WO2024130254A2 - A multi-antigenic rna sars-cov-2 vaccine and associated methods - Google Patents

A multi-antigenic rna sars-cov-2 vaccine and associated methods Download PDF

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WO2024130254A2
WO2024130254A2 PCT/US2023/084666 US2023084666W WO2024130254A2 WO 2024130254 A2 WO2024130254 A2 WO 2024130254A2 US 2023084666 W US2023084666 W US 2023084666W WO 2024130254 A2 WO2024130254 A2 WO 2024130254A2
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Prior art keywords
peptide
sars
cov
vaccine composition
cell
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PCT/US2023/084666
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French (fr)
Inventor
David James Ryan
Alfred E. Slanetz
Farzonai MUZAFFAR
Wolfgang Fritz KLIETMANN
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Geneius Biotechnology, Inc.
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Publication of WO2024130254A2 publication Critical patent/WO2024130254A2/en

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Definitions

  • COVID-19 cortivirus disease 2019
  • COVID-19 is a highly contagious infection with a spectrum of symptoms from subclinical malaise to severe clinical disease and death.
  • Symptoms of COVID-19 include headache, loss of smell and taste, nasal congestion, cough, muscle pain, fever, diarrhea, and coagulation disorders, disseminated intravascular coagulation, and thromboembolism.
  • the present technology comprises a vaccine composition comprising a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • S Spike
  • M VME1
  • N NCAP
  • the present technology comprises a vaccine composition comprising a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • the present technology comprises a vaccine composition comprising a nucleotide sequence encoding a Spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding a NCAP (N) peptide, a nucleotide sequence encoding, 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 a Nsp6 peptide.
  • the peptide length is less than 30 amino acids.
  • the peptide length is about 5 amino acids to about
  • the peptide length is about 14 amino acids.
  • the nucleotide sequences are deoxyribonucleotide (DNA) sequences.
  • the nucleotide sequences are ribonucleotide (RNA) sequences.
  • the peptide does not comprise an active site.
  • the peptide does not fold into tertiary peptide structures.
  • the nucleotide sequences are present in a lipid composition.
  • the lipid composition comprises a lipid nanoparticle.
  • the lipid nanoparticle comprises an antigen presenting cell (APC) targeting molecule.
  • APC antigen presenting cell
  • the APC is a dendritic cell (DC).
  • DC dendritic cell
  • the APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
  • each of the peptides are present on a single peptide chain.
  • each of the peptides are present on different peptide chains
  • each of the nucleotide sequences are present in a polycistronic sequence.
  • each of the nucleotide sequences are present on different nucleotide chains.
  • the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the SARS-CoV-2 antigen is an antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS-CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV- 2 lambda antigen, and a SARS-CoV-2 mu antigen.
  • the vaccine composition comprises an adjuvant.
  • the vaccine composition is a multivalent vaccine composition.
  • the present technology comprises a method of generating a vaccine composition, comprising the steps of (i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject that has successfully cleared a virus; (ii) exposing the sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (v) calculating a difference in the T cell population quantity between (i) and (iii); (vi) comparing the difference in (iv) to a threshold value; and (iv) including (a) a peptide of the one or more antigens in (ii), or (b) a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v).
  • PBMC peripheral blood mononuclear cell
  • the present technology comprises a method of generating a T cell composition that specifically recognizes one or more viral antigens, comprising the steps of (i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (iv) calculating a difference in the T cell population quantity between (i) and (iii); (v) comparing the difference in (iv) to a threshold value; (vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v
  • the methods further comprise (ix) screening the expanded T cells for reactivity to interferon gamma (I FNy) or interleukin 4 (IL4).
  • the T cell composition is formulated for administration to the second subject.
  • the T cell composition is formulated in a vaccine composition.
  • the T cell population comprises CD4+ T cells or CD8+ T cells.
  • the T cell population comprises T helper cells.
  • the T helper cells comprise Th1 or Th2 cells.
  • the T cell composition comprises CD4+ T cells or
  • the T cell composition comprises T helper cells.
  • the T helper cells comprise Th1 or Th2 cells.
  • the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
  • APC antigen presenting cell
  • the APC is a dendritic cell (DC).
  • DC dendritic cell
  • the DC cells are stimulated with IL4 or Granulocyte-
  • GM-CSF Macrophage Colony-Stimulating Factor
  • the present technology comprises a T cell composition immunogenic to viral antigens, generated by the steps of: (i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (iv) calculating a difference in the T cell population quantity between (i) and (iii); (v) comparing the difference in (iv) to a threshold value; (vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v); (vii) expanding the T cells in
  • the T cell composition is formulated for administration to the second subject.
  • the T cell composition is formulated in a vaccine composition.
  • the T cell population comprises CD4+ T cells or CD8+ T cells.
  • the T cell population comprises T helper cells.
  • the T helper cells comprise Th1 or Th2 cells.
  • the T cell composition comprises CD4+ T cells or
  • the T cell composition comprises T helper cells.
  • the T helper cells comprise Th1 or Th2 cells.
  • FIGS. 1 A-1 C illustrate antigen recognition patterns of viral peptides in T- cells from subjects who have successfully cleared SARS-CoV-2 relative to T-cells from subjects who have not been infected with SARS-CoV-2. Shown are Activation Induced Marker (AIM) results for CD4+ T-cells (FIG. 1A and 1 C (right)) and CD8+ T-cells (FIG. 1 B and 1 C (left)).
  • AIM Activation Induced Marker
  • FIGS. 2A and 2B illustrate changes in antigen-specific CD4+ T-cells (FIG. 2A) and CD8+ T-cells (FIG. 2B) following exposure to different viral antigens (X-axis) in samples from subjects who have successfully cleared SARS-CoV-2, samples from subjects not previously exposed to SARS-CoV-2 (i.e. , a naive subject), control samples primed with dendritic cell (DC) antigen presentation, and peripheral blood mononuclear cell (PBMC) controls.
  • X-axis viral antigens
  • FIGS. 3A-3I illustrate changes in T-cell response measured by interleukin 4 (IL4) and interferon gamma (IFNy) production from in Th1 cells at day 14 (left) and Th2 cells at day 21 (right).
  • Samples were incubated using different media mixtures (media mixtures 1 , 2, and 3) supplemented with different components
  • A refers to Spike (S) protein supplementation
  • B refers to a mixture of the Spike (S), VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8) supplementation
  • C refers to a mixture of VME1 (M), NCAP (N), ORF3a (3a), 0RF7a (7a), and ORF8 (8) supplementation
  • DMSO refers to media supplemented with DMSO as a negative control.
  • FIG. 3A a media control
  • FIG. 3B a Spike peptide from a common cold coronavirus
  • FIG. 3C a first SARS-CoV-2 Spike (S) peptide
  • FIG. 3D a second SARS- CoV-2 S peptide
  • FIG. 3E a SARS-CoV-2 VME1 (M) peptide
  • N SARS-CoV- 2 NCAP (N) peptide
  • FIG. 3F a SARS-CoV-2 3a peptide
  • FIG. 3H a SARS-CoV-2 7a peptide
  • FIG. 3I a SARS-CoV-2 8 peptide
  • FIG. 4 illustrates T-cell memory measured as a percentage of CD3 + CD62L + CD197 + T-cell populations in cells stimulated with different peptide antigens using DC cells (left bars) relative to cells that were stimulated with the same peptide antigens in the absence of DC cells (right bars).
  • FIG. 5 illustrates HLA types that bind SARS-CoV-2 Alpha, Delta, and Omicron peptides, and where the changes occur in the full protein sequence.
  • the dark shaded bands represent peptides binding with strong affinity, ⁇ 60 nM.
  • the light shaded bands represent peptides with medium affinities between > 60 nM to 185 nM. These sequences are conserved if they are found among the SARS-CoV-2 variants, alpha, delta, and omicron.
  • the white bands show the change in the binding of HLA types to peptides or the loss of the HLA type for a peptide altogether.
  • the white bands represent sequences where a major mutation for a viral variant is found.
  • FIG. 6 Illustrates an RNA cassette used to produce an mRNA polycistronic construct.
  • 1 signal peptide for MHC Class I or Class 2; 2: 30 amino acid or less antigen; 3: linker sequence.
  • FIG. 7 Illustrates lipid nanoparticles delivering green fluorescent protein (GFP) RNA to DCs.
  • GFP green fluorescent protein
  • FIG. 8 illustrates lipid nanoparticles delivering SARS-CoV-2 RNA to DCs.
  • FIG. 9 illustrates GFP levels in DCs transfected with nanoparticles comprising GFP RNA and a Mannose, CD180, CD209, or HLA-DR targeting ligand relative to cells formulated with LNP only.
  • the term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. In some embodiments, such variation may be as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
  • binding refers to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.
  • conserved epitope refers to conserved proteins with epitopes identified by the methods of the present technology that are conserved across the multiple strains of the virus.
  • pathogen refers to a bacterium, virus, or other microorganism that may cause disease.
  • Other microorganisms may include fungi, mold, parasites, and prions.
  • the “peptides” of the present technology may be (a) naturally-occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
  • peptide as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids.
  • the amino acids forming all or a part of a peptide may 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, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone.
  • peptide also includes dimers or multimers of peptides.
  • a “manufactured” peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method.
  • the term “peptide” includes peptides containing a variable number of amino acid residues, optionally with non-amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
  • amino acids are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid.
  • the key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group.
  • the various a-amino acids differ in the side-chain moiety that is attached to the a-carbon.
  • the “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User's Guide, G. A. Grant, editor, W.H.
  • 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 described generally 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.
  • L-Phe or “IPhe” is L- phenylalanine
  • D-Phe or “dPhe” is D-phenylalanine
  • dVal is D-valine
  • dPro is D-proline
  • D-/L-Phe or “d/IPhe” is D-phenylalanine, L-phenylalanine, or combinations thereof
  • Phe is also D-phenylalanine, L-phenylalanine, or combinations thereof, and so on.
  • Non-standard amino acids are “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; Tie is tert-Leucine; Nva is norvaline; Orn is ornithine; Bip is biphenyl amino acid; and so on.
  • 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 (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as “residues.”
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • polynucleotide refers to a linear sequence of nucleotides.
  • nucleotide typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof.
  • nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid.
  • a nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid.
  • Examples of such analogues include, without limitation, phosphorothiolates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptidenucleic acids (PNAs).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identity nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • identity in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • sequences are then said to be “substantially identity.”
  • This definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • administering include delivery of therapies (e.g., vaccine compositions) of the present technology to a subject either by local or systemic administration.
  • Administration may be parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • composition refers to a mixture of the active ingredient with other chemical components, such as pharmaceutically acceptable carriers and/or excipients.
  • a “pharmaceutically acceptable carrier” of the first or the second pharmaceutical composition refers to a carrier or diluent that does not cause significant irritation to an organism, does not abrogate the biological activity and properties of the administered active ingredient, and/or does not interact in a deleterious manner with the other components of the composition in which it is contained.
  • carrier encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • 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; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, N.J.)-
  • An “excipient” of the first or the second pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples, without
  • the present technology comprises vaccine compositions which provide viral antigens and promote immunological memory.
  • the vaccine compositions elicit an immune response, thereby triggering the recognition, response, and or elimination of the viral antigens.
  • the vaccine compositions of the present technology may induce an immune response in a subject to the viral antigens.
  • the vaccine compositions of the present technology may be administered to a subject to treat, prevent, and/or reduce a disease and/or other conditions associated with an infection.
  • the viral antigens of the present technology may be comprised of protein and/or peptide antigens, glycoprotein antigens, or lipid antigens.
  • the vaccine composition comprises one or more nucleotide sequences which encode viral antigens.
  • the viral antigens of the present technology may be an arterivirus, a mesonivirus, a ronivirus, a torovirus, or a coronavirus antigen.
  • the viral antigen is selected from the group consisting of a cytomegaloviral antigen, an Epstein-Barr viral antigen, a hepatitis B viral antigen, a human papillomaviral antigen, an adenoviral antigen, a herpes viral antigen, a human immunodeficiency viral antigen, an influenza viral antigen, a human respiratory syncytial viral antigen, a vaccinia viral antigen, a varicella-zoster viral antigen, a yellow fever viral antigen, an Ebola viral antigen, a coronaviral antigen, an Eastern equine encephalitis viral antigen, a Polyomaviral antigen hominisl (BKV), a SV40 and a Zika viral antigen
  • the viral antigen is an antigen from a virus comprising a variant of interest (VOI), variant of concern (VOC) or variant of high consequence (VOHC), as defined by the US Center for Disease Control and the World Health Organization (WHO).
  • VI variant of interest
  • VOC variant of concern
  • VOHC variant of high consequence
  • WHO World Health Organization
  • the antigen may be a viral antigen that is associated with and/or is capable of pandemic-like infection rates.
  • 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).
  • the coronavirus antigen is a Wuhan strain antigen.
  • the coronavirus antigen is a common cold coronavirus antigen.
  • common cold coronaviruses include Human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKLH ).
  • the coronavirus antigen is a SARS-CoV-2 antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS- CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV-2 lambda antigen, and a SARS-CoV-2 mu antigen.
  • SARS-CoV-2 antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen,
  • the present technology comprises vaccine compositions having nucleotide sequences that encode viral antigens.
  • Any one of the nucleotide sequences may promote a response by an immune cell, an immune response, or immunological memory against the viral antigen which it encodes (e.g., generation of a CD4+ T-cell memory response, a CD8+ T-cell memory response, central memory T-cells (TCM), effector memory T-cells (TEM), cross-reactivity of memory T-cells, or the maintenance of memory T-cells).
  • each of the nucleotide sequences promote an immune response or immunological memory against one or more of the viral antigens to which the nucleotide sequence corresponds to.
  • the nucleotide sequences may comprise a Deoxyribonucleic Acid (DNA) sequence.
  • the one or more nucleotides sequences comprise a Ribonucleic Acid (RNA) sequence.
  • the RNA sequence is a messenger RNA (mRNA) sequence.
  • the RNA sequence is a circular (circRNA) sequence. The circRNA may have increased half-life and/or increased expression relative to a non-circular RNA.
  • the RNA sequence is a messenger CRISPR RNA (crRNA) sequence.
  • the RNA sequence is a self-replicating RNA.
  • 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., a S peptide). In some embodiments, the nucleotide sequence encodes an S peptide antigen that is a S subunit 1 (S1 ) peptide or a S subunit 2 (S2) peptide.
  • S Spike
  • S2 S subunit 1
  • S2 S subunit 2
  • the vaccine composition comprises a nucleotide sequence encoding a membrane protein or a peptide thereof.
  • the nucleotide sequence encodes a VME1 (M) protein or peptide thereof (i.e., a M peptide).
  • the vaccine composition comprises a nucleotide sequence encoding a nucleocapsid protein or a peptide thereof.
  • the nucleotide sequence encodes a NCAP (N) protein or peptide thereof (i.e., a N peptide).
  • the vaccine composition comprises a nucleotide sequence that corresponds to an open reading frame (ORF) or a portion thereof.
  • the vaccine composition comprises a nucleotide sequence encoding an ORF peptide.
  • the ORF peptide is a ORF7a (7a) peptide, an ORF3a (3a) peptide, or an ORF8 (8) peptide.
  • the vaccine composition comprises a nucleotide sequence that encodes a nonstructural protein or a peptide thereof.
  • the nucleotide sequence encodes an Nsp6 peptide.
  • the vaccine composition comprises a first nucleotide sequence encoding a first antigen and a second nucleotide sequence encoding a second antigen, the first and second antigens each independently selected from the group consisting of a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, a 8 peptide, and a Nsp6 peptide.
  • the vaccine composition comprises a nucleotide sequence encoding an S peptide, a nucleotide sequence encoding a M peptide, a nucleotide sequence encoding a N peptide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding a 8 peptide, and a nucleotide sequence encoding a Nsp6 peptide.
  • the vaccine composition comprises a nucleotide sequence listed in Table 1.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NO: 1 -67. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence are each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 79-100. In some embodiments, the vaccine composition comprises a nucleotide sequence each 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.
  • the vaccine composition comprises a nucleotide sequence each 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.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 79-100.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each 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.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each 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.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
  • the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
  • the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -1 13.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119.
  • 60%, 65%, 70%, 75% In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-1 19. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-1 19.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 114-1 19.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125.
  • the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125.
  • the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 120- 125.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 120-125.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 120-125.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 126- 139.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 126-139.
  • the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 126-139.
  • the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity to SEQ ID NO: 140.
  • the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 141 .
  • the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 141 .
  • the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 141 .
  • each of the nucleotide sequences in the vaccine composition are present on different nucleotide chains.
  • each of the nucleotide sequences in the vaccine composition are present in a polycistronic sequence.
  • the polycistronic sequence comprises a nucleotide linker sequence. Nucleotide linker sequences may be used to connect or separate different nucleotide sequences that encode antigens, create spacers, enhance nucleotide sequence packaging into lipid compositions, or to facilitate molecular manipulations.
  • the polycistronic sequence comprises two or more nucleotide linker sequence.
  • the polycistronic nucleotide sequence comprises a modified nucleotide (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).
  • UTR untranslated region
  • the polycistronic nucleotide sequence may further comprise a nucleotide sequence that promotes localization of the nucleotide sequence or encodes a peptide that promotes localization of the peptide (e.g., a signal peptide).
  • the nucleotide sequence promotes localization to cells comprising a human leukocyte antigen molecule (HLA).
  • HLA human leukocyte antigen molecule
  • the HLA is a HLA-A molecule, a HLA- B molecule, or a HLA-C molecule.
  • the nucleotide sequence promotes localization to cells comprising a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the MHC molecule is an MHC Class I molecule or an MHC Class II molecule.
  • Localization patterns for nucleotides and peptides of the present technology may be assessed using predictive localization software, including but not limited to TargetP, WoLF PSORT, DeepLoc, CELLO, YLoc, BaCelLo, and LocTree3.
  • the polycistronic sequence may comprise a nucleotide linker sequence that is nonimmunogenic or comprises low immunogenicity (e.g., polyG linker sequence).
  • the nucleotide linker sequence may disrupt an active site in a viral peptide encoded by a nucleotide sequence of the present technology.
  • the nucleotide linker sequence may prevent tertiary structure formation of the viral antigen and/or may prevent function of the viral antigen.
  • the polycistronic sequences comprise a nucleotide linker sequence.
  • the nucleotide linker sequence may be encoded by an RNA sequence.
  • the RNA sequence may be present in the vaccine composition.
  • the polycistronic sequence comprises a nucleotide linker sequence 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% Identity 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% Identity 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% Identity to SEQ. ID NO: 142.
  • the vaccine compositions do not comprise a nucleotide linker sequence.
  • the present technology comprises vaccine compositions having one or more viral antigens that are a protein or peptide antigen.
  • the vaccine composition comprises two different peptide antigens. Any one of the protein or peptide antigens may promote an immune response or immunological memory. In some embodiments, each of the protein or peptide antigens promote an immune response or immunological memory.
  • the viral antigen comprises a S protein or a peptide thereof (i.e., a S peptide). In some embodiments, the viral antigen comprises an S peptide antigen that is a S1 peptide or a S2 peptide.
  • the viral antigen comprises a membrane protein or a peptide thereof. In some embodiments, the viral antigen comprises a M protein or peptide thereof (i.e., a M peptide). [0127] In some embodiments, the viral antigen comprises a nucleocapsid protein or a peptide thereof. In some embodiments, the viral antigen comprises a N protein or peptide thereof (i.e., a N peptide).
  • the viral antigen corresponds to an ORF peptide.
  • the ORF peptide corresponds to a 7a peptide, a 3a peptide, or an 8 peptide.
  • the viral antigen comprises a nonstructural protein or a peptide thereof (i.e., a Nsp6 peptide).
  • the vaccine composition comprises 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, Nsp6.
  • the vaccine composition comprises a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • the vaccine compositions of the present technology comprise a first antigen and a second antigen, each present on different peptide chains. In some embodiments, the vaccine compositions comprise 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 the tertiary folding or creation of functional viral domains.
  • the first and second antigens are each independently selected from the group consisting of a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • the vaccine composition comprises a peptide listed in Table 3.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209.
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 143-209.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 143-209.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 143-209.
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 210-220.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 210-220.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 210-220.
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 .
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 .
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 .
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 .
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 .
  • the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 .
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 .
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281.
  • the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 .
  • the first peptide and the second peptide comprise or consist of different amino acid sequences.
  • the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282.
  • the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282 [0149] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283.
  • the vaccine compositions of the present technology further comprise a signal peptide.
  • signal peptides include peptides that promote localization to cellular compartments (e.g., endoplasmic reticulum, mitochondria, nucleus, or peroxisomes), signal peptides that promote peptide secretion, and signal peptides that promote cleavage.
  • the signal peptide promotes localization and/or binding to cells comprising an MHC Class I molecule. In some embodiments, the signal peptide promotes localization and/or binding to cells comprising an MHC Class II molecule. In some embodiments, the cells comprising an MHC Class II molecule 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 a HLA. In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule.
  • Peptide binding for peptides of the present technology may be assessed using predictive binding software, including but not limited to NetMHC, NetMHCpan, NetMHCHpan, Immune Epitope Database and Analysis Resource (IEDB), SYFPEITHI, Stabilized Matrix Method (SMM), ProPred-l, ProPred, and NetCTLpan.
  • predictive binding software including but not limited to NetMHC, NetMHCpan, NetMHCHpan, Immune Epitope Database and Analysis Resource (IEDB), SYFPEITHI, Stabilized Matrix Method (SMM), ProPred-l, ProPred, and NetCTLpan.
  • vaccines of the present technology may comprise about 8 to about 10 peptides of about 25 to about 30 amino acids to induce a Class I HLA restricted CD8 response and about 8 to about 10 peptides of about 15 amino acids for Class II HLA restricted CD4 responses.
  • Peptides of the present technology of about 25 to about 30 amino acids in length are not expected to mimic an viral active site or to recombine with a wild type virus.
  • the linkers positioned between the peptides may further prevent formation of viral active sites and/or the randomization of sequences encoding the peptides prevent creation of an viral active sites. This approach differs from vaccines which induce the humoral immune response and/or encode a complete spike protein, including the RBD which binds the ACE receptor, and stabilizes the tertiary structure with proline.
  • the vaccine compositions of the present technology further comprise peptides having a peptide linker sequence.
  • Peptide linker sequences may be used to connect or separate different antigens, enhance peptide packaging into various compositions, or to facilitate molecular manipulations.
  • the peptide linker sequence may be nonimmunogenic or comprises low immunogenicity.
  • the peptide linker sequence comprises a specific cleavage site linker (e.g., a Furan cleavage site; 2A linker peptides).
  • the specific cleavage site linker may comprise a protease recognition site that is selectively cleaved by certain proteases.
  • the peptide linker sequence comprises a targeting or tagging linker that permits the localization, detection, or purification of peptides.
  • the peptide linker sequence may comprise a flexible linker (e.g., a glycine and serine rich linker) or a rigid linker (e.g., a glycine rich linker or an alpha-helical structure linker).
  • a flexible linker e.g., a glycine and serine rich linker
  • a rigid linker e.g., a glycine rich linker or an alpha-helical structure linker
  • Positioning the peptide linker sequence between peptides having fewer than 50 amino acids, for example, about 20, about 25, about 30, or about 35 amino acids, may disrupt an active site in a viral antigen.
  • the peptide linker sequence may prevent tertiary structure formation of the viral antigen and/or may prevent function of the viral antigen.
  • the linked peptides are derived from different proteins. In other embodiments, the linked peptides are derived from the same protein and are discontinuous. Without intending to be limiting, the linked peptides of the present technology may also prevent tertiary structure formation of the viral antigen and/or may also prevent function of the viral antigen.
  • the vaccine compositions comprise a peptide linker that is encoded by an 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).
  • the nucleotide sequence is an RNA sequence.
  • the peptide linker sequence comprises an amino acid sequence listed in Table 2.
  • the peptide linker sequence comprises an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Identity to SEQ. ID NO: 349.
  • the peptide linker sequence comprises an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Identity to SEQ. ID NO: 349.
  • the peptide linker sequence comprises an amino acid sequence least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Identity to SEQ. ID NO: 349.
  • the vaccine compositions do not comprise a peptide linker sequence.
  • the vaccine compositions comprise a peptide that is about 5 to about 50 amino acids, about 50 to about 100 amino acids, about 100 to about 200 amino acids, about 200 to about 500 amino acids, about 500 to about 1000 amino acids, about 1000 to about 2500 amino acids, about 2500 to about 5000 amino acids, about 5000 to about 10000 amino acids, about 10000 to about 100000 amino acids, or more.
  • the vaccine compositions comprise a peptide that is more than about 30 amino acids.
  • the vaccine compositions comprise a peptide that is less than about 30 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 30 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is about 25 to about 30 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 25 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 25 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 20 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 20 amino acids.
  • the vaccine compositions comprise a peptide that is less than about 15 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 15 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 10 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 10 amino acids.
  • the vaccine compositions comprise one or more peptides of the present technology and one or more nucleotide sequences of the present technology (e.g., a nucleotide sequence encoding a viral peptide).
  • the vaccine compositions of the present technology comprise two or more peptides of the present technology and one or more nucleotide sequences of the present technology.
  • the vaccine compositions of the present technology comprise two or more nucleotide sequences of the present technology and one or more peptides of the present technology.
  • the vaccine compositions comprise two or more nucleotide sequences of the present technology and two or more peptides of the present technology.
  • Vaccines of the present technology which may lack a tertiary structure may generate T cell epitopes without forming an active viral enzyme and/or an active virus function, in whole or in part, following delivery to a subject.
  • TH2 helper cells formed in the subject following delivery of the vaccine may induce or otherwise promote a B cell antibody response in addition to a cytotoxic T cell response.
  • the nucleotide sequences e.g., nucleotide sequences encoding a viral antigen
  • a lipid composition such as a lipid nanoparticle.
  • the lipid composition may comprise proteolipids (e.g., protamine), carrier proteins, and/or small molecules.
  • the lipid composition may comprise a single lipid group or multiple lipid groups.
  • lipid groups include cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)ylated lipids, ionizable lipids, helper lipids, stealth lipids, or cholesterols.
  • Nonlimiting 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-trimethyl ammonium propane, DOTAP 1 ,2-Dioleoyl-3- trimethyalammoniumpropane, and DC-Cholesterol 3p-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol.
  • Nonlimiting 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,
  • helper lipids include cholesterol (1 F?,3aS,3bS,7S,9aF?,9bS,11 aF?)-9a,1 1 a-Dimethyl-1 -[(2F?)-6-methylheptan-2-yl]-
  • Nonlimiting examples of stealth lipids include PEGIG (R)-2,3- bis(myristoyloxy)propyl-1 -(methoxy poly (ethylene glycol) 2000) carbamate and ALC- 0159 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide.
  • the nanoparticles may be polymer based, metal (e.g., silver, gold, palladium, titanium, zinc, or copper) based, silica based, or lipid based.
  • the nanoparticles are a multilayer nanoparticle.
  • multilayer nanoparticles e.g., a nanoparticle having two or more polymer based layers; a nanoparticle having two or more metal based layers; a nanoparticle having two or more silica based layers; a nanoparticle having two or more lipid-based layers; or a nanoparticle having a first layer 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 selected from the group consisting of a polymer- based layer, a metal based layer, a silica-based layer, and a lipid-based layer, wherein the second layer composition is different from the first layer composition).
  • lipid nanoparticles incorporate antigen presenting cell (APC) moieties, such as DC binding moieties.
  • APC binding moieties include antibodies and/or single-chain variable fragment (scFv) to surface molecules expressed on dendritic cells (e.g., CD1 a, CD1 c (BDCA1 ), CD1 1 b (ITGAM), CD1 1 c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC-205), CD206 (MRC1 ), CD209 (DC-SIGN), FceR1 , HLA-DR, TLR2 (e.g., glycolipids and phospholipids), TLR3 (double-stranded RNA), TLR4 (lipopolysaccharide), TLR7/8 (single-stranded RNA), and TLR9 (unmethylated CpG DNA)
  • scFv single-chain
  • the APC binding moieties are conjugated to a lipid in the lipid composition.
  • the APC binding moieties may be identified or designed using methods including, but not limited to, critical process parameters (CPPs), high- throughput sequencing, or predictive modeling.
  • CPPs critical process parameters
  • high- throughput sequencing or predictive modeling.
  • the APC moieties may be a ligand that binds a surface marker (e.g., peptide, glycoprotein, carbohydrate) of the APC cell (e.g., a DC, a B cell, a T cell, a macrophage).
  • a surface marker e.g., peptide, glycoprotein, carbohydrate
  • the surface marker is a molecule that is upregulated during maturation of the APC cell.
  • lipid nanoparticles comprise opsonization elements.
  • the opsonization elements may engage APCs (e.g., bind or interact with APCs).
  • the opsonization elements may induce the phagocytic process of macrophages, neutrophils, and dendritic cells.
  • the opsonization elements incorporate mannose or other carbohydrates.
  • the lipid nanoparticles comprise Fc binding fragments.
  • the Fc binding fragments bind Fc receptors on APCs.
  • the lipid nanoparticle incorporates complement factors C3b, C4 and or C1q.
  • the present technology comprises cell compositions that are immunogenic to antigens and promote immunological memory.
  • the cell composition may be generated such that the cells are immunogenic to specific antigens (e.g., viral antigens). Additionally, the cell compositions may stimulate an immune reaction leading to the production of an immune response. Nonlimiting examples of immune responses include the production of antibodies or activation of lymphatic cells (e.g., T-cells).
  • the cell compositions of the present technology induce an immune response in a subject to one or more antigens.
  • the cell compositions of the present technology may be administered to a subject to treat, prevent, and/or reduce a disease and/or other conditions associated with an infection.
  • the cell composition may be immunogenic to a protein and/or peptide of the present technology.
  • the cell composition is immunogenic to a nucleotide sequence of the present technology (e.g., a nucleotide sequence encoding a peptide of the present technology).
  • the cell composition is immunogenic to one or more peptides of the present technology and one or more nucleotide sequences of the present technology (e.g., a nucleotide sequence encoding a viral peptide).
  • the cell composition is immunogenic to two or more peptides of the present technology and one or more nucleotide sequences of the present technology.
  • the cell composition is immunogenic to two or more nucleotide sequences of the present technology and one or more peptides of the present technology. In some embodiments, the cell composition is immunogenic to two or more nucleotide sequences of the present technology and two or more peptides of the present technology.
  • the cell compositions of the present technology may be generated from a subject sample (e.g., a biological sample).
  • the subject sample may 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.
  • PBMC peripheral blood mononuclear cell
  • lymphoid tissue sample e.g., lymph node, spleen, or tonsil tissue
  • mucosal sample e.g., mucosal tissue
  • bone marrow sample e.g., a bone marrow sample
  • CSF cerebrospinal fluid
  • synovial fluid sample e.g., a synovial fluid sample.
  • the cell composition is a lymphatic cell composition.
  • the cell composition may comprise one or more of T-cells, B cells, Natural Killer (NK) cells, Dendritic Cells (DCs), macrophages, or granulocytes.
  • NK Natural Killer
  • DCs Dendritic Cells
  • macrophages or granulocytes.
  • the cell composition is a T-cell composition.
  • T-cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells).
  • Th cells include Th1 cells, Th2 cells, Th17 cells.
  • the cell composition comprises one or more CD4+ cell subsets.
  • the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.
  • the Th1 (CD4+) T-cells produce cytokines, including IFNy, and generate a CD8+ T-cell (e.g., cytotoxic T-cell) response.
  • Th2 (CD4+) T-cells produce cytokines, including IL4, and generate a high affinity antibody immune response, including class switching to IgG and IgA.
  • the cell composition is reactive to one or more cytokines and/or chemokines (e.g., activation, differentiation, signaling, and/or function of an immune cell is stimulated in the presence of the cytokines or chemokines).
  • Nonlimiting examples of cytokines and chemokines include interleukins (ILs) (e.g., IL- 2, IL-4, IL-6, IL-8, IL-10, or IL-12), tumor necrosis factors (TNFs) (e.g., TNFa or TNFp), interferons (INFs) (e.g., INFy, INFa, or INFP), chemokines (e.g., CXCL8, CXCL10, or CCL2), colony-stimulating factors (CSFs) (e.g., granulocyte-macrophage CSF or granulocyte CSF), or Transforming Growth Factor-beta (e.g., TGFP).
  • ILs interleukins
  • TNFa or TNFp tumor necrosis factors
  • INFs interferons
  • chemokines e.g., CXCL8, CXCL10, or CCL2
  • CSFs colony-stimulating
  • the T cell composition is present in a vaccine composition or an infusion.
  • compositions of the present technology comprise a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutically acceptable carrier and/or excipient may comprise aqueous or non-aqueous carriers that may facilitate dissolution of ac component of the compositions (e.g., peptides or nucleotide sequences).
  • Nonlimiting examples of pharmaceutically acceptable carriers and/or excipients include sucrose, polysorbate 80, monobasic sodium phosphate, monohydrate, dibasic sodium phosphate, dihydrate.
  • the composition may comprise a filler or diluent (e.g., lactose, mannitol, and microcrystalline cellulose), a disintegrant (e.g., croscarmellose sodium, crospovidone, starches), a buffer (e.g., phosphate buffer or acetate buffers), a solvent (e.g., water, alcohol, glycerin), or a stabilizer (e.g., sugars or gelatins); a preservative (e.g., thimerosal).
  • a filler or diluent e.g., lactose, mannitol, and microcrystalline cellulose
  • a disintegrant e.g., croscarmellose sodium, crospovidone, starches
  • a buffer e.g., phosphate buffer or acetate buffers
  • a solvent e.g., water, alcohol, glycerin
  • a stabilizer e.g.
  • compositions of the present technology comprise adjuvants.
  • Adjuvants may stimulate or enhance an immune response upon administration of the composition.
  • Nonlimiting 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.
  • the composition is sterilized (e.g., sterilization by filtration).
  • the compositions of the present technology comprise features that promote binding to and/or co-localization with an HLA (e.g., HLA- A, HLA-B, or HLA-C), an APC (e.g., a peptide or nucleotide sequence that targets or binds to an MHC Class II molecule), and/or a T-cell receptor (TCR).
  • the compositions comprise a molecule that binds a receptor on an APC (e.g., dendritic cells).
  • the molecule is a carbohydrate.
  • the carbohydrate is a mannose carbohydrate.
  • the mannose carbohydrate may be selected from the group consisting of D-mannose, mannose-6- phosphate (M6P), a mannans, a mannose polymer, and a mannose receptor ligand.
  • M6P mannose-6- phosphate
  • the molecule that binds a receptor on an APC is selected from the group consisting of Mannose, CD180, CD209, and HLA-DR.
  • compositions of the present technology comprise a hydrophobic tail.
  • the hydrophobic tail may act as a targeting moiety, promoting binding to APCs.
  • the hydrophobic tail comprises a fatty acid, a phospholipid, a cholesterol, a retinoid, a steroid, an alkyl chain, or a nonpolar amino acid side chain.
  • the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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
  • the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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 compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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
  • the composition comprises a lyophilized drug product (lyophilized powder or lyophilized cake of the peptides or nucleotide sequences).
  • the lyophilized drug product is reconstituted (e.g., using water or saline) prior to administration.
  • the reconstituted composition provides a solution having a pH of about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about
  • the reconstituted composition provides 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
  • the reconstituted composition provides a solution having 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
  • the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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.
  • the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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 1 1 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.
  • the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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 1 1 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,
  • the composition or the solution is diluted for administration.
  • diluents include sodium chloride, water (e.g., sterile water for injection (SWFI)), dextrose solutions, bacteriostatic solutions, or Ringer’s solutions (e.g., solutions comprising electrolytes, lactated Ringer’s solution).
  • the present technology comprises methods of generating the vaccine compositions and the cell compositions of the present technology.
  • the methods of generating the vaccine compositions and/or the cell compositions of the present technology comprise a step of quantifying a lymphocyte cell (e.g., T-cell, B cell, NK cells, DC, macrophage, or granulocyte) population in a sample from a subject that has successfully cleared a virus (e.g., elimination of active infection, reduction of viral load, resolution of a symptom associated with the virus, establishment of immune memory against the virus).
  • a lymphocyte cell e.g., T-cell, B cell, NK cells, DC, macrophage, or granulocyte
  • Quantifying the lymphocyte cell population may comprise methods including, but not limited to, flow cytometry, immunohistochemistry, immunofluorescence, protein quantification and/or detection methods (e.g., assessing levels of lymphocyte cell makers), or gene expression quantification methods (e.g., assessing expression levels of transcripts associated with lymphocyte cells).
  • the lymphocyte cell population is a T-cell population.
  • T-cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells).
  • Th cells include Th1 cells, Th2 cells, Th17 cells.
  • the T-cell composition comprises one or more CD4+ cell subsets.
  • 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 may 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.
  • PBMC peripheral blood mononuclear cell
  • lymphoid tissue sample e.g., lymph node, spleen, or tonsil tissue
  • mucosal sample e.g., mucosal tissue
  • bone marrow sample e.g., a bone marrow sample
  • CSF cerebrospinal fluid
  • synovial fluid sample e.g., a synovial fluid sample.
  • the subject that has successfully cleared a virus has or had mild symptoms associated with viral infection or no symptoms associated with viral infection).
  • the virus may be an arterivirus, a mesonivirus, a ronivirus, a torovirus, or a coronavirus.
  • the virus is selected from the group consisting of a cytomegalovirus, an Epstein-Barr virus, a hepatitis B virus, a human papillomavirus, an adenovirus, a herpes virus, a human immunodeficiency virus, an influenza virus, a human respiratory syncytial virus, a vaccinia virus, a varicella-zoster virus, a yellow fever virus, an Ebola virus, a coronavirus, an Eastern equine encephalitis virus, a Polyomavirus hominisl (BKV), a SV40 and a Zika virus.
  • the virus comprises a variant of interest ( VOI) , variant of concern (VOC) or variant of high consequence (VOHC), as defined by the US Center for Disease Control and the World Health Organization (WHO).
  • the virus may be a virus that is associated with and/or is capable of pandemic-like infection rates.
  • 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 ( I B V)), or a deltacoronavirus (e.g., Spotted green pigeon coronavirus HKU13 or White-eye coronavirus HKU16).
  • the coronavirus antigen is a Wuhan strain antigen.
  • the coronavirus is a common cold coronavirus.
  • common cold coronaviruses include Human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV- HKU1 ).
  • the coronavirus is a SARS-CoV-2 strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV- 2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV-2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS-CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
  • SARS-CoV-2 strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV- 2 gamma , a SARS-CoV-2 delta , a SARS-CoV
  • the methods of generating the vaccine compositions and/or cell compositions of the present technology comprise a step of exposing a sample to one or more antigens from the virus. Exposing the sample may comprise direct exposure (e.g., inoculating the sample with a protein antigen, peptide antigen, lipid antigen, glycoprotein antigen, or a nucleotide sequence that produces an antigen), or indirect exposure. Indirect exposure may comprise exposing the sample to the one or more antigens using an antigen-presenting cell (APC).
  • APCs comprise DCs (e.g., follicular DCs), macrophages, B cells, monocytes, and Langerhans cells.
  • the methods comprise quantifying the lymphocyte population before, during, and/or after exposure to the one or more viral antigens.
  • the lymphocyte population is quantified 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 the one or more viral antigens.
  • 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 the one or more viral antigens.
  • the lymphocyte population is 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 the one or more viral antigens.
  • quantifying the lymphocyte cell population comprises a normalization step.
  • the methods comprise a step of calculating a difference in the lymphocyte cell population quantity between two different time points.
  • the two different time points comprise a time point before exposure to the one or more viral antigens and a time point after exposure to the one or more viral antigens.
  • the two different time points comprise a time point before exposure to the one or more viral antigens and a time point during exposure to the one or more viral antigens.
  • the two different time points comprise a time point during exposure to the one or more viral antigens and a time point after exposure to the one or more viral antigens.
  • calculating the difference in the lymphocyte cell population between two different time points further comprises comparing the difference to a threshold value.
  • the threshold value may be useful in determining whether lymphocyte cell populations change in response to the one or more viral antigens.
  • the one or more viral antigens or a nucleotide sequence encoding the one or more viral antigens is included in a vaccine composition if the difference in the lymphocyte cell population exceeds the threshold value.
  • the methods of generating cell compositions of the present technology comprise a step of exposing a second sample to one or more antigens from the virus.
  • the one or more viral antigens or a nucleotide sequence encoding the one or more viral antigens are used to expose a second sample from a second subject if the difference in the lymphocyte cell population exceeds the threshold value.
  • the second sample may comprise a biological sample.
  • 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.
  • PBMC peripheral blood mononuclear cell
  • lymphoid tissue sample e.g., lymph node, spleen, or tonsil tissue
  • mucosal sample e.g., mucosal tissue
  • bone marrow sample e.g., a bone marrow sample
  • CSF cerebrospinal fluid
  • synovial fluid sample e.g., a synovial fluid sample.
  • the second subject may comprise a subject that has never been exposed to the virus (i.e., a naive subject), a subject that has never been infected with the virus, a subject that is infected with the virus, or a subject that has symptoms associated with infection with the virus.
  • the lymphocyte cell population is expanded in the second sample after exposure to the one or more viral antigens or the nucleotide sequence encoding the one or more viral antigens.
  • methods that may be used to expand the lymphocyte cell population include cytokine stimulation, cell culture methods (e.g., addition of nutrients or growth factors to promote proliferation; co-culture systems); or bioreactors, antibody stimulation.
  • the expanded lymphocyte cell population is isolated and/or may be screened for reactivity to a cytokine and/or chemokine (e.g., IFNy, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGFp, CXCL12, CCL19) formulation for administration (e.g., in a vaccine composition).
  • a cytokine and/or chemokine e.g., IFNy, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGFp, CXCL12, CCL19
  • peptide sequences corresponding to one or more of the identified immunodominant viral antigens were synthesized, and DCs were loaded with peptides associated with all or portions of the viral antigens (e.g., SARS-CoV-2 S, M, N, 3a, 7a, 8 and nsp6) and then used to prime subject T-cells.
  • a diverse CD4 + and CD8 + T-cell response was produced in PBMCs from subjects naive for SARS-CoV-2 infection against these antigens with a comparable response pattern to that of the T- cells from the subjects who had successfully cleared SARS-CoV-2 infection, as measured by an activation included marker (AIM) assay (FIGs. 2A and 2B).
  • AIM activation included marker
  • PBMCs peripheral blood cells
  • Monocyte-derived dendritic cells were produced using a cell culture plastic adhesion method. NonadherenT-cells were removed and frozen to use as a T- cell source in later cultures.
  • Differentiation media comprising complete DC culture media was supplemented with 800 U/mL human IL-4, and 500 U/mL human GM-CSF. Cells were cultured in differentiation media for 5 days with media exchanges everyday starting one day after initial culture. On day 5, differentiation media was removed, and maturation media was added overnight.
  • the maturation media was comprised of IL-6, IL1 p, TNFa, and Prostaglandin E2 (PGE2).
  • Prostaglandin E2 may be substituted with Polyinosinic:polycytidylic acid (Polyl:C), which binds toll-like receptor 3 (TLR3) (e.g., TLR3 on DCs).
  • Peptides resuspended in DMSO were added such that each peptide was at the final concentration of 0.1 pg/mL.
  • Cells were plated into a well plate. The culture was moved to a humidified incubator (5% CO2 37°C). Every two days half of the medium was exchanged for fresh media without disturbing the cells. On day 7, the process was repeated by thawing PBMCs and combining with new DCs and peptides, adding to current culture.
  • a CD3/CD28/CD2 T-Cell activator mix was added to cultures (15 pl/mL) with fresh medium containing cytokines and placed back into a 5% CO237°C humidified incubator.
  • Activation induced markers (AIM) assays and phenotyping flow cytometry assays were conducted on days 14, 21 , and 28. Cell count and viability were also quantified. One million cells per well were plated in 2 separate well plates for AIM assays and phenotype flow cytometry assays. A stimulation with an equimolar amount of dimethyl sulfoxide (DMS)O was performed as negative control for both assays, and cells were stained with antibody cocktails for 15 min at room temperature in the dark. After the final wash, cells were resuspended in fluorescence-activated cell sorting (FACS buffer) and were quantified and analyzed using FlowJo Flow Cytometry Analysis software (TreeStar).
  • FACS buffer fluorescence-activated cell sorting
  • T-cells reactive to viral antigens were plated (1 x10 5 per well) on enzyme- linked immunosorbent spot (ELISpot) plates comprising IFNy and IL4 (FIGs. 3A-3I). After 24-hour incubation, the plates were washed and incubated with a second antibody against IFNy which enzymatically catalyzes a first color and anti IL-4 which enzymatically catalyzes a second color. After drying, the first color spots and the second color spots were counted. Each spot represented a single T-cell secreting the analyte cytokine, where Th1 cells secreted IFNy and Th2 cells secrete IL-4. These assays were performed on T-cells at day 14 and day 21 post stimulation. T-cell immunological memory was then measured as a percentage of CD3+CD62L+CD197+ T-cell populations (FIG. 4).
  • T-cell antigens most likely to be reactive with approximately 50% accuracy were identified by using an MHC class I binding predictor MHCnetpan on the full SARS- CoV-2 amino acid sequence for the top 1 % (71 most common MHC alleles, HLA-A, B, C) in the population (Table 4). The frequency of subjects expressing at least two of the MHC alleles represents approximately 90% of the population. The antigens selected may be immunogenic and may provide protection in a high percentage of the population (FIG. 5).
  • RNA vaccine was produced using an RNA construct designed to express S, M, N, 3a, 7a, 8, and Nsp6 antigens (FIG. 6). All reagents used were derived from sources that did not contain contaminants and were produced with defined media and not natural sources. The RNA underwent a purification process. Following purification, the RNA may be encapsulated in lipid nanoparticles, or with cationic proteolipids such as protamine, with/or carrier proteins and small molecules.
  • the mRNA construct comprised a linker sequence (SEQ ID NO: 10) was intercalated between the nucleotide sequences which encoded viral antigens.
  • This linker may comprise low immunogenicity as indicated by use of the NetMHC MHC I binding affinity tool.
  • the MHC I binding predictions were done with IEDB analysis resource using NetMHCpan method.
  • the viral antigen sequences of interest were contained in the areas in which the binding affinity is below nM binding (Table 5) where lower rank indicates better binding.
  • Table 7 Examples of Peptide Selected for Spike Table 8: Examples of Peptide Selected for 0RF3a
  • the peptides selected were combined into peptides of 30 amino acids which were placed between the linker sequences and scrambled peptides from different viral peptides (e.g., in the Se construct or different parts of the protein) on either side of the linker sequence. This length provided an optimal number antigens, but also does not allow for the tertiary folding or creation of functional domains of the viral peptide.
  • This vaccine design comprises nucleotide sequences expressing antigens of all SARS-CoV-2 variants to date, including XBB (BA.2.10). Although the vaccine was designed using antigens from the alpha strain of SARS-CoV-2, only 1.69% of the antigens selected by our method across all 7 proteins had mutated across the alpha, delta and omicron variants, so that 98.31 % of the antigens are still capable of producing an effective immune response against the newest strains (Table 13). Additionally, 28/34 mutations in the antigens between alpha and omicron strains are in S and only 7 are present in antigens selected from all other viral peptides combined.
  • Macaque monkeys were injected intramuscularly on day 0 and day 21 in 3 groups including 6 animals each.
  • Group 1 was administered a vaccine generated from the sequences of the SARS-CoV- 2 alpha strain with 200 ug of mRNA conserved antigen + 100 ug of mRNA for Spike vaccine for two injections.
  • Group 2 was administered a vaccine generated from the sequences of the SARS-CoV-2 alpha strain with 200 ug of mRNA conserved antigen alone.
  • Group 3 was administered a vaccine generated from the sequences of the SARS-CoV-2 alpha strain with 100ug of mRNA for Spike antigen alone.
  • the mRNA antigen vaccines were comprised of 6 of the designated conserved antigens. Blood was drawn on days 14 and 35 to test for T cell gamma interferon production in response to the injected antigens. Antigen specific T cell frequency was determined by pulsing with antigen specific peptides and screened using the ELISpot assay for INFy. The results showed that T cell spots that equate to each T cells’ IFNy release, in a Th-1 response to each target antigen multivalent target.
  • RNA encoding vaccine antigens may enhance the delivery of RNA encoding vaccine antigens to DC’s.
  • the lipids were used to encapsulate and deliver an RNA encoding green fluorescent protein (GFP) in vitro.
  • DCs were analyzed 24 hours after nanoparticle delivery vis flow cytometry to assess GFP expression.
  • DCs were either exposed to nanoparticles carrying GFP RNA as a control or nanoparticles carrying SARS-CoV-2 RNA encoding viral peptides (FIG. 7 and FIG. 8).
  • Nanoparticles were designed to target DCs using mannose, CD180, CD209, or HLA-DR-targeting ligands. Nanoparticles with targeting ligands were more efficient in RNA delivery, as measured by GFP levels (FIG. 9).
  • Macaque monkeys were administered nanoparticles comprising 6 SARS- CoV-2 viral antigens, via a vaccine.
  • Controls comprised nanoparticles having no targeting ligand and/or no viral antigen load.
  • DC targeted lipid nanoparticles resulted in greater protection of Macaques when challenged with Omicron XBB at lower concentrations of RNA than the protection achieved with non-targeted lipid nanoparticles (Table 15).
  • a vaccine composition comprising a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • a vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen,
  • the first and second antigens each independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • a vaccine composition comprising a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
  • a vaccine composition comprising a nucleotide sequence encoding a Spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding a NCAP (N) peptide, a nucleotide sequence encoding, 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 a Nsp6 peptide.
  • nucleotide sequences are deoxyribonucleotide (DNA) sequences.
  • the Spike (S) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity 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% identity to one or more of SEQ ID NOs: 243-255;
  • the 7a peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%
  • a vaccine composition comprising two or more antigens selected from the group consisting of a Spike (S) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 210-220; a NCAP (N) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 243-255; a 7a peptide at least about 60%, 65%,
  • APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
  • CoV-2 antigen is an antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS-CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV-2 lambda antigen, and a SARS-CoV-2 mu antigen.
  • a vaccine composition comprising an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282.
  • a vaccine composition comprising an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283.
  • a vaccine composition comprising a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 140.
  • a vaccine composition comprising a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 141.
  • APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
  • PBMC peripheral blood mononuclear cell
  • a method of generating a T cell composition that specifically recognizes one or more viral antigens comprising the steps of
  • PBMC peripheral blood mononuclear cell
  • T helper cells comprise Th1 or Th2 cells.
  • T cell composition comprises CD4+ T cells or CD8+ T cells.
  • T cell composition comprises T helper cells.
  • T helper cells comprise Th1 or Th2 cells.
  • APC antigen presenting cell
  • the SARS-CoV-2 is strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV-2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV- 2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS- CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
  • any one of embodiments 41 -58, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
  • the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
  • a T cell composition immunogenic to viral antigens generated by the steps of:
  • PBMC peripheral blood mononuclear cell
  • T cell composition of embodiment 61 further comprising (ix) screening the expanded T cells for reactivity to interferon gamma (IFNy) or interleukin 4 (IL4).
  • IFNy interferon gamma
  • IL4 interleukin 4
  • T cell composition of embodiment 66, wherein the T helper cells comprise Th1 or Th2 cells.
  • T cell composition of any one of embodiments 61 -70, wherein the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
  • APC antigen presenting cell
  • DC dendritic cell
  • T cell composition of any one of embodiments 61 -76, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
  • any one of embodiments 61 -76, wherein the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.

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Abstract

The present technology provides multivalent vaccine compositions and Tcell compositions comprising viral antigens and associated methods. In some embodiments, the viral antigens are SARS-CoV-2 antigens. The vaccine compositions and the T cell compositions may comprise 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

A MULTI-ANTIGENIC RNA SARS-COV-2 VACCINE AND
ASSOCIATED METHODS
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/387,721 , filed on December 16, 2022. The contents of this provisional application are incorporated by reference in their entirety.
SEQUENCE LISTING INCORPORATED BY REFERENCE
[0002] This application contains an ST.26 compliant Sequence Listing, which is submitted concurrently in xml format via Patent Center and is hereby incorporated by reference in its entirety. The .xml copy, created on December 16, 2023, is named 1406308004WQ00.xml and is 0.331 MB (331 KB) (339,888 bytes) in size.
BACKGROUND
[0003] SARS-CoV-2 virus causes Coronavirus disease (COVID-19). COVID-19 is a highly contagious infection with a spectrum of symptoms from subclinical malaise to severe clinical disease and death. Symptoms of COVID-19 include headache, loss of smell and taste, nasal congestion, cough, muscle pain, fever, diarrhea, and coagulation disorders, disseminated intravascular coagulation, and thromboembolism.
[0004] To date, vaccines, antibodies, and immune therapies for COVID-19 have focused primarily the Spike Protein (S) and may result in a relatively narrow response. Naturally arising and circulating variants of SARS-CoV-2 S protein exhibit altered antigenicity. These variants occur due to adaption in immune-experienced hosts, especially during prolonged infection. This also reduced the therapeutic efficacy of supplemented antibody mediated immunity, such as convalescent sera or therapeutic monoclonal antibodies. Vaccines solely directed at the S protein therefore become less effective over time.
SUMMARY
[0005] In some embodiments, the present technology comprises a vaccine composition comprising a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0006] In some embodiments, the present technology comprises a vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen, the first and second antigens each independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0007] In some embodiments, the present technology comprises a vaccine composition comprising a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0008] In some embodiments, the present technology comprises a vaccine composition comprising a nucleotide sequence encoding a Spike (S) peptide, a nucleotide sequence encoding a VME1 (M) peptide, a nucleotide sequence encoding a NCAP (N) peptide, a nucleotide sequence encoding, 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 a Nsp6 peptide.
[0009] In some embodiments, the peptide length is less than 30 amino acids.
[0010] In some embodiments, the peptide length is about 5 amino acids to about
20 amino acids.
[0011] In some embodiments, the peptide length is about 9 amino acids.
[0012] In some embodiments, the peptide length is about 14 amino acids.
[0013] In some embodiments, the nucleotide sequences are deoxyribonucleotide (DNA) sequences.
[0014] In some embodiments, the nucleotide sequences are ribonucleotide (RNA) sequences.
[0015] In some embodiments, the peptide does not comprise an active site.
[0016] In some embodiments, the peptide does not fold into tertiary peptide structures. [0017] In some embodiments, the nucleotide sequences are present in a lipid composition.
[0018] In some embodiments, the lipid composition comprises a lipid nanoparticle.
[0019] In some embodiments, the lipid nanoparticle comprises an antigen presenting cell (APC) targeting molecule.
[0020] In some embodiments, the APC is a dendritic cell (DC).
[0021] In some embodiments, the APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
[0022] In some embodiments, each of the peptides are present on a single peptide chain.
[0023] In some embodiments, the single chain comprises one or more linker sequences.
[0024] In some embodiments, each of the peptides are present on different peptide chains
[0025] In some embodiments, each of the nucleotide sequences are present in a polycistronic sequence.
[0026] In some embodiments, the polycistronic sequence comprises one or more linker sequences.
[0027] In some embodiments, each of the nucleotide sequences are present on different nucleotide chains.
[0028] In some embodiments, the first antigen or the second antigen comprises a coronavirus antigen peptide.
[0029] In some embodiments, one or more of the antigens comprise a coronavirus peptide.
[0030] In some embodiments, the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.
[0031] In some embodiments, the SARS-CoV-2 antigen is an antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS-CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV- 2 lambda antigen, and a SARS-CoV-2 mu antigen.
[0032] In some embodiments, the vaccine composition comprises an adjuvant.
[0033] In some embodiments, the vaccine composition is a multivalent vaccine composition.
[0034] In some embodiments, the present technology comprises a method of generating a vaccine composition, comprising the steps of (i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject that has successfully cleared a virus; (ii) exposing the sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (v) calculating a difference in the T cell population quantity between (i) and (iii); (vi) comparing the difference in (iv) to a threshold value; and (iv) including (a) a peptide of the one or more antigens in (ii), or (b) a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v).
[0035] In some embodiments, the present technology comprises a method of generating a T cell composition that specifically recognizes one or more viral antigens, comprising the steps of (i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (iv) calculating a difference in the T cell population quantity between (i) and (iii); (v) comparing the difference in (iv) to a threshold value; (vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v); (vii) expanding the T cells in the second sample after the exposure in (vi); and (viii) isolating the expanded T cells.
[0036] In some embodiments, the methods further comprise (ix) screening the expanded T cells for reactivity to interferon gamma (I FNy) or interleukin 4 (IL4). [0037] In some embodiments, the T cell composition is formulated for administration to the second subject.
[0038] In some embodiments, the T cell composition is formulated in a vaccine composition.
[0039] In some embodiments, the T cell population comprises CD4+ T cells or CD8+ T cells.
[0040] In some embodiments, the T cell population comprises T helper cells.
[0041] In some embodiments, the T helper cells comprise Th1 or Th2 cells.
[0042] In some embodiments, the T cell composition comprises CD4+ T cells or
CD8+ T cells.
[0043] In some embodiments, the T cell composition comprises T helper cells.
[0044] In some embodiments, the T helper cells comprise Th1 or Th2 cells.
[0045] In some embodiments, the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
[0046] In some embodiments, the APC is a dendritic cell (DC).
[0047] In some embodiments, the DC cells are stimulated with IL4 or Granulocyte-
Macrophage Colony-Stimulating Factor (GM-CSF).
[0048] In some embodiments, the present technology comprises a T cell composition immunogenic to viral antigens, generated by the steps of: (i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus; (ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens; (iv) calculating a difference in the T cell population quantity between (i) and (iii); (v) comparing the difference in (iv) to a threshold value; (vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v); (vii) expanding the T cells in the second sample after the exposure in (vi); and (viii) isolating the expanded T cells. [0049] In some embodiments, the T cell compositions further comprises (ix) screening the expanded T cells for reactivity to interferon gamma (IFNy) or interleukin 4 (IL4).
[0050] In some embodiments, the T cell composition is formulated for administration to the second subject.
[0051] In some embodiments, the T cell composition is formulated in a vaccine composition.
[0052] In some embodiments, the T cell population comprises CD4+ T cells or CD8+ T cells.
[0053] In some embodiments, the T cell population comprises T helper cells.
[0054] In some embodiments, the T helper cells comprise Th1 or Th2 cells.
[0055] In some embodiments, the T cell composition comprises CD4+ T cells or
CD8+ T cells.
[0056] In some embodiments, the T cell composition comprises T helper cells.
[0057] In some embodiments, the T helper cells comprise Th1 or Th2 cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIGS. 1 A-1 C illustrate antigen recognition patterns of viral peptides in T- cells from subjects who have successfully cleared SARS-CoV-2 relative to T-cells from subjects who have not been infected with SARS-CoV-2. Shown are Activation Induced Marker (AIM) results for CD4+ T-cells (FIG. 1A and 1 C (right)) and CD8+ T-cells (FIG. 1 B and 1 C (left)).
[0059] FIGS. 2A and 2B illustrate changes in antigen-specific CD4+ T-cells (FIG. 2A) and CD8+ T-cells (FIG. 2B) following exposure to different viral antigens (X-axis) in samples from subjects who have successfully cleared SARS-CoV-2, samples from subjects not previously exposed to SARS-CoV-2 (i.e. , a naive subject), control samples primed with dendritic cell (DC) antigen presentation, and peripheral blood mononuclear cell (PBMC) controls.
[0060] FIGS. 3A-3I illustrate changes in T-cell response measured by interleukin 4 (IL4) and interferon gamma (IFNy) production from in Th1 cells at day 14 (left) and Th2 cells at day 21 (right). Samples were incubated using different media mixtures (media mixtures 1 , 2, and 3) supplemented with different components (A refers to Spike (S) protein supplementation, B refers to a mixture of the Spike (S), VME1 (M), NCAP (N), ORF3a (3a), ORF7a (7a), and ORF8 (8) supplementation, and C refers to a mixture of VME1 (M), NCAP (N), ORF3a (3a), 0RF7a (7a), and ORF8 (8) supplementation and DMSO refers to media supplemented with DMSO as a negative control. Each sample was exposed to either a media control (FIG. 3A), a Spike peptide from a common cold coronavirus (FIG. 3B), a first SARS-CoV-2 Spike (S) peptide (FIG. 3C), a second SARS- CoV-2 S peptide (FIG. 3D), a SARS-CoV-2 VME1 (M) peptide (FIG. 3E), a SARS-CoV- 2 NCAP (N) peptide (FIG. 3F), a SARS-CoV-2 3a peptide (FIG. 3G), a SARS-CoV-2 7a peptide (FIG. 3H), and a SARS-CoV-2 8 peptide (FIG. 3I).
[0061] FIG. 4 illustrates T-cell memory measured as a percentage of CD3+CD62L+CD197+ T-cell populations in cells stimulated with different peptide antigens using DC cells (left bars) relative to cells that were stimulated with the same peptide antigens in the absence of DC cells (right bars).
[0062] FIG. 5 illustrates HLA types that bind SARS-CoV-2 Alpha, Delta, and Omicron peptides, and where the changes occur in the full protein sequence. The dark shaded bands represent peptides binding with strong affinity, < 60 nM. The light shaded bands represent peptides with medium affinities between > 60 nM to 185 nM. These sequences are conserved if they are found among the SARS-CoV-2 variants, alpha, delta, and omicron. The white bands show the change in the binding of HLA types to peptides or the loss of the HLA type for a peptide altogether. The white bands represent sequences where a major mutation for a viral variant is found.
[0063] FIG. 6. Illustrates an RNA cassette used to produce an mRNA polycistronic construct. 1 : signal peptide for MHC Class I or Class 2; 2: 30 amino acid or less antigen; 3: linker sequence.
[0064] FIG. 7 Illustrates lipid nanoparticles delivering green fluorescent protein (GFP) RNA to DCs.
[0065] FIG. 8 illustrates lipid nanoparticles delivering SARS-CoV-2 RNA to DCs. [0066] FIG. 9 illustrates GFP levels in DCs transfected with nanoparticles comprising GFP RNA and a Mannose, CD180, CD209, or HLA-DR targeting ligand relative to cells formulated with LNP only.
DETAILED DESCRIPTION
[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present technology belongs. For the purposes of the present technology, the following terms are defined below.
[0068] 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.
[0069] The term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. In some embodiments, such variation may be as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
[0070] The terms “bind,” “binding,” “complex,” and “complexing,” refer to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.
[0071] The term “conserved epitope” refers to conserved proteins with epitopes identified by the methods of the present technology that are conserved across the multiple strains of the virus.
[0072] The term “pathogen” refers to a bacterium, virus, or other microorganism that may cause disease. Other microorganisms may include fungi, mold, parasites, and prions.
[0073] The “peptides” of the present technology may be (a) naturally-occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
[0074] The term “peptide” as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids. The amino acids forming all or a part of a peptide may 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, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone. The term “peptide” also includes dimers or multimers of peptides. A “manufactured” peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method. The term “peptide” includes peptides containing a variable number of amino acid residues, optionally with non-amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
[0075] By employing chemical synthesis, a useful means of production, it is possible to introduce various amino acids which do not naturally occur along the chain, modify the N- or C-terminus, and the like, thereby providing for improved stability and formulation, resistance to protease degradation, and the like.
[0076] “Amino acids” are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group. The various a-amino acids differ in the side-chain moiety that is attached to the a-carbon. The “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User's Guide, G. A. Grant, editor, W.H. Freeman & Co., New York (1992), the teachings of which are incorporated herein by reference, including the text and table set forth at pages 11 through 24. As set forth 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 described generally 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.
[0077] In the peptides of the present technology, conventional amino acid residues have their conventional meaning as given 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; “Gin” is glutamine; “Glu” is glutamic acid; “His” is histidine; “He” 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 “Vai” is valine. Unless otherwise indicated, all amino acids abbreviations represent either isomer, i.e., the L-isomer, the D-isomer, or combinations thereof may be used. Thus, for example, “L-Phe” or “IPhe” is L- phenylalanine; “D-Phe” or “dPhe” is D-phenylalanine; dVal is D-valine; dPro is D-proline; “D-/L-Phe” or “d/IPhe” is D-phenylalanine, L-phenylalanine, or combinations thereof; “Phe” is also D-phenylalanine, L-phenylalanine, or combinations thereof, and so on. Non-standard amino acids are “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; Tie is tert-Leucine; Nva is norvaline; Orn is ornithine; Bip is biphenyl amino acid; and so on.
[0078] 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 (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as “residues.”
[0079] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term “polynucleotide” refers to a linear sequence of nucleotides. The term “nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. Nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogues include, without limitation, phosphorothiolates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptidenucleic acids (PNAs).
[0080] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identity nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0081] The terms “identity” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identity.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
[0082] The terms “administering” or “administer” include delivery of therapies (e.g., vaccine compositions) of the present technology to a subject either by local or systemic administration. Administration may be parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
[0083] As used herein, a “composition,” a “vaccine composition,” or a “pharmaceutical composition” refers to a mixture of the active ingredient with other chemical components, such as pharmaceutically acceptable carriers and/or excipients.
[0084] As used herein, a “pharmaceutically acceptable carrier” of the first or the second pharmaceutical composition refers to a carrier or diluent that does not cause significant irritation to an organism, does not abrogate the biological activity and properties of the administered active ingredient, and/or does not interact in a deleterious manner with the other components of the composition in which it is contained. The term “carrier” encompasses any excipient, binder, diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21 st 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; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, N.J.)- An “excipient” of the first or the second pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Vaccine compositions
[0085] The present technology comprises vaccine compositions which provide viral antigens and promote immunological memory. The vaccine compositions elicit an immune response, thereby triggering the recognition, response, and or elimination of the viral antigens. In some embodiments, the vaccine compositions of the present technology may induce an immune response in a subject to the viral antigens. In some embodiments, the vaccine compositions of the present technology may be administered to a subject to treat, prevent, and/or reduce a disease and/or other conditions associated with an infection.
[0086] The viral antigens of the present technology may be comprised of protein and/or peptide antigens, glycoprotein antigens, or lipid antigens. In some embodiments, the vaccine composition comprises one or more nucleotide sequences which encode viral antigens.
[0087] The viral antigens of the present technology may be an arterivirus, a mesonivirus, a ronivirus, a torovirus, or a coronavirus antigen. In some embodiments, the viral antigen is selected from the group consisting of a cytomegaloviral antigen, an Epstein-Barr viral antigen, a hepatitis B viral antigen, a human papillomaviral antigen, an adenoviral antigen, a herpes viral antigen, a human immunodeficiency viral antigen, an influenza viral antigen, a human respiratory syncytial viral antigen, a vaccinia viral antigen, a varicella-zoster viral antigen, a yellow fever viral antigen, an Ebola viral antigen, a coronaviral antigen, an Eastern equine encephalitis viral antigen, a Polyomaviral antigen hominisl (BKV), a SV40 and a Zika viral antigen. In some embodiments, the viral antigen is an antigen from a virus comprising a variant of interest (VOI), variant of concern (VOC) or variant of high consequence (VOHC), as defined by the US Center for Disease Control and the World Health Organization (WHO). [0088] In some embodiments, the antigen may be a viral antigen that is associated with and/or is capable of pandemic-like infection rates.
[0089] 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.
[0090] In some embodiments, the coronavirus antigen is a common cold coronavirus antigen. Nonlimiting examples of common cold coronaviruses include Human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV-HKLH ).
[0091] In some embodiments, the coronavirus antigen is a SARS-CoV-2 antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS- CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV-2 lambda antigen, and a SARS-CoV-2 mu antigen.
[0092] Nucleotides
[0093] The present technology comprises vaccine compositions having nucleotide sequences that encode viral antigens. Any one of the nucleotide sequences may promote a response by an immune cell, an immune response, or immunological memory against the viral antigen which it encodes (e.g., generation of a CD4+ T-cell memory response, a CD8+ T-cell memory response, central memory T-cells (TCM), effector memory T-cells (TEM), cross-reactivity of memory T-cells, or the maintenance of memory T-cells). In some embodiments, each of the nucleotide sequences promote an immune response or immunological memory against one or more of the viral antigens to which the nucleotide sequence corresponds to. The nucleotide sequences may comprise a Deoxyribonucleic Acid (DNA) sequence. In some embodiments, the one or more nucleotides 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 (circRNA) sequence. The circRNA may have increased half-life and/or increased expression relative to a non-circular RNA. In some embodiments, the RNA sequence is a messenger CRISPR RNA (crRNA) sequence. In some embodiments, the RNA sequence is a self-replicating RNA.
[0094] 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., a S peptide). In some embodiments, the nucleotide sequence encodes an S peptide antigen that is a S subunit 1 (S1 ) peptide or a S subunit 2 (S2) peptide.
[0095] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a membrane protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes a VME1 (M) protein or peptide thereof (i.e., a M peptide).
[0096] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding a nucleocapsid protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes a NCAP (N) protein or peptide thereof (i.e., a N peptide).
[0097] In some embodiments, the vaccine composition comprises a nucleotide sequence that corresponds to an open reading frame (ORF) or a portion thereof. In some embodiments, the vaccine composition comprises a nucleotide sequence encoding an ORF peptide. In some embodiments, the ORF peptide is a ORF7a (7a) peptide, an ORF3a (3a) peptide, or an ORF8 (8) peptide.
[0098] In some embodiments, the vaccine composition comprises a nucleotide sequence that encodes a nonstructural protein or a peptide thereof. In some embodiments, the nucleotide sequence encodes an Nsp6 peptide.
[0099] In some embodiments, the vaccine composition comprises a first nucleotide sequence encoding a first antigen and a second nucleotide sequence encoding a second antigen, the first and second antigens each independently selected from the group consisting of a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, a 8 peptide, and a Nsp6 peptide.
[0100] In some embodiments, the vaccine composition comprises a nucleotide sequence encoding an S peptide, a nucleotide sequence encoding a M peptide, a nucleotide sequence encoding a N peptide, a nucleotide sequence encoding a 3a peptide, a nucleotide sequence encoding a 7a peptide, a nucleotide sequence encoding a 8 peptide, and a nucleotide sequence encoding a Nsp6 peptide.
[0101] In some embodiments, the vaccine composition comprises a nucleotide sequence listed in Table 1.
Table 1 : Nucleotide Sequences
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
[0102] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NO: 1 -67. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67.
[0103] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence are each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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 of different nucleotide sequences, in whole or in part.
[0104] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 68-78.
[0105] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence each having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence each having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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 of different nucleotide sequences, in whole or in part.
[0106] In some embodiments, the vaccine composition comprises a nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 79-100. In some embodiments, the vaccine composition comprises a nucleotide sequence each 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. In some embodiments, the vaccine composition comprises a nucleotide sequence each 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.
[0107] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence each 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. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each 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. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
[0108] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -113.
[0109] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 101 -1 13. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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 of different nucleotide sequences, in whole or in part.
[0110] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119. 60%, 65%, 70%, 75%, In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-1 19. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-1 19.
[0111] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 114-1 19. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119. In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 14-119. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part. [0112] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 120-125.
[0113] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 120-125. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
[0114] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139. In some embodiments, the vaccine composition comprises a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 126-139.
[0115] In some embodiments, the vaccine composition comprises a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence and the second nucleotide sequence each about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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, the first nucleotide sequence and the second nucleotide sequence each at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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, the first nucleotide sequence and the second nucleotide sequence each at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 126-139. In some embodiments, the first nucleotide sequence and the second nucleotide sequence comprise or consist of different nucleotide sequences, in whole or in part.
[0116] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity to SEQ ID NO: 140.
[0117] In some embodiments, the vaccine composition comprises a nucleotide sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity to SEQ ID NO: 141 .
[0118] In some embodiments, each of the nucleotide sequences in the vaccine composition are present on different nucleotide chains.
[0119] In some embodiments, each of the nucleotide sequences in the vaccine composition are present in a polycistronic sequence. In some embodiments, the polycistronic sequence comprises a nucleotide linker sequence. Nucleotide linker sequences may be used to connect or separate different nucleotide sequences that encode antigens, create spacers, enhance nucleotide sequence packaging into lipid compositions, or to facilitate molecular manipulations. In some embodiments, the polycistronic sequence comprises two or more nucleotide linker sequence.
[0120] In some embodiments, the polycistronic nucleotide sequence comprises a modified nucleotide (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).
[0121] The polycistronic nucleotide sequence may further comprise a nucleotide sequence that promotes localization of the nucleotide sequence or encodes a peptide that promotes localization of the peptide (e.g., a signal peptide). In some embodiments, the nucleotide sequence promotes localization to cells comprising a human leukocyte antigen molecule (HLA). In some embodiments, the HLA is a HLA-A molecule, a HLA- B molecule, or a HLA-C molecule. In some embodiments, the nucleotide sequence promotes localization to cells comprising a major histocompatibility complex (MHC) molecule. In some embodiments, the MHC molecule is an MHC Class I molecule or an MHC Class II molecule. Localization patterns for nucleotides and peptides of the present technology may be assessed using predictive localization software, including but not limited to TargetP, WoLF PSORT, DeepLoc, CELLO, YLoc, BaCelLo, and LocTree3.
[0122] The polycistronic sequence may comprise a nucleotide linker sequence that is nonimmunogenic or comprises low immunogenicity (e.g., polyG linker sequence). The nucleotide linker sequence may disrupt an active site in a viral peptide encoded by a nucleotide sequence of the present technology. In some embodiments, the nucleotide linker sequence may prevent tertiary structure formation of the viral antigen and/or may prevent function of the viral antigen. In some embodiments, the polycistronic sequences comprise a nucleotide linker sequence. The nucleotide linker sequence may be encoded by an RNA sequence. The RNA sequence may be present in the vaccine composition. In some embodiments, the polycistronic sequence comprises a nucleotide linker sequence 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% Identity 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% Identity 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% Identity to SEQ. ID NO: 142.
[0123] In some embodiments, the vaccine compositions do not comprise a nucleotide linker sequence.
Table 2: Linker Sequences
Figure imgf000051_0001
Peptides:
[0124] The present technology comprises vaccine compositions having one or more viral antigens that are a protein or peptide antigen. In some embodiments, the vaccine composition comprises two different peptide antigens. Any one of the protein or peptide antigens may promote an immune response or immunological memory. In some embodiments, each of the protein or peptide antigens promote an immune response or immunological memory.
[0125] In some embodiments, the viral antigen comprises a S protein or a peptide thereof (i.e., a S peptide). In some embodiments, the viral antigen comprises an S peptide antigen that is a S1 peptide or a S2 peptide.
[0126] In some embodiments, the viral antigen comprises a membrane protein or a peptide thereof. In some embodiments, the viral antigen comprises a M protein or peptide thereof (i.e., a M peptide). [0127] In some embodiments, the viral antigen comprises a nucleocapsid protein or a peptide thereof. In some embodiments, the viral antigen comprises a N protein or peptide thereof (i.e., a N peptide).
[0128] 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.
[0129] In some embodiments, the viral antigen comprises a nonstructural protein or a peptide thereof (i.e., a Nsp6 peptide).
[0130] In some embodiments, the vaccine composition comprises 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, Nsp6.
[0131] In some embodiments, the vaccine composition comprises a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0132] In some embodiments, the vaccine compositions of the present technology comprise a first antigen and a second antigen, each present on different peptide chains. In some embodiments, the vaccine compositions comprise 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 the tertiary folding or creation of functional viral domains.
[0133] In some embodiments, the first and second antigens are each independently selected from the group consisting of a S peptide, a M peptide, a N peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide. In some embodiments, the vaccine composition comprises a peptide listed in Table 3.
Table 3: Peptide Sequences
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
[0134] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 143-209.
[0135] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 143-209. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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.
[0136] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 210-220.
[0137] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 210-220. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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.
[0138] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242.
[0139] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 221 -242. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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. [0140] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255.
[0141] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 243-255. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of 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.
[0142] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 . In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 . [0143] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 . In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 256-261 . some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.
[0144] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267.
[0145] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 262-267. some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.
[0146] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 . In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 .
[0147] In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 . In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281. In some embodiments, the vaccine composition comprises a first peptide and a second peptide, the first peptide and the second peptide each having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of one or more of SEQ ID NOs: 268-281 . some embodiments, the first peptide and the second peptide comprise or consist of different amino acid sequences.
[0148] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282 [0149] In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283. In some embodiments, the vaccine composition comprises a peptide having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283.
[0150] In some embodiments, the vaccine compositions of the present technology further comprise a signal peptide. Nonlimiting examples of signal peptides include peptides that promote localization to cellular compartments (e.g., endoplasmic reticulum, mitochondria, nucleus, or peroxisomes), signal peptides that promote peptide secretion, and signal peptides that promote cleavage.
[0151] In some embodiments, the signal peptide promotes localization and/or binding to cells comprising an MHC Class I molecule. In some embodiments, the signal peptide promotes localization and/or binding to cells comprising an MHC Class II molecule. In some embodiments, the cells comprising an MHC Class II molecule 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 a HLA. In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. Peptide binding for peptides of the present technology may be assessed using predictive binding software, including but not limited to NetMHC, NetMHCpan, NetMHCHpan, Immune Epitope Database and Analysis Resource (IEDB), SYFPEITHI, Stabilized Matrix Method (SMM), ProPred-l, ProPred, and NetCTLpan.
[0152] Without intending to be limited by any particular theory, vaccines of the present technology may comprise about 8 to about 10 peptides of about 25 to about 30 amino acids to induce a Class I HLA restricted CD8 response and about 8 to about 10 peptides of about 15 amino acids for Class II HLA restricted CD4 responses. Peptides of the present technology of about 25 to about 30 amino acids in length are not expected to mimic an viral active site or to recombine with a wild type virus. In some embodiments, the linkers positioned between the peptides may further prevent formation of viral active sites and/or the randomization of sequences encoding the peptides prevent creation of an viral active sites. This approach differs from vaccines which induce the humoral immune response and/or encode a complete spike protein, including the RBD which binds the ACE receptor, and stabilizes the tertiary structure with proline.
[0153] In some embodiments, the vaccine compositions of the present technology further comprise peptides having a peptide linker sequence. Peptide linker sequences may be used to connect or separate different antigens, enhance peptide packaging into various compositions, or to facilitate molecular manipulations. The peptide linker sequence may be nonimmunogenic or comprises low immunogenicity.
[0154] In some embodiments, the peptide linker sequence comprises a specific cleavage site linker (e.g., a Furan cleavage site; 2A linker peptides). The specific cleavage site linker may comprise a protease recognition site that is selectively cleaved by certain proteases.
[0155] In some embodiments, the peptide linker sequence comprises a targeting or tagging linker that permits the localization, detection, or purification of peptides.
[0156] The peptide linker sequence may comprise a flexible linker (e.g., a glycine and serine rich linker) or a rigid linker (e.g., a glycine rich linker or an alpha-helical structure linker).
[0157] Positioning the peptide linker sequence between peptides having fewer than 50 amino acids, for example, about 20, about 25, about 30, or about 35 amino acids, may disrupt an active site in a viral antigen. In some embodiments, the peptide linker sequence may prevent tertiary structure formation of the viral antigen and/or may prevent function of the viral antigen. In some embodiments, the linked peptides are derived from different proteins. In other embodiments, the linked peptides are derived from the same protein and are discontinuous. Without intending to be limiting, the linked peptides of the present technology may also prevent tertiary structure formation of the viral antigen and/or may also prevent function of the viral antigen.
[0158] In some embodiments, the vaccine compositions comprise a peptide linker that is encoded by an 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. [0159] 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 each having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Identity 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% Identity to SEQ. ID NO: 349. the peptide linker sequence comprises an amino acid sequence least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% Identity to SEQ. ID NO: 349.
[0160] In some embodiments, the vaccine compositions do not comprise a peptide linker sequence.
[0161] In some embodiments, the vaccine compositions comprise a peptide that is about 5 to about 50 amino acids, about 50 to about 100 amino acids, about 100 to about 200 amino acids, about 200 to about 500 amino acids, about 500 to about 1000 amino acids, about 1000 to about 2500 amino acids, about 2500 to about 5000 amino acids, about 5000 to about 10000 amino acids, about 10000 to about 100000 amino acids, or more.
[0162] In some embodiments, the vaccine compositions comprise a peptide that is more than about 30 amino acids.
[0163] In some embodiments, the vaccine compositions comprise a peptide that is less than about 30 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 30 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is about 25 to about 30 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 25 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 25 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 20 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 20 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 15 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 15 amino acids. In some embodiments, the vaccine compositions comprise a peptide that is less than about 10 amino acids. In some embodiments, each of the peptides in the vaccine compositions are less than about 10 amino acids.
[0164] In some embodiments, the vaccine compositions comprise one or more peptides of the present technology and one or more nucleotide sequences of the present technology (e.g., a nucleotide sequence encoding a viral peptide). In some embodiments, the vaccine compositions of the present technology comprise two or more peptides of the present technology and one or more nucleotide sequences of the present technology. In some embodiments the vaccine compositions of the present technology comprise two or more nucleotide sequences of the present technology and one or more peptides of the present technology. In some embodiments, the vaccine compositions comprise two or more nucleotide sequences of the present technology and two or more peptides of the present technology.
[0165] Vaccines of the present technology which may lack a tertiary structure may generate T cell epitopes without forming an active viral enzyme and/or an active virus function, in whole or in part, following delivery to a subject. In some embodiments, TH2 helper cells formed in the subject following delivery of the vaccine may induce or otherwise promote a B cell antibody response in addition to a cytotoxic T cell response.
Lipid Compositions
[0166] In some embodiments, the nucleotide sequences (e.g., nucleotide sequences encoding a viral antigen) and/or the peptides of the present technology are present in a lipid composition, such as a lipid nanoparticle. The lipid composition may comprise proteolipids (e.g., protamine), carrier proteins, and/or small molecules.
[0167] The lipid composition may comprise a single lipid group or multiple lipid groups. Nonlimiting examples of lipid groups include cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)ylated lipids, ionizable lipids, helper lipids, stealth lipids, or cholesterols.
[0168] Nonlimiting 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-trimethyl ammonium propane, DOTAP 1 ,2-Dioleoyl-3- trimethyalammoniumpropane, and DC-Cholesterol 3p-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol. [0169] Nonlimiting 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,31 Z)-heptatriaconta-6,9,2823icol23htraen-19-yl4-(dimethylamino) butanoate, and DODMA 1 ,2-Dioleyloxy-3-dimethylamino propane.
[0170] Nonlimiting examples of helper lipids include cholesterol (1 F?,3aS,3bS,7S,9aF?,9bS,11 aF?)-9a,1 1 a-Dimethyl-1 -[(2F?)-6-methylheptan-2-yl]-
2, 3, 3a, 3b, 4, 6, 7, 8, 9, 9a, 9b, 10,1 1 ,11 a-tetradecahydro -1 /7-cyclopen23icoll23hrenethren- 7-ol DSPC 1 ,2-distearoyl-sn-glycero-3-phosphocholine, and DOPE 1 ,2-Dimyristoyl-sn- glycerophosphoethanolamine.
[0171] Nonlimiting examples of stealth lipids include PEGIG (R)-2,3- bis(myristoyloxy)propyl-1 -(methoxy poly (ethylene glycol) 2000) carbamate and ALC- 0159 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide.
[0172] In some embodiments, the nanoparticles may be polymer based, metal (e.g., silver, gold, palladium, titanium, zinc, or copper) based, silica based, or lipid based.
[0173] In some embodiments, the nanoparticles are a multilayer nanoparticle. Nonlimiting examples of multilayer nanoparticles (e.g., a nanoparticle having two or more polymer based layers; a nanoparticle having two or more metal based layers; a nanoparticle having two or more silica based layers; a nanoparticle having two or more lipid-based layers; or a nanoparticle having a first layer 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 selected from the group consisting of a polymer- based layer, a metal based layer, a silica-based layer, and a lipid-based layer, wherein the second layer composition is different from the first layer composition).
[0174] In some embodiments, lipid nanoparticles incorporate antigen presenting cell (APC) moieties, such as DC binding moieties. Nonlimiting examples of APC binding moieties include antibodies and/or single-chain variable fragment (scFv) to surface molecules expressed on dendritic cells (e.g., CD1 a, CD1 c (BDCA1 ), CD1 1 b (ITGAM), CD1 1 c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC-205), CD206 (MRC1 ), CD209 (DC-SIGN), FceR1 , HLA-DR, TLR2 (e.g., glycolipids and phospholipids), TLR3 (double-stranded RNA), TLR4 (lipopolysaccharide), TLR7/8 (single-stranded RNA), and TLR9 (unmethylated CpG DNA)). In some embodiments, the APC binding moieties are conjugated to a lipid in the lipid composition. The APC binding moieties may be identified or designed using methods including, but not limited to, critical process parameters (CPPs), high- throughput sequencing, or predictive modeling.
[0175] The APC moieties may be a ligand that binds a surface marker (e.g., peptide, glycoprotein, carbohydrate) of the APC cell (e.g., a DC, a B cell, a T cell, a macrophage). In some embodiments, the surface marker is a molecule that is upregulated during maturation of the APC cell.
[0176] In some embodiments, lipid nanoparticles comprise opsonization elements. The opsonization elements may engage APCs (e.g., bind or interact with APCs). The opsonization elements may induce the phagocytic process of macrophages, neutrophils, and dendritic cells. In some embodiments, the opsonization elements incorporate mannose or other carbohydrates.
[0177] In some embodiments, the lipid nanoparticles comprise Fc binding fragments. The Fc binding fragments bind Fc receptors on APCs.
[0178] In some embodiments, the lipid nanoparticle incorporates complement factors C3b, C4 and or C1q.
Cell Compositions
[0179] The present technology comprises cell compositions that are immunogenic to antigens and promote immunological memory. The cell composition may be generated such that the cells are immunogenic to specific antigens (e.g., viral antigens). Additionally, the cell compositions may stimulate an immune reaction leading to the production of an immune response. Nonlimiting examples of immune responses include the production of antibodies or activation of lymphatic cells (e.g., T-cells). In some embodiments, the cell compositions of the present technology induce an immune response in a subject to one or more antigens. In some embodiments, the cell compositions of the present technology may be administered to a subject to treat, prevent, and/or reduce a disease and/or other conditions associated with an infection.
[0180] The cell composition may be immunogenic to a protein and/or peptide of the present technology. In some embodiments, the cell composition is immunogenic to a nucleotide sequence of the present technology (e.g., a nucleotide sequence encoding a peptide of the present technology). In some embodiments, the cell composition is immunogenic to one or more peptides of the present technology and one or more nucleotide sequences of the present technology (e.g., a nucleotide sequence encoding a viral peptide). In some embodiments, the cell composition is immunogenic to two or more peptides of the present technology and one or more nucleotide sequences of the present technology. In some embodiments the cell composition is immunogenic to two or more nucleotide sequences of the present technology and one or more peptides of the present technology. In some embodiments, the cell composition is immunogenic to two or more nucleotide sequences of the present technology and two or more peptides of the present technology.
[0181] The cell compositions of the present technology may be generated from a subject sample (e.g., a biological sample). The subject sample may 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.
[0182] In some embodiments, the cell composition is a lymphatic cell composition. The cell composition may comprise one or more of T-cells, B cells, Natural Killer (NK) cells, Dendritic Cells (DCs), macrophages, or granulocytes.
[0183] In some embodiments, the cell composition is a T-cell composition. T-cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells). Nonlimiting examples of Th cells include Th1 cells, Th2 cells, Th17 cells.
[0184] In some embodiments, the cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.
[0185] In some embodiments, the Th1 (CD4+) T-cells produce cytokines, including IFNy, and generate a CD8+ T-cell (e.g., cytotoxic T-cell) response. In some embodiments, Th2 (CD4+) T-cells produce cytokines, including IL4, and generate a high affinity antibody immune response, including class switching to IgG and IgA. [0186] In some embodiments, the cell composition is reactive to one or more cytokines and/or chemokines (e.g., activation, differentiation, signaling, and/or function of an immune cell is stimulated in the presence of the cytokines or chemokines). Nonlimiting examples of cytokines and chemokines include interleukins (ILs) (e.g., IL- 2, IL-4, IL-6, IL-8, IL-10, or IL-12), tumor necrosis factors (TNFs) (e.g., TNFa or TNFp), interferons (INFs) (e.g., INFy, INFa, or INFP), chemokines (e.g., CXCL8, CXCL10, or CCL2), colony-stimulating factors (CSFs) (e.g., granulocyte-macrophage CSF or granulocyte CSF), or Transforming Growth Factor-beta (e.g., TGFP).
[0187] In some embodiments, the T cell composition is present in a vaccine composition or an infusion.
[0188] Other Composition Features & Formulations:
[0189] In some embodiments, the compositions of the present technology (e.g., vaccine compositions or cell compositions) comprise a pharmaceutically acceptable carrier and/or excipient. The pharmaceutically acceptable carrier and/or excipient may comprise aqueous or non-aqueous carriers that may facilitate dissolution of ac component of the compositions (e.g., peptides or nucleotide sequences). Nonlimiting examples of pharmaceutically acceptable carriers and/or excipients include sucrose, polysorbate 80, monobasic sodium phosphate, monohydrate, dibasic sodium phosphate, dihydrate. The composition may comprise a filler or diluent (e.g., lactose, mannitol, and microcrystalline cellulose), a disintegrant (e.g., croscarmellose sodium, crospovidone, starches), a buffer (e.g., phosphate buffer or acetate buffers), a solvent (e.g., water, alcohol, glycerin), or a stabilizer (e.g., sugars or gelatins); a preservative (e.g., thimerosal).
[0190] In some embodiments, the compositions of the present technology comprise adjuvants. Adjuvants may stimulate or enhance an immune response upon administration of the composition. Nonlimiting 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.
[0191] In some embodiments, the composition is sterilized (e.g., sterilization by filtration). [0192] In some embodiments, the compositions of the present technology comprise features that promote binding to and/or co-localization with an HLA (e.g., HLA- A, HLA-B, or HLA-C), an APC (e.g., a peptide or nucleotide sequence that targets or binds to an MHC Class II molecule), and/or a T-cell receptor (TCR). In some embodiments, the compositions comprise a molecule that binds a receptor on an APC (e.g., dendritic cells). In some embodiments, the molecule is a carbohydrate. In some embodiments, the carbohydrate is a mannose carbohydrate. The mannose carbohydrate may be selected from the group consisting of D-mannose, mannose-6- phosphate (M6P), a mannans, a mannose polymer, and a mannose receptor ligand. In some embodiments, the molecule that binds a receptor on an APC is selected from the group consisting of Mannose, CD180, CD209, and HLA-DR.
[0193] In some embodiments, the compositions of the present technology comprise a hydrophobic tail. The hydrophobic tail may act as a targeting moiety, promoting binding to APCs. In some embodiments, the hydrophobic tail comprises a fatty acid, a phospholipid, a cholesterol, a retinoid, a steroid, an alkyl chain, or a nonpolar amino acid side chain.
[0194] In some embodiments, the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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.
[0195] In some embodiments, the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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.
[0196] In some embodiments, the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology 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.
[0197] In some embodiments, the composition comprises a lyophilized drug product (lyophilized powder or lyophilized cake of the peptides or nucleotide sequences). In some embodiments, the lyophilized drug product is reconstituted (e.g., using water or saline) prior to administration.
[0198] In some embodiments, the reconstituted composition provides a solution having a pH of about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about
6.4, about 6.5, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about
7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about
8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about
8.9, or about 9.0.
[0199] In some embodiments, the reconstituted composition provides 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.
[0200] In some embodiments, the reconstituted composition provides a solution having 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 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.
[0201] In some embodiments, the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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.
[0202] In some embodiments, the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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 1 1 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.
[0203] In some embodiments, the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is 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 1 1 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.
[0204] In some embodiments, the composition or the solution is diluted for administration. Nonlimiting examples of diluents include sodium chloride, water (e.g., sterile water for injection (SWFI)), dextrose solutions, bacteriostatic solutions, or Ringer’s solutions (e.g., solutions comprising electrolytes, lactated Ringer’s solution).
Associated Methods
[0205] The present technology comprises methods of generating the vaccine compositions and the cell compositions of the present technology.
[0206] Quantifying Lymphocyte Cell Populations
[0207] In some embodiments, the methods of generating the vaccine compositions and/or the cell compositions of the present technology comprise a step of quantifying a lymphocyte cell (e.g., T-cell, B cell, NK cells, DC, macrophage, or granulocyte) population in a sample from a subject that has successfully cleared a virus (e.g., elimination of active infection, reduction of viral load, resolution of a symptom associated with the virus, establishment of immune memory against the virus). Quantifying the lymphocyte cell population may comprise methods including, but not limited to, flow cytometry, immunohistochemistry, immunofluorescence, protein quantification and/or detection methods (e.g., assessing levels of lymphocyte cell makers), or gene expression quantification methods (e.g., assessing expression levels of transcripts associated with lymphocyte cells).
[0208] In some embodiments, the lymphocyte cell population is a T-cell population. T-cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells). Nonlimiting examples of Th cells include Th1 cells, Th2 cells, Th17 cells. [0209] In some embodiments, the T-cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Th1 cells, Th2 cells, and Th17 cells.
[0210] The sample from the subject may 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.
[0211 ] In some embodiments, the subject that has successfully cleared a virus has or had mild symptoms associated with viral infection or no symptoms associated with viral infection). The virus may be an arterivirus, a mesonivirus, a ronivirus, a torovirus, or a coronavirus. In some embodiments, the virus is selected from the group consisting of a cytomegalovirus, an Epstein-Barr virus, a hepatitis B virus, a human papillomavirus, an adenovirus, a herpes virus, a human immunodeficiency virus, an influenza virus, a human respiratory syncytial virus, a vaccinia virus, a varicella-zoster virus, a yellow fever virus, an Ebola virus, a coronavirus, an Eastern equine encephalitis virus, a Polyomavirus hominisl (BKV), a SV40 and a Zika virus. In some embodiments, the virus comprises a variant of interest ( VOI) , variant of concern (VOC) or variant of high consequence (VOHC), as defined by the US Center for Disease Control and the World Health Organization (WHO).
[0212] In some embodiments, the virus may be a virus that is associated with and/or is capable of pandemic-like infection rates.
[0213] 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 ( I B V)), 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.
[0214] In some embodiments, the coronavirus is a common cold coronavirus. Nonlimiting examples of common cold coronaviruses include Human coronaviruses 229E (HCoV-229E), NL63 (HCoV-NL63), OC43 (HCoV-OC43), and HKU1 (HCoV- HKU1 ).
[0215] In some embodiments, the coronavirus is a SARS-CoV-2 strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV- 2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV-2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS-CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
[0216] Exposing the Sample to a Viral Antigen
[0217] In some embodiments, the methods of generating the vaccine compositions and/or cell compositions of the present technology comprise a step of exposing a sample to one or more antigens from the virus. Exposing the sample may comprise direct exposure (e.g., inoculating the sample with a protein antigen, peptide antigen, lipid antigen, glycoprotein antigen, or a nucleotide sequence that produces an antigen), or indirect exposure. Indirect exposure may comprise exposing the sample to the one or more antigens using an antigen-presenting cell (APC). Nonlimiting examples of APCs comprise DCs (e.g., follicular DCs), macrophages, B cells, monocytes, and Langerhans cells.
[0218] In some embodiments, the methods comprise quantifying the lymphocyte population before, during, and/or after exposure to the one or more viral antigens.
[0219] In some embodiments, the lymphocyte population is quantified 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 the 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 the one or more viral antigens. In some embodiments, the lymphocyte population is 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 the one or more viral antigens. [0220] In some embodiments, quantifying the lymphocyte cell population comprises a normalization step.
[0221] In some embodiments, the methods comprise a step of calculating a difference in the lymphocyte cell population quantity between two different time points. In some embodiments, the two different time points comprise a time point before exposure to the one or more viral antigens and a time point after exposure to the one or more viral antigens. In some embodiments, the two different time points comprise a time point before exposure to the one or more viral antigens and a time point during exposure to the one or more viral antigens. In some embodiments, the two different time points comprise a time point during exposure to the one or more viral antigens and a time point after exposure to the one or more viral antigens.
[0222] In some embodiments, calculating the difference in the lymphocyte cell population between two different time points further comprises comparing the difference to a threshold value. The threshold value may be useful in determining whether lymphocyte cell populations change in response to the one or more viral antigens.
[0223] In some embodiments, the one or more viral antigens or a nucleotide sequence encoding the one or more viral antigens is included in a vaccine composition if the difference in the lymphocyte cell population exceeds the threshold value.
[0224] Expansion of Lymphocyte cells
[0225] In some embodiments, the methods of generating cell compositions of the present technology comprise a step of exposing a second sample to one or more antigens from the virus. In some embodiments, the one or more viral antigens or a nucleotide sequence encoding the one or more viral antigens are used to expose a second sample from a second subject if the difference in the lymphocyte cell population exceeds the threshold value. The second sample may comprise 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.
[0226] The second subject may comprise a subject that has never been exposed to the virus (i.e., a naive subject), a subject that has never been infected with the virus, a subject that is infected with the virus, or a subject that has symptoms associated with infection with the virus.
[0227] In some embodiments, the lymphocyte cell population is expanded in the second sample after exposure to the one or more viral antigens or the nucleotide sequence encoding the one or more viral antigens. Nonlimiting examples of methods that may be used to expand the lymphocyte cell population include cytokine stimulation, cell culture methods (e.g., addition of nutrients or growth factors to promote proliferation; co-culture systems); or bioreactors, antibody stimulation. In some embodiments, the expanded lymphocyte cell population is isolated and/or may be screened for reactivity to a cytokine and/or chemokine (e.g., IFNy, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGFp, CXCL12, CCL19) formulation for administration (e.g., in a vaccine composition).
Examples
Identification of Viral Antigens
[0228] To identify which viral antigens were most likely to produce a response for pilot experiments, the SARS-CoV-2 antigen response pattern of T-cells of subject who has successfully cleared SARS-CoV-2 infection with minimal side effects was compared to T-cells obtained from subjects naive for SARS-CoV-2 infection. These antigens included CoV-2 S, M, N, 3a, 7a, 8, and Nsp6 (FIGs. 1 A-1 C).
[0229] To test this concept, peptide sequences corresponding to one or more of the identified immunodominant viral antigens were synthesized, and DCs were loaded with peptides associated with all or portions of the viral antigens (e.g., SARS-CoV-2 S, M, N, 3a, 7a, 8 and nsp6) and then used to prime subject T-cells. A diverse CD4+ and CD8+ T-cell response was produced in PBMCs from subjects naive for SARS-CoV-2 infection against these antigens with a comparable response pattern to that of the T- cells from the subjects who had successfully cleared SARS-CoV-2 infection, as measured by an activation included marker (AIM) assay (FIGs. 2A and 2B). Subjects showed higher response for DC-mediated viral antigen presentation, whereas for PBMCs and Naive donors, no response for all antigens was observed. This suggests that stimulating T-cells with SARS-CoV-2 specific viral antigens via DCs may induce a robust sensitized T-cell population. This also suggests that a protective immune response to a virus may be generated prior to viral infection. Generation of DCs and T-Cell Culture
[0230] Whole blood samples from subjects naive for SARC-CoV-2 infection were obtained by blood draw or apheresis, and PBMCs were isolated from the blood by Ficoll separation. Monocyte-derived dendritic cells (DCs), were produced using a cell culture plastic adhesion method. NonadherenT-cells were removed and frozen to use as a T- cell source in later cultures. Differentiation media comprising complete DC culture media was supplemented with 800 U/mL human IL-4, and 500 U/mL human GM-CSF. Cells were cultured in differentiation media for 5 days with media exchanges everyday starting one day after initial culture. On day 5, differentiation media was removed, and maturation media was added overnight. The maturation media was comprised of IL-6, IL1 p, TNFa, and Prostaglandin E2 (PGE2). Prostaglandin E2 may be substituted with Polyinosinic:polycytidylic acid (Polyl:C), which binds toll-like receptor 3 (TLR3) (e.g., TLR3 on DCs).
[0231] On day 6 of differentiation and maturation of adherent T-cells (typically monocytes) to DCs, the harvested DCs were combined with the SARS-CoV-2 viral peptides S, M, N, 3a, 7a, 8, and S+ (all peptides combined). The nonadherent T-cell fraction was thawed and combined with DCs in the ratio of 2:1 nonadherent T-cells (T- cells) to DCs. The total volume was 1 mL at a cell density of 3x106 cells/mL using complete Cellgenix GMP DC media supplemented with a cytokine cocktail. Peptides resuspended in DMSO were added such that each peptide was at the final concentration of 0.1 pg/mL. Cells were plated into a well plate. The culture was moved to a humidified incubator (5% CO2 37°C). Every two days half of the medium was exchanged for fresh media without disturbing the cells. On day 7, the process was repeated by thawing PBMCs and combining with new DCs and peptides, adding to current culture. On day 14, a CD3/CD28/CD2 T-Cell activator mix was added to cultures (15 pl/mL) with fresh medium containing cytokines and placed back into a 5% CO237°C humidified incubator.
Activation Induced Markers Assay and Phenotyping by Flow Cytometry
[0232] Activation induced markers (AIM) assays and phenotyping flow cytometry assays were conducted on days 14, 21 , and 28. Cell count and viability were also quantified. One million cells per well were plated in 2 separate well plates for AIM assays and phenotype flow cytometry assays. A stimulation with an equimolar amount of dimethyl sulfoxide (DMS)O was performed as negative control for both assays, and cells were stained with antibody cocktails for 15 min at room temperature in the dark. After the final wash, cells were resuspended in fluorescence-activated cell sorting (FACS buffer) and were quantified and analyzed using FlowJo Flow Cytometry Analysis software (TreeStar).
Profiling Th1 and Th2 T-cells
[0233] T-cells reactive to viral antigens were plated (1 x105 per well) on enzyme- linked immunosorbent spot (ELISpot) plates comprising IFNy and IL4 (FIGs. 3A-3I). After 24-hour incubation, the plates were washed and incubated with a second antibody against IFNy which enzymatically catalyzes a first color and anti IL-4 which enzymatically catalyzes a second color. After drying, the first color spots and the second color spots were counted. Each spot represented a single T-cell secreting the analyte cytokine, where Th1 cells secreted IFNy and Th2 cells secrete IL-4. These assays were performed on T-cells at day 14 and day 21 post stimulation. T-cell immunological memory was then measured as a percentage of CD3+CD62L+CD197+ T-cell populations (FIG. 4).
Selection of T-Cell Antigens for Vaccine Incorporation
[0234] T-cell antigens most likely to be reactive with approximately 50% accuracy were identified by using an MHC class I binding predictor MHCnetpan on the full SARS- CoV-2 amino acid sequence for the top 1 % (71 most common MHC alleles, HLA-A, B, C) in the population (Table 4). The frequency of subjects expressing at least two of the MHC alleles represents approximately 90% of the population. The antigens selected may be immunogenic and may provide protection in a high percentage of the population (FIG. 5).
Table 4: Most Common HLA alleles with a population frequency greater than 1 %
Figure imgf000083_0001
Figure imgf000084_0001
Manufacturing of GMP RNA Vaccines
[0235] An RNA vaccine was produced using an RNA construct designed to express S, M, N, 3a, 7a, 8, and Nsp6 antigens (FIG. 6). All reagents used were derived from sources that did not contain contaminants and were produced with defined media and not natural sources. The RNA underwent a purification process. Following purification, the RNA may be encapsulated in lipid nanoparticles, or with cationic proteolipids such as protamine, with/or carrier proteins and small molecules.
[0236] The mRNA construct comprised a linker sequence (SEQ ID NO: 10) was intercalated between the nucleotide sequences which encoded viral antigens. This linker may comprise low immunogenicity as indicated by use of the NetMHC MHC I binding affinity tool. The MHC I binding predictions were done with IEDB analysis resource using NetMHCpan method. The viral antigen sequences of interest were contained in the areas in which the binding affinity is below nM binding (Table 5) where lower rank indicates better binding.
Table 5: Linker Sequence Binding Ability to the Top 1 % HLA-ABC Binders
Figure imgf000085_0001
Figure imgf000086_0001
[0237] Strong binding peptides selected from the viral peptides identified for the RNA vaccine were 60nM or below for the most common HLA. The linker did not bind having 40,000 nM or above for all of the HLA. (Tables 6-12)
Table 6: Examples of Peptide Selected for NSP6
Figure imgf000086_0002
Figure imgf000087_0001
Table 7: Examples of Peptide Selected for Spike
Figure imgf000087_0002
Table 8: Examples of Peptide Selected for 0RF3a
Figure imgf000088_0001
Table 9: Examples of Peptide Selected for VME1
Figure imgf000088_0002
Figure imgf000089_0001
Table 10: Examples of Peptide Selected for ORF7a
Figure imgf000089_0002
Figure imgf000090_0001
Table 11 : Examples of Peptide Selected for 0RF8
Figure imgf000090_0002
Figure imgf000091_0001
Table 12: Examples of Peptide Selected for NCAP
Figure imgf000091_0002
Figure imgf000092_0001
[0238] The peptides selected were combined into peptides of 30 amino acids which were placed between the linker sequences and scrambled peptides from different viral peptides (e.g., in the Se construct or different parts of the protein) on either side of the linker sequence. This length provided an optimal number antigens, but also does not allow for the tertiary folding or creation of functional domains of the viral peptide.
[0239] This vaccine design comprises nucleotide sequences expressing antigens of all SARS-CoV-2 variants to date, including XBB (BA.2.10). Although the vaccine was designed using antigens from the alpha strain of SARS-CoV-2, only 1.69% of the antigens selected by our method across all 7 proteins had mutated across the alpha, delta and omicron variants, so that 98.31 % of the antigens are still capable of producing an effective immune response against the newest strains (Table 13). Additionally, 28/34 mutations in the antigens between alpha and omicron strains are in S and only 7 are present in antigens selected from all other viral peptides combined.
Table 13: Common Mutations for 3 Viral Variants
Figure imgf000092_0002
Figure imgf000093_0001
Assessing Vaccine Compositions in a Primate Model
[0240] To assess the vaccine compositions in a primate model, Macaque monkeys were injected intramuscularly on day 0 and day 21 in 3 groups including 6 animals each. Group 1 was administered a vaccine generated from the sequences of the SARS-CoV- 2 alpha strain with 200 ug of mRNA conserved antigen + 100 ug of mRNA for Spike vaccine for two injections. Group 2 was administered a vaccine generated from the sequences of the SARS-CoV-2 alpha strain with 200 ug of mRNA conserved antigen alone. Group 3 was administered a vaccine generated from the sequences of the SARS-CoV-2 alpha strain with 100ug of mRNA for Spike antigen alone. The mRNA antigen vaccines were comprised of 6 of the designated conserved antigens. Blood was drawn on days 14 and 35 to test for T cell gamma interferon production in response to the injected antigens. Antigen specific T cell frequency was determined by pulsing with antigen specific peptides and screened using the ELISpot assay for INFy. The results showed that T cell spots that equate to each T cells’ IFNy release, in a Th-1 response to each target antigen multivalent target. At day 35 post challenge, half of the animals (N=3) in each Group were challenged with SARS-Cov-2 alpha strain or with Omicron XBB strain. The animals were then followed for 30 days for pulmonary symptoms, weight loss and, starting on Day 42 and every 7 days thereafter, Nasal swab sampling and antigen testing for viral peptide was used to screen for active SARS-CoV-
2 virus.
[0241] After 30 days, none of the animals vaccinated with the SARS-CoV-2 alpha strain and challenged with the SARS-CoV-2 alpha had weight loss, pulmonary infection, or presence of viral peptides. The identity results are observed by challenge with the XBB strain for animals in Group 1 and Group 2. However, 2 of 3 of the animals in Group
3 who only received the vaccine to the SARS-CoV-2 alpha Spike as the vaccination had weight loss, pulmonary infection, and presence of viral peptides (Table 14). This suggests that the conserved proteins are protective across viral strains and may be usually in dampening viral stain evolution. Table 14: Animals Dosed with Alpha Strain Vaccine
Figure imgf000095_0001
[0242] Development of a DC targeted nanoparticle may enhance the delivery of RNA encoding vaccine antigens to DC’s. The lipids were used to encapsulate and deliver an RNA encoding green fluorescent protein (GFP) in vitro. DCs were analyzed 24 hours after nanoparticle delivery vis flow cytometry to assess GFP expression. DCs were either exposed to nanoparticles carrying GFP RNA as a control or nanoparticles carrying SARS-CoV-2 RNA encoding viral peptides (FIG. 7 and FIG. 8). Nanoparticles were designed to target DCs using mannose, CD180, CD209, or HLA-DR-targeting ligands. Nanoparticles with targeting ligands were more efficient in RNA delivery, as measured by GFP levels (FIG. 9).
[0243] Macaque monkeys were administered nanoparticles comprising 6 SARS- CoV-2 viral antigens, via a vaccine. Controls comprised nanoparticles having no targeting ligand and/or no viral antigen load. DC targeted lipid nanoparticles resulted in greater protection of Macaques when challenged with Omicron XBB at lower concentrations of RNA than the protection achieved with non-targeted lipid nanoparticles (Table 15).
Table 15: Infection Rates in Animals Administered Lipid Nanoparticle Compositions
Figure imgf000095_0002
Figure imgf000096_0001
Additional Embodiments
[0244] The present technology includes, but is not limited to, the following specific embodiments set forth herein below in paragraphs [0241 ]-[0319]:
[0245] 1. A vaccine composition comprising a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0246] 2. A vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen,
[0247] the first and second antigens each independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0248] 3. A vaccine composition comprising a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
[0249] 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 a NCAP (N) peptide, a nucleotide sequence encoding, 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 a Nsp6 peptide.
[0250] 5. The vaccine composition of any one of the preceding embodiments, wherein the peptide length is less than 30 amino acids. [0251] 6. The vaccine composition of embodiment 5, wherein the peptide length is about 5 amino acids to about 20 amino acids.
[0252] 7. The vaccine composition of embodiment 6, wherein the peptide length is about 9 amino acids.
[0253] 8. The vaccine composition of embodiment 6, wherein the peptide length is about 14 amino acids.
[0254] 9. The vaccine composition of embodiment 2 or 4, wherein the nucleotide sequences are deoxyribonucleotide (DNA) sequences.
[0255] 10. The vaccine composition of embodiment 2 or 4, wherein the nucleotide sequences are ribonucleotide (RNA) sequences.
[0256] 11. The vaccine composition of any one of the preceding embodiments, wherein the peptide does not comprise an active site.
[0257] 12. The peptide composition of any one of the preceding embodiments, wherein the peptide does not fold into tertiary peptide structures.
[0258] 13. The vaccine composition of any one of the preceding embodiments, wherein the Spike (S) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity 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% identity 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% identity to one or more of SEQ ID NOs: 256-261 ; the 8 peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 268-281 .
[0259] 14. A vaccine composition comprising two or more antigens selected from the group consisting of a Spike (S) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 210-220; a NCAP (N) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity to one or more of SEQ ID NOs: 256-261 ; an 8 peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 262-267; or a Nsp6 peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 268-281 .
[0260] 15. A vaccine composition comprising two or more nucleotide sequences selected from the group consisting of a first nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67; a second nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity 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% identity to one or more of SEQ ID NOs: 114-1 19; a sixth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 126-139.
[0261 ] 16. The vaccine composition of any one of embodiments 2, 4, 9, or 15, wherein the nucleotide sequences are present in a lipid composition.
[0262] 17. The vaccine composition of embodiment 16, wherein the lipid composition comprises a lipid nanoparticle.
[0263] 18. The vaccine composition of embodiment 17, wherein the lipid nanoparticle comprises an antigen presenting cell (APC) targeting molecule.
[0264] 19. The vaccine composition of embodiment 18, wherein the APC is a dendritic cell (DC).
[0265] 20. The vaccine composition of embodiment 18 or 19, wherein the
APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
[0266] 21. The vaccine composition of embodiment 1 , 3, 13, or 14, wherein each of the peptides are present on a single peptide chain.
[0267] 22. The vaccine composition of embodiment 21 , wherein the single chain comprises one or more linker sequences.
[0268] 23. The vaccine composition of embodiment 1 , 3, 13, or 14, wherein each of the peptides are present on different peptide chains.
[0269] 24. The vaccine composition of embodiment 2 or 4, wherein each of the nucleotide sequences are present in a polycistronic sequence.
[0270] 25. The vaccine composition of embodiment 24, wherein the polycistronic sequence comprises one or more linker sequences.
[0271] 26. The vaccine composition of embodiment 2, 4, 9, or 15, wherein each of the nucleotide sequences are present on different nucleotide chains. [0272] 27. The vaccine composition of any one of embodiments 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigen peptide.
[0273] 28. The vaccine composition of embodiment 1 or 2, wherein one or more of the antigens comprise a coronavirus peptide.
[0274] 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.
[0275] 30. The vaccine composition of embodiment 29, wherein the SARS-
CoV-2 antigen is an antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS-CoV-2 zeta antigen, a SARS-CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS-CoV-2 kappa antigen, a SARS-CoV-2 lambda antigen, and a SARS-CoV-2 mu antigen.
[0276] 31 . A vaccine composition comprising an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 282.
[0277] 32. A vaccine composition comprising an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 283.
[0278] 33. A vaccine composition comprising a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 140.
[0279] 34. A vaccine composition comprising a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 141.
[0280] 35. The vaccine composition of embodiment 33 or 34, wherein the nucleotide sequence is present in a lipid composition.
[0281] 36. The vaccine composition of embodiment 35, wherein the lipid composition comprises a lipid nanoparticle. [0282] 37. The vaccine composition of embodiment 36, wherein the lipid nanoparticle comprises an antigen presenting cell (APC) targeting molecule.
[0283] 38. The vaccine composition of embodiment 37, wherein the APC is a dendritic cell (DC).
[0284] 39. The vaccine composition of embodiment 37 or 38, wherein the
APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
[0285] 40. The vaccine composition of any one of the preceding embodiments, further comprising an adjuvant.
[0286] 41. A method of generating a vaccine composition, comprising the steps of
(i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject that has successfully cleared a virus;
(ii) exposing the sample to one or more antigens from the virus;
(iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(v) calculating a difference in the T cell population quantity between (i) and (iii);
(vi) comparing the difference in (iv) to a threshold value; and
(iv) including (a) a peptide of the one or more antigens in (ii), or (b) a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v).
[0287] 42. The method of embodiment 41 , wherein the vaccine composition is a multivalent vaccine composition.
[0288] 43. A method of generating a T cell composition that specifically recognizes one or more viral antigens, comprising the steps of
(i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus;
(ii) exposing the first sample to one or more antigens from the virus; (iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(iv) calculating a difference in the T cell population quantity between (i) and (iii);
(v) comparing the difference in (iv) to a threshold value;
(vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v);
(vii) expanding the T cells in the second sample after the exposure in (vi); and
(viii) isolating the expanded T cells.
[0289] 44. The method of embodiment 43, further comprising (ix) screening the expanded T cells for reactivity to interferon gamma (IFNy) or interleukin 4 (IL4).
[0290] 45. The method of embodiment 43, wherein the T cell composition is formulated for administration to the second subject.
[0291] 46. The method of embodiment 45, wherein the T cell composition is formulated in a vaccine composition.
[0292] 47. The method of any one of embodiments 41 -46, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.
[0293] 48. The method of any one of embodiments 41 -47, wherein the T cell population comprises T helper cells.
[0294] 49. The method of embodiment 48, wherein the T helper cells comprise Th1 or Th2 cells.
[0295] 50. The method of embodiment 43 or 47, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.
[0296] 51 . The method of embodiment 43 or 47, wherein the T cell composition comprises T helper cells.
[0297] 52. The method of embodiment 51 , wherein the T helper cells comprise Th1 or Th2 cells. [0298] 53. The method of any one of embodiments 41 -47, wherein the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
[0299] 54. The method of embodiment 53, wherein the APC is a dendritic cell
(DC).
[0300] 55. The method of embodiment 54, wherein the DC cells are stimulated with IL4 or Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
[0301] 56. The method of any one of embodiments 41 -55, wherein the virus is a coronavirus.
[0302] 57. The method of embodiment 56, wherein the coronavirus is SARS-
CoV-2.
[0303] 58. The method of embodiment 57, wherein the SARS-CoV-2 is strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV-2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV- 2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS- CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
[0304] 59. The method of any one of embodiments 41 -58, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
[0305] 60. The method of any one of embodiments 41 -58, wherein the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
[0306] 61 . A T cell composition immunogenic to viral antigens, generated by the steps of:
(i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus;
(ii) exposing the first sample to one or more antigens from the virus;
(iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(iv) calculating a difference in the T cell population quantity between (i) and (iii); (v) comparing the difference in (iv) to a threshold value;
(vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v);
(vii) expanding the T cells in the second sample after the exposure in (vi); and
(viii) isolating the expanded T cells.
[0307] 62. The T cell composition of embodiment 61 , further comprising (ix) screening the expanded T cells for reactivity to interferon gamma (IFNy) or interleukin 4 (IL4).
[0308] 63. The T cell composition of embodiment 61 or embodiment 48, wherein the T cell composition is formulated for administration to the second subject.
[0309] 64. The T cell composition of embodiment 63, wherein the T cell composition is formulated in a vaccine composition.
[0310] 65. The T cell composition of any one of embodiments 61 -64, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.
[0311] 66. The T cell composition of any one of embodiments 61 -65, wherein the T cell population comprises T helper cells.
[0312] 67. The T cell composition of embodiment 66, wherein the T helper cells comprise Th1 or Th2 cells.
[0313] 68. The T cell composition of any one of embodiments 61 -67, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.
[0314] 69. The T cell composition of any one of embodiments 61 -68, wherein the T cell composition comprises T helper cells.
[0315] 70. The T cell composition of embodiment 69, wherein the T helper cells comprise Th1 or Th2 cells.
[0316] 71 . The T cell composition of any one of embodiments 61 -70, wherein the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC). [0317] 72. The T cell composition of embodiment 71 , wherein the APC is a dendritic cell (DC).
[0318] 73. The T cell composition of embodiment 72, wherein the DC cells are stimulated with IL4 or Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
[0319] 74. The T cell composition of any one of embodiments 61 -73, wherein the virus is a coronavirus.
[0320] 75. The T cell composition of embodiment 74, wherein the coronavirus is SARS-CoV-2.
[0321 ] 76. The T cell composition of embodiment 75, wherein the SARS-CoV-
2 is strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV-2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS- CoV-2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS-CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
[0322] 77. The T cell composition of any one of embodiments 61 -76, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
[0323] 78. The T cell composition of any one of embodiments 61 -76, wherein the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.

Claims

I/We claim:
1 . A vaccine composition comprising a first antigen and a second antigen, each of the first antigen and the second antigen independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
2. A vaccine composition comprising (i) a first nucleotide sequence encoding a first antigen and (ii) a second nucleotide sequence encoding a second antigen, the first and second antigens each independently selected from the group consisting of a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a Nsp6 peptide.
3. A vaccine composition comprising a Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, and a 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 a NCAP (N) peptide, a nucleotide sequence encoding, 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 a Nsp6 peptide.
5. The vaccine composition of any one of the preceding claims, wherein the peptide length is less than 30 amino acids.
6. The vaccine composition of claim 5, wherein the peptide length is about 5 amino acids to about 20 amino acids.
7. The vaccine composition of claim 6, wherein the peptide length is about 9 amino acids.
8. The vaccine composition of claim 6, wherein the peptide length is about 14 amino acids.
9. The vaccine composition of claim 2 or 4, wherein the nucleotide sequences are deoxyribonucleotide (DNA) sequences.
10. The vaccine composition of claim 2 or 4, wherein the nucleotide sequences are ribonucleotide (RNA) sequences.
1 1 . The vaccine composition of any one of the preceding claims, wherein the peptide does not comprise an active site.
12. The peptide composition of any one of the preceding claims, wherein the peptide does not fold into tertiary peptide structures.
13. The vaccine composition of any one of the preceding claims, wherein the Spike (S) peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity 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% identity 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% identity to one or more of SEQ ID NOs: 256-261 ; the 8 peptide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 268-281 .
14. A vaccine composition comprising two or more antigens selected from the group consisting of a Spike (S) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 210-220; a NCAP (N) peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity to one or more of SEQ ID NOs: 256-261 ; an 8 peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 262-267; or a Nsp6 peptide at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 268-281 .
15. A vaccine composition comprising two or more nucleotide sequences selected from the group consisting of a first nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to one or more of SEQ ID NOs: 1 -67; a second nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity 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% identity 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% identity to one or more of SEQ ID NOs: 1 14-119; a sixth nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity 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% identity to one or more of SEQ ID NOs: 126-139.
16. The vaccine composition of any one of claims 2, 4, 9, or 15, wherein the nucleotide sequences are present in a lipid composition.
17. The vaccine composition of claim 16, wherein the lipid composition comprises a lipid nanoparticle.
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 a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
21. The vaccine composition of claim 1 , 3, 13, or 14, wherein each of the peptides are present on a single peptide chain.
22. The vaccine composition of claim 21 , wherein the single chain comprises one or more linker sequences.
23. The vaccine composition of claim 1 , 3, 13, or 14, wherein each of the peptides are present on different peptide chains.
24. The vaccine composition of claim 2 or 4, wherein each of the nucleotide sequences are present in a polycistronic sequence.
25. The vaccine composition of claim 24, wherein the polycistronic sequence comprises one or more linker sequences.
26. The vaccine composition of claim 2, 4, 9, or 15, wherein each of the nucleotide sequences are present on different nucleotide chains.
27. The vaccine composition of any one of claims 1 or 2, wherein the first antigen or the second antigen comprises a coronavirus antigen peptide.
28. The vaccine composition of claim 1 or 2, wherein one or more of the antigens comprise a coronavirus peptide.
29. The vaccine composition of claim 27 or 28, wherein the coronavirus peptide is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.
30. The vaccine composition of claim 29, wherein the SARS-CoV-2 antigen is an antigen selected from the group consisting of a SARS-CoV-2 alpha antigen, a SARS-CoV2 beta antigen, a SARS-CoV-2 gamma antigen, a SARS-CoV-2 delta antigen, a SARS-CoV-2 omicron antigen, a SARS-CoV-2 epsilon antigen, a SARS- CoV-2 zeta antigen, a SARS-CoV-2 eta antigen, a SARS-CoV-2 iota antigen, a SARS- CoV-2 kappa antigen, a SARS-CoV-2 lambda antigen, and a 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% identity 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% identity 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% identity 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% identity to SEQ ID NO:
141 .
-I OS-
35. The vaccine composition of claim 33 or 34, wherein the nucleotide sequence is present in a lipid composition.
36. The vaccine composition of claim 35, wherein the lipid composition comprises a lipid nanoparticle.
37. The vaccine composition of claim 36, wherein the lipid nanoparticle comprises an antigen presenting cell (APC) targeting molecule.
38. The vaccine composition of claim 37, wherein the APC is a dendritic cell (DC).
39. The vaccine composition of claim 37 or 38, wherein the APC targeting molecule is selected from the group consisting of a Mannose, a CD180, a CD209, or a HLA-DR targeting molecule.
40. The vaccine composition of any one of the preceding claims, further comprising an adjuvant.
41 . A method of generating a vaccine composition, comprising the steps of
(i) quantifying a T cell population in a peripheral blood mononuclear cell (PBMC) sample from a subject that has successfully cleared a virus;
(ii) exposing the sample to one or more antigens from the virus;
(iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(v) calculating a difference in the T cell population quantity between (i) and (iii);
(vi) comparing the difference in (iv) to a threshold value; and
(iv) including (a) a peptide of the one or more antigens in (ii), or (b) a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v).
42. The method of claim 41 , wherein the vaccine composition is a multivalent vaccine composition.
43. A method of generating a T cell composition that specifically recognizes one or more viral antigens, comprising the steps of
(i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus;
(ii) exposing the first sample to one or more antigens from the virus;
(iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(iv) calculating a difference in the T cell population quantity between (i) and (iii);
(v) comparing the difference in (iv) to a threshold value;
(vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v);
(vii) expanding the T cells in the second sample after the exposure in (vi); and
(viii) isolating the expanded T cells.
44. The method of claim 43, further comprising (ix) screening the expanded T cells for reactivity to interferon gamma (I FN y) or interleukin 4 (IL4).
45. The method of claim 43, wherein the T cell composition is formulated for administration to the second subject.
46. The method of claim 45, wherein the T cell composition is formulated in a vaccine composition.
47. The method of any one of claims 41 -46, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.
48. The method of any one of claims 41 -47, wherein the T cell population comprises T helper cells.
49. The method of claim 48, wherein the T helper cells comprise Th1 or Th2 cells.
-no-
50. The method of claim 43 or 47, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.
51 . The method of claim 43 or 47, wherein the T cell composition comprises T helper cells.
52. The method of claim 51 , wherein the T helper cells comprise Th1 or Th2 cells.
53. The method of any one of claims 41 -47, wherein the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
54. The method of claim 53, wherein the APC is a dendritic cell (DC).
55. The method of claim 54, wherein the DC cells are stimulated with IL4 or Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
56. The method of any one of claims 41 -55, wherein the virus is a coronavirus.
57. The method of claim 56, wherein the coronavirus is SARS-CoV-2.
58. The method of claim 57, wherein the SARS-CoV-2 is strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV-2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV-2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS-CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
59. The method of any one of claims 41 -58, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
-in-
60. The method of any one of claims 41 -58, wherein the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
61 . A T cell composition immunogenic to viral antigens, generated by the steps of:
(i) quantifying a T cell population in a first peripheral blood mononuclear cell (PBMC) sample from a first subject that has successfully cleared a virus;
(ii) exposing the first sample to one or more antigens from the virus;
(iii) quantifying the same T cell population in (i) after exposure of the one or more peptide antigens;
(iv) calculating a difference in the T cell population quantity between (i) and (iii);
(v) comparing the difference in (iv) to a threshold value;
(vi) exposing a second PBMC sample from a second subject to a peptide of the one or more antigens in (ii) or a nucleotide sequence encoding the one or more antigens in (ii) in the vaccine composition if the difference in (iv) exceeds the threshold value in (v);
(vii) expanding the T cells in the second sample after the exposure in (vi); and
(viii) isolating the expanded T cells.
62. The T cell composition of claim 61 , further comprising (ix) screening the expanded T cells for reactivity to interferon gamma (I FNy) or interleukin 4 (IL4).
63. The T cell composition of claim 61 or claim 48, wherein the T cell composition is formulated for administration to the second subject.
64. The T cell composition of claim 63, wherein the T cell composition is formulated in a vaccine composition.
65. The T cell composition of any one of claims 61 -64, wherein the T cell population comprises CD4+ T cells or CD8+ T cells.
66. The T cell composition of any one of claims 61 -65, wherein the T cell population comprises T helper cells.
67. The T cell composition of claim 66, wherein the T helper cells comprise Th1 or Th2 cells.
68. The T cell composition of any one of claims 61 -67, wherein the T cell composition comprises CD4+ T cells or CD8+ T cells.
69. The T cell composition of any one of claims 61 -68, wherein the T cell composition comprises T helper cells.
70. The T cell composition of claim 69, wherein the T helper cells comprise Th1 or Th2 cells.
71 . The T cell composition of any one of claims 61 -70, wherein the exposure of the one or more antigens in (ii) comprises an antigen presenting cell (APC).
72. The T cell composition of claim 71 , wherein the APC is a dendritic cell (DC).
73. The T cell composition of claim 72, wherein the DC cells are stimulated with IL4 or Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
74. The T cell composition of any one of claims 61 -73, wherein the virus is a coronavirus.
75. The T cell composition of claim 74, wherein the coronavirus is SARS-CoV- 2.
76. The T cell composition of claim 75, wherein the SARS-CoV-2 is strain selected from the group consisting of a SARS-CoV-2 alpha , a SARS-CoV2 beta , a SARS-CoV-2 gamma , a SARS-CoV-2 delta , a SARS-CoV-2 omicron , a SARS-CoV-
-US- 2 epsilon , a SARS-CoV-2 zeta , a SARS-CoV-2 eta , a SARS-CoV-2 iota , a SARS- CoV-2 kappa , a SARS-CoV-2 lambda , and a SARS-CoV-2 mu.
77. The T cell composition of any one of claims 61 -76, wherein the one or more antigens comprise Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
78. The T cell composition of any one of claims 61 -76, wherein the one or more antigens comprise a nucleotide sequence encoding Spike (S) peptide, a VME1 (M) peptide, a NCAP (N) peptide, a 3a peptide, a 7a peptide, an 8 peptide, or a Nsp6 peptide.
PCT/US2023/084666 2022-12-16 2023-12-18 A multi-antigenic rna sars-cov-2 vaccine and associated methods WO2024130254A2 (en)

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