Modified vaccine design development

Abstract

Provided herein are isolated polynucleotides encoding a polypeptide comprising an antigen protein fused to a signal sequence and a transmembrane domain, and optionally fused to a ferritin. Also provided herein are isolated polynucleotides encoding alphaviral non-structural proteins nsp1, nsp2, nsp3, and nsp4 and polypeptides comprising an antigen protein fused to a signal sequence and a transmembrane domain, and optionally fused to a ferritin. The antigen may be influenza. Polynucleotides, such as RNAs, can be used as vaccines against influenza infection.

Description

Technical Field

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

[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 serious complications from influenza. There are two main types of influenza (flu) viruses: type A and type B. Influenza A and B viruses that circulate routinely among humans (human influenza viruses) are responsible for the annual seasonal influenza epidemics.

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

[0004] Different types of flu vaccines are available, including quadrivalent flu vaccines that protect against four different flu viruses; high-dose flu vaccines, which contain four times the amount of antigens (the part of the vaccine that helps your body build protection against the flu virus) as regular flu shots and are specifically licensed for use in people 65 and older; cell-based flu vaccines, which are grown in cultured cells of mammalian origin rather than in eggs; live attenuated influenza vaccine [LAIV] nasal spray flu vaccines given as a nasal spray; flu vaccinations approved for use in people 18 to 64 years of age via a jet injector; adjuvanted vaccines, which are prepared with ingredients added to the vaccine that help produce a stronger immune response and are specifically licensed for use in people 65 and older; and recombinant flu vaccines, which are produced using a method that does not require egg-grown vaccine viruses (https: / / www.cdc.gov / flu / about / index.html). Data on vaccine effectiveness (VE) were obtained from the March CDC Morbidity and Mortality Weekly Report. VE was calculated using data from 3,363 children and adults with acute respiratory tract infection (ARI) enrolled in the U.S. Influenza Vaccine Effectiveness Network from seven different study sites across the United States from October 2021 to February 2022. Only a 16% VE was observed from the 2021-22 seasonal influenza vaccine that protected the U.S. population from infection with the most common influenza virus A(H3N2) currently in circulation. More specifically, in outpatients receiving medical care, the VE for mild to moderate ARI associated with influenza A(H3N2) viruses was 16%, which was not significantly effective. In addition, the VE for outpatients receiving medical care with ARI associated with influenza A virus types was even less significantly effective at 14%. The low vaccine efficacy among the most common influenza strains in circulation is of particular concern (https: / / www.clinicaltrialsarena.com / comment / us-flu-vaccine-efficacy / ).

[0005] More promising influenza vaccines are expected, which have long-term high efficacy. Summary of the invention

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

[0007] In another aspect, the present disclosure relates to novel polynucleotides encoding the above-mentioned novel antigenically active proteins / polypeptides capable of inducing protection against antigens.

[0008] On the other hand, the present disclosure relates to a novel alphavirus replicon (self-amplifying RNA "saRNA") that can express the above-mentioned antigenically active protein / polypeptide. The alphavirus replicon includes polynucleotides such as RNA encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4, and a polynucleotide encoding the above-mentioned antigenically active protein / polypeptide as a target gene.

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

[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, preventing or treating influenza in a subject comprises administering an effective amount of the above-mentioned polypeptide or polynucleotide, such as saRNA, to a subject in need thereof.

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

[0012] In a further aspect, the present disclosure relates to novel polynucleotides encoding polypeptides comprising an antigenic protein fused to a signal sequence and a transmembrane domain, and optionally fused to ferritin. The polynucleotide may be a saRNA comprising polynucleotides 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 fused to ferritin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [ Figure 1 ] Representative constructs of saRNA disclosed in this application.

[0014] [ Figure 2 ] Schematic diagram of constructs F1, F2, F4 and F5.

[0015] [ Figure 3 ] Western blot results of lysates of cells transfected with saRNA.

[0016] [ Figure 4 ] FACS analysis of HA antigen expressed on cells transfected with saRNA. DETAILED DESCRIPTION

[0017] As used herein, "influenza" is intended to refer to the Orthomyxoviridae (a group of RNA viruses). Influenza viruses are classified as type A, type B, type C, and type D. These major types generally produce similar symptoms, but are antigenically completely unrelated, so infection with one type does not confer immunity against the other types. A viruses cause influenza pandemics, while B viruses cause smaller local outbreaks. C viruses cause only mild respiratory illness in humans. Influenza D viruses are not known to infect humans and have only been observed in pigs and cattle.

[0018] Influenza A viruses are classified into subtypes, and both subtypes of influenza B and influenza A are further divided into strains. Subtypes of influenza A are mainly distinguished based on two surface antigens (foreign 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. Strains of influenza B and strains of influenza A subtypes are further distinguished by variations in gene sequences.

[0019] 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 up to 10% of the amino acids are deleted, substituted and / or added based on a naturally occurring viral envelope protein or a fragment thereof.

[0020] As used herein, a "transmembrane domain (TM)" is a protein derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. In one aspect, the membrane-bound or transmembrane protein is a protein heterologous to influenza. Examples of membrane-bound or transmembrane proteins may include α, β or ζ chains of T cell receptors, CD28, CD3ε, 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 IL-1-28 receptors, RANTES receptors (CCR1, CCR3, CCR5), MIP-1 receptors, PF4 receptors, M-CSF receptors, and NAP-2 receptors belonging to GPCR chemokine receptors; hemagglutinin (HA). On the other hand, the membrane-bound or transmembrane protein is a protein derived from influenza virus.

[0021] Examples of transmembrane proteins may also include the following: 5-lipoxygenase activating protein, ABC transporter, 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-induced protein, immunoglobulin, insulin receptor, integrin, ion channel, MAPEG, MFS, MinK family, MPP, peptidase AD, peptidase family M48, peptidase MA, jagged protein, receptor kinase, SNARE complex, sulfatase, TNF receptor, transmembrane protein 14, transporter, T ROBP, VEGF receptor, aldehyde dehydrogenase, ammonia and urea transporter, FMN-linked oxidoreductase, transmembrane protein containing leucine-rich repeats (LRR), leukotriene C4 synthase, lysosome-associated membrane glycoprotein, major intrinsic protein (MIP) / FNT superfamily, microsomal prostaglandin E synthase, N-(deoxy)ribosyltransferase-like membrane protein, neutral / alkaline ceramidase, oligosaccharyltransferase, pentameric ligand-gated ion channel, rhodopsin-like receptor and pump, single-helical ATPase regulatory factor, squalene / phytoene synthase, stearoyl-CoA desaturase 1, Stannin (SNN) membrane protein, T cell surface glycoprotein CD3ζ chain, triangular tetratricopeptide repeat (TPR) α-helical repeat protein, transmembrane protein with NAD(P)-binding Rossmann fold domain.

[0022] In addition, monotypic / peripheral proteins or other integral proteins and peptides attached to the lipid bilayer can also be used as transmembrane proteins. Examples may include α / β-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 (group E) 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, saposin, synuclein, and transcription factor tubby.

[0023] As used herein, "ferritin" refers to any one of mammalian-derived ferritin, amphibian-derived ferritin, bacterial-derived ferritin, or plant-derived ferritin, or a combination of at least two of them. Ferritin of mammalian origin or ferritin of bacterial origin is preferred. Preferred ferritin of mammalian origin includes any one of human-derived ferritin, mouse-derived ferritin, or horse spleen ferritin, or a combination of at least two of them. Preferred ferritin of amphibian origin includes bullfrog. Preferred ferritin of bacterial origin includes Helicobacter pylori ferritin or Escherichia coli ferritin. Preferred sources of ferritin include any one of natural extracts, artificially synthesized products, or genetic engineering technology products, or a combination of at least two of them. In one embodiment, a Helicobacter pylori (H. pylori)-bullfrog hybrid ferritin can be used, for example, one constructed by fusing residues 2-9 of the bullfrog (Ranacates beiana) ferritin lower subunit (UniProt: P07797, with N8Q mutation to eliminate a potential N-glycosylation site) with a H. pylori non-heme ferritin (UniProt: Q9ZLI1, residues 3-167) with an I7E mutation to retain a conserved salt bridge with 6R of bullfrog ferritin, which is found in human and bullfrog ferritin (6R and 14E in both human light chain and bullfrog smaller subunit ferritin) (Kanekiyo et al., Cell, 2015 Aug 27;162(5):1090-1100).

[0024] As used herein, "nucleoside" refers to a molecule composed of guanine (G), adenine (A), thymine (T), uridine (U), cytidine (C), or modified nucleosides thereof.

[0025] Modified nucleosides include, but are not limited to, pseudouridine, N1-methylpseudouridine, 5-methyluridine, pseudocytidine, N1-methylpseudouridine, and 5-methylcytidine.

[0026] Pseudouridine or pseudocytidine is an isomer of uridine or cytidine in which uracil or cytosine is attached via a carbon-carbon rather than a nitrogen-carbon glycosidic bond.

[0027] In one embodiment, the modified nucleoside is independently selected from N1-methyl pseudouridine or 5-methylcytidine. In one embodiment, the mRNA or saRNA substantially comprises 100% modified cytidine (e.g., 5-methylcytidine) 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.

[0028] The expression "transmembrane domain" used in the present disclosure includes at least one or more transmembrane regions of a membrane-bound or transmembrane protein. In addition, the transmembrane domain may also include a membrane-proximal domain (JMD) and / or a cytoplasmic tail of a membrane-bound or transmembrane protein.

[0029] Alternatively, in some embodiments, the membrane spaning domain is synthetic. In some aspects, the synthetic membrane spaning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic membrane spaning domain.

[0030] Preferred transmembrane domains may be those derived from influenza virus hemagglutinin (HA), CD80, Toll-like receptor 4 (TLR4). Specific examples may include proteins consisting of the transmembrane domain and cytoplasmic tail of influenza virus hemagglutinin "HA (TM / CT)"; proteins consisting of the transmembrane domain and cytoplasmic tail of human CD80; proteins consisting of a transmembrane domain (TM) and a Toll / interleukin-1 receptor domain (TIR), and proteins consisting of a proximal membrane domain (JMD).

[0031] 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. The signal sequence is about 9 to 200 nucleotides or 3-70 amino acids in length, and is optionally incorporated into the coding region or the 5' or N-terminus of the protein. Some signal sequences are cleaved from the protein, for example, by a signal peptidase after the protein is transported to the desired site.

[0032] The signal sequence is not limited and can be selected from a variety of sequences. In some embodiments, the signal sequence can be the signal sequence of a target protein to be expressed by the alphavirus replicon.

[0033] In some embodiments, the signal sequence of influenza HA, COVID-19, or IL-2, and in particular influenza HA, may be used.

[0034] Influenza structural proteins, transmembrane domains and / or signal sequences can be fused directly or indirectly. In one embodiment, one or two linkers can be inserted therebetween.

[0035] Influenza structural proteins, transmembrane domains and / or signal sequences can also be truncated and replaced by short linkers. In some embodiments, viral structural proteins, transmembrane domains and / or signal sequences comprise one or more peptide linkers.

[0036] The antigen protein, signal sequence, transmembrane domain and optional ferritin may be further fused to a T cell epitope. A "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, a T cell epitope may be derived from a virus to be treated.

[0037] The expression "PADRE" used in the present disclosure refers to the Pan HLA DR binding epitope, which is a peptide that universally activates antigen-specific CD4+ T cells. The amino acid sequence of PADRE is AKFVAAWTLKAAA.

[0038] The viral structural proteins, signal sequence and transmembrane domain, and optionally ferritin and at least one universal epitope such as PADRE can be fused directly or indirectly. In one embodiment, one or two linkers can be inserted in between.

[0039] Viral structural proteins, signal sequences and transmembrane domains, as well as optional ferritin and at least one or more T cell target epitopes, can also be truncated and replaced by short linkers. In some embodiments, viral structural proteins, transmembrane domains and / or signal sequences comprise one or more peptide linkers.

[0040] Examples of short linkers are composed of 2 to 25 amino acids (e.g., 2, 3, 4, 5, or 6 amino acids). Typically, its length is 2 to 15 amino acids, such as SG, GS, SGG, GGS, SGSG, and TRGGS. In some cases, the linker can consist of only one amino acid, such as glycine (G), serine (S), and cysteine ​​(C).

[0041] When the influenza structural protein is chemically conjugated to the transmembrane domain and / or signal sequence by a chemical cross-linking agent, examples of the cross-linking agent 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-linking agents may also be used.

[0042] IgG-derived substances can also be used as linkers. Examples of IgG-derived substances include IgG1 to IgG4, which contain (i) all (hinge-CH 2 CH 3 )(ii) Half (hinge-CH 3 ) and (iii) short (only 12aa hinge). A preferred example is IgG4-CH 3 .

[0043] "Alphavirus structural protein" refers to a polypeptide or fragment thereof having at least about 80% amino acid sequence identity with 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 with Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus, Mukambu Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus (CHIKV), Agnang-Nyang Virus, Ross River Virus, Bama Forest Virus, Geta Virus, Lushan Virus, Bebaru Virus, Mayaro Virus, Una Virus, Ora Virus, Vodaro River Virus, Babanki Virus, Zilagachi Virus, Highland J Virus, Fort Morgan Virus, Ndumu Virus, or Bogi River Virus. The wild-type amino acid sequence of the alphavirus structural protein can be obtained from GenBank.

[0044] "Alphavirus structural protein" refers to a polypeptide or fragment thereof having at least about 80% amino acid sequence identity with 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 with Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus, Mukambu Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEEV), Sindbis Virus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus (CHIKV), Agnang-Nyang Virus, Ross River Virus, Bama Forest Virus, Geta Virus, Lushan Virus, Bebaru Virus, Mayaro Virus, Una Virus, Ora Virus, Vodaro River Virus, Babanki Virus, Zilagachi Virus, Highland J Virus, Fort Morgan Virus, Ndumu Virus, or Bogi River Virus. The wild-type amino acid sequence of the alphavirus structural protein can be obtained from GenBank.

[0045] 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.

[0046] "Alphavirus replicon" refers to an RNA molecule that can direct its own in vivo amplification in a target cell. The replicon encodes one or more polymerases (nsp1, nsp2, nsp3, nsp4) that catalyze RNA amplification, and contains a cis RNA sequence required for replication, which is recognized and utilized by the encoded one or more polymerases. Alphavirus replicons typically contain the following ordered elements: 5'UTR, sequences encoding alphavirus nonstructural proteins (nsp1, nsp2, nsp3, nsp4), 3'UTR, and polyadenylation signals. Alphavirus replicons also contain one or more viral subgenomic promoters that direct the expression of target genes. Those sequences may have one or more mutations taught in prior art references.

[0047] The alphavirus replicon provided by the present disclosure may have Figure 1 The constructs shown in .

[0048] In the present disclosure, “comprises, comprising, containing” and “having”, etc. may have the meanings ascribed to them in U.S. patent law, and may represent “includes, including”, etc.; “consisting essentially of or consists essentially” also has the meanings ascribed to them in U.S. patent law, and the terms are open-ended, allowing for more than what is described, as long as the basic or novel features described are not altered by the presence of more than what is described, but excluding prior art implementations.

[0049] "Fragment" refers to 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 a reference nucleic acid molecule or polypeptide. A fragment may 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.

[0050] "Reference" refers to a standard or control condition.

[0051] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or all of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is typically at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer near or therebetween.

[0052] Sequence identity is typically measured using sequence analysis software (e.g., sequence analysis software packages, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, wherein probability scores between e<"3> and e<"100> indicate closely related sequences.

[0053] An "effective amount" refers to the amount of a pharmaceutical agent required to improve the symptoms of a disease relative to an untreated patient. The effective amount of one or more active compounds used to practice the present invention to prevent or treat a disease varies depending on 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.

[0054] You can do this by taking 10 3 -10 10 Infectious unit (IU) or 0.01-500 μg, preferably 10 5 -10 10 IU or 0.1-100 μg, for example, 10 7 -10 9 IU or 1-50 μg is administered systemically, for example intramuscularly, subcutaneously or intravenously 1-4 times to achieve satisfactory results. The replicon may preferably be formulated in a vaccine composition suitable for administration in a conventional manner.

[0055] "Subject" refers to a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0056] As used herein, the terms "treat, treating, treatment" and the like refer to the alleviation or improvement of a disorder and / or symptoms associated therewith. It should be understood that, although not excluded, the treatment of a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0057] 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.

[0058] Unless specifically stated or obvious from the context, as used herein, the term "or" should be construed as being inclusive.

[0059] As used herein, the terms "a," "an," and "the" are to be construed as referring to the singular or the plural, unless specifically stated or clear from the context.

[0060] In the present description and claims, the term "about" encompasses values ​​of ±20%, ±10% or ±5% of the relevant numerical value.

[0061] One skilled in the art will recognize that the polynucleotide sequences described in the specification and claims will list "T" in representative DNA sequences, but where the sequence represents RNA, the "T" will be replaced with "U".

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

[0063] The term "vector" refers to a means by which a nucleic acid sequence can be propagated and / or transferred between organisms, cells or cell components. Vectors include plasmids, viruses, phages, proviruses, phagemids, transposons, artificial chromosomes, etc. that replicate autonomously or can be integrated into the chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide containing both DNA and RNA in the same strand, a polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, etc. that does not replicate autonomously. In many, but not all common embodiments, the vector of the present invention is a plasmid or a bacmid.

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

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

[0066] There are a variety of expression systems for generating the constructs of the present invention. Expression vectors for generating constructs include, but are not limited to, chromosomal, episomal and viral derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as alphaviruses (e.g., Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV)), baculoviruses, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses, and vectors derived from combinations thereof.

[0067] The constructs and / or vectors used herein comprise an alphavirus polynucleotide encoding nonstructural proteins nsp1, nsp2, nsp3, and nsp4, and a target gene encoding a polypeptide comprising an antigen, such as a viral structural protein fused to a signal sequence and a transmembrane domain as described above. Specific examples of constructs or vectors include Figure 1 as shown in .

[0068] The vector can be, for example, a phage, plasmid, virus or retroviral vector. The construct and / or vector comprising nucleotides should be operably connected to a suitable promoter, for example, CMV promoter, bacteriophage λPL promoter, E. coli lac, phoA and tac promoter, SV40 early and late promoters and the promoter of retroviral LTR are non-limiting examples. Depending on the host cell and / or the required expression rate, other suitable promoters will be known to those skilled in the art. The expression construct will further include sites for transcription initiation, termination, and (in the transcription region) a ribosome bind site for translation. The coding portion of the transcript expressed by the construct will preferably include a translation initiation codon at the start and a termination codon appropriately positioned at the end of the polypeptide to be translated.

[0069] The vector will preferably contain 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 cultivation in Escherichia coli and other bacteria. In the vector, viral vectors are preferred, such as baculovirus, poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoon pox virus, swine pox virus, etc.), adenovirus (e.g., canine adenovirus), herpes virus and retrovirus. Other vectors that can be used together 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. Preferred eukaryotic vectors are pFastBacl pWINEO, pSV2CAT, pOG44, pXT1 and pSG, pSVK3, pBPV, pMSG and pSVL. Other suitable vectors will be readily apparent to those skilled in the art.

[0070] Recombinant constructs can be prepared and used for transfection, which can express viral proteins (including those described herein) into eukaryotic cells and / or prokaryotic cells. Thus, in one embodiment, the disclosure provides a host cell comprising a vector (or multiple vectors) containing nucleic acids encoding alphavirus structural proteins (including capsid, E3, E2, 6K and E1 or portions thereof), and (under conditions that allow the formation of alphavirus replicon particles) containing nucleic acids encoding alphavirus nsp1, nsp2, nsp3 and nsp4 and at least one viral gene of interest.

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

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

[0073] Depending on the selected vector and host cell, the construct is produced by growing the host cell transfected by the vector under conditions whereby the recombinant protein is expressed and the alphavirus replicon is produced, and a construct comprising the alphavirus replicon packaged with particles of the alphavirus structural protein is formed. In one embodiment, the invention includes a method for producing a construct comprising a vector comprising a polynucleotide encoding the alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4 and at least one target gene encoding a polypeptide (the polypeptide comprising a viral structural protein fused to a signal sequence and / or a transmembrane domain), and at least one vector encoding at least one alphavirus structural protein, each co-transfected into a suitable host cell, and expressing the alphavirus structural protein under conditions that allow the formation of the construct. In another embodiment, the eukaryotic cell is selected from yeast, insect, amphibian, avian or mammalian cells. The selection of appropriate growth conditions is within the skill of a person of ordinary skill in the art or a person having the skill.

[0074] Methods for growing cells that produce alphavirus replicon particles of the present invention include, but are not limited to, batch, fed-batch, continuous and perfusion cell culture techniques. In one embodiment, cells co-transfected with a vector encoding an alphavirus replicon and a vector comprising a polypeptide encoding a capsid, and a vector comprising a polynucleotide encoding an envelope protein (e.g., those derived from CHIKV or VEEV) are grown in a bioreactor or fermentation chamber, where the cells proliferate and express proteins (e.g., recombinant proteins) for purification and separation. Typically, cell culture is carried out under sterile, controlled temperature and atmospheric conditions. A bioreactor is a chamber for culturing 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.RTM., Wave Biotech, Bridgewater, NJ, the contents of the cited literature are incorporated herein by reference). In another embodiment, the pre-sterilized plastic bag is about 10L to 1000L bag.

[0075] In another embodiment, RNA molecules, such as alphavirus replicons, can be generated from template DNA sequences by conventional procedures known in the art. The in vitro transcription (IVT) method allows template-directed synthesis of RNA molecules. The IVT method allows the synthesis of large quantities of RNA transcripts. Typically, IVT utilizes a DNA template that contains a promoter sequence upstream of the target sequence. The promoter sequence is most commonly of phage origin, such as T7, T3 or SP6 promoter sequences, but many other promoter sequences can be tolerated, including those designed from scratch. Transcription of the DNA template is generally best achieved using an RNA polymerase corresponding to a specific phage promoter sequence. Exemplary RNA polymerases include, but are not limited to, especially T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. IVT typically initiates at dsDNA, but can continue on a single strand. Kits for in vitro transcription, such as the T7 transcription kit (RiboMax TM Express Large Scale RNA production System, Promega (WI USA)).

[0076] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates, including any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, 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), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutritional supplements, similar materials and combinations thereof, as will be known to those of ordinary skill in the art. The pH and exact concentrations of the various components in the vaccine composition are adjusted according to well-known parameters.

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

[0078] In some embodiments, the vaccine composition or formulation of the present disclosure comprises a lipid delivery system, such as a liposome, a lipoplex, a lipid nanoparticle, or any combination thereof. The polynucleotides described herein, such as alphavirus replicons, can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. Liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficacy of polynucleotide-directed protein production because these formulations can increase cell transfection of polynucleotides; and / or increase the translation of encoded proteins. Liposomes, lipoplexes, or lipid nanoparticles can also be used to increase the stability of polynucleotides.

[0079] Liposomes are artificially prepared vesicles that may mainly contain lipid bilayers and can be used as delivery vehicles for administering drug formulations. Liposomes may have different sizes. The diameter of multilamellar vesicles (MLVs) may be hundreds of nanometers and may include a series of concentric bilayers separated by narrow aqueous compartments. The diameter of small single cell vesicles (SUVs) may be less than 50 nm, while the diameter of large unilamellar vesicles (LUVs) may be between 50 and 500 nm. Liposome design may include, but is not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissues or activation events, such as, but not limited to, endocytosis. Liposomes may contain low pH or high pH to improve the delivery of drug formulations.

[0080] The formation of liposomes may depend on the entrapped drug formulation and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped material and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimal size, polydispersity and shelf life of the vesicles for the intended application, and batch-to-batch reproducibility and scale-up production of safe and effective liposome products, etc.

[0081] In some embodiments, a polynucleotide, such as an alphavirus replicon described herein, can be encapsulated by liposomes and / or it can be contained within an aqueous core, which can then be encapsulated by liposomes.

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

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

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

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

[0086] LNPs contain pH-sensitive ionizable cationic lipids that attract anionic nucleic acids to form the core of self-assembled nanoparticles to ensure high encapsulation. At physiological pH, LNPs are neutral, which eliminates the toxicity mechanisms seen for permanent cationic molecules.

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

[0088] This replicon-based vaccine technology is a unique platform technology for vaccination because RNA can self-amplify to produce vaccine antigens and deliver to organelles. In addition, this replicon-based vaccine technology overcomes challenges typically associated with DNA-based vaccines, such as the risk of genomic integration or the high doses and devices required for administration, such as electroporation, and is expected to yield higher immunogenicity at minimal doses relative to mRNA technology based on a self-replicating system.

[0089] According to the present invention, novel antigenic active proteins / polypeptides can also be used to generate antibodies for diagnosis and protection against antigens while minimizing the possibility of ADE. Proteins / polypeptides disclosed herein include minimal sequences encoding RBD fused to signal sequences and / or transmembrane domain (TMD) sequences, which are intended to maximize immunogenicity and minimize ADE.

[0090] The present invention will be described in detail with reference to the following examples, however, the examples are not intended to limit the scope of the present application. Example 1

[0091] Each gene encoding constructs 1-12 shown below was synthesized by Integrated DNA Technologies, Inc. (https: / / www.idtdna.com / pages). 1. Construct 1 [Chemical Structure 1] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGD FIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGN SYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRP KVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGsgESQVRQQFskdiekllne qvnkemqssnlymsmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks*(SEQ ID NO:1) 2. Construct 2 [Chemical Structure 2] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGD FIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGN SYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRP KVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI*(SEQ ID NO:2) 3. Construct 3 [Chemical Structure 3] MKAILVVLLYTFATANALHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGD FIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGN SYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRP KVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI*(SEQ ID NO:3) 4. Construct 4 [Chemical Structure 4] mfvflvllplvssLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELRE QLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSY INDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGR MNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGsgESQVRQQFskdiekllneqvnkemqssnly msmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks(SEQ ID NO:4) 5. Construct 5 [Chemical Structure 5] mfvflvllplvssLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELRE QLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSY INDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGR MNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:5) 6. Construct 6 [Chemical Structure 6] MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCK LRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLS SVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIND KGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNY YWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPF QNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQ NEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGF LDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTC MESVKNGTYDYPKYSEEAKLNREEIDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI*(SEQ ID NO:6) 7. Construct 7 [Chemical Structure 7] MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCK LRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLS SVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIND KGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNY YWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPF QNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQ NEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGF LDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTC MESVKNGTYDYPKYSEEAKLNREEIDsgESQVRQQFskdiekllneqvnkemqssnlymsmsswcythsldg aglflfdhaaeeyehakliiflnennvpvqltsisapehkfegltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrk(SEQ ID NO:7) Constructs 8-14 Constructs 8-14 correspond to constructs 1-7 further fused to PADRE (T epitope) at the C-terminus, respectively. (SEQ ID NO: 8-14) Influenza A virus HA1: DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPK LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQS(SEQ ID NO:15) Influenza A virus HA2: GLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKM NTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYE KVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEID(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: LHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFE RFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEV LVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAG(SEQ ID NO:19) sg: connector 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): ESQVRQQFSkdiekllneqvnkemqssnlymsmsswcythsldgaglflfdhaaeeyehakkliiflnennvpvqltsisapehkf egltqifqkayeheqhisesinnivdhaikskdhatfnflqwyvaeqheeevlfkdildkielignenhglyladqyvkgiaksrks(SEQ ID NO:22) PADRE:AKFVAAWTLKAAA(SEQ ID NO:23) Example 2 Preparation of replicon vector

[0092] A schematic construction of the alphavirus replicon is shown in Figure 1 As Figure 1 The promoter in the expression vector was T7 promoter.

[0093] The VEEV full-length replicon plasmid vector was prepared by the procedure disclosed in WO 2019 / 124441. Each of the constructs 1-14 prepared in Example 1 was used as the target gene. The nucleotides encoding the constructs were cloned into the VEEV replicon vector under the control of the SG promoter. VEEV replicon plasmids encoding the various fragments were generated by inserting AscI and SbfI restriction sites to obtain the full-length VEEV TC-83 replicon plasmid.

[0094] The nucleotide sequences of the SG promoter, 5'UTR, 3'UTR and poly(A) tail are as follows. RNA sequences were obtained by using these DNA sequences as templates. SG promoter:cctgaatggactacgacatagtctagtccgccaag(SEQ ID NO:24) 5'UTR:ataggcggcgcatgagagaagcccagaccaattacctacccaaa(SEQ ID NO:25) 3'UTR: gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgcatgccttaaaattttttattttatttttcttttcttttccga atcggattttgtttttaatatttc (SEQ ID NO: 26) Polyadenylation tail: aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO: 27)

[0095] The amino acid sequence of VEEV TC-83 replicon nsP1-4 is as follows. MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTIL DIGSAPARMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAV MSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTP FMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSV GSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAA TMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRI VVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFR RHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDIQEA KCAADEAKEVREAEELRAALPPLAADFEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYA GEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPV QDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQ CVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKK DLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLR ALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFY DKRMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTR KGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKILTAKYPGNFTATIEE WQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETD KAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSR RYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKL KGRTVLVVGEKLSVPGKKVDWLSDQPEATFRARLDLGIPGDVPKYDIVFINVRTPYKYHHY QQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSH EETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGC APSYHVVRGDIATATEGVIIN AANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGD KQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRD KKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLE GTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPSTLPCLCI HAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETP PVEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEA DIHGSPSVSSSSWSIPHASDFDVDSLSILDLDGASVTSGAVSAETNSYFARSMEFRARPVPA PRTVFRNPPHPAPRTRTPPLAHSRASSRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSL VSNPPGVNRVITREEFEAFVAQQQXRFDAGA YIFSSDTGQGHLQQKSVRQTVLSEVVLERT ELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRRVENMKAITARRILQGLGHYLKAEG KVECYRTLHPVPLYSSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGA SCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPV LDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNM LQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATADLCGIHRELVRRLNAVL LPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFFDKSEDDAMALTALMILEDLGVDAEL LTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFI GDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVAD PLKRLFKLGKPLAVDDEHDDDRRRALHEESTRWNRVGILPELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG(SEQ ID NO:28)

[0096] The amino acid sequence corresponding to nsp3 is underlined.

[0097] In this example, the amino acid sequence of nsp3 corresponding to 1330-1886 in SEQ ID NO:28 was replaced with the sequence shown below. The underlined sequence is different from SEQ ID NO:28. APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGA AKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQS LNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPK SSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILG K SMSSI RSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCS QPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEE EDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSA ETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA (SEQ ID NO:29) Example 3 Preparation of self-amplifying RNA (saRNA)

[0098] Plasmids with influenza HA variant sequences placed downstream of the T7 promoter were linearized by digestion with Nrul or BspQ1 restriction enzymes at 37°C or 50°C for 3 hours. The linearized plasmid was then purified using Wizard Plus SV Miniprep DNA Purification System (Promega), and saRNA was transcribed in vitro using T7 RiboMAX Express Large-Scale RNA Production System (Promega). After DNase treatment, saRNA was purified with RNeasy midi kit (Qiagen), and then modified with Vaccinia Capping System (New England Biolabs [NEB]) by adding 7-methylguanosine caps using NEB Capping protocol (NEB, M20280). After the capped saRNA was purified using Monarch kit (NEB).

[0099] Western blotting HEK293T cells were transfected with 0.5 of saRNA using lipofectamine (Promega). Cells were collected 18 hours after transfection, lysed in cell lysis buffer (Cell Signaling Technology) and fractionated by SDS-PAGE (Any kD acrylamide gel, Bio-Rad). Proteins were detected by western blot 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 ChemiDocTMXRS+ (Bio-Rad) and Image LabTM Software (Bio-Rad).

[0100] The results for plasmid vectors with constructs 6, 13, 3 and 10 as target genes are shown in Figure 3 In. Figure 2 As shown in , these constructs correspond to F01, F02, F04 and F05, respectively. The results of the Western blot assay indicated that cells transfected with saRNA-influenza variants expressed influenza antigens with the expected molecular weight.

[0101] Flow cytometric analysis To analyze cell surface proteins, transfected HEK293T cells were collected, 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; BioLegend). The levels of surface proteins were assessed using an Attune acoustic focusing flow cytometer (Applied Biosystems). As a control, untransfected HEK293T cells were used. The results are shown in Figure 4 In. Figure 4 Among them, the R3 fraction contains antigen-positive cells and the R5 fraction contains highly antigen-positive cells in the R3 fraction.

[0102] Flow cytometry analysis indicated that influenza antigens were expressed on the surface of cells transfected with saRNA-influenza variants. In previous studies, the proportion of cells in the R5 fraction correlated with immunogenicity against antigens in animal model experiments. Example 4

[0103] 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 harvested 48-96 hours after transfection. The replicon particles were purified using an ion exchange column. HEK293T or Vero cells were infected with dilutions of the purified particle formulation to determine the infection titer. The purified replicon particles were used to produce antigens for diagnosis and vaccination. Example 5 Preparation of mRNA or self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs)

[0104] The plasmid vector containing the DNA sequence encoding construct 1-14 prepared in Example 2 was used as the target gene. The plasmid vector was linearized and used as a template. T7 in vitro transcription was based on the T7 transcription kit (RiboMax TM Express Large Scale RNA production System, Promega, (WI USA)) provided protocol. Linear DNA template is mixed with T7 enzyme and rNTP to synthesize RNA. In order to synthesize RNA containing modified nucleotides, modified NTPs such as 5-methyl-cytidine triphosphate and N1-methyl-pseudouridine triphosphate are added to the in vitro transcription reaction mixture. The purified RNA product is capped using vaccinia capping enzyme to obtain self-amplified RNA.

[0105] The obtained mRNA or saRNA is encapsulated in lipid nanoparticles to obtain mRNA or saRNA particles.

Claims

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

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 of any one of claims 1-4, wherein the ferritin is derived from Helicobacter pylori or Helicobacter pylori-bullfrog 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 of any one of claims 1-6, wherein the transmembrane domain and / or signal sequence is fused to the antigen protein via a linker.

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

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

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

11. The vector of claim 10, comprising a promoter, a 5'UTR, a polynucleotide encoding alphavirus nonstructural proteins nsp1, nsp2, nsp3 and nsp4, an SG promoter, a target gene encoding a polypeptide, a 3'UTR and a polyadenylation tail, wherein the polypeptide comprises an antigen protein fused to a signal sequence and a transmembrane domain.

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

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

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

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

16. The use of claim 15, wherein the medicament is for inducing immune modulation in a subject.

17. The use of claim 15, wherein the medicament is for treating or preventing a condition caused by an infection in a subject.

18. A method of inducing immune modulation in a subject, the method comprising administering to a subject in need thereof an immunologically effective amount of the vaccine composition of any one of claims 12-14.

19. A method for treatment, prevention and / or immunization against an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of the vaccine composition of any one of claims 12 to 14.

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

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