Coronavirus vaccine

Abstract

To provide a vaccine against coronavirus infection, especially, COVID-19 infection.SOLUTION: Provided herein is an isolated polynucleotide, which encodes alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and a polypeptide comprising a coronavirus protein fused to a signal sequence and / or transmembrane domain. The coronavirus protein may be the receptor binding domain of the S1 subunit of coronavirus spike (S) protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates generally to the field of vaccines and to methods and compositions for treating and / or immunizing against viral infections. 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 animal reservoirs 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. Sometimes, these viruses can jump to humans and cause disease, called spillover events. Of the seven known coronaviruses that make humans sick, four cause only mild to moderate illness. Three can cause more severe, 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 an animal reservoir 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 global pandemic by the World Health Organization on March 11, 2020. (https: / / www.niaid.nih.gov / diseases-conditions / coronaviruses)

[0004] Most humans infected with the COVID-19 virus experience mild to moderate respiratory illness and recover without needing special treatment. Older people and those with underlying conditions such as cardiovascular disease, diabetes, chronic respiratory disease and cancer are more likely to develop severe illness. There is currently no specific vaccine or treatment for COVID-19. (https: / / www.who.int / health-topics / coronavirus#tab=tab_1)

[0005] Because vaccines are administered to large numbers of healthy people, they usually have a higher standard of safety than drugs administered to people who are already sick. With regard to SARS-CoV-2 (COVID-19) vaccines, researchers' main safety concern is to avoid a phenomenon called disease enhancement, in which infected humans who have been vaccinated develop a more severe form of the disease than those who have not been vaccinated. In a study of an experimental SARS vaccine reported in 2004, vaccinated ferrets developed inflammation that damaged their livers after infection with the virus.

[0006] Antibody-dependent enhancement (ADE) is a mechanism by which some viruses, such as dengue virus, feline coronavirus, and HIV, can infect host cells that display Fc receptors as an alternative strategy, thereby exploiting the functions of the antiviral humoral immune response. Although there is debate as to whether ADE occurs during SARS-coronavirus infection, it has been reported that antibodies against the spike protein of SARS-coronavirus may induce the ADE effect.

[0007] Peter Hotez, a vaccine scientist at Baylor College of Medicine, Houston, Texas, believes that potential vaccines should first be tested in animals to rule out disease enhancement before moving to humans. He says he understands the reasons for pushing a SARS-CoV-2 (COVID-19) vaccine quickly into human trials, but adds that the possibility of the vaccine enhancing disease means "you're not sure this is the vaccine you want." (Non-Patent Document 1) [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2019 / 124441 [Non-patent literature]

[0009] [Non-Patent Document 1] Nature 579, 481 (2020) Summary of the Invention [Problem to be solved by the invention]

[0010] Thus, there is a need in the art for safer vaccines for coronaviruses that may avoid the ADE effect. [Means for solving the problem]

[0011] The present disclosure relates to novel antigenically active proteins / polypeptides that can induce protection against coronaviruses while minimizing the likelihood of ADE. The proteins / polypeptides disclosed herein include coronavirus structural proteins fused to a signal sequence and / or a transmembrane domain. The coronavirus structural proteins may be spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, small envelope protein (E) or combinations thereof. Specific examples include the S1 and / or S2 subunits of the spike protein, in particular the receptor binding domain of the S1 subunit.

[0012] In another aspect, the present disclosure relates to novel polynucleotides encoding the novel antigenically active proteins / polypeptides discussed above that are capable of inducing protection against coronaviruses.

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

[0014] In yet another aspect, the disclosure relates to a vaccine comprising the polypeptide or polynucleotide discussed above. In particular, the disclosure provides a vaccine comprising a polynucleotide encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 and a polypeptide comprising a coronavirus structural protein fused to a signal sequence and / or a transmembrane domain. The vaccine can be used to prevent and / or treat a subject from coronavirus infection.

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

[0016] In yet another aspect, the present disclosure relates to the use of the polypeptides or polynucleotides discussed above for the manufacture of a medicament. [Brief description of the drawings]

[0017]

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[0018] As used herein, "coronavirus" refers to a single-stranded, positive-sense RNA virus belonging to the family Coronaviridae. Exemplary coronavirus 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 variants. Known variants can include COVID-19 E484K_N501Y_K417T variant (Brazilian variant), E484K_N501Y_K417N variant (South African variant), and E484K variant. The coronavirus genome encodes a number of nonstructural proteins and four major structural proteins, including spike (S), nucleocapsid (N), membrane (M), and minor envelope (E). 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 a structural protein as used herein is the coronavirus RBD. The COVID-19 spike protein, the S1 and S2 subunits of the spike protein and the RBD and mutant forms thereof have been identified and published (Wrapp et al., Science 10.1126 / science.abb2507(2020), Z. Wang et al., (preprint) bioRxiv 2021.01.15.426911; doi: https: / / doi.org / 10.1101 / 2021.01.15.426911).

[0019] 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. 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.

[0020] As used herein, a "transmembrane domain (TM)" is a protein that is either derived from a natural source or from a synthetic source. When the source is natural, the domain in some embodiments is derived from any membrane-associated or transmembrane protein. In one embodiment, the membrane-associated or transmembrane protein is a heterologous protein to COVID-19. 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.

[0021] 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.

[0022] 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.

[0023] The term "transmembrane domain" as used in this disclosure includes at least the transmembrane region of a membrane-associated or transmembrane protein. In addition, the transmembrane domain can also include the juxtamembrane domain (JMD) and / or the cytoplasmic tail of a membrane-associated or transmembrane protein.

[0024] 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.

[0025] 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 consisting of 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 TM of COVID-19 spike (S).

[0026] 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.

[0027] 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.

[0028] The coronavirus structural proteins, transmembrane domains and / or signal sequences may be directly or indirectly fused, in one embodiment with one or two linkers intervening between them.

[0029] Additionally, coronavirus structural proteins, transmembrane domains and / or signal sequences can be truncated and replaced with short linkers, hi some embodiments, coronavirus structural proteins, transmembrane domains and / or signal sequences include one or more peptide linkers.

[0030] 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).

[0031] When the coronavirus structural protein is chemically conjugated to the transmembrane domain and / or signal sequence 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.

[0032] Substances derived from IgG can also be used as linkers. Examples of IgG-derived substances include IgG1-IgG4, including (i) full (hinge-CH2CH3), (ii) half (hinge-CH3) and (iii) short (only 12aa hinge). A preferred example is IgG4-CH3.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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 order of elements: 5'UTR, sequences encoding alphavirus nonstructural proteins (nsp1, nsp2, nsp3, nsp4), 3'UTR, and polyA signal. Alphavirus replicons also contain one or more viral subgenomic promoters that direct the expression of a gene of interest. These sequences can have one or more mutations as taught in the prior art.

[0037] Alphavirus replicons provided by this disclosure can have the construct shown in FIG.

[0038] 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.

[0039] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full 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.

[0040] "Reference" means a standard or control condition.

[0041] 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.

[0042] 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”.

[0043] "Effective amount" refers to the amount of agent required to ameliorate the symptoms of a disease in an untreated patient. The effective amount of an active compound used to practice the present invention 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.

[0044] 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 9 It can be obtained in amounts of IU or 1-50 μg. The replicon can be preferably formulated into a vaccine composition suitable for administration in a conventional manner.

[0045] "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.

[0046] 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.

[0047] 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.

[0048] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0049] 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.

[0050] Those of skill in the art will understand that the polynucleotide sequences described herein and in the claims contain a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0051] Any vaccine composition or method provided herein can be combined with any one or more of the other vaccine compositions and methods provided herein.

[0052] 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. A vector can also be a naked RNA polynucleotide, naked DNA polynucleotide, a polynucleotide 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., which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the invention are plasmids or bacmids.

[0053] 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.

[0054] 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.

[0055] There are various expression systems for generating the construct of the present invention.The 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, e.g., SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus-derived vectors, and combinations thereof.

[0056] Constructs and / or vectors used herein include 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 a transmembrane domain, as discussed above. Specific examples of constructs or vectors are shown in FIG.

[0057] 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 and a termination codon at the beginning, appropriately arranged at the end of the polypeptide to be translated.

[0058] 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 invention 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.

[0059] Recombinant constructs capable of expressing viral proteins, including those described herein, can be prepared and used to transfect 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 at least one of the coronavirus genes of interest under conditions that permit the formation of alphavirus replicon particles.

[0060] 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.

[0061] 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.

[0062] Depending on the vector and host cell selected, constructs are generated by growing host cells transfected with the vector under conditions in which the recombinant proteins are expressed and an alphavirus replicon is created, and a construct is formed that contains an alphavirus replicon packaged with particles of alphavirus structural proteins. In one embodiment, the invention includes a method of generating a construct, which involves co-transfecting vectors containing polynucleotides encoding 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.

[0063] Methods for growing cells that produce alphavirus replicon particles of the invention 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 from CHIKV or VEEV, are grown in a bioreactor or fermentation chamber that expands the cells and expresses 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 the cited references are incorporated herein by reference). In another embodiment, the pre-sterilized plastic bag is a bag of about 10 L to 1000 L.

[0064] 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)].

[0065] 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.

[0066] An encapsulating material refers to a 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).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] In some embodiments, polynucleotides such as the alphavirus replicons described herein can be formulated in lipid nanoparticles (LNPs).

[0074] 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%.

[0075] 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.

[0076] 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.

[0077] This replicon-based vaccine technology is a unique platform technology for vaccination because RNA can self-amplify to produce vaccine antigens and can be delivered to cellular organs.In addition, 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, such as electroporation, and is expected to have higher immunogenicity with the minimum dose based on the self-replicating system, which is superior to mRNA technology.

[0078] According to the present invention, 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 and / or a transmembrane domain (TMD) sequence intended to maximize immunogenicity and minimize ADE.

[0079] 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. [Example 1]

[0080] Each of the genes encoding constructs 1 to 8 shown below was synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages).

[0081] 1. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and HA (flexible domain-transmembrane (TM)-cytoplasmic tail (Cyt)).

[0082] [ka] MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 1)

[0083] hIL-2 signal sequence: MYRMQLLSCIALSLALVTNS (SEQ ID NO:2) COVID19-RBD: VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS (SEQ ID NO: 3) HA (flexible-TM-Cyt): GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMC SNGSLQCRICI (SEQ ID NO: 4) hIL-2: human interleukin-2 HA (Flexible-TM-Cyt): A polypeptide of influenza virus

[0084] 2. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and HA (flexible-TM-Cyt)

[0085] [ka] MYRMQLLSCIALSLALVTNS C VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS C GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 5) "C" in the schematic diagram: linker (cysteine) The linkers in the amino acid sequence are underlined.

[0086] 3. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and HA (flexible-TM-Cyt) using a linker

[0087] [ka] MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS GS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 6) "GS" in the schematic diagram: linker (GGATCC) The linkers are underlined in the amino acid sequence. Human IgG4 CH3: GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7)

[0088] 4. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and HA (flexible-TM-Cyt) using a linker

[0089] [ka] MYRMQLLSCIALSLALVTNS C VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS CGS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GS GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 8) "C", "CGS" and "GS" in the schematic diagram: linkers The linkers are underlined in the amino acid sequence.

[0090] 5. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and human TLR4 [TM-Toll / Interleukin-1 receptor domain (TIR)]

[0091] [ka] MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGS KTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 9) Human TLR4 (TM-TIR): KTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 10)

[0092] 6. Construct of COVID-19-RBD gene sequence fused to hIL-2 signal sequence and human TLR4 (TM-TIR)

[0093] [ka] MYRMQLLSCIALSLALVTNS C VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS CGSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 11) "C" and "CGS" in the schematic diagram: linkers The linkers are underlined in the amino acid sequence.

[0094] 7. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and human TLR4 (TM-TIR) using a linker

[0095] [ka] MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS GS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 12) "GS" in the schematic diagram: linker The linkers are underlined in the amino acid sequence.

[0096] 8. Construct of COVID-19-RBD sequence fused to hIL-2 signal sequence and human TLR4 (TM-TIR) using a linker

[0097] [ka] MYRMQLLSCIALSLALVTNS C VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS CGS GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK GSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 13) "C", "CGS" and "GS" in the schematic diagram: linkers The linkers are underlined in the amino acid sequence.

[0098] Information regarding the hIL-2 signal sequence, COVID19-RBD, human IgG4 CH3 and HA (Flexible-TM-Cyt) is also available from the National Center for Biotechnology Information website and from the UniProt website. hIL-2 signal sequence https: / / www.ncbi.nlm.nih.gov / protein / P60568 UniProtKB / Swiss-Prot:P60568.1 COVID19-RBD https: / / www.ncbi.nlm.nih.gov / nuccore / NC_045512 NCBI reference sequence: NC_045512.2 HA(Flexible-TM-Cyt) https: / / www.ncbi.nlm.nih.gov / protein / P03452 UniProtKB / Swiss-Prot:P03452.2 Human IgG4 https: / / www.uniprot.org / uniprot / P01861 UniProtKB:P01861 [Example 2]

[0099] Each of the genes encoding constructs A to O shown below was synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages).

[0100] [ka]

[0101] Construct A: SS1-RBD1(330-521) (SEQ ID NO: 37) Construct B: SS2-RBD1(330-521)-TM2 (SEQ ID NO: 38) Construct C: SS1-RBD1(330-521)-GS-Linker-GS-TM1 (SEQ ID NO: 39) Construct D: SS1-RBD1(330-521)-GS-Linker-GS-TM2 (SEQ ID NO: 40) Construct E: SS1-RBD2(330-530)-GS-Linker-GS-TM1 (SEQ ID NO: 41) Construct F: SS1-RBD2(330-530)-GS-Linker-GS-TM2 (SEQ ID NO: 42) Construct G: SS2-RBD1(330-521)-GS-Linker-GS-TM1 (SEQ ID NO: 43) Construct H: SS2-RBD1(330-521)-GS-Linker-GS-TM2 (SEQ ID NO: 44) Construct I: SS2-RBD2(330-530)-GS-Linker-GS-TM1 (SEQ ID NO: 45) Construct J: SS2-RBD2(330-530)-GS-Linker-GS-TM2 (SEQ ID NO: 46) Construct K: SS3-RBD3(327-531)-TM2 (SEQ ID NO: 47) Construct L: SS1-RBD2(330-521)-TM2 (SEQ ID NO: 48) Construct M: SS1-RBD3(327-531)-C-TM3 (SEQ ID NO: 49) Construct N: SS3-RBD3(327-531)-TM3 (SEQ ID NO: 50) Construct O: SS3-RBD1(330-521)-TM3 (SEQ ID NO:51)

[0102] Signal sequence SS1: COVID-19 signal sequence (1-15): MFVFLVLLPLVSSQC (SEQ ID NO: 14) SS2: hIL-2 signal sequence: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 15) SS3: COVID-19 signal sequence (1-13): MFVFLVLLPLVSS (SEQ ID NO: 16)

[0103] COVID-19-RBD RBD1: COVID-19 RBD amino acid positions 330 and 521 RBD2: COVID-19 RBD amino acid positions 330 and 530 RBD3: COVID-19 RBD amino acid positions 327 and 531

[0104] RBD1: PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP (SEQ ID NO: 17)

[0105] RBD2: PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS (SEQ ID NO: 18)

[0106] RBD3: VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST (SEQ ID NO: 19)

[0107] Linker Human IgG4-CH3 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20)

[0108] Transmembrane-cytoplasmic (TM-Cyt) TM1: human CD80 * (TM-Cyt): LLPSWAIT LISVNGIFVI CCLTYCFAPR CRERRRNERL RRESVRPV (SEQ ID NO: 21) * CD80:https: / / www.ncbi.nlm.nih.gov / protein / NP_005182 TM2:HA (Flexible-TM-Cyt): GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 22) TM3: COVID19 (JMD-TM): GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 23)

[0109] The juxtamembrane domain (JMD) of the spike protein is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses (Howard et al., JOURNAL OF VIROLOGY, Mar. 2008, p. 2883-2894 Vol. 82, No. 6, the contents of which are incorporated herein by reference). JMD: GKYEQYIKWPWYIWL (SEQ ID NO: 24) TM:GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDED-DSEPVLKGVKLHYT (SEQ ID NO: 25) [Example 3]

[0110] Preparation of replicon vectors. The schematic construct of an alphavirus replicon is shown in FIG.

[0111] Each gene of interest in DNA prepared in Examples 1 and 2 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 a full-length VEEV TC-83 replicon construct. An example of a full-length VEEV TC-83 plasmid vector is shown in FIG. 2.

[0112] 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: 26) 5'UTR: ataggcggcgcatgagagaagcccagaccaattacctacccaaa (SEQ ID NO: 27) 3'UTR: gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc (SEQ ID NO: 28) Poly A tail: aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 29)

[0113] The amino acid sequences of the VEEV TC-83 replicons nsP1-4 are as follows: APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSWSIPHASDFDVDSLSILDTLDGASVTSGAVSAETNSYFARSMEFRARPVPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO:30) The amino acid sequence corresponding to nsp3 is underlined.

[0114] In this example, the amino acid sequence of nsp3 corresponding to 1330 to 1886 in SEQ ID NO: 30 was replaced with the sequence shown below. The underlined sequences were different from SEQ ID NO: 30. APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDR LTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILG K SMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO: 31) [Example 4]

[0115] Preparation of alphavirus replicon particles 10 μg of the full-length replicon plasmid prepared in Example 3, 1 μg of VEEV Env expression plasmid, and 1 μg of VEEV capsid NLS mutant (or 1 μg of VEEV capsid expression plasmid) were transfected into HEK293T cells. The supernatant was collected 48 to 96 hours after transfection. The replicon particles were purified by using an ion exchange column. HET293T or Vero cells were infected with dilutions of the purified particle preparation to determine the infectious titer. The purified replicon particles are used to generate antigens for diagnosis and vaccination. [Example 5]

[0116] Expression of RBD in HEK293T cells 293T cells (6.25x10^5 cells / well) were transfected with Fectopro transfection reagent (Polyplus) and 2ug of each replicon plasmid. Western blotting was performed 72 hours after transfection on the supernatant (left) directly without particle purification or on the cell lysate fraction (right) using antiserum reactive with Covid-19-RBD as the primary antibody and goat anti-rabbit immunoglobulin linked to horseradish peroxidase as the secondary antibody. Western blotting was also performed to confirm the expression of antigens from construct K and construct O in the cell lysate fraction 72 hours after transfection using antiserum reactive with Covid-19-RBD as the primary antibody and goat anti-rabbit immunoglobulin linked to horseradish peroxidase as the secondary antibody.

[0117] The results are shown in Figure 3. The abbreviations stand for the following: ssRBD:Construct A hIL-2ssRBD-TM: Construct 1 (Mouse HA) ssRBD-Linker-TM: Construct C (TM1: Mouse CD80, Linker: Mouse IgG4-CH3)

[0118] Mouse: CD80 (TM-Cyt) TLVLFGAGFGAVITVVVIVVIIKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL (SEQ ID NO: 32)

[0119] Mouse IgG4-CH3 GRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 33)

[0120] Expression was confirmed for all tested particles in cells, and as expected, the ssRBD without the transmembrane sequence was secreted into the cell culture medium, whereas each RBD construct linked to a transmembrane sequence was retained intracellularly and not secreted into the medium. [Example 6]

[0121] immunogenicity The following replicon particle vaccines prepared in Example 4 were used. VRepssRBD: Construct A packaging VEEV particles VRepsshIL2RBDHA: Construct 1 packaging VEEV particles VRepssRBDmIgG4CD80: VEEV replicon particles packaged in construct C (TM1: mouse CD80, linker: mouse IgG4-CH3) Control: VEEV replicon without the gene of interest DNA vaccine: DNA expressing the COVID-19 spike protein

[0122] Mice were immunized intramuscularly three times (days 0, 7 and 14) with the indicated replicon particles (RBD or RBD-Linker-TM, 10^7IU / dose), control replicon particles or DNA vaccine (20ug / dose) and sera were collected on days 14 (7 days after the second immunization) and 35 (21 days after the third immunization). These sera were tested in ELISA assays performed against COVID-19-Spike S1 antigen or RBD antigen. Symbols indicate the mean of five mice in each group and error bars indicate sem. Curve fits were calculated by Prism software. The results are shown in Figure 4 (4-1 and 4-2) and Figure 5 (5-1 and 5-2).

[0123] Figures 4 and 5 show that significantly higher antibody titers were achieved against the recombinant coronavirus RBD and spike S1 after immunization with the replicon vaccine. In contrast, the DNA vaccine induced very low antibody titers. [Example 7]

[0124] immunogenicity The following replicon particle vaccines prepared in Example 4 were used. CD80:Construct C packaging of VEEV particles HA No.1: Construct K packaging VEEV particles Control: PBS

[0125] Mice were immunized intramuscularly with the indicated replicon vaccine (CD80 or HA No.1, 10^5 IU / dose) or PBS, and sera were collected 7 days after immunization. These sera were subjected to ELISA analysis performed against COVID-19-Spike S1 antigen. Symbols indicate the mean of five mice in each group, error bars indicate sem. These results are shown in Figure 6.

[0126] FIG. 6 shows that significantly higher antibody titers were achieved against the recombinant coronavirus spike S1 after immunization with the replicon vaccine. [Example 8]

[0127] Preparation of self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs) A vector containing the DNA sequence encoding construct C prepared in Example 3 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)]. RNA was synthesized by mixing the linear DNA template with T7 enzyme and rNTPs. The purified RNA product was capped using vaccinia capping enzyme, resulting in self-amplified RNA.

[0128] The resulting saRNA was encapsulated in lipid nanoparticles to obtain saRNA(RBD-CD80TM) particles. Lipid nanoparticles without RNA were used as a control. [Example 9]

[0129] immunity The saRNA formulated in LNP prepared in Example 8 was used. Immune dose: saRNA(RBD-CD80TM) 0.3ug saRNA(RBD-CD80TM) 1ug saRNA(RBD-CD80TM) 10ug No RNA (control) The indicated amounts of saRNA formulated in LNPs (Genvoy) or no RNA (control) were used. Mice (n=5 per group) aged 4-6 weeks were immunized by intramuscular injection on days 0 and 28 with saRNA formulated in LNPs.

[0130] Mice were bled on days 14, 28 and 46. Antibody titers against S1 protein in serum from immunized mice were measured by ELISA. Plates were coated with S1 protein (Sinobio 100ng / ml). Results are shown in Figure 7. [Example 10]

[0131] The COVID-19 RBD can be derived from a COVID-19 variant. As shown in Figure 1, the following amino acid sequence of a construct containing a COVID-19 signal sequence, a COVID-19 RBD derived from a COVID-19 variant, and human HA (flexible-TM-Cyt) is also used to create an alphavirus replicon. In this example, the gene of interest is replaced with a polynucleotide encoding the following amino acid sequence: Self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs) is created in the same manner as in Example 8.

[0132] Construct derived from COVID-19 E484K_N501Y_K417T variant (Brazilian variant) MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 34)

[0133] Construct derived from COVID-19 E484K_N501Y_K417N variant (South African variant) MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 35)

[0134] Constructs derived from the COVID-19 E484K variant MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 36)

Claims

1. An isolated polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 and a polypeptide comprising a coronavirus protein fused to a signal sequence and / or a transmembrane domain.

2. 2. The polynucleotide of claim 1, wherein the coronavirus protein is a spike (S) protein, a nucleocapsid (N) protein, a membrane (M) protein, a minor envelope (E) protein, or a combination thereof.

3. 2. The polynucleotide of claim 1, wherein the coronavirus protein is a spike (S) protein.

4. 2. The polynucleotide of claim 1, wherein the coronavirus protein is the S1 subunit and / or the S2 subunit of the spike (S) protein.

5. The polynucleotide of claim 4, wherein the coronavirus protein is the S1 subunit in the spike (S) protein.

6. The polynucleotide of claim 5 , wherein the coronavirus protein is the receptor binding domain of the S1 subunit.

7. The polynucleotide of any one of claims 1 to 6, wherein the transmembrane domain is derived from influenza hemagglutinin (HA), CD80 or TLR4.

8. The polynucleotide of any one of claims 1 to 6, wherein the transmembrane domain is a modified transmembrane domain derived from a coronavirus structural protein.

9. 9. The polynucleotide of claim 8, wherein the modified transmembrane domain comprises the juxtamembrane and transmembrane domains of COVID-19 spike (S).

10. The polynucleotide of any one of claims 1 to 9, wherein the coronavirus protein is fused to a signal sequence and a transmembrane domain.

11. The polynucleotide according to any one of claims 1 to 10, wherein the signal sequence is derived from human IL-2.

12. The polynucleotide of any one of claims 1 to 10, wherein the signal sequence is derived from a COVID-19 spike protein.

13. The polynucleotide of any one of claims 1 to 12, wherein the transmembrane domain and / or the signal sequence are fused to the coronavirus protein by a linker.

14. The polynucleotide of claim 13, wherein the linker is IgG4CH3 and / or a short linker.

15. The polynucleotide of any one of claims 1 to 14, wherein the coronavirus is COVID-19.

16. 16. The polynucleotide of claim 15, wherein the polypeptide encodes an amino acid sequence selected from SEQ ID NOs: 1, 5, 6, 8, 9, 11, 12, 13, 34, 35 and 36.

17. The polynucleotide of claim 15, wherein the polypeptide encodes an amino acid sequence selected from SEQ ID NOs: 37-51.

18. The polynucleotide of any one of claims 1 to 17, which is RNA.

19. The polynucleotide according to any one of claims 1 to 17, which is DNA.

20. A vector comprising the polynucleotide according to any one of claims 1 to 19.

21. 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 a coronavirus protein fused to a signal sequence and / or a transmembrane domain, 3'UTR and including a polyA tail, The vector of claim 20.

22. A vaccine composition comprising the polynucleotide or the vector according to any one of claims 1 to 21 and a pharma- ceutically acceptable carrier.

23. 23. The vaccine composition of claim 22, wherein the pharma- ceutically acceptable carrier is a delivery vehicle.

24. 24. The vaccine composition of claim 23, wherein the delivery vehicle is a particle comprised of one or more alphavirus structural proteins or a lipid delivery system.

25. Use of the polynucleotide or the vector according to any one of claims 1 to 21 for the manufacture of a medicament.

26. 26. The use according to claim 25, wherein the medicament is for inducing immunomodulation in a subject.

27. 26. The use according to claim 25, wherein the medicament is for treating or preventing a subject from a condition caused by a coronavirus infection.

28. A method of immunomodulating in a subject in need thereof, comprising administering to said subject an immunologically effective amount of the vaccine composition according to any one of claims 22 to 24.

29. Administering to a subject in need thereof an effective amount of the vaccine composition according to any one of claims 22 to 24. A method of treating, preventing and / or immunizing against a coronavirus virus infection in said subject.

30. A polypeptide comprising a coronavirus structural protein fused to a signal sequence and / or a heterologous transmembrane domain.

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