Vaccine

Abstract

The present disclosure relates to methods of inducing a pan sand coronavirus variant immune response to treat and prevent coronavirus infection.

Description

Technical Field

[0001] The present disclosure relates to immunogenic compositions that can be used to generate a pan-sarbecoronavirus immune response as part of a prime-boost vaccination regimen. Background Art

[0002] Many vaccines are approved for the treatment of COVID-19, but data suggest that vaccination effectiveness may vary depending on the variant. Since SARS-CoV-2 was first reported, multiple variants of concern have evolved. Therefore, vaccines targeting multiple SARS-CoV-2 strains could be of great benefit for immunization against COVID-19.

[0003] However, a phenomenon known as "original antigenic sin" has been observed. This refers to the recall of dominant epitopes from the initial priming vaccine in booster vaccinations, even when the booster vaccine targets a different variant. This poses a challenge to achieving broad neutralizing responses against multiple variants.

[0004] Nanoparticles displaying antigens have been proposed as vaccines to elicit broadly neutralizing responses against highly mutated viruses such as sarbecoronavirus or influenza virus.

[0005] The strategy is based on the idea that virus-like particles (VLPs) can display multiple variants of viral-derived antigens, such as SARS-CoV-2. Displaying multiple antigens can generate immune responses against conserved epitopes, thereby limiting the possibility of immune escape caused by mutations.

[0006] This approach has been successfully used in many in vivo and preclinical vaccination studies. For example, Boyoglu-Barnum et al. (Nature, 592; 623-628 (2021)) showed that quadrivalent HA-VLPs induce broad protection against influenza. Cohen et al. (Science, 377, eabq0839 (2022)) showed that nanoparticles displaying eight different sabie coronavirus spike receptor binding domains can protect against multiple sabie coronaviruses in an animal model. Therefore, this approach is promising.

[0007] However, these examples of broad neutralization responses were achieved using protein-based nanoparticle vaccines, which require expensive and complex manufacturing processes. This is in contrast to mRNA vaccines, which are generally cheaper to manufacture and have more robust manufacturing processes, which is very advantageous when vaccines need to be modified to address immune escape. One disadvantage associated with mRNA is the general size limit of the vaccine that can be administered. For example, it can be challenging to develop an mRNA vaccine that encodes up to eight different antigen sequences, each fused to a multimerization element that enables the formation of a VLP.

[0008] Therefore, it is desirable to develop a vaccine that combines the advantages of mRNA technology with the broad neutralization achieved with multivalent antigen nanoparticles. Summary of the Invention

[0009] The present disclosure relates to an immunogenic composition that can be used to generate a pan-Sabecoronavirus immune response as part of a prime-boost vaccination regimen. The composition comprises mRNA encoding a single spike protein from a Sabeicoronavirus variant and a coding sequence for a multimerization unit that forms a multimeric complex upon assembly in vivo.

[0010] Some examples of the present disclosure are summarized below. This list is merely illustrative and not an exhaustive list of all examples provided by the present disclosure.

[0011] 1. A method of inducing a pan-Sabemicoronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine against one or more first Sabemicoronavirus variants (Variant 1), the method comprising administering to the individual one or more doses of a second SARS-CoV-2 vaccine, wherein the second SARS-CoV-2 vaccine comprises mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a Sabemicoronavirus variant that is different from Variant 1 (Variant 2), wherein the S protein is encoded as an S protein-multimerization subunit fusion, and wherein the method induces a pan-Sabemicoronavirus variant 1 and variant 2 immune response in the individual and induces an immune response against one or more additional Sabemicoronavirus variants that are different from Variant 1 and Variant 2.

[0012] 2. The method of clause 1, wherein the S protein-multimerization subunit fusion is a protein-ferritin subunit fusion.

[0013] 3. The method of clause 1 or clause 2, wherein the mRNA of the second SARS-CoV-2 vaccine is formulated in lipid nanoparticles (LNPs).

[0014] 4. The method of any one of clauses 1 to 3, wherein variant 2 is SARS-CoV-2 Omicron BA.4 / 5.

[0015] 5. The method according to clause 4, wherein the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID NO.11.

[0016] 6. A method according to any one of clauses 1 to 3, wherein variant 2 is SARS-CoV-2OmicronXBB.1.5.

[0017] 7. The method of clause 6, wherein the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID NO. 17.

[0018] 8. The method of any one of clauses 1 to 7, wherein the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabich coronavirus S protein in the form of a fusion protein capable of assembling in vivo to form nanoparticles.

[0019] 9. The method of any one of clauses 1 to 8, wherein the method induces an immune response against one or more additional Sabich coronavirus variants that differ mutationally from variant 1 and / or variant 2.

[0020] 10. The method of any one of clauses 1 to 9, wherein the second SARS-CoV-2 vaccine is a monovalent vaccine.

[0021] 11. A method according to any one of clauses 1 to 9, wherein the second SARS-CoV-2 vaccine is a bivalent vaccine comprising additional mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimeric subunit fusion and is derived from a Sabeucoronavirus variant that is different from variant 2 and the same as or different from variant 1.

[0022] 12. The method of any one of clauses 1 to 11, wherein the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 4 months after the last dose of the first SARS-CoV-2 vaccine.

[0023] 13. The method of clause 12, wherein the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 6 months after the last dose of the first SARS-CoV-2 vaccine.

[0024] 14. An immunogenic composition comprising mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a first Sabicorina coronavirus variant for use in inducing a pan-Sabicorina coronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine comprising or encoding an immunogen from a second Sabicorina coronavirus variant,

[0025] wherein the S protein is encoded as an S protein-multimerized subunit fusion, and

[0026] The immunogenic composition is used to induce an immune response against the first Sabei coronavirus variant and the second Sabei coronavirus variant and against at least a third Sabei coronavirus variant.

[0027] 15. An immunogenic composition for use according to clause 14, wherein the S protein-multimerization subunit fusion is a protein-ferritin subunit fusion.

[0028] 16. An immunogenic composition for use according to clause 14 or 15, wherein the mRNA is formulated in lipid nanoparticles (LNPs).

[0029] 17. An immunogenic composition for use according to any one of clauses 14 to 16, wherein the first Sabicolaou coronavirus variant is SARS-CoV-2 Omicron BA.4 / 5.

[0030] 18. The immunogenic composition for use according to clause 17, wherein the mRNA comprises or consists of the mRNA sequence of SEQ ID NO. 11.

[0031] 19. An immunogenic composition for use according to any one of clauses 14 to 16, wherein the first Sabicolaou coronavirus variant is SARS-CoV-2 Omicron XBB.1.5.

[0032] 20. The immunogenic composition for use according to clause 19, wherein the mRNA comprises or consists of the mRNA sequence of SEQ ID NO. 17.

[0033] 21. An immunogenic composition for use according to any one of clauses 14 to 20, wherein the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabei coronavirus S protein in the form of a fusion protein capable of assembling in vivo to form nanoparticles.

[0034] 22. An immunogenic composition for use according to any one of clauses 14 to 21 wherein the third Sabecovoravirus variant differs from the first and / or second Sabecovoravirus variant in mutations.

[0035] 23. An immunogenic composition for use according to any one of clauses 14 to 22, wherein the immunogenic composition is a monovalent composition.

[0036] 24. An immunogenic composition for use according to any one of clauses 14 to 22, wherein the immunogenic composition is a bivalent composition further comprising mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimeric subunit fusion and is derived from a Sabei coronavirus variant that is different from the first Sabei coronavirus variant and is the same as or different from the second Sabei coronavirus variant. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0038] Figure 1 shows the improved pan-variant immunogenicity in vaccinated mice of mRNA molecules according to the present disclosure encoding the Wuhan D614G antigen fused to ferritin via a linker (Group 2), the BA.4 / 5 antigen fused to ferritin via a linker (Group 3), or a combination of the two (Wuhan D614G and BA.4 / 5 antigens fused to ferritin via a linker; Group 4), compared to encoding two native spike antigens (Wuhan D614G and BA.4 / 5; Group 1). Figure 1a Neutralizing antibody titers against BA.4 / 5 pseudovirus at day 180 are shown. Figure 1b It is shown that after the third vaccination (such as Figure 2 Neutralizing antibody titers 14 days after administration (shown on day 231) are shown. The numbers under each data bar indicate the geometric mean titer. Figure 1c Shown are neutralizing antibody titers measured 14 days after the third vaccination with mRNA molecules encoding native XBB.1.5 spike protein or XBB.1.5 spike protein as VLP antigen, as measured in vaccinated mice (vs. Figure 2 The numbers within each data bar indicate the geometric mean titer.

[0039] Figure 2 Schematic diagram showing the design of the boost vaccination mouse study.

[0040] Figure 3 Shown are neutralizing antibody titers against a panel of variant pseudoviruses elicited by either mRNA-native Delta or mRNA-VLP Delta vaccines in nonhuman primates 14 days after the second mRNA immunization.

[0041] Figure 4 Shown is the persistence of neutralizing antibody titers against Delta variant pseudovirus elicited by mRNA-native Delta or mRNA-VLP Delta vaccines in nonhuman primates up to 196 days after the first mRNA immunization.

[0042] Figure 5 Shown are the frequencies of spike-specific memory B cells generated by mRNA-natural Delta or mRNA-VLP Delta vaccines in non-human primates before and 28 days after the second immunization.

[0043] Figure 6 Shown are counts of long-lived antibody-secreting cells in the bone marrow of nonhuman primates 196 days after the first immunization with mRNA-native Delta or mRNA-VLP Delta vaccines.

[0044] Figure 7 Shown are the frequencies of spike-specific memory CD4+ T cells generated by mRNA-native Delta or mRNA-VLP Delta vaccines in non-human primates 28 days after the second immunization.

[0045] Figure 8 Schematic diagram showing the design of the booster vaccination nonhuman primate study.

[0046] Figure 9 Shown are neutralizing antibody titers against the original Wuhan_D614G variant pseudovirus elicited by mRNA-native XBB.1.5 or mRNA-VLP XBB.1.5 vaccines in vaccinated nonhuman primates measured before the third immunization (day 246) and 14 days after the third immunization (day 260).

[0047] Figure 10 Shown are neutralizing antibody titers against Omicron BA.4 / 5 variant pseudoviruses elicited by mRNA-native XBB.1.5 or mRNA-VLP XBB.1.5 vaccines in vaccinated nonhuman primates measured before the third immunization (day 246) and 14 days after the third immunization (day 260).

[0048] Figure 11Shown are neutralizing antibody titers against Omicron XBB.1.5 variant pseudoviruses elicited by mRNA-native XBB.1.5 or mRNA-VLP XBB.1.5 vaccines in vaccinated nonhuman primates measured before the third immunization (day 246) and 14 days after the third immunization (day 260).

[0049] Figure 12 Shown are the fold increases in neutralizing antibody (nAb) titers 14 days after the third immunization against the different variant pseudoviruses tested in vaccinated nonhuman primates given either mRNA-native XBB.1.5 or mRNA-VLP XBB.1.5 vaccines. DETAILED DESCRIPTION

[0050] All cited references are incorporated herein by reference in their entirety.

[0051] Many modifications and other examples of the disclosure set forth herein will occur to those skilled in the art having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it should be understood that the disclosure is not limited to the specific examples disclosed, and that modifications and other examples are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0052] Units, prefixes and symbols can be expressed in a form accepted by its International System of Units (SI). Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively. Numerical ranges include numerical values ​​that limit the range. Amino acids can be represented herein by their commonly known three letter symbols or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be represented by their commonly accepted single-letter codes. The terms defined below are more completely defined by reference to the entire specification.

[0053] definition

[0054] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which can be single-stranded or double-stranded, and which can contain non-natural or altered nucleotides, such as modified uridine. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include analogs of RNA or DNA made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated and / or capped polynucleotides) as equivalents. Nucleic acids are typically connected via phosphate bonds to form nucleic acid sequences or polynucleotides, although many other connections are known in the art (e.g., phosphorothioate, borane phosphate, etc.).

[0055] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present disclosure are antibody-based, in some aspects, the polypeptides may exist as single chains or associated chains.

[0056] "5'-untranslated region (5'-UTR)" has the common meaning recognized by those skilled in the art. It is the region of a nucleic acid molecule located 5' of a coding sequence and is not translated into protein. The 5'-UTR generally begins at the transcription start site and ends before the start codon of the coding sequence.

[0057] "3'-untranslated region (3'-UTR)" has the common meaning recognized by those skilled in the art. It is the region of a nucleic acid molecule located 3' from the coding sequence and is not translated into protein. The 3'-UTR is generally 3' from the coding sequence. If the molecule contains a polyadenylation signal, the 3'-UTR is generally located between the coding sequence and the polyadenylation signal.

[0058] A "coding sequence" is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). A coding sequence typically encodes a polypeptide. The coding sequences disclosed herein are operably linked to the 5' and 3' UTRs described herein.

[0059] "Messenger RNA (mRNA)" is any RNA that encodes (at least one) protein (naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. Those skilled in the art will understand that, unless otherwise indicated, the nucleic acid sequences set forth in this application may list "T" in representative DNA sequences, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with a "U." Therefore, any DNA disclosed and identified herein by a particular sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence that is complementary to the DNA, wherein each "T" of the DNA sequence is replaced with a "U."

[0060] "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or its derivatives in combination with an organic base (e.g., purine or pyrimidine) or its derivatives (also referred to herein as "nucleobase"). A nucleic acid may comprise one or more regions of linked nucleosides. Such regions may have variable backbone bonds. The bonds may be standard phosphodiester bonds, in which case the nucleic acid will comprise a region of nucleotides ("nucleotide" refers to a nucleoside, including a phosphate group).

[0061] "Expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); and (3) translation of the RNA into a polypeptide or protein.

[0062] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient is effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. The composition may be sterile.

[0063] As used herein, the terms "subject," "individual," and "patient" are used interchangeably. A subject can be an animal. In some aspects, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some aspects, the subject is a human.

[0064] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] It should be understood that wherever aspects are described herein using the language "comprising," other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprising," "including," "containing," and "having" and the like may mean "including," "comprising," and the like; "consisting essentially of" is open-ended, allowing for the presence of more than those listed, as long as the basic or novel characteristics of the listed ones are not changed by the presence of the more than those listed, but excluding prior art aspects.

[0066] Unless otherwise specified or obvious from the context, the term "or" as used herein should be understood to be inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0067] Coronavirus vaccine

[0068] The recent Covid-19 pandemic has led to an urgent need for improved vaccines targeting the coronavirus of interest. So far, several variants of SARS-CoV-2 have been identified, some of the most infectious of which are Delta variants and Omicron variants. The vaccines currently approved for the treatment of SARS-CoV-2 all cover stimulation immunity against SARS-CoV-2 spike protein. Although many mutations in the receptor binding domain of the spike protein have been identified in the new variants of SARS-CoV-2, it is believed that this leads to increased vaccine resistance in the emerging variants (Zhao, J. et al., Environmental research, (2022), 206 (112240)). Studies estimate that the efficacy of current vaccines against the Delta variant of SARS-CoV-2 is about one-fifth to one-third of the efficacy against the Alpha variant (Planas, D. et al., Nature, (2021), 596, 276-280).

[0069] Therefore, there is an ongoing need for improved vaccines overall, including those for the prevention and treatment of coronaviruses.

[0070] Coronavirus virions contain numerous glycosylated spike (S) proteins that protrude from the virion surface. These S proteins form a trimer structure and mediate viral entry into host cells, making them a prime target for vaccine design.

[0071] The coronavirus spike protein is 1,273 amino acids long and consists of a signal peptide and S1 and S2 subunits. The S1 subunit contains the receptor binding domain (RBD), which recognizes and binds to the specific host cell receptor, angiotensin-converting enzyme 2 (ACE2). The S2 subunit mediates viral-cell membrane fusion.

[0072] Therefore, in some examples, the nucleic acid molecule comprises a sequence encoding an S protein or an antigenic fragment thereof. When delivered to a host cell, the S protein is translated and processed in the host cell, resulting in the trimerized S protein being displayed on the host cell surface.

[0073] In some examples, the nucleic acid molecule comprises sequences encoding CoV S protein and ferritin, wherein the CoV S protein and ferritin assemble to form nanoantigen particles.

[0074] The S protein may be in a pre-fusion conformation. In addition, the S protein may include K986P and / or V987P mutations. These mutations stabilize the S protein in a pre-fusion conformation (Wrapp D et al., Science (2020), 367: 1260-1263).

[0075] In one example, the nucleic acid molecules of the present disclosure encode an antigenic fragment thereof that is a receptor binding domain (RBD).

[0076] Vaccines according to the present disclosure comprise nucleic acid molecules encoding SARS-CoV antigens. The antigens may be antigens as described anywhere herein. In one example, the SARS-CoV antigens may be selected from any SARS-CoV-2 antigen or an immunogenic fragment thereof. In one example, the nucleic acid molecule is an mRNA sequence encoding a SARS-CoV-2 antigen selected from one or more of the following variants: Wuhan, Alpha, Beta, Delta, and Omicron, optionally selected from subvariants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, and XBB.1.5. In one example, the nucleic acid molecule is an mRNA sequence encoding a SARS-CoV-2 spike (S) protein or an immunogenic fragment thereof, wherein the SARS-CoV-2 spike (S) protein or an immunogenic fragment thereof is selected from one or more of the following variants: Wuhan, Alpha, Beta, Delta and Omicron, optionally selected from subvariants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1 and XBB.1.5.

[0077] The vaccine also comprises a nucleic acid sequence encoding a multimerization unit. In one example, the multimerization unit is ferritin. The multimerization unit can be a scaffold for the antigen particle. In some examples, the ferritin is Helicobacter pylori ferritin. In some examples, the nucleic acid sequence encoding ferritin can be modified to remove native glycosylation sites. In some examples, the nucleic acid molecule comprises a sequence encoding an antigen protein and ferritin, wherein the antigen protein and ferritin assemble to form nanoantigen particles.

[0078] The vaccine according to the present disclosure further comprises a sequence encoding a linker. The linker may be encoded between the antigen and the multimerization unit (optionally ferroin) such that the antigen is fused to the multimerization unit in the encoded molecule.

[0079] In one example, the vaccine comprises RNA 5'- and 3'-UTR sequences and mRNA coding sequences.

[0080] Multimerization unit

[0081] Vaccines as described herein include nucleic acid molecules encoding fusion proteins, and the fusion proteins include vaccine antigens connected to multimerization units. In some instances, such multimerization units confer desired properties on antigens encoded by nucleic acid molecules. For example, examples show that multimerization units improve the immunogenicity of antigens (e.g., COVID spike proteins) compared to the immunogenicity of the same antigens not expressed with multimerization units. In addition, multimerization units provided herein improve pan-variant reactions against antigens. For example, nucleic acid molecules comprising coding sequences encoding COVID spike protein-multimerization unit fusion proteins provided herein, when administered as booster vaccines, induce a wider immune response against SARs-CoV-2 variants compared to single spike proteins.

[0082] In some instances, the polymerizing unit is a protein that can self-assemble into protein nanoparticles that are highly symmetrical, stable, and structurally organized with a diameter of 10 nm to 150 nm, a highly suitable size range for optimal interaction with various cells of the immune system. In some instances, viral proteins or virus-like particles (VLPs) can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some instances, the polymerizing unit is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of approximately 22 nm, and it lacks nucleic acid and is therefore non-infectious (Lopez-Sagaseta, J. et al., Computational and Structural Biotechnology Journal 14 (2016) 58-68). In some instances, the polymerizing unit is hepatitis B core antigen (HBcAg) that self-assembles into particles with a diameter of 24 nm to 31 nm, which is similar to the viral core obtained from human liver infected with HEY. HBcAg self-assembles to produce two types of nanoparticles of different sizes, with diameters of 300 Å and 360 Å, corresponding to 180 or 240 protomers. In some examples, the antigen is fused to HBsAG or HBcAG to promote the self-assembly of antigen-displaying nanoparticles.

[0083] In some examples, the multimerization unit is selected from the group consisting of the self-assembling proteins ferritin, lumazine synthase, and encapsulin.

[0084] Ferritin is a protein whose main function is to store iron in cells. Ferritin consists of 24 subunits, each of which consists of four α-helical bundles, which self-assemble into a quaternary structure with octahedral symmetry (Cho KJ et al., J Mol Biol. 2009; 390: 83-98). Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin consists of 24 identical protomers, while in animals, there are ferritin light chains and heavy chains, which can be assembled alone or in different ratios into particles of 24 subunits (Granier T. et al., J Biol Inorg Chem. 2003; 8: 105-111; Lawson DM et al., Nature. 1991; 349: 541-544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Therefore, ferritin nanoparticles are very suitable for carrying and exposing antigens.

[0085] Dioxetine synthase (LS) is also very suitable as a nanoparticle platform for antigen display. LS (which is responsible for the penultimate catalytic step in riboflavin biosynthesis) is an enzyme present in a variety of organisms (including archaea, bacteria, fungi, plants and eubacteria) (Weber SEFlavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). The LS monomer is 150 amino acids long and consists of a β-sheet and a series of α-helices on both sides. Many different quaternary structures of LS have been reported, illustrating its morphological versatility: from homopentamers to symmetrical assemblies of 12 pentamers forming a capsid with a diameter of 150A. Even LS cages with more than 100 subunits have been described (Zhang X. et al., J Mol Biol. 2006; 362: 753-770).

[0086] Encapsulin, a novel protein cage nanoparticle isolated from the thermophilic bacterium Thermotoga maritima, can also be used as a platform for presenting antigens on the surface of self-assembled nanoparticles. Encapsulin is assembled from 60 copies of the same 31 kDa monomer with a thin, icosahedral T=1 symmetrical cage structure with an inner and outer diameter of 20 nm and 24 nm, respectively (Sutter M. et al., Nat Struct Mol Biol. 2008, 15: 939-947). Although the exact function of encapsulin in Thermotoga maritima is not yet clearly understood, its crystal structure has recently been solved, and it is speculated that its function is to serve as a cellular compartment that encapsulates proteins involved in the oxidative stress response (such as DyP (dye-decolorizing peroxidase) and Flp (ferritin-like protein) (Rahman pour R. et al., FEES J. 2013, 280: 2097-2104).

[0087] Linker

[0088] Vaccine disclosed herein can comprise nucleic acid that encodes fusion protein.In this example, each domain of fusion protein (for example, antigen and multimerization unit) can be separated by the coding sequence that encodes connexon sequence.In some instances, connexon can be glycine-serine connexon.

[0089] In some examples, the glycine-serine linker has the following amino acid sequence: GSGGSG (SEQ ID NO: 4). In some examples, the glycine-serine linker is encoded by SEQ ID NO: 5.

[0090] The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs disclosed herein (e.g., encoded by the nucleic acid molecules provided herein). The skilled artisan will also appreciate that other polycistronic constructs (nucleic acid molecules encoding more than one antigen / polypeptide within the same molecule) may be suitable for use as provided herein.

[0091] In various examples, vaccines according to the present disclosure comprise a promoter, a 5'-UTR and a 3'-UTR (flanking the antigen coding sequence), and a polyadenylation signal in the 5' to 3' transcriptional direction.

[0092] In one embodiment, the vaccine described herein further comprises a 5'-cap structure, optionally a cap 1 structure. Suitable cap structures and methods for generating suitable cap structures are disclosed in WO 2017 / 053297 and Tusup et al., "Design of in vitro Transcribed mRNA Vectors for Research and Therapy," Chim Int J Chem. 2019; 73(5): 391–394, both of which are hereby incorporated by reference. 5'-capping of the polynucleotide can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure, according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; 35G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to generate a "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The cap 1 structure can be generated using both a vaccinia virus capping enzyme and a 2'-O transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure, followed by 2'-O methyltransferase 2'-O-methylation of the 5'-penultimate nucleotide. The cap 3 structure can be generated from the cap 2 structure, followed by 2'-O methyltransferase 2'-O-methylation of the 5'-penultimate nucleotide. The enzyme can be derived from a recombinant source. Additional suitable means for generating suitable cap structures are disclosed in WO2016 / 193226, which is hereby incorporated by reference.

[0093] In one example, the vaccine of the present disclosure comprises a promoter, which is any promoter for a DNA-dependent RNA polymerase, for example, T7 (optionally comprising or consisting of the sequence TAATACGACTC ACTATAAGG (SEQ ID NO: 15)), T3, SP6, or Syn5 RNA polymerase.

[0094] In some examples, the vaccines disclosed herein comprise a polyadenylation signal (poly A tail). The poly A tail is a long sequence of adenine residues located at the 3' end of the molecule. The role of the poly A tail is twofold. The poly A tail is essential for translation, where poly (A) binding protein (PABP) recruits translation factors to enhance translation levels. In addition, the poly A tail increases the stability of the nucleic acid molecule by binding the poly (A) in the mRNA through PABP and protecting it from exonuclease digestion. In mRNA, the poly A tail is also known to play a key role in the transport of mRNA from the nucleus to the ribosome (Shlake, T. et al., RNA Biol., (2012), 9 (11), 1319-1330). In one example, the nucleic acid molecule of the present disclosure comprises a poly A tail of about 50 to about 500 adenosine nucleotides. For example, the poly A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosines. In some instances, the poly A tail contains 50 to 250 adenosines. In some instances, the poly A tail contains 60 to 100 adenosines. In some instances, the poly A tail contains 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 adenosines. In some instances, the poly A tail contains 77 adenosines. In some instances, the poly A tail contains 74 adenosines.

[0095] In one example of the present disclosure, the vaccine comprises a split poly(A) tail. The split poly(A) tail may comprise at least two adenosine-containing elements, optionally each having 30 to 60 adenosines, separated by a spacer optionally having 1 to 25 nucleotides.

[0096] leader sequence

[0097] In some instances, the antigen encoding sequence disclosed herein comprises a leader sequence. The leader sequence can encode a signal peptide. In some instances, the signal peptide is fused to the expressed therapeutic protein. In such instances, the leader sequence and the gene of interest are in the same open reading frame (ORF).

[0098] Signal peptides (comprising the N-terminal 15-60 amino acids of a protein) are typically required for transmembrane transfer on the secretory pathway and control the entry of most proteins into the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs the ribosome to the rough endoplasmic reticulum (ER) and initiates transport of the growing peptide chain across it for processing. ER processing produces the mature protein, where the signal peptide is typically cleaved by a resident signal peptidase, at least for secretory proteins.

[0099] The signal peptide can be 15 to 60 amino acids in length. For example, the signal peptide can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. In some examples, the signal peptide is 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-55 In some examples, the signal peptide has the following sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 8).

[0100] Nucleosides and nucleotides

[0101] In some examples, the vaccines provided herein comprise nucleic acids (preferably mRNA) that are not chemically modified and comprise the standard RNA nucleotides adenine (A), uracil (U), guanine (G), or cytosine (C).

[0102] In some instances, the vaccine comprises a nucleic acid comprising modified nucleotides. Many modified nucleotides are known in the art, such as disclosed in WO2007 / 024708, which is hereby incorporated by reference. Modifications can include naturally occurring modifications or non-naturally occurring modifications. Modifications can include those modifications of nucleotides and / or nucleosides, sugars, backbones, or nucleobase proteins well known in the art.

[0103] In some examples, the nucleic acid molecules herein can include natural (ie, standard) nucleotides or nucleosides, non-natural or naturally occurring modified nucleotides or nucleosides, or any combination thereof.

[0104] In one example, the RNA can comprise standard A, G, and C nucleotides as well as modified U nucleotides.

[0105] In some examples, nucleic acid molecules comprising modified nucleosides or nucleotides (e.g., "modified RNA nucleic acid molecules") exhibit reduced immunogenicity in a cell or organism relative to an unmodified RNA nucleic acid molecule comprising the same sequence.

[0106] In some examples, the modified nucleosides provided herein (e.g., RNA nucleic acid molecules, such as mRNA) include N1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C) and / or pseudouridine (Ψ). In some examples, the modified nucleotides in the nucleic acid molecules (e.g., RNA nucleic acid molecules, such as mRNA) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine and / or 5-methoxycytidine. In some examples, the RNA nucleic acid molecules include a combination of at least two (e.g., 2, 3, 4 or more) of any of the above-mentioned modified nucleobases.

[0107] In some examples, the nucleic acid molecules provided herein comprise N1-methyl-pseudouridine (m1Ψ) at one or more or all uridine positions of the nucleic acid molecule.

[0108] In some examples, the nucleic acid molecule comprises 5-methoxy-uridine (mo5U) at one or more or all uridine positions of the nucleic acid molecule.

[0109] In some examples, the nucleic acid molecule comprises from about 1% to about 100% modified nucleotides (relative to the total nucleotide content or relative to one or more types of nucleotides (i.e., any one or more of A, G, U, T, or C). In some examples, the nucleic acid molecule comprises any intermediate percentage of modified nucleotide content. For example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 0% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. The remaining percentages are made up of unmodified A, G, U, T, or C.

[0110] Nucleic acid molecules can contain a minimum of 1% and a maximum of 100% modified nucleotides, or any intermediate percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, nucleic acids can contain modified pyrimidines, such as modified uracils or cytosines. In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracils in the nucleic acid are replaced by modified uracils (e.g., 5-substituted uracils). Modified uracils can be replaced by compounds having a single unique structure, or can be replaced by multiple compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some examples, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the nucleic acid are replaced by modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced by a compound having a single unique structure, or can be replaced by multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures).

[0111] In some instances, the nucleic acid molecule is mRNA in which uridine is replaced by a compound having a single unique structure. In some instances, the single unique structure is N1-methyl-pseudouridine. In some instances, the nucleic acid molecule comprises at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% N1-methyl-pseudouridine.

[0112] In some instances, the mRNA comprises modified nucleobases. In some instances, the modified nucleobases are modified adenine (A), cytosine (C), uracil (U), and guanine (G).

[0113] In some examples, the modified nucleobase is a modified U. In some examples, the modified U is 1-methylpseudouridine (m1Ψ) and pseudouridine (Ψ), such as disclosed in WO 2007 / 024708, which is hereby incorporated by reference.

[0114] In one example, the nucleic acid molecule of the present disclosure comprises a UTR sequence comprising 5-methoxy-uridine (mo5U) at one or more or all uridine positions of the nucleic acid molecule. The molecule can comprise at least a 25% ratio of modified uridine to unmodified uridine (including 25% to 50%, or at least 50%).

[0115] The examples show that modification of uridine in the 5'-UTR sequence derived from the 5'-UTR of human CHIT1 and in the 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS) leads to a particularly significant increase in translation.

[0116] In one example, the nucleic acid molecule of the present disclosure comprises a sequence comprising N1-methyl-pseudouridine (m1Ψ) at one or more or all uridine positions of the nucleic acid molecule. The molecule can comprise a ratio of at least 75% modified uridine to unmodified uridine (including 100%).

[0117] manufacture

[0118] Vaccines of the present disclosure can be manufactured according to in vitro transcription. In vitro transcription of RNA is known in the art and is described in International Publication WO2014 / 152027, which is incorporated herein by reference in its entirety. In some instances, RNA of the present disclosure is prepared according to any one or more of the methods described in WO2018 / 053209 and WO2019 / 036682, each of which is incorporated herein by reference. In summary, a DNA template is generated, typically as a linearized plasmid, followed by in vitro transcription to synthesize RNA, which is then capped.

[0119] The 5' cap can be added by a multi-step enzymatic reaction or via co-transcriptional addition. In co-transcriptional capping, a cap analog (such as AG) is added directly to the in vitro transcription mixture. Alternatively, enzymatic capping using vaccinia virus capping enzyme is performed separately from in vitro transcription.

[0120] After purification, the mRNA product can be encapsulated in lipid nanoparticles (LNPs).

[0121] Composition

[0122] The present disclosure also provides a pharmaceutical composition comprising a nucleic acid molecule or LNP as defined anywhere herein and a pharmaceutical carrier.

[0123] The present disclosure further provides a composition comprising a first nucleic acid molecule according to the present disclosure, wherein the disease-associated antigen is a Delta variant S protein. In one example, the disease-associated antigen of the first nucleic acid molecule is a Wuhan variant S protein.

[0124] In one example, the composition according to the present disclosure may further comprise a second nucleic acid molecule encoding an Omicron variant S protein (optionally variant BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1 or XBB.1.5).

[0125] In one example, the second nucleic acid molecule encodes the Omicron variant S protein BA.4 / 5.

[0126] In one example, the second nucleic acid molecule encodes the Omicron variant S protein XBB.1.5.

[0127] In all examples of the present disclosure, the sequence encoding the antigen can be further optimized through mutation to increase protein stability (such as the structure of the CoV spike protein or RBD), maximize protein translation and reduce unwanted side effects.

[0128] In some examples of the present disclosure, the sequence encoding the antigen can be optimized through mutation to increase the stability of the trimeric S protein and / or eliminate the furin cleavage site.

[0129] In some examples of the present disclosure, the disease-associated antigen is an antigenic fragment of the CoV S protein consisting of amino acid residues 1 to 1162 of the CoV S protein.

[0130] The composition may comprise an effective amount of a nucleic acid molecule as defined herein. The effective amount of the nucleic acid molecule to be used therapeutically will depend, for example, on the therapeutic goal, route of administration, and the patient's condition. In one example, an effective amount of a nucleic acid molecule as defined anywhere herein within the pharmaceutical composition can effectively treat or prevent a disease associated with a coronavirus infection.

[0131] Said composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. Pharmaceutically acceptable carriers can include one or more excipients. Pharmaceutically acceptable excipients are known and are included in carriers, excipients or stabilizers that are nontoxic to the cells or mammals exposed thereto at the dosage and concentration employed. In the context of the present disclosure, any suitable carrier can be used, and such carriers are well known in the art. The selection of carrier will be determined in part by the specific site to which the composition can be applied and the ad hoc method for applying the composition. The physiologically acceptable excipient can be an aqueous pH buffer solution. Examples of physiologically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM .

[0132] The composition may optionally be sterile. The composition may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The composition may be prepared according to conventional techniques described in, for example, "Remington: The Science and Practice of Pharmacy," 21st edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0133] The composition can be administered intravenously. The composition can also be administered parenterally or subcutaneously.

[0134] The method of administering the pharmaceutical composition as defined herein includes but is not limited to parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and mucosal (e.g., intranasal and oral routes). In a specific example, the pharmaceutical composition is intranasal, intramuscular, intravenous or subcutaneous administration. The composition can be administered by any convenient route, such as by infusion or bolus, absorbed by epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. Each dose may or may not be administered by the same route of administration.

[0135] Delivery system

[0136] Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., nucleic acid molecules as disclosed herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, constructing the nucleic acid as part of a retroviral or other vector, etc. In addition, pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. The present disclosure relates to nucleic acid molecules that can be suitable for use as vaccine vectors.

[0137] Lipid nanoparticles (LNPs) can be used as the platform for vaccine vector delivery. LNPs can include ionizable cationic lipids, cholesterol, phospholipids (such as distearoylphosphatidylcholine) and polyethylene glycol (PEG)-lipids. Ionizable cationic lipids participate in nanoparticle packaging by interacting with negatively charged RNA molecules. After administration, LNPs are quickly removed from the tissue of injection, and are therefore unlikely to induce inflammation and tissue damage.

[0138] Therefore, in one example of the present disclosure, the nucleic acid molecules as described anywhere herein are packaged into a delivery system. In one example, the delivery system is an LNP. Therefore, the present disclosure also relates to an LNP comprising a nucleic acid molecule as described anywhere herein.

[0139] In some instances, the LNP comprises a nucleic acid molecule as described anywhere herein, wherein the nucleic acid molecule encodes an antigenic protein. In some instances, the LNP comprises one or more nucleic acid molecules as described anywhere herein, wherein the molecule encodes a CoV S protein.

[0140] Uses of vaccine compositions

[0141] After patient administration, coding sequence will be transcribed and translated (in the case of DNA sequence) and translated (in the case of RNA sequence) into its encoded antigen protein or antigen protein fragment.The generation of these antigen proteins or antigen protein fragments will stimulate immune response, thereby causing the generation of neutralizing antibodies.After being infected by corresponding infectious agent, the presence of neutralizing antibodies and memory B cells will increase the speed of immune response, thereby minimizing the severity and time length of symptom onset.

[0142] The vaccine vectors of the present disclosure can be used as prophylactic therapy against target antigens that cause disease. In one example of the present disclosure, the vaccine vectors can be used to prevent CoV, particularly SARS-CoV-2.

[0143] The vaccine vectors of the present disclosure can also be used as treatments for target antigens in infected subjects. In one example of the present disclosure, the vaccine vectors can be used to treat CoV, particularly SARS-CoV-2.

[0144] Methods for inducing immune responses to Pan-Sabinabul coronavirus variants

[0145] According to a first aspect, the present disclosure provides a method for inducing a pan-Sabemicoronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine against one or more first Sabemicoronavirus variants (variant 1), the method comprising administering to the individual one or more doses of a second SARS-CoV-2 vaccine, wherein the second SARS-CoV-2 vaccine comprises mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a Sabemicoronavirus variant that is different from variant 1 (variant 2), wherein the S protein is encoded as an S protein-multimerization subunit fusion, and wherein the method induces a pan-variant immune response against Sabemicoronavirus variant 1 and variant 2 in the individual and induces an immune response against one or more additional Sabemicoronavirus variants that are different from variant 1 and variant 2.

[0146] The phrase "pan-Sabe coronavirus immune response" refers to an immune response generated in response to a challenge with multiple Sabae coronavirus variants. In a preferred embodiment, the Sabae coronavirus variants are SARS-CoV-2 variants, including but not limited to: Wuhan D614G, Alpha, Beta, Delta, and Omicron, optionally subvariants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, and XBB.1.5.

[0147] In one example, variant 2 is SARS-CoV-2 Omicron BA.4 / 5. In one example, variant 2 is SARS-CoV-2 Omicron XBB.1.5. In one example, variant 1 is SARS-CoV-2 Wuhan D614G. In one example, variant 1 is SARS-CoV-2 Delta.

[0148] In one example, the method induces an immune response against one or more additional sabie coronavirus variants that are different in mutation from variant 1 and / or variant 2. As used herein, the term "different in mutation" refers to that the SARS-CoV2 variants have separate and different mutational pedigrees. For example, SARS-CoV-2 variants that are different in mutation can be specified according to the Pango pedigree nomenclature. Pango pedigrees are designated to help track SARS-CoV-2 in detail. They represent branches within a phylogenetic tree defined by both at least one evolutionary event (non-synonymous mutation, insertion / deletion, or recombination event) and an epidemiologically significant event.

[0149] In one example, the immune response is a neutralizing response, which is determined by the presence of neutralizing antibodies (NAb). Methods for detecting the presence of NAb in a sample obtained from an individual (including, for example, microneutralization assays, enzyme-linked immunosorbent assays (ELISAs), and rapid lateral flow assays) will be apparent to the skilled person, and any suitable method may be used. Kuan-Ting Lui et al. (Vir uses (2022) 14 (7) 1560) provide an overview of international standards for detecting SARS-CoV-2 neutralizing antibodies, and the contents of that publication are incorporated herein in their entirety.

[0150] The immune response is preferably a protective immune response, meaning that the immune response provides protection to the individual against infection or disease caused by the SARS-CoV-2 variant. Protection may mean that the individual has no symptoms of infection, or that any symptoms of infection experienced after vaccination are milder or shorter-lasting than those experienced without the vaccine.

[0151] Preferably, the subject is a human, and may be an infant, child, adolescent or adult.

[0152] The multimerization unit may be selected from the following self-assembling proteins: ferritin, lumazine synthase, and encapsulin. In a preferred embodiment, the protein-multimerization subunit fusion is an S protein-ferritin subunit fusion.

[0153] In one example, the second SARS-CoV-2 vaccine comprises another monovalent immunogenic composition comprising mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimerized subunit fusion and is derived from a Sabeucoronavirus variant that is different from variant 2 and the same as or different from variant 1.

[0154] In one example, the second SARS-CoV-2 vaccine comprises mRNA formulated in lipid nanoparticles (LNPs).

[0155] In one example, the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID No.11.

[0156] In one example, the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID No.17.

[0157] In one example, the first SARS-CoV-2 vaccine comprises a protein antigen that is not provided in the form of a virus-like particle (VLP). In this example, the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabeucoronavirus S protein in the form of a fusion protein that can assemble in vivo to form nanoparticles. As used herein, "first" generally refers to a previous SARS-CoV-2 vaccine that is different in composition from the compositions disclosed herein. In other words, it describes a previous vaccine that encodes or delivers an S-protein antigen or an immunogenic fragment or immunogenic variant thereof. Depending on the vaccination status of the subject, this can refer to a prime vaccine or a subsequent booster vaccine administered before the "second" vaccine disclosed herein.

[0158] In one example, the second SARS-CoV-2 vaccine is a monovalent vaccine, meaning that the vaccine composition administered to the individual being vaccinated contains mRNA encoding a single spike (S) protein antigen or an immunogenic fragment or immunogenic variant thereof.

[0159] In alternative examples, the second SARS-CoV-2 vaccine is a bivalent vaccine comprising an additional mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimerized subunit fusion and is derived from a Sabei coronavirus variant that is different from variant 2 and the same as or different from variant 1. In some examples, the additional mRNA of the second SARS-CoV2 vaccine comprises the mRNA sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0160] As needed, one or more doses of each of the first and second SARS-CoV-2 vaccines can be administered to an individual as part of an approved prime-boost vaccination regimen. In one example, the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 4 months after the last dose of the first SARS-CoV-2 vaccine. In another example, the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 6 months after the last dose of the first SARS-CoV-2 vaccine.

[0161] A second aspect of the present disclosure provides an immunogenic composition comprising mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a first sabemi coronavirus variant, for inducing a pan-sabemi coronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine, the first SARS-CoV-2 vaccine comprising or encoding an immunogen from a second sabemi coronavirus variant, wherein the S protein is encoded as an S protein-multimerized subunit fusion, and wherein the immunogenic composition is used to induce an immune response against the first sabemi coronavirus variant and the second sabemi coronavirus variant and against at least a third sabemi coronavirus variant.

[0162] The phrase "pan-Sabecoronavirus immune response" refers to an immune response generated in response to a challenge with multiple Sabaecoronavirus variants. In one example, the Sabaecoronavirus variants are SARS-CoV-2 variants, including but not limited to: WuhanD614G, Alpha, Beta, Delta, and Omicron, optionally BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, and XBB.1.5.

[0163] In one example, the first Sabae coronavirus variant is SARS-CoV-2 Omicron BA.4 / 5. In one example, the first Sabae coronavirus variant is SARS-CoV-2 Omicron XBB.1.5. In one example, the second variant is SARS-CoV-2 Wuhan D614G. In one example, variant 1 is SARS-CoV-2 Delta.

[0164] Preferably, the third sabeccovirus variant is different from the first and / or second sabeccovirus variant in mutation. As used herein, the term "different in mutation" means that the SARS-CoV2 variant has a separate and different mutational pedigree. For example, SARS-CoV-2 variants that are different in mutation can be specified according to the Pango pedigree nomenclature. Pango pedigrees are designated to help track SARS-CoV-2 in detail. They represent clades within a phylogenetic tree defined by both at least one evolutionary event (non-synonymous mutation, insertion / deletion, or recombination event) and an epidemiologically significant event.

[0165] In one example, the immune response is a neutralizing response, which is determined by the presence of neutralizing antibodies (NAb). Methods for detecting the presence of NAb in a sample obtained from an individual (including, for example, microneutralization assays, enzyme-linked immunosorbent assays (ELISAs), and rapid lateral flow assays) will be apparent to the skilled person, and any suitable method may be used. Kuan-Ting Lui et al. (Vir uses (2022) 14 (7) 1560) provide an overview of international standards for detecting SARS-CoV-2 neutralizing antibodies, and the contents of that publication are incorporated herein in their entirety.

[0166] The immune response is preferably a protective immune response, meaning that the immune response provides protection to the individual against infection or disease caused by the SARS-CoV-2 variant. Protection may mean that the individual has no symptoms of infection, or that any symptoms of infection experienced after vaccination are milder or shorter-lasting than those experienced without the vaccine.

[0167] Preferably, the subject is a human, and may be an infant, child, adolescent or adult.

[0168] In one example, the S protein-multimerization subunit fusion is a S protein-ferritin subunit fusion.

[0169] In one example, the mRNA is formulated in lipid nanoparticles (LNPs).

[0170] In another example, the mRNA comprises or consists of the mRNA sequence of SEQ ID NO. 17.

[0171] In another example, the mRNA comprises or consists of the mRNA sequence of SEQ ID NO. 17.

[0172] In another example, the first SARS-CoV-2 vaccine comprises a protein antigen that is not provided in the form of a virus-like particle (VLP). In this case, the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabei coronavirus S protein in the form of a fusion protein that can assemble in vivo to form nanoparticles.

[0173] In one example, the immunogenic composition is a monovalent composition, meaning that it comprises mRNA encoding a single spike (S) protein antigen or an immunogenic fragment or immunogenic variant thereof.

[0174] In an alternative example, the immunogenic composition is a bivalent composition comprising an additional mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimerized subunit fusion and is derived from a Sabei coronavirus variant that is different from the first Sabei coronavirus variant and the same or different from the second Sabei coronavirus variant. In some examples, the additional mRNA of the bivalent composition comprises the mRNA sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0175] Example

[0176] The following examples further illustrate the present disclosure but should not be construed as limiting its scope in any way.

[0177] Example 1: Efficacy of mRNA vaccine vectors containing antigen-linker-ferritin sequences

[0178] The overall goal of this study was to determine the immunogenicity of a candidate SARS-CoV-2 mRNA vaccine in mice. The mRNA vaccine encodes a stabilized spike (S) protein-ferritin subunit fusion protein that, when expressed, assembles into nanoparticles for high-concentration antigen display.

[0179] method

[0180] Mouse studies 1

[0181] Groups of naive BALB / c mice (n=6 per group) were injected intramuscularly in the thigh muscle with 50 μl of LNP-encoded mRNA vaccine encoding the Wuhan D614G spike protein twice (28 days apart). Approximately 150 days later, serum was collected and the neutralizing antibody level against the BA.4 / 5 variant was assessed using a SARS-CoV-2 pseudovirus-based neutralization assay ( Figure 1a ).

[0182] Approximately 200 days later, mice received a third immunization with an mRNA vaccine formulated with LNPs encoding the nanoparticles disclosed herein. Fourteen days later, sera were collected and the levels of neutralizing antibodies against a panel of SARS-CoV-2 variants were assessed using a SARS-CoV-2 pseudovirus-based neutralization assay ( Figure 1b ).

[0183] On day 200, mice were divided into four groups and administered one or two mRNAs encoding antigens, as specified in Table 1 below. Groups 1 and 4 were administered a bivalent vaccine encoding both the Wuhan original and BA.4 / 5 spike (S) proteins. The Group 4 vaccine encoded the antigen as a ferritin-linker fusion protein (virus-like particle, i.e., nanoparticle or "VLP"). Alternatively, Groups 2 and 3 were administered vaccines encoding monovalent antigens (Group 2 - Wuhan S protein, Group 3 - BA.4 / 5 S protein), both as VLPs.

[0184] Table 1

[0185]

[0186]

[0187] Mouse study 2

[0188] Groups of unvaccinated BALB / c mice (n=6 per group) were injected intramuscularly in the thigh muscle twice (28 days apart) with 50 μl of LNP-formulated mRNA vaccine encoding the Wuhan D614G spike protein.

[0189] Approximately 300 days later, the mice were divided into three groups: Group 1 was administered PBS, Group 2 was administered a monovalent vaccine encoding the Omicron XBB.1.5 spike protein, and Group 3 was administered a monovalent vaccine encoding the Omicron XBB.1.5 spike protein in the form of VLPs.

[0190] After 14 days, serum was collected and the levels of neutralizing antibodies against a panel of SARS-CoV-2 variants were assessed using a SARS-CoV-2 pseudovirus-based neutralization assay ( Figure 1c ).

[0191] result

[0192] Mouse studies simulating booster vaccination campaigns1 showed that Group 3 and Group 4 VLP-based vaccines were more able to overcome the antigenic sin experienced by mice administered the Group 1 vaccine, in which the neutralizing antibody response was dominated by the original variant used as the inoculum in the original "prime" vaccination campaign (e.g., Wuhan "D614G"). This is likely due to the fact that mice were initially challenged with the Wuhan D614G S protein in the first vaccination campaign, and therefore memory recall of the antibody pool neutralizing this S protein dominated the Group 1 vaccine response.

[0193] Group 1 (and Group 2) mice showed minimal neutralization responses against BA.4 / 5 ( Figure 1b ), even though Group 1 animals were challenged with the mRNA encoding the BA.4 / 5S protein. In contrast, vaccinated mice in Groups 3 and 4 exhibited a 14-fold and 23-fold increase in mean neutralization responses to BA 4 / 5 compared to Group 1 mice. Furthermore, Group 3 and Group 4 mice also exhibited low-level neutralization responses against the newly emerged variant of interest, BQ.1.1, while no responses were detected against this variant in Group 1 or Group 2 mice.

[0194] Similar results were obtained in mouse study 2, where the mean neutralization response to BA.4 / 5 was increased by 4.2-fold, the mean neutralization response to XBB.1.5 was increased by 1.7-fold, and the mean neutralization response to XBB.1.16 was increased by 2.9-fold in group 3 mice compared to group 2 mice.

[0195] In summary, the data therefore suggest that broadly neutralizing responses can be achieved from a single vaccine construct when mRNA is used to prime the VLP antigen. This could lead to better efficacy against a variety of existing and potential future variants of concern without the need to independently generate boosters each time a new dominant variant emerges.

[0196] Example 2: mRNA vaccine vector containing antigen-linker-ferritin sequence in non-human primates Immunogenicity

[0197] The overall aim of this study was to determine the immunogenicity of a candidate SARS-CoV-2 mRNA vaccine in nonhuman primates (NHPs).

[0198] method

[0199] SARS-CoV-2 seronegative NHPs were immunized with two doses (4 weeks apart) of 10 μg of either the mRNA-natural Delta vaccine or the mRNA-VLP Delta vaccine. Neutralizing antibody titers were measured two weeks after the second immunization (day 42) against a panel of SARS-Co V-2 reporter viruses (original D614G, Delta, BA.1, BA.2, or BA.4 / 5) and approximately 6.5 months after the first immunization against the Delta reporter virus. The frequency of long-lived antibody-secreting cells in bone marrow biopsies of vaccinated NHPs was measured using a B cell ELISpot assay with recombinant Delta antigen.

[0200] result

[0201] Two weeks after the second immunization, the mRNA-VLP Delta vaccine elicited neutralizing antibodies against D614G, Delta, BA.1, BA.2, or BA.4 / 5 reporter viruses, with geometric mean titers (GMTs) that were 15-, 9-, 29-, 18-, and 7-fold higher than those elicited by the mRNA-natural Delta vaccine, respectively (all p ≤ 0.002; Figure 3 Furthermore, 196 days after the first administration, the neutralizing antibody titer of mRNA-VLP Delta was 3.5 times higher than that of mRNA-native Delta (GMT 236 and 68, respectively; Figure 4 The mRNA-VLP Delta vaccine also induced a statistically significant 1-fold increase in long-lived antibody-secreting cells compared to the mRNA-natural Delta vaccine (per 10 6 The ratio of bone marrow cells was 16.9 to 8.8; Figure 5 ).

[0202] In summary, the data demonstrate that broadly neutralizing responses can be achieved from a single vaccine construct when mRNA is used to prime the VLP antigen, as observed in Example 1. The data also demonstrate that the mRNA-VLPDelta vaccine elicits a more potent and broader neutralizing antibody response in NHPs compared to the mRNA-natural Delta vaccine. In addition, the mRNA-VLP Delta vaccine maintains higher neutralizing antibody titers for ≥6 months and generates a greater number of long-lived antibody-secreting cells compared to the mRNA-natural Delta vaccine.

[0203] Example 3: mRNA vaccine vector containing antigen-linker-ferritin sequence in vaccinated non-human primates Enhanced ability in long-legged animals

[0204] The overall aim of this study was to determine the boosting capacity of candidate SARS-CoV-2 mRNA vaccines in vaccinated nonhuman primates (NHPs).

[0205] method

[0206] NHPs were first immunized with two doses (3 weeks apart) of the mRNA-natural original Wuhan_D614G vaccine to establish baseline anti-spike immunity. The animals were then rested for approximately 7.5 months, at which time they were given a third mRNA-natural XBB.1.5 or mRNA-VLP XBB.1.5 booster vaccination ( Figure 8 Neutralizing antibody titers were measured before (day 246) and 14 days after (day 260) the third booster vaccination against a panel of SARS-CoV-2 reporter viruses (original Wuhan_D614G, Omicron BA.4 / 5, and Omicron XBB.1.5).

[0207] result

[0208] Two weeks after the third immunization, a 30 μg dose of the mRNA-VLP XBB.1.5 vaccine elicited neutralizing antibodies against D614G, BA.4 / 5, and XBB.1.5 reporter viruses, with geometric mean titers (GMTs) comparable to those elicited by a 30 μg dose of the mRNA-native XBB.1.5 vaccine ( Figures 9 to 12 Furthermore, immunization of animals with 10 μg or 5 μg of the mRNA-VLP XBB.1.5 vaccine elicited neutralizing antibody titers comparable to those induced by a 30 μg dose of either the mRNA-native XBB.1.5 or the mRNA-VLP XBB.1.5 vaccine ( Figures 9 to 12 ).

[0209] In summary, the data demonstrate that broad neutralizing responses are achieved with a single low booster dose of mRNA-VLP vaccine when mRNA is used to prime the VLP antigen.

[0210] sequence

[0211] SARS-CoV-2 Wuhan D614G spike protein-linker-ferritin RNA sequence (SEQ ID NO: 1)

[0212] AUGCCCCUCCUUCCUUCCUUCCCUCCUUUUGGGCUGGAGCGCUGGCUCUCUCAGUGUGUUAAUCUCACCACCAGAACCCAGCUGCCCCUGCCUAUACCAAUUCCUUCAGGUGUGUCUGUGUCUCUUGCUUUUUUUCUAACGUGACCUGGUUCCACGCCAUCCACGUGAGCGGCACCAUUGGCCAAUGGCCAAAGGAGUUCGACAAUCCAGUGCUGCUGCCCUUUUAACGAUGGCGUGUUACUUCGCCUUCACGAGAAGUCUAACAUCCGCGGCUGGAUCUUGGCCACACUGGACAGCAAGCCACAGUCCUCCUGCUGUACUCGUA

[0213] GGCAAGCAGGGCAAUUUAGAGAACCUGCGGGAGUUCGUGUUUAAGA

[0214] AUAUCGAUGGCUACUUCAAGAUCUACUCCAAGCACACCCCCAUCAA

[0215] CCUGGUGCGGGACCUGCCACAGGGCUUCUCUGCCCUGGAGCCACUG

[0216] GUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGC

[0217] UGGCCCUGCACAGAGCUACCUGACACCAGGCGACAGCUCCUCUGG

[0218] AUGGACCGCAGGAGCAGCAGCCUACUAUUGUGGGCUAUCUGCCAGCCC

[0219] CGGACCUUCCUGCUGAAGUACAACGAGAAUGGCACCAUCACAGACG

[0220] CCGUGGAUUGCGCCCUGGAUCCCUGUCUGAGACAAAGGUGACACU

[0221] GAAGAGUUUUACCGUGGAGAAGGGCAUCUAUCAGACAAGCAAUUUC

[0222] AGGGUGCAGCCUACCGAGUCCAUCGUGCGCUUUCCAAUAUCACAA

[0223] ACCUGUGCCCUUUUGGCGAGGUGUCAACGCCACCAGAAUCGCCAG

[0224] CGUGUACGCCUGGAAUAGGAAGCGCAUCUCCAACUGCGUGGCCGAC

[0225] UAUUCUGGCUGUAACAGCGCCUCCUUUCUCUACCUUUAAGUGCU

[0226] AUGGCGUGAGCCCCACAAAGCUGAAUGACCUGUGCUUUACCAACGU

[0227] GUACGCCGAUUCCUUCGUGAUCAGGGGCGACGAGGUGCGCCAGAUC

[0228] GCACCAGGACAGACAGGACAAGAUCGCAGACUACAAUUAAAGCUGC

[0229] CUGACGUUUCACCCGGCUGCGUGAUCGCCUGGAACUUAACAUCU

[0230] GGAUAGCAAAGUGGGCGGCAACUACAAUUAUCUGUACCGGCUGUUU

[0231] AGAAAGUCUAAUCUGAAGCCAUCGAGCGGACAUCUCCACAGAG

[0232] UCUACCAGGCCGGCUCUACCCCCUGCAAUGGCGUGGAGGGCUUUAA

[0233] CUGUUAUUUCCCUCUGCAGAGCUACGGCUUCCAGCCAACCAACGGC

[0234] GUGGGCUAUCAGCCCUACAGAGUGGUGGUGCUGCUUUUGAGCUGC

[0235] UGCACGCACCUGCAACAGUGUGCGGACCAAAAAGAGCACCAAUCU

[0236] GGUGAAGAACAAGUGCGUGACUUCAACUUCAACGGCCUGACCGGA

[0237] ACAGGCGUGCUGACCGAGUCCAACAAGAAGUUCCUGCCUUUUCAGC

[0238] AGUUCGGCAGGGACAUCGCAGAUACCACAGACGCCGUGCGCGACCCC

[0239] UCAGACCCUGGAGAUCCUGGAUAUCACACCAUGCUCCUUCGGGCGGC

[0240] GUGUCUGUGAUCACACCAGGCACCAAUACAAGCAACCAGGUGGCCG

[0241] UGCUGUAUCAGGGCGUGAAUUGUACCGAGGUGCCAGUGGCAAUCCA

[0242] CGCAGAUCAGCUGACCCCUACAUGGCGGGGUGUACUCUACCGGCAGC

[0243] AACGUGUUCCAGACAAGCCGGAUGCCUGAUCGGAGCAGAGCACG

[0244] UGAACAAUAGCUAUGAGUGCGACAUCCCUAUCGGCGCCGGCAUCUG

[0245] UGCCUCCUACCAGACCCAGACAAACUCCCCCAGGGUCUGCAUCAUCU

[0246] GUGGCAAGCCAGUCCAUCAUCGCCUAUACCAUGAGCCUGGGCGCCG

[0247] AGAAUUCCGUGGCCUACUCCAACAAUUCUAUCGCCAUCCCUACCAA

[0248] CUUCACAAUCUCCGUGACCACAGAGAUCCUGCCAGUGAGCAUGACC

[0249] AAGACAUCCGUGGACUGCACAAUGUAUAUCUGUGGCGAUUCCACCG

[0250] AGUGCUCUAACCUGCUGCUGCAGUACGGCAGCUUUUGUACCCAGCU

[0251] GAAUCGCGCCCUGACAGGAAUCGCAGUGGAGCAGGAUAAGAACACA

[0252] CAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCACCCA

[0253] UCAAGGACUUUGGCGGCUUCAAUUUUUCCCAGAUCCUGCCCGAUCC

[0254] UAGCAAGCCAUCCAAGAGGUCUUUUAUCGAGGACCUGCUGUUCAAC

[0255] AAGGUGACCCUGGCCGAUGCCGGCUUCAUCAAGCAGUAUGGCGAUU

[0256] GCCUGGGCGACAUCGCAGCCCGCGACCUGAUCUGUGCCCAGAAGUU

[0257] UAAUGGCCUGACCGUGCUGCCUCCACUGCUGACAGAUGAGAUGAUC

[0258] GCCCAGUACACAUCUGCCCUGCUGGCAGGAACCAUCACAAGCGGAU

[0259] GGACCUUCGGCGCAGGAGCCGCCCUGCAGAUCCCCUUUGCCAUGCA

[0260] GAUGGCCUAUCGGUUCAACGGCAUCGGCGUGACCCAGAAUGUGCUG

[0261] UACGAGAACCAGAAGCUGAUCGCCAAUCAGUUUAACUCCGCCAUCG

[0262] GCAAGAUCCAGGACUCUCUGAGCAGCACAGCAAGCGCCCUGGGCAA

[0263] GCUGCAGGAGUGUGGUGAAUCAGAACGCCCAGGCCCUGAAUACCCUG

[0264] GUGAGCAGCUGUCUAGCAACUUCGGCGCCAUCUCCUCUGUGCUGA

[0265] AUGAUAUCCUGAGCAGGCUGGACCCUCCAGAGGCCGAAGUGCAGAU

[0266] CGAUCGGCUGAUCACUGGUAGGCUGCAGAGUCUUGCAGACAAUAUUGUC

[0267] ACCCAGCAGCUGAUCAGCAGCGAGUUCGAGCUUCACCUAACU

[0268] UAGCUGCCACAAAGAUGAGUGAGUGCGUGCUGGCCAGUCCAAGAG

[0269] GGUGGAUUUCUGCGGAAAAGGCUAAUCAUCUCAUGUCCUUCCCCCAA

[0270] AGCGCACCCCACGGGGGUUCUUCCUGCACGUAACGUAUGUGCCCG

[0271] CUCAGGAGAACUUUACUACUGCCCCGCCAUCUGCCACGAUGG

[0272] GAAAGCUCACUUCCCGAGAGAAGGAGUAUUCGUUUCCAAUGGGACG

[0273] CAUUGGUUCGUCACCCAGCGGAACUUUUACGAACCACAGAUCAUUA

[0274] CCACGGAUAUACAUUUGUGAGCGGGGAAUUGCGAUGUGGUGUGAUUG

[0275] GAAUUGUAAUAACACAGUGUACGAUCCAGUCAGAAUUAGA

[0276] CUCAAUUAAGGAGGAGUGGAUAAAGAUUCAUAAAAAUGGGUUCAGG

[0277] UGGAUCAGGUGUAUAGAAAAAACUCUCCUCAAUGAACAAGUAAAUAA

[0278] GGAGAUGCAAAGUUCUAACCUGUACAUGAGCAUGUCUUCUUGGUGU

[0279] UACACCCAUAGCCUCGAUGGAGCGGAUUGUUCCUUUUUGACCACG

[0280] CUGCGGAGGAGUAUGAGCAUGCUAAAAAGCUGAUAAUAUUUCUCA

[0281] ACGAGAAUAAUGUUCCAGUGCAAUUGACAAGUAUAUCCGCCCCUGA

[0282] GCAUAAGUUGAAGGGCUCACACAAAUUUUCCAAAAGGCAUACGAA

[0283] CACGAACAGCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUG

[0284] CAAUCAAGUCCAAAGAUCACGCCACGUUUAAUUUCCUCCAGUGGUA

[0285] UGUAGCUGAGCAACAUGAGGAAGGAAGAGGUGUUGUUUAAGGAUAUUCU

[0286] UGAUAAAAUUGAACUUAUUGGAAAAUGAACCAUGGCCUCUAUCU

[0287] UGCGGACCAAUACGUCAAGGGAAUUGCCAAGUCCCGCAAGAGU

[0288] SARS-CoV-2 Delta-linker-ferritin RNA sequence (SEQ ID NO: 2)

[0289] SARS-CoV-2 Omicron BA.4 / 5-linker-ferritin RNA sequence (SEQ ID NO: 3)

[0290] AUGCCCCUCCUUCCUUCCUUCCUUCCUCCUUUUGGGCUGGAGCGCUGGCCUCUCUCAGUGUGUGUUAAAUCUCAUACCCGCCAGCAGCUACCACCACCUGCCUAUACAAACUCCUUCCCGGUGUCUAUUACCCCGACAAGGUUUUUCGAUCCUCUCUUCCAGUUCUUCAUCCACCCAGGAUCUUUUCCUUCCCAACGUCCUUGGUUUCAUCAUGCUAUUCAUGUGUCAGGGAACUAAAAGGUUCGACAAUCCCGUACUGCUUUUCCAACGACGGGGUGUAUUUCGCAUCAACAGAAAAAAGCAACAUUAUCAGAGGUGGAUUUUGGCACUACACUCGACUCAAAAGCCCAAAGUUUGCUUAUUGUUAAUACGCAACGAAUUGCUGUGAAUUCCAGUUUUGGUUAAUACCUAUAAAGGAACAAUAAGU CCUGGAUGGAGUCAGAGUCAGAGUCUACAUCAUCAGCAAACAAUUGCACUUUUGAGUACGUAUCUCAGCCAUUCUGAUGGACCUGGAAGGCAAGCAAGGGAACUUAAAAAAUCUCCGAGAGUUUUGUUUCAAAAACUAGAUGGUUAAUUCAAGAUCUAUUCUAAAACACACCCGAUUAAUCUUGGCCGGGACCUGCCCCAGGUUUCUCUGCCCUGAGCCCCUUGUAGA. CUUGCCCAUGGGAUAAAUAUUACUAGGUUUCAGACACUGCUUGCGCUGCACAGGUCAUACCUCACUCCUGGGGAUUCAUCAAGCGGCUGGACUGCAGGUGCCGCUGCAUACUACGUGGGAAUACCUCAACCCCGAACUUUUUUGCUCAAGUACAAUGAGAAUGGGACGAUAACUGAUGCGUAGACUGCGCACUCGAUCCUGACUGAGACGAAGUGCACUGAGAGA

[0291] UUCACGGUGGAAAAGGGUAUAUAUCAAACAUCCAUUUCCGAGUCC

[0292] AGCCCACUGAAAGUAUUGUUAGGUUCCCGAACAUCACAAAUCUCUG

[0293] UCCAUUUGACGAGGUAUUUAACGCGACACGCUUUGCUUCCGUGUAU

[0294] GCGUGGAAUAGAAGCGCAUUAGCAACUGUGUCGCGGAUUAUUCAG

[0295] UCUUGUACAACUUUGGCCCUUUUUUCGCAUUUAAAUGCUACGGGU

[0296] CAGCCCCACAAAGCUGAAUGAUCUCUGCUUCACAAAUGUAUAUUGCA

[0297] GAUAGCUUUGUCACGAGGUAACGAAGAUUCUCAGAUGCUCCCCG

[0298] GCCAAACGGGAAACAUAGCUGACUAUAACUAUAAGCUCCCAGACGA

[0299] UUUUACGGGGUUGUGUCAUUGCAUGGAACUCCAAUAAAUUGGAUUCA

[0300] AAGGUAGGUGGUAAUUAUAACUACAGGUUAUGGCUGUUCAGGAAGU

[0301] GUARANTEED

[0302] AGCGGGCAAUAAACCCUGCAACGGAGUCGCGGGGUGUUAAUUGCUAC

[0303] UUCCCAUUGCAGAGUUACGGCUUUCGGCCGACUUAUGGCGUCGAC

[0304] AUCAACCUUACAGGGUAGUUGUCUGUCUUUCGAACUCCUGCAUGC

[0305] UCCGGCAACGGUGUGUGGUCCAAGAAAAGUACUAACCUUGUCAAA

[0306] AAUAAGUGUGUGAACUUCAAUUUUAACGGUCUUACAGGUACUGGAG

[0307] UAUUGACUGAGUCAAAUAAGAAGUUUCUUCCAUUCCAACAAUUCGG

[0308] CCGCGAUAUAGCUGAUACAACGGAUGCCGUCCGAGACCCUCAGACUC

[0309] UGGAGAUCCUGGACAUAACUCCAUGUUCUUUUGGAGGCGUUAGUGU

[0310] GAUUACCCCAGGCACUAAUACCUCAAACCAAGUAGCCGUCCUGUAUC

[0311] AAGGAGUCAAUUGUACUGAGGUCCCUGUGGCCAUCCAUGCGGACCA

[0312] ACUGACACCCACAUGCGCGUAUAUAGCACAGGAUCAAACGUAUUC

[0313] CAGACUCGGCGCGGGUUGUCUGAUCGGGGCGAAUACGUAAAACAAUA

[0314] GCUAUGAGUGUGUAUAUACCUAUUGGAGCUGGGAUUUGCGCAAGCUA

[0315] UCAAACAAACGAAAUCCCACGGAUCGGCAUCAUCAGUUGCAUCUC

[0316] AAUCUAUUAUCGCUUAUACAAAUGUCUCUUGGCGCUGAGAACUCCGU

[0317] UGCUUACAGCAAUAAUAGCAUUGCCAUACCGACGAACUUUACUAUU

[0318] AGCGUAACGACUGAGUACUCCCGGUGUCUAUGACAAAAAACUAGCG

[0319] UUGAUUGUACAAUGUACAUUUGUGGGGAUAGUACAGUGGCUCAAA

[0320] CUUGCUUCCAGUACGGGUCAUUCUGUACCCAACUUAACGCGCAU

[0321] UGACUGGUAUUGCCGUCGAGCAAGACAAAACACACAAGAGGUUUUU

[0322] CGCUCAAGUAAAACAAAUUUACAAAACGCCCCCUAUAAAAAUAUUUU

[0323] GGCGGUUUCAACUUUUCUCAAAUUCUGCCCGAUCCUUCUAAGCCUA

[0324] GUAAGCGAAGCUUCAUUGAGAGACCUGCUUUUCAACAAAGUAACCUU

[0325] GGCUGACGCGGGUUUCAUAAAACAGUAUGGAGACUGUCUGGGAC

[0326] AUAGCCGCAAGGGACUUGAUUUGUGCCCAAAAUUCAACGGACUGA

[0327] CAGUUCUUCCUCCCACUUUUGACUGAUGAGAUGAUAGCACAAUACAC

[0328] UUCUGCUUUGCUGGCCGGUACAAUAACGAGUGGGUGGACCUUCGGG

[0329] GCUGGGGCUGCCCUCCAAAUACCCCUUCGCUAUGCAAAUGGCAUAUCG

[0330] AUUUAAUGGCAUCGGGGGUUACACAGAACGUCCUGUAUGAAACCAA

[0331] AAGCUCAUUGCCAACCAGUUUAACUCAGCAAUCGGAAAAAUUCAGG

[0332] ACAGCCUGAGUAGUACCGCCAGCGCCUUGGGAAAGUUGCAGGAUGU

[0333] CGUCAACCAUAACGCCCAAGCAUUGAAUACGUUGGUCAAACAGCUC

[0334] UCCUCUAGUUCGGUGCUAUAUCCUCACAGUCCUCAACGAUAUCCUGA

[0335] GCAGGCUCGAUCCCCCUGAGGCCGAAGUGCAGAUCGAUCGGCUGAUC

[0336] ACUGGUAGGCUGCAGAGUCUGCAGACAUAUGUCACCCAGCAGCUGA

[0337] UCAGAGCAGCCGAGAUUCGAGCUUCAGCUAACUUAGCCACAAA

[0338] GAUGAGUGAGUGCGUGCUGGGCCAGUCCAAGAGGGUGGAUUUCUGC

[0339] GGAAAAGGCUAUCAUCUCAUGUCCUUCCCCCCAAAGCGCACCCCACGG

[0340] GGUUGUCUUCCUGCACGUAACGUAUGUGCCCGCUCAGGAGAAGAAC

[0341] UUUACUACUGCCCCCCGCCAUCUGCCACGAUGGGAAAGCUCACUUCCC

[0342] GAGAGAAGGAGUAUUCGUUUCCAAUGGGACGCAUUGGUUCGUCACC

[0343] CAGCGGAACUUUUACGAACCACAGAUCAUUACCACGGAUAAUACAU

[0344] UUGAGGCGGGAAUUGCGAUGUGGUGAUUGGAAUUGUAAAUAACAC

[0345] AGUGUACGAUCCACUGCAGUCAGAAUUAGACUCAAUUAAGGAGGAG

[0346] UUGGAUAAGAUCAUAAAAAAUGGGUUCAGGUGGAUCAGGUGAUAUAG

[0347] AAAAAACUCCUCAAUGACAAGUAAAUAAAGGAGAUGCAAGUUCUAA

[0348] CCUGUACAUGACAUGUCUUCUGGUGUUACACCCCAUAGCCUCGAU

[0349] GGAGCGGGAUUGUUCCUUUUUGACCACGCUGCGGAGGAGUAUGAGC

[0350] AUGCUAAAAAGCUGAUAAUAUUUCUCAACGAGAAUAAUGUUCCAGU

[0351] GCAAUUGACAAGUAUAUCCGCCCUGAGCAUAAGUUUGAAGGGCUC

[0352] ACACAAAUUUUUCCAAAGGCAUACGAACGAACAGCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUGCAAUAUCAAGUCCACGCCACGUUUAAUUUCCUCCAGUGGUAUUGCUAGCAACAUGAGGAAGAAGGUGUUGUUUAAGGAUAUAUAUUGAUAAGGAUAUUCUUGUAAAAAAUUGACUUAUUGGAAAUGAGAACCAUGGCUCUAUCUUGCGGACCAAUACGUCAAGGGAAUUGCCAAUGCCCGCAAGU

[0353] Linker amino acid sequence: GSGGSG (SEQ ID NO: 4) 。

[0354] Linker RNA sequence: GGUUCAGGUGGAUCAGGU (SEQ ID NO: 5)

[0355] Ferritin subunit, RNA sequence (SEQ ID NO: 6) :

[0356] GAUAUAGAAAAACUCUCCUCAAUGACAAGUAAAUAAAGGAGAUGCAAAGUUCUAACCUGACAUGAUCUUCUUGGUUACACCCAUAGCCUCGAUGGAGCGGGAUUGUUCCUUUUUGACCACGUGGAGUAUUGUAUGAGCAUGCUAAAAAGCUGUAUAUUUCUCAACGAGAAAUAUUUUCCAGUGCAAUUGACAAGUAUUCCCCCCUGAGCAUAAAGCUAUUAAGGUCUCACACACAAAUUUUUCCAGAGCACAUUAGCGAGUCUAUUAACAACAUUGUGUUGUACUAUCAUGCCAAAUCAAGUCCAAAGUCACCGCACGUUUAAUAUUCCUCCUGUGGUAUUGUAUGUAGCUGAGCAACAUGAGGAAGGAAGGUGUUGUUUAAGGAUAUAUUCUUCCAGUUGUAUAAAAUUGACUUAUUGAAAUUGAAAUUGAAAUUGAAAUUGAAAUUGAAAUUGAAAUUGAAAUUGGAAUGAACCAUGGCCUCUAUCUUGCGACAUACGUCAAGGGAAUUGCCAAUGCCCGCAAGUCCCGCAAGU

[0357] Ferritin subunit, protein sequence (SEQ ID NO: 7)

[0358] DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLLFDHAAEEYEHAKKLIIFNLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINIVDHAISKDHATFNFFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0359] Leader sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 8) 。

[0360] Sequence of the mRNA construct encoding the SARS-CoV-2 Wuhan D614G spike-ferritin fusion protein (SEQ ID NO:9)

[0361]

[0362] The sequence of the mRNA construct encoding the SARS-CoV-2 Delta spike-ferritin fusion protein (SEQ ID NO:10)

[0363]

[0364] mRNA encoding the SARS-CoV-2 Omicron BA.4 / 5 spike-ferritin fusion protein A construct sequence Column (SEQ ID NO: 11)

[0365]

[0366] SARS-CoV2 Wuhan D614G spike-ferritin fusion protein polypeptide sequence (SEQ ID NO: 12)

[0367] MPLLLLLPLLWAGALA SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSS

[0368] VLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTE

[0369] KSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHK

[0370] NNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI

[0371] DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLT

[0372] PGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET

[0373] KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVY

[0374] AWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV

[0375] IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN

[0376] YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN

[0377] GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTG

[0378] VLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT

[0379] NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLI

[0380] GAEHVNNSYECDIPIGAGICASYQTQTNSPGSASSVASQSIIAYTMSLGAEN

[0381] SVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQY

[0382] GSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILP

[0383] DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL

[0384] TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGI

[0385] GVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL

[0386] NTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQ

[0387] LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVV

[0388] FLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE

[0389] PQIITTDNTFVSGNCDVVIGIVNNTVYDPLQSELDSIKEELDKIHKN

[0390] SARS-CoV-2 Delta spike-ferritin fusion protein polypeptide sequence (SEQ ID NO: 13)

[0391] MPLLLLLPLLWAGALA

[0392] SARS-CoV-2 Omicron BA.4 / 5 spike-ferritin fusion protein polypeptide sequence (SEQ ID NO:14)(former (The guide sequence is underlined)

[0393] MPLLLLLPLLWAGALA

[0394] T7 promoter (SEQ ID NO: 15) TAATACGACTCACTATAAGG

[0395] SARS-CoV-2 XBB.1.5 spike protein-linker-ferritin RNA sequence (SEQ ID NO: 16)

[0396] AUGCCCCUCCUUCUCCUUUCCCCCUCCUUUGGGCUGGAGCGCUGGCCUCUCAGUGUGUAAAUCUCAUUACCCGCACGCAGAGCUAUCCAAUUCCUUCACACGGGGCGGUGUACUAUCCCGACAAGGUGUUAUAGAUCUAGCGUGCUGCACUCCCACACAGGAUCUUCUGCCCUUU CUUUUCUAACGUGACCUGGUUCCCACGCCAUCCACGUGAGCGGCACAAUGGCACAAAGAGGUUCGACAAUCCAGCCCUGCCCUUUAACGAUGGCGUGUACUCGCCCUCCACCGAGAAGUCUAACAUCAUCCGGGCUGGAUUUUGGCACCACACUGGACAGCAAGACACAGUCCC UGCUGAUCGUGAACAAUGCCACCAACGUGGUCAUCAAGGUGUGGCGAGUUCCAGUUUUGUAAUGAUCCAUUCCUGGACGUGUAUCAGAAGAACAAUAAGUCUUGGAUGGAGAGCGAGUUCCGGUGUAUUCCUGUGCCAACAAUUGCACAUUUGAGUACUGUCCCAGCCCUUCCUGAUGGACCUGGAGGGCAAGGAGGGCAAUUUUUAAGAAUAUCGAUGGCUACUUCAAGAAUCUCGUACUCUACUCCAGCCACCCCCAUCCACCCAUCCACCCAUCGAGCGGACCUGCCAUGGCCUACAGGGCUUCUCUGCCCAUCGCCAUCGCCAUCGCCAUCGACAUCAACAU

[0397] CACCCGGUUUCAGACACUGCUGGCCCUGCACAGAGCUACCUGACAC

[0398] CAGUGGACAGCUCCUCUGGCUGGACCGCAGGAGCAGCAGCCUACUAU

[0399] GUGGGCUAUCUGCAGCCCCGGACCUUCCUGCUGAGAUACAGAGAA

[0400] UGGCACCAUCACAGACGCCGUGGAUUGCGCCCUGGAUCCCCUGUCUG

[0401] AGACAAAGUGUACACUGAAGAGUUUUACCGUGGAGAAGGGCAUCUA

[0402] UCAGACAAGCAAUUUCAGGGUGCAGCCUACCGAGUCCAUCGUGCGCU

[0403] UUCCCAAUAUCACAAACCUGUGCCCUUUUCACGAGGUGUUCAACGCC

[0404] ACCACCUUCGCCAGCGUGUACGCCUGGAAUAGGAAGCGCAUCUCCAA

[0405] CUGCGUGGCCGACUAUUCUGUGAUCUACAACUUCGCCCCCUUCUCUG

[0406] CCUUUAAGUGCUAUGGCGUGAGCCCCCACAAAGCUGAAUGACCUGUGC

[0407] UUUACCAACGUGUACGCCGAUUCCUUCGUGAUCAGGGGGCAACGAGGU

[0408] GAGCCAGAUCGCCACCAGGACAGACAGGCAACAUCGCAGACUACAAUU

[0409] AUAAGCUGCCUGACGAUUCACCCGGCUGCGUGAUCGCCUGGAACUCU

[0410] AACAAGCUGGAUAGCAAACCCAGCGGCAACUACAAUUACUGUACCG

[0411] GCUGUUUAGAAGUCUAAGCUGAAGCCAUGCAUGCAUGCA

[0412] CAGAGAUCUACCAGGCCCGGCAACAAGCCCUGCAAUGGCGUGGCCGGC

[0413] CCAAACUGUUAUGCCCAUUGCAGAGUUACGGCUUUCGGCCGACUUA

[0414] UGGCGUCGGACAUCAACCUUACAGGGUAGUUGUCUCUUUCGAAC

[0415] UCCUGCAUGCUCCGGCAACGGUGUGUGGUCCCAAGAAAAGUACUAAC

[0416] CUUGUCAAAAAUAAGUGUGUGAACUUCAAUUUUAACGGUCUUACAG

[0417] GUACUGGAGUAUUGACUGAGUCAAAUAAGAAGAUUUCUUCCAUUCCAUUCCA

[0418] ACAAUUCGGCCGCGAUAUAGCUGAUAACAACGGAUGCCGUCCGAGACC

[0419] CUCAGACUCUGGAGAUCCUGGACAUAACUCCAUGUUCUUUUGGAGGC

[0420] GUUAGUGUGAUAUACCCCAGGCACUAAUACCUCAAACCAAGUAGCCGU

[0421] CCUGAUCAAGGAGUCAAUUGUACUGAGGUCCCCUGUGGCCAUCAUG

[0422] CGGACCAACUGACACCACCAUGGCGCGUAUAUAGCACAGGAUCAAAC

[0423] GUAUUCCAGACUCGGCGCGGGUUGUCUGAUCGGGGCGAAUACGUAA

[0424] CAAUAGCUAUGAGUGUGUAUAUACCUAUUGGAGCUGGGAUUUGCGCA

[0425] AGCUAUCAAACACAAACGAAAUCCCACGGAUCGGCAUCAUCAGUUGC

[0426] AUCUCAAUUAUAUUCGCUUAUAUACAUGUCUCUUGGGCGCUGAGAACU

[0427] CCGUUGCUUACAGCAAUAAUAGCAUUGCCAUACCGACGAACUUUACU

[0428] AUUAGCGUAACGACUGAGAUACUCCCGGGUCUAUGACAAAAAACUAG

[0429] CGUUGAUGUCAAAUGUACAUUGUGGGGAUAGUACAGAGUGCUCA

[0430] AACUUGCUUCCAGUACGGGUCAUCUGUACCCAACUUAAACGCGC

[0431] AUUGACUGGUAUUGCCGUCGAGCAAGACAAAACACACAAGAGGUUU

[0432] UCGCUCAAGUAAAACAAAUUUACAAAACGCCCCCUAUAAAAAUAUUUU

[0433] GGCGGUUUCAACUUUUCUCAAAUUCUGCCCGAUCCUUCUAAGCCUAG

[0434] UAAGCGAAGCUUCAUUGAAGACCUGCUUUUCAACAAAGUAACCUUGG

[0435] CUGACGCGGGUUUCAUAAACAGUAUGGAGACUGUCUCGGGGACAUA

[0436] GCCGCAAGGGACUUGAUUUGUGCCCAAAAUUCAACGGACUGACAGU

[0437] UCUCCUCCACUUUUGACUGAUGAGAGAUAUAGCACAAUACACUUCUG

[0438] CUUUGCUGGCCGGUACAAUAACGAGUGGUGGACCCUUCGGGGCUGG

[0439] GCUGCCCUCCAAAUACCCCUUCGCUAUGCAAAUGGCAUAUCGAUUUA

[0440] UGGCAUCGGGGUUACACAGAACGUCCUGUAUGAAACCAAAAGCUCA

[0441] UUGCCAACCAGUUUAACUCAGCAAUCGGAAAAAUUCAGGACAGCCUG

[0442] AGUAGUACCGCCAGGCCCCUUGGGAAAAGUGCAGGAUGUCGUCAACCA

[0443] UAACGCCCAAGCAUUGAAUACGUUGGUCAAACAGCUCUCCUCUAGU

[0444] UCGGUGCUAUAUACCUCAGUCCUCACGAUAUCCUGAGCAGGCUCGAU

[0445] CCCCCUGAGGCCGAAGUGCAGAUCGAUCGGCUGAUCACUGGUAGGCU

[0446] GCAGAGUCUGCAGACAUAUGUCACCCAGCAGCUGAUCAGAGCAGCCG

[0447] AGAUUCGAGCUUCAGCUAACUUAGCUGCCCAAAAAUGAGUGAGUGC

[0448] GUGCUGGCCAGUCCAAGAGGGUGGAUUUCUGCGGAAAAGGCUAUCA

[0449] UCUCAUGUCCUUCCCCCAAAGCGCACCCCACGGGUUGUCUUCCUGC

[0450] ACGUAACGUAUUGUGCCCGCUCAGAGAAACUUACUACUCGCCCCC

[0451] GCCAUCUGCCACGAUGGGAAAGCUCACUUCCCGAGAGAAGGAGUAUU

[0452] CGUUUCCAAUGGGACGCAUUGGUUCGUCACCCAGCGGAACUUUACG

[0453] AACCACAGAUCAUUACCACGGAUAAUACAUUUGUGAGCGGGAAUUGC

[0454] GAUGUGGUGAUUGGAAUUGUAAAUAACACAGUGUACGAUCCACUGC

[0455] AGUCAGAAUUAGACUCAAUUAAGGAGGAGUUGGAUAAGAUUCAUAA

[0456] AAAUGGUUCAGGUGGAUCAGGUGAUAUAGAAAAACUCCUCAAUGAA

[0457] CAAGUAAAUAAGGAGAUGCAAAGUUCUAACCUGUACAUGAGCAUGU

[0458] CUUCUUGGUGUUACACCCAUAGCCUCGAUGGAGCGGGAUUGUUCCUUUUUGACCACGCUGCGGAGGAGUAUGAGCAUGCUAAAAAGCUGAUAAUAUUUCUCAACGAGAAUAAUGUUCCAGUGCAAUUGACAAGUAUAUCCGCCCCUGAGCAUAAGUUUGAAGGGCUCACACAAAUUUUCCAAAAGGCAUACGAACACGAACAGCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUGCAAUCAAGUCCAAAGAUCACGCCACGUUUAAUUUCCUCCAGUGGUAUGUAGCUGAGCAACAUGAGGAAGAAGUGUUGUUUAAGGAUAUUCUUGAUAAAAUUGAACUUAUUGGAAAUGAGAACCAUGGCCUCUAUCUUGCGGACCAAUACGUCAAGGGAAUUGCCAAGUCCCGCAAGAGU

[0459] The sequence of the mRNA construct encoding the SARS-CoV-2 XBB.1.5 spike-ferritin fusion protein (SEQ ID NO:17)

[0460] AGGAUUGUGCUGCAUCAAGCUUGCCGCCACCAUGCCCUCCUUCUCCUUCCUUCCUUCCUUUGGGCUGGAGCGCUGGCUCUCUCAGUGUGUUAAAUCUCAUACCUACCGCAGCGAGCUAUACCAAUUCCUUCCUCACACAGGGGCGGUGUAUUCCCGACAAGGGUUUUAGUAUUACUAGAUCGUGCUGCCUUCCACACUCCACACAGGUUCCUUCCUUCCUUUUUUUAACGUGACCUGGUUCCACGCAUCCACGUGAGCGGCACCAAUGGCACAAAGAGGUUCGACAAUCCAUCCCUGCCCUUUUAACGAUGGCGUGUUACUUCGCCUUCCACCGAGAAGUCUAACAUACAUCCGCGGCUGGAUCUUGGCCACACGUGCAUCGUGAUCGUGAUCGUGAACAAUGCCACACAGGUGUCAUCAACGUGGUGCGAGUUCCAGUUUUUAAUGAUCCAAUUCCAGUUUGGA GGAGAGCGAGUUCCGGUGUGUAUUCCUCUGCCAACAAUUGCACAUUUGAGUACGUGUCCAGCCCUUCCUGAUGGACCUGGAGGGAGGCAAUUUCAAGAACCUGCGGAGGUUCGUUUUAAGAAUAUCGAUGGCUACUUCUACUACUCCAAGCCACCCCCAUCAGCACGGAGCGGACCUGCCACGGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUGUGUCCUCCACAGAAGCUACCUGACACGUGGACAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAGCCAAGUACAACGAGAAUGGCACCAUCACAGAGCCAUGAUUGCGCCCGGAUGAUUGCGCCCUGGAUCCCUGUCUGACACAAAGUGUACACACAGACGCCGUGGAUUGCGCCCUGGAUCCCUGUCUGACACAAAGUGUACACACUAGUAGUACACUGCAAGGUUUAACCGUGGGAAGGGCAUCUACAGACAAG

[0461] CAAUUUCAGGGUGCAGCCUACCGAGUCCAUCGUGCGCUUUCCCAAUA

[0462] UCACAAACCUGUGCCCUUUUCACGAGGGUUUCAACGCCACCACCUUC

[0463] GCCAGCGUGUACGCCUGGAAUAGGAAGCGCAUCUCCAACUGCGUGG

[0464] CCGACUAUUCUGAUCUACAACUUCGCCCCCUUCUCGCCUUUAAG

[0465] UGCUAUGGCGUGAGCCCCACAAAGCUGAAUGACCUGUGCUUUACCA

[0466] ACGUGUACGCCGAUUCCUUCGUGAUCAGGGCAACGAGGUGAGCCA

[0467] GAUCGCACCAGGACAGACAGGACAACUCGCAGACUACAAUUAAAG

[0468] CUGCCUGACGAUUUCACCGGCUGCGUGAUCGCCUGGAACUCUAACAA

[0469] GCUGGAUAGCAAACCCAGCGGCAACUAACAAUAUUCUGUACCGGCUG

[0470] UUUAGAAAGUCUAAGCUGAAGCCAUUCGAGCGGACAUCUCCACAG

[0471] AGAUCUACCAGGCCGGCAACAAGCCCUGCAAUGGCGUGGCCGGCCCA

[0472] AACUGUUAUGCCCAUUGCAGAGUUACGGCUUUCGGCCGACUUAUG

[0473] GCGUCGGACAUCAACCUUACAGGGUAGUUGUCUCUCUUCGAACU

[0474] CCUGCAUGCUCCGGCAACGGUGUGUGGUCCCAAGAAAAGUACUAAC

[0475] CUUGUCAAAAAUAAGUGUGUGAACUUCAAUUUUAACGGUCUUACAG

[0476] GUACUGGAGUAUUGACUGAGUCAAAUAAGAAGUUUCUUCCAUUCCA

[0477] ACAAUUCGGCCGCGAUAUAGCUGAUACAACGGAUGCCGUCCGAGAC

[0478] CCUCAGACUCUGGAGAUCCUGGACAUAACUCCAUGUUCUUUUGGAG

[0479] GCGUUAGUGUGAUUACCCCAGGCACUAAUACCUCAAACCAAGUAGC

[0480] CGUCCUGUAUCAAGGAGUCAAUUGUACUGAGGUCCCUGUGGCCAUC

[0481] CAUGCGGACCAACUGACACCCACAUGGCGCGUAUAUAGCACAGGAUC

[0482] AAACGUAUUCCAGACUCGCGCGGGUUGUCUGAUCGGGGCGGAAUAC

[0483] GUAAACAAUAGCUAUGAGUGUGAUAUACCUAUUGGAGCUGGGAUUU

[0484] GCGCAAGCUAUCAAACACAAACGAAAUCCCACGGAUCGGCAUCAUCA

[0485] GUUGCAUCUCAAUCUAUUAUCGCUUAUACAAUGUCUCUUGGCGCUG

[0486] AGAACUCCGUUGCUUACAGCAAUAAUAGCAUUGCCAUACCGACGAA

[0487] CUUUACUAUUAGCGUAACGACUGAGAUACUCCCGGUGUCUAUGACA

[0488] AAAACUAGCGUUGAUUGUACAAAUGUACAUUGUGGGGAUAGUACAG

[0489] AGUGCUCAAACUUGCUUCUCCAGUACGGGUCAUUCUGUACCCAACU

[0490] UAAACGCGCAUUGACUGGUAUUGCCGUCGAGCAAGACAAAACACA

[0491] CAAGAGGUUUUCGCUCAAGUAAAACAAAUUUACAAAACCCCCUA

[0492] UAAAAUAUUUUGGCGUUUCAACUUUUCUCAAAUUCUGCCCGAUCC

[0493] UUCUAAGCCUAGUAAGCGAAGCUUCAUUGAAGACCUGCUUUUCAAC

[0494] AAAGUAACCUUGGCUGACGCGGGUUUCAUAAAACAGUAUGGAGACU

[0495] GUCUCGGGGACAUAGCCGCAAGGACUUGAUUUGUGCCCAAAAUU

[0496] CAACGGACUGACAGUUUCCUCCCACUUUUGACUGAUGAGAUGAUAU

[0497] GCACAAUACACUUCUGCUUUGCUGGCCGGUACAAUAACGAGUGGGU

[0498] GGACCUUCGGGGCUGGGCUGCCCUCCAAAUACCCUUCGCUAUGCAA

[0499] AUGGCAUAUCGAUUUAAUGGCAUCGGGUUACACAGAACGUCCUGU

[0500] AUGAAAACCAAAAGCUCAUUGCCAACCAGUUUAACUCAGCAAUCGG

[0501] AAAAAAUUCAGGACAGCCUGAGUAGUACCGCCAGCGCCUUGGGAAAG

[0502] UUGCAGGAUGUCGUCAACCAUAACGCCCAAGCAUUGAAUACGUUGG

[0503] UCAAACAGCUCUCUCUAAGUUCGGUGCUAUAUCCUCACAGUCCUCAAC

[0504] GAUAUCCUGAGCAGGCUCGAUCCCCCUGAGGCCGAAGUGCAGAUCG

[0505] AUCGGCUGAUCACUGGUAGGCUGCAGAGUCUGCAGACAUAUUGUCAC

[0506] CCAGCAGCUGAUCAGAGCAGCCGAGAUUCGAGCUUCAGCUAACUUA

[0507] GCUGCCACAAAGAUGAGUGAGUGCGUGCUGGGCCAGUCCAAGAGGG

[0508] UGGAUUUCUGCGGAAAAGGCUAUCAUCUCAUGUCCUUCCCCCCAAAG

[0509] CGCACCCCACGGGUUGUCUUCCUGCACGUAAACGUAUGUGCCCGCUC

[0510] AGGAGAAGAACUUUACUACUGCCCCCGCCAUCUGCCACGAUGGGAA

[0511] AGCUCACUUCCCGAGAGAAGGAGUAUUCGUUUCCAAUGGGACGCAU

[0512] UGGUUCGUCACCCAGCGGAACUUUUACGAACCACAGAUCAUUACCAC

[0513] GGAUAAUACAUUUGUGAGCGGGGAAUUGCGAUGUGGUGUGAUUGGAAUU

[0514] GUAAAUAACACAGUGUACGAUCCACUGCAGUCAGAAUUAGACUCAA

[0515] UUAAGGAGGAGUGGAUAAGAUUCAUAAAAAUUGGUUCAGGUGGAUC

[0516] AGGUGAUAUAGAAAAACUCCUCAAUGAACAGUAAAAUAAGGAGAUG

[0517] CAAGUUCUAACCUGUACAUGAGCAUGUCUUCUUGGUGUUACACCCC

[0518] AUAGCCUCGAUGGAGCGGGAUUGUUCCUUUUUGACCACGCUGCGGGA

[0519] GGAGUAUGAGCAUGCUAAAAAGCUGAUAUAUUUUCUCCAACGAGAAU

[0520] AAUGUUCCAGUGCAAUUGACAAGUAUAUCCGCCCUGAGCAUAAGU

[0521] UUGAAGGGCUCACACCAAAAUUUUCCAAAAGGCAUACGAACCAGAACA

[0522] GCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUGCAAUCAAG

[0523] UCCAAAGAUCACGCCACGUUUAAUUUCCUCCAGUGGUAUGUAGCUG

[0524] AGCAACAUGAGGAAGAAAGGUGUUGUUUAAGGAUAUUCUUGUAAAAU

[0525] UGAUUUUGGAAAUGGAACCAUGGCCUCUAUCUUGGCGGACCAA

[0526] UACGUCAAGGGAAUUGCCAAGUCCCGCAAGUUGAUGAUAAUAGG

[0527] ACUAGUGGAUCCAACUGGAGACUGGGUGAAGUGACUACCAGAAAG

[0528] UGAGGAAGCCUAAAAUAAACCUAGCGUACGUAAAAAUUGGAAAGAAC

[0529] CUAGCGUACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0530] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0531] SARS-CoV2 Omicron XBB.1.5 spike-ferritin fusion protein polypeptide sequence (leader sequence is underlined) (SEQ ID NO: 18)

[0532] MPLLLLLPLLWAGALA SQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKP

[0533] FERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLS

[0534] FELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQF

[0535] GRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNC

[0536] TEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGA

[0537] GICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTIS

[0538] VTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVE

[0539] QDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKV

[0540] TLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSAL

[0541] LAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQF

[0542] NSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLN

[0543] DILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE

[0544] CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAI

[0545] CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI

[0546] VNNTVYDPLQSELDSIKEELDKIHKNGSGGSGDIEKLLNEQVNKEMQSSNL

[0547] YMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS

[0548] APEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAE

[0549] QHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

Claims

1. A method of inducing a pan-Sabemicoronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine against one or more first Sabemicoronavirus variants (Variant 1), the method comprising administering to the individual one or more doses of a second SARS-CoV-2 vaccine, wherein the second SARS-CoV-2 vaccine comprises mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a Sabei coronavirus variant (variant 2) that is different from variant 1, wherein the S protein is encoded as an S protein-multimeric subunit fusion, and wherein the method induces a pan-variant immune response in the individual against Sabich coronavirus variant 1 and variant 2, and induces an immune response against one or more additional Sabich coronavirus variants that are different from variant 1 and variant 2.

2. The method of claim 1, wherein the S protein-multimerization subunit fusion is a protein-ferritin subunit fusion.

3. The method of claim 1 or claim 2, wherein the mRNA of the second SARS-CoV-2 vaccine is formulated in lipid nanoparticles (LNPs).

4. The method according to any one of claims 1 to 3, wherein variant 2 is SARS-CoV-2 Omicron.

5. The method of claim 4, wherein variant 2 is selected from subvariants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1 or XBB.1.

5.

6. The method of claim 5, wherein the subvariant is BA.4 / 5.

7. The method of claim 5, wherein the subvariant is XBB.1.

5.

8. The method according to any one of claims 1 to 7, wherein the S protein or immunogenic fragment or immunogenic variant thereof comprises K986P and / or V987P mutations.

9. The method of claim 6, wherein the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID NO.

11.

10. The method of claim 7, wherein the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID NO.

17.

11. The method of any one of claims 1 to 10, wherein the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabich coronavirus S protein in the form of a fusion protein capable of assembling in vivo to form nanoparticles.

12. The method of any one of claims 1 to 11, wherein the method induces an immune response against one or more additional Sabich coronavirus variants that differ mutationally from variant 1 and / or variant 2.

13. The method of any one of claims 1 to 12, wherein the second SARS-CoV-2 vaccine is a monovalent vaccine.

14. The method of any one of claims 1 to 12, wherein the second SARS-CoV-2 vaccine is a bivalent vaccine comprising an additional mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimerized subunit fusion and is derived from a Sabeucoronavirus variant that is different from variant 2 and the same as or different from variant 1.

15. The method of claim 14, wherein the S protein encoded by the additional mRNA is selected from the group consisting of WuhanD614G, Alpha, Beta or Delta variant S proteins.

16. The method of claim 15, wherein the additional mRNA of the second SARS-CoV-2 vaccine comprises the mRNA sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

17. The method of any one of claims 1 to 16, wherein the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 4 months after the last dose of the first SARS-CoV-2 vaccine is administered.

18. The method of claim 17, wherein the first dose of the second SARS-CoV-2 vaccine is administered to the individual at least 6 months after administration of the last dose of the first SARS-CoV-2 vaccine.

19. An immunogenic composition comprising mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof from a first Sabicorina coronavirus variant for use in inducing a pan-Sabicorina coronavirus variant immune response in an individual who has previously received one or more doses of a first SARS-CoV-2 vaccine comprising or encoding an immunogen from a second Sabicorina coronavirus variant, wherein the S protein is encoded as an S protein-multimeric subunit fusion, and wherein the immunogenic composition is used to induce an immune response against the first Sabei coronavirus variant and the second Sabei coronavirus variant and against at least a third Sabei coronavirus variant.

20. The immunogenic composition for use according to claim 19, wherein the S protein-multimerization subunit fusion is a protein-ferritin subunit fusion.

21. The immunogenic composition for use according to claim 19 or 20, wherein the mRNA is formulated in lipid nanoparticles (LNPs).

22. The immunogenic composition for use according to any one of claims 19 to 21 , wherein the first Sabicolaou coronavirus variant is SARS-CoV-2 Omicron.

23. An immunogenic composition for use according to claim 22, wherein the SARS-CoV-2 Omicron variant is selected from subvariants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1 or XBB.1.

5.

24. An immunogenic composition for use according to claim 23, wherein the subvariant is BA.4 / 5.

25. An immunogenic composition for use according to claim 23, wherein the subvariant is XBB.1.

5.

26. The immunogenic composition for use according to any one of claims 19 to 25, wherein the S protein or immunogenic fragment or immunogenic variant thereof comprises the K986P and / or V987P mutations.

27. The immunogenic composition for use according to claim 24, wherein the mRNA comprises or consists of the mRNA sequence of SEQ ID NO.

11.

28. The immunogenic composition for use according to claim 25, wherein the mRNA comprises or consists of the mRNA sequence of SEQ ID NO.

17.

29. The immunogenic composition for use according to any one of claims 19 to 28, wherein the first SARS-CoV-2 vaccine does not comprise a nucleic acid encoding the Sabich coronavirus S protein in the form of a fusion protein capable of assembling in vivo to form nanoparticles.

30. The immunogenic composition for use according to any one of claims 19 to 29, wherein the third Sabecovoravirus variant differs from the first and / or second Sabecovoravirus variant in mutations.

31. The immunogenic composition for use according to any one of claims 19 to 30, wherein the immunogenic composition is a monovalent composition.

32. The immunogenic composition for use according to any one of claims 19 to 30, wherein the immunogenic composition is a bivalent composition further comprising a second mRNA encoding a single spike (S) protein or an immunogenic fragment or immunogenic variant thereof, wherein the S protein is encoded as an S protein-multimerized subunit fusion and is derived from a Sabei coronavirus variant that is different from the first Sabei coronavirus variant and is the same as or different from the second Sabei coronavirus variant.

33. The immunogenic composition for use according to claim 32, wherein the S protein or immunogenic fragment or immunogenic variant thereof encoded by the second mRNA is from a variant selected from Wuhan D614G, Alpha, Beta or Delta SARS-CoV2.

34. The immunogenic composition for use according to claim 33, wherein the second mRNA comprises the mRNA sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

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