Multi-antigen RNA SARSCOV-2 vaccines and related methods

Abstract

The present technology provides multivalent vaccine compositions and T cell compositions comprising viral antigens and related methods. In some embodiments, the viral antigen is an SARS-CoV-2 antigen. The vaccine composition and the T cell composition may include any one of a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, an ORF7a (7a) peptide, an ORF3a (3a) peptide, an ORF8 (8) peptide, and an Nsp6 peptide.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 387,721, filed December 16, 2022. The entire contents of that provisional application are incorporated herein by reference.

[0003] By referencing the merged sequence list

[0004] This application contains a sequence list conforming to ST.26, which was filed concurrently with the PatentCenter in XML format and is incorporated herein by reference in its entirety. This .xml copy was created on December 16, 2023, and is named 1406308004WQ00.xml, with a size of 0.331MB (331KB) (339,888 bytes). Background Technology

[0005] The SARS-CoV-2 virus causes coronavirus disease (COVID-19). COVID-19 is a highly contagious infection with symptoms ranging from subclinical malaise to severe clinical illness and death. Symptoms of COVID-19 include headache, loss of smell and taste, nasal congestion, cough, muscle pain, fever, diarrhea, and coagulation disorders, disseminated intravascular coagulation, and thromboembolism.

[0006] To date, vaccines, antibodies, and immunotherapies for COVID-19 have primarily focused on the spike protein (S), potentially leading to a relatively narrow response. Naturally occurring and spreading variants of the SARS-CoV-2 S protein exhibit altered antigenicity. These variants arise from adaptations in immune-experienced hosts, particularly during prolonged infection. This also reduces the effectiveness of supplemental antibody-mediated immunizations, such as convalescent serum or therapeutic monoclonal antibodies. Consequently, vaccines targeting only the S protein become less effective over time. Summary of the Invention

[0007] In some embodiments, the present technology includes a vaccine composition comprising a first antigen and a second antigen, each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide, and Nsp6 peptide.

[0008] In some embodiments, the present technology includes a vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen, wherein the first antigen and the second antigen are each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

[0009] In some embodiments, the technology includes a vaccine composition comprising spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide, and Nsp6 peptide.

[0010] In some embodiments, the present technology includes a vaccine composition comprising a nucleotide sequence encoding a spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding an NCAP (N) peptide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding an octapeptide, and a nucleotide sequence encoding an Nsp6 peptide.

[0011] In some implementations, the peptide length is less than 30 amino acids.

[0012] In some implementations, the peptide length is from about 5 amino acids to about 20 amino acids.

[0013] In some implementations, the peptide length is approximately 9 amino acids.

[0014] In some implementations, the peptide length is approximately 14 amino acids.

[0015] In some implementations, the nucleotide sequence is a deoxyribonucleotide (DNA) sequence.

[0016] In some implementations, the nucleotide sequence is a ribonucleotide (RNA) sequence.

[0017] In some implementations, the peptide does not contain an active site.

[0018] In some implementations, the peptide does not fold into a tertiary peptide structure.

[0019] In some implementations, the nucleotide sequence is present in the lipid composition.

[0020] In some embodiments, the lipid composition comprises lipid nanoparticles.

[0021] In some implementations, the lipid nanoparticles contain antigen-presenting cell (APC) targeting molecules.

[0022] In some implementations, APCs are dendritic cells (DCs).

[0023] In some implementations, the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209, or HLA-DR targeting molecules.

[0024] In some implementations, each peptide exists on a single peptide chain.

[0025] In some implementations, a single chain contains one or more connector sequences.

[0026] In some implementations, each peptide exists on a different peptide chain.

[0027] In some implementations, each nucleotide sequence is present in a polycistronic sequence.

[0028] In some implementations, the polycistronic sequence includes one or more connector sequences.

[0029] In some implementations, each nucleotide sequence exists on a different nucleotide chain.

[0030] In some implementations, the first or second antigen includes a coronavirus antigen peptide.

[0031] In some implementations, one or more of the antigens include coronavirus peptides.

[0032] In some implementations, the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

[0033] In some implementations, the SARS-CoV-2 antigen is selected from the group consisting of SARS-CoV-2α (alpha) antigen, SARS-CoV-2β (beta) antigen, SARS-CoV-2γ (gamma) antigen, SARS-CoV-2δ (delta) antigen, SARS-CoV-2ο (omicron) antigen, SARS-CoV-2ε (epsilon) antigen, SARS-CoV-2ζ (zeta) antigen, SARS-CoV-2η (eta) antigen, SARS-CoV-2ι (iota) antigen, SARS-CoV-2κ (kappa) antigen, SARS-CoV-2λ (lambda) antigen, and SARS-CoV-2μ (mu) antigen.

[0034] In some implementations, the vaccine composition contains an adjuvant.

[0035] In some implementations, the vaccine composition is a multivalent vaccine composition.

[0036] In some embodiments, the present technology includes a method for preparing a vaccine composition, the method comprising the steps of: (i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject who has successfully cleared a virus; (ii) exposing the sample to one or more antigens derived from the virus; (iii) quantifying the same T cell population from (i) after exposure to one or more peptide antigens; (v) calculating the difference in the number of T cell populations between (i) and (iii); (vi) comparing the difference in (iv) to a threshold; and (iv) if the difference in (iv) exceeds the threshold in (v), then including in the vaccine composition a peptide of one or more antigens described in (a) or (ii), or (b) a nucleotide sequence encoding one or more antigens described in (ii).

[0037] In some embodiments, the technology includes a method for preparing a T-cell composition that specifically recognizes one or more viral antigens, the method comprising the steps of: (i) quantifying a T-cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject who has successfully cleared a virus; (ii) exposing the first sample to one or more antigens from a virus; (iii) quantifying the same T-cell population from (i) after exposure to one or more peptide antigens; (iv) calculating the difference in the number of T-cell populations between (i) and (iii); (v) comparing the difference in (iv) to a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to a peptide of one or more antigens described in (ii) or a nucleotide sequence encoding one or more antigens described in (ii) in a vaccine composition; (vii) amplifying T-cells in the second sample after exposure in (vi); and (viii) isolating the amplified T-cells.

[0038] In some implementations, the method further includes (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

[0039] In some embodiments, the T-cell composition is formulated for administration to a second subject.

[0040] In some implementations, the T-cell composition is formulated into the vaccine composition.

[0041] In some implementations, the T cell population includes CD4+ T cells or CD8+ T cells.

[0042] In some implementations, the T cell population includes T helper cells.

[0043] In some implementations, T helper cells include Th1 or Th2 cells.

[0044] In some embodiments, the T cell composition includes CD4+ T cells or CD8+ T cells.

[0045] In some embodiments, the T cell composition includes T helper cells.

[0046] In some implementations, T helper cells include Th1 or Th2 cells.

[0047] In some implementations, exposure to one or more antigens in (ii) includes antigen-presenting cells (APCs).

[0048] In some implementations, APCs are dendritic cells (DCs).

[0049] In some implementations, dendritic cells (DCs) are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0050] In some embodiments, the technology includes a T-cell composition immunogenic to a viral antigen, which is generated by the following steps: (i) quantifying a T-cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject who has successfully cleared the virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T-cell population from (i) after exposure to one or more peptide antigens; (iv) calculating the difference in the number of T-cell populations between (i) and (iii); (v) comparing the difference in (iv) to a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to a peptide of one or more antigens described in (ii) or a nucleotide sequence encoding one or more antigens described in (ii) in the vaccine composition; (vii) amplifying T-cells in the second sample after exposure in (vi); and (viii) isolating the amplified T-cells.

[0051] In some embodiments, the T-cell composition further includes (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

[0052] In some embodiments, the T-cell composition is formulated for administration to a second subject.

[0053] In some implementations, the T-cell composition is formulated into the vaccine composition.

[0054] In some implementations, the T cell population includes CD4+ T cells or CD8+ T cells.

[0055] In some implementations, the T cell population includes T helper cells.

[0056] In some implementations, T helper cells include Th1 or Th2 cells.

[0057] In some embodiments, the T cell composition includes CD4+ T cells or CD8+ T cells.

[0058] In some embodiments, the T cell composition includes T helper cells.

[0059] In some implementations, T helper cells include Th1 or Th2 cells. Attached Figure Description

[0060] Figures 1A-1C This diagram illustrates the antigen recognition patterns of viral peptides in T cells from subjects who successfully cleared SARS-CoV-2, relative to T cells from uninfected subjects. The figure shows CD4+ T cells (…). Figure 1A and Figure 1C (right)) and CD8+ T cells ( Figure 1B and Figure 1C (Left) Results of activation-inducing marker (AIM).

[0061] Figure 2A and Figure 2B The images show antigen-specific CD4+ T cells (X-axis) after exposure to different viral antigens (samples from subjects who had successfully cleared SARS-CoV-2, subjects who had not previously been exposed to SARS-CoV-2 (i.e., subjects who had not been infected with SARS-CoV-2), control samples induced by dendritic cell (DC) antigen presentation, and peripheral blood mononuclear cell (PBMC) controls. Figure 2A ) and CD8+ T cells ( Figure 2B ) changes.

[0062] Figures 3A-3I The changes in T cell responses, measured by the production of interleukin-4 (IL4) and interferon-γ (IFNγ) in Th1 cells (left) on day 14 and Th2 cells (right) on day 21, are shown. Samples were incubated with different culture medium mixtures (culture medium mixtures 1, 2, and 3) supplemented with different components (A represents spike (S) protein supplement, B represents a mixture of spike (S), VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8) supplements, C represents a mixture of VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8) supplements, and DMSO represents culture medium supplemented with DMSO as a negative control). Each sample was exposed to a culture medium control ( Figure 3A), the spike peptide of the common cold coronavirus ( Figure 3B ), the first SARS-CoV-2 spike (S) peptide Figure 3C ), the second SARS-CoV-2 S peptide ( Figure 3D ), SARS-CoV-2 VME1(M) peptide ( Figure 3E ), SARS-CoV-2 NCAP(N) peptide ( Figure 3F ), SARS-CoV-23a peptide ( Figure 3G ), SARS-CoV-27a peptide ( Figure 3H ) and SARS-CoV-28 peptide ( Figure 3I ).

[0063] Figure 4 The left bar shows CD3 levels in cells stimulated with different peptide antigens using DC cells. + CD62L + CD197 + T cell memory is measured as a percentage of the T cell population, relative to the percentage of cells stimulated with the same peptide antigen in the absence of dendritic cells (right bar).

[0064] Figure 5 The diagram shows the HLA types that bind to the SARS-CoV-2 α, δ, and o (omicron) peptides, and the locations where changes occur within the intact protein sequences. Dark shading bands represent peptide binding with strong affinity (<60 nM). Light shading bands represent peptides with moderate affinity, ranging from >60 nM to 185 nM. These sequences are conserved if found in SARS-CoV-2 variants α, δ, and o. White bands indicate changes in HLA type binding to peptides or complete loss of binding between the peptide and HLA type. White bands represent sequences in which major mutations of viral variants are found.

[0065] Figure 6 The RNA cassette used to generate mRNA polycistronic constructs is shown. 1: MHC1 or 2 class signal peptide; 2: antigen of 30 or fewer amino acids; 3: adapter sequence.

[0066] Figure 7 Lipid nanoparticles for delivering green fluorescent protein (GFP) RNA to DCs are shown.

[0067] Figure 8 Lipid nanoparticles for delivering SARS-CoV-2 RNA to DCs are shown.

[0068] Figure 9The levels of GFP in DCs transfected with nanoparticles containing GFP RNA and mannose, CD180, CD209, or HLA-DR targeting ligands are shown relative to cells formulated with LNP alone. Invention Details

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this art pertains. For the purposes of this art, the following terms are defined as follows.

[0071] This article uses the articles “one” and “a” to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one element or more elements.

[0072] The term "about" means a quantity, level, value, number, frequency, percentage, size, quantity, weight, or length that varies at a level acceptable in the art. In some embodiments, such variation can be as high as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of said value.

[0073] The terms “bind,” “binding,” “complex,” and “complexing” refer to all types of physical and chemical binding, reaction, complexation, attraction, chelation, etc.

[0074] The term "conserved epitope" refers to a conserved protein that has an epitope that is conserved in multiple strains of the virus and has been identified by the methods of this technique.

[0075] The term "pathogen" refers to bacteria, viruses, or other microorganisms that can cause disease. Other microorganisms may include fungi, molds, parasites, and prions.

[0076] The “peptide” of this technology may be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of a larger molecule, (e) produced by a combination of the methods (a) to (d) above, or (f) produced by any other method of producing peptides.

[0077] As used herein, the term "peptide" includes any structure consisting of two or more amino acids, including chemical modifications and derivatives of amino acids. All or part of the amino acids forming a peptide can be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post-translational modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, etc. Therefore, the term "peptide" includes pseudopeptides and peptide mimics, including structures with a non-peptide backbone. The term "peptide" also includes dimers or polymers of peptides. "Manufactured" peptides include peptides produced by chemical synthesis, recombinant DNA technology, biochemical or enzymatic fragmentation of larger molecules, combinations of the foregoing methods, or generally by any other method. The term "peptide" includes peptides containing a variable number of amino acid residues, optionally having non-amino acid residue groups at the N-terminus and C-terminus, including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, etc.

[0078] By employing the useful production method of chemical synthesis, various amino acids not naturally present in the chain can be introduced, and the N-terminus or C-terminus can be modified, thereby providing improved stability and formulation performance, resistance to protease degradation, etc.

[0079] An "amino acid" is a molecule containing an amino group, a carboxylic acid group, and a side chain unique to each amino acid. The main elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, and the general formula is H₂N—CHR—COOH, where R represents a side chain group. Different α-amino acids differ in the side chain portion attached to the α-carbon. In this technique, "amino acid" includes known naturally occurring protein amino acids, which are represented by common three-letter abbreviations and single-letter abbreviations. See *Synthetic Peptides: A User's Guide*, edited by GA Grant, WH Freeman & Co., New York (1992), whose teachings are incorporated herein by reference, including the text and tables shown on pages 11–24. As noted above, the term "amino acid" also includes naturally occurring protein amino acids, non-protein amino acids, post-translational modified amino acids, enzyme-synthesized amino acids, derived amino acids, stereoisomers and modifications designed to mimic the structure of amino acids, etc. Modified and uncommon amino acids are generally described in Synthetic Peptides: A User's Guide (see above); Hruby et al., Journal of Biochemistry 268: 249-262 (1990); and Toniolo, International Journal of Peptide and Protein Research 35: 287-300 (1990); the teachings of all these sources are incorporated herein by reference.

[0080] In the peptides of this technology, conventional amino acid residues have their conventional meanings, as given in Chapter 2400 of the 8th edition of the Manual of Patent Examining Procedure. Therefore, "Ala" is alanine; "Arg" is arginine; "Asn" is asparagine; "Asp" is aspartic acid; "Cys" is cysteine; "Gin" is glutamine; "Glu" is glutamic acid; "His" is histidine; "Ile" is isoleucine; "Leu" is leucine; "Lys" is lysine; "Met" is methionine; "Phe" is phenylalanine; "Pro" is proline; "Ser" is serine; "Thr" is threonine; "Trp" is tryptophan; "Tyr" is tyrosine; and "Val" is valine. Unless otherwise stated, all amino acid abbreviations represent isomers, i.e., L-isomers, D-isomers, or combinations thereof may be used. Therefore, for example, "L-Phe" or "IPhe" is L-phenylalanine; "D-Phe" or "dPhe" is D-phenylalanine; dVal is D-valine; dPro is D-proline; "D- / L-Phe" or "d / IPhe" is D-phenylalanine, L-phenylalanine, or a combination thereof; "Phe" is also D-phenylalanine, L-phenylalanine, or a combination thereof, and so on. Non-standard amino acids include: "Nle" for ortholeucine; "Nal" for naphthalenealanine; "D-Nal" for D-naphthalenealanine; D-Nal(2') or DNal(2') for D-2'-naphthalenealanine; L-Nal(2') or LNal(2') for L-2'-naphthalenealanine; L-Nal(1') for L-1'-naphthalenealanine; D-Nal(1') or DNal(T) for D-1'-naphthalenealanine; Tle for tertiary leucine; Nva for orthovaline; Orn for ornithine; Bip for biphenyl amino acid; and so on.

[0081] Amino acids, including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post-translational modified amino acids, enzyme-synthesized amino acids, derived amino acids, constructs or structures designed to mimic amino acids (peptide mimics), etc., are all of the above and are sometimes referred to as "residues" in this document.

[0082] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, their polymers, and their complements. The term “polynucleotide” refers to a linear sequence of nucleotides. The term “nucleotide” generally refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of polynucleotides described herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Nucleic acid as used herein also refers to nucleic acids having the same basic chemical structure as naturally occurring nucleic acids. Such analogs have modified sugars and / or modified ring substituents but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid mimics are compounds having a structure different from the general chemical structure of nucleic acids but functioning in a manner similar to naturally occurring nucleic acids. Examples of such mimics include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0083] The "sequence identity percentage" is determined by comparing two best-aligned sequences within a comparison window. This comparison window may contain additions or deletions (i.e., gaps) in the polynucleotide or polypeptide sequence portion compared to the reference sequence (which does not contain additions or deletions) to achieve optimal alignment. The percentage is calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in both sequences, obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and then multiply the result by 100 to obtain the sequence identity percentage.

[0084] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or percentage "identity" refers to the fact that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned within a comparison window or specified region to obtain maximum correspondence using one of the following sequence comparison algorithms or by manual alignment and visual inspection (i.e., 60% identity within a specific region, for example, the entire polypeptide sequence of the present invention or a single domain of the polypeptide of the present invention, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity). Such sequences are referred to as "substantially identical." This definition also refers to the complementary sequence of the test sequence. Optionally, identity is present in a region of at least about 50 nucleotides in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides in length.

[0085] The term "administering" or "administer" includes delivering a therapy of this technology (e.g., a vaccine composition) to a subject by local or systemic administration. The route of administration may be parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration.

[0086] As used herein, “composition,” “vaccine composition,” or “pharmaceutical composition” refers to a mixture of an active ingredient with other chemical components (e.g., pharmaceutically acceptable carriers and / or excipients).

[0087] As used herein, a “pharmaceutically acceptable carrier” for the first or second pharmaceutical composition means a carrier or diluent that does not cause significant irritation to a living organism, does not eliminate the bioactivity and properties of the applied active ingredient, and / or does not interact harmfully with other components of the composition comprising it. The term “carrier” encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations. The choice of carrier for a composition will depend on the intended route of administration of the composition. Pharmaceutically acceptable carriers comprising these materials and the preparation of formulations are described, for example, in Remington Pharmaceutical Sciences, 21st edition, edited by the University of the Science of Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which is incorporated herein by reference in its entirety. Some examples of physiologically acceptable carriers include antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as... (ICI, Inc.; Bridgewater, NJ), polyethylene glycol (PEG) and PLURONICS TM (BASF; Florham Park, NJ). An “excipient” in a first or second pharmaceutical composition refers to an inert substance added to the composition to further facilitate administration of the compound. Examples of excipients include (but are not limited to) calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0088] vaccine composition

[0089] This technology includes vaccine compositions that provide viral antigens and promote immune memory. The vaccine composition elicits an immune response, thereby triggering the recognition, response, and / or elimination of the viral antigen. In some embodiments, the vaccine composition of this technology can induce an immune response in a subject to a viral antigen. In some embodiments, the vaccine composition of this technology can be administered to a subject to treat, prevent, and / or alleviate infection-related diseases and / or other conditions.

[0090] The viral antigens of this technology may include protein and / or peptide antigens, glycoprotein antigens, or lipid antigens. In some embodiments, the vaccine composition comprises one or more nucleotide sequences encoding a viral antigen.

[0091] The viral antigen of this technology can be an arteritis virus, a medium-sized arteritis virus, a circovirus, a porcine circovirus, or a coronavirus antigen. In some embodiments, the viral antigen is selected from the group consisting of cytomegalovirus antigen, Epstein-Barr virus antigen, hepatitis B virus antigen, human papillomavirus antigen, adenovirus antigen, herpesvirus antigen, human immunodeficiency virus antigen, influenza virus antigen, human respiratory syncytial virus antigen, vaccinia virus antigen, varicella-zoster virus antigen, yellow fever virus antigen, Ebola virus antigen, coronavirus antigen, eastern equine encephalitis virus antigen, human polyomavirus antigen (BKV), SV40, and Zika virus antigen. In some embodiments, the viral antigen is an antigen derived from a virus that includes a Target Variant (VOI), Variant of Concern (VOC), or High Consequence Variant (VOHC) as defined by the U.S. Centers for Disease Control and Prevention and the World Health Organization (WHO).

[0092] In some implementations, the antigen may be a viral antigen that is associated with and / or capable of generating a pandemic-like infection rate.

[0093] In some implementations, the coronavirus antigen is selected from the group consisting of alpha coronaviruses (e.g., human coronavirus 229E (HCoV-229E) or human coronavirus NL63 (HCoV-NL63)), beta coronaviruses (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or bat coronaviruses), gamma coronaviruses (e.g., swine coronavirus HKU15 or avian coronavirus infectious bronchitis virus (IBV)), or delta coronaviruses (e.g., spotted green pigeon coronavirus HKU13 or white-eye coronavirus HKU16).

[0094] In some implementations, the coronavirus antigen is a common cold coronavirus antigen. Non-limiting examples of common cold coronaviruses include human coronavirus 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKLH).

[0095] In some implementations, the coronavirus antigen is the SARS-CoV-2 antigen, which is an antigen selected from the group consisting of SARS-CoV-2α antigen, SARS-CoV-2β antigen, SARS-CoV-2γ antigen, SARS-CoV-2δ antigen, SARS-CoV-2ο antigen, SARS-CoV-2ε antigen, SARS-CoV-2ζ antigen, SARS-CoV-2η antigen, SARS-CoV-2ι antigen, SARS-CoV-2κ antigen, SARS-CoV-2λ antigen, and SARS-CoV-2μ antigen.

[0096] Nucleotides

[0097] This technology includes vaccine compositions having nucleotide sequences encoding viral antigens. Any nucleotide sequence can promote an immune cell response, immune reaction, or immune memory against the viral antigen it encodes (e.g., generating a CD4+ T cell memory response, a CD8+ T cell memory response, central memory T cells (TCM), effector memory T cells (TEM), memory T cell cross-reactivity, or maintenance of memory T cells). In some embodiments, each nucleotide sequence promotes an immune response or immune memory against one or more viral antigens corresponding to that nucleotide sequence. The nucleotide sequence may include a deoxyribonucleic acid (DNA) sequence. In some embodiments, one or more nucleotide sequences include a ribonucleic acid (RNA) sequence. In some embodiments, the RNA sequence is a messenger RNA (mRNA) sequence. In some embodiments, the RNA sequence is a circular (circRNA) sequence. Circular RNA may have an increased half-life and / or increased expression compared to non-circular RNA. In some embodiments, the RNA sequence is a messenger CRISPR RNA (crRNA) sequence. In some embodiments, the RNA sequence is self-replicating RNA.

[0098] In some embodiments, the vaccine composition comprises one or more nucleotide sequences. In some embodiments, the vaccine composition comprises two or more nucleotide sequences. In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a spike (S) protein or a peptide thereof (i.e., an S-peptide). In some embodiments, the nucleotide sequence encodes an S-peptide antigen, which is an S subunit 1 (S1) peptide or an S subunit 2 (S2) peptide.

[0099] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a membrane protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes the VME1(M) protein or a peptide thereof (i.e., the M peptide).

[0100] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a nucleocapsid protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes an NCAP(N) protein or a peptide thereof (i.e., an N-peptide).

[0101] In some embodiments, the vaccine composition comprises a nucleotide sequence corresponding to an open reading frame (ORF) or a portion thereof. In some embodiments, the vaccine composition comprises a nucleotide sequence encoding an ORF peptide. In some embodiments, the ORF peptide is an ORF7a (7a) peptide, an ORF3a (3a) peptide, or an ORF8 (8) peptide.

[0102] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a non-structural protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes an Nsp6 peptide.

[0103] In some embodiments, the vaccine composition comprises a first nucleotide sequence encoding a first antigen and a second nucleotide sequence encoding a second antigen, wherein the first antigen and the second antigen are each independently selected from the group consisting of S peptide, M peptide, N peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

[0104] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding an S peptide, a nucleotide sequence encoding an M peptide, a nucleotide sequence encoding an N peptide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding an octapeptide, and a nucleotide sequence encoding an Nsp6 peptide.

[0105] In some embodiments, the vaccine composition comprises the nucleotide sequences listed in Table 1.

[0106] Table 1: Nucleotide Sequences

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] In some embodiments, the vaccine composition comprises a nucleotide sequence, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67.

[0135] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of different nucleotide sequences.

[0136] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with each of one or more of SEQ ID NO:68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with each of one or more of SEQ ID NO:68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with each of one or more of SEQ ID NO:68-78.

[0137] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:68-78. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:68-78. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:68-78. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0138] In some embodiments, the vaccine composition comprises a nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100.

[0139] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0140] In some embodiments, the vaccine composition comprises a nucleotide sequence, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113.

[0141] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0142] In some embodiments, the vaccine composition comprises nucleotide sequences, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119. In some embodiments, the vaccine composition comprises nucleotide sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119.

[0143] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0144] In some embodiments, the vaccine composition comprises a nucleotide sequence, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125.

[0145] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0146] In some embodiments, the vaccine composition comprises a nucleotide sequence, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139.

[0147] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139. In some implementations, the first nucleotide sequence and the second nucleotide sequence may contain all or part of different nucleotide sequences or may be composed of all or part of different nucleotide sequences.

[0148] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:140. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:140. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:140.

[0149] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:141. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:141. In some embodiments, the vaccine composition comprises a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:141.

[0150] In some implementations, each nucleotide sequence in the vaccine composition is present on a different nucleotide chain.

[0151] In some embodiments, each nucleotide sequence in the vaccine composition is present as a polycistronic sequence. In some embodiments, the polycistronic sequence includes a nucleotide linker sequence. The nucleotide linker sequence can be used to link or separate different nucleotide sequences encoding antigens, generate spacers, enhance the packaging of nucleotide sequences into lipid compositions, or facilitate molecular manipulation. In some embodiments, the polycistronic sequence includes two or more nucleotide linker sequences.

[0152] In some embodiments, the polycistronic nucleotide sequence includes a modified nucleotide (e.g., a 5' cap). In some embodiments, the polycistronic nucleotide sequence includes a polyA tail or an untranslated region (UTR) (e.g., a 5' UTR or a 3' UTR).

[0153] The polycistronic nucleotide sequence may also include a nucleotide sequence that facilitates nucleotide sequence localization or a nucleotide sequence encoding a peptide that facilitates peptide localization (e.g., a signal peptide). In some embodiments, the nucleotide sequence facilitates localization to cells containing human leukocyte antigen molecules (HLA). In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. In some embodiments, the nucleotide sequence facilitates localization to cells containing major histocompatibility complex (MHC) molecules. In some embodiments, the MHC molecule is an MHC class I molecule or an MHC class II molecule. The localization patterns of nucleotides and peptides of this technology can be evaluated using predictive localization software, including but not limited to TargetP, WoLF PSORT, DeepLoc, CELLO, YLoc, BaCelLo, and LocTree3.

[0154] The polycistronic sequence may contain a non-immunogenic or low-immunogenic nucleotide linker sequence (e.g., a polyG linker sequence). The nucleotide linker sequence can disrupt the active site in a viral peptide encoded by the nucleotide sequence of this technology. In some embodiments, the nucleotide linker sequence can prevent the formation of the tertiary structure of the viral antigen and / or can inhibit the function of the viral antigen. In some embodiments, the polycistronic sequence contains a nucleotide linker sequence. The nucleotide linker sequence may be encoded by an RNA sequence. The RNA sequence may be present in the vaccine composition. In some embodiments, the polycistronic sequence contains a nucleotide linker sequence, which is a sequence listed in Table 2. In some embodiments, the nucleotide linker sequence has about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:142. In some embodiments, the nucleotide adapter sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:142. In some embodiments, the nucleotide adapter sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:142.

[0155] In some implementations, the vaccine composition does not contain nucleotide adapter sequences.

[0156] Table 2: Connector Sequence

[0157]

[0158] peptides:

[0159] This technology includes vaccine compositions having one or more viral antigens, said viral antigens being protein or peptide antigens. In some embodiments, the vaccine composition comprises two different peptide antigens. Either protein or peptide antigen may promote an immune response or immune memory. In some embodiments, each protein or peptide antigen promotes an immune response or immune memory.

[0160] In some embodiments, the viral antigen includes the S protein or a peptide thereof (i.e., the S peptide). In some embodiments, the viral antigen includes the S peptide antigen, which is either the S1 peptide or the S2 peptide.

[0161] In some embodiments, the viral antigen includes a membrane protein or a peptide thereof. In some embodiments, the viral antigen includes an M protein or a peptide thereof (i.e., the M peptide).

[0162] In some embodiments, the viral antigen includes a nucleocapsid protein or a peptide thereof. In some embodiments, the viral antigen includes an N protein or a peptide thereof (i.e., an N-peptide).

[0163] In some embodiments, the viral antigen corresponds to the ORF peptide. In some embodiments, the ORF peptide corresponds to the 7a peptide, 3a peptide, or octapeptide.

[0164] In some implementations, the viral antigen comprises a non-structural protein or a peptide thereof (i.e., the Nsp6 peptide).

[0165] In some embodiments, the vaccine composition comprises a first antigen and a second antigen, each independently selected from the group consisting of S, M, N, 3a, 7a, 8, and Nsp6.

[0166] In some embodiments, the vaccine composition comprises S-peptide, M-peptide, N-peptide, 3a-peptide, 7a-peptide, 8-peptide, and Nsp6-peptide.

[0167] In some embodiments, the vaccine composition of this technology comprises a first antigen and a second antigen, each present on a different peptide chain. In some embodiments, the vaccine composition comprises a first antigen and a second antigen, each present on a single peptide chain. In some embodiments, the single peptide chain is not permitted to ternary fold or generate a functional viral domain.

[0168] In some embodiments, the first and second antigens are each independently selected from the group consisting of S-peptide, M-peptide, N-peptide, 3a-peptide, 7a-peptide, 8-peptide, and Nsp6-peptide. In some embodiments, the vaccine composition comprises peptides listed in Table 3.

[0169] Table 3: Peptide Sequences

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209.

[0180] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209. In some implementations, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0181] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220.

[0182] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220. In some implementations, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0183] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242.

[0184] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242. In some implementations, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0185] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255.

[0186] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255. In some implementations, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0187] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence, each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence, each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence, each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261.

[0188] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261. In some embodiments, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0189] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267.

[0190] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267. In the implementation scheme, the first peptide and the second peptide include different amino acid sequences or are composed of different amino acid sequences.

[0191] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281.

[0192] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, each having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281. In some embodiments, the first peptide and the second peptide contain different amino acid sequences or are composed of different amino acid sequences.

[0193] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:282. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:282. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:282.

[0194] In some embodiments, the vaccine composition comprises peptides having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of SEQ ID NO:283. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of SEQ ID NO:283. In some embodiments, the vaccine composition comprises peptides having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of SEQ ID NO:283.

[0195] In some embodiments, the vaccine composition of this technology further comprises a signal peptide. Non-limiting examples of signal peptides include peptides that promote localization to cellular compartments (e.g., endoplasmic reticulum, mitochondria, nucleus, or peroxisomes), signal peptides that promote peptide secretion, and signal peptides that promote cleavage.

[0196] In some embodiments, the signal peptide promotes localization and / or binding to cells containing MHC class I molecules. In some embodiments, the signal peptide promotes localization and / or binding to cells containing MHC class II molecules. In some embodiments, the cells containing MHC class II molecules are APC cells. In some embodiments, APC cells are DCs. In some embodiments, the signal peptide promotes localization and / or binding to cells containing HLA. In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. Peptide binding of the peptides of this technology can be assessed using predictive binding software, including but not limited to NetMHC, NetMHCpan, NetMHCHpan, the Immunoeptope Database and Analysis Resource (IEDB), SYFPEITHI, the Stability Matrix Method (SMM), ProPred-1, ProPred, and NetCTLpan.

[0197] Not limited by any particular theory, vaccines of this technology may contain approximately 8 to 10 peptides of approximately 25 to 30 amino acids each to induce a class I HLA-restricted CD8 response, and approximately 8 to 10 peptides of approximately 15 amino acids each to induce a class II HLA-restricted CD4 response. It is anticipated that peptides of approximately 25 to 30 amino acids in length will not mimic viral active sites or recombine with wild-type viruses. In some embodiments, linkers located between peptides may further prevent the formation of viral active sites and / or randomization of the sequence encoding the peptides may prevent the generation of viral active sites. This approach differs from vaccines that induce humoral immune responses and / or encode the complete spike protein, including RBDs that bind to the ACE receptor and stabilize the tertiary structure with proline.

[0198] In some embodiments, the vaccine composition of this technology further comprises a peptide having a peptide linker sequence. The peptide linker sequence can be used to link or separate different antigens, enhance peptide packaging into various compositions, or facilitate molecular manipulation. The peptide linker sequence can be non-immunogenic or have low immunogenicity.

[0199] In some embodiments, the peptide linker sequence includes a specific cleavage site linker (e.g., a furan cleavage site; a 2A linker peptide). The specific cleavage site linker may include a protease recognition site that is selectively cleaved by certain proteases.

[0200] In some implementations, the peptide linker sequence contains a targeting or tagging linker that allows the peptide to be located, detected, or purified.

[0201] Peptide linker sequences can include flexible linkers (e.g., glycine- and serine-rich linkers) or rigid linkers (e.g., glycine-rich linkers or α-helical linkers).

[0202] Positioning peptide linker sequences between peptides having fewer than 50 amino acids (e.g., about 20, about 25, about 30, or about 35 amino acids) can disrupt active sites in viral antigens. In some embodiments, the peptide linker sequences can prevent the formation of tertiary structures of viral antigens and / or can prevent the function of viral antigens. In some embodiments, the linked peptides are derived from different proteins. In other embodiments, the linked peptides are derived from the same protein and are discontinuous. Not to be limiting, the linked peptides of this technology can also prevent the formation of tertiary structures of viral antigens and / or can also prevent the function of viral antigens.

[0203] In some embodiments, the vaccine composition comprises a peptide linker encoded by a nucleotide sequence (e.g., a nucleotide sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ.ID NO 142). In some embodiments, the nucleotide sequence is an RNA sequence.

[0204] In some embodiments, the peptide linker sequence comprises the amino acid sequences listed in Table 2. In some embodiments, the peptide linker sequence comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:349. In some embodiments, the peptide linker sequence comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:349. The peptide linker sequence comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:349.

[0205] In some implementations, the vaccine composition does not contain peptide linker sequences.

[0206] In some embodiments, the vaccine composition comprises peptides of about 5 to about 50 amino acids, about 50 to about 100 amino acids, about 100 to about 200 amino acids, about 200 to about 500 amino acids, about 500 to about 1000 amino acids, about 1000 to about 2500 amino acids, about 2500 to about 5000 amino acids, about 5000 to about 10000 amino acids, about 10000 to about 10000 amino acids, or more amino acids.

[0207] In some implementations, the vaccine composition contains a peptide of more than about 30 amino acids.

[0208] In some embodiments, the vaccine composition comprises a peptide of fewer than about 30 amino acids. In some embodiments, each peptide in the vaccine composition comprises fewer than about 30 amino acids. In some embodiments, the vaccine composition comprises a peptide of about 25 to about 30 amino acids. In some embodiments, the vaccine composition comprises a peptide of fewer than about 25 amino acids. In some embodiments, each peptide in the vaccine composition comprises fewer than about 25 amino acids. In some embodiments, the vaccine composition comprises a peptide of fewer than about 20 amino acids. In some embodiments, each peptide in the vaccine composition comprises fewer than about 20 amino acids. In some embodiments, the vaccine composition comprises a peptide of fewer than about 15 amino acids. In some embodiments, each peptide in the vaccine composition comprises fewer than about 15 amino acids. In some embodiments, the vaccine composition comprises a peptide of fewer than about 10 amino acids. In some embodiments, each peptide in the vaccine composition comprises fewer than about 10 amino acids.

[0209] In some embodiments, the vaccine composition comprises one or more peptides of the present invention and one or more nucleotide sequences of the present invention (e.g., nucleotide sequences encoding viral peptides). In some embodiments, the vaccine composition comprises two or more peptides of the present invention and one or more nucleotide sequences of the present invention. In some embodiments, the vaccine composition comprises two or more nucleotide sequences of the present invention and one or more peptides of the present invention. In some embodiments, the vaccine composition comprises two or more nucleotide sequences of the present invention and two or more peptides of the present invention.

[0210] The vaccine of this technology may lack tertiary structure, and upon delivery to a subject, it may generate T-cell epitopes without fully or partially forming active viral enzymes and / or active viral function. In some embodiments, TH2 helper cells formed in the subject after vaccine delivery may induce or otherwise promote B-cell antibody responses, in addition to inducing cytotoxic T-cell responses.

[0211] lipid compositions

[0212] In some embodiments, the nucleotide sequence (e.g., a nucleotide sequence encoding a viral antigen) and / or peptide of this technology are present in the lipid composition, such as lipid nanoparticles. The lipid composition may comprise proteolipids (e.g., protamine), carrier proteins, and / or small molecules.

[0213] Lipid compositions may contain a single lipid group or multiple lipid groups. Non-limiting examples of lipid groups include cationic lipids, anionic lipids, neutral lipids, PEGylated lipids, ionizable lipids, accessory lipids, stealthlipids, or cholesterol.

[0214] Non-limiting examples of lipids include DOSPA 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate, DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTAP 1,2-dioleoyl-3-trimethylammonium propane, and DC-cholesterol 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol.

[0215] Non-limiting examples of ionizable lipids include SM-1029-heptadecyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, ALC-03154-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptadecyl-6,9,2823icol23htraen-19-yl4-(dimethylamino)butanoate, and DODMA 1,2-dioleyloxy-3-dimethylaminopropane.

[0216] Non-limiting examples of accessory lipids include cholesterol (1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylhept-2-yl]-2,3,3a,3b,4,6,7,8,9,9a,9b,10,11,11a-tetradecano-1H-cyclopentadien-23-isopropyl-23-en-7-ol, DSPC 1,2-distearate-sn-glycero-3-phosphocholine, and DOPE 1,2-dimyristoyl-sn-glycerophosphate ethanolamine.

[0217] Non-limiting examples of occult lipids include PEGIG(R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)carbamate and ALC-01592-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide.

[0218] In some implementations, the nanoparticles may be polymer-based, metal-based (e.g., silver, gold, palladium, titanium, zinc, or copper), silica-based, or lipid-based.

[0219] In some embodiments, the nanoparticles are multilayer nanoparticles. Non-limiting examples of multilayer nanoparticles include (e.g., nanoparticles having two or more polymer-based layers; nanoparticles having two or more metal-based layers; nanoparticles having two or more silica-based layers; nanoparticles having two or more lipid-based layers; or nanoparticles having a first layer selected from the group consisting of polymer-based layers, metal-based layers, silica-based layers, and lipid-based layers, and a second layer selected from the group consisting of polymer-based layers, metal-based layers, silica-based layers, and lipid-based layers, wherein the composition of the second layer differs from the composition of the first layer).

[0220] In some embodiments, lipid nanoparticles are incorporated into antigen-presenting cell (APC) portions, such as DC-binding portions. Non-limiting examples of APC-binding portions include antibodies and / or single-stranded variable fragments (scFvs) against surface molecules expressed on dendritic cells (e.g., CD1a, CD1c (BDCA1), CD11b (ITGAM), CD11c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC-205), CD206 (MRC1), CD209 (DC-SIGN), FceR1, HLA-DR, TLR2 (e.g., glycolipids and phospholipids), TLR3 (double-stranded RNA), TLR4 (lipopolysaccharide), TLR7 / 8 (single-stranded RNA), and TLR9 (unmethylated CpG DNA)). In some embodiments, the APC-binding moiety is conjugated to lipids in the lipid composition. The APC-binding moiety can be identified or designed using methods including, but not limited to, critical process parameters (CPP), high-throughput sequencing, or predictive models.

[0221] The APC moiety can be a ligand that binds to surface markers (e.g., peptides, glycoproteins, carbohydrates) of APC cells (e.g., DCs, B cells, T cells, macrophages). In some embodiments, the surface markers are molecules upregulated during APC cell maturation.

[0222] In some embodiments, the lipid nanoparticles comprise a conditioning element. This conditioning element can bind to (e.g., bind to or interact with) APCs. The conditioning element can induce phagocytosis by macrophages, neutrophils, and dendritic cells. In some embodiments, the conditioning element comprises mannose or other carbohydrates.

[0223] In some embodiments, the lipid nanoparticles contain an Fc-binding fragment. The Fc-binding fragment binds to the Fc receptor on the APC.

[0224] In some implementations, the lipid nanoparticles are doped with complement factors C3b, C4, and / or C1q.

[0225] Cell composition

[0226] This technology includes cellular compositions that are immunogenic to antigens and promote immune memory. Cellular compositions can be produced such that cells are immunogenic to a specific antigen (e.g., a viral antigen). Furthermore, the cellular compositions can stimulate an immune response, thereby generating an immune response. Non-limiting examples of an immune response include antibody production or activation of lymphocytes (e.g., T cells). In some embodiments, the cellular compositions of this technology induce an immune response against one or more antigens in a subject. In some embodiments, the cellular compositions of this technology can be administered to a subject to treat, prevent, and / or alleviate infection-related diseases and / or other conditions.

[0227] The cell composition may be immunogenic to the proteins and / or peptides of this technology. In some embodiments, the cell composition is immunogenic to the nucleotide sequences of this technology (e.g., nucleotide sequences encoding peptides of this technology). In some embodiments, the cell composition is immunogenic to one or more peptides of this technology and one or more nucleotide sequences of this technology (e.g., nucleotide sequences encoding viral peptides). In some embodiments, the cell composition is immunogenic to two or more peptides of this technology and one or more nucleotide sequences of this technology. In some embodiments, the cell composition is immunogenic to two or more nucleotide sequences of this technology and one or more peptides of this technology. In some embodiments, the cell composition is immunogenic to two or more nucleotide sequences of this technology and two or more peptides of this technology.

[0228] The cell composition of this technology can be generated from subject samples (e.g., biological samples). Subject samples can be circulating fluid samples (e.g., peripheral blood mononuclear cell (PBMC) samples), lymphoid tissue samples (e.g., lymph node, spleen, or tonsil tissue), mucosal samples (e.g., mucosal tissue), bone marrow samples, cerebrospinal fluid (CSF) samples, or synovial fluid samples.

[0229] In some embodiments, the cell composition is a lymphocyte composition. The cell composition may comprise one or more of T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages, or granulocytes.

[0230] In some embodiments, the cell composition is a T cell composition. T cells may include helper T cells (Th cells) (e.g., CD4+ cells), cytotoxic T cells (e.g., CD8+ cells), regulatory T cells (Treg), memory T cells, and follicular helper T cells (Tfh cells). Non-limiting examples of Th cells include Th1 cells, Th2 cells, and Th17 cells.

[0231] In some embodiments, the cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.

[0232] In some embodiments, Th1(CD4+) T cells produce cytokines, including IFNγ, and generate a CD8+ T cell (e.g., cytotoxic T cell) response. In some embodiments, the Th2(CD4+) T system produces cytokines, including IL4, and generates a high-affinity antibody immune response, including class switching to IgG and IgA.

[0233] In some embodiments, the cellular composition is responsive to one or more cytokines and / or chemokines (e.g., stimulating the activation, differentiation, signal transduction, and / or function of immune cells in the presence of cytokines or chemokines). Non-limiting examples of cytokines and chemokines include interleukins (ILs) (e.g., IL-2, IL-4, IL-6, IL-8, IL-10, or IL-12), tumor necrosis factor (TNF) (e.g., TNFα or TNFβ), interferons (INFs) (e.g., INFγ, INFα, or INFβ), chemokines (e.g., CXCL8, CXCL10, or CCL2), colony-stimulating factors (CSFs) (e.g., granulocyte-macrophage CSF or granulocyte CSF), or transforming growth factor-β (e.g., TGFβ).

[0234] In some implementations, the T-cell composition is present in the vaccine composition or infusion.

[0235] Other composition characteristics and formulations:

[0236] In some embodiments, the compositions of this technology (e.g., vaccine compositions or cell compositions) comprise pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers and / or excipients may include aqueous or non-aqueous carriers that can promote the solubility of components of the composition (e.g., peptide or nucleotide sequences). Non-limiting examples of pharmaceutically acceptable carriers and / or excipients include sucrose, polysorbate 80, sodium dihydrogen phosphate monohydrate, and disodium hydrogen phosphate dihydrate. The composition may comprise fillers or diluents (e.g., lactose, mannitol, and microcrystalline cellulose), disintegrants (e.g., croscarmellose sodium, crospovidone, starch), buffers (e.g., phosphate buffer or acetate buffer), solvents (e.g., water, ethanol, glycerol), or stabilizers (e.g., sugar or gelatin); and preservatives (e.g., thimerosal).

[0237] In some embodiments, the compositions of this technology comprise adjuvants. Adjuvants can stimulate or enhance an immune response when the composition is administered. Non-limiting examples of adjuvants include aluminum salts, oil-in-water emulsions (e.g., MF59 or AS03), pathogen mimics (e.g., CpG oligonucleotides, monophospholipid A), squalene, virions, and liposomes.

[0238] In some embodiments, the composition is sterilized (e.g., by filtration sterilization).

[0239] In some embodiments, the compositions of this technology comprise features that promote binding and / or co-localization with HLA (e.g., HLA-A, HLA-B, or HLA-C), APCs (e.g., peptide or nucleotide sequences that target or bind to MHC class II molecules), and / or T-cell receptors (TCRs). In some embodiments, the composition comprises a molecule that binds to a receptor on an APC (e.g., dendritic cells). In some embodiments, the molecule is a carbohydrate. In some embodiments, the carbohydrate is a mannose carbohydrate. The mannose carbohydrate may be selected from the group consisting of D-mannose, mannose-6-phosphate (M6P), mannan, mannose polymers, and mannose receptor ligands. In some embodiments, the molecule that binds to the receptor on an APC is selected from the group consisting of mannose, CD180, CD209, and HLA-DR.

[0240] In some embodiments, the composition of this technology includes a hydrophobic tail. The hydrophobic tail can serve as a targeting portion to facilitate binding with APC. In some embodiments, the hydrophobic tail includes fatty acids, phospholipids, cholesterol, retinoids, steroids, alkyl chains, or nonpolar amino acid side chains.

[0241] In some embodiments, the composition comprises one or more peptides of the present technology (e.g., antigenic peptides) or nucleotide sequences encoding peptides of the present technology, in amounts of about 0.05% w / v or w / w, about 0.1% w / v or w / w, about 1% w / v or w / w, about 10% w / v or w / w, about 20% w / v or w / w, about 30% w / v or w / w, about 40% w / v or w / w, about 50% w / v or w / w, about 60% w / v or w / w, about 70% w / v or w / w, about 80% w / v or w / w, about 90% w / v or w / w, about 95% w / v or w / w, or about 99% w / v or w / w.

[0242] In some embodiments, the composition comprises one or more peptides of the present technology (e.g., antigenic peptides) or nucleotide sequences encoding peptides of the present technology, in amounts of at least 0.05% w / v or w / w, at least 0.1% w / v or w / w, at least 1% w / v or w / w, at least 10% w / v or w / w, at least 20% w / v or w / w, at least 30% w / v or w / w, at least 40% w / v or w / w, at least 50% w / v or w / w, at least 60% w / v or w / w, at least 70% w / v or w / w, at least 80% w / v or w / w, at least 90% w / v or w / w, at least 95% w / v or w / w, or at least 99% w / v or w / w.

[0243] In some embodiments, the composition comprises one or more peptides of the present technology (e.g., antigenic peptides) or nucleotide sequences encoding peptides of the present technology, in amounts of at least about 0.05% w / v or w / w, at least about 0.1% w / v or w / w, at least about 1% w / v or w / w, at least about 10% w / v or w / w, at least about 20% w / v or w / w, at least about 30% w / v or w / w, at least about 40% w / v or w / w, at least about 50% w / v or w / w, at least about 60% w / v or w / w, at least about 70% w / v or w / w, at least about 80% w / v or w / w, at least about 90% w / v or w / w, at least about 95% w / v or w / w, or at least about 99% w / v or w / w.

[0244] In some embodiments, the composition comprises a lyophilized pharmaceutical product (lyophilized powder or lyophilized cake of a peptide or nucleotide sequence). In some embodiments, the lyophilized pharmaceutical product is reconstituted prior to administration (e.g., using water or saline).

[0245] In some embodiments, the reconstituted composition provides a solution having a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0246] In some embodiments, the reconstituted composition provides a solution having a pH value of at least 5.0, at least 5.1, at least 5.2, at least 5.3, at least 5.4, at least 5.5, at least 5.6, at least 5.7, at least 5.8, at least 5.9, at least 6.0, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, at least 6.7, at least 6.8, at least 6.9, at least 7.0, at least 7.1, at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, at least 8.0, at least 8.1, at least 8.2, at least 8.3, at least 8.4, at least 8.5, at least 8.6, at least 8.7, at least 8.8, at least 8.9, or at least 9.0.

[0247] In some embodiments, the reconstituted composition provides having at least about 5.0, at least about 5.1, at least about 5.2, at least about 5.3, at least about 5.4, at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.7, at least about 6.8, at least about 6.9, to A solution with a pH value of at least about 7.0, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8.0, at least about 8.1, at least about 8.2, at least about 8.3, at least about 8.4, at least about 8.5, at least about 8.6, at least about 8.7, at least about 8.8, at least about 8.9, or at least about 9.0.

[0248] In some embodiments, the reconstructed composition provides a solution having a peptide or nucleotide sequence of the present technology, said peptide or nucleotide sequence at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, or about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, or about 100 mg / mL.

[0249] In some embodiments, the reconstructed composition provides a solution having a peptide or nucleotide sequence of the present technology, said peptide or nucleotide sequence having a concentration of at least 1 mg / mL, at least 2 mg / mL, at least 3 mg / mL, at least 4 mg / mL, at least 5 mg / mL, at least 6 mg / mL, at least 7 mg / mL, at least 8 mg / mL, at least 9 mg / mL, at least 10 mg / mL, at least 11 mg / mL, at least 12 mg / mL, at least 13 mg / mL, at least 14 mg / mL, at least 15 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, or at least 40 mg / mL, at least 50 mg / mL, at least 60 mg / mL, at least 70 mg / mL, at least 80 mg / mL, at least 90 mg / mL, or at least 100 mg / mL.

[0250] In some embodiments, the reconstructed composition provides a solution having a peptide or nucleotide sequence of the present technology, said peptide or nucleotide sequence having a concentration of at least about 1 mg / mL, at least about 2 mg / mL, at least about 3 mg / mL, at least about 4 mg / mL, at least about 5 mg / mL, at least about 6 mg / mL, at least about 7 mg / mL, at least about 8 mg / mL, at least about 9 mg / mL, at least about 10 mg / mL, at least about 11 mg / mL, at least about 12 mg / mL, at least about 13 mg / mL, at least about 14 mg / mL, at least about 15 mg / mL, at least about 20 mg / mL, at least about 25 mg / mL, at least about 30 mg / mL, or at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, at least about 80 mg / mL, at least about 90 mg / mL, or at least about 100 mg / mL.

[0251] In some embodiments, the composition or solution is diluted to administer the drug. Non-limiting examples of diluents include sodium chloride, water (e.g., sterile water for injection (SWFI)), glucose solution, antibacterial solution, or Ringer's solution (e.g., solution containing electrolytes, lactated Ringer's solution).

[0252] Related methods

[0253] This technology includes methods for producing vaccine compositions and cell compositions of this technology.

[0254] Quantitative analysis of lymphocyte populations

[0255] In some embodiments, methods for generating vaccine compositions and / or cell compositions of this technology include the step of quantifying a population of lymphocytes (e.g., T cells, B cells, NK cells, dendritic cells, macrophages, or granulocytes) in samples from subjects who have successfully cleared the virus (e.g., eliminated active infection, reduced viral load, resolved virus-related symptoms, or established immune memory against the virus). Quantification of the lymphocyte population may include, but is not limited to, methods such as flow cytometry, immunohistochemistry, immunofluorescence, protein quantification and / or detection methods (e.g., assessing the level of lymphocyte markers) or gene expression quantification methods (e.g., assessing the expression level of lymphocyte-associated transcripts).

[0256] In some implementations, the lymphocyte population is a T cell population. T cells may include helper T cells (Th cells) (e.g., CD4+ cells), cytotoxic T cells (e.g., CD8+ cells), regulatory T cells (Treg), memory T cells, and follicular helper T cells (Tfh cells). Non-limiting examples of Th cells include Th1 cells, Th2 cells, and Th17 cells.

[0257] In some embodiments, the T cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.

[0258] Samples from subjects can be circulating fluid samples (e.g., peripheral blood mononuclear cell (PBMC) samples), lymphoid tissue samples (e.g., lymph node, spleen, or tonsil tissue), mucosal samples (e.g., mucosal tissue), bone marrow samples, cerebrospinal fluid (CSF) samples, or synovial fluid samples.

[0259] In some implementations, subjects who have successfully cleared the virus have or have had mild symptoms associated with viral infection or no symptoms associated with viral infection. The virus may be an arteritis virus, medium-sized arteritis virus, circovirus, porcine circovirus, or coronavirus. In some implementations, the virus is selected from the group consisting of cytomegalovirus, Epstein-Barr virus, hepatitis B virus, human papillomavirus, adenovirus, herpesvirus, human immunodeficiency virus, influenza virus, human respiratory syncytial virus, vaccinia virus, varicella-zoster virus, yellow fever virus, Ebola virus, coronavirus, eastern equine encephalitis virus, human polyomavirus (BKV), SV40, and Zika virus. In some implementations, the virus includes the Target Variant (VOI), Variant of Concern (VOC), or High Consequence Variant (VOHC) as defined by the U.S. Centers for Disease Control and Prevention and the World Health Organization (WHO).

[0260] In some embodiments, the virus may be a virus associated with a pandemic-like infection rate and / or a virus capable of generating a pandemic-like infection rate.

[0261] In some implementations, the coronavirus is selected from the group consisting of alpha coronaviruses (e.g., human coronavirus 229E (HCoV-229E) or human coronavirus NL63 (HCoV-NL63)), beta coronaviruses (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or bat coronaviruses), gamma coronaviruses (e.g., swine coronavirus HKU15 or avian coronavirus infectious bronchitis virus (IBV)), or delta coronaviruses (e.g., spotted green pigeon coronavirus HKU13 or white-eye coronavirus HKU16).

[0262] In some implementations, the coronavirus is a common cold coronavirus. Non-limiting examples of common cold coronaviruses include human coronavirus 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKU1).

[0263] In some implementations, the coronavirus is a SARS-CoV-2 strain selected from the group consisting of SARS-CoV-2α, SARS-CoV-2β, SARS-CoV-2γ, SARS-CoV-2δ, SARS-CoV-2ο, SARS-CoV-2ε, SARS-CoV-2ζ, SARS-CoV-2η, SARS-CoV-2ι, SARS-CoV-2κ, SARS-CoV-2λ, and SARS-CoV-2μ.

[0264] Expose the sample to viral antigens

[0265] In some embodiments, methods for generating vaccine compositions and / or cell compositions of this technology include the step of exposing a sample to one or more antigens derived from a virus. Exposure to the sample may include direct exposure (e.g., incubating the sample with a protein antigen, peptide antigen, lipid antigen, glycoprotein antigen, or a nucleotide sequence that produces the antigen) or indirect exposure. Indirect exposure may include exposing the sample to one or more antigens using antigen-presenting cells (APCs). Non-limiting examples of APCs include dendritic cells (DCs) (e.g., follicular dendritic cells (DCs)), macrophages, B cells, monocytes, and Langerhans cells.

[0266] In some implementations, the method includes quantifying lymphocyte populations before, during, and / or after exposure to one or more viral antigens.

[0267] In some embodiments, lymphocyte populations are quantified approximately 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15 or 20 weeks before and / or after exposure to one or more viral antigens. In some embodiments, lymphocyte populations are quantified at least 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15 or 20 weeks before and / or after exposure to one or more viral antigens. In some implementations, lymphocyte populations are quantified at least approximately 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15 or 20 weeks before and / or after exposure to one or more viral antigens.

[0268] In some implementations, quantifying lymphocyte populations includes standardized procedures.

[0269] In some embodiments, the method includes the step of calculating the difference in lymphocyte population counts between two different time points. In some embodiments, the two different time points include a time point prior to exposure to one or more viral antigens and a time point after exposure to one or more viral antigens. In some embodiments, the two different time points include a time point prior to exposure to one or more viral antigens and a time point during exposure to one or more viral antigens. In some embodiments, the two different time points include a time point during exposure to one or more viral antigens and a time point after exposure to one or more viral antigens.

[0270] In some implementations, calculating the difference in lymphocyte populations between two different time points also includes comparing that difference to a threshold. This threshold may help determine whether the lymphocyte population has changed due to one or more viral antigens.

[0271] In some implementations, if the difference in lymphocyte populations exceeds a threshold, one or more viral antigens or nucleotide sequences encoding one or more viral antigens are included in the vaccine composition.

[0272] Lymphocyte expansion

[0273] In some embodiments, the method of generating the cellular composition of this technology includes the step of exposing a second sample to one or more antigens derived from a virus. In some embodiments, if the difference in the lymphocyte population exceeds a threshold, the second sample from a second subject is exposed using one or more viral antigens or nucleotide sequences encoding one or more viral antigens. The second sample may include a biological sample. In some embodiments, the second sample is a circulating fluid sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), a lymphoid tissue sample (e.g., lymph node, spleen, or tonsil tissue), a mucosal sample (e.g., mucosal tissue), a bone marrow sample, a cerebrospinal fluid (CSF) sample, or a synovial fluid sample.

[0274] The second subject may include subjects who have never been exposed to the virus (i.e., subjects who have never been infected with the virus). Subjects include those who have never been infected with the virus, those who have been infected with the virus, and those who have symptoms related to viral infection.

[0275] In some embodiments, lymphocyte populations are amplified in a second sample after exposure to one or more viral antigens or nucleotide sequences encoding one or more viral antigens. Non-limiting examples of methods that can be used to amplify lymphocyte populations include cytokine stimulation, cell culture methods (e.g., adding nutrients or growth factors to promote proliferation; co-culture systems); or bioreactors, antibody stimulation. In some embodiments, the amplified lymphocyte populations are isolated and / or screened for responsiveness to formulations of cytokines and / or chemokines (e.g., IFNγ, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGFβ, CXCL12, CCL19) for administration (e.g., in a vaccine composition). Example

[0276] Identification of viral antigens

[0277] To determine which viral antigens were most likely to elicit a response in a pilot study, the SARS-CoV-2 antigen response pattern of T cells from subjects who successfully cleared SARS-CoV-2 infection with minimal side effects was compared with that of T cells obtained from subjects who had never been infected with SARS-CoV-2. These antigens included CoV-2S, M, N, 3a, 7a, 8, and Nsp6 (…). Figures 1A-1C ).

[0278] To validate this concept, peptide sequences corresponding to one or more identified immunodominant viral antigens were synthesized, and peptides associated with all or some viral antigens (e.g., SARS-CoV-2S, M, N, 3a, 7a, 8, and nsp6) were loaded into dendritic cells (DCs) and then used to elicit T cells in subjects. The activation-inducible marker (AIM) was used to determine... Figure 2A and 2B Measurable PBMCs in subjects not infected with SARS-CoV-2 produced diverse CD4+ responses to these antigens. + and CD8 + The T-cell response pattern was comparable to that of subjects who successfully cleared SARS-CoV-2 infection. Subjects showed a higher response to DC-mediated viral antigen presentation, while no response to all antigens was observed for PBMCs and naive donors. This suggests that stimulation of T cells with SARS-CoV-2-specific viral antigens via dendritic cells (DCs) may induce a robust population of sensitized T cells. This also suggests that a protective immune response against the virus may be generated prior to viral infection.

[0279] Production of DC and T cell cultures

[0280] Whole blood samples were obtained from subjects uninfected with SARC-CoV-2 via aspiration or apheresis, and PBMCs were isolated from the blood using the Ficoll separation method. Monocyte-derived dendritic cells (DCs) were generated using a cell culture plastic adhesion method. Non-adherent T cells were removed and frozen for use as a T cell source in subsequent cultures. The differentiation medium consisted of complete DC medium supplemented with 800 U / mL human IL-4 and 500 U / mL human GM-CSF. Cells were cultured in differentiation medium for 5 days, with the medium changed daily starting from day 1 after initial culture. On day 5, the differentiation medium was removed, and maturation medium was added overnight. The maturation medium contained IL-6, IL-1β, TNFα, and prostaglandin E2 (PGE2). Prostaglandin E2 could be replaced with polyinosinic acid:polycytidylic acid (Polyl:C), which binds to Toll-like receptor 3 (TLR3) (e.g., TLR3 on DCs).

[0281] On day 6 of differentiation and maturation of adherent T cells (usually monocytes) into dendritic cells (DCs), harvested DCs were conjugated with SARS-CoV-2 viral peptides S, M, N, 3a, 7a, 8, and S+ (all peptide combinations). Non-adhesive T cells were partially thawed and conjugated with DCs at a ratio of 2:1. The total volume was 1 mL, and the cell density was 3 x 10⁻⁶ cells / DCs. 6Cells / mL were cultured in intact Cellgenix GMP DC medium supplemented with a cytokine mixture. Peptides suspended in DMSO were added to achieve a final concentration of 0.1 pg / mL for each peptide. Cells were seeded into wells. The cultures were transferred to a humidified incubator (5% CO2, 37°C). Every two days, half of the medium was replaced with fresh medium without disturbing the cells. On day 7, this process was repeated, thawing PBMCs and binding them with new DCs and peptides, and adding them to the current culture. On day 14, a CD3 / CD28 / CD2 T cell activator mixture was added to the culture in fresh medium containing cytokines (15 pg / mL), and the culture was then returned to the humidified incubator at 5% CO2, 37°C.

[0282] Activation-inducing marker detection and flow cytometry phenotypic analysis

[0283] Activation-Induced Marker (AIM) assays and phenotypic flow cytometry assays were performed on days 14, 21, and 28. Cell number and viability were quantified. Cells were seeded in two separate wells, one million cells per well, for both AIM and phenotypic flow cytometry analyses. An equimolar amount of dimethyl sulfoxide (DMSO) stimulation was used as a negative control in both assays, and cells were stained with an antibody mixture at room temperature for 15 minutes in the dark. After a final wash, cells were resuspended in fluorescently activated cell sorting buffer (FACS) and quantified and analyzed using FlowJo flow cytometry software (TreeStar).

[0284] Th1 and Th2 T cell analysis

[0285] T cells that react with viral antigens (1 x 10 cells per well) 5 ) Inoculated onto enzyme-linked immunosorbent assay (ELISpot) plates containing IFNγ and IL4. Figures 3A-3I After 24 hours of incubation, the culture plates were washed and incubated with a secondary antibody against IFNγ (which enzymatically catalyzes the first color) and a secondary antibody against IL-4 (which enzymatically catalyzes the second color). After drying, the first and second colored spots were counted. Each spot represents a T cell secreting an analyte cytokine, with Th1 cells secreting IFNγ and Th2 cells secreting IL-4. These assays were performed on T cells on days 14 and 21 post-stimulation. T cell immune memory was then measured as the percentage of the CD3+CD62L+CD197+ T cell population (…). Figure 4 ).

[0286] Selection of T-cell antigens for vaccine incorporation

[0287] By using the MHC class I binding predictor MHCnetpan to predict the full amino acid sequence of SARS-CoV-2, the top 1% (71 most common MHC alleles, HLA-A, B, C) of T-cell antigens most likely to elicit a response in the population were identified, with an accuracy of approximately 50% (Table 4). Subjects expressing at least two MHC alleles accounted for approximately 90% of the population. The selected antigens are likely to be immunogenic and could provide protection for a large population. Figure 5 ).

[0288] Table 4: Most common HLA alleles, with a population frequency greater than 1%.

[0289]

[0290]

[0291] GMP RNA vaccine production

[0292] RNA vaccines are produced using RNA constructs designed to express S, M, N, 3a, 7a, 8, and Nsp6 antigens. Figure 6 All reagents used were derived from contaminant-free sources and produced using defined culture media rather than from natural sources. The RNA underwent a purification process. After purification, the RNA could be encapsulated in lipid nanoparticles or encapsulated together with cationic protein lipids (such as protamine), carrier proteins, and small molecules.

[0293] An mRNA construct containing the adapter sequence (SEQ ID NO: 10) is inserted between nucleotide sequences encoding viral antigens. This adapter may have low immunogenicity, as indicated by the NetMHC MHCI binding affinity tool. MHCI binding was predicted using the NetMHCpan method via IEDB analysis resources. Viral antigen sequences of interest are contained in regions with binding affinity below the nM level (Table 5), where lower levels indicate better binding.

[0294] Table 5: Binding ability of the linker sequence to the first 1% of HLA-ABC conjugates

[0295]

[0296]

[0297] Strong binding peptides selected from viral peptides identified for RNA vaccines are 60 nM or less for the most common HLAs. Linkers of 40,000 nM or higher cannot bind to all HLAs. (Table 6-12)

[0298] Table 6: Examples of peptides selected for NSP6

[0299]

[0300]

[0301] Table 7: Examples of peptides selected for spikes

[0302]

[0303] Table 8: Examples of peptides selected for ORF3a

[0304]

[0305] Table 9: Examples of peptides selected for VME1

[0306]

[0307]

[0308] Table 10: Examples of peptides selected for ORF7a

[0309]

[0310]

[0311] Table 11: Examples of peptides selected for ORF8

[0312]

[0313]

[0314] Table 12: Examples of peptides selected for NCAP

[0315]

[0316]

[0317] Selected peptides were combined into 30-amino acid peptides, which were then placed between the linker sequence and randomized peptides from different viral peptides located on either side of the linker sequence (e.g., in the Se construct or in different parts of the protein). This length provides an optimal amount of antigen but does not allow for the tertiary folding or generation of functional domains of the viral peptides.

[0318] The vaccine design incorporates nucleotide sequences expressing antigens from all SARS-CoV-2 variants to date, including XBB (BA.2.10). Although the vaccine was designed using antigens from the SARS-CoV-2 α strain, only 1.69% of the antigens screened across all seven proteins were mutated between the α, δ, and omicron variants, meaning that 98.31% of the antigens were still capable of generating an effective immune response against the most recent strains (Table 13). Furthermore, 28 / 34 mutations in the antigens between the α and omicron strains occurred in S, compared to only seven in all other combinations of viral peptides selected from the other variants.

[0319] Table 13: Common mutations in the three viral variants

[0320]

[0321]

[0322] Evaluation of vaccine compositions in primate models

[0323] To evaluate the vaccine composition in a primate model, three groups of rhesus monkeys (n=6 per group) were administered intramuscular injections on days 0 and 21. Group 1 received a vaccine derived from a SARS-CoV-2α strain sequence, consisting of 200 μg of conserved mRNA antigen plus 100 μg of mRNA targeting the spike antigen, administered twice. Group 2 received a vaccine derived from a SARS-CoV-2α strain sequence containing only 200 μg of conserved mRNA antigen. Group 3 received a vaccine derived from a SARS-CoV-2α strain sequence containing only 100 μg of mRNA targeting the spike antigen. The mRNA antigen vaccine consisted of six specified conserved antigens. Blood samples were drawn on days 14 and 35 to detect T-cell interferon-gamma (IFNγ) production in response to the injected antigens. Antigen-specific T-cell frequencies were determined by pulses with antigen-specific peptides, and INFγ was screened using ELISpot analysis. Results showed that in the Th-1 response against a multivalent target antigen, T-cell specks corresponded to IFNγ release per T cell. On day 35 post-challenge, half of the animals in each group (N=3) were challenged with either the SARS-CoV-2α strain or the Omicron XBB strain. The animals were then followed up for 30 days to observe their lung symptoms and weight loss. Starting from day 42, nasal swabs were collected every 7 days for viral peptide antigen testing to screen for live SARS-CoV-2 virus.

[0324] Thirty days later, none of the animals vaccinated with the SARS-CoV-2α strain or challenged with SARS-CoV-2α showed weight loss, lung infection, or the presence of viral peptides. Consistent results were observed when animals in groups 1 and 2 were challenged with the XBB strain. However, in group 3, two out of the three animals vaccinated only with the SARS-CoV-2α spike protein showed weight loss, lung infection, and the presence of viral peptides (Table 14). This suggests that conserved proteins provide protection among viral strains and can generally inhibit viral evolution.

[0325] Table 14: Animals vaccinated with the α strain vaccine

[0326]

[0327] Develop nanoparticles targeting dendritic cells (DCs) or enhancing the delivery of RNA encoding vaccine antigens to DCs. Liposomes were used to encapsulate and deliver RNA encoding green fluorescent protein (GFP) in vitro. DCs were analyzed by flow cytometry 24 hours after nanoparticle delivery to assess GFP expression. Dendritic cells were exposed to nanoparticles carrying GFP RNA as a control, or exposed to nanoparticles carrying SARS-CoV-2 RNA encoding a viral peptide. Figure 7 and Figure 8 The nanoparticles were designed to target dendritic cells using mannose, CD180, CD209, or HLA-DR targeting ligands. Nanoparticles with targeting ligands were more efficient in RNA delivery, as measured by GFP levels. Figure 9 ).

[0328] Nanoparticles containing six SARS-CoV-2 viral antigens were administered to rhesus monkeys via vaccine. Controls included nanoparticles without targeting ligands and / or without viral antigen loading. When challenged with Omicron XBB at lower concentrations of RNA, lipid nanoparticles targeting dendritic cells (DCs) provided stronger protection to rhesus monkeys than non-targeting lipid nanoparticles (Table 15).

[0329] Table 15: Infection rate in animals treated with lipid nanoparticle compositions

[0330]

[0331] Additional Implementation Plan

[0332] This technology includes, but is not limited to, the specific implementation schemes described in paragraphs

[0241] -

[0319] below:

[0333] 1. A vaccine composition comprising a first antigen and a second antigen, wherein the first antigen and the second antigen are each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

[0334] 2. A vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen.

[0335] The first antigen and the second antigen are each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

[0336] 3. A vaccine composition comprising spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

[0337] 4. A vaccine composition comprising a nucleotide sequence encoding a spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding an NCAP (N) peptide, a nucleotide sequence encoding a nucleotide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding an octapeptide, and a nucleotide sequence encoding an Nsp6 peptide.

[0338] 5. The vaccine composition according to any one of the foregoing embodiments, wherein the peptide length is less than 30 amino acids.

[0339] 6. The vaccine composition according to embodiment 5, wherein the peptide length is from about 5 amino acids to about 20 amino acids.

[0340] 7. The vaccine composition according to embodiment 6, wherein the peptide length is about 9 amino acids.

[0341] 8. The vaccine composition according to embodiment 6, wherein the peptide length is about 14 amino acids.

[0342] 9. The vaccine composition according to embodiment 2 or 4, wherein the nucleotide sequence is a deoxyribonucleic acid (DNA) sequence.

[0343] 10. The vaccine composition according to embodiment 2 or 4, wherein the nucleotide sequence is a ribonucleotide (RNA) sequence.

[0344] 11. The vaccine composition according to any one of the foregoing embodiments, wherein the peptide does not contain an active site.

[0345] 12. The peptide composition of any one of the foregoing embodiments, wherein the peptide does not fold into a tertiary peptide structure.

[0346] 13. The vaccine composition according to any one of the foregoing embodiments, wherein

[0347] The spike (S) peptide has a content of at least about 60%, 65%, 70% with one or more of SEQ ID NO:143-209.

[0348] The sameness of %, 75%, 80%, 85%, 90%, 95%, 99%, or 100%;

[0349] The VME1(M) peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220;

[0350] The NCAP(N) peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242;

[0351] The 3a peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255;

[0352] The 7a peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261;

[0353] The 8 peptides have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267; or

[0354] The Nsp6 peptide has a content of at least about 60%, 65%, 70% with one or more of SEQ ID NO:268-281.

[0355] The sameness of % , 75% , 80% , 85% , 90% , 95% , 99% or 100%.

[0356] 14. A vaccine composition comprising two or more antigens, said antigens being selected from the group consisting of:

[0357] Spike (S) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209;

[0358] VME1(M) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220;

[0359] NCAP(N) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242;

[0360] Having at least about 60%, 65%, 70%, 75%, 80% of one or more of SEQ ID NO:243-255

[0361] 3a peptides with 85%, 90%, 95%, 99%, or 100% identity;

[0362] Having at least about 60%, 65%, 70%, 75%, or 80% of one or more of SEQ ID NO:256-261

[0363] 7a peptides with 85%, 90%, 95%, 99%, or 100% identity;

[0364] Having at least about 60%, 65%, 70%, 75%, or 80% of one or more of SEQ ID NO:262-267

[0365] 85%, 90%, 95%, 99%, or 100% identity octapeptides; or,

[0366] Nsp6 peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281.

[0367] 15. A vaccine composition comprising two or more nucleotide sequences, said nucleotide sequences being selected from the group consisting of:

[0368] A first nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67;

[0369] A second nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:68-78;

[0370] A third nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100;

[0371] A fourth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113;

[0372] A fifth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119;

[0373] A sixth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125; or,

[0374] A seventh nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139.

[0375] 16. The vaccine composition according to any one of embodiments 2, 4, 9 or 15, wherein the nucleotide sequence is present in the lipid composition.

[0376] 17. The vaccine composition of embodiment 16, wherein the lipid composition comprises lipid nanoparticles.

[0377] 18. The vaccine composition of embodiment 17, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule.

[0378] 19. The vaccine composition of embodiment 18, wherein the APC is a dendritic cell (DC).

[0379] 20. The vaccine composition according to embodiment 18 or 19, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209 or HLA-DR targeting molecules.

[0380] 21. The vaccine composition according to embodiments 1, 3, 13 or 14, wherein each peptide is present on a single peptide chain.

[0381] 22. The vaccine composition of embodiment 21, wherein the single chain comprises one or more adapter sequences.

[0382] 23. The vaccine composition according to embodiments 1, 3, 13 or 14, wherein each peptide is present on a different peptide chain.

[0383] 24. The vaccine composition according to embodiment 2 or 4, wherein each nucleotide sequence is present in a polycistronic sequence.

[0384] 25. The vaccine composition of embodiment 24, wherein the polycistronic sequence comprises one or more adapter sequences.

[0385] 26. The vaccine composition according to embodiments 2, 4, 9 or 15, wherein each nucleotide sequence is present on a different nucleotide chain.

[0386] 27. The vaccine composition according to any one of embodiments 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigen peptide.

[0387] 28. The vaccine composition according to embodiment 1 or 2, wherein one or more of the antigens comprises coronavirus peptides.

[0388] 29. The vaccine composition of embodiment 27 or 28, wherein the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

[0389] 30. The vaccine composition of embodiment 29, wherein the SARS-CoV-2 antigen is selected from the group consisting of SARS-CoV-2α antigen, SARS-CoV-2β antigen, SARS-CoV-2γ antigen, SARS-CoV-2δ antigen, SARS-CoV-2ο antigen, SARS-CoV-2ε antigen, SARS-CoV-2ζ antigen, SARS-CoV-2η antigen, SARS-CoV-2ι antigen, SARS-CoV-2κ antigen, SARS-CoV-2λ antigen and SARS-CoV-2μ antigen.

[0390] 31. A vaccine composition comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:282.

[0391] 32. A vaccine composition comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:283.

[0392] 33. A vaccine composition comprising a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:140.

[0393] 34. A vaccine composition comprising a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:141.

[0394] 35. The vaccine composition according to embodiment 33 or 34, wherein the nucleotide sequence is present in the lipid composition.

[0395] 36. The vaccine composition of embodiment 35, wherein the lipid composition comprises lipid nanoparticles.

[0396] 37. The vaccine composition of embodiment 36, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule.

[0397] 38. The vaccine composition of embodiment 37, wherein the APC is a dendritic cell (DC).

[0398] 39. The vaccine composition according to embodiment 37 or 38, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209 or HLA-DR targeting molecules.

[0399] 40. The vaccine composition according to any one of the foregoing embodiments further comprises an adjuvant.

[0400] 41. A method for producing a vaccine composition, comprising the following steps:

[0401] (i) T cell population from peripheral blood mononuclear cell (PBMC) samples from subjects who have successfully cleared the virus.

[0402] Quantitative analysis;

[0403] (ii) Exposing the sample to one or more antigens from the virus;

[0404] (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens;

[0405] (v) Calculate the difference in T cell population size between (i) and (iii);

[0406] (vi) Compare the differences in (iv) to the threshold; and

[0407] (iv) If the difference in (iv) exceeds the threshold in (v), then the vaccine composition includes a peptide of one or more antigens described in (a) or (ii), or (b) a nucleotide sequence encoding one or more antigens described in (ii).

[0408] 42. The method of embodiment 41, wherein the vaccine composition is a multivalent vaccine composition.

[0409] 43. A method for generating a T-cell composition that specifically recognizes one or more viral antigens, comprising the following steps:

[0410] (i) Quantification of T cell populations from the first peripheral blood mononuclear cell (PBMC) sample of the first subject who had successfully cleared the virus;

[0411] (ii) Expose the first sample to one or more antigens from the virus;

[0412] (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens;

[0413] (iv) Calculate the difference in T cell population numbers between (i) and (iii);

[0414] (v) Compare the differences in (iv) with the threshold;

[0415] (vi) If the difference in (iv) exceeds the threshold in (v), then a second PBMC sample from the second subject is exposed to a peptide or encoding one or more antigens described in (ii) of the vaccine composition.

[0416] The nucleotide sequence of one or more antigens;

[0417] (vii) followed exposure in (vi) to amplify T cells in the second sample; and

[0418] (viii) Isolate the expanded T cells.

[0419] 44. The method of implementation scheme 43 further includes (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

[0420] 45. The method of embodiment 43, wherein the T-cell composition is formulated for administration to a second subject.

[0421] 46. ​​The method of embodiment 45, wherein the T-cell composition is formulated in a vaccine composition.

[0422] 47. The method of any one of embodiments 41-46, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.

[0423] 48. The method of any one of embodiments 41-47, wherein the T cell population includes T helper cells.

[0424] 49. The method of embodiment 48, wherein the T helper cells include Th1 or Th2 cells.

[0425] 50. The embodiment of 43 or 47, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.

[0426] 51. The method of embodiment 43 or 47, wherein the T cell composition comprises T helper cells.

[0427] 52. The method of embodiment 51, wherein the T helper cells include Th1 or Th2 cells.

[0428] 53. The method of any one of embodiments 41-47, wherein exposure to one or more antigens in (ii) includes antigen-presenting cells (APCs).

[0429] 54. The method of embodiment 53, wherein the APC is a dendritic cell (DC).

[0430] 55. The method of embodiment 54, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0431] 56. The method of any one of embodiments 41-55, wherein the virus is a coronavirus.

[0432] 57. The method of implementation scheme 56, wherein the coronavirus is SARS-CoV-2.

[0433] 58. The method of embodiment 57, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2α, SARS-CoV-2β, SARS-CoV-2γ, SARS-CoV-2δ, SARS-CoV-2ο, SARS-CoV-2ε, SARS-CoV-2ζ, SARS-CoV-2η, SARS-CoV-2ι, SARS-CoV-2κ, SARS-CoV-2λ and SARS-CoV-2μ.

[0434] 59. The method of any one of embodiments 41-58, wherein the one or more antigens comprise spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide or Nsp6 peptide.

[0435] 60. The method of any one of embodiments 41-58, wherein the one or more antigens comprise a nucleotide sequence encoding a spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide, or Nsp6 peptide.

[0436] 61. A T-cell composition immunogenic to a viral antigen, which is produced by the following steps:

[0437] (i) Quantification of T cell populations from the first peripheral blood mononuclear cell (PBMC) sample of the first subject who had successfully cleared the virus;

[0438] (ii) Expose the first sample to one or more antigens from the virus;

[0439] (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens;

[0440] (iv) Calculate the difference in T cell population numbers between (i) and (iii);

[0441] (v) Compare the differences in (iv) with the threshold;

[0442] (vi) If the difference in (iv) exceeds the threshold in (v), then a second PBMC sample from the second subject is exposed to a peptide or encoding one or more antigens described in (ii) of the vaccine composition.

[0443] The nucleotide sequence of one or more antigens;

[0444] Following exposure in (vii), T cells in the second sample were amplified; and

[0445] (viii) Isolate the expanded T cells.

[0446] 62. The T cell composition of embodiment 61 further includes (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

[0447] 63. The T-cell composition of embodiment 61 or embodiment 48, wherein the T-cell composition is formulated for administration to a second subject.

[0448] 64. The T-cell composition of embodiment 63, wherein the T-cell composition is formulated in a vaccine composition.

[0449] 65. The T cell composition according to any one of embodiments 61-64, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.

[0450] 66. The T cell composition according to any one of embodiments 61-65, wherein the T cell population includes T helper cells.

[0451] 67. The T cell composition of embodiment 66, wherein the T helper cells include Th1 or Th2 cells.

[0452] 68. The T cell composition according to any one of embodiments 61-67, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.

[0453] 69. The T cell composition according to any one of embodiments 61-68, wherein the T cell composition comprises T helper cells.

[0454] 70. The T cell composition of embodiment 69, wherein the T helper cells comprise Th1 or Th2 cells.

[0455] 71. The T-cell composition according to any one of embodiments 61-70, wherein exposure to one or more antigens in (ii) includes antigen-presenting cells (APCs).

[0456] 72. The T cell composition of embodiment 71, wherein the APC is a dendritic cell (DC).

[0457] 73. The T cell composition of embodiment 72, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0458] 74. The T-cell composition according to any one of embodiments 61-73, wherein the virus is a coronavirus.

[0459] 75. The T-cell composition of embodiment 74, wherein the coronavirus is SARS-CoV-2.

[0460] 76. The T-cell composition of embodiment 75, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2α, SARS-CoV-2β, SARS-CoV-2γ, SARS-CoV-2δ, SARS-CoV-2ο, SARS-CoV-2ε, SARS-CoV-2ζ, SARS-CoV-2η, SARS-CoV-2ι, SARS-CoV-2κ, SARS-CoV-2λ, and SARS-CoV-2μ.

[0461] 77. The T-cell composition according to any one of embodiments 61-76, wherein the one or more antigens include spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide or Nsp6 peptide.

[0462] 78. The T-cell composition according to any one of embodiments 61-76, wherein the one or more antigens comprise a nucleotide sequence encoding a spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide, or Nsp6 peptide.

Claims

1. A vaccine composition comprising a first antigen and a second antigen, wherein the first antigen and the second antigen are each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

2. A vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen. The first antigen and the second antigen are each independently selected from the group consisting of spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

3. A vaccine composition comprising spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide and Nsp6 peptide.

4. A vaccine composition comprising a nucleotide sequence encoding a spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding an NCAP (N) peptide, a nucleotide sequence encoding a nucleotide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding an octapeptide, and a nucleotide sequence encoding an Nsp6 peptide.

5. The vaccine composition of any one of the preceding claims, wherein the peptide length is less than 30 amino acids.

6. The vaccine composition of claim 5, wherein the peptide length is from about 5 amino acids to about 20 amino acids.

7. The vaccine composition of claim 6, wherein the peptide length is about 9 amino acids.

8. The vaccine composition of claim 6, wherein the peptide length is about 14 amino acids.

9. The vaccine composition of claim 2 or 4, wherein the nucleotide sequence is a deoxyribonucleic acid (DNA) sequence.

10. The vaccine composition of claim 2 or 4, wherein the nucleotide sequence is a ribonucleotide (RNA) sequence.

11. The vaccine composition of any one of the preceding claims, wherein the peptide does not contain an active site.

12. The peptide composition of any one of the preceding claims, wherein the peptide does not fold into a tertiary peptide structure.

13. The vaccine composition according to any one of the preceding claims, wherein The spike (S) peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209; The VME1(M) peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220; The NCAP(N) peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242; The 3a peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255; The 7a peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261; The 8 peptides have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267; or, The Nsp6 peptide has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281.

14. A vaccine composition comprising two or more antigens, said antigens being selected from the group consisting of: Spike (S) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:143-209; VME1(M) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:210-220; NCAP(N) peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:221-242; 3a peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:243-255; 7a peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:256-261; An octapeptide having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:262-267; or, Nsp6 peptides having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:268-281.

15. A vaccine composition comprising two or more nucleotide sequences, said nucleotide sequences being selected from the group consisting of: A first nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:1-67; A second nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:68-78; A third nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:79-100; A fourth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:101-113; A fifth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:114-119; A sixth nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:120-125; or, A seventh nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with one or more of SEQ ID NO:126-139.

16. The vaccine composition according to any one of claims 2, 4, 9 or 15, wherein the nucleotide sequence is present in the lipid composition.

17. The vaccine composition of claim 16, wherein the lipid composition comprises lipid nanoparticles.

18. The vaccine composition of claim 17, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule.

19. The vaccine composition of claim 18, wherein the APC is a dendritic cell (DC).

20. The vaccine composition of claim 18 or 19, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209, or HLA-DR targeting molecules.

21. The vaccine composition of claim 1, 3, 13 or 14, wherein each peptide is present on a single peptide chain.

22. The vaccine composition of claim 21, wherein the single chain comprises one or more adapter sequences.

23. The vaccine composition of claim 1, 3, 13 or 14, wherein each peptide is present on a different peptide chain.

24. The vaccine composition of claim 2 or 4, wherein each nucleotide sequence is present in a polycistronic sequence.

25. The vaccine composition of claim 24, wherein the polycistronic sequence comprises one or more adapter sequences.

26. The vaccine composition of claim 2, 4, 9 or 15, wherein each nucleotide sequence is present on a different nucleotide chain.

27. The vaccine composition of any one of claims 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigen peptide.

28. The vaccine composition of claim 1 or 2, wherein one or more of the antigens comprises coronavirus peptides.

29. The vaccine composition of claim 27 or 28, wherein the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

30. The vaccine composition of claim 29, wherein the SARS-CoV-2 antigen is selected from the group consisting of SARS-CoV-2α antigen, SARS-CoV-2β antigen, SARS-CoV-2γ antigen, SARS-CoV-2δ antigen, SARS-CoV-2O antigen, SARS-CoV-2ε antigen, SARS-CoV-2ζ antigen, SARS-CoV-2η antigen, SARS-CoV-2ι antigen, SARS-CoV-2κ antigen, SARS-CoV-2λ antigen, and SARS-CoV-2μ antigen.

31. A vaccine composition comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

282.

32. A vaccine composition comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

283.

33. A vaccine composition comprising a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

140.

34. A vaccine composition comprising a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

141.

35. The vaccine composition of claim 33 or 34, wherein the nucleotide sequence is present in the lipid composition.

36. The vaccine composition of claim 35, wherein the lipid composition comprises lipid nanoparticles.

37. The vaccine composition of claim 36, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule.

38. The vaccine composition of claim 37, wherein the APC is a dendritic cell (DC).

39. The vaccine composition of claim 37 or 38, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209, or HLA-DR targeting molecules.

40. The vaccine composition of any one of the preceding claims further comprises an adjuvant.

41. A method for producing a vaccine composition, comprising the following steps: (i) Quantification of T cell populations in peripheral blood mononuclear cell (PBMC) samples from subjects who have successfully cleared the virus; (ii) Exposing the sample to one or more antigens from the virus; (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens; (v) Calculate the difference in T cell population size between (i) and (iii); (vi) Compare the differences in (iv) with the threshold; as well as (iv) If the difference in (iv) exceeds the threshold in (v), then the vaccine composition includes a peptide of one or more antigens described in (a) or (ii), or (b) a nucleotide sequence encoding one or more antigens described in (ii).

42. The method of claim 41, wherein the vaccine composition is a multivalent vaccine composition.

43. A method for generating a T-cell composition that specifically recognizes one or more viral antigens, comprising the following steps: (i) Quantification of T cell populations from the first peripheral blood mononuclear cell (PBMC) sample of the first subject who had successfully cleared the virus; (ii) Expose the first sample to one or more antigens from the virus; (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens; (iv) Calculate the difference in T cell population numbers between (i) and (iii); (v) Compare the differences in (iv) with the threshold; (vi) If the difference in (iv) exceeds the threshold in (v), a second PBMC sample from the second subject is exposed to a peptide of one or more antigens described in (ii) or a nucleotide sequence encoding one or more antigens described in (ii) in the vaccine composition; Following exposure in (vii), T cells in the second sample were amplified; as well as (viii) Isolate the expanded T cells.

44. The method of claim 43, further comprising (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

45. The method of claim 43, wherein the T-cell composition is formulated for administration to a second subject.

46. ​​The method of claim 45, wherein the T-cell composition is formulated in a vaccine composition.

47. The method of any one of claims 41-46, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.

48. The method of any one of claims 41-47, wherein the T cell population comprises T helper cells.

49. The method of claim 48, wherein the T helper cells comprise Th1 or Th2 cells.

50. The embodiment of claim 43 or 47, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.

51. The method of claim 43 or 47, wherein the T cell composition comprises T helper cells.

52. The method of claim 51, wherein the T helper cells comprise Th1 or Th2 cells.

53. The method of any one of claims 41-47, wherein exposure to one or more antigens in (ii) comprises antigen-presenting cells (APCs).

54. The method of claim 53, wherein the APC is a dendritic cell (DC).

55. The method of claim 54, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF).

56. The method of any one of claims 41-55, wherein the virus is a coronavirus.

57. The method of claim 56, wherein the coronavirus is SARS-CoV-2.

58. The method of claim 57, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2α, SARS-CoV-2β, SARS-CoV-2γ, SARS-CoV-2δ, SARS-CoV-2ο, SARS-CoV-2ε, SARS-CoV-2ζ, SARS-CoV-2η, SARS-CoV-2ι, SARS-CoV-2κ, SARS-CoV-2λ, and SARS-CoV-2μ.

59. The method of any one of claims 41-58, wherein the one or more antigens comprise spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide or Nsp6 peptide.

60. The method of any one of claims 41-58, wherein the one or more antigens comprise a nucleotide sequence encoding a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or an Nsp6 peptide.

61. A T-cell composition immunogenic to a viral antigen, which is produced by the following steps: (i) Quantification of T cell populations from the first peripheral blood mononuclear cell (PBMC) sample of the first subject who had successfully cleared the virus; (ii) Expose the first sample to one or more antigens from the virus; (iii) Quantify the same T cell population as in (i) after exposure to one or more peptide antigens; (iv) Calculate the difference in T cell population numbers between (i) and (iii); (v) Compare the differences in (iv) with the threshold; (vi) If the difference in (iv) exceeds the threshold in (v), a second PBMC sample from the second subject is exposed to a peptide of one or more antigens described in (ii) or a nucleotide sequence encoding one or more antigens described in (ii) in the vaccine composition; Following exposure in (vii), T cells in the second sample were amplified; as well as (viii) Isolate the expanded T cells.

62. The T-cell composition of claim 61 further comprises (ix) screening the responsiveness of expanded T cells to interferon-γ (IFNγ) or interleukin-4 (IL4).

63. The T-cell composition of claim 61 or claim 48, wherein the T-cell composition is formulated for administration to a second subject.

64. The T-cell composition of claim 63, wherein the T-cell composition is formulated in a vaccine composition.

65. The T cell composition of any one of claims 61-64, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.

66. The T cell composition of any one of claims 61-65, wherein the T cell population comprises T helper cells.

67. The T cell composition of claim 66, wherein the T helper cells comprise Th1 or Th2 cells.

68. The T cell composition of any one of claims 61-67, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.

69. The T cell composition of any one of claims 61-68, wherein the T cell composition comprises T helper cells.

70. The T cell composition of claim 69, wherein the T helper cells comprise Th1 or Th2 cells.

71. The T-cell composition of any one of claims 61-70, wherein exposure to one or more antigens in (ii) comprises antigen-presenting cells (APCs).

72. The T cell composition of claim 71, wherein the APC is a dendritic cell (DC).

73. The T cell composition of claim 72, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF).

74. The T-cell composition of any one of claims 61-73, wherein the virus is a coronavirus.

75. The T-cell composition of claim 74, wherein the coronavirus is SARS-CoV-2.

76. The T-cell composition of claim 75, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2α, SARS-CoV-2β, SARS-CoV-2γ, SARS-CoV-2δ, SARS-CoV-2ο, SARS-CoV-2ε, SARS-CoV-2ζ, SARS-CoV-2η, SARS-CoV-2ι, SARS-CoV-2κ, SARS-CoV-2λ, and SARS-CoV-2μ.

77. The T-cell composition of any one of claims 61-76, wherein the one or more antigens comprise spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide or Nsp6 peptide.

78. The T-cell composition of any one of claims 61-76, wherein the one or more antigens comprise a nucleotide sequence encoding a spike (S) peptide, VME1 (M) peptide, NCAP (N) peptide, 3a peptide, 7a peptide, 8 peptide, or Nsp6 peptide.

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