An efficient vaccine

Abstract

Provided herein is an isolated polynucleotide encoding a polypeptide comprising structural proteins nsp1, nsp2, nsp3 and nsp4 and an antigenic protein fused to a signal sequence, a transmembrane domain and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope. The polynucleotide is useful for producing a vaccine against viral infections, particularly COVID-19 infections, treating cancer and / or inflammatory diseases.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of efficient vaccines comprising alphavirus replicons and antigens, as well as methods and compositions for treating and / or immunizing against the antigens. In particular, the present disclosure relates to vaccines for coronaviruses, such as SARS-CoV-2 (COVID-19). [Background technology]

[0002] Coronaviruses are a large family of viruses that typically cause mild-to-moderate upper respiratory tract illnesses, similar to the common cold, but over the past two decades, three novel coronaviruses have emerged from zoonotic sources and cause severe and widespread illness and death.

[0003] There are hundreds of coronaviruses, most of which circulate among animals such as pigs, camels, bats, and cats. Occasionally, these viruses can infect humans and cause disease in so-called spillover events. Of the seven known coronaviruses that cause human illness, four cause only mild to moderate illness. Three can cause more severe and even fatal illness. SARS coronavirus (SARS-CoV) emerged in November 2002 and caused Severe Acute Respiratory Syndrome (SARS). The virus had disappeared by 2004. Middle East Respiratory Syndrome (MERS) is caused by MERS coronavirus (MERS-CoV). Transmitted from a zoonotic source in camels, MERS was identified in September 2012 and continues to cause sporadic and localized outbreaks. The third novel coronavirus to emerge this century is called SARS-CoV-2. The virus causes coronavirus disease 2019 (COVID-19), which emerged from China in December 2019 and was declared a pandemic by the World Health Organization on March 11, 2020. (Coronaviruses: https: / / www.niaid.nih.gov / diseases-conditions / coronaviruses)

[0004] Most people infected with the SARS-CoV-2 virus experience mild to moderate respiratory illness and recover without needing special treatment. Older people and people with underlying conditions such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop severe illness. Anyone can get COVID-19, and any age can become seriously ill or die. (Coronavirus disease (COVID-19): https: / / www.who.int / health-topics / coronavirus#tab=tab _1)

[0005] Omicron (B.1.1.529 variant) was first reported to WHO from South Africa on 24 November 2021 as a novel SARS-CoV-2 variant.

[0006] Preliminary evidence suggests that there may be an increased risk of reinfection with Omicron compared to other variants of concern (i.e., people who have previously been infected with COVID-19 may be more susceptible to reinfection with Omicron). (Update on Omicron: https: / / www.who.int / news / item / 28-11-2021-update-on-omicron)

[0007] To date, several vaccines against COVID-19 have been approved. In particular, mRNA vaccines have shown efficacy of over 95%. However, more vaccines need to be supplied to the world to prevent this pandemic. In order to supply the world with an efficient vaccine against any variant, further vaccine development is needed, especially vaccines based on technologies that can rapidly produce an efficient vaccine against any variant. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2021 / 21068 Summary of the Invention [Problem to be solved by the invention]

[0009] One of the objectives of the present disclosure is to provide a new design concept for developing vaccines against antigens, such as viral antigens, cancer antigens, etc., that are effective for preventing or treating infectious diseases or cancer. [Means for solving the problem]

[0010] The present disclosure relates to novel antigenically active proteins / polypeptides capable of inducing an immune response against an antigen. The proteins / polypeptides disclosed herein comprise an antigenic protein fused to a signal sequence, a transmembrane domain and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope. The antigenic protein can be a protein derived from a coronavirus structural protein or a fragment thereof.

[0011] Coronavirus structural proteins may include spike (S) protein, nucleocapsid (N) protein, membrane (M) protein and small envelope protein (E). Antigenic proteins may be derived from any of the structural proteins or combinations thereof. Specific examples of antigenic proteins include the S1 and / or S2 subunits of the spike protein, particularly the receptor binding domain (RBD) of the S1 subunit.

[0012] In another aspect, the present disclosure relates to novel polynucleotides encoding the above-mentioned novel antigenically active proteins / polypeptides capable of inducing protection against antigenic proteins.

[0013] In another aspect, the present disclosure relates to novel alphavirus replicons capable of expressing the above-mentioned antigenically active proteins / polypeptides, which comprise a polynucleotide, such as an RNA, encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding the above-mentioned antigenically active protein / polypeptide as a gene of interest.

[0014] In yet another aspect, the present disclosure relates to a vaccine comprising the above-mentioned polypeptide or polynucleotide. In particular, the present disclosure provides a vaccine comprising a polynucleotide encoding an alphavirus nonstructural protein nsp1, nsp2, nsp3 and nsp4, and a polypeptide comprising an antigenic protein fused to a signal sequence, a transmembrane domain and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope. The CD4+ T cell epitope may be a universal epitope. When the antigenic protein is a protein derived from a viral structural protein, the vaccine can be used to prevent and / or treat a subject from a viral infection.

[0015] In yet another aspect, the present disclosure relates to a method for immunizing, preventing or treating a subject against a viral infection comprising administering to a subject in need thereof an effective amount of the above-mentioned polypeptide or polynucleotide.

[0016] In yet another aspect, the present disclosure relates to the use of the aforementioned polypeptide or polynucleotide for the manufacture of a medicament.

[0017] In a further aspect, the disclosure relates to novel polynucleotides encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 and polypeptides comprising antigenic peptides, the polynucleotides comprising modified nucleosides (e.g., N1-methyl-pseudouridine or 5-methyl-cytidine or both).

[0018] This efficient vaccine design is also applied to develop vaccines for the treatment of cancer or inflammatory diseases. [Brief description of the drawings]

[0019] [Figure 1] Alphavirus replicon constructs. [Diagram 2]ELISA for RBD proteins of SARS-CoV-2 mutants. [Diagram 3] ACE inhibition assay [Figure 4] Effects of polynucleotides with modified nucleosides on innate immunity. [Diagram 5] Effect of vaccine compositions on body weight in hamsters exposed to COVID-19 wild-type or gamma variant. [Figure 6] Effect of vaccine compositions on quantitative RT-PCR assays of SARS-CoV-2 in hamsters exposed to SARS-CoV-2 wild-type or gamma variant [Figure 7] Effect of vaccine compositions on histopathological analysis of lungs in hamsters exposed to SARS-COV-2 wild-type or gamma mutant strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] As used herein, "antigen" refers to a molecule that can be bound by an antibody or a T cell receptor (TCR). Antigens can additionally be recognized by the immune system and / or induce humoral and / or cellular immune responses that result in activation of B and / or T lymphocytes. Antigens as used herein include, but are not limited to, viruses, allergens, autoantigens, haptens, cancer antigens (i.e., tumor antigens) and infectious disease antigens such as Mycobacterium tuberculosis, as well as small organic molecules such as abused drugs (such as nicotine), and fragments and derivatives thereof. Furthermore, antigens as used in this disclosure can be peptides, proteins, domains, carbohydrates, alkaloids, lipids such as steroid hormones, or small molecules and fragments and derivatives thereof, autoantibodies, and cytokines themselves. As used herein, "antigenic peptide" refers to a protein or peptide that can act as an antigen.

[0021] As used herein, a "virus" may be, but is not limited to, severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, hepatitis B virus (HBV), influenza, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, or a virus of the family Orthymyxoviridae.

[0022] As used herein, "coronavirus" refers to a single-stranded, positive-sense RNA virus belonging to the family Coronaviridae. Exemplary Coronaviridae viruses include, but are not limited to, SARS-Cov, MERS-Cov, and SARS-CoV-2 (COVID-19). SARS-CoV-2 (COVID-19) can include known and unknown mutant forms. Known mutations include the SARS-CoV-2 E484K_N501Y_K417T mutation (gamma, Brazilian valiant), T478K_L452R (delta, Indian valiant), E484K_N501Y_K417N mutation (beta, South African valiant), E484K mutation and K417N_S477N_T478K_E484A_Q493K_G446S_N501Y_Y505H_G496S_Q498R_N440K_G339D_S375F_S373P_S371L mutation (Omicron). The coronavirus genome encodes a number of non-structural proteins and four major structural proteins including spike (S), nucleocapsid (N), membrane (M) and small envelope (E) proteins. The large envelope glycoprotein, spike (S) protein, is composed of S1 and S2 subunits. The "receptor binding domain (RBD)" is located in the S1 subunit. A preferred example of an antigenic protein as used herein is the coronavirus RBD, a fragment thereof or a mutant form thereof. The COVID-19 spike protein, the S1 and S2 subunits and RBD of the spike protein and its mutant forms have been identified and published (Wrapp et al., Science Vol. 367, issue 6483(2020) pp.1260-1263 10.1126 / science.abb2507, Z. Wang et al., Nature Vol 592(2021) pp616-622, the contents of which are incorporated herein by reference).

[0023] As used herein, a "coronavirus structural protein" may be a naturally occurring viral structural protein or a modified protein thereof. The modified protein may be a fragment of a naturally occurring viral structural protein or a mutant thereof. In one embodiment, the modified protein has at least 70%, 75%, 80%, 85%, 90%, 95% or 98% amino acid sequence identity with a naturally occurring viral structural protein or a fragment thereof. In one embodiment, the modified protein is a mutant in which at most 10% of amino acids are deleted, substituted and / or added to a naturally occurring viral envelope protein or a fragment thereof.

[0024] As used herein, a "transmembrane domain (TM)" is a protein that is either derived from a natural or synthetic source. When natural in origin, the domain in some embodiments is derived from any membrane-bound or transmembrane protein. In one embodiment, the membrane-bound or transmembrane protein is a heterologous protein relative to the origin of the antigenic peptide. Examples of membrane-bound or transmembrane proteins include the alpha, beta or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; Toll-like receptors (TLRs), such as TLR1-TLR10 in humans and TLR1-TLR9, TLR11-TLR13 in mice; interleukin (IL) receptors, such as the IL-1-28 receptor, RANTES receptor (CCR1, CCR3, CCR5), MIP-1 receptor, PF4 receptor, M-CSF receptor and NAP-2 receptor, which belong to the GPCR chemokine receptors; hemagglutinin (HA). In another embodiment, the membrane-bound or transmembrane protein is a protein derived from COVID-19, such as the COVID-19 spike protein.

[0025] Examples of transmembrane proteins include: 5-lipoxygenase activating protein, ABC transporters, ACBP, amyloid beta (A4), Bcl-2 inhibitor, BNIP, CAAX protease, cytochrome P450, E-NPP, EPHA1, EPHA2, EPHA3, EPHA4, fatty acid desaturase, gamma secretase, glucose transporter, glycophorin, GPCR, HER2 / ErbB2, HER3 / ErbB3, HER4 / ErbB4, HSD-11β, hypoxia-inducible protein, immunoglobulins, insulin receptor, integrins, insulin receptors ... On-channels, MAPEG, MFS, MinK family, MPP, peptidase AD, peptidase family M48, peptidase MA, protein jagged, receptor kinases, SNARE complexes, sulfatases, TNF receptors, transmembrane protein 14, transporters, TROBP, VEGF receptors, aldehyde dehydrogenases, ammonia and urea transporters, FMN-linked oxidoreductases, leucine-rich repeat (LRR)-containing transmembrane proteins, leukotriene C4 synthase, lysosome-associated membrane glycoproteins, major integral proteins Also included are the Membrane Intrinsic Protein (MIP) / FNT superfamily, microsomal prostaglandin E synthase, N-(deoxy)ribosyltransferase-like membrane protein, neutral / alkaline ceramidase, oligosaccharyltransferase, pentameric ligand-gated ion channels, rhodopsin-like receptors and pumps, single-helical ATPase regulators, squalene / phytoene synthase, stearoyl-CoA desaturase 1, stannin (SNN) membrane proteins, T-cell surface glycoprotein CD3 zeta chain, tetratricopeptide repeat (TPR) alpha-helical repeat proteins, transmembrane proteins with NAD(P)-binding Rossmann fold domains.

[0026] In addition, monotypic / peripheral proteins attached to the lipid bilayer or other integral proteins and peptides can also be used as transmembrane proteins. Examples include alpha / beta-hydrolases, annexins, Bet V1-like proteins, C1 domain-containing proteins, C2 domain-containing proteins, CoA-dependent acyltransferases, CRAL-TRIO domain-containing proteins, DNase I-like proteins, fibrinogen, FYVE / PHD zinc finger proteins, galactose-binding domain-like proteins, glycolipid transfer proteins, immunoglobulin-like superfamily (E-set) proteins, lipocalins, lipoxygenases, PGBD superfamily, PH domain-like proteins, phosphatidylinositol 3- / 4-kinases, PLC-like phosphodiesterases, phosphotyrosine protein phosphatase II, P-loop-containing nucleoside triphosphate hydrolases, protein kinase superfamily, PX domain-containing proteins, saposins, synucleins, and the transcription factor Tabby.

[0027] In one embodiment of the present disclosure, the transmembrane domain comprises at least the transmembrane region of a membrane-associated or transmembrane protein. In addition, the transmembrane domain can also comprise the juxtamembrane domain (JMD) and / or the cytoplasmic tail of the membrane-associated or transmembrane protein.

[0028] Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0029] Preferred transmembrane domains may be derived from influenza virus hemagglutinin (HA), CD80, Toll-like receptor 4 (TLR4) or COVID-19 spike protein. Specific examples include a protein comprising the flexible juxtamembrane region or flexible linker, transmembrane domain and cytoplasmic tail of influenza virus hemagglutinin "HA (flexible-TM-Cyt)", a protein consisting of the transmembrane domain and cytoplasmic tail of human CD80, a protein consisting of the transmembrane domain (TM) and Toll / Interleukin-1 receptor domain (TIR), and a protein consisting of the juxtamembrane domain (JMD) and transmembrane domain of COVID-19 spike (S) protein.

[0030] As used herein, "nucleoside" refers to a molecule comprised of guanine (G), adenine (A), thymine (T), uridine (U), cytidine (C), or modified nucleosides thereof.

[0031] Modified nucleosides include, but are not limited to, pseudouridine, N1-methyl-pseudouridine, 5-methyl-uridine, pseudocytidine, N1-methyl-pseudocytidine and 5-methyl-cytidine.

[0032] Pseudouridine or pseudocytidine is an isomer of uridine or cytidine in which uracil or cytosine is attached through a carbon-carbon rather than a nitrogen-carbon glycosidic bond.

[0033] In one embodiment, the modified nucleoside is independently selected from N1-methyl-pseudouridine or 5-methyl-cytidine. In one embodiment, substantially 100% of the cytidine and uridine contained in the mRNA or saRNA are modified cytidine (e.g., 5-methyl-cytidine) and modified uridine (e.g., N1-methyl-pseudouridine), respectively. In one embodiment, 100% of the cytidines are modified cytidines, 80% of the uridines are modified uridines, and in another embodiment, 50% of the cytidines are modified cytidines, and 50% of the uridines are modified uridines. In yet another embodiment, less than 100% of the uridines in the saRNA are modified uridines.

[0034] As used herein, a "signal sequence" (sometimes referred to as a signal peptide, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) is a polynucleotide or polypeptide, depending on the context. Signal sequences are about 9 to 200 nucleotides or 3-70 amino acids in length and are incorporated at the 5' or N-terminus of a coding region or protein, as appropriate. Some signal sequences are cleaved from the protein, e.g., by a signal peptidase, after the protein has been transported to a desired site.

[0035] In some embodiments, the signal sequence of IL-2 can be used, particularly human IL-2. In another embodiment, the signal sequence of the COVID-19 spike protein can be used.

[0036] As used herein, a CD4+ T cell epitope is a peptide that binds to a major histocompatibility complex (MHC) class II molecule (MHC-II) and elicits a CD4+ T cell immune response. The CD4+ T cell epitope may be a peptide derived from the virus from which the antigen protein is derived. CD4+ T cell epitopes derived from viral proteins can be identified by known epitope mapping procedures. In the present disclosure, a peptide consisting of multiple CD4+ T cell epitopes linked by a linker such as "AA" can be used as a "CD4+ T cell epitope". Alternatively, the CD4+ T cell epitope may be a Pan-DR epitope (PADRE).

[0037] The term "PADRE" as used in this disclosure refers to a Pan HLA DR binding epitope or universal epitope, a peptide that activates almost all CD4+ T cells regardless of HLA haplotype. Various peptides have been identified as PADREs, and any of the known PADREs can be used in this disclosure. One example of a PADRE is AKFVAAWTLKAAA (SEQ ID NO: 1).

[0038] As used herein, a CD8+ T cell epitope is a peptide that binds to a major histocompatibility complex (MHC) class I molecule (MHC-I) and induces a CD8+ T cell immune response. A CD8+ T cell epitope can be a peptide derived from a virus from which an antigen protein is derived. CD8+ T cell epitopes derived from viral proteins can be identified by known epitope mapping procedures. In the present disclosure, a peptide consisting of multiple CD8+ T cell epitopes linked by a linker such as "AA" can be used as a "CD8+ T cell epitope".

[0039] The antigen protein, the signal sequence, the transmembrane domain, and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope may be directly or indirectly fused, and in one embodiment, may be interposed between them by one or two linkers.

[0040] Also, at least one peptide selected from a coronavirus structural protein, a signal sequence, a transmembrane domain, and a CD4+ T cell epitope and a CD8+ T cell epitope can be truncated and replaced with a short linker, hi some embodiments, the coronavirus structural protein, the transmembrane domain and / or the signal sequence comprises one or more peptide linkers.

[0041] Examples of short linkers consist of 2 to 25 amino acids (e.g., 2, 3, 4, 5 or 6 amino acids). Usually, it is 2 to 15 amino acids in length, such as SG, GS, SGG, GGS SGSG and TRGGS. In certain circumstances, the linker may consist of only one amino acid, such as glycine (G), serine (S) and cysteine ​​(C).

[0042] When the coronavirus structural protein is chemically conjugated to at least one peptide selected from a signal sequence, a transmembrane domain, and a CD4+ T cell epitope and a CD8+ T cell epitope through a chemical cross-linker, examples of the cross-linker include, but are not limited to, SMPH, sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, and SIA. Commercially available chemical cross-linkers can also be used.

[0043] IgG-derived substances can also be used as linkers. Examples of IgG-derived substances include: (i) complete (hinge-CH 2 CH 3 ) (ii) Half (hinge-CH 3) and (iii) IgG1 to IgG4 containing a short (only 12 aa hinge). A preferred example is IgG4-CH 3 It is.

[0044] As used herein, "alphavirus" refers to an RNA-containing virus belonging to the Togaviridae family of viruses. Exemplary togaviruses include, but are not limited to, Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki forest virus, Middleburg virus, Chikungunya virus (CHIKV), O'nyong-nyong virus, Ross River virus, Barmah forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragati virus, Highland J virus, Fort Morgan virus, Ndum virus, Boggy Creek virus, and Okelbo virus.

[0045] By "alphavirus structural protein" is meant a polypeptide or fragment thereof having at least about 80% amino acid sequence identity to a naturally occurring viral capsid or envelope protein. In one embodiment, the alphavirus structural protein has at least about 85%, 90%, 95% or more amino acid sequence identity to Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEEV), Sindbis virus, Semliki Forest virus, Middleburg virus, Chikungunya virus (CHIKV), O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Babanki virus, Kiziragati virus, Highland J virus, Fort Morgan virus, Ndum virus, or Boggy Creek virus. Wild-type amino acid sequences for alphavirus structural proteins can be obtained from GenBank.

[0046] In specific embodiments, the alphavirus is CHIKV, such as CHIKV strain 37997 or LR2006 OPY-1.In other embodiments, the alphavirus is VEEV, such as VEEV strain TC-83.

[0047] By "alphavirus replicon" is meant an RNA molecule capable of directing its own amplification in vivo in a target cell. The replicon contains the cis-RNA sequences required for replication that are recognized and utilized by the encoded polymerases, as well as encoding the polymerases (nspl, nsp2, nsp3, nsp4) that catalyze RNA amplification. Alphavirus replicons usually contain the following ordered elements: 5'UTR, sequences encoding alphavirus nonstructural proteins (nsp1, nsp2, nsp3, nsp4), 3'UTR, and polyA tail. Alphavirus replicons also contain one or more viral subgenomic (SG) promoters that direct the expression of a gene of interest. These sequences can have one or more mutations as taught in the prior art.

[0048] Alphavirus replicons provided by this disclosure can have the construct shown in FIG.

[0049] In this disclosure, "comprises," "comprising," "containing," "having," and the like, may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. patent law, but the terms are open-ended, thus permitting the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited.

[0050] By "fragment" is meant a portion of a protein, polypeptide or polynucleotide. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0051] "Reference" means a standard or control condition.

[0052] A "reference sequence" is a defined sequence that is used as a standard for sequence comparison. A reference sequence may be a subset or the entirety of a particular sequence; for example, it may be a segment of a full-length cDNA or gene sequence, or it may be a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer number therebetween or thereabout.

[0053] Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity can use the BLAST program, where closely related sequences are indicated by a probability score between e<”3” and e<”100”.

[0054] "Effective amount" refers to the amount of agent required to ameliorate the symptoms of a disease in an untreated patient. The effective amount of active compound used to practice the present disclosure for the prevention or treatment of a disease varies according to the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0055] Satisfactory efficacy can be achieved by administering 1 to 4 systemic doses, e.g., intramuscular, subcutaneous, or intravenous, in a dose of 10 3 ~10 10 Infectious units (IU) or 0.01 to 500 μg per dose, preferably 10 5 ~10 10 IU or 0.1-100 μg, e.g. 10 per dose 7 ~10 9IU or in amounts of 1-50. The replicons are preferably formulated into vaccine compositions suitable for administration in conventional manners. The present disclosure provides vaccine compositions comprising the alphavirus replicons disclosed herein.

[0056] "Subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0057] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms. Although not intended to prevent, it is understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0058] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of or susceptible to developing the disorder or condition.

[0059] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0060] Unless specifically stated otherwise or clear from the context, as used herein, the terms "a," "an," and "the" are understood to be in the singular or in the plural.

[0061] Those of skill in the art will recognize that the polynucleotide sequences described herein and in the claims recite a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0062] Any vaccine composition or method provided herein can be combined with any one or more of the other vaccine compositions and methods provided herein.

[0063] The term "vector" refers to a means by which a nucleic acid sequence can be propagated and / or introduced between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, etc., which can replicate autonomously or integrate into a host cell's chromosome. Vectors can also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, poly-lysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, etc., but which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the present disclosure are plasmids or bacmids.

[0064] Typically, the nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is under the transcriptional regulatory control of the promoter and / or enhancer.

[0065] The method of transfection and the selection of the expression vehicle will depend on the host system selected.Transfection methods are described, for example, in Ausubel et al. (supra); expression vehicles can be selected, for example, from those provided in Cloning Vectors: A Laboratory Manual (PH Pouwels et al., 1985, Supp. 1987).The references cited in this paragraph are incorporated herein by reference.

[0066] There are various expression systems for generating the construct of the present disclosure.Expression vectors useful for generating the construct include, but are not limited to, chromosomal, episomal, and viral vectors, such as bacterial plasmid-derived, bacteriophage-derived, transposon-derived, yeast episome-derived, insertion element-derived, yeast chromosomal element-derived, alphavirus (e.g., Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV)), baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus-derived vectors, and combinations thereof.

[0067] The constructs and / or vectors used herein comprise an alphavirus polynucleotide encoding the nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and a gene of interest encoding a polypeptide comprising a coronavirus structural protein fused to a signal sequence and / or transmembrane domain, as described above.

[0068] The vector may be, for example, a phage, plasmid, virus, or retrovirus vector. The construct and / or vector containing the nucleotide is required to be operably linked to a suitable promoter, such as, but not limited to, the CMV promoter, the phage lambda PL promoter, the E. coli lac, phoA and tac promoters, the SV40 early and late promoters, and the promoter of a retrovirus LTR. Other suitable promoters are known to those skilled in the art depending on the host cell and / or the desired expression rate. The expression construct further contains sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably includes a translation initiation codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0069] The vector preferably contains at least one selection marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Among the vectors, preferred are viral vectors such as baculovirus, poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpes virus and retrovirus. Other vectors that can be used with the present disclosure include vectors for use in bacteria, including pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among the preferred eukaryotic vectors are pFastBacl pWINEO, pSV2CAT, pOG44, pXTl and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readily apparent to those skilled in the art.

[0070] Recombinant constructs can be prepared and used to transfect and express viral proteins, including those described herein, in eukaryotic and / or prokaryotic cells. Thus, in one embodiment, the disclosure provides a host cell comprising a vector (or vectors) containing nucleic acid encoding alphavirus structural proteins including capsid, E3, E2, 6K, and El or portions thereof, and a vector comprising nucleic acid encoding alphavirus nsp1, nsp2, nsp3 and nsp4, and a gene of interest encoding a polypeptide comprising a signal sequence, a coronavirus RBD, a transmembrane domain, and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope under conditions that allow for the formation of alphavirus replicon particles.

[0071] In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into an insect cell. In a preferred embodiment, the cell is an insect cell. In another embodiment, the insect cell is an Sf9 cell.

[0072] One particular bacterial expression system for producing polypeptides is the E. coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of T7 regulatory signals, expression of the polypeptide is achieved by inducing expression of T7 RNA polymerase in the host cell. This is usually achieved by using a host strain that expresses T7 RNA polymerase in response to IPTG induction. Once produced, the recombinant polypeptide is then isolated according to standard methods known in the art, such as those described herein.

[0073] Depending on the vector and host cell selected, the construct is produced by growing a host cell transfected with the vector under conditions in which the recombinant proteins are expressed and the alphavirus replicon is produced, forming a construct comprising an alphavirus replicon packaged in particles of alphavirus structural proteins. In one embodiment, a method of producing a construct is provided which involves co-transfecting a vector comprising a polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest encoding a polypeptide comprising a coronavirus structural protein fused to a signal sequence and / or transmembrane domain, and at least one vector each encoding at least one alphavirus structural protein, into a suitable host cell and expressing said alphavirus structural protein under conditions that allow for construct formation. In another embodiment, the eukaryotic cell is selected from the group consisting of yeast, insect, amphibian, avian or mammalian cells. Selection of appropriate growth conditions is within the skill of the art or the skill of a person skilled in the art.

[0074] Methods for growing cells producing alphavirus replicon particles of the present disclosure include, but are not limited to, batch, batch-fed, continuous and perfusion cell culture techniques. In one embodiment, cells co-transfected with a vector encoding an alphavirus replicon, a vector comprising a polypeptide encoding a capsid, and a vector comprising a polynucleotide encoding an envelope protein, such as that from CHIKV or VEEV, are grown in a bioreactor or fermentation chamber that allows the cells to grow and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is performed in a sterile environment under controlled temperature and atmospheric conditions. A bioreactor is a chamber used to culture cells in which environmental conditions such as temperature, atmosphere, agitation and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ, the contents of which are incorporated herein by reference). In another embodiment, the pre-sterilized plastic bag is about a 10 L to 1000 L bag.

[0075] In another embodiment, RNA molecules such as alphavirus replicons can be created from a template DNA sequence by conventional procedures known in the art. In vitro transcription (IVT) methods allow for template-directed synthesis of RNA molecules. IVT methods allow for the synthesis of large amounts of RNA transcripts. Generally, IVT utilizes a DNA template that includes a promoter sequence upstream of the sequence of interest. The promoter sequence is most commonly of bacteriophage origin, such as the T7, T3, or SP6 promoter sequences, but many other promoter sequences can be tolerated, including those designed de novo. Transcription of the DNA template is usually optimally performed by using an RNA polymerase that corresponds to a specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT generally initiates with dsDNA, but can proceed on a single strand. Kits for in vitro transcription such as the T7 transcription kit [RiboMax™ Express Large Scale RNA production System, Promega (WI USA)].

[0076] As used herein, the term "pharmaceutical acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates, and includes, as known to those skilled in the art, any aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, and Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, infusion and nutritional supplements, such materials and combinations thereof. The pH and exact concentrations of the various components in the vaccine composition are adjusted according to well-known parameters.

[0077] Encapsulating material refers to the delivery vehicle in which the polynucleotide or vector is packaged, such as replicon particles (e.g., the alphavirus replicon particles described in U.S. Patent Publication No. 2019 / 0185822, the contents of which are incorporated by reference) and lipid delivery systems (e.g., liposomes).

[0078] In some embodiments, the vaccine composition or formulation of the present disclosure comprises a lipid delivery system, such as liposomes, lipoplexes, lipid nanoparticles, or any combination thereof. A polynucleotide, such as an alphavirus replicon described herein, can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. Liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficiency of polynucleotide-directed protein production, as such formulations can enhance cell transfection by the polynucleotide and / or enhance translation of the encoded protein. Liposomes, lipoplexes, or lipid nanoparticles can also be used to enhance the stability of the polynucleotide.

[0079] Liposomes are artificially prepared vesicles that can be composed primarily of lipid bilayers and can be used as delivery vehicles for administration of pharmaceutical formulations. Liposomes can be of various sizes. Multilamellar vesicles (MLVs) can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments. Small single cell vesicles (SUVs) can be smaller than 50 nm in diameter, and large unilamellar vesicles (LUVs) can be between 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve attachment of liposomes to non-healthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes can contain low or high pH values ​​to improve delivery of pharmaceutical formulations.

[0080] The formation of liposomes may be influenced by factors such as the pharmaceutical agent and liposomal components to be encapsulated, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any further processes involved during application and / or delivery of the vesicles, the size, polydispersity and shelf life of the vesicles optimal for the intended application, and the batch-to-batch reproducibility and scale-up productivity of a safe and efficient liposomal product.

[0081] In some embodiments, a polynucleotide such as an alphavirus replicon described herein may be encapsulated by a liposome and / or contained in an aqueous core that can then be encapsulated by a liposome.

[0082] In some embodiments, polynucleotides such as the alphavirus replicons described herein can be formulated in cationic oil-in-water emulsions, where the emulsion particles contain an oily core and cationic lipids that can interact with the polynucleotide, thereby tethering the molecule to the emulsion particle. In some embodiments, polynucleotides described herein can be formulated in water-in-oil emulsions that contain a continuous hydrophobic phase in which a hydrophilic phase is dispersed.

[0083] In some embodiments, polynucleotides such as the alphavirus replicons described herein can be formulated in lipid polycation complexes. By way of non-limiting example, the polycation can include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine and / or polyarginine, and cationic peptides.

[0084] In some embodiments, polynucleotides such as the alphavirus replicons described herein can be formulated in lipid nanoparticles (LNPs).

[0085] Lipid nanoparticle formulations usually comprise one or more lipids. In some embodiments, lipid is cationic or ionizable lipid. In some embodiments, lipid nanoparticle formulations further comprise other components, including phospholipids, structural lipids, quaternary amine compounds, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids. In some embodiments, the amount of cationic and ionizable lipid in lipid composition ranges from about 0.01 mol% to about 99 mol%.

[0086] LNPs contain pH-sensitive, ionizable cationic lipids that attract anionic nucleic acids to form the core of the self-assembling nanoparticles, thus ensuring high encapsulation. At physiological pH, LNPs are neutral, eliminating the toxicity mechanisms seen with molecules that are constantly cationic.

[0087] These same pH-sensitive lipids are involved in responding to the acidic environment of the endosome and triggering the rupture of the endosome and the release of nucleic acids into the cell.

[0088] This replicon-based vaccine technology is a unique platform technology for vaccination because RNA can self-amplify to generate vaccine antigens and can be delivered to cellular organs. Moreover, this replicon-based vaccine technology overcomes the challenges commonly associated with DNA-based vaccines, such as the risk of genome integration or the high dose and device required for administration, e.g., electroporation, and is expected to have higher immunogenicity with a minimum dose based on a self-replicating system, which is superior to mRNA technology. Also, the inventors have designed antigens based on a receptor binding domain (RBD) with a signal sequence, a transmembrane domain, and at least one peptide selected from CD4+ T cell epitopes and CD8+ T cell epitopes. A preferred embodiment includes a universal epitope of CD4+ T cells, such as PADRE, to enhance immunogenicity.

[0089] In one embodiment, the vaccine composition may be monovalent, containing only one alphavirus replicon, hi another embodiment, the vaccine composition may be bivalent and contain two alphavirus replicons encoding antigenic proteins from different strains or variants.

[0090] According to the present disclosure, the novel antigenically active proteins / polypeptides are also useful for generating antibodies for diagnosis and protection against coronaviruses while minimizing the possibility of ADE. The proteins / polypeptides disclosed herein comprise a minimal sequence encoding the coronavirus RBD fused to a signal sequence, a transmembrane domain (TMD) and at least one peptide selected from a CD4+ T cell epitope and a CD8+ T cell epitope intended to maximize immunogenicity and minimize ADE.

[0091] Such efficient vaccine design may also be applied to the development of vaccines against cancer or inflammatory diseases.

[0092] The present invention will now be described in detail with reference to the following examples, which are not intended to limit the scope of this application. EXAMPLES

[0093] The gene encoding Construct 1 shown below was synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages).

[0094] Construct 1 [ka] The full amino acid sequence of Construct 1 is as follows: MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICIAKFVAAWTLKAAA (SEQ ID NO: 2) Signal sequence COVID19(1-13): MFVFLVLLPLVSS (SEQ ID NO: 3) COVID-19-RBD [Brazil (Gamma) Variant]: VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST (SEQ ID NO: 4) HA Linker: GVKLESMGIY (SEQ ID NO:5) HA TM: QILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 6) PADRE: AKFVAAWTLKAAA (SEQ ID NO: 1) EXAMPLES

[0095] The genes encoding constructs 2 and 3 shown below were synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages). Construct 2 [ka] Total Amino Acids: MFVFLVLLPLVSSVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLKSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICIAKFVAAWTLKAAA* (SEQ ID NO: 7) Signal sequence: MFVFLVLLPLVSS (SEQ ID NO: 3) COVID-19-RBD (Omicron variant): VRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLKSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKST (SEQ ID NO: 8) HA Linker: GVKLESMGIY (SEQ ID NO:5) HA TM: QILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 6) PADRE: AKFVAAWTLKAAA (SEQ ID NO: 1) Construct 3 [ka] Total Amino Acids: MFVFLVLLPLVSSVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVI AWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLKSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLV LLVSLGAISFWMCSNGSLQCRICIAKFVAAWTLKAAAgaaVYFLQSINFNAASPRWYFYYLNARLNEVAKNLKFIAGLIAIVNAAANFKDQVILLGAAAVLQSGFRKGAAAYYQLYSTQLNLITGRLQSLKLQLPQGTTLNAAKTFPPTEPKKAAGDAALALLLLNAAAMEVTPSGTWLGAGVAMPNLYKKAATLACFVLAAVNAAGLMWLSYFIGLWLLWPVTLNKQFDTYNLW* (SEQ ID NO: 9) Linker: gaa CD8 epitopes: VYFLQSINFNAASPRWYFYYLNARLNEVAKNLKFIAGLIAIVNAAANFKDQVILLGAAAVLQSGFRKGAAAYYQLYSTQLNLITGRLQSLKLQLPQGTTLNAAKTFPPTEPKKAAGDAALALLLLNAAAMEVTPSGTWLGAGVAMPNLYKKAATLACFVLAAVNAAGLMWLSYFIGLWLLWPVTLNKQFDTYNLW (SEQ ID NO: 10) EXAMPLES

[0096] Preparation of replicon vectors The schematic construct of the alphavirus replicon is shown in FIG.

[0097] The gene of the target DNA prepared in Example 1 was cloned into a VEEV replicon vector under the control of a subgenomic (SG) promoter. VEEV replicon plasmids encoding each fragment were generated by insertion at the AscI and SbfI restriction sites to obtain the full-length VEEV TC-83 replicon construct.

[0098] The nucleotide sequences of the SG promoter, 5'UTR, 3'UTR and polyA tail are as follows: RNA sequences were obtained by using those DNA sequences as templates. SG promoter: cctgaatggactacgacatagtctagtccgccaag (SEQ ID NO: 11) 5'UTR: ataggcggcgcatgagagaagcccagaccaattacctacccaaa (SEQ ID NO: 12) 3'UTR: gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc (SEQ ID NO: 13) Poly A tail: aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 14)

[0099] The amino acid sequences of the VEEV TC-83 replicons nsP1-4 are as follows: APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADV AIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERV QRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPPVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSWSIPHAS DFDVDSLSILDTLDGASVTSGAVSAETNSYFARSSMEFRARPVPAPRTVFRNPPHPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO:15) The amino acid sequence corresponding to nsp3 is underlined. EXAMPLES

[0100] Preparation of self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs) A vector containing the DNA sequence encoding constructs 1-3 prepared in Examples 1 and 2 was used. The DNA was linearized and used as a template. T7 in vitro transcription was performed based on the protocol provided by the T7 transcription kit [RiboMax™ Express Large Scale RNA production System, Promega, (WI USA)]. The linear DNA template was mixed with T7 enzyme and rNTPs to synthesize RNA. For the synthesis of RNA containing modified nucleotides, modified NTPs such as 5-methyl-cytidine and N1-methyl-pseudouridine triphosphate were added to the in vitro transcription reaction mixture. The purified RNA product was capped using vaccinia capping enzyme, resulting in self-amplified RNA.

[0101] In the same manner as above, saRNA containing a target gene encoding a control construct "RBD(Wuhan)-TM" consisting of the COVID19 signal sequence, COVID-19-RBD of the Wuhan strain, HA linker and HA TM, and saRNA containing a target gene encoding a construct "RBD(Brazil)-TM" consisting of the COVID19 signal sequence, COVID-19-RBD of the Brazilian (gamma) variant, HA linker and HA TM were also obtained.

[0102] The amino acid sequence of "RBD(Wuhan)-TM" is as follows: MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 16)

[0103] In the above, the amino acid sequence of COVID-19-RBD of the Wuhan strain is as follows: VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST (SEQ ID NO: 17)

[0104] The resulting saRNA was encapsulated in lipid nanoparticles to obtain saRNA particles, which were used in the following examples. EXAMPLES

[0105] immunity Methods: Golden Syrian hamsters (n=12 per group) were immunized twice intramuscularly at weeks 0 and 4 with 10ug of saRNA LNPs expressing SARS-CoV-2 spike RBD (Wuhan strain) with HA transmembrane (RBD(Wuhan)-TM), RBD Brazil (gamma) with HA transmembrane [RBD(Brazil)-TM] and construct 1 of Example 1, i.e. RBD with HA transmembrane + PADRE [Brazil (gamma)][RBD(Brazil)-TM-PADRE], or PBS (placebo). Antibody titers of serum from immunized hamsters 4 weeks after the second immunization (day 55) were assessed by ELISA against SARS-CoV-2 RBD proteins of the Wuhan strain, Brazil variant, Indo-delta variant, Indo-delta + variant and Colombian Mu variant. Results are shown in Figure 2.

[0106] Results: Antibodies in sera immunized with RBD[Brazil(gamma)]-TM-PADRE induced more than three-fold higher antibodies (IC50 titers) against each RBD variant compared to antibodies in sera immunized with RBD[Brazil(gamma)]-TM. EXAMPLES

[0107] ACE2 inhibition assay Methods: Using a commercially available kit (Genscript L00847), the inhibitory effect of serum from hamsters immunized with the vaccine prepared in Example 4 on the binding of RBD mutants to ACE2 was tested. The serum was incubated with RBD mutants conjugated to horseradish peroxidase at 37°C for 30 minutes. The mixture was added to an ACE2-coated 96-well plate and incubated at 37°C for 15 minutes. After washing the plate, TMB substrate was added and incubated at room temperature for 15 minutes. The absorbance at 450 nm was measured with a microplate reader. The inhibition was calculated from the absorbance according to the following formula: Inhibition (%) = (1-absorbance of sample / average absorbance of negative control) x 100 The results are shown in Figure 3.

[0108] RESULTS:All vaccines had inhibitory effects against the SARS-CoV-2 Omicron BA.2 variant and the Wuhan strain, with the Brazilian Gamma-PADRE vaccine showing the highest inhibitory effect. EXAMPLES

[0109] Effects of modified nucleosides on innate immunity Methods: THP-1 Dual cells (InvivoGen) were used, which feature secreted luciferase (Luca gene) under the control of the minimal promoter of interferon stimulated gene (ISG) 54 combined with IFN-Stimulated Response Elements (ISRE). The IFN regulatory factor (IRF) induction effect of construct 1 (sample 1) of Example 1 and construct 1 (sample 2) of Example 1, provided that 5-methyl-cytidine was used instead of cytidine, were tested. The subcultured THP-1 Dual cells were harvested and the cell suspension was diluted with culture medium (RPMI-1640 + 10% FBS) to 5.6 × 10 5A concentration of 180 cells / mL was prepared. 180 μL of this cell suspension was seeded into each well of a 96-well culture plate. Then, 20 μL of phosphate-buffered saline (control) or a mixture of Lipofectamine (Lipofectamine™ RNAiMAX Transfection Reagent) and Sample 1 or Sample 2 was added per well. The cells were incubated at 37° C., 5% CO 2 The mixture was incubated under reduced pressure for 24 hours.

[0110] 20 μL of culture supernatant was aliquoted into another 96-well white plate, 50 μL of foaming reagent (QUANTI-Luc, InvivoGen) solution was added to each well, and luminescence intensity was measured immediately after addition.

[0111] The fold increase in each sample was calculated from the luminescence intensity according to the formula below and used as an index of IRF induction. Increase fold = (luminescence intensity of sample) / (luminescence intensity of control) The results are shown in Figure 4.

[0112] Compared to sample 1 (using cytidine), sample 2 (using 5-methyl-cytidine, a modified nucleoside) induced lower IRFs, demonstrating a clear reduction in the innate immune stimulatory effect. EXAMPLES

[0113] Animal testing Golden Syrian hamsters (N=6 per group) were injected intramuscularly twice with 10ug of saRNA prepared in Example 4. The saRNAs used in this example were RBD(Wuhan)-TM, RBD(Brazil)-TM, and RBD(Brazil)-TM-PADRE at weeks 0 and 4. Hamsters were exposed to live SARSCoV-2 Wuhan strain (WT) or Brazil strain (gamma) mutant on day 56.

[0114] Weight measurement method: Body weights were recorded daily after challenge and the mean percentage change in body weight was recorded from study day 56 to the end of study day 64.

[0115] result: The results are shown in Figure 5. The group immunized with RBD(gamma)-TM-PADRE prevented weight loss in hamsters to a similar level as the RBD(gamma)-TM or RBD(WT)-TM immunized groups in WT strain-exposed hamsters (left panel). On the other hand, no weight loss was observed in RBD(gamma)-TM-PADRE-exposed animals, whereas the other two groups exhibited mild weight loss (right panel).

[0116] Quantitative RT-PCR assay for SARS-CoV-2 method: Oral swabs were taken from anesthetized animals at selected time points post-exposure and tested for SARS-CoV2 RNA copies per mL. The lower limit of detection for this assay is 50 RNA copies / mL.

[0117] result: The results are shown in Figure 6. A greater reduction in oral swab viral RNA load was observed in the RBD(gamma)-TM-PADRE immunized group compared to the RBD(gamma)-TM or RBD(WT)-TM immunized groups after challenge with either the WT strain (left panel) or the gamma form (right panel).

[0118] histology method: At necropsy, the left lung was harvested and placed in 10% neutral buffered formalin for histopathological analysis. Tissue sections were trimmed and processed for hematoxylin and eosin (H&E) stained slides.

[0119] result: The results are shown in Figure 7. In the group immunized with RBD(gamma)-TM-PADRE, the occurrence of pathological findings was prevented in lung tissue after each challenge with either the WT strain (third row from the left, top) or the gamma type (third row from the left, bottom).

Claims

1. Alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and Polypeptides including antigenic proteins 1. An isolated polynucleotide encoding a polypeptide comprising:

2. The polynucleotide of claim 1 , wherein the antigenic protein is a protein derived from a virus, a cancer, or a cytokine.

3. The polynucleotide of claim 1, wherein the antigen protein comprises at least one selected from a signal sequence, a receptor binding domain (RBD), a transmembrane domain, a CD4+ T cell epitope, and a CD8+ T cell epitope.

4. 3. The polynucleotide of claim 2, wherein the antigenic protein is a protein derived from a virus or bacterium selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola virus, HIV, hepatitis B virus (HBV), influenza virus, hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), chikungunya virus, respiratory syncytial virus (RSV), dengue virus, orthomyxoviridae virus, and Mycobacterium tuberculosis.

5. The polynucleotide of claim 4 , wherein the antigenic protein is a protein derived from a coronavirus.

6. The polynucleotide of claim 3 , wherein the antigenic protein comprises the receptor binding domain (RBD) of the coronavirus S1 subunit.

7. The polynucleotide of claim 5, wherein the coronavirus is SARS-CoV2.

8. The polynucleotide of claim 7, wherein the antigenic protein comprises a receptor binding domain (RBD) of the S1 subunit of SARS-CoV2.

9. The polynucleotide of claim 3 , wherein the transmembrane domain is derived from influenza hemagglutinin (HA), CD80, or a modified transmembrane domain derived from the antigenic protein.

10. 9. The method of claim 8, wherein the transmembrane domain is derived from influenza hemagglutinin (HA). The polynucleotide described in

11. The polynucleotide of claim 3 , wherein the antigenic protein comprises a receptor binding domain, a transmembrane domain and a CD4+ T cell epitope.

12. The polynucleotide of claim 3 , wherein the antigenic protein comprises a CD8+ T cell epitope.

13. The polynucleotide of claim 1 , wherein the polynucleotide is RNA.

14. The polynucleotide of claim 3, wherein the CD4+ T cell epitope is a Pan-DR epitope (PADRE).

15. A vector comprising the polynucleotide according to any one of claims 1 to 14.

16. promoter, 5'UTR, A polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4; SG promoter, A gene of interest encoding a polypeptide comprising an antigen protein; 3'UTR and including a polyA tail, The vector described in claim 15.

17. A vaccine composition comprising the polynucleotide according to any one of claims 1 to 14 or a vector containing said polynucleotide, and a pharma- ceutically acceptable delivery vehicle.

18. 18. The vaccine composition of claim 17, wherein the polynucleotide or vector is combined with one or more other vaccine compositions.

19. 18. A vaccine composition according to claim 17 for treating, preventing and / or immunising against an antigen.

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