O-glycosites mutated S / T to A and glycosite 1194 mutated N to Q, all in one), and S-(deg-RBD-122-165-234) (S protein with two N-glycosites in the RBD mutated N to Q and two O-glycosites mutated S / T to A and glycosites 122, 165, and 234 mutated N to Q, all in one).

[0030] In one embodiment, immunization with an exemplary coronavirus vaccine of the present disclosure results in the accumulation of misfolded S protein in the endoplasmic reticulum, as described herein. In one embodiment, immunization with an exemplary coronavirus vaccine of the present disclosure results in the upregulation of BiP / GRP78, XBP1 and p-eIF2α, resulting in cell apoptosis and CD8 + Induce a T cell response. In one embodiment, immunization with a coronavirus vaccine of the present disclosure can enhance expression of class I major histocompatibility complex (MHC I), as described herein.

[0031] In some embodiments, exemplary CoVs described herein include, but are not limited to, SARS-CoV, MERS-CoV, and SARS-CoV-2. In some embodiments, exemplary coronaviruses (CoVs) described herein include, but are not limited to, alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, and omicron-SARS-CoV2 and variants thereof.

[0032] In some embodiments, the disclosure provides a linear DNA comprising a promoter, a 5' untranslated region, a 3' untranslated region, an expression plasmid with or without S-2P, and a poly(A) tail signal sequence, where the putative sequon NXS / T has been changed to QXS / T, and the O-glycosite has been changed from S / T to A on the expression plasmid.

[0033] In some embodiments, the S-2P expression plasmid comprises the S gene of SARS-CoV-2 encoding the pre-fusion form of S with proline substitutions at K968 and V969.

[0034] In some embodiments, the disclosure provides mRNA prepared by in vitro translation from the above-mentioned DNA.

[0035] In another aspect, the disclosure provides a vector comprising the modified nucleic acid molecule described above.

[0036] In another aspect, the disclosure provides a host cell comprising the modified nucleic acid molecule described above.

[0037] In another aspect, the disclosure provides a modified spike protein as described above.

[0038] In another aspect, the disclosure provides a method for delivering mRNA for in vivo production of a protein, the delivery method comprising: administering to a subject a composition comprising an mRNA of the invention encoding a protein, wherein the mRNA is encapsulated within a lipid nanoparticle, and wherein administering the composition results in expression of the protein encoded by the mRNA.

[0039] In some embodiments, the mRNA described herein can be used as a vaccine, either alone or in combination with other vaccines. Thus, the present disclosure provides a combo vaccine comprising the mRNA vaccine of the present disclosure and one or more additional vaccines. The additional vaccines are selected from one or more of COVID-19 vaccines, influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines, and any combination thereof.

[0040] In another aspect, the present disclosure provides a guanidine-based nanoparticle for use as a carrier for delivering to a subject a modified nucleic acid molecule according to any one of claims 1 to 25. In one embodiment, the nanoparticle is a liposome or a polymersome.

[0041] In another aspect, the present disclosure provides an mRNA nanocluster comprising an mRNA vaccine as described herein formulated in a lipid nanoparticle. In one embodiment, the lipid nanoparticle is a biodegradable lipid nanoparticle.

[0042] In some embodiments, the lipid nanoparticles described herein are guanidine-based polymers.In one embodiment, the present disclosure provides mRNA nanoclusters, comprising lipid nanoparticles encapsulating mRNA vaccines described herein, wherein the lipid nanoparticles comprise guanidine-based polymer units, and the guanidine base and zwitterionic groups of the polymer are attached to the lipid tail of the polymer, and the guanidine-based polymer is attached to the mRNA, thereby forming a salt bridge between the guanidinium group and the phosphate in the mRNA.Examples of guanidine-based polymers include, but are not limited to, P1, P2, P3, Pb and Pz, as shown below.

[0043] [ka] (Wherein, R is

[0044] [ka] It is.)

[0045] In some embodiments, the guanidine-based polymers form copolymers, such as P1 / P3 copolymers, P2 / P3 copolymers, P1 / Pb copolymers, P2 / Pb copolymers, P1 / Pz copolymers, and P2 / Pz copolymers.

[0046] In some embodiments, the guanidine-based and zwitterionic lipid nanoparticles are selected from the group consisting of P1 and / or P2 and Pz

[0047] [ka] (In the formula, R is

[0048] [ka] It is.) Contains a mixture of.

[0049] In some embodiments, the mRNA nanoclusters described herein have a nanoparticle / mRNA (N / P) ratio of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 30, about 40, about 50 or about 100.

[0050] In some embodiments, the mRNA nanoclusters described herein have a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

[0051] In some embodiments, the present disclosure is directed to nanoparticle / nanocluster compositions comprising nanoparticles having attached thereto a coronavirus vaccine of the present disclosure. In one embodiment, the nanoparticles are lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles such as gold nanoparticles, liposomes, immunostimulating complexes, virus-like particles, or self-assembling proteins. In further embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0052] In some embodiments, the present disclosure provides a vaccine composition comprising an mRNA vaccine, mRNA nanocluster / nanocluster or nanoparticle composition described herein.

[0053] In some embodiments, the disclosure is directed to antibodies and CD8+ T cells elicited by the vaccines described herein that have broader protection against alpha, beta, gamma, delta and omicron variants.

[0054] In some embodiments, the disclosure provides a method of immunizing a subject, the method comprising administering a vaccine composition described herein. The disclosure also provides a method of preventing or treating a coronavirus infection, the method comprising administering an effective amount of an mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition described herein to a subject infected with or at risk of infection with a coronavirus. In one embodiment, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition described herein may be used in a method of boosting an adaptive immune response.

[0055] In some embodiments, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition described herein is administered at an initial dose and two, three or four booster doses. In some embodiments, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition is administered at an initial dose and at least one booster dose about 1 month, about 2 months, about 3 months, about 4 months, about 5 months or about 6 months after the initial dose. In some embodiments, the provided composition is administered at a second booster dose about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 1 year after the initial dose.

[0056] In some embodiments, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition is administered in a single dose or in more than one dose. In one embodiment, the dose may include or exclude 5 μg to 50 μg of mRNA. In some embodiments, the dose is about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, or 50 μg.

[0057] In some embodiments, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition is administered via intravenous, intramuscular, intradermal or subcutaneous routes, or by injection or nasal spray.

[0058] In some embodiments, the disclosure provides methods for preparing broadly protective vaccines and antibodies against SARS-CoV-2. In one embodiment, the method includes generating a vaccine using native or glycoengineered S protein RNA or DNA, while the protein expressed in antigen presenting cells, including folded or unfolded forms, is processed and presented to T cells.

[0059] These and other aspects will become apparent from the following description of preferred embodiments taken in conjunction with the following drawings, in which variations and modifications may be effected without departing from the spirit and scope of the novel concepts of the present disclosure.

[0060] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, and the invention of the disclosure can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0061] [Figure 1] conserved epitopes in S protein variants, 10 of which are shielded by glycans. [Diagram 2] Identification of N- and O-glycosites and mutations in variants. All 24 glycosites are highly conserved among the 6 million S protein sequences. [Diagram 3] Analysis of S protein expression after transfection with mRNA at 48 hours by Western blot. Filters were probed with anti-S and anti-β-actin monoclonal antibodies. [Figure 4] Humoral immune responses in BALB / c mice are shown as serum anti-S WT (A), S2 (B), RBD (C), deglycosylated S (D), deglycosylated S2 (E) and deglycosylated RBD (F) protein-specific IgG endpoint titers analyzed by ELISA. Mean ± SD of five independent experiments. *P<0.001. [Diagram 5]Glycosylation modulated the specificity of induced antibodies and influenced the breadth of mRNA vaccine protection. End-point titers of alpha (A), beta (B), gamma (C) and delta (D) S protein-specific IgG antibodies determined by ELISA. Mean ± SD of five independent experiments. *P<0.001, **P<0.05. [Figure 6] Neutralization curves of pseudovirus variants are shown for WT (A), alpha (B), beta (C), gamma (D), delta (E) and omicron (F). Mean ± SD of five independent experiments. *P<0.001, **P<0.05. [Figure 7] Glycosylation influenced CD8+ T cell responses. Splenocytes isolated from immunized mice were incubated with full-length WT S (A), RBD (B) and S2 (C) peptide pools and GrzB-secreting cells were measured by Elispot. CD4+ (D) and CD8+ (E) T cells were isolated and incubated with bone marrow-derived dendritic cells and full-length WT S peptide pools and IFNγ-secreting T cells were measured by flow cytometry. (A-E) Mean ± SD of 5 independent experiments. *P<0.001. [Figure 8] Glycosylation affects cytokine production. Splenocytes isolated from mRNA-vaccinated mice were incubated with full-length WT S peptide pools, and then (A) INFγ, (B) IL-2, (C) IL-4, (D) IL-6, (E) IL-12, and (F) IL-13 were measured. (A-F) Mean ± SD of five independent experiments. *P<0.001, **P<0.05. [Figure 9] Response of deglycosylated S protein to mRNA deletion and unfolded protein to generate deglycosylated S protein. Analysis of deglycosylated S protein expression through plasmid-transfected and MG132-treated HEK293T cells by Western blot. Filters were probed with anti-S and anti-GAPDH monoclonal antibodies. [Figure 10]In vitro translated deglycosylated S variants were monitored by ELISA at various incubation times as indicated in the figure. Mean ± SD of three independent experiments. *P<0.001. [Figure 11] HEK293 cells were transfected with mRNA vaccines for 48 hours, and the plasma membrane (A), cytosol (no ER) (B) and ER (C) were isolated and analyzed for the amount of S protein by Western blot. Filters were probed with anti-S, anti-Na / K ATPase, anti-SERCA2 and anti-GAPDH monoclonal antibodies. [Figure 12] Western blot analysis of UPR marker proteins BiP / GRP78, XBP1 and p-eIF2α after transfection of HEK293 cells with deglycosylated S protein variant mRNA vaccine for 48 hours. Filters were probed with anti-BiP, anti-XBP1, anti-p-eIF2α and anti-β-actin monoclonal antibodies. [Figure 13] Analysis of apoptotic cells via APO-BrdU TUNEL assay after transfection of HEK293 cells with mRNA vaccines for different times as indicated. Mean ± SD of three independent experiments. *P<0.001. [Figure 14] Analysis of MHC I expression by flow cytometry of DCs after incubation with mRNA vaccine variants. Mean ± SD of three independent experiments. *P<0.001. [Figure 15]Schematic representation of SARS-CoV-2 spike and vaccine design: WT (A), S-(deg-RBD) (B), S-(deg-S2) (C), S-(deg-S2-1194) (D), S-(deg-RBD-801) (E), S-(deg-RBD-1194) (F) and S-(deg-RBD-122-165-234) (G). NTD, N-terminal domain (residues 14-305). RBD, receptor binding domain (residues 319-541). FP, fusion peptide (residues 788-806). HR1, heptapeptide repeat 1 (residues 912-984). HR2, heptapeptide repeat 2 (residues 1163-1213). TM, transmembrane domain (residues 1213-1237). CT, cytoplasmic domain (residues 1237-1273). S2 subunit (residues 686-1273). 2P, (K986P, V987P). ψ, N-glycosylation site. φ, O-glycosylation site. [Figure 16] Glycosylation on S2 regulated the secretion of soluble prefusion SARS-CoV-2 spike protein. After transfecting HEK293 cells with an mRNA vaccine encoding a soluble prefusion version of variant S, the location of S was determined by Western blot. Filters were probed with anti-S and anti-GAPDH monoclonal antibodies. [Figure 17] mRNA vaccines affected MHC II expression on DCs. Analysis of MHC II expression by flow cytometry of DCs after incubation with mRNA vaccine variants. Mean ± SD of three independent experiments. *P<0.001. [Figure 18] Characterization of immune responses from specific glycosite-deleted S mRNA vaccines. Humoral immune responses in BALB / c mice were shown as protein-specific IgG titers from sera against S WT (A), RBD (B) and deglycosylated RBD (C) analyzed by ELISA. [Figure 19]Characterization of immune responses from specific glycosite-deleted S mRNA vaccines. Neutralization curves of pseudovirus variants are shown with WT (A), alpha (B), beta (C) gamma (D) and delta (E). [Figure 20] Characterization of immune responses from specific glycosite-deleted S mRNA vaccines. (A) Splenocytes isolated from immunized mice were incubated with a full-length WT S peptide pool, and GrzB-secreting cells were measured by Elispot. (B) CD8+ T cells were isolated from immunized mice and incubated with bone marrow-derived DCs and a full-length WT S peptide pool, and IFNγ-secreting T cells were measured by flow cytometry. (A-J) Means ± SD of five independent experiments. *P<0.001. [Figure 21] Protein expression levels of specific glycosite-deleted S. (A) Analysis of various S protein expression in HEK293T cells transfected with plasmids and treated with MG132 by Western blot. (B) Analysis of S protein expression in HEK293T cells after transfection with mRNA-LNP at 48 hours by Western blot. Filters were probed with anti-S and anti-GAPDH monoclonal antibodies. [Figure 22] Characterization of immune responses from specific glycosite-deleted S mRNA vaccines. Splenocytes isolated from immunized mice were incubated with RBD (A) and S2 (B) peptide pools, and GrzB-secreting cells were measured by Elispot. (C) CD4+ T cells were isolated from immunized mice and incubated with bone marrow-derived DCs and full-length WT S peptide pools, and IFNγ-secreting T cells were measured by flow cytometry. (A-C) Means ± SD of five independent experiments. *P<0.001. [Diagram 23] The guanidine group-containing propagator P1 and the multivalent display propagator P2 facilitate the attachment of the mRNA to the polymer by forming a strong salt bridge between the guanidinium and the phosphate in the mRNA. [Figure 24]Design structure of initiator (I0), propagator (P1, P2, Pb, P3, Pz) and polymer reaction process. [Diagram 25] (A) Agarose gel electrophoresis assay of copolymers with GFP mRNA at an N / P ratio of 10. (B) Particle size of the mRNA complexes imaged by TEM. [Figure 26A] Fluorescence images of GFP expression in HEK293T cells transfected with poly(disulfide). GFP mRNA complexed with P1, P3, P1 / P3 and PEI at N / P ratio = 10. [Figure 26B] Fluorescence images of GFP expression in HEK293T cells transfected with poly(disulfide). GFP mRNA complexed with P1 / P3, P2 / P3, P1 / Pb, and P2 / Pb P1 / Pz at N / P ratio = 10. [Figure 26C] Fluorescence images of GFP expression in HEK293T cells transfected with poly(disulfide)-complexed GFP mRNA with various N / P ratios. [Figure 27] Agarose gel electrophoresis assay of spike mRNA-polymer complexes at various N / P ratios. [Figure 28] Chemiluminescence imaging of spike protein expression mediated by spike mRNA-polymer complexes in HEK293T cells. [Figure 29] Cell viability assay of HEK293T cells after treatment with various ratio amounts of polyGu / spike-mRNA complexes. The ratios varied from 0.01 to 1. Error bars represent standard error (mean ± SD, n = 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The terms used herein generally have their ordinary meaning in the art, in the context of the present invention and in the specific context in which each term is used. With respect to the description of the present invention, certain terms used to describe the present invention are discussed below or elsewhere in this specification to provide further guidance to the practitioner. For convenience, certain terms may be highlighted, for example, using italics and / or quotation marks. The use of highlighting does not affect the scope and meaning of a term; the scope and meaning of a term is the same in the same context, whether or not it is highlighted. It is clear that the same thing can be described in multiple ways. As a result, alternative words and synonyms can be used for any one or more of the terms discussed in this specification, and no special significance is given to a term, whether or not it is elaborated or discussed in detail in this specification. Synonyms are provided for certain terms. The description of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed in this specification, is merely illustrative and does not limit the scope and meaning of the present invention or the scope and meaning of any term exemplified in any way. Likewise, the present invention is not limited to the various embodiments presented herein.

[0063] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0064] As used herein, the terms "spike protein" and "spike glycoprotein" and "coronavirus spike protein" are used interchangeably.

[0065] As used herein, the terms "wild-type (natural) coronavirus spike protein," "wild-type (natural) coronavirus spike glycoprotein," "wild-type (natural) spike glycoprotein," and "wild-type (natural) spike protein" are used interchangeably.

[0066] As used herein, the terms "treat," "treatment," and "treating" refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or disease; ameliorating or reducing the occurrence of symptoms of an infection or disease; or a combination thereof.

[0067] As used herein, the term "preventing", which is used interchangeably with "prophylaxis", can mean preventing an infection altogether, or preventing the onset of symptoms of that infection; delaying the onset of an infection or its symptoms; or reducing the severity of an infection or its symptoms that subsequently occurs.

[0068] As used herein, an "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory antibodies and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA) or microneutralization assays.

[0069] As used herein, the term "vaccine" refers to an immunogenic agent, such as an immunogen derived from a coronavirus (with or without an adjuvant), used to induce an immune response against a coronavirus that provides protective immunity (e.g., immunity that protects a subject against infection with a coronavirus and / or reduces the severity of a condition caused by infection with a coronavirus). The protective immune response can include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of immunogen administered to a subject to create protective immunity.

[0070] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2-14 years), an infant (birth-2 years), or a newborn (up to 2 months). In certain embodiments, the subject is up to 4 months old, or up to 6 months old. In some embodiments, the adult is about 65 years old or older, or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who is contemplating pregnancy. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0071] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the U.S. Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other pharmacopoeias generally recognized for use in mammals, and more particularly for use in humans. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0072] The outbreak of SARS-CoV-2, which causes COVID-19, has resulted in a global pandemic. The current clinical management of SARS-CoV-2 infection includes prevention, control measures, and supportive care. To halt the current pandemic and possible future recurrences, it is important to fully understand this virus and develop rapid diagnostic methods, therapeutic treatments, and prophylactic vaccines to combat this dangerous pathogen. Most of the vaccine and antibody development efforts have focused primarily on the extensively glycosylated S protein of SARS-CoV-2, which is a key mediator for viral entry into host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface. Similar to many other viral fusion proteins, the S protein of SARS-CoV-2 utilizes a glycan coat to shield the S protein backbone in both pre- and post-fusion conformations to evade the host immune response. However, it is still unclear how post-translational modifications affect the translated immunogens after mRNA vaccination, but among post-translational modification events, glycosylation plays an important role in regulating protein folding, structure and function. The present disclosure aims to develop mono-GlcNAc decorated and glycosite-engineered variants (removal of non-essential glycosites via reverse genetics replacing Asn with Gln) for full-length S protein and its subunits including S1 or S2 and RBD domain as vaccine candidates for immune studies to generate antigen-specific neutralizing antibodies.

[0073] It is believed that the development of innovative strategies and broadly protective vaccines to combat CoV infections may lead to important discoveries with medical implications that would not otherwise be emphasized. The principles and strategies developed in this disclosure provide a universal coronavirus mRNA vaccine against various CoVs and their variants.

[0074] Immunogenic peptides derived from coronavirus spike proteins To date, more than 8 million S protein sequences have been reported, including the highly transmissible D614G mutant and those from the UK and South Africa, with more than 1,000 mutation sites within its 1,273 amino acid sequence. In addition, all glycosites on S protein are highly conserved, and the conserved peptide epitopes on S protein are largely shielded by glycans. This poses a great challenge in developing broadly effective antibodies and vaccines to combat the coming virus strains. The present disclosure develops a more effective vaccine design strategy using S protein with modified glycosylation as an immunogen to better expose highly conserved epitopes for vaccine design to induce broadly protective immune responses.

[0075] The present disclosure finds that removing the glycan shield on viral surface glycoproteins to expose more conserved epitopes is a highly effective approach for vaccine design against SARS-CoV-2. Since a single Asn-linked GlcNAc residue is the minimal component of the N-glycan required for glycoprotein folding and stabilization, it is envisioned that trimming the N-glycan to leave a single GlcNAc on the S protein of SARS-CoV-2 will not affect its folding but will facilitate maximal exposure of the protein backbone while maintaining its structural integrity to elicit a robust, protein-specific immune response.

[0076] By removing the glycan shield on the spike protein of SARS-CoV-2, the present disclosure provides immunogenic peptides, which are: TESIVRFPNITNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), GPKKSTNLVKNKC (SEQ ID NO: 47), GPKKSTNLVKNKC (SEQ ID NO: 48), GPKKSTNLVKNKC (SEQ ID NO: 49), GPKKSTNLVKNKC (SEQ ID NO: 50), GPKKSTNLVKNKC (SEQ ID NO: 51), GPKKSTNLVKNKC (SEQ ID NO: 52), GPKKSTNLVKNKC (SEQ ID NO: 53), GPKKSTNLVKNKC (SEQ ID NO: 54), GPKKSTNLVKNKC (SEQ ID NO: 55), GPKKSTNLVKNKC (SEQ ID NO: 56), GPKKSTNLVKNKC (SEQ ID NO: 57), GPKKSTNLVKNKC (SEQ ID NO: 58), GPKKSTNLVKNKC (SEQ ID NO: 59), GPKKSTNLVKNKC (SEQ ID NO: 60), GPKKSTNLVKNKC (SEQ ID NO: 61), GPKKSTNLVKNKC (SEQ ID NO: 62), GPKKSTNLVKNKC (SEQ ID NO: 63), GPKKSTNLVKNKC (SEQ ID NO: 64), GPKKSTNLVKNKC (SEQ ID NO: 65), GPKKSTNLVKNKC No. 47), NCDVVIGIV[N]NTVY (SEQ ID NO: 48), PELDSFKEELDKYFK[N]HTS (SEQ ID NO: 49), VNIQKEIDRLNEVA (SEQ ID NO: 50), NL[N]ESLIDLQ (SEQ ID NO: 51) and LGKYEQYIKWP (SEQ ID NO: 52), or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95% or 90% identity to any of SEQ ID NOs: 41 to 52.

[0077] In some embodiments, the immunogenic peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 41-43 and 45-51.

[0078] The amino acid sequences of SEQ ID NOs: 41 to 52 can be used, individually or in combination, as antigens capable of stimulating an immune response against coronaviruses.

[0079] Conventional methods, such as chemical synthesis or recombinant techniques, can be used to generate the immunogenic peptides described herein.

[0080] The immunogenic peptide or an expression vector capable of expressing the immunogenic peptide can be mixed with a pharma- ceutically acceptable carrier to form an immunogenic composition, which can be administered to a subject in need thereof to prevent or treat coronavirus infection.

[0081] The composition can be formulated with a pharma- ceutically acceptable carrier, such as phosphate buffered saline, bicarbonate solution, and / or adjuvants.Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are known in the art.The composition can be prepared as an injectable, solution, emulsion, or other suitable formulation.

[0082] Examples of adjuvants include, but are not limited to, alum precipitate, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG, QS21, monophosphoryl lipid A / trehalose dicorynomycolate adjuvant, Corynebacterium parvum and tRNA-containing water-in-oil emulsions, and other substances that perform the task of enhancing immune responses by mimicking a specific set of evolutionarily conserved molecules, including liposomes, lipopolysaccharide (LPS), molecular cages of antigens, components of bacterial cell walls, and plasma membrane-entrapped nucleic acids such as double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Other examples include cholera toxin, E. coli heat-labile enterotoxin, liposomes, immune stimulating complexes (ISCOMs), immunostimulatory sequence oligodeoxynucleotides, and aluminum hydroxide. The composition can also include a polymer to facilitate in vivo delivery.

[0083] Coronavirus mRNA vaccine Coronaviruses (CoVs) infect humans and animals and cause a variety of diseases, including respiratory, intestinal, renal and neurological diseases. CoVs use their spike glycoprotein (S), the primary target of neutralizing antibodies, to bind their receptors and mediate membrane fusion and viral entry. The coronavirus spike protein is highly conserved among all human coronaviruses (CoVs) and is involved in receptor recognition, viral attachment and entry into host cells. Similarly, the S protein of SARS-CoV-2 is also highly conserved with the S proteins of CoVs. The S protein of SARS-CoV-2 has three major immunogenic domains: the N-terminal domain (NTD), the receptor binding domain (RBD) and the subunit 2 domain (S2). Previous studies show that neutralizing antibodies (NAbs) that recognize the RBD are highly protective against SARS-CoV-2 and other coronaviruses, and the S protein is highly glycosylated (24 glycosites per monomer) and frequently mutated, with millions of sequences reported by GISAID.The most conserved regions of the SARS-CoV-2 S protein are located in the RBD and S2 domains, which are largely shielded by glycans (Han-Yi Huang et al. Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2. bioRxiv preprint.doi:www.biorxiv.org / content / 10.1101 / 2021.05.25.445523v2.full), and antibodies that recognize these regions could provide broad-spectrum protection against SARS-CoV-2 variants (Maximilian M Sauer. et al. Structural basis for broad coronavirus neutralization. Nat Struct Mol Biol. 28(6):478-486 (2021) 1314; C. Wang. et al. A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies. Nat Commun. 12(1):1715 (2021)). Glycosylation on target antigens or pathogens modulated the induction of antibodies, but it is still unclear whether glycosylation affects T cell responses. Indeed, it is difficult or impossible to express S protein from a plasmid with deletion of certain glycosylation sites (Han-Yi Huang et al. Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2. bioRxiv preprint.doi:www.biorxiv.org / content / 10.1101 / 2021.05.25.445523v2.full).

[0084] The present disclosure surprisingly finds that using mRNA technology to remove the glycan shield, thereby better exposing conserved regions, is an effective strategy for broad-spectrum vaccine design. In the present disclosure, mRNA of coronavirus spike protein (e.g., S protein of SARS-CoV-2) with specific glycosite mutations is used as an immune model to investigate how glycosite mutant mRNA affects protein expression and immune response.

[0085] Thus, the disclosure provides modified nucleic acid molecules encoding modified spike proteins that include one or more amino acid substitutions of asparagine (N) to glutamine (Q) in an N-linked glycosylation sequon (NXS / T), where X is any amino acid residue except proline and S / T represents a serine or threonine residue.

[0086] The modified nucleic acid molecule may be mRNA or single- or double-stranded DNA and may be used as an immunogen or vaccine against a pathogen. In one embodiment, the pathogen is a CoV. Examples of CoVs include, but are not limited to, SARS-CoV, MERS-CoV, and SARS-CoV-2. Examples of SARS-CoV-2 include, but are not limited to, alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, and omicron-SARS-CoV2 and variants thereof.

[0087] Compared to the wild-type spike proteins of the Wuhan and Delta strains (e.g., SEQ ID NOs: 2, 16, 18, and 20), the modified spike proteins described herein comprise one or more amino acid deletions or additions at the N-linked glycosylation sequon (NXS / T) to eliminate the N-linked glycan sequon. Alternatively, the modified spike proteins described herein comprise one or more amino acid substitutions of S / T to alanine (A) at the O-linked glycosylation site to eliminate the O-linked glycosylation site.

[0088] The coronavirus spike protein mRNA can be used as a coronavirus vaccine, which has one or more glycosylated mutations or variants in the receptor binding domain (RBD), subunit 1 (S1) domain, or subunit 2 (S2) domain.

[0089] The mutations of CoVs or variants thereof described herein may be deletions, additions, or substitutions. In some embodiments, the coronavirus spike protein mRNA has one or more mutations in the glycosites in the RBD, S1, or S2 with one or more exchanges of N to Q or S / T to A or combinations thereof. A mutation in an N-glycosite is a change of the putative sequon NXS / T to QXS / T and / or a change of S / T to A in an O-glycosite.

[0090] Glycosites with an N to Q exchange include, but are not limited to, the following: S-(deg-RBD): An S protein with two N-glycosides in the RBD mutated from N to Q and two O-glycosides mutated from S / T to A, all (e.g., SEQ ID NO: 4, 22, 24, or 26); S-(deg-S2): S protein with all 9 glycosites in S2 mutated from N to Q (e.g., SEQ ID NO: 6, 28, 30, or 32); S-(deg-S2-1194): S protein with 8 glycosites in S2 mutated from N to Q except for glycosite 1194 (e.g., SEQ ID NO: 8 or 34); S-(deg-RBD-801): An S protein with two N glycosites in the RBD mutated from N to Q and two O-glycosides mutated from S / T to A and glycosite 801 mutated from N to Q, all (e.g., SEQ ID NO: 10 or 36); S-(deg-RBD-1194): An S protein (e.g., SEQ ID NO: 12 or 38) with two N glycosites in the RBD mutated from N to Q and two O-glycosides mutated from S / T to A and glycosite 1194 mutated from N to Q, all together; and S-(deg-RBD-122-165-234): An S protein with two N-glycosides in the RBD mutated from N to Q and two O-glycosides mutated from S / T to A and glycosites 122, 165 and 234 all mutated from N to Q (e.g., SEQ ID NO: 14 or 40).

[0091] In further embodiments, the S-(deg-RBD) mRNA or DNA has the sequence of SEQ ID NO: 3, 21, 23 or 25, the S-(deg-S2) mRNA or DNA has the sequence of SEQ ID NO: 5, 27, 29 or 31, the S-(deg-S2-1194) mRNA or DNA has the sequence of SEQ ID NO: 7 or 33, the S-(deg-RBD-801) mRNA or DNA has the sequence of SEQ ID NO: 9 or 35, the S-(deg-RBD-1194) mRNA or DNA has the sequence of SEQ ID NO: 11 or 37 and the S-(deg-RBD-122-165-234) mRNA or DNA has the sequence of SEQ ID NO: 13 or 39.

[0092] The present disclosure provides a linear DNA comprising a promoter, a 5' untranslated region, a 3' untranslated region, an expression plasmid with or without S-2P, and a poly(A) tail signal sequence, where the putative sequon NXS / T has been changed to QXS / T and the O-glycosite has been changed from S / T to A on the expression plasmid. In one embodiment, the S-2P expression plasmid comprises the S gene of SARS-CoV-2 encoding the pre-fusion form of S with proline substitutions at K968 and V969.

[0093] The mRNA can be prepared by in vitro translation from the DNA described above using vectors containing the modified nucleic acid molecules described herein and host cells containing the vectors. The target spike protein gene is synthetically produced and inserted into a plasmid or small, circular piece of DNA. Plasmids are used to generate mRNA vaccines because they are easy to replicate (copy) and ensure that they contain the target gene sequence. The two strands of the plasmid DNA are then separated. RNA polymerase, a molecule that transcribes RNA from DNA, uses the spike protein gene to create a single mRNA molecule. Finally, other molecules degrade the remaining plasmid to ensure that only the mRNA is packaged as the vaccine. The speed and efficiency of this process allows large amounts of mRNA to be made in a short period of time.

[0094] The present disclosure relates to a method for the preparation of monoGlcNAc-decorated S proteins (S mg ) immunization with wild-type S protein, which has glycans at all N-glycosylations trimmed to N-acetylglucosamine (GlcNAc), induced broadly protective antibodies and CD4 + T cell responses and CD8 + The present disclosure also found that the single B cell technique was used to detect and induced T cell responses in S protein. Further studies show that most of the conserved epitopes of the S protein are located in the RBD and HR2 domains of the S2 subunit, but these conserved epitopes are largely shielded by glycans, thus evading immune responses. Thus, removal of the shielded glycans exposes more of the conserved epitopes, inducing a broader and stronger immune response. The present disclosure also uses single B cell techniques to detect and elucidate the function of the S protein in the HR2 domains of the RBD and S2 subunits. mgBy screening B cells from immunized mice, we identify broadly neutralizing monoclonal antibodies targeting highly conserved regions in the RBD that were not induced upon immunization with fully glycosylated S protein. We further demonstrate that removal of the glycan shield from the S protein is an effective strategy for developing broadly protective vaccines against SARS-CoV-2 variants. To translate this discovery into mRNA vaccine design, we focus here on studying SARS-CoV-2 spike mRNAs with specific glycosite mutations in the RBD, S1 or S2 or combinations thereof with N to Q and S / T to A exchanges, and investigating their protein expression and immune response and breadth of protection.

[0095] Immunization with such mRNA results in the accumulation of misfolded spike protein in the endoplasmic reticulum, leading to upregulation of BiP / GRP78, XBP1 and p-eIF2α, inducing cell apoptosis and CD8 T cell responses. In addition, dendritic cells (DCs) incubated with S2 glycosite-deleted mRNA vaccines enhanced the expression of class I major histocompatibility complex (MHC I). Furthermore, removing glycosites that affect the stability of spike protein reduced antibody production and enhanced CD8+ T cell responses. The present disclosure provides an mRNA vaccine with a broad therapeutic spectrum that is unlikely to be achieved using proteins expressed as antigens.

[0096] mRNA nanoclusters and nanoparticles In one aspect, the present disclosure provides mRNA nanoclusters comprising the mRNA vaccines described herein formulated in lipid nanoparticles.

[0097] Biodegradable lipid nanoparticles may be used as lipid nanoparticles. In one embodiment, the biodegradable lipid nanoparticles are guanidine-based polymers.

[0098] In another embodiment, the present disclosure provides an mRNA nanocluster comprising a biodegradable lipid nanoparticle encapsulating an mRNA vaccine as described herein, wherein the biodegradable lipid nanoparticle comprises a guanidine-based and zwitterionic unit, the guanidine-based and zwitterionic groups are attached to the lipid tail of a polymer, and the guanidine-based group attaches to the mRNA, thereby forming a salt bridge between the guanidinium group and the phosphate in the mRNA. Examples of guanidine-based polymers include, but are not limited to, P1, P2, P3, Pb, and Pz as described herein.

[0099] The present disclosure provides guanidine-based lipid nanoparticles as carriers for mRNA nanovaccine formulations. The polymers produce efficient delivery of mRNA to antigen-presenting cells and exhibit a strong ability of endosomal escape. Timely degradation of poly(disulfide) by intracellular glutathione also minimizes cytotoxicity compared to other non-degradable nanocarriers.

[0100] In another embodiment, the mRNA nanoclusters have a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

[0101] The coronavirus mRNA vaccines of the present disclosure can also be attached to nanoparticles.

[0102] mRNA nanoclusters and nanoparticles are particles between 1 and 100 nanometers (nm) in size that can be used as substrates for immobilizing ligands. Nanoparticles can be, for example, lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles such as gold nanoparticles, liposomes, immune stimulating complexes (ISCOMs), virus-like particles (VLPs) or self-assembling proteins.

[0103] In one embodiment, lipid nanoparticle formulations typically comprise one or more lipids. In some embodiments, lipids are cationic lipids or ionized lipids. In some embodiments, lipid nanoparticle (LNP) formulations further comprise other components, including phospholipids, structured lipids, quaternary amine compounds, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids.

[0104] The mRNA vaccines described herein can be encapsulated in liposomes or polymersomes.

[0105] Conventional liposomes are produced by one-step lipid bilayer formation, and therefore both the inner and outer membranes of the liposome are usually made of the same components. Examples of liposome components include, but are not limited to, DSPC, DOTAP, DMG, PEGylated DMG, cholesterol, and combinations thereof. In one embodiment, mRNA liposomes are produced by mixing mRNA and lipid components at room temperature in the ratios described herein.

[0106] Polymersomes, as disclosed herein, are enclosures that are self-assembled from amphiphilic block copolymers. These amphiphilic block copolymers are macromolecules that include at least one hydrophobic polymer block and at least one hydrophilic polymer block. Upon hydration, these amphiphilic block copolymers self-assemble into enclosures by the hydrophobic blocks associating with each other to minimize direct exposure to water and form the inner surface of the enclosure, while the hydrophilic blocks face outward to form the outer surface of the enclosure. The hydrophobic core of these water-soluble polymersomes can provide an environment to solubilize additional hydrophobic molecules. Thus, these water-soluble polymersomes can function as carrier polymers for hydrophobic molecules encapsulated within the polymersomes. Furthermore, the self-assembly of amphiphilic block polymers occurs in the absence of stabilizers that would otherwise provide colloidal stability and prevent aggregation. In one embodiment, mRNA liposomes are produced by mixing mRNA and polymer at room temperature in the ratios described herein.

[0107] Vaccines, combo vaccines, vaccine compositions, methods and therapeutic uses The mRNA described herein can be used as a vaccine, either alone or in combination with other vaccines. Thus, the present disclosure provides a combo vaccine comprising the mRNA vaccine of the present disclosure and one or more additional vaccines. The additional vaccines are selected from one or more of COVID-19 vaccines, influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines, and any combination thereof.

[0108] The present disclosure also provides a vaccine composition comprising the mRNA vaccine, mRNA nanocluster, or mRNA nanoparticle described herein. The present disclosure also provides a method of preventing or treating a coronavirus infection, the method comprising administering to a subject an mRNA vaccine, mRNA nanocluster, or mRNA nanoparticle, or a vaccine composition described herein. In one embodiment, the subject is infected or at risk of being infected with a coronavirus.

[0109] The mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition described herein may be administered as an initial dose or as two, three or four booster doses. In some embodiments, the mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition is administered as an initial dose and at least one booster dose about 1 month, about 2 months, about 3 months, about 4 months, about 5 months or about 6 months after the initial dose. In some embodiments, the provided compositions are administered as a second booster dose about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 1 year after the initial dose.

[0110] The mRNA vaccine, mRNA nanocluster or mRNA nanoparticle or vaccine composition is administered in a single dose or in more than one dose. In one embodiment, the dose may include or exclude 5 μg to 50 μg. In some embodiments, the dose is about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, or 50 μg.

[0111] The vaccine composition preferably comprises a pharma- ceutically acceptable vaccine, carrier or diluent. The vaccine composition may be formulated using any suitable method. Formulation using standard pharma- ceutically acceptable carriers and / or excipients may be performed using routine methods in the pharmaceutical art. The exact nature of the formulation will depend on several factors, including the vaccine to be administered and the desired route of administration. Suitable types of formulations are described in detail in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0112] The vaccine or pharmaceutical compositions described herein may be administered by any route. Suitable routes include, but are not limited to, nasal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, transdermal and oral / buccal routes.

[0113] The composition may be prepared together with a physiologically acceptable carrier or diluent. Usually, such a composition is prepared as a liquid suspension of nanoparticles. The nanoparticles may be mixed with an excipient that is pharma-ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, and the like, and combinations thereof.

[0114] Suitable compositions wherein the carrier is a liquid for administration, for example, as a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.

[0115] In addition, if desired, the pharmaceutical composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and / or pH buffering agents.

[0116] Without intending to limit the scope of the present invention, exemplary devices, apparatus, methods and related results according to embodiments of the present invention are presented below. Titles or subtitles may be used in the examples for the convenience of the reader, but please note that the titles or subtitles are in no way intended to limit the scope of the present invention. Furthermore, although certain theories are proposed and disclosed herein, whether they are correct or incorrect, they are in no way intended to limit the scope of the present invention, as long as the present invention is practiced according to the present invention regardless of any specific theory or scheme of action. EXAMPLES

[0117] Materials and Methods Cell lines. Human embryonic kidney cells (HEK293) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD) containing 10% heat-inactivated fetal bovine serum (FBS) (Thermo Scientific) and antibiotics (penicillin G 100 U / ml and streptomycin 100 gm / ml).

[0118] Antibodies and proteins. Rabbit anti-SARS-CoV-2 S polyclonal antibody and SARS-CoV-2 full-length S, S2, RBD and variant proteins (expressed in 293T cells) were purchased from Sino Biologicals (Beijing, China). Mouse monoclonal anti-β-actin, GAPDH and rabbit monoclonal anti-MHC II antibodies were purchased from Millipore. Rabbit monoclonal anti-Na / K ATPase was obtained from ABcan. Mouse monoclonal anti-SERCA2 and rabbit monoclonal anti-MHC I antibodies were obtained from Invitrogen. Rabbit monoclonal anti-BiP / GRP78, XBP1 and p-eIF2α antibodies were purchased from ABclonal. All commercial antibodies were validated for specificity by the companies and by the inventors via Western blot. To obtain deglycosylated proteins, S, RBD, S1 or S2 proteins were deglycosylated in a buffer containing PNGase F (Sigma) in the dark at 37° C. for 24 h. After deglycosylation, samples were purified and checked by Western blot.

[0119] mRNA vaccines and formulations of deglycosylated S protein. The prefusion state of S, the codon-optimized S gene of SARS-CoV-2, was synthesized by GenScript, cloned into pcDNA3.1 or pVax, and in one embodiment, stabilized by proline substitutions at K968 and V969 (S-2P). A soluble version of S was constructed that terminates at glutamine Q1208 of S-2P, followed by a T4 fibritin (foldon) trimerization motif, a thrombin cleavage site, and a 6xHis tag at the C-terminus. To mutate the N-glycosite, the putative sequon NXS / T was changed to QXS / T by using site-directed mutagenesis on the S-2P expression plasmid, and the O-glycosite was changed from S / T to A. To obtain the mRNA vaccine, linear DNA containing the T7 promoter, 5' untranslated region, 3' untranslated region, S-2P and poly(A) tail signal sequence was amplified for 1 h at 37°C by using TOOLS Ultra High Fidelity DNA Polymerase (BIOTOOLS Co., Ltd., Taipei, Taiwan) with 1 μl of DNA template included in mMESSAGE mMACHINE® Kit (Thermo Scientific) according to the manufacturer's protocol. The mRNA was purified by RNA clean-up kit (BioLabs) according to the manufacturer's protocol and stored at -80°C until further use. For the formulation of mRNA-LNP, the mRNA was encapsulated into LNP using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 was rapidly mixed with an ethanolic lipid mixture containing ionized cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid. The composition of the LNPs was DSPC (Sigma), cholesterol (Sigma), DOTAP (Sigma) and DMG-PEG 2000 (Sigma). The mRNA-LNPs were characterized and then stored at -80°C at a concentration of 1 mg / ml.HEK293 cells were transfected with 10 μg of mRNA-LNP in six-well plates for 48 h, and then total cell lysates were harvested to monitor the expression of S by Western blot.

[0120] Animals and immunization. 6-8 week old BALB / c mice (n=5) were immunized intramuscularly with 50 μg of mRNA-LNP in PBS containing 300 mM sucrose. Animals were immunized at week 0 and boosted with a second vaccination at week 2, and serum samples and spleens were taken from each mouse 1 week after the booster immunization. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Academia Sinica.

[0121] Serum IgG titer determination. IgG titers were determined using anti-S protein ELISA. Plates were coated with 50 ng / well of variant S protein as shown in Figures 4 and 5, and then blocked with 5% skim milk. Serum from immunized mice and HRP-conjugated secondary antibody were added sequentially. Peroxidase substrate solution (TMB) and 1M H 2 SO 4 A stop solution was used and the absorbance (OD 450 nm) was read using a microplate reader.

[0122] Pseudovirus neutralization assay for serology studies. Pseudoviruses were constructed by the RNAi Core Facility at Academia Sinica. Briefly, pseudotyped lentiviruses carrying the S protein or variants of SARS-CoV-2 were generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91, pLAS2w.Fluc.Ppuro, and pcDNA3.1-nCoV-SΔ18.HEK-293T cells were seeded 1 day before transfection, following which the plasmids were delivered to the cells by TransITR-LT1 transfection reagent (Mirus). Culture medium was refreshed at 16 h and harvested at 48 and 72 h post-transfection. Cell debris was removed by centrifugation and the supernatant was passed through a 0.45 μm syringe filter (Pall Corporation). Pseudotyped lentiviruses were then stored at -80 °C. To estimate lentivirus titer by AlarmaBlue assay (Thermo Scientific), transducing units (TU) of pseudotyped lentivirus were estimated by using cell viability assay. HEK-293T cells expressing human ACE2 gene were seeded in 96-well plates one day before lentivirus transduction. To determine the titer of pseudotyped lentivirus, various amounts of lentivirus were added to polybrene-containing culture medium (final concentration 8 μg / ml) (Sigma), and spin infection was performed at 1,100 × g for 30 min at 37 °C in 96-well plates. After 16 h of incubation, the virus and polybrene-containing culture medium were removed and replaced with fresh complete DMEM containing 2.5 μg / ml puromycin (Sigma). After 48 h of treatment with puromycin, the culture medium was removed and cell viability was detected by using AlarmaBlue reagent according to the manufacturer's instructions. The viability of uninfected cells was set as 100% and virus titers were determined by plotting viable cells against diluted virus dose.

[0123] For neutralization assays, heat-inactivated serum or antibodies were serially diluted at the desired dilution and incubated with 1,000TU of SARS-CoV-2 pseudotyped lentivirus in DMEM for 1 hour at 37°C. The mixture was then inoculated with 10,000 HEK-293T cells stably expressing the human ACE2 gene in a 96-well plate. The culture medium was replaced with fresh complete DMEM (supplemented with 10% FBS and penicillin / streptomycin 100U / ml) 16 hours after infection and cultured continuously again for 48 hours. The expression level of the luciferase gene was measured by using the Bright-Glo™ Luciferase Assay System (Promega). Relative light units (RLU) were detected by Tecan i-control (Infinite 500). The percentage of inhibition was calculated as the ratio of the RLU reduction in the presence of diluted serum to the RLU value of the serum-free control. The formula is shown below: (RLU 対照 -RLU 血清 ) / RLU 対照 .

[0124] Informatics analysis of SARS-CoV-2 S protein. 1,117,474 S protein sequences of SARS-CoV-2 and their variants were extracted from the Global Initiative on Sharing Avian Influenza Database (GISAID version: April 18, 2021). 3D structural models of S protein with representative glycan profiles were constructed by CHARMM-GUI and OpenMM programs. The transmembrane region of spike protein defined by UniProt was used in this study. The input of CHARMM-GUI includes PDB file 6VSB_1_1_1, representative glycan profile and parameter settings. Relative solvent accessibility (RSA) of spike protein both with and without representative glycans is calculated by FreeSASA program. A probe radius of 7.2 Å was used in the FreeSASA program to mimic the average size of hypervariable loops in the complementarity determining regions (CDRs) of antibodies. The RSA value of each residue used in this study was the average RSA value from the three protein chains. The definition of exposed / buried residues was the same as in the study by Kajander, T. et al.

[0125] Measurement of GrzB and IFNγ secreting cells. A total of 5 × 10 5 Splenocytes were restimulated ex vivo with a mixture of full-length S, RBD and S2 peptides (final concentration 0.1 μg / ml per peptide) (Sino Biologicals) in a GrzB ELISpot assay (R&D Systems) according to the manufacturer's instructions, and spots were counted. For T cell subtyping, CD8 + T cells and CD4 + T cells were isolated from splenocyte suspensions using the Dynabeads Untouched Mouse CD4 and CD8 Cells kit (Invitrogen) according to the manufacturer's instructions. + T cells or CD8 + T cells (1×10 55 × 10 syngeneic bone marrow-derived DCs (Sino Biologicals) loaded with a full-length WT S peptide mixture (final concentration 0.1 μg / ml) 4 The purity of the isolated T cell subsets was determined by flow cytometry and consisted of CD4 + T cells or CD8 + T cells 1 x 10 5 For flow cytometry, cells were cultured at 10 6 The cells were suspended at a density of 1000 cells / ml, and the antibody used in this study was anti-IFNγ (Abcam). The fluorescence intensity of the cells was analyzed using a FACS Canto (BD Biosciences) and FCS Express 3.0 software.

[0126] Measurement of IFNγ and other cytokines. IFNγ, IL-2, IL-4, IL-6, IL-12 and IL-13 were measured by using ELISA kits according to the manufacturer's protocols (IFN-γ: Boster Biological Technology Co., Ltd.; IL-2, IL-4, IL-6, IL-12 and IL-13: R&D Systems).

[0127] DNA plasmid transfection and MG132 treatment. After seeding HEK293 cells in 6-well plates, the cells were transfected with 3 μg of each plasmid by TransIT®-LT1 Transfection Reagent (Mirus), and then incubated with MG-132 (MedChemExpress) or DMSO 1 μM at 37° C. for 24 h. Total lysates were harvested and the expression of variant S was analyzed by Western blot.

[0128] In vitro translation. In vitro translation was performed with a plasmid encoding S-2P using the glycoprotein expression in Human IVT System (Thermo) according to the manufacturer's instructions. Expression of S protein at different incubation periods was monitored by SARS-COV-2 spike protein ELISA kit (ABclonal) according to the manufacturer's protocol.

[0129] Detection of unfolded protein response. HEK293 cells were transfected with 10 μg of S mRNA using TransIT®-mRNA Transfection Kit (Mirus) for 48 hours, and then plasma membrane and ER were isolated by Minute™ ER Enrichment Kit (Invent Biotech) according to the manufacturer's protocol. Plasma membrane, cytosol and ER S protein were analyzed by Western blot. Total lysates were harvested and UPR markers XBP1, BiP / GRP78 and p-eIF2α were monitored by Western blot. Apoptotic cells were measured by APO™-BrdU TUNEL Assay Kit (Thermo) according to the manufacturer's instructions.

[0130] Soluble version of S-2P expression. HEK293 cells were transfected with 10 μg of mRNA encoding a soluble version of variant S-2P using the TransIT®-mRNA Transfection Kit (Mirus) for 72 hours, and S protein was purified from cell supernatants using Ni-NTA affinity columns (GE Healthcare). Purified proteins and total lysates were monitored for S protein levels by Western blot.

[0131] The mRNA vaccine induced MHC I / II expression on DCs. DCs were isolated from mice by using the M-pluriBead Cell Separation kit (pluriSelect) according to the procedure from the company, incubated with 10 μg of mRNA-LNPs in DC culture medium (RPMI1640 supplemented with 20 ng / mL mouse GM-CSF (R&D Systems), 10% FBS, 50 μM 2-ME, 100 units / mL penicillin, and 100 μg / mL streptomycin) at 37° C. for 48 hours, and then analyzed for MHC I and MHC II expression by flow cytometry.

[0132] Statistics and reproducibility. All data are presented as the mean ± standard error of the mean. The number of samples and replicates per experiment are indicated as described in the figure legends. Comparisons between groups were determined using Student's t-test. Differences were reported using the * P<0.001, ** P<0.05 was considered significant. All data were analyzed using GraphPad Prism 6 software.

[0133] [Example 1] Glycosylation of S protein affected antibody production As part of our efforts to identify potential conserved epitopes as targets for antibody development and next-generation vaccine design, as well as for the design of universal vaccines with broadly protective immune responses, we performed S protein mutation analysis from the available 218,516 sequences of the S protein of SARS-CoV-2. The S protein has 1,273 amino acids, and among the 218,516 sequences analyzed, there are 1,149 variable amino acid positions, including 613 in the S1 domain (672 amino acids), 524 in the S2 domain (588 amino acids), and 134 in the RBD (152 amino acids); however, the mutation rate is less than 0.1% at 1,076 sites, while it is greater than 0.1% at 73 sites. Conserved sequences can be found in the S1, S2, and RBD regions, with the longest one being from R983-I1013 near the HR1 domain of the S2 region. All 22 N-glycosylation sites are highly conserved among SARS-CoV-2 variants. Further analysis on a larger number of sequences (approximately 6 million) shows a similar distribution of conserved epitopes, seven of which are in the RBD and five in HR2, with 10 of the conserved epitopes being shielded by glycans (Figure 1). We believe that removing the glycan shield on viral surface glycoproteins to expose more conserved epitopes is a highly effective and universal approach for vaccine design against SARS-CoV-2. Since a single GlcNAc residue linked to Asn is the minimal component of N-glycans required for glycoprotein folding and stabilization, it is therefore envisioned that trimming the N-glycan to leave a single GlcNAc on the S protein of SARS-CoV-2 will not affect its folding but will facilitate maximum exposure of the protein backbone while maintaining its structural integrity to elicit a robust and protein-specific immune response.

[0134] Based on our preliminary results, full-length S, S1, S2 and RBD, fully glycosylated (unmodified) and mono-GlcNAc decorated (on all A-glycosites), will be used as immunogens for immunization studies. We will generate S, S1, S2 and RBD subunits that retain the essential glycosites and lack specific glycosites found in our preliminary studies above, as well as their mono-GlcNAc decorated variants, as immunogens for immunization. Antisera will be tested for their interaction with representative S protein variants and neutralizing activity against pseudovirus-mediated infections, and those with broadly protective activity will be analyzed by epitope mapping and CD4 + T cell responses and CD8 +Further investigations will include adjuvant effects on T cell responses. Mono-GlcNAc decorated variants are made by removing the heterogeneous glycan layer on the N-glycosylation sites of full-length S, S1, S2 and RBD, which are produced using the more versatile and well-documented CHO, HEK293 or Gnt1-deficient HEK293 cell line expression systems. The glycans of the S protein expressed in these cell lines can be trimmed with endoglycosidases to generate the desired protein with mono-GlcNAc at all N-glycosylation sites. Those from the latter (Gnt1-deficient HEK293) are high mannose type and can be digested with endoglycosidase H (Endo-H) to generate the desired protein with mono-GlcNAc at all N-glycosylation sites. O-glycans are not modified at all, as they are important for viral entry, but can be trimmed with a cocktail of exoglycosidases if necessary. The monoGlcNAc decorated full-length and truncated S protein have been studied for their structural integrity. Immunogens containing the fully glycosylated and monoGlcNAc proteins as well as the glycosite-modified S protein (by replacing Asn with Gln as shown in reverse genetic studies) have been used for immunization of mice to identify antibodies targeting different domains on the S protein with broad neutralizing activity. The specificity of serum antibodies is checked by the full and monoGlcNAc decorated glycosite-modified S protein and its truncated forms. In addition, an array of synthetic peptides with or without monoGlcNAc decoration or glycopeptides obtained from protease digestion of the monoGlcNAc decorated S protein have been used to evaluate binding specificity and CD8 expression in transgenic mice with humanized ACE2 receptor. + T cell responses are studied. Sera from immune mice are further evaluated for neutralizing activity using a pseudovirus neutralization assay developed in the inventor's laboratory. Figure 2 shows the identification of N-glycosides and O-glycosides and mutations in variants. All 24 glycosites are highly conserved among 6 million S protein sequences.

[0135] The S protein is frequently mutated and highly glycosylated with 22 N-glycosides and 2 O-glycosides (2 N-glycosides and 2 O-glycosides in RBD and 6 N-glycosides in S2), thereby evading the host immune response (Figure 16). To study whether and how the mRNA vaccine of S protein with mutated glycosites affected the expression and immune response of S protein, we mutated multiple N-glycosides (N to Q in NXS / T sequon) and O-glycosides (S / T to A) in the RBD or S2 regions of the mRNA, respectively. After confirming the expression of the variant pre-fusion S protein in HEK293 cell line (Figure 3), the mRNA encoding the S protein or S protein with glycosite mutations was encapsulated into LNPs to form mRNA-LNPs for immunization of mice. Sera from mice immunized with mRNA in which all S2 N-glycosylated sites were mutated (S-(deg-S2)) or all S2 N-glycosylated sites except N1194 were mutated (S-(S2-1194)) showed lower IgG titers against the fully glycosylated WT S protein (Fig. 4A), S2 (Fig. 4B), RBD (Fig. 4C) or deglycosylated S protein (Fig. 4D), but had higher IgG titers against the deglycosylated S2 antigen in ELISA assays compared to unmodified mRNA (Fig. 4E). However, mice immunized with mRNA lacking all RBD glycosites (S-(deg-RBD)) elicited slightly lower IgG titers against the fully glycosylated RBD and higher IgG titers recognizing the deglycosylated RBD antigen (Figure 4F), suggesting that glycosylation on the S protein affected antibody production and its binding specificity.In addition, immunization with S-(deg-RBD), S-(deg-S2) or S-(S2-1194) mRNA elicited higher IgG titers against the alpha (Figure 5A), beta (Figure 5B), gamma (Figure 5C), delta and omicron (Figure 5D) variants, suggesting that glycosylation of the S protein modulates the specificity of antibodies produced by mRNA vaccines. To analyze the effect of glycosite mutations on the neutralizing activity of antibodies produced from immunized mice, pseudovirus neutralization assays were performed. The results showed that mRNA vaccines with deletions of glycosites in RBD or S2 produced antibodies with reduced neutralizing activity against the WT pseudovirus (Figure 5), but produced antibodies with better neutralizing activity than the WT against the four variants of concern (Figure 6 and Table 1). To further understand this observation, sequence analysis revealed that the mutation of glycosites in RBD or S2 exposed more conserved epitopes to elicit immune responses (Figure 6), and the RBD and S2 domains contain most of the highly conserved sequences in the S protein (seven in RBD and five in HR2 of S2). These results suggest that the mutation of certain glycosites in the mRNA of the S protein vaccine affects the production and specificity of antibodies and immune responses.

[0136] DNA or RNA sequence of WT S (Wuhan strain) (from 5' to 3' end):

[0137] [ka] TIFF2025081478000007.tif219169

[0138] Protein sequence of WT S (Wuhan strain) (from N-terminus to C-terminus):

[0139] [ka] TIFF2025081478000009.tif139167

[0140] DNA or RNA sequence of WT S (strain Delta) (5' to 3' end):

[0141] [ka] TIFF2025081478000011.tif209168

[0142] Protein sequence of WT S (strain Delta) (N- to C-terminus):

[0143] [ka] TIFF2025081478000013.tif131168

[0144] DNA or RNA sequence of WT S (Wuhan strain S-2P strain) (from 5' to 3' end):

[0145] [ka] TIFF2025081478000015.tif211168

[0146] Protein sequence of WT S (Wuhan strain S-2P) (N-terminus to C-terminus):

[0147] [ka] TIFF2025081478000017.tif139168

[0148] DNA or RNA sequence of WT S (strain DeltaS-2P) (5' to 3' end):

[0149] [ka] TIFF2025081478000019.tif211167

[0150] Protein sequence of WT S (strain Delta S-2P) (N-terminus to C-terminus):

[0151] [ka] TIFF2025081478000021.tif140168

[0152] DNA or RNA sequence of S-(deg-RBD) of the Wuhan strain (from 5' to 3' end):

[0153] [ka] TIFF2025081478000023.tif218168

[0154] Protein sequence of S-(deg-RBD) from Wuhan strain (N-terminus to C-terminus):

[0155] [ka] TIFF2025081478000025.tif147168

[0156] DNA or RNA sequence of S-(deg-RBD) of Delta strain (5' to 3' end):

[0157] [ka] TIFF2025081478000027.tif225168

[0158] Protein sequence of S-(deg-RBD) from Delta strain (N-terminus to C-terminus):

[0159] [ka]

[0160] DNA or RNA sequence of S-(deg-RBD) of Wuhan S-2P strain (5' to 3' end):

[0161] [ka] TIFF2025081478000030.tif243168

[0162] Protein sequence of S-(deg-RBD) from Wuhan S-2P strain (from N-terminus to C-terminus):

[0163] [ka]

[0164] DNA or RNA sequence of S-(deg-RBD) of DeltaS-2P strain (5' to 3' end):

[0165] [ka] TIFF2025081478000033.tif255167

[0166] Protein sequence of S-(deg-RBD) of DeltaS-2P strain (N-terminus to C-terminus):

[0167] [ka]

[0168] DNA or RNA sequence of S-(deg-S2) of the Wuhan strain (from the 5' end to the 3' end):

[0169] [ka] TIFF2025081478000036.tif247168TIFF2025081478000037.tif13166

[0170] Protein sequence of S-(deg-S2) from Wuhan strain (from N-terminus to C-terminus):

[0171] [ka]

[0172] DNA or RNA sequence of S-(deg-S2) of Delta strain (5' to 3' end):

[0173] [ka] TIFF2025081478000040.tif253167TIFF2025081478000041.tif14167

[0174] Protein sequence of S-(deg-S2) from Delta strain (N-terminus to C-terminus):

[0175] [ka]

[0176] DNA or RNA sequence of S-(deg-S2) of Wuhan S-2P (from 5' to 3' end):

[0177] [ka] TIFF2025081478000044.tif250166TIFF2025081478000045.tif14166

[0178] Protein sequence of S-(deg-S2) of Wuhan S-2P (from N-terminus to C-terminus):

[0179] [ka]

[0180] DNA or RNA sequence of S-(deg-S2) of the delta S-2P strain (5' to 3' end):

[0181] [ka] TIFF2025081478000048.tif253166TIFF2025081478000049.tif21166

[0182] Protein sequence of S-(deg-S2) from strain deltaS-2P (N- to C-terminus):

[0183] [ka]

[0184] DNA or RNA sequence of Wuhan strain S-(S2-1194) (5' to 3' end):

[0185] [ka] TIFF2025081478000052.tif250167TIFF2025081478000053.tif37167

[0186] Protein sequence of Wuhan strain S-(S2-1194) (N- to C-terminus):

[0187] [ka]

[0188] DNA or RNA sequence of S-(S2-1194) of Wuhan S-2P strain (5' to 3' end):

[0189] [ka] TIFF2025081478000056.tif251168TIFF2025081478000057.tif43167

[0190] Protein sequence of S-(S2-1194) from Wuhan S-2P strain (N- to C-terminus):

[0191] [ka]

[0192] DNA or RNA sequence of S-(deg-RBD-801) from the Wuhan strain (5' to 3' end):

[0193] [ka] TIFF2025081478000060.tif64167

[0194] Protein sequence of S-(deg-RBD-801) from Wuhan strain (N- to C-terminus):

[0195] [ka]

[0196] DNA or RNA sequence of S-(deg-RBD-801) of Wuhan S-2P strain (5' to 3' end):

[0197] [ka] TIFF2025081478000063.tif80167

[0198] Protein sequence of S-(deg-RBD-801) from Wuhan S-2P strain (N- to C-terminus):

[0199] [ka] TIFF2025081478000065.tif13166

[0200] DNA or RNA sequence of S-(deg-RBD-1194) from the Wuhan strain (5' to 3' end):

[0201] [ka] TIFF2025081478000067.tif96167

[0202] Protein sequence of S-(deg-RBD-1194) from Wuhan strain (N- to C-terminus):

[0203] [ka] TIFF2025081478000069.tif29166

[0204] DNA or RNA sequence of S-(deg-RBD-1194) of Wuhan S-2P strain (5' to 3' end):

[0205] [ka] TIFF2025081478000071.tif115167

[0206] Protein sequence of S-(deg-RBD-1194) from Wuhan S-2P strain (N- to C-terminus):

[0207] [ka] TIFF2025081478000073.tif44167

[0208] DNA or RNA sequence of S-(deg-RBD-122-165-234) of the Wuhan strain (from the 5' end to the 3' end):

[0209] [ka] TIFF2025081478000075.tif122167

[0210] Protein sequence of S-(deg-RBD-122-165-234) from Wuhan strain (N-terminus to C-terminus):

[0211] [ka] TIFF2025081478000077.tif50167

[0212] DNA or RNA sequence of S-(deg-RBD-122-165-234) of Wuhan S-2P strain (5' to 3' end):

[0213] [ka] TIFF2025081478000079.tif130168

[0214] Protein sequence of S-(deg-RBD-122-165-234) from Wuhan S-2P strain (N-terminus to C-terminus):

[0215] [ka] TIFF2025081478000081.tif58167

[0216] [Table 1]

[0217] [Example 5] Glycosylation affected cellular and cytokine responses To characterize the T cell response, splenocytes from immunized mice were isolated and incubated with a peptide pool of S protein and granzyme B (GrzB)-secreting T cells were measured by ELISPOT analysis. S-(deg-S2) and S-(S2-1194) were shown to induce more GrzB-secreting cells than WT and S-(deg-RBD) after incubation with full-length WT S (Figure 7A), RBD (Figure 7B) and S2 peptide (Figure 7C), suggesting that glycosylation on S2 regulated the T cell response. CD4 + T cells and CD8 + To further study the effect on T cells, isolated T cells were incubated with bone marrow-derived dendritic cells (DCs) and the WT S peptide pool, and IFNγ-secreting T cells were measured by flow cytometry. Among all vaccinated mice, IFNγ-secreting CD4 + There was no significant difference in the number of T cells (Figure 7D), but S-(deg-S2) or S-(S2-1194) mRNA vaccines elicited more IFNγ-secreting CD8 + T cells, indicating that glycosylation of S2 mediates the expression of CD8 + 7E ), suggesting that it modulated T cell responses.

[0218] To analyze cytokine expression, media from splenocytes incubated with full-length WT S peptide pools were measured by ELISA. It was shown that splenocytes from S-(deg-S2)-immunized and S-(S2-1194)-immunized mice secreted higher levels of T-helper-1 (TH1) cytokines (IFNγ, IL-2, and IL-12) (Figure 8A, B, and E), whereas splenocytes from WT-immunized and S-(deg-RBD)-immunized mice secreted higher levels of T-helper-2 (TH2) cytokines (IL-4, IL-6, and IL-13) (Figure 8C, D, and F). Overall, all RNA vaccines with glycosylation mutations increased antibody, CD4 +T cell responses and CD8 + Although T cell responses and relative cytokines were induced, stronger IFNγ-producing CD8+ T cells were observed in mice immunized with S-(deg-S2) and S-(S2-1194). + These results suggest that glycosylation on S2 regulated T cell responses and cytokine expression.

[0219] [Example 6] Deglycosylation on S2 induced the unfolded protein response To investigate how glycosylation on S2 affected immune responses, HEK293 cells were transfected with a variant prefusion stabilized S protein expression plasmid. It was shown that S-(deg-S2) and S-(S2-1194) were not expressed well, but the levels of S-(deg-S2) and S-(S2-1194) proteins were restored to some extent after treatment with MG132, a proteasome inhibitor (Figure 9). In vitro translation assays showed that mutation of glycosites in the mRNA sequence did not affect translation efficiency (Figure 10). These results suggest that removal of glycosylation from S2 caused degradation of the translated protein in vivo. Since misfolded or unfolded S proteins can accumulate in the ER for refolding or destruction through ER-associated degradation, to study whether the removal of glycosylation on S2 led to the expression of misfolded or unfolded proteins, the distribution of S proteins was examined by separating the plasma membrane and the endoplasmic reticulum (ER). After transfection of mRNA into HEK293 cells for 48 hours, all variant S proteins were present in the plasma membrane, cytosol, and ER. However, WT and S-(deg-RBD) proteins were more abundant in the plasma membrane, whereas S-(deg-S2) and S-(S2-1194) proteins were more abundant in the ER (Figure 11). In addition, HEK293 cells were transfected with mRNA encoding the soluble prefusion versions of S-(deg-S2) and S-(S2-1194), but the proteins could not be secreted into the culture medium (Figure 16). This suggests that deglycosylation of S2 affected the folding of S protein. Because an increase in unfolded S protein in the ER is thought to activate the unfolded protein response (UPR), the UPR marker proteins BiP / GRP78, XBP1 and p-eIF2α were examined in RNA-transfected HEK293 cells at 48 h.The results showed that BiP / GRP78 and XBP1 were upregulated, and the level of p-eIF2α was stronger in S-(deg-S2) and S-(S2-1194) transfected cells than in WT and S-(deg-RBD) groups (Figure 12). In addition, removal of glycosylation on S2 induced more apoptotic cells than WT and S-(deg-RBD) did (Figure 13), suggesting that deglycosylation of S2 induced a higher level of ER stress than WT and S-(deg-RBD) proteins, and that glycosylation on S2 affected protein folding to regulate the expression of UPR.

[0220] [Example 7] Glycosylation on S2 affected MHC I expression on dendritic cells (DCs).

[0221] To study whether the UPR leads to biased immune responses, major histocompatibility complex class I (MHC I) and class II (MHC II), which are essential for the presentation of internalized molecules after processing, were measured on DCs by flow cytometer. After incubating DCs with mRNA vaccine variants, MHC I / II was upregulated among all vaccines, and S-(deg-S2) or S-(S2-1194) mRNA vaccines induced more MHC I-expressing DCs than WT and S-(deg-RBD) did (Figures 14 and 17), suggesting that the UPR regulated the expression of MHC I on DCs.

[0222] [Example 8] Glycosites in the spike protein affect the stability of the S protein and subsequently the CD8 + Affects T cell responses.

[0223] To study which glycosites regulated the host immune response, we used the S-(deg-RBD) vaccine as a model system because it induced the same level of antibodies as the WT and had better neutralizing activity against the four variants of concern than the WT. Here, we removed the RBD glycosites in S2 and glycosite N-801 (S-(deg-RBD-801)) or glycosite N-1194 (S-(deg-RBD-1194)), especially glycosite N-1194, which is involved in the integrity of the S protein and its binding affinity, because those glycosites were associated with the expression of the S protein. Since glycosites N-122, N-165 and N-234 regulated the structure of RBD and influenced the neutralizing activity of the antibody, we removed these glycosites to form the S-(deg-RBD-122-165-234) vaccine (Figure 15). After transfecting HEK293 cells with the variant pre-fusion stabilized S protein expression plasmid, it was shown that S-(deg-RBD-801) and S-(deg-RBD-122-165-234) were not expressed well, and S-(deg-RBD-1194) dramatically reduced protein expression, but the protein levels of these variants were restored to some extent after treatment with MG132, suggesting that the vaccine variants induced degradation of the translated protein (Figure 21A). After confirming the expression of variant pre-fusion S proteins from mRNA-LNP in HEK293 cell line (Figure 21B), mice were immunized with various vaccines. S-(deg-RBD-801), S-(deg-RBD-1194) and S-(deg-RBD-122-165-234) induced lower IgG titers against fully glycosylated WT S (Figure 18A) and RBD protein (Figure 18B), whereas S-(deg-RBD-801) and S-(deg-RBD-122-165-234) had higher IgG titers against deglycosylated RBD antigen compared to unmodified mRNA in ELISA assay (Figure 18C). This suggests that glycosylation on these glycosites regulated antibody production.To analyze the neutralizing activity of antibodies produced from immunized mice, pseudovirus neutralization assays showed that the S-(deg-RBD-801), S-(deg-RBD-1194) and S-(deg-RBD-122-165-234) mRNA vaccines produced antibodies with reduced neutralizing activity against the WT pseudovirus (Figure 19), but in this case had better neutralizing activity against the four variants of concern than the WT (Figure 19 and Table 2).

[0224] [Table 2]

[0225] To characterize the T cell response, splenocytes from immunized mice were incubated with peptide pools of S, RBD, and S2 proteins, and granzyme B (GrzB)-secreting T cells were then measured by ELISPOT analysis. It was shown that S-(deg-RBD-801), S-(deg-RBD-1194), and S-(deg-RBD-122-165-234) induced more GrzB-secreting T cells than WT did with all peptide pools, but especially S-(deg-RBD-1194) (Figure 20 and Figures 22A, 22B). Isolated CD4 + T cells and CD8 + After incubating T cells with bone marrow-derived DCs and WT S peptide pools and measuring IFNγ-secreting T cells by flow cytometry, IFNγ-secreting CD4+ T cells were detected among all vaccinated mice. + Although there was no significant difference in the number of T cells (Figure 22C), S-(deg-RBD-801), S-(deg-RBD-1194), and S-(deg-RBD-122-165-234) mRNA vaccines induced more IFNγ-secreting CD8 + This indicates that these vaccines induce stronger CD8 T cells. +Overall, S-(deg-RBD-801), S-(deg-RBD-1194) and S-(deg-RBD-122-165-234) reduced protein expression levels in the plasmid system and induced fewer antibodies, but still induced CD8 T cell responses (Figure 20). + Enhanced T cell responses, especially in S-(deg-RBD-1194) CD8 + These results suggest that glycosylation on glycosites involved in the folding or stability of S modulated the host immune response.

[0226] Example 9. Design and synthesis of guanidine-based poly(disulfide).

[0227] We designed a series of polymers and guanidine-based and / or zwitterionic head groups attached to lipid tails to explore their ability to deliver spiked mRNA. As shown in Figure 23, the guanidine-group-containing propagator P1 and multivalent display propagator P2 facilitate the attachment of mRNA to the polymer by forming a strong salt bridge between guanidinium and phosphate in the mRNA. When the mRNA-encapsulating polymer reaches the cytoplasm, the disulfide linker could be degraded by intracellular glutathione to release the mRNA. In addition, the degraded disulfide monomer also reduces cytotoxicity by avoiding the accumulation of high molecular weight polymer inside the cell.

[0228] To prepare the polymers, monoguanidine-containing disulfide monomers were synthesized according to previously reported procedures (Gasparini, G.; Bang, E.-K.; Molinard, G.; Tulumello, DV; Ward, S.; Kelley, SO; Roux, A.; Sakai, N.; Matile, S., J. Am. Chem. Soc. 2014, 136, 6069-6074), and tri-guanidine disulfide monomers were synthesized from nitrilotriacetic acid linkers to provide trimeric guanidine monomers. Propagator Pb, which contains two distorted disulfides beside the guanidine group, was designed to form a branched configuration of the polymer. Propagators P3 and Pz were designed as spacers, which can facilitate the escape of the trapped molecules from the endosome.

[0229] FIG. 24 illustrates the design structure of the initiator (I0), propagator (P1, P2) and the polymerization / depolymerization process.

[0230] Polymerization of P1, P2, P3 and Pb was carried out in degassed aqueous solution at room temperature. Briefly, 200 mM propagator P in the presence of 5 mM initiator 1 and 1M pH 7 TEOA buffer was vigorously stirred for 30 min. Termination was performed by adding 0.5 M iodoacetamide. To screen the optimal polymer for efficient intracellular delivery of mRNA, copolymerization of various propagators was performed and their encapsulation capacity and transfection efficiency were evaluated by GFP encoding mRNA in HEK293T cells. Copolymers (P1 / P3) and (P2 / P3) were prepared in a ratio of 2:1, and (P1 / Pb) and (P2 / Pb) were prepared in a ratio of 4:1.

[0231] To screen the optimal polymer for best encapsulation and efficient intracellular delivery of mRNA, eight types of synthetic homopolymers and heterocopolymers were tested for their ability to encapsulate GFP mRNA. As shown in Figure 25A, all copolymers that possessed guanidine groups were able to inhibit the translocation of GFP mRNA at N / P ratio = 10. Notably, P2 / P3 and P2 / Pb copolymers with triguanidine moieties provided higher ability to form complexes with mRNA, which was comparable to the results mediated by polyethyleneimine (PEI), a traditionally used transfection agent. The average size of the resulting nanocluster P2 / P3-mRNA complexes was approximately 90 nm by TEM (Figure 25B). In addition, branched polymers P1 / Pb and P2 / Pb also showed similar ability for encapsulating mRNA. Branched guanidinium has attracted attention due to its good accessibility and flexibility to functional materials.

[0232] Next, we evaluated the transfection efficiency of GFP-mRNA in HEK293T cells by using various copolymers. First, we found that P1 / P3 copolymer exhibited good ability to transfect mRNA at N / P=10, which was more efficient than P1 or P3 alone and PEI (Figure 26A). The results showed that neither P1 nor P3 polymers were desirable for intracellular delivery, while the bifunctional copolymer P1 / P3 greatly transfected mRNA. The random spacing of guanidine groups by copolymerization with diethylenetriamine spacer may be suitable to match the phosphate charge of nucleotides. Then, we studied the influence of various copolymers on transfection activity. In Figure 26B, the combination of P2 / P3 showed improved GFP expression. This indicated that the triguanidine group enhanced the encapsulation of mRNA and also liberated mRNA when delivered to the cytoplasm. However, branched poly(disulfide) P1 / Pb and P2 / Pb did not show satisfactory release of mRNA cargo, whereas complete complexation with mRNA was achieved by using P2 / Pb (Figure 26A). We further optimized the N / P ratio (1, 5, 10 and 20) utilizing the outstanding copolymer P2 / P3. The best performance for transfection was found to be 10 (Figure 26C).

[0233] Based on the results of GFP mRNA, wild-type spike mRNA was prepared and encapsulated by polyGu at various N / P ratios (Figure 27). PolyGu showed sufficient ability to neutralize the charge of spike mRNA, and successfully achieved this ability at an N / P ratio of 1.

[0234] Next, we transfected spike mRNA into HEK293T cells and performed Western blotting. HEK293T cells were transfected with 3 μg of spike mRNA. 48 hours after transfection, cells were analyzed for spike expression via Western blotting using spike-specific antibodies. The results showed a prominent band of SARS-Cov-2 spike at approximately 250 kDa, while PBS buffer containing spike mRNA was used as a negative control (Figure 28), proving the feasibility of polyGu as a nanocarrier for mRNA transfection in vitro. In addition, polyGu exhibited no obvious cytotoxicity up to 10 μg (50 μg / mL) (Figure 29). Meanwhile, lipid nanoparticles (LNPs) exhibited much higher toxicity to cells with high complex loading.

[0235] In conclusion, we developed a series of poly(disulfides) and demonstrated that the combination of guanidyl groups and zwitterionic spacers exhibited great efficiency for in vitro mRNA delivery. Efficient intracellular delivery through the thiol-mediated uptake pathway by the strained disulfides is cleavable under intracellular glutathione. In addition, the degradation of the polymer also minimizes cytotoxicity compared to the commonly used LNPs against SARS-Cov-2.

[0236] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0237] Although the present invention has been described in some detail by way of illustration and example to enable a clear understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this invention, that certain changes and modifications can be made to the present invention without departing from the spirit and scope of the appended claims.

Claims

1. A modified nucleic acid molecule encoding a modified spike protein comprising one or more amino acid substitutions of asparagine (N) to glutamine (Q) in the N-linked glycosylation sequon (N-X-S / T), where X is any amino acid residue except proline and S / T represents a serine or threonine residue.

2. 2. The modified nucleic acid molecule of claim 1, comprising a deletion or addition of one or more amino acids in an N-linked glycosylation sequon (N-X-S / T) to remove an N-linked glycan sequon.

3. 2. The modified nucleic acid molecule of claim 1, comprising one or more amino acid substitutions of S / T to alanine (A) at an O-linked glycosylation site to eliminate the O-linked glycosylation site.

4. A modified nucleic acid molecule which is an mRNA or which is double-stranded or single-stranded DNA.

5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein is derived from the SARS-CoV-2 spike protein.

6. 5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the spike protein comprises an amino acid sequence of SEQ ID NO: 2, 16, 18 or 20, or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence of SEQ ID NO: 2, 16, 18 or 20.

7. The modified nucleic acid molecule of claim 6, wherein the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, 16, 18 or 20 is an mRNA comprising a nucleotide sequence of SEQ ID NO: 1, 15, 17 or 19, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 1, 15, 17 or 19, respectively.

8. 5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO: 4, 22, 24 or 26, and wherein the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site.

9. The modified nucleic acid molecule of claim 8, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:4, 22, 24 or 26 comprises a nucleotide sequence of SEQ ID NO:3, 21, 23 or 25, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:3, 21, 23 or 25, respectively.

10. 5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO: 6, 28, 30 or 32, and wherein the modified spike protein comprises an S2 subunit lacking a glycosylation site.

11. The modified nucleic acid molecule of claim 10, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 6, 28, 30 or 32 comprises a nucleotide sequence of SEQ ID NO: 5, 27, 29 or 31, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 5, 27, 29 or 31, respectively.

12. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 8 or 34, and the modified spike protein comprises an S2 subunit consisting of a single glycosylation site. In some embodiments, the single glycosylation site is at position N1194.

13. The modified nucleic acid molecule of claim 12, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 8 or 34 comprises a nucleotide sequence of SEQ ID NO: 7 or 33, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 7 or 33, respectively.

14. 5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO: 10 or 36, and the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site and an amino acid substitution of N801 to Q801.

15. The modified nucleic acid molecule of claim 14, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 10 or 36 comprises a nucleotide sequence of SEQ ID NO: 9 or 35, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 9 or 35, respectively.

16. 5. The modified nucleic acid molecule of any one of claims 1 to 4, wherein the modified spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 38, and the modified spike protein comprises a receptor binding domain (RBD) lacking a glycosylation site and an amino acid substitution of N1194 to Q1194.

17. The modified nucleic acid molecule of claim 16, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 12 or 38 comprises a nucleotide sequence of SEQ ID NO: 11 or 37, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 11 or 37, respectively.

18. 5. The modified nucleic acid molecule of any of claims 1-4, wherein the modified spike protein described herein comprises the amino acid sequence of SEQ ID NO: 14 or 40, and the modified spike protein comprises a modified receptor binding domain (RBD) lacking a glycosylation site, and amino acid substitutions of N122 to Q122, N165 to Q165, and N234 to Q234.

19. The modified nucleic acid molecule of claim 18, wherein the modified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 14 or 40 comprises a nucleotide sequence of SEQ ID NO: 13 or 39, respectively, or a nucleotide sequence having at least about 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO: 13 or 39, respectively.

20. A modified nucleic acid molecule according to any one of claims 1 to 4, wherein the modified spike protein comprises an S1 subunit lacking a glycosylation site.

21. A modified nucleic acid molecule according to any one of claims 1 to 4, wherein the modified spike protein comprises both an S1 subunit and an S2 subunit lacking a glycosylation site.

22. 5. The modified nucleic acid molecule of claim 1, wherein the S-(deg-RBD) mRNA has the sequence of SEQ ID NO: 3, 21, 23 or 25, the S-(deg-S2) mRNA or DNA has the sequence of SEQ ID NO: 5, 27, 29 or 31, the S-(deg-S2-1194) mRNA or DNA has the sequence of SEQ ID NO: 7 or 33, the S-(deg-RBD-801) mRNA or DNA has the sequence of SEQ ID NO: 9 or 35, the S-(deg-RBD-1194) mRNA or DNA has the sequence of SEQ ID NO: 11 or 37, and the S-(deg-RBD-122-165-234) mRNA or DNA has the sequence of SEQ ID NO: 13 or 39.

23. A modified nucleic acid molecule according to any one of claims 1 to 22, which may be used as a coronavirus mRNA vaccine.

24. The modified nucleic acid molecule according to any one of claims 1 to 23, wherein immunization with the modified nucleic acid molecule causes upregulation of BiP / GRP78, XBP1 and p-eIF2α, inducing cell apoptosis and CD8 T cell responses.

25. The modified nucleic acid molecule of any one of claims 1 to 23, wherein the coronavirus (CoV) is selected from the group consisting of alpha-CoV, beta-CoV, gamma-CoV, delta-CoV2 and omicron-CoV2.

26. 24. A combo vaccine comprising the mRNA vaccine of claim 23 and one or more additional vaccines.

27. 27. The combo vaccine of claim 26, wherein the additional vaccine is selected from one or more of a COVID-19 vaccine, an influenza (flu) vaccine, an adenovirus vaccine, an anthrax vaccine, a cholera vaccine, a diphtheria vaccine, a hepatitis A or B vaccine, an HPV vaccine, a measles vaccine, a mumps vaccine, a smallpox vaccine, a rotavirus vaccine, a tuberculosis vaccine, a pneumococcal vaccine, and a Haemophilus influenzae type b vaccine, and any combination thereof.

28. A nanoparticle which is guanidine-based and is used as a carrier for delivering a modified nucleic acid molecule according to any one of claims 1 to 25 to a subject.

29. 29. The nanoparticle of claim 28, which is a liposome or a polymersome.

30. 28. An mRNA nanocluster comprising the vaccine of claim 26 or 27 formulated in a lipid nanoparticle.

31. The mRNA nano-cluster of claim 30, wherein the lipid nanoparticle is a biodegradable lipid nanoparticle.

32. 32. The mRNA nanocluster of claim 30 or 31, wherein the lipid nanoparticle is a guanidine-based polymer.

33. An mRNA nanocluster comprising lipid nanoparticles encapsulating an mRNA vaccine, the biodegradable lipid nanoparticles comprising guanidine-based and zwitterionic units, the guanidine-based and zwitterionic groups attached to lipid tails, the guanidine-based groups attaching to the mRNA, thereby forming salt bridges between the guanidinium groups and phosphates in the mRNA.

34. The guanidine-based polymer is P1, P2, P3, Pb or Pz: 【Chemistry 1】 (Wherein, R is 【Chemistry 2】 It is.) The nanoparticle of claim 28 or 29 or the mRNA nanocluster of any one of claims 30 to 33,

35. The nanoparticle of claim 28 or 29 or the mRNA nanocluster of any one of claims 30 to 34, wherein the guanidine-based polymer forms a copolymer, such as P1 / P3 copolymer, P2 / P3 copolymer, P1 / Pb copolymer, P2 / Pb copolymer, P1 / Pz copolymer and P2 / Pz copolymer.

36. The guanidine-based and zwitterionic nanoparticles are selected from P1 and / or P2 and Pz 【Chemistry 3】 (In the formula, R= 【Chemistry 4】 ） The nanoparticle of claim 28 or 29 or the mRNA nanocluster of any one of claims 30 to 35, which is a mixture of

37. The nanoparticle of claim 28 or 29 or the mRNA nanocluster of any one of claims 30 to 36, having a nanoparticle / mRNA (N / P) ratio of about 1 to about 100.

38. A nanoparticle according to claim 28 or 29 or an mRNA nanocluster according to any one of claims 30 to 37, having a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

39. A nanoparticle composition comprising nanoparticles having the mRNA vaccine of claim 23 attached thereto.

40. A method of preventing or treating a coronavirus infection, comprising administering to a subject infected with or at risk of infection with a coronavirus an effective amount of a modified nucleic acid molecule of any one of claims 1 to 25, a nanoparticle of claims 28 or 29, an mRNA nanocluster of any one of claims 30 to 38, or a nanoparticle composition of claim 39.

41. A method of boosting an adaptive immune response, comprising administering to a subject an effective amount of a modified nucleic acid molecule of any one of claims 1 to 25, a nanoparticle of claim 28 or 29, an mRNA nanocluster of any one of claims 30 to 38, or a nanoparticle composition of claim 39.

42. The method of claim 41 or 42, wherein the modified nucleic acid molecule of any one of claims 1 to 25, the nanoparticle of claim 28 or 29, the mRNA nanocluster of any one of claims 30 to 38 or the nanoparticle composition of claim 39 is administered as an initial dose and two, three or four booster doses.

43. The method of claim 41 or 42, wherein the modified nucleic acid molecule of any one of claims 1 to 25, the nanoparticle of claim 28 or 29, the mRNA nanocluster of any one of claims 30 to 38, or the nanoparticle composition of claim 39 is administered as an initial dose and at least one booster dose about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months after the initial dose.

44. The method of claim 41 or 42, wherein the dose is in the range of 5 μg to 50 μg of the modified nucleic acid molecule according to any one of claims 1 to 25.

45. 43. The method of claim 41 or 42, wherein the dose is 30 μg.

46. The method of claim 41 or 42, wherein the modified nucleic acid molecule of any one of claims 1 to 25, the nanoparticle of claim 28 or 29, the mRNA nanocluster of any one of claims 30 to 38 or the nanoparticle composition of claim 39 is administered via intravenous, intramuscular, intradermal or subcutaneous routes, or by injection or nasal spray.

47. TESIVRFPNITNNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), GPKKSTNLVKNKC (SEQ ID NO: 47), NCDVVIGIVNNTVY (SEQ ID NO: 48), PELDSFK An immunogenic peptide comprising at least one amino acid sequence selected from the group consisting of EELDKYFKNHTS (SEQ ID NO:49), VNIQKEIDRLNEVA (SEQ ID NO:50), NLNESLIDLQ (SEQ ID NO:51) and LGKYEQYIKWP (SEQ ID NO:52), or an amino acid sequence having at least about 99%, 98%, 97%, 96%, 95% or 90% identity to any of SEQ ID NOs:41-52.

48. 48. The immunogenic peptide of claim 47, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 41-43 and 45-51.

49. An immunogenic composition comprising an immunogenic peptide according to claim 47 or 48.

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