Development of engineered vaccine designs

Abstract

Provided herein is an isolated polynucleotide that encodes a polypeptide that includes an antigen protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. Also provided herein is an isolated polynucleotide that encodes an alphavirus nonstructural protein nsp1, nsp2, nsp3, and nsp4, and a polypeptide that includes an antigen protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. The antigen may be influenza. The polynucleotide, such as RNA, is useful as a vaccine against influenza infection.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of novel vaccine design, and methods and compositions for treating and / or immunizing against antigens. In particular, the present disclosure relates to influenza vaccines. [Background technology]

[0002] Influenza (flu) is a contagious respiratory illness caused by influenza viruses that infect the nose, throat, and lungs. Some people, such as the elderly, young children, and people with certain health conditions, are at higher risk for severe influenza complications. There are two main types of influenza (flu) viruses, types A and B. Influenza A and B viruses, which circulate routinely in humans (human influenza viruses), are responsible for the annual seasonal influenza epidemics.

[0003] The first and most important step in preventing influenza is to get the influenza vaccine each year. The influenza vaccine has been shown to reduce the risk of influenza-related illness and serious influenza complications that can lead to hospitalization or even death.

[0004] Various types of influenza vaccines are available, including the following: Quadrivalent influenza vaccines, which protect against four different types of influenza viruses; high-dose influenza vaccines, where the high-dose vaccine contains four times the amount of antigen (the part of the vaccine that helps the body develop a defense against influenza viruses) of the regular influenza vaccination and are specifically licensed for people 65 years of age and older; cell-based influenza vaccines, where the cell-based vaccine is grown in cultured cells of mammalian origin rather than in chicken eggs; nasal spray influenza vaccines, where live attenuated influenza vaccine [LAIV] is given as a nasal spray; influenza vaccination via a jet injector, which is approved for use in people 18 to 64 years of age; adjuvanted influenza vaccines, which are manufactured with ingredients added to the vaccine that help the adjuvanted influenza vaccine generate a stronger immune response and are specifically licensed for people 65 years of age and older; recombinant influenza vaccines, where the recombinant influenza vaccine is produced using a method that does not require the vaccine virus to be cultured from eggs (https: / / www.cdc.gov / flu / about / index.html). Data on vaccine effectiveness (VE) was taken from the March CDC Morbidity and Mortality Weekly Report. VE was calculated using data from 3,363 children and adults with acute respiratory infections (ARI) enrolled in the US Influenza Vaccine Effectiveness Network across seven different US research sites from October 2021 to February 2022. A VE of only 16% was observed from the 2021-22 seasonal influenza vaccine in protecting people in the US from infection with the most common influenza virus currently circulating, A(H3N2). More specifically, the VE for mild-to-moderate ARI associated with influenza A(H3N2) virus in outpatients receiving medical care was 16%, which was not significantly more effective. Furthermore, the VE for outpatients with ARI associated with the influenza A virus type they received was even less effective at 14%.Of particular concern is the low efficacy of the vaccine against the most common circulating influenza strains (https: / / www.clinicaltrialsarena.com / comment / us-flu-vaccine-efficacy / ). Summary of the Invention [Problem to be solved by the invention]

[0005] The hope is for a more potent influenza vaccine with high long-term efficacy. [Means for solving the problem]

[0006] The present disclosure relates to novel antigenically active proteins / polypeptides capable of inducing protection against antigens. The proteins / polypeptides disclosed herein include antigen proteins fused to a signal sequence and a transmembrane domain, and optionally to ferritin.

[0007] 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 antigens.

[0008] In another aspect, the present disclosure relates to novel alphavirus replicons (self-amplifying RNA "saRNA") capable of expressing the antigenically active proteins / polypeptides described above. The alphavirus replicons comprise a polynucleotide, such as an RNA, encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding the antigenically active protein / polypeptide described above as a gene of interest.

[0009] 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 a polypeptide comprising an antigenic protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. The vaccine preferably comprises a saRNA comprising a polynucleotide encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and a polypeptide comprising an antigenic protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. In a preferred embodiment, the antigen is an influenza antigen. The vaccine can be used to prevent and / or treat a subject from influenza infection.

[0010] In yet another aspect, the present disclosure relates to a method for inducing and / or enhancing an immune response to an antigen. In a preferred embodiment, the method for immunizing a subject against influenza, preventing or treating influenza comprises administering to a subject in need thereof an effective amount of the above-mentioned polypeptide or polynucleotide such as saRNA.

[0011] In yet another aspect, the present disclosure relates to the use of the aforementioned polypeptide or polynucleotide for the manufacture of a medicament.

[0012] In a further aspect, the disclosure relates to novel polynucleotides encoding a polypeptide comprising an antigenic protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. The polynucleotide may be a saRNA comprising a polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and a polypeptide comprising an antigenic protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. [Brief description of the drawings]

[0013] [Figure 1]Representative constructs of the saRNA disclosed in this application. [Diagram 2] Schematic diagram of constructs F1, F2, F4 and F5. [Diagram 3] Western blotting results of lysates of cells transfected with saRNA. [Figure 4] FACS analysis of HA antigen expressed on saRNA-transfected cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As used herein, "influenza" refers to the family Orthomyxoviridae (a group of RNA viruses). Influenza viruses are classified into types A, B, C, and D. These major types generally produce similar symptoms but are antigenically completely unrelated, so that infection with one type does not confer any immunity to the others. A viruses cause influenza pandemics, while B viruses cause smaller localized outbreaks. C viruses cause only mild respiratory disease in humans. Influenza D virus is not known to infect humans and has only been observed in pigs and cattle.

[0015] Influenza A viruses are classified into subtypes, and both influenza B and influenza A subtypes are further divided into strains. Influenza A subtypes are differentiated primarily based on two surface antigens (external proteins) - hemagglutinin (H) and neuraminidase (N). Examples of influenza A subtypes include H1N1, H5N1, and H3N2. Influenza B viruses are subdivided into two main lineages: B / Yamagata and B / Victoria. Influenza B strains and influenza A subtype strains are further differentiated by genetic sequence variations.

[0016] As used herein, "influenza 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.

[0017] 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 influenza. 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 an influenza virus.

[0018] 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.

[0019] 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.

[0020] As used herein, "ferritin" refers to any one or a combination of at least two of mammalian ferritin, amphibian ferritin, bacterial ferritin, or plant ferritin. Mammalian ferritin or bacterial ferritin is preferred. Preferred mammalian ferritin includes any one or a combination of at least two of human ferritin, mouse ferritin, or horse spleen ferritin. Preferred amphibian ferritin includes bullfrog. Preferred bacterial ferritin includes Helicobacter pylori ferritin or Escherichia coli ferritin. Preferred sources of ferritin include any one or a combination of at least two of natural extract products, artificially synthesized products, or genetic engineering products. In one embodiment, a H. pylori-billfrog hybrid ferritin can be used, such as a H. pylori-billfrog hybrid ferritin (Kanekiyo et al., Cell. 2015 Aug 27; 162(5): 1090-1100) constructed by fusing residues 2-9 of the lower subunit of Rana catesbeiana ferritin (UniProt:P07797, with an N8Q mutation that eliminates a potential N-glycosylation site) to H. pylori non-heme ferritin with an I7E mutation (UniProt:Q9ZLI1, residues 3-167) to ensure a conserved salt bridge found in human and bullfrog ferritins (6R and 14E in both human light chain ferritin and bullfrog lower subunit ferritin) with 6R of bullfrog ferritin.

[0021] As used herein, "nucleoside" refers to a molecule comprised of guanine (G), adenine (A), thymine (T), uridine (U), cytidine (C), or modified nucleosides thereof.

[0022] Modified nucleosides include, but are not limited to, pseudouridine, N1-methyl-pseudouridine, 5-methyl-uridine, pseudocytidine, N1-methyl-pseudocytidine and 5-methyl-cytidine.

[0023] 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.

[0024] In one embodiment, the modified nucleoside is independently selected from N1-methyl-pseudouridine or 5-methyl-cytidine.In one embodiment, the mRNA or saRNA comprises substantially 100% modified cytidine (e.g., 5-methyl-cytidine) and 100% modified uridine (e.g., N1-methyl-pseudouridine), 100% modified cytidine and 80% modified uridine, and 50% modified cytidine and 50% modified uridine.In one embodiment, the saRNA comprises less than 100% modified uridine.

[0025] 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.

[0026] 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.

[0027] Preferred transmembrane domains may be derived from influenza virus hemagglutinin (HA), CD80, Toll-like receptor 4 (TLR4). Specific examples include a protein consisting of the transmembrane domain and cytoplasmic tail of influenza virus hemagglutinin "HA(TM / CT)", 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).

[0028] 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.

[0029] The signal sequence is not limited and can be selected from a variety of sequences. In some embodiments, the signal sequence may be a target protein expressed by an alphavirus replicon.

[0030] In some embodiments, the signal sequence of influenza HA, COVID-19 or IL-2, especially influenza HA, may be used.

[0031] The influenza structural proteins, transmembrane domains and / or signal sequences may be directly or indirectly fused, and in one embodiment may be separated by one or two linkers.

[0032] Also, influenza structural proteins, transmembrane domains and / or signal sequences can be truncated and replaced with short linkers, hi some embodiments, the viral structural proteins, transmembrane domains and / or signal sequences include one or more peptide linkers.

[0033] The antigen protein, signal sequence, transmembrane domain and optionally ferritin may further be fused to a T cell epitope. The "T cell epitope" may be a CD4+ T cell target epitope, a CD8+ T cell target epitope, or a Pan-DR epitope (PADRE). As used herein, the T cell epitope may be derived from the virus to be treated.

[0034] The term "PADRE" as used in this disclosure refers to a Pan HLA DR binding epitope, a peptide that universally activates antigen-specific CD4+ T cells. The amino acid sequence of PADRE is AKFVAAWTLKAAA.

[0035] The viral structural proteins, signal sequence and transmembrane domain and optionally ferritin and at least one universal epitope such as PADRE may be directly or indirectly fused, in one embodiment with one or two linkers intervening therebetween.

[0036] Also, the viral structural proteins, signal sequences and transmembrane domains and optionally ferritin and at least one or more T cell target epitopes can be truncated and replaced with short linkers. In some embodiments, the viral structural proteins, transmembrane domains and / or signal sequences include one or more peptide linkers.

[0037] 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).

[0038] When the influenza structural protein is chemically conjugated to the transmembrane domain and / or signal sequence through a chemical cross-linker, examples of cross-linkers 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 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 literature.

[0044] Alphavirus replicons provided by this disclosure can have the construct shown in FIG.

[0045] 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.

[0046] 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.

[0047] "Reference" means a standard or control condition.

[0048] 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.

[0049] 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”.

[0050] "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.

[0051] 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 7 ~10 9It 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.

[0052] "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.

[0053] 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.

[0054] 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.

[0055] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0056] 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.

[0057] In this specification and claims, the term "about" covers values ​​of ±20%, ±10% or ±5% of the associated numerical value.

[0058] 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."

[0059] Any vaccine composition or method provided herein can be combined with any one or more of the other vaccine compositions and methods provided herein.

[0060] The term "vector" refers to a means by which a nucleic acid sequence can be propagated and / or transferred 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., which do not replicate autonomously. In many, but not all, common embodiments, the vectors of the invention are plasmids or bacmids.

[0061] Typically, a nucleic acid molecule to be expressed is "operably linked" to a promoter and / or enhancer and is subject to the transcriptional regulatory control of the promoter and / or enhancer.

[0062] 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.

[0063] 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.

[0064] 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 an antigen, such as a viral structural protein, fused to a signal sequence and a transmembrane domain, as described above. An exemplary construct or vector is shown in FIG.

[0065] 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.

[0066] 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.

[0067] 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 viral gene of interest under conditions that permit the formation of alphavirus replicon particles.

[0068] 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.

[0069] 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.

[0070] 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 into a suitable host cell vectors containing polynucleotides encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, and at least one gene of interest encoding a polypeptide comprising a viral structural protein fused to a signal sequence and / or transmembrane domain, and at least one vector each encoding at least one alphavirus structural protein, 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.

[0071] Methods for growing cells that produce the 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 polynucleotide 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 the cited references are incorporated herein by reference). In another embodiment, the pre-sterilized plastic bag is about a 10 L to 1000 L bag.

[0072] 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. A kit for in vitro transcription such as the T7 transcription kit (RiboMax™ Express Large Scale RNA production System, Promega, WI USA) may be used.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In some embodiments, polynucleotides such as the alphavirus replicons described herein can be formulated in lipid nanoparticles (LNPs).

[0082] 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%.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] According to the present invention, the novel antigenically active proteins / polypeptides are also useful for generating antibodies for diagnosis and protection against the antigen, while minimizing the possibility of ADE. The proteins / polypeptides disclosed herein comprise a minimal sequence encoding the RBD fused to a signal sequence and / or to a transmembrane domain (TMD) sequence, intended to maximize immunogenicity and minimize ADE.

[0087] 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]

[0088] Each gene encoding construct 1 to 12 shown below was synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages).

[0089] 1. Construct 1 [ka] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGsgESQVRQQFskdiekllneqvnkemqssnlymsmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks* (SEQ ID NO: 1)

[0090] 2. Construct 2 [ka] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI* (SEQ ID NO: 2)

[0091] 3. Construct 3 [ka] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI* (SEQ ID NO: 3)

[0092] 4. Construct 4 [ka] mfvflvllplvssLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGsgESQVRQQFskdiekllneqvnkemqssnlymsmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks (SEQ ID NO: 4)

[0093] 5. Construct 5 [ka] mfvflvllplvssLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 5)

[0094] 6. Construct 6 [ka] *(SEQ ID NO:6)

[0095] 7. Construct 7 [ka] (SEQ ID NO:7)

[0096] Constructs 8-14 Constructs 8 to 14 correspond to constructs 1 to 7, respectively, further fused to PADRE (T epitope) at the C-terminus. (SEQ ID NOs: 8 to 14)

[0097] Influenza A virus HA1: DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQS (SEQ ID NO: 15) Influenza A virus HA2: GLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEID (SEQ ID NO: 16) COVID-19 signal sequence: MFVFLVLLPLVSS (SEQ ID NO: 17) Influenza A virus HA1 signal sequence: MKAILVVLLYTFATANA (SEQ ID NO: 18) Influenza A virus HA1 head: LHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAG (SEQ ID NO: 19) sg:linker TM / CT derived from influenza A virus [A / Puerto Rico / 8 / 1934 (H1N1)]: GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 20) or TM / CT derived from influenza A virus [A / California / 07 / 2009 (H1N1)]: GVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 21) Ferritin (Helicobacter pylori-Bullfrog hybrid ferritin): ESQVRQQFSkdiekllneqvnkemqssnlymsmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks (SEQ ID NO: 22) PADRE:AKFVAAWTLKAAA (SEQ ID NO:23) [Example 2]

[0098] Preparation of replicon vectors A schematic of the construct of the alphavirus replicon is shown in Figure 1. As the promoter in Figure 1, the T7 promoter was used.

[0099] A VEEV full-length replicon plasmid vector was prepared by the procedure disclosed in WO2019 / 124441. Each of constructs 1 to 14 prepared in Example 1 was used as the gene of interest. Nucleotides encoding the constructs were cloned into a VEEV replicon vector under the control of the SG promoter. VEEV replicon plasmids encoding each fragment were generated by inserting AscI and SbfI restriction sites to obtain a full-length VEEV TC-83 replicon plasmid.

[0100] 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: 24) 5'UTR: ataggcggcgcatgagagaagcccagaccaattacctacccaaa (SEQ ID NO: 25) 3'UTR: gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc (SEQ ID NO: 26) PolyA tail: aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 27)

[0101] The amino acid sequences of the VEEV TC-83 replicons nsP1-4 are as follows: APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADV AIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERV QRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPPVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSWSIPHAS DFDVDSLSILDTLDGASVTSGAVSAETNSYFARSSMEFRARPVPAPRTVFRNPPHPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO:28) The amino acid sequence corresponding to nsp3 is underlined.

[0102] In this example, the amino acid sequence of nsp3 corresponding to 1330 to 1886 of SEQ ID NO:28 was replaced with the sequence shown below. The underlined sequences differed from SEQ ID NO:28. APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDR LTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILG K SMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO: 29) [Example 3]

[0103] Preparation of self-amplifying RNA (saRNA) Plasmids with influenza HA variant sequences placed downstream from the T7 promoter were linearized by digestion with Nrul or BspQ1 restriction enzymes at 37°C or 50°C for 3 hours. The linearized plasmids were then purified using Wizard Plus SV Miniprep DNA Purification System (Promega) and saRNA was in vitro transcribed using T7 RiboMAX Express Large-Scale RNA Production System (Promega). After DNase treatment, saRNA was purified with RNeasy midi kit (Qiagen) and subsequently modified by addition of a 7-methylguanosine cap with the Vaccinia Capping System [New England Biolabs (NEB)] using the NEB Capping protocol (NEB, M20280). The capped saRNA was purified using Monarch kit (NEB).

[0104] Western blotting HEK293T cells were transfected with saRNA 0.5 using Lipofectamine (Promega). Cells were harvested 18 hours after transfection, lysed with cell lysis buffer (Cell Signaling Technology), and fractionated by SDS-PAGE (Any kD acrylamide gel, Bio-Rad). Proteins were detected by Western blotting using anti-IAV H1N1 (A / California / 07 / 2009) hemagglutinin, rabbit polyclonal antibody (1:5000 dilution; Sino Biological), and horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:2000; Santa Cruz Biotechnology). Protein bands were visualized by enhanced chemiluminescence using ChemiDoc™ XRS+ (Bio-Rad) and Image Lab™ Software (Bio-Rad).

[0105] The results of plasmid vectors carrying constructs 6, 13, 3 and 10 as the gene of interest are shown in Figure 3. As shown in Figure 2, these constructs correspond to F01, F02, F04 and F05, respectively. The results of the Western blot assay indicate that cells transfected with saRNA-Flu variants express influenza antigens of the expected molecular weight.

[0106] Flow cytometry analysis For cell surface protein analysis, transfected HEK293T cells were harvested, washed with PBS, and stained with anti-IAV H1N1 (A / California / 07 / 2009) hemagglutinin, rabbit polyclonal antibody, and donkey anti-rabbit IgG secondary PE (1:200 dilution; Bio Legend). Surface protein levels were assessed using an Attune acoustic focusing cytometer (applied biosystems). Non-transfected HEK293T cells were used as a control. The results are shown in Figure 4. In Figure 4, the R3 fraction contains antigen-positive cells, and the R5 fraction contains highly antigen-positive cells among the R3 fraction.

[0107] Flow cytometry analysis indicated that influenza antigens were expressed on the surface of cells transfected with saRNA-Flu variants, and in previous studies, the proportion of cells in the R5 fraction correlated with immunogenicity to the antigen in animal model studies. [Example 4]

[0108] Preparation of alphavirus replicon particles 10 μg of the full-length replicon plasmid prepared in Example 2, 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. HEK293T cells or Vero cells were infected with dilutions of the purified particle preparation to measure the infectious titer. The purified replicon particles are used to generate antigens for diagnosis and vaccination. [Example 5]

[0109] Preparation of mRNA or self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs) A plasmid vector containing a DNA sequence encoding constructs 1-14 as the gene of interest prepared in Example 2 was used. The plasmid vector 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.

[0110] The obtained mRNA or saRNA was encapsulated in lipid nanoparticles to obtain mRNA particles or saRNA particles.

Claims

1. 1. An isolated polynucleotide comprising: A polypeptide comprising an antigen protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin. A polynucleotide encoding the

2. The polynucleotide of claim 1 , wherein the antigenic protein is an influenza protein.

3. The polynucleotide of claim 1 , wherein the influenza protein is influenza virus hemagglutinin 1 (HA1) and / or influenza virus hemagglutinin 2 (HA2).

4. The polynucleotide of claim 1 , wherein the influenza protein is the head region of HA1.

5. The polynucleotide according to any one of claims 1 to 4, wherein the ferritin is derived from Helicobacter pylori or is a Helicobacter pylori-Rana catesbeiana hybrid ferritin.

6. The polynucleotide of any one of claims 1 to 5, further encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4.

7. The polynucleotide according to any one of claims 1 to 6, wherein the transmembrane domain and / or signal sequence are fused to the antigen protein by a linker.

8. The polynucleotide according to any one of claims 1 to 7, wherein the polynucleotide is RNA.

9. The polynucleotide according to any one of claims 1 to 7, wherein the polynucleotide is DNA.

10. A vector comprising the polynucleotide according to any one of claims 1 to 10.

11. promoter, 5'UTR, A polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4; SG promoter, a gene of interest encoding said polypeptide comprising an antigenic protein fused to a signal sequence and a transmembrane domain; 3'UTR and including a polyA tail, The vector described in claim 10.

12. A vaccine composition comprising the polynucleotide or vector of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

13. The vaccine composition of claim 12, wherein the pharma- ceutically acceptable carrier is a delivery vehicle.

14. 15. The vaccine composition of claim 14, wherein the delivery vehicle is a particle comprised of one or more alphavirus structural proteins or a lipid delivery system.

15. Use of the polynucleotide or vector according to any one of claims 1 to 11 for the manufacture of a medicament.

16. The use according to claim 15, wherein the medicament is for inducing immunomodulation in a subject.

17. 16. The use according to claim 15, wherein the medicament is for treating or preventing a subject from a condition caused by an infection.

18. Administering an immunologically effective amount of the vaccine composition according to any one of claims 12 to 14 to a subject in need thereof. A method of inducing immunomodulation in said subject.

19. A method of treating, preventing and / or immunizing against an antigen in a subject, comprising administering to a subject in need thereof an effective amount of the vaccine composition according to any one of claims 12 to 14. method.

20. A polypeptide comprising an antigen protein fused to a signal sequence and a transmembrane domain, and optionally to ferritin.

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