Multi-antigenic RNA SARS-COV-2 vaccines and related methods

Abstract

The present technology provides multivalent vaccine compositions and T cell compositions, and related methods, that include a viral antigen. In some embodiments, the viral antigen is a SARS-CoV-2 antigen. The vaccine compositions and T cell compositions may include each 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

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 387,721, filed December 16, 2022, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING INCORPORATED BY REFERENCE This application contains a Sequence Listing in accordance with ST.26, which is being simultaneously filed via the Patent Center in xml format and is hereby incorporated by reference in its entirety. The .xml copy, created on December 16, 2023, is named 1406308004WO00.xml and is 0.331 MB (331 KB) (339,888 bytes) in size. [Background technology]

[0002] The SARS-CoV-2 virus causes coronavirus disease 2019 (COVID-19). COVID-19 is a highly contagious infectious disease with a wide range of symptoms, from asymptomatic 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 coagulopathy, disseminated intravascular coagulation, and thromboembolism. To date, vaccines, antibodies, and immunotherapies for COVID-19 have primarily focused on the spike (S) protein, resulting in relatively narrow responses. Naturally occurring and circulating SARS-CoV-2 S protein variants exhibit antigenic changes. These variants arise due to adaptation in immune-experienced hosts, particularly those during prolonged infection. This also reduces the therapeutic efficacy of added antibody-mediated immunity, such as convalescent serum or monoclonal antibody preparations. Vaccines, which are generated exclusively against the S protein, become less effective over time. Summary of the Invention

[0003] In some embodiments, the present technology includes a vaccine composition comprising a first antigen and a second antigen, wherein each of the first antigen and the second antigen is independently selected from the group consisting of a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. 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 a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. In some embodiments, the present technology includes vaccine compositions comprising a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. 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 8 peptide, and a nucleotide sequence encoding an Nsp6 peptide.

[0004] In some embodiments, the peptide is less than 30 amino acids in length. In some embodiments, the peptide is from about 5 amino acids to about 20 amino acids in length. In some embodiments, the peptide is about 9 amino acids in length. In some embodiments, the peptide is about 14 amino acids in length.

[0005] In some embodiments, the nucleotide sequence is a deoxyribonucleotide (DNA) sequence. In some embodiments, the nucleotide sequence is a ribonucleotide (RNA) sequence. In some embodiments, the peptide does not comprise an active site. In some embodiments, the peptide does not fold into a tertiary peptide structure. In some embodiments, the nucleotide sequence is present in a lipid composition. In some embodiments, the lipid composition comprises lipid nanoparticles. In some embodiments, the lipid nanoparticle comprises an antigen-presenting cell (APC) targeting molecule. In some embodiments, the APC is a dendritic cell (DC). In some embodiments, the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209, or an HLA-DR targeting molecule. In some embodiments, each peptide is present on a single peptide chain. In some embodiments, the single chain comprises one or more linker sequences. In some embodiments, each of the peptides is present on a different peptide chain. In some embodiments, each of the nucleotide sequences is present within a polycistronic sequence. In some embodiments, the polycistronic sequence comprises one or more linker sequences. In some embodiments, each of the nucleotide sequences is present on a different nucleotide strand.

[0006] In some embodiments, the first antigen or the second antigen comprises a coronavirus antigenic peptide. In some embodiments, one or more of the antigens comprises a coronavirus peptide. In some embodiments, the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen. In some embodiments, the SARS-CoV-2 antigen is an antigen selected from the group consisting of SARS-CoV-2 alpha antigen, SARS-CoV-2 beta antigen, SARS-CoV-2 gamma antigen, SARS-CoV-2 delta antigen, SARS-CoV-2 omicron antigen, SARS-CoV-2 epsilon antigen, SARS-CoV-2 zeta antigen, SARS-CoV-2 eta antigen, SARS-CoV-2 iota antigen, SARS-CoV-2 kappa antigen, SARS-CoV-2 lambda antigen, and SARS-CoV-2 mu antigen.

[0007] In some embodiments, the vaccine composition comprises an adjuvant. In some embodiments, the vaccine composition is a multivalent vaccine composition. In some embodiments, the present technology includes a method of producing a vaccine composition comprising: (i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject who has successfully cleared the virus; (ii) exposing the sample to one or more antigens from the virus; (iii) quantifying the same T cell population of (i) after exposure to one or more peptide antigens; (v) calculating the difference in the amount of the T cell population between (i) and (iii); (vi) comparing the difference in (iv) with a threshold; and if the difference in (iv)(iv) exceeds the threshold in (v), including peptides of the one or more antigens of (a)(ii) or nucleotide sequences encoding the one or more antigens of (b)(ii) in the vaccine composition.

[0008] In some embodiments, the present technology comprises a method for generating a T cell composition that specifically recognizes one or more viral antigens, comprising: (i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject who has been successfully cleared of the virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population of (i) after exposure to one or more peptide antigens; (iv) calculating the difference in the amount of the T cell population between (i) and (iii); (v) comparing the difference in (iv) to a threshold; and (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens of (ii) in a vaccine composition or a nucleotide sequence encoding the one or more antigens of (ii); (vii) expanding the T cells in the second sample after exposure in (vi); and (viii) isolating the expanded T cells.

[0009] In some embodiments, the method further comprises (ix) screening the expanded T cells for responsiveness to interferon gamma (IFNγ) or interleukin 4 (IL4). In some embodiments, the T cell composition is formulated for administration to a second subject. In some embodiments, the T cell composition is formulated into a vaccine composition. In some embodiments, the T cell population comprises CD4+ T cells or CD8+ T cells. In some embodiments, the T cell population comprises helper T cells. In some embodiments, the helper T cells comprise Th1 or Th2 cells. In some embodiments, the T cell composition comprises CD4+ T cells or CD8+ T cells. In some embodiments, the T cell composition comprises helper T cells. In some embodiments, the helper T cells comprise Th1 or Th2 cells.

[0010] In some embodiments, (ii) exposing the one or more antigens comprises antigen-presenting cells (APCs). In some embodiments, the APC is a dendritic cell (DC). In some embodiments, the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the technology of the present invention comprises a T cell composition immunogenic against a viral antigen produced by: (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 of (i) after exposure to one or more peptide antigens; (iv) calculating the difference in the amount of the T cell population between (i) and (iii); (v) comparing the difference in (iv) to a threshold; and (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens of (ii) or a nucleotide sequence encoding the one or more antigens of (ii) in a vaccine composition; (vii) expanding the T cells in the second sample after exposure in (vi); and (viii) isolating the expanded T cells. In some embodiments, the T cell composition further comprises (ix) screening the expanded T cells for responsiveness to interferon gamma (IFNγ) or interleukin 4 (IL4).

[0011] In some embodiments, the T cell composition is formulated for administration to a second subject. In some embodiments, the T cell composition is formulated into a vaccine composition. In some embodiments, the T cell population comprises CD4+ T cells or CD8+ T cells. In some embodiments, the T cell population comprises helper T cells. In some embodiments, the helper T cells comprise Th1 or Th2 cells. In some embodiments, the T cell composition comprises CD4+ T cells or CD8+ T cells. In some embodiments, the T cell composition comprises helper T cells. In some embodiments, the helper T cells comprise Th1 or Th2 cells. [Brief explanation of the drawings]

[0012] [Figures 1A-1C] The antigen recognition patterns of viral peptides in T cells from subjects who successfully cleared SARS-CoV-2 are illustrated compared to T cells from subjects not infected with SARS-CoV-2. Activation-induced marker (AIM) results are shown for CD4+ T cells (Figures 1A and 1C (right)) and CD8+ T cells (Figures 1B and 1C (left)). [Figure 2A-2B] Changes in antigen-specific CD4+ T cells (Figure 2A) and CD8+ T cells (Figure 2B) after exposure to various viral antigens (X-axis) are illustrated in samples from subjects who were successfully cleared of SARS-CoV-2, samples from subjects not previously exposed to SARS-CoV-2 (i.e., naive subjects), control samples stimulated with dendritic cell (DC) antigen presentation, and peripheral blood mononuclear cell (PBMC) controls. [Figures 3A-3I]Figure 1 illustrates changes in T cell responses, as measured by interleukin 4 (IL4) and interferon gamma (IFNγ) production by Th1 cells at day 14 (left) and Th2 cells at day 21 (right). Samples were incubated using various media mixtures (media mixtures 1, 2, and 3) supplemented with various components (A refers to the addition of spike (S) protein; B refers to the addition of a mixture of spike (S), VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8); C refers to the addition of a mixture of VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8); DMSO refers to media supplemented with DMSO as a negative control). Each sample was exposed to either a medium control (Figure 3A), a spike peptide derived from a common cold coronavirus (Figure 3B), the first SARS-CoV-2 spike (S) peptide (Figure 3C), the second SARS-CoV-2 S peptide (Figure 3D), the SARS-CoV-2 VME1(M) peptide (Figure 3E), the SARS-CoV-2 NCAP(N) peptide (Figure 3F), the SARS-CoV-2 3a peptide (Figure 3G), the SARS-CoV-2 7a peptide (Figure 3H), and the SARS-CoV-2 8 peptide (Figure 3I). [Figure 4] DC cells (left bar) were used to stimulate with various peptide antigens, and T cell memory, measured as a percentage of the CD3+CD62L+CD197+ T cell population in cells, is illustrated compared to cells stimulated with the same peptide antigens in the absence of DC cells (right bar). [Figure 5]HLA types binding to SARS-CoV-2 alpha, delta, and omicron peptides are illustrated, along with the changes occurring in the full protein sequence. Darkly shaded bands represent peptides that bind with strong affinity (<60 nM). Lightly shaded bands represent peptides with moderate affinity (>60 nM-185 nM). Such sequences are conserved when found in SARS-CoV-2 variants alpha, delta, and omicron. White bands collectively indicate changes in HLA type-peptide binding or a decrease in HLA type binding to the peptide. White bands represent sequences where major mutations were found in viral variants. [Figure 6] The following are examples of RNA cassettes used to generate the mRNA polycistronic constructs: 1: MHC class I or class II signal peptide, 2: antigen of 30 amino acids or less, 3: linker sequence. [Figure 7] Illustrates lipid nanoparticles that deliver green fluorescent protein (GFP) RNA to DCs. [Figure 8] Illustrates lipid nanoparticles that deliver SARS-CoV-2 RNA to DCs. [Figure 9] GFP levels in DCs transfected with nanoparticles containing GFP RNA and mannose, CD180, CD209, or HLA-DR targeting ligands are illustrated compared to cells formulated with LNPs alone. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless defined otherwise, 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 invention belongs. For purposes of the present technology, the following terms are defined below.

[0014] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The term "about" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, mass, or length that varies at a level acceptable in the art. In some embodiments, such variation can be up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, mass, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. The terms "bind" and "binding" and "complex" refer to any kind of physical and chemical bonding, reaction, combination, attraction, chelating, and the like. The term "conserved epitope" refers to a conserved protein having an epitope identified by the methods of the present technology that is conserved across multiple viral strains. The term "pathogen" refers to a bacterium, virus, or other microorganism that can cause disease. Other microorganisms can include fungi, molds, parasites, and prions.

[0015] A "peptide" according to the technology of the present invention can be (a) a naturally occurring peptide, (b) a peptide produced by chemical synthesis, (c) a peptide produced by recombinant DNA technology, (d) a peptide produced by biochemical or enzymatic cleavage of a polymer, (e) a peptide produced by a method resulting from a combination of the methods (a) to (d) listed above, or (f) a peptide produced by any other means for producing a peptide. The term "peptide," as used herein, includes any structure composed of two or more amino acids, including chemical modifications and derivatives of amino acids. Because the amino acids forming all or part of a peptide can be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, the term "peptide" also includes pseudopeptides and peptidomimetics, including structures with non-peptide backbones. The term "peptide" also includes peptide dimers or multimers. "Artificial" peptides include peptides produced by chemical synthesis, recombinant DNA technology, biochemical or enzymatic cleavage of polymers, combinations of the foregoing, or generally by any other method. The term "peptide" includes peptides containing a variable number of amino acid residues, optionally with non-amino acid residues at the N- and C-termini, such groups including, inter alia, acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups.

[0016] Chemical synthesis is a useful production method, and by introducing various amino acids that do not naturally occur between chains and modifying the N- or C-terminus, it is possible to improve stability, formulation feasibility, and resistance to protease degradation.

[0017] An "amino acid" is a molecule containing an amine group, a carboxylic acid group, and a side chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, and have the general formula HN-CHR-COOH, where R represents the side chain group. Various amino acids differ in the side chain moiety attached to the α-carbon. "Amino acids" according to the present technology include the known naturally occurring protein amino acids, which are referred to by both the common three-letter abbreviations and the one-letter abbreviations. See generally, Synthetic Peptides: A User's Guide, GA Grant, editor, WH Freeman & Co., New York (1992), the teachings of which, including the text and tables on pages 11-24, are incorporated herein by reference. As noted above, the term "amino acid" also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are generally described in Synthetic Peptides: A User's Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, Int. J. Peptide Protein Res. 35:287-300 (1990), the teachings of all of which are incorporated herein by reference.

[0018] In peptides according to the present technology, conventional amino acid residues have their conventional meanings as set forth in Chapter 2400, of the Manual of Patent Examining Procedure, 8th Ed. Thus, "Ala" is alanine, "Arg" is arginine, "Asn" is asparagine, "Asp" is aspartic acid, "Cys" is cysteine, "Gln" 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 indicated, all amino acid abbreviations represent either isomer, i.e., L-isomer, D-isomer, or any combination thereof that may be used. Thus, for example, "L-Phe" or "lPhe" is L-phenylalanine, "D-Phe" or "dPhe" is D-phenylalanine, dVal is D-valine, dPro is D-proline, "D- / L-Phe" or "d / lPhe" is D-phenylalanine, L-phenylalanine, or a combination thereof, "Phe" is also D-phenylalanine, L-phenylalanine, or a combination thereof, etc. For non-standard amino acids, "Nle" is norleucine, "Nal" is naphthylalanine, "D-Nal" is D-naphthylalanine, D-Nal(2') or DNal(2') is D-2'-naphthylalanine, L-Nal(2') or LNal(2') is L-2'-naphthylalanine, L-Nal(1') is L-1'-naphthylalanine, D-Nal(1') or DNal(T) is D-1'-naphthylalanine, Tle is tert-leucine, Nva is norvaline, Orn is ornithine, Bip is a biphenyl amino acid, etc.

[0019] Amino acids, including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids (peptidomimetics), and the like, including all of the above, may be referred to herein as "residues." "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form and their complements.

[0020] The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit, i.e., monomer, of a polynucleotide. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of polynucleotides discussed herein include single- and double-stranded DNA, single- and double-stranded RNA (including siRNA), and hybrid molecules containing mixtures of single- 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 sugar and / or modified ring substituents but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid mimetics refer to compounds that have a structure different from the general chemical structure of nucleic acids but function similarly to naturally occurring nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0021] "Percentage of sequence identity" is determined by comparing a comparison window of two optimally aligned sequences, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) when comparing optimally aligned two sequences to a reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The term "identity" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., for example, 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region of an entire polypeptide sequence of the invention or of an individual domain of a polypeptide of the invention), when compared and aligned over a specified region as measured by maximum correspondence over a comparison window or by manual alignment and visual inspection using one of the following sequence comparison algorithms: (i) a sequence comparison algorithm for a nucleic acid sequence of at least about 50 nucleotides in length, or (ii) a region of at least about 100 to 500 or 1000 nucleotides in length, or more preferably over a region of at least about 50 nucleotides in length.

[0022] The term "administering" or "administer" includes delivery of a therapy (e.g., a vaccine composition) according to the technology of the present invention to a subject by either local or systemic administration. Administration can be parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration.

[0023] As used herein, a "composition," "vaccine composition," or "pharmaceutical composition" refers to a mixture of an active ingredient with other chemical components, such as pharmaceutically acceptable carriers and / or excipients.

[0024] As used herein, a "pharmaceutically acceptable carrier" of a first or second pharmaceutical composition refers to a carrier or diluent that does not cause significant irritation to an organism, does not inhibit the biological activity and properties of the administered active ingredient, and / or does not adversely interact with other components of the composition. The term "carrier" includes 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 use in a composition depends on the intended route of administration of the composition. Pharmaceutically acceptable carriers and the preparation of formulations containing such materials are described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in 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) polypeptides, 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 dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions that form salts, such as sodium, and / or non-ionic surfactants such as TWEEN® (ICI, Inc., Bridgewater, NJ), polyethylene glycol (PEG), and PLURONICS™ (BASF, Florham Park, NJ). An "excipient" of a first or second pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0025] Vaccine Composition The present technology includes vaccine compositions that provide viral antigens and promote immunological memory. The vaccine compositions induce an immune response that results in the recognition, response, and / or elimination of the viral antigen. In some embodiments, vaccine compositions according to the present technology can induce an immune response in a subject against a viral antigen. In some embodiments, vaccine compositions according to the present technology can be administered to a subject to treat, prevent, and / or reduce disease and / or other symptoms associated with an infectious disease. Viral antigens according to the present technology can consist of protein and / or peptide antigens, glycoprotein antigens, or lipid antigens. In some embodiments, the vaccine composition comprises one or more nucleotide sequences encoding the viral antigen.

[0026] Viral antigens according to the present technology can be arterivirus, mesonivirus, ronivirus, torovirus, or coronavirus antigens. 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, polyomavirus hominis 1 (BKV) antigen, SV40, and Zika virus antigen. In some embodiments, the viral antigen is derived from a virus containing a Variant of Interest (VOI), Variant of Concern (VOC), or Variant of High Community Impact (VOHC) as defined by the Centers for Disease Control and the World Health Organization (WHO).

[0027] In some embodiments, the antigen may be a viral antigen associated with and / or capable of rating a pandemic-like infection. In some embodiments, the coronavirus antigen is selected from the group consisting of an alphacoronavirus (e.g., human coronavirus 229E (HCoV-229E) or human coronavirus NL63 (HCoV-NL63)), a betacoronavirus (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 a bat coronavirus), a gammacoronavirus (e.g., porcine coronavirus HKU15 or avian coronavirus infectious bronchitis virus (IBV)), or a deltacoronavirus (e.g., spotted green pigeon coronavirus HKU13 or white-eye coronavirus HKU16). In some embodiments, the coronavirus antigen is a Wuhan strain antigen.

[0028] In some embodiments, the coronavirus antigen is a common cold coronavirus antigen. Non-limiting examples of common cold coronaviruses include human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKU1). In some embodiments, the coronavirus antigen is a SARS-CoV-2 antigen selected from the group consisting of SARS-CoV-2 alpha antigen, SARS-CoV-2 beta antigen, SARS-CoV-2 gamma antigen, SARS-CoV-2 delta antigen, SARS-CoV-2 omicron antigen, SARS-CoV-2 epsilon antigen, SARS-CoV-2 zeta antigen, SARS-CoV-2 eta antigen, SARS-CoV-2 iota antigen, SARS-CoV-2 kappa antigen, SARS-CoV-2 lambda antigen, and SARS-CoV-2 mu antigen.

[0029] nucleotide The present technology includes vaccine compositions having nucleotide sequences encoding viral antigens. Any one of the nucleotide sequences can promote a response, immune response, or immunological memory by immune cells to the encoded viral antigen (e.g., a CD4+ T cell memory response, a CD8+ T cell memory response, central memory T cells (TCM), effector memory T cells (TCM), expression of memory T cell cross-reactivity, or maintenance of memory T cells). In some embodiments, each nucleotide sequence promotes an immune response or immunological memory to one or more of the viral antigens to which the nucleotide sequence corresponds. The nucleotide sequence can comprise a deoxyribonucleic acid (DNA) sequence. In some embodiments, one or more nucleotide sequences comprise 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 RNA (circRNA) sequence. CircRNAs may have an increased half-life and / or increased expression compared to non-circular RNAs. In some embodiments, the RNA sequence is a messenger CRISPR RNA (crRNA) sequence. In some embodiments, the RNA sequence is self-replicating RNA.

[0030] 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. In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a membrane protein or a peptide thereof, hi some embodiments, the nucleotide sequence encodes a VME1(M) protein or a peptide thereof (i.e., an M peptide). In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a nucleocapsid protein or a peptide thereof, hi some embodiments, the nucleotide sequence encodes an NCAP (N) protein or a peptide thereof (i.e., an N peptide). In some embodiments, the vaccine composition comprises a nucleotide sequence corresponding to an open reading frame (ORF) or 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.

[0031] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a nonstructural protein or peptide thereof, hi some embodiments, the nucleotide sequence encodes the Nsp6 peptide. 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 an S peptide, an M peptide, an N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. 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 8 peptide, and a nucleotide sequence encoding an Nsp6 peptide. In some embodiments, the vaccine composition comprises a nucleotide sequence listed in Table 1.

[0032] [Table 1] JPEG2026501447000003.jpg225152 JPEG2026501447000004.jpg224152 JPEG2026501447000005.jpg224151 JPEG2026501447000006.jpg224151 JPEG2026501447000007.jpg224151 JPEG2026501447000008.jpg222153 JPEG2026501447000009.jpg224153 JPEG2026501447000010.jpg226152 JPEG2026501447000011.jpg221152 JPEG2026501447000012.jpg222152 JPEG2026501447000013.jpg218151 JPEG2026501447000014.jpg224152 JPEG2026501447000015.jpg222152 JPEG2026501447000016.jpg224152 JPEG2026501447000017.jpg222151 JPEG2026501447000018.jpg223152 JPEG2026501447000019.jpg224152 JPEG2026501447000020.jpg223152 JPEG2026501447000021.jpg223152 JPEG2026501447000022.jpg223152 JPEG2026501447000023.jpg222151 JPEG2026501447000024.jpg223152 JPEG2026501447000025.jpg223151 JPEG2026501447000026.jpg223152 JPEG2026501447000027.jpg223152 JPEG2026501447000028.jpg117152

[0033] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 1-67, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 1-67. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 1-67.

[0034] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 1-67. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 1-67. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity, respectively, to one or more of SEQ ID NOs: 1-67. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0035] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 68-78, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 68-78.

[0036] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity, respectively, to one or more of SEQ ID NOs: 68-78. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0037] In some embodiments, the vaccine composition comprises a nucleotide sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 79-100, respectively. 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 to one or more of SEQ ID NOs: 79-100, respectively. 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 to one or more of SEQ ID NOs: 79-100, respectively.

[0038] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 79-100. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 79-100. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to one or more of SEQ ID NOs: 79-100. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0039] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 101-113, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 101-113. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 101-113.

[0040] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 101-113. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 101-113. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical, respectively, to one or more of SEQ ID NOs: 101-113. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0041] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 114-119, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 114-119. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 114-119.

[0042] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 114-119. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 114-119. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical, respectively, to one or more of SEQ ID NOs: 114-119. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0043] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 120-125, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 120-125. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 120-125.

[0044] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 120-125. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 120-125. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical, respectively, to one or more of SEQ ID NOs: 120-125. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0045] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to one or more of SEQ ID NOs: 126-139, respectively. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 126-139.

[0046] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical, respectively, to one or more of SEQ ID NOs: 126-139. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist, in whole or in part, of different nucleotide sequences.

[0047] In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to SEQ ID NO: 140. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 140. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 140. In some embodiments, the vaccine composition comprises a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity, respectively, to SEQ ID NO: 141. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 141. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 141.

[0048] In some embodiments, each of the nucleotide sequences in the vaccine composition is present on a different nucleotide strand. In some embodiments, each of the nucleotide sequences in the vaccine composition is present within a polycistronic sequence. In some embodiments, the polycistronic sequence comprises a nucleotide linker sequence. The nucleotide linker sequence can be used to connect or separate various nucleotide sequences encoding antigens, to generate spacers, to enhance the packaging of nucleotide sequences into lipid compositions, or to facilitate molecular manipulation. In some embodiments, the polycistronic sequence comprises two or more nucleotide linker sequences.

[0049] In some embodiments, the polycistronic nucleotide sequence comprises modified nucleotides (e.g., a 5' cap). In some embodiments, the polycistronic nucleotide sequence comprises a polyA tail or an untranslated region (UTR) (e.g., a 5' UTR or a 3' UTR).

[0050] The polycistronic nucleotide sequence may further comprise a nucleotide sequence that promotes localization of the nucleotide sequence or encodes a peptide (e.g., a signal peptide) that promotes localization of the peptide. In some embodiments, the nucleotide sequence promotes localization to cells that contain a human leukocyte antigen (HLA) molecule. 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 promotes localization to cells that contain a major histocompatibility complex (MHC) molecule. 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 according to the technology of the present invention may be evaluated using predictive localization software, including, but not limited to, TargetP, WoLF PSORT, DeepLoc, CELLO, YLoc, BaCelLo, and LocTree3.

[0051] The polycistronic sequence may include a nucleotide linker sequence (e.g., a poly-G linker sequence) that is non-immunogenic or has low immunogenicity. The nucleotide linker sequence may disrupt an active site within a viral peptide encoded by a nucleotide sequence according to the present technology. In some embodiments, the nucleotide linker sequence may interfere with the tertiary structure formation of a viral antigen and / or may interfere with the function of the viral antigen. In some embodiments, the polycistronic sequence includes a nucleotide linker sequence. The nucleotide linker sequence may be encoded by an RNA sequence. The RNA sequence may be present in a vaccine composition. In some embodiments, the polycistronic sequence includes a nucleotide linker sequence that is a sequence listed in Table 2. In some embodiments, the nucleotide linker sequence is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 142. In some embodiments, the nucleotide linker sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 142. In some embodiments, the nucleotide linker sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 142. In some embodiments, the vaccine composition does not include a nucleotide linker sequence.

[0052] [Table 2]

[0053] peptide: The present technology includes vaccine compositions having one or more viral antigens that are protein or peptide antigens. In some embodiments, the vaccine compositions include two different peptide antigens. Any one of the protein or peptide antigens can promote an immune response or immune memory. In some embodiments, each of the protein or peptide antigens promotes an immune response or immune memory. In some embodiments, the viral antigen comprises an S protein or a peptide thereof (i.e., an S peptide). In some embodiments, the viral antigen comprises an S peptide antigen, which is an S1 peptide or an S2 peptide. In some embodiments, the viral antigen comprises a membrane protein or a peptide thereof, hi some embodiments, the viral antigen comprises an M protein or a peptide thereof (ie, an M peptide). In some embodiments, the viral antigen comprises a nucleocapsid protein or a peptide thereof, hi some embodiments, the viral antigen comprises an N protein or a peptide thereof (i.e., an N peptide). In some embodiments, the viral antigen corresponds to an ORF peptide. In some embodiments, the ORF peptide corresponds to a 7a peptide, a 3a peptide, or an 8 peptide. In some embodiments, the viral antigen comprises a nonstructural protein or peptide thereof (ie, an Nsp6 peptide).

[0054] In some embodiments, the vaccine composition comprises a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of S, M, N, 3a, 7a, 8, and Nsp6. In some embodiments, the vaccine composition comprises an S peptide, an M peptide, an N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. In some embodiments, a vaccine composition according to the present technology comprises a first antigen and a second antigen, each present on a different peptide chain. In some embodiments, a vaccine composition comprises a first antigen and a second antigen, each present on a single peptide chain. In some embodiments, the single peptide chain does not allow for tertiary folding or the generation of functional viral domains.

[0055] In some embodiments, the first antigen and the second antigen are each independently selected from the group consisting of an S peptide, an M peptide, an N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an Nsp6 peptide. In some embodiments, the vaccine composition comprises a peptide listed in Table 3.

[0056] [Table 3] JPEG2026501447000031.jpg221151 JPEG2026501447000032.jpg221151 JPEG2026501447000033.jpg221152 JPEG2026501447000034.jpg221152 JPEG2026501447000035.jpg220152 JPEG2026501447000036.jpg221152 JPEG2026501447000037.jpg220151 JPEG2026501447000038.jpg197151

[0057] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209, respectively. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209.

[0058] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0059] In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220, respectively. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220.

[0060] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0061] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242, respectively. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242.

[0062] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more of SEQ ID NOs: 221-242. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0063] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255, respectively. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255.

[0064] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0065] In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261, respectively. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261.

[0066] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0067] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267, respectively. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267.

[0068] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0069] In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281, respectively. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281. In some embodiments, a vaccine composition comprises a peptide having an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281.

[0070] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, wherein the first peptide and the second peptide each have an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281. In some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.

[0071] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to SEQ ID NO: 282. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 282. In some embodiments, the vaccine composition comprises a peptide having 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.

[0072] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical, respectively, to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having 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.

[0073] In some embodiments, vaccine compositions according to the present technology further comprise a signal peptide. Non-limiting examples of signal peptides include peptides that promote localization to an intracellular compartment (e.g., the endoplasmic reticulum, mitochondria, nucleus, or peroxisome), signal peptides that promote secretion of the peptide, and signal peptides that promote cleavage. In some embodiments, the signal peptide promotes localization and / or binding to cells comprising MHC class I molecules. In some embodiments, the signal peptide promotes localization and / or binding to cells comprising MHC class II molecules. In some embodiments, the cells comprising MHC class II molecules are APC cells. In some embodiments, the APC cells are DCs. In some embodiments, the signal peptide promotes localization and / or binding to cells comprising HLA. In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. Peptide binding to peptides according to the technology of the present invention may be assessed using predictive binding software, including, but not limited to, NetMHC, NetMHCpan, NetMHCIIpan, Immune Epitope Database and Analysis Resource (IEDB), SYFPEITHI, Stabilized Matrix Method (SMM), ProPred-1, ProPred, and NetCTLpan.

[0074] Without intending to be limited to any particular theory, vaccines provided by the technology of the present invention may include about 8 to about 10 peptides of about 25 to about 30 amino acids each to induce a Class I HLA-restricted CD8 response, and about 8 to about 10 peptides of about 15 amino acids each to induce a Class II HLA-restricted CD4 response. Peptides provided by the technology of the present invention, which are about 25 to about 30 amino acids in length, are not expected to mimic viral active sites or recombine with wild-type viruses. In some embodiments, the formation of viral active sites may be further prevented by linkers positioned between the peptides and / or by randomization of the peptide-encoding sequence. This approach differs from vaccines that encode the entire spike protein, including the RBD that induces a humoral immune response and / or binds the ACE receptor and stabilizes the tertiary structure with prolines.

[0075] In some embodiments, vaccine compositions according to the present technology further comprise a peptide having a peptide linker sequence. The peptide linker sequence may be used to combine or separate various antigens, enhance packaging of the peptide into various compositions, or facilitate molecular manipulation. The peptide linker sequence has the potential to be non-immunogenic or have low immunogenicity. In some embodiments, the peptide linker sequence comprises a specific cleavage site linker (e.g., a furin cleavage site; a 2A linker peptide). The specific cleavage site linker may contain a protease recognition site that is selectively cleaved by a particular protease. In some embodiments, the peptide linker sequence comprises a targeting or tagging linker that allows for localization, detection, or purification of the peptide. Peptide linker sequences can include flexible linkers (eg, glycine- and serine-rich linkers) or fixed linkers (eg, glycine-rich linkers or alpha-helical linkers).

[0076] Placing a peptide linker sequence 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 within viral antigens. In some embodiments, the peptide linker sequence may interfere with tertiary structure formation and / or function of the viral antigen. In some embodiments, the connecting peptides are derived from different proteins. In other embodiments, the connecting peptides are derived from the same protein and are non-contiguous. While not intended to be limiting, connecting peptides according to the technology of the present invention may also interfere with tertiary structure formation and / or function of the viral antigen.

[0077] 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 to SEQ ID NO: 142). In some embodiments, the nucleotide sequence is an RNA sequence. In some embodiments, the peptide linker sequence comprises an amino acid sequence 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, respectively, to SEQ ID NO: 349. In some embodiments, the peptide linker sequence comprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 349. The peptide linker sequence comprises an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 349.

[0078] In some embodiments, the vaccine composition does not include a peptide linker sequence. In some embodiments, the vaccine composition comprises a peptide 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 10,000 amino acids, about 10,000 to about 100,000 amino acids, or more. In some embodiments, the vaccine composition comprises a peptide of more than about 30 amino acids. In some embodiments, the vaccine composition comprises a peptide of fewer than about 30 amino acids. In some embodiments, each of the peptides in the vaccine composition is 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 of the peptides in the vaccine composition is 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 of the peptides in the vaccine composition is 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 of the peptides in the vaccine composition is 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 of the peptides in the vaccine composition is fewer than about 10 amino acids.

[0079] In some embodiments, a vaccine composition comprises one or more peptides according to the technology of the present invention and one or more nucleotide sequences according to the technology of the present invention (e.g., nucleotide sequences encoding viral peptides). In some embodiments, a vaccine composition according to the technology of the present invention comprises two or more peptides according to the technology of the present invention and one or more nucleotide sequences according to the technology of the present invention. In some embodiments, a vaccine composition according to the technology of the present invention comprises two or more nucleotide sequences according to the technology of the present invention and one or more peptides according to the technology of the present invention. In some embodiments, a vaccine composition comprises two or more nucleotide sequences according to the technology of the present invention and two or more peptides according to the technology of the present invention. Vaccines according to the present technology, which may lack tertiary structure, may generate T cell epitopes after delivery to a subject without, in whole or in part, forming active viral enzymes and / or functions. In some embodiments, TH2 helper cells formed in a subject after delivery of the vaccine may induce or otherwise enhance a B cell antibody response in addition to a cytotoxic T cell response.

[0080] lipid composition In some embodiments, the nucleotide sequences (e.g., nucleotide sequences encoding viral antigens) and / or peptides of the present technology are present in a lipid composition, e.g., lipid nanoparticles. The lipid composition may include a proteolipid (e.g., protamine), a carrier protein, and / or a small molecule. The lipid composition may comprise a single lipid group or multiple lipid groups, non-limiting examples of which include cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)-modified lipids, ionized lipids, helper lipids, stealth lipids, or cholesterol. Non-limiting examples of lipids include DOSPA 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTAP 1,2-Dioleoyl-3-trimethylammoniumpropane, and DC-cholesterol 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol.

[0081] Non-limiting examples of ionizable lipids include SM-102 9-heptadecanyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, ALC-0315 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, and DODMA 1,2-dioleyloxy-3-dimethylaminopropane.

[0082] Non-limiting examples of helper lipids include cholesterol (1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-2,3,3a,3b,4,6,7,8,9,9a,9b,10,11,11a-tetradecahydro-1H-cyclopentenyl-23threnethren-7-ol, DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine, and DOPE 1,2-dimyristoyl-sn-glycerophosphoethanolamine.

[0083] Non-limiting examples of stealth lipids include PEGIG(R)-2,3-bis(myristoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)carbamate and ALC-0159 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0084] In some embodiments, the nanoparticles can be polymer-based, metal (eg, silver, gold, palladium, titanium, zinc, or copper)-based, silica-based, or lipid-based. In some embodiments, nanoparticle is multi-layer nanoparticle.Non-limiting examples of multi-layer nanoparticle include the nanoparticle that has two or more polymer-based layers; the nanoparticle that has two or more metal-based layers; the nanoparticle that has two or more silica-based layers; the nanoparticle that has two or more lipid-based layers; or the nanoparticle that has a first layer that is selected from the group consisting of a polymer-based layer, a metal-based layer, a silica-based layer and a lipid-based layer, and a second layer that is selected from the group consisting of a polymer-based layer, a metal-based layer, a silica-based layer and a lipid-based layer, and the composition of the second layer is different from the composition of the first layer. In some embodiments, the lipid nanoparticles incorporate an antigen-presenting cell (APC) moiety, e.g., a DC-binding moiety. Non-limiting examples of APC-binding moieties include antibodies and / or single-chain variable fragments (scFv) against surface molecules expressed on dendritic cells (e.g., CD1a, CD1c (BDCA1), CD11b (ITGAM), CD11c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172α (SIRPα), CD180 (RP105), CD205 (DEC-205), CD206 (MRC1), CD209 (DC-SIGN), FcεR1, 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 a lipid in the lipid composition. The APC binding moiety may be identified or designed using methods including, but not limited to, critical process parameters (CPPs), high-throughput sequencing, or predictive modeling.

[0085] The APC moiety can be a ligand that binds to a surface marker (e.g., a peptide, glycoprotein, carbohydrate) of an APC cell (e.g., DC, B cell, T cell, macrophage). In some embodiments, the surface marker is a molecule that is upregulated during APC cell maturation. In some embodiments, the lipid nanoparticles comprise opsonization elements. The opsonization elements can associate with (e.g., bind to or interact with) APCs. The opsonization elements can induce the phagocytic process of macrophages, neutrophils, and dendritic cells. In some embodiments, the opsonization elements incorporate mannose or other carbohydrates. In some embodiments, the lipid nanoparticle comprises an Fc binding fragment, which binds to an Fc receptor on an APC. In some embodiments, the lipid nanoparticles incorporate complement factors C3b, C4, and / or C1q.

[0086] cell composition The present technology includes cell compositions that are immunogenic against an antigen and promote immunological memory. Cell compositions can be generated such that the cells are immunogenic against a particular antigen (e.g., a viral antigen). In addition, the cell compositions can produce an immune response by stimulating an immune reaction. Non-limiting examples of an immune response include the production of antibodies or the activation of lymphoid cells (e.g., T cells). In some embodiments, cell compositions according to the present technology induce an immune response against one or more antigens in a subject. In some embodiments, cell compositions according to the present technology can be administered to a subject to treat, prevent, and / or reduce disease and / or other symptoms associated with an infectious disease.

[0087] The cellular composition may be immunogenic against proteins and / or peptides according to the technology of the present invention. In some embodiments, the cellular composition is immunogenic against a nucleotide sequence according to the technology of the present invention (e.g., a nucleotide sequence encoding a peptide according to the technology of the present invention). In some embodiments, the cellular composition is immunogenic against one or more peptides according to the technology of the present invention and one or more nucleotide sequences according to the technology of the present invention (e.g., a nucleotide sequence encoding a viral peptide). In some embodiments, the cellular composition is immunogenic against two or more peptides according to the technology of the present invention and one or more nucleotide sequences according to the technology of the present invention. In some embodiments, the cellular composition is immunogenic against two or more nucleotide sequences according to the technology of the present invention and one or more peptides according to the technology of the present invention. In some embodiments, the cellular composition is immunogenic against two or more nucleotide sequences according to the technology of the present invention and two or more peptides according to the technology of the present invention.

[0088] Cell compositions according to the present technology can be generated from a subject sample (e.g., a biological sample), which can be a circulatory 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. In some embodiments, the cell composition is a lymphoid cell composition. The cell composition may include one or more of T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages, or granulocytes. In some embodiments, the cell composition is a T cell composition. T cells can 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. In some embodiments, the cell composition comprises one or more CD4+ cell subsets, hi some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.

[0089] In some embodiments, Th1 (CD4+) T cells produce cytokines including IFNγ and drive CD8+ T cell (e.g., cytotoxic T cell) responses. In some embodiments, Th2 (CD4+) T cells produce cytokines including IL4 and drive high-affinity antibody immune responses, including class switching to IgG and IgA.

[0090] In some embodiments, the cell composition is responsive to one or more cytokines and / or chemokines (e.g., immune cell activation, differentiation, signaling, and / or function are stimulated in the presence of the cytokine or chemokine). Non-limiting examples of cytokines and chemokines include interleukins (IL) (e.g., IL-2, IL-4, IL-6, IL-8, IL-10, or IL-12), tumor necrosis factors (TNF) (e.g., TNFα or TNFβ), interferons (INF) (e.g., INFγ, INFα, or INFβ), chemokines (e.g., CXCL8, CXCL10, or CCL2), colony-stimulating factors (CSF) (e.g., granulocyte-macrophage CSF or granulocyte CSF), or transforming growth factor beta (e.g., TGFβ). In some embodiments, the T cell composition is present in a vaccine composition or in an infusion.

[0091] Other composition characteristics and formulations: In some embodiments, compositions (e.g., vaccine compositions or cell compositions) according to the present technology include a pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers and / or excipients can include aqueous or non-aqueous carriers that can facilitate dissolution of the components of the composition (e.g., peptides or nucleotide sequences). Non-limiting examples of pharmaceutically acceptable carriers and / or excipients include sucrose, polysorbate 80, monobasic sodium phosphate monohydrate, dibasic sodium phosphate dihydrate. The composition can also include a filler or diluent (e.g., lactose, mannitol, and microcrystalline cellulose), a disintegrant (e.g., croscarmellose sodium, crospovidone, starch), a buffer (e.g., phosphate buffer or acetate buffer), a solvent (e.g., water, alcohol, glycerin), or a stabilizer (e.g., sugar or gelatin); a preservative (e.g., thimerosal).

[0092] In some embodiments, the compositions of the present technology include an adjuvant. The adjuvant can stimulate or enhance the immune response upon administration of the composition. Non-limiting examples of adjuvants include aluminum salts, oil-in-water emulsions (e.g., MF59 or AS03), pathogen mimics (e.g., CpG oligonucleotides, monophosphoryl lipid A), squalene, virosomes, and liposomes. In some embodiments, the composition is sterilized (eg, sterilized by filtration).

[0093] In some embodiments, compositions according to the present technology comprise the feature of promoting binding and / or co-localization with HLA (e.g., HLA-A, HLA-B, or HLA-C), APC (e.g., a peptide or nucleotide sequence that targets or binds to an MHC class II molecule), and / or T cell receptor (TCR). In some embodiments, the composition comprises a molecule that binds to a receptor on an APC (e.g., a dendritic cell). In some embodiments, the molecule is a carbohydrate. In some embodiments, the carbohydrate is a mannose carbohydrate. The mannose carbohydrate is selected from the group consisting of D-mannose, mannose-6-phosphate (M6P), mannan, mannose polymer, and mannose receptor ligand. In some embodiments, the molecule that binds to a receptor on an APC is selected from the group consisting of mannose, CD180, CD209, and HLA-DR. In some embodiments, compositions according to the present technology comprise a hydrophobic tail. The hydrophobic tail may act as a targeting moiety to promote binding to APCs. In some embodiments, the hydrophobic tail comprises a fatty acid, a phospholipid, cholesterol, a retinoid, a steroid, an alkyl chain, or a nonpolar amino acid side chain.

[0094] In some embodiments, the composition comprises one or more peptides (e.g., antigenic peptides) according to technology of the present invention or nucleotide sequences encoding peptides according to technology of the present invention in an amount of about 0.05% w / v or w / w of the composition, about 0.1% w / v or w / w of the composition, about 1% w / v or w / w of the composition, about 10% w / v or w / w of the composition, about 20% w / v or w / w of the composition, about 30% w / v or w / w of the composition, about 40% w / v or w / w of the composition, about 50% w / v or w / w of the composition, about 60% w / v or w / w of the composition, about 70% w / v or w / w of the composition, about 80% w / v or w / w of the composition, about 90% w / v or w / w of the composition, about 95% w / v or w / w of the composition, or about 99% w / v or w / w of the composition. In some embodiments, the composition comprises one or more peptides (e.g., antigenic peptides) according to technology of the present invention or nucleotide sequences encoding peptides according to technology of the present invention in an amount of at least 0.05% w / v or w / w of the composition, at least 0.1% w / v or w / w of the composition, at least 1% w / v or w / w of the composition, at least 10% w / v or w / w of the composition, at least 20% w / v or w / w of the composition, at least 30% w / v or w / w of the composition, at least 40% w / v or w / w of the composition, at least 50% w / v or w / w of the composition, at least 60% w / v or w / w of the composition, at least 70% w / v or w / w of the composition, at least 80% w / v or w / w of the composition, at least 90% w / v or w / w of the composition, at least 95% w / v or w / w of the composition, or at least 99% w / v or w / w of the composition.

[0095] In some embodiments, the composition comprises one or more peptides (e.g., antigenic peptides) according to technology of the present invention or nucleotide sequences encoding peptides according to technology of the present invention in an amount of at least about 0.05% w / v or w / w of the composition, at least about 0.1% w / v or w / w of the composition, at least about 1% w / v or w / w of the composition, at least about 10% w / v or w / w of the composition, at least about 20% w / v or w / w of the composition, at least about 30% w / v or w / w of the composition, at least about 40% w / v or w / w of the composition, at least about 50% w / v or w / w of the composition, at least about 60% w / v or w / w of the composition, at least about 70% w / v or w / w of the composition, at least about 80% w / v or w / w of the composition, at least about 90% w / v or w / w of the composition, at least about 95% w / v or w / w of the composition, or at least about 99% w / v or w / w of the composition.

[0096] In some embodiments, the composition comprises a lyophilized formulation (a lyophilized powder or cake of the peptide or nucleotide sequence), which in some embodiments is reconstituted (e.g., with water or saline) prior to administration. In some embodiments, reconstitution of the composition produces 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.

[0097] In some embodiments, reconstitution of the composition produces a solution having a pH 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.

[0098] In some embodiments, reconstitution of the composition results in a pH of 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, at least about 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.

[0099] In some embodiments, reconstitution of the composition produces a solution having 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 of the peptide according to the present technology or the nucleotide sequence according to the present technology. In some embodiments, reconstitution of the composition produces a solution having a concentration of peptides according to the present technology or nucleotide sequences according to the present technology 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.

[0100] In some embodiments, reconstitution of the composition produces a solution having a concentration of a peptide according to the present technology or a nucleotide sequence according to the present technology 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. In some embodiments, the composition or solution is diluted for administration. Non-limiting examples of diluents include sodium chloride, water (e.g., sterile water for injection (SWFI)), dextrose solution, bacteriostatic solution, or Ringer's solution (e.g., a solution containing electrolytes, lactated Ringer's solution).

[0101] Related Methods The present technology includes methods for producing vaccine compositions and cell compositions according to the present technology.

[0102] Quantification of lymphocyte cell populations In some embodiments, methods of producing vaccine and / or cell compositions according to the technology of the present invention include quantifying lymphoid cell (e.g., T cell, B cell, NK cell, DC, macrophage, or granulocyte) populations in a sample from a subject that has successfully cleared a virus (e.g., cleared active infection, reduced viral load, resolved symptoms associated with the virus, established immunological memory against the virus). Quantifying lymphoid cell populations can include methods including, but not limited to, flow cytometry, immunohistochemistry, immunofluorescence, protein quantification, and / or detection methods (e.g., assessing lymphoid cell marker levels) or gene expression quantification methods (e.g., assessing expression levels of transcripts associated with lymphoid cells).

[0103] In some embodiments, the lymphocyte cell population is a T cell population. T cells can 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. In some embodiments, the T cell composition comprises one or more CD4+ cell subsets, hi some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells. The sample from the subject can be a circulatory 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.

[0104] In some embodiments, subjects who have successfully cleared a virus have or have had mild symptoms associated with the viral infection, or no symptoms associated with the viral infection. The virus can be an arterivirus, mesonivirus, ronivirus, torovirus, or coronavirus. In some embodiments, 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, polyomavirus hominis 1 (BKV), SV40, and Zika virus. In some embodiments, the virus comprises a variant of interest (VOI), variant of concern (VOC), or variant of very high societal impact (VOHC), as defined by the U.S. Centers for Disease Control and the World Health Organization (WHO). In some embodiments, the virus may be one that is associated with and / or capable of rating as a pandemic-like infection.

[0105] In some embodiments, the coronavirus is selected from the group consisting of an alphacoronavirus (e.g., human coronavirus 229E (HCoV-229E) or human coronavirus NL63 (HCoV-NL63)), a betacoronavirus (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 a bat coronavirus), a gammacoronavirus (e.g., porcine coronavirus HKU15 or avian coronavirus infectious bronchitis virus (IBV)), or a deltacoronavirus (e.g., spotted green pigeon coronavirus HKU13 or white-eyed coronavirus HKU16). In some embodiments, the coronavirus is a Wuhan strain. In some embodiments, the coronavirus is a common cold coronavirus. Non-limiting examples of common cold coronaviruses include human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKU1).

[0106] In some embodiments, the coronavirus is a SARS-CoV-2 strain selected from the group consisting of SARS-CoV-2 alpha, SARS-CoV2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 omicron, SARS-CoV-2 epsilon, SARS-CoV-2 zeta, SARS-CoV-2 eta, SARS-CoV-2 iota, SARS-CoV-2 kappa, SARS-CoV-2 lambda, and SARS-CoV-2 mu.

[0107] Exposure of the sample to viral antigens In some embodiments, methods of producing vaccine compositions and / or cell compositions according to the technology of the present invention include exposing a sample to one or more antigens derived from a virus. Exposing the sample may include direct exposure (e.g., inoculating the sample with a protein antigen, a peptide antigen, a lipid antigen, a glycoprotein antigen, or a nucleotide sequence that produces an 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 DCs (e.g., follicular DCs), macrophages, B cells, monocytes, and Langerhans cells. In some embodiments, the methods involve quantification of lymphocyte populations before, during, and / or after exposure to one or more viral antigens.

[0108] In some embodiments, the lymphocyte population is quantified at about 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, the lymphocyte population is 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 embodiments, lymphocyte populations are quantified at least about 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, the quantification of the lymphocyte cell population comprises a normalization step.

[0109] In some embodiments, the method includes calculating a difference in the amount of a lymphocyte cell population between two different time points. In some embodiments, the two different time points include a time point before 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 before 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. In some embodiments, calculating the difference in lymphocyte cell population between two different time points further comprises comparing the difference to a threshold value, which can be useful in determining whether the lymphocyte cell population changes in response to one or more viral antigens. In some embodiments, if the difference in lymphocyte cell population exceeds a threshold, one or more viral antigens or nucleotide sequences encoding one or more viral antigens are included in the vaccine composition.

[0110] Lymphocyte cell proliferation In some embodiments, the method for generating a cell composition according to the present technology includes exposing a second sample to one or more antigens derived from a virus. In some embodiments, if the difference in lymphocyte cell population exceeds a threshold, one or more viral antigens or nucleotide sequences encoding one or more viral antigens are used to expose the second sample from the second subject. The second sample may include a biological sample. In some embodiments, the second sample is a circulatory 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. The second subject may include a subject who has never been exposed to the virus (i.e., a naive subject), a subject who has never been infected with the virus, a subject who is infected with the virus, or a subject who has symptoms associated with infection with the virus.

[0111] In some embodiments, the lymphocyte cell population in the second sample is expanded 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 expand the lymphocyte cell population 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 expanded lymphocyte cell population can be isolated and / or screened for responsiveness to cytokine and / or chemokine (e.g., IFNγ, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGFβ, CXCL12, CCL19) formation for administration (e.g., via a vaccine composition). [Example]

[0112] Identification of viral antigens To identify viral antigens most likely to elicit a response in a pilot study, we compared the SARS-CoV-2 antigen response patterns of T cells from subjects who successfully cleared SARS-CoV-2 infection with minimal side effects with T cells from subjects naive to SARS-CoV-2 infection. Such antigens included CoV-2 S, M, N, 3a, 7a, 8, and Nsp6 (Figures 1A-1C). To test this concept, we synthesized peptide sequences corresponding to one or more of the identified immunodominant viral antigens, loaded DCs with peptides binding to all or part of the viral antigens (e.g., SARS-CoV-2 S, M, N, 3a, 7a, 8, and nsp6), and then used them to stimulate subject T cells. PBMCs from subjects naive to SARS-CoV-2 infection evoked diverse CD4+ and CD8+ T cell responses to these antigens, with response patterns comparable to those of T cells from subjects who successfully cleared SARS-CoV-2 infection, as measured by activation-induced marker (AIM) assays (Figures 2A and 2B). Subjects demonstrated high responses to DC-mediated viral antigen presentation, whereas no responses to any antigen were observed in PBMCs or naive donors. This suggests that DC-mediated stimulation of T cells with SARS-CoV-2-specific viral antigens can induce robust priming of T cell populations. This also suggests that a protective immune response against the virus can be mounted prior to viral infection.

[0113] Generation of DC and T cell cultures Whole blood samples from subjects naive to SARS-CoV-2 infection were obtained by blood collection or apheresis, and PBMCs were isolated from the blood by Ficoll separation. Monocyte-derived dendritic cells (DCs) were generated using a cell culture plastic adherence method. Nonadherent T cells were removed and frozen for subsequent culture as a T cell source. Differentiation medium containing complete DC culture medium was 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 daily medium changes beginning 1 day after initial culture. On day 5, differentiation medium was removed and maturation medium was added overnight. The maturation medium consisted of IL-6, IL1β, TNFα, and prostaglandin E2 (PGE2). Prostaglandin E2 can substitute for polyinosinic:polycytidylic acid (poly-I:C), which binds to toll-like receptor 3 (TLR3) (e.g., TLR3 on DCs).

[0114] On day 6 after differentiation and maturation of adherent T cells (typically monocytes) into DCs, collected DCs were mixed with SARS-CoV-2 viral peptides S, M, N, 3a, 7a, 8, and S+ (all peptides mixed). The non-adherent T cell fraction was thawed and mixed with DCs at a 2:1 ratio of non-adherent T cells (T cells) to DCs. A total volume of 1 mL and a cell density of 3x10 cells were cultured using complete Cellgenix GMP DC medium supplemented with a cytokine cocktail. 6 The final concentration of each peptide was 0.1 μg / mL. Peptides resuspended in DMSO were added to the wells of the wells. Cells were seeded into well plates. 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, the process was repeated by thawing PBMCs, mixing them with fresh DCs and peptides, and adding them to the treatment cultures. On day 14, a CD3 / CD28 / CD2 T cell activation mix was added to the cultures (15 μl / mL) along with fresh medium containing cytokines, and the cultures were returned to a humidified incubator (5% CO2, 37°C).

[0115] Activation-induced marker assay and flow cytometric phenotyping Activation-induced marker (AIM) assays and phenotyping flow cytometry assays were performed on days 14, 21, and 28. Cell counts and viability were also quantified. For AIM assays and phenotyping flow cytometry assays, 1 million cells were seeded per well in two separate well plates. In both assays, stimulation with an equimolar amount of dimethyl sulfoxide (DMS)O was performed as a negative control, and cells were stained with an antibody cocktail for 15 minutes at room temperature in the dark. After a final wash, cells were resuspended in fluorescence-activated cell sorting (FACS) buffer and quantified and analyzed using FlowJo flow cytometry analysis software (TreeStar).

[0116] Th1 and Th2 T cell profiling T cells reactive to viral antigens (1 × 10 per well) 5 ) were plated onto enzyme-linked immunosorbent spot (ELISpot) plates containing IFNγ and IL-4 (Figures 3A-3I). After 24 h of incubation, the plates were washed and incubated with a secondary antibody against IFNγ, which enzymatically catalyzed the first color development, and anti-IL-4, which enzymatically catalyzed the second color development. After drying, the first and second color-developed spots were counted. Each spot represented a single T cell secreting the cytokine analyte, where Th1 cells secreted IFNγ and Th2 cells secreted IL-4. Such assays were performed on T cells 14 and 21 days after stimulation. T cell immune memory was then measured as a percentage of the CD3+CD62L+CD197+ T cell population (Figure 4).

[0117] Selection of T cell antigens for incorporation into vaccines Using the MHC class I binding predictor MHCnetpan against the top 1% of the population of complete SARS-CoV-2 amino acid sequences (71 most common MHC alleles, HLA-A, B, C), we identified T cell antigens most likely to be reactive with approximately 50% accuracy (Table 4). The frequency of subjects expressing at least two of the MHC alleles is approximately 90% of the population. The selected antigens have the potential to be immunogenic and confer protection in a high percentage of the population (Figure 5).

[0118] [Table 4]

[0119] GMP RNA vaccine manufacturing RNA constructs designed to express the S, M, N, 3a, 7a, 8, and Nsp6 antigens were used to generate RNA vaccines (Figure 6). All reagents used were derived from contaminant-free sources and were produced using defined media and non-natural sources. The RNA was subjected to a purification process. After purification, the RNA can be encapsulated with lipid nanoparticles or cationic proteolipids, such as protamine, and / or carrier proteins and small molecules.

[0120] An mRNA construct containing a linker sequence (SEQ ID NO: 10) was inserted between the nucleotide sequences encoding the viral antigen. This linker has the potential for low immunogenicity, as shown by the use of the NetMHC MHC I binding affinity tool. MHC I binding predictions were performed using the NetMHCpan method using the IEDB analysis resource. The viral antigen sequence of interest was contained in the region with binding affinity below nM binding (Table 5). Here, lower ranking indicates better binding.

[0121] [Table 5]

[0122] Among the viral peptides identified for RNA vaccines, the strongest binding peptides were below 60 nM for the most common HLAs, whereas the linkers that did not bind had binding densities above 40,000 nM for all HLAs (Tables 6-12).

[0123] [Table 6]

[0124] [Table 7]

[0125] [Table 8]

[0126] [Table 9]

[0127] [Table 10]

[0128] [Table 11]

[0129] [Table 12]

[0130] Selected peptides were mixed into 30-amino acid peptides, which were placed between a linker sequence and scrambled peptides derived from different viral peptides (e.g., in different parts of the Se construct or protein) on either side of the linker sequence. This length provided the optimal number of antigens, but also did not allow tertiary folding of the viral peptides or the generation of functional domains.

[0131] This vaccine design encompasses nucleotide sequences expressing antigens from all SARS-CoV-2 variants to date, including XBB (BA.2.10). Although we designed the vaccine using antigens from the alpha strain of SARS-CoV-2, only 1.69% of the antigens selected by our method across all seven proteins mutated across the alpha, delta, and omicron variants, with 98.31% of the antigens still capable of generating an effective immune response against the latest strains (Table 13). In addition, 28 / 34 mutations in antigens from the alpha to omicron strains are mutations in S, and only 7 are present in antigens selected from all other peptides in the mix.

[0132] [Table 13] JPEG2026501447000049.jpg206149

[0133] Evaluation of vaccine compositions in primate models To evaluate the vaccine composition in a primate model, three groups of six macaques were injected intramuscularly on days 0 and 21. Group 1 received two injections of a vaccine derived from SARS-CoV-2 alpha strain sequences, consisting of 200 μg of mRNA preserved antigen plus 100 μg of spike vaccine mRNA. Group 2 received a vaccine derived from SARS-CoV-2 alpha strain sequences consisting of 200 μg of mRNA preserved antigen alone. Group 3 received a vaccine derived from SARS-CoV-2 alpha strain sequences consisting of 100 μg of spike antigen mRNA alone. The mRNA antigen vaccine consisted of six designated preserved antigens. Blood samples were collected on days 14 and 35 to test for T cell gamma interferon production in response to antigen injection. Antigen-specific T cell frequencies were determined by pulse application of antigen-specific peptides, and IFNγ was screened using an ELISpot assay. Results showed that T cell spots equivalent to the IFNγ release of each T cell were multivalently targeted in Th-1 responses to each target antigen. On day 35 post-challenge, half of the animals (N=3) in each group were challenged with either the SARS-CoV-2 alpha or Omicron XBB strain. Animals were then followed for 30 days for pulmonary symptoms and weight loss. Starting on day 42, they were screened for active SARS-CoV-2 virus every 7 days using nasal swab sampling and antigen testing against viral peptides. After 30 days, none of the animals vaccinated with the SARS-CoV-2 alpha strain and challenged with SARS-CoV-2 alpha had weight loss, lung infection, or the presence of viral peptides. Identical results were observed in animals in groups 1 and 2 challenged with the XBB strain. However, two of three animals in group 3, vaccinated only with a vaccine against SARS-CoV-2 alpha spike, had weight loss, lung infection, and the presence of viral peptides (Table 14). This suggests that conserved proteins are protective across viral strains and may be attenuated during normal viral strain evolution.

[0134] [Table 14]

[0135] Advances in DC-targeting nanoparticles may improve the delivery of RNA encoding vaccine antigens to DCs. These lipids were used to encapsulate and deliver RNA encoding green fluorescent protein (GFP) in vitro. 24 hours after nanoparticle delivery, DCs were analyzed by flow cytometry to assess GFP expression. DCs were exposed to either nanoparticles carrying GFP RNA as a control or nanoparticles carrying SARS-CoV-2 RNA encoding viral peptides (Figures 7 and 8). Nanoparticles were designed to target DCs using mannose, CD180, CD209, or HLA-DR targeting ligands. For RNA delivery, nanoparticles carrying targeting ligands were more efficient, as measured by GFP levels (Figure 9).

[0136] Macaques were vaccinated with nanoparticles containing six SARS-CoV-2 viral antigens. Controls included nanoparticles without targeting ligands and / or loaded with no viral antigens. When exposed to Omicron XBB at low concentrations of RNA, DC-targeted lipid nanoparticles conferred greater protection to macaques than did non-targeted lipid nanoparticles (Table 15).

[0137] [Table 15] Further embodiments The technology of the present invention includes, but is not limited to, the following specific embodiments described in sections

[0241] -

[0319] herein below.

[0138] 1. A vaccine composition comprising a first antigen and a second antigen, wherein each of the first antigen and the second antigen is independently selected from the group consisting of a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an 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, A vaccine composition, 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. 3. A vaccine composition comprising a spike (S) peptide, a VME1 (M) peptide, an NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and an 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 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding an 8 peptide, and a nucleotide sequence encoding an Nsp6 peptide.

[0139] 5. A vaccine composition according to any one of the preceding embodiments, wherein the peptide is less than 30 amino acids in length. 6. The vaccine composition of embodiment 5, wherein the peptide is from about 5 amino acids to about 20 amino acids in length. 7. The vaccine composition of embodiment 6, wherein the peptide is about 9 amino acids in length. 8. The vaccine composition of embodiment 6, wherein the peptide is about 14 amino acids in length. 9. The vaccine composition of embodiment 2 or 4, wherein the nucleotide sequence is a deoxyribonucleotide (DNA) sequence.

[0140] 10. The vaccine composition of embodiment 2 or 4, wherein the nucleotide sequence is a ribonucleotide (RNA) sequence. 11. A vaccine composition according to any one of the preceding embodiments, wherein the peptide does not comprise an active site. 12. The peptide composition of any one of the preceding embodiments, wherein the peptide does not fold into a tertiary peptide structure.

[0141] 13. The spike (S) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209; the VME1(M) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220; the NCAP (N) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242; 3a peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255; the 7a peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261; 8. The peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267; or the Nsp6 peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281; The vaccine composition of any one of the preceding embodiments.

[0142] 14. A spike (S) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209; a VME1(M) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220; NCAP (N) peptides at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242; a 3a peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255; a 7a peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261; 8 peptides at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267; or Nsp6 peptides at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281. A vaccine composition comprising two or more antigens selected from the group consisting of:

[0143] 15. A first nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 1-67; a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 68-78; a third nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 79-100; a fourth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 101-113; a fifth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 114-119; a sixth nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 120-125; or A seventh nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 126-139. 1. A vaccine composition comprising two or more nucleotide sequences selected from the group consisting of:

[0144] 16. The vaccine composition of any one of embodiments 2, 4, 9, or 15, wherein the nucleotide sequence is present in a lipid composition. 17. A vaccine composition described in embodiment 16, wherein the lipid composition comprises lipid nanoparticles. 18. The vaccine composition of embodiment 17, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule. 19. The vaccine composition of embodiment 18, wherein the APC is a dendritic cell (DC).

[0145] 20. The vaccine composition of embodiment 18 or 19, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209 or an HLA-DR targeting molecule. 21. The vaccine composition of embodiment 1, 3, 13 or 14, wherein each of the peptides is present on a single peptide chain. 22. The vaccine composition of embodiment 21, wherein the single chain comprises one or more linker sequences. 23. The vaccine composition of embodiment 1, 3, 13, or 14, wherein each of the peptides is present on a different peptide chain. 24. The vaccine composition of embodiment 2 or 4, wherein each of the nucleotide sequences is present within a polycistronic sequence.

[0146] 25. The vaccine composition of embodiment 24, wherein the polycistronic sequence comprises one or more linker sequences. 26. The vaccine composition of embodiment 2, 4, 9, or 15, wherein each of the nucleotide sequences is present on a different nucleotide strand. 27. The vaccine composition of embodiment 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigenic peptide. 28. The vaccine composition of embodiment 1 or 2, wherein one or more of the antigens comprises a coronavirus peptide. 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.

[0147] 30. The vaccine composition of embodiment 29, wherein the SARS-CoV-2 antigen is an antigen selected from the group consisting of SARS-CoV-2 alpha antigen, SARS-CoV-2 beta antigen, SARS-CoV-2 gamma antigen, SARS-CoV-2 delta antigen, SARS-CoV-2 omicron antigen, SARS-CoV-2 epsilon antigen, SARS-CoV-2 zeta antigen, SARS-CoV-2 eta antigen, SARS-CoV-2 iota antigen, SARS-CoV-2 kappa antigen, SARS-CoV-2 lambda antigen, and SARS-CoV-2 mu antigen. 31. A vaccine composition comprising an amino acid sequence 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 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 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 at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 141.

[0148] 35. A vaccine composition described in embodiment 33 or 34, wherein the nucleotide sequence is present in a lipid composition. 36. A vaccine composition described in embodiment 35, wherein the lipid composition comprises lipid nanoparticles. 37. The vaccine composition of embodiment 36, wherein the lipid nanoparticles comprise an antigen-presenting cell (APC) targeting molecule. 38. The vaccine composition of embodiment 37, wherein the APC is a dendritic cell (DC). 39. The vaccine composition of embodiment 37 or 38, wherein the APC targeting molecule is selected from the group consisting of mannose, CD180, CD209 or an HLA-DR targeting molecule.

[0149] 40. The vaccine composition of any one of the preceding embodiments, further comprising an adjuvant. 41. (i) quantifying the T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject who has successfully cleared the virus; (ii) exposing the sample to one or more antigens from the virus; (iii) quantitating the same T cell population of (i) after challenge with one or more peptide antigens; (v) calculating the difference in the amount of T cell population between (i) and (iii); (vi) comparing the difference of (iv) with a threshold value; (iv) if the difference in (iv) exceeds the threshold in (v), including peptides of one or more antigens in (a)(ii) or nucleotide sequences encoding one or more antigens in (b)(ii) in a vaccine composition; A method for producing a vaccine composition comprising:

[0150] 42. The method of embodiment 41, wherein the vaccine composition is a multivalent vaccine composition. 43. (i) quantifying the 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 a virus; (iii) quantitating the same T cell population of (i) after challenge with one or more peptide antigens; (iv) calculating the difference in the amount of T cell population between (i) and (iii); (v) comparing the difference of (iv) with a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens in (ii) or nucleotide sequences encoding the one or more antigens in (ii) in a vaccine composition; (vii) expanding the T cells in the second sample after exposure of (vi); (viii) isolating the expanded T cells; A method for generating a T cell composition that specifically recognizes one or more viral antigens, comprising: 44. The method of embodiment 43, further comprising (ix) screening the expanded T cells for responsiveness to interferon gamma (IFNγ) or interleukin 4 (IL4).

[0151] 45. The method of embodiment 43, wherein the T cell composition is formulated for administration to a second subject. 46. ​​The method of embodiment 45, wherein the T cell composition is formulated into a vaccine composition. 47. The method of any one of embodiments 41 to 46, wherein the T cell population comprises CD4+ T cells or CD8+ T cells. 48. The method of embodiments 41 to 47, wherein the T cell population comprises helper T cells. 49. The method of embodiment 48, wherein the helper T cells comprise Th1 or Th2 cells.

[0152] 50. The method of embodiment 43 or 47, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells. 51. The method of embodiment 43 or 47, wherein the T cell composition comprises helper T cells. 52. The method of embodiment 51, wherein the helper T cells comprise Th1 or Th2 cells. 53. A method according to any one of embodiments 41 to 47, wherein the exposure of one or more antigens in (ii) comprises an antigen-presenting cell (APC). 54. The method of embodiment 53, wherein the APC is a dendritic cell (DC).

[0153] 55. The method of embodiment 54, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF). 56. The method of any one of embodiments 41 to 55, wherein the virus is a coronavirus. 57. The method of embodiment 56, wherein the coronavirus is SARS-CoV-2. 58. The method of embodiment 57, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2 alpha, SARS-CoV2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 omicron, SARS-CoV-2 epsilon, SARS-CoV-2 zeta, SARS-CoV-2 eta, SARS-CoV-2 iota, SARS-CoV-2 kappa, SARS-CoV-2 lambda, and SARS-CoV-2 mu. 59. The method of any one of embodiments 41 to 58, wherein the one or more antigens comprise 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.

[0154] 60. The method of any one of embodiments 41 to 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. (i) quantifying the 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 a virus; (iii) quantitating the same T cell population of (i) after challenge with one or more peptide antigens; (iv) calculating the difference in the amount of T cell population between (i) and (iii); (v) comparing the difference of (iv) with a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens in (ii) or nucleotide sequences encoding the one or more antigens in (ii) in a vaccine composition; (vii) expanding the T cells in the second sample after exposure of (vi); (viii) isolating the expanded T cells; A T cell composition immunogenic against a viral antigen, produced by

[0155] 62. (ix) A T cell composition described in embodiment 61, further comprising a step of screening the expanded T cells for responsiveness to interferon gamma (IFNγ) or interleukin 4 (IL4). 63. The T cell composition of embodiment 61 or 48, wherein the T cell composition is formulated for administration to a second subject. 64. The T cell composition of embodiment 63, wherein the T cell composition is formulated into a vaccine composition.

[0156] 65. A T cell composition described in any one of embodiments 61 to 64, wherein the T cell population comprises CD4+ T cells or CD8+ T cells. 66. A T cell composition described in any one of embodiments 61 to 65, wherein the T cell composition comprises helper T cells. 67. A T cell composition described in embodiment 66, wherein the helper T cells comprise Th1 or Th2 cells. 68. A T cell composition described in any one of embodiments 61 to 67, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells. 69. A T cell composition described in any one of embodiments 61 to 68, wherein the T cell composition comprises helper T cells.

[0157] 70. The T cell composition of embodiment 69, wherein the helper T cells comprise Th1 or Th2 cells. 71. A T cell composition described in any one of embodiments 61 to 70, wherein the exposure of one or more antigens in (ii) comprises an antigen-presenting cell (APC). 72. The T cell composition of embodiment 71, wherein the APC is a dendritic cell (DC). 73. The T cell composition of embodiment 72, wherein the DC cells are stimulated with IL4 or granulocyte-macrophage colony-stimulating factor (GM-CSF). 74. A T cell composition described in any one of embodiments 61 to 73, wherein the virus is a coronavirus.

[0158] 75. The T cell composition of embodiment 74, wherein the coronavirus is SARS-CoV-2. 76. The T cell composition of embodiment 75, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2 alpha, SARS-CoV2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 omicron, SARS-CoV-2 epsilon, SARS-CoV-2 zeta, SARS-CoV-2 eta, SARS-CoV-2 iota, SARS-CoV-2 kappa, SARS-CoV-2 lambda, and SARS-CoV-2 mu.

[0159] 77. A T cell composition described in any one of embodiments 61 to 76, wherein the one or more antigens comprise 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. 78. A T cell composition described in any one of embodiments 61 to 76, 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.

Claims

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

2. 1. A vaccine composition comprising: (i) a first nucleotide sequence encoding a first antigen; and (ii) a second nucleotide sequence encoding a second antigen, A vaccine composition, 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.

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

5. The vaccine composition of any one of claims 1 to 4, wherein the peptide is less than 30 amino acids in length.

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

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

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

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

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

11. The vaccine composition according to any one of claims 1 to 10, wherein the peptide does not contain an active site.

12. The peptide composition of any one of claims 1 to 11, wherein the peptide does not fold into a tertiary peptide structure.

13. the spike (S) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209; the VME1(M) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs:210-220; the NCAP(N) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242; the 3a peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs:243-255; the 7a peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs:256-261; the 8 peptides are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267; or 13. The vaccine composition of any one of claims 1 to 12, wherein the Nsp6 peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281.

14. a spike (S) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 143-209; a VME1(M) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 210-220; an NCAP (N) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 221-242; a 3a peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 243-255; a 7a peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 256-261; 8 peptides at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 262-267; or Nsp6 peptides at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 268-281 A vaccine composition comprising two or more antigens selected from the group consisting of:

15. a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 1-67; a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 68-78; a third nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 79-100; a fourth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 101-113; a fifth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 114-119; a sixth nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 120-125; or A seventh nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one or more of SEQ ID NOs: 126-139.

1. A vaccine composition comprising two or more nucleotide sequences selected from the group consisting of:

16. 16. The vaccine composition of claim 2, 4, 9 or 15, wherein the nucleotide sequence is present in a lipid composition.

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

18. 18. The vaccine composition of claim 17, wherein the lipid nanoparticle comprises 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 an HLA-DR targeting molecule.

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

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

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

24. 5. The vaccine composition of claim 2 or 4, wherein each of the nucleotide sequences is present within a polycistronic sequence.

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

26. 16. The vaccine composition of claim 2, 4, 9 or 15, wherein each of the nucleotide sequences is present on a different nucleotide strand.

27. 3. The vaccine composition of claim 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigenic peptide.

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

29. 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. 30. The vaccine composition of claim 29, wherein the SARS-CoV-2 antigen is an antigen selected from the group consisting of SARS-CoV-2 alpha antigen, SARS-CoV-2 beta antigen, SARS-CoV-2 gamma antigen, SARS-CoV-2 delta antigen, SARS-CoV-2 omicron antigen, SARS-CoV-2 epsilon antigen, SARS-CoV-2 zeta antigen, SARS-CoV-2 eta antigen, SARS-CoV-2 iota antigen, SARS-CoV-2 kappa antigen, SARS-CoV-2 lambda antigen, and SARS-CoV-2 mu 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. 35. The vaccine composition of claim 33 or 34, wherein the nucleotide sequence is present in a lipid composition.

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

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

38. 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 an HLA-DR targeting molecule.

40. 40. The vaccine composition of any one of claims 1 to 39, further comprising an adjuvant.

41. (i) quantifying 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) quantitating the same T cell population of (i) after exposure to one or more of the peptide antigens; (v) calculating the difference in the amount of the T cell population between (i) and (iii); (vi) comparing the difference of (iv) with a threshold; (iv) if the difference in (iv) exceeds the threshold in (v), including peptides of one or more of the antigens in (a)(ii) or nucleotide sequences encoding one or more of the antigens in (b)(ii) in the vaccine composition; A method for producing a vaccine composition comprising:

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

43. (i) quantifying the T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject who has been successfully cleared of virus; (ii) first exposing said sample to one or more antigens from said virus; (iii) quantitating the same T cell population of (i) after exposure to one or more of the peptide antigens; (iv) calculating the difference in the amount of the T cell population between (i) and (iii); (v) comparing the difference of (iv) with a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens of (ii) or nucleotide sequences encoding the one or more antigens of (ii) in the vaccine composition; (vii) expanding the T cells in the second sample after the exposing of (vi); (viii) isolating the expanded T cells; 1. A method for generating a T cell composition that specifically recognizes one or more viral antigens, comprising:

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

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

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

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

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

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

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

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

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

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

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

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

58. 58. The method of claim 57, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 omicron, SARS-CoV-2 epsilon, SARS-CoV-2 zeta, SARS-CoV-2 eta, SARS-CoV-2 iota, SARS-CoV-2 kappa, SARS-CoV-2 lambda, and SARS-CoV-2 mu.

59. 59. The method of any one of claims 41 to 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. 59. The method of any one of claims 41 to 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. (i) quantifying the T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject who has been successfully cleared of virus; (ii) first exposing said sample to one or more antigens from said virus; (iii) quantitating the same T cell population of (i) after exposure to one or more of the peptide antigens; (iv) calculating the difference in the amount of the T cell population between (i) and (iii); (v) comparing the difference of (iv) with a threshold; (vi) if the difference in (iv) exceeds the threshold in (v), exposing a second PBMC sample from a second subject to peptides of the one or more antigens of (ii) or nucleotide sequences encoding the one or more antigens of (ii) in the vaccine composition; (vii) expanding the T cells in the second sample after the exposing of (vi); (viii) isolating the expanded T cells; A T cell composition immunogenic against a viral antigen, produced by

62. 62. The T cell composition of claim 61, further comprising (ix) screening the expanded T cells for responsiveness to interferon gamma (IFNγ) or interleukin 4 (IL4).

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

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

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

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

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

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

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

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

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

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

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

76. 76. The T cell composition of claim 75, wherein the SARS-CoV-2 is a strain selected from the group consisting of SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 omicron, SARS-CoV-2 epsilon, SARS-CoV-2 zeta, SARS-CoV-2 eta, SARS-CoV-2 iota, SARS-CoV-2 kappa, SARS-CoV-2 lambda, and SARS-CoV-2 mu.

77. 77. The T cell composition of any one of claims 61 to 76, wherein the one or more antigens comprise 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.

78. 77. The T cell composition of any one of claims 61 to 76, 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.

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