T cell-based SARS-COV-2 vaccine

Abstract

The present disclosure relates to coronavirus vaccines and methods for use thereof.

Description

[0001] T CELL-BASED SARS-COV-2 VACCINE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to, and the benefit of, U.S. Provisional Patent Application No.63 / 612,412, filed December 20, 2023, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government Support under Grant No. R01AI139092 and R01AI157975 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on December 20, 2024, as an .XML file entitled “10013- 105WO1.xml” created on December 17, 2024, and having a file size of 85,714 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD The present disclosure relates to coronavirus vaccines and methods for use thereof. BACKGROUND Coronavirus disease 2019 (COVID-19) emerged in December 2019. The virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), that causes COVID-19 was sequenced and published in Jan 2020. SARS-CoV-2 caused a pandemic, with consequences much more severe than its close relative SARS-CoV-1, which was identified in 2002. Global collaborative efforts have been made by pharmaceutical companies, academic laboratories and governmental agencies, resulting in rapid development of vaccines to prevent SARS-CoV-2 infection or symptoms. Many of the vaccines are aimed at inducing immune responses (mostly neutralizing antibodies) to spike (S) protein of SARS-CoV-2, and the S protein sequence in the initial vaccines was from the original virus strain. Some of the vaccines have been approved in a number of countries, which are successful in reducing COVID-19. However, SARS-CoV-2 variants of concern have been reported, e.g., Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.l), Delta (B.1.617.2), and Omicron (BA.1.1.529 and its subvariants). It is expected that more variant strains will come up as the virus keeps mutating when replicating. The variant strains have deletions or substitutions in the S protein. Given the impending evolution of SARS-CoV-2 variant strains, there is a need to address the aforementioned problems mentioned above by developing improved vaccines against SARS- CoV-2 variants. The vaccines, compositions, and methods disclosed herein address these and other needs. SUMMARY The present disclosure provides DNA vaccines, vectors, and compositions thereof for preventing SARS-CoV-2 infection. The present disclosure also provides methods of inducing T cell-based immune responses using the DNA vaccines, vectors, and compositions thereof. The present disclosure also provides methods of preventing a SARS-CoV-2 infection using the DNA vaccines, vectors, and compositions thereof. In one aspect, disclosed herein is a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the spike protein is split and rearranged into three parts, and wherein the spike protein is fused to the ubiquitin protein. In one aspect, disclosed herein is a vector comprising the DNA vaccine of any preceding aspect. In one aspect, disclosed herein is a method of inducing a T cell-based response to a SARS- CoV-2 virus in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the spike protein is split and rearranged into three parts, and wherein the spike protein is fused to the ubiquitin protein. In one aspect, disclosed herein is a method of preventing a SARS-CoV-2 infection in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein. In one aspect, disclosed herein is a composition comprising the DNA vaccine of any preceding aspect, an adjuvant, and a pharmaceutically acceptable carrier. In some embodiments, the adjuvant comprises Imiquimod, Aluminum (hereinafter Alum), monophosphoryl lipid A (MPL), MF59, Poly(I:C), Montanide ISA51, AddaVax, or a combination thereof. In some embodiments, the DNA vaccine of any preceding aspect further encodes at least one cytotoxic T lymphocyte (CTL) epitope. In some embodiments, the protein of any preceding aspect comprises a mutation that can improve protein degradation. In some embodiments, the protein degradation-enhancing mutation comprises a glycine (G) residue at amino acid position 76 of the protein replaced by an alanine (A) residue. In some embodiments, the method of any preceding aspect further comprises inducing the T cell-based response against an original SARS-CoV-2 (WT-S), Alpha, Beta, Gamma, Delta, or Omicron SARS-CoV-2 variants, or subvariants thereof. In some embodiments, the T cell-based response comprises a response from CD8+T cells, CD4+T cells, or a combination thereof. In some embodiments, the vaccine of any preceding aspect provides protection without inducing an antibody response against the spike (S) protein. BRIEF DESCRIPTION OF FIGURES The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIGS.1A, 1B, and 1C show the SARS-CoV-2 T cell-based Ub-S DNA vaccine design and antigen expression. Figure 1A shows a schematic diagram of plasmid design. The gene sequence encoding for the spike (S) protein was split into three parts (denoted S-a, S-b, and S-c). The 30 nucleotide bases before and after any cleaved site were placed back to preserve any epitopes that may have been disrupted. The open-reading frame gene encoding for a human monomer of ubiquitin (Ub) was placed immediately upstream the rearranged S sequence. The gene encoding a glycine at the 76thresidue was modified to encode an alanine to enhance the stability of the Ub-S complex. Figure 1B shows the plasmids were digested with EcoRI and NotI and run on an agarose gel. Figure 1C shows that the 293T cells were transfected with the Ub-S plasmid encoding ubiquitinated and rearranged S protein, a Ub-S unmodified plasmid encoding ubiquitinated original S protein, or a plasmid encoding original S protein (no ubiquitin), overnight. The cells were allowed to stably express plasmid for 36 h. After this period, MG132 was added overnight (right). The cell lysate was analyzed via Western blot for the expression of SARS-CoV-2 S protein and beta-Actin as a control. Lanes were run on the same respective gels, but are noncontiguous. FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show that the SARS-CoV-2 Ub-S DNA vaccine induced strong T cell responses without antibodies in BALB / c mice. Mice were immunized with Ub-S DNA vaccine (plus Imiquimod adjuvant), full-length S protein vaccine control (plus Alum + MPL adjuvants), or PBS without adjuvant (background control), and boosted twice with the same immunogens plus the respective adjuvants at 3-week intervals. Ten days post- 3rddose, splenocytes from the mice were tested for S-specific T cell responses, and sera were tested for IgG antibodies specific to the S, N-terminal domain (NTD), receptor-binding domain (RBD), S1, or S2 of SARS-CoV-2. SARS-CoV-2 S-specific CD8+(Figures 2A and 2B) and CD4+(Figures 2C and 2D) T cells were analyzed by flow cytometry. Splenocytes were stimulated with pooled peptides (final concentration 5 μg / ml) from the SARS-CoV-2 S protein predicted to contain both CTL and T helper cell epitopes, and IFN-γ and TNF-α-producing CD45+CD8+T cells and IFN-γ and TNF-α-producing CD45+CD4+T cells were stained for respective cell surface and intracellular cytokine markers. Figures 2E, 2F, 2G, 2H, and 2I shows the serum SARS-CoV-2 S, NTD, RBD, S1, or S2-specific IgG antibodies tested by ELISA. The ELISA plates were respectively coated with SARS-CoV-2 full-length S protein or its fragments (1 μg / ml), and antibody (Ab) titers were calculated as the endpoint dilution that remained positively detectable. Data are expressed as mean ± standard deviation of the mean (s.e.m) of mice in each group (n=5). * and ** indicate significant difference among indicated groups. Experiments were repeated twice, and similar results were obtained. FIGS.3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show that the SARS-CoV-2 Ub-S DNA vaccine formulated with different adjuvants induced strong and durable T cell responses without antibodies in C57BL / 6 (B6) mice. Mice were immunized with Ub-S DNA vaccine, unmodified S DNA (S DNA) control, full-length S protein (S Protein) vaccine control, or PBS control in the presence of either Imiquimod adjuvant or Alum plus MPL adjuvants, and boosted with the same immunogens plus the respective adjuvants twice at 3-week intervals and once at 6 months. PBS without adjuvant was included as a background control. Splenocytes collected 10 days post-last dose from mice immunized with each immunogen plus Imiquimod adjuvant (A-D) or Alum + MPL adjuvants (E-H) were analyzed for SARS-CoV-2 S-specific CD8+(Figures 3A and 3B or 3E and 3F) and CD4+(Figures 3C and 3D or 3G and 3H) T cells by flow cytometry. Splenocytes were stimulated with pooled peptides (final concentration 5 μg / ml) from SARS-CoV-2 S protein predicted to contain B6 mouse CTL and Th cell epitopes, and IFN-γ or TNF-α-producing CD45+CD8+and CD45+CD4+T cells were stained for respective cell surface and intracellular cytokine markers. Sera collected 10 days post-3rddose from mice immunized with each immunogen plus Imiquimod adjuvant (Figures 3I, 3J, 3K, 3L, and 3M) or Alum + MPL adjuvants (Figures 3N, 3O, 3P, 3Q, and 3R) were detected for SARS-CoV-2 S, NTD, RBD, S1, or S2-specific IgG antibodies by ELISA. The ELISA plates were respectively coated with SARS- CoV-2 full-length S protein, as well as NTD, RBD, S1, or S2 fragment (1 μg / ml), and IgG antibody (Ab) titers were calculated as the endpoint dilution that remained positively detectable. Data are expressed as mean ± s.e.m of mice in each group (n=4-5) (five mice / group were used for antibody detection and four of them for T cell detection). *, ** and *** indicate significant difference among indicated groups. Experiments were repeated twice, and similar results were obtained. FIGS.4A and 4B show the SARS-CoV-2 Ub-S DNA vaccine induced protection against high-dose SARS-CoV-2 infection in mice.4-6-month-old hACE2-Tg mice were immunized with Ub-S DNA vaccine (plus Imiquimod adjuvant), full-length S protein (S Protein) vaccine control (plus Alum + MPL adjuvants), or PBS without adjuvant (background control). Two weeks post- 3rddose of immunization, mice were I.N. challenged with SARS-CoV-2 (strain 2019n-CoV / USA- WA1 / 2020, 5,000 PFU / mouse), and recorded for survival (Figure 4A) and weight changes (Figure 4B) for 14 days post-infection (p.i.). The data in Figure 4B are presented as mean + s.e.m. of mice in each group (n=7). ** indicates significant difference between S protein and PBS or Ub-S DNA and PBS groups. Experiments were repeated once, and similar results were obtained. FIGS. 5A, 5B, 5C, 5D, 5E, and 5F show the SARS-CoV-2 Ub-S DNA vaccine induced cross-protection against SARS-CoV-2 Omicron-BA5 infection without antibodies in mice. 10- week-old BALB / c mice were immunized with Imiquimod-adjuvanted Ub-S DNA vaccine, unmodified S DNA (S DNA) control, full-length S protein (S protein) control, or PBS control, and boosted twice with the same immunogens at 3-week intervals. Sera were collected from the mice before challenge, and then the mice were I.N. challenged with SARS-CoV-2 (Omicron-BA5 variant, 50,000 PFU / mouse) (Figures 5A, 5B, 5C, 5D, and 5E). Sere were analyzed for SARS- CoV-2 S, NTD, RBD, S1, or S2-specific IgG antibodies by ELISA. The ELISA plates were respectively coated with SARS-CoV-2 full-length S protein, NTD, RBD, S1, or S2 fragment (1 μg / ml), and IgG antibody (Ab) titers were calculated as the endpoint dilution that remained positively detectable. Figure 5F shows the viral titers in the lungs of challenged mice 2 days post- infection (p.i.). Data are presented as mean ± s.e.m. of mice in each group (n=5). ** and *** indicate significant difference between Ub-S DNA and other groups. Experiments were repeated once, and similar results were obtained. FIGS.6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the SARS-CoV-2 DNA vaccine-induced T cells provided protection against SARS-CoV-2 infection. hACE2-Tg mice were immunized with Imiquimod-adjuvanted Ub-S DNA vaccine or PBS control. Two months post-3rddose of immunization, mice were intraperitoneally injected with anti-mouse-CD4 (IgG2b, for depleting CD4+T cells), anti-mouse-CD8a (IgG2b, for depleting CD8+T cells), or IgG2b isotype control antibody (Iso Ctrl, without depleting CD4+and depleting CD8+T cells) (200 μg / mouse) at -2, -1, and 1 days. CD8+or CD4+T cell-depleted mice were I.N. challenged with SARS-CoV-2 (strain 2019n-CoV / USA-WA1 / 2020, 2,000 PFU / mouse), and recorded for weight changes (Figures 6A, 6B, and 6E) and survival (Figures 6C, 6D, and 6F) for 14 days post-infection (p.i.). PBS control mice receiving isotype control antibody were included as controls (Figure 6G and 6H). The data (Figures 6A, 6B, 6E, and 6G) are presented as mean + s.e.m. of mice in each group (n=3-5: one group had 3 mice as 2 mice accidently died before virus infection; all other groups had 5 mice / group). There were significant weight loss between the Ub-S DNA-immunized mice receiving isotype control antibody (Figure 6E) and anti-CD8 depletion antibody (Figure 6A) (***) or anti-CD4 depletion antibody (Figure 6B) (*), as well as the mice immunized with Ub-S DNA (Figure 6E) or PBS (Figure 6G) and received isotype control antibody (***). Experiments were repeated once, and similar results were obtained. FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H show the MHC class I epitope mutations in SARS-CoV-2 variants of concern. The S proteins of five variant strains were aligned with SARS- CoV-2 S protein of original strain to search for the predicted and confirmed CD8 T-cell epitope mutations. The results of the top six alleles with the highest occurring frequencies (see Table 1) for HLA-A, HLA-B, and HLA-C are shown above (Figures 7A, 7B, 7C, 7D, 7E, and 7F). Ten epitopes with the highest scoring IC50 values for each allele were chosen using TepiTool, a prediction analysis resource from the Immune Epitope Database (IEDB). Total 60 epitopes from HLA-A, B and C, respectively, were used for analysis for mutations. Total 286 confirmed epitopes from IEDB were analyzed for mutations for all MHC class I alleles (Figures 7G and 7H). Mutations found were sorted by single residue mutations, single residue deletions, or multiple mutations that encompass any combination of single residue mutations or deletions. FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, and 8H show the MHC class II epitope mutations in SARS-CoV-2 variants of concern. S proteins of five variant strains were aligned with SARS-CoV- 2 S protein to search for predicted and confirmed CD4+T-cell epitope mutations. The results of the top six alleles with the highest occurring frequencies (see Table 2) for HLA-DRB1, HLA- DQA1 / DQB1, and HLA-DPA1 / DPB1 are shown above (Figures 8A, 8B, 8C, 8D, 8E, and 8F). Ten epitopes of S protein from the original strain with the highest scoring IC50values for each allele were chosen using TepiTool, a prediction analysis resource from the Immune Epitope Database (IEDB). Total 60 epitopes from HLA-DR, DQ and DP, respectively, were used for analysis for mutations. Total 160 confirmed epitopes from IEDB were analyzed for mutations for all MHC class II alleles (Figure 8G and 8H). Mutations found were sorted by single residue mutations, single residue deletions, or multiple mutations that can encompass any combination of single residue mutations or deletions. FIGS.9A and 9B show the SARS-CoV-2 DNA vaccine induced protection against SARS- CoV-2 challenge with reduced viral titers. B6 mice were immunized with Ub-S DNA vaccine, full- length S protein vaccine control, or PBS control. Mouse sera were collected two weeks after last dose of immunization, and the mice were I.N. infected with SARS-CoV-2 (mouse-adapted strain N501YMA30, 5,000 PFU / mouse). Figure 9A shows that the SARS-CoV-2 titers were detected in the lung by plaque assay after 2 days p.i. Statistical difference between SARS-CoV-2 Ub-S DNA and PBS control was performed using unpaired student t test and GraphPad Prism 9 statistical software. * indicates P < 0.05. Figure 9B shows that the SARS-CoV-2 RBD-specific IgG antibodies were detected in the sera of mice before SARS-CoV-2 infection. The data are presented as mean ± s.e.m. of mice in each group (n = 4). Experiments were repeated once, and similar results were obtained. DETAILED DESCRIPTION The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. The following definitions are provided for the full understanding of terms used in this specification. The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%. As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. It should be noted that “composition” and “therapeutic composition” are used interchangeably throughout the disclosure. The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction below, above, or in between the given ranges as compared to native or control levels. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. An "effective amount" is an amount sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. “Effective amount” encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent, or diagnose a symptom or sign of a medical condition or disorder (e.g., SARS- CoV-2 infection). Unless dictated otherwise, explicitly or by context, an “effective amount” is not limited to a minimal amount sufficient to ameliorate a condition. The severity of a disease or disorder, as well as the ability of a treatment to prevent, treat, or mitigate, the disease or disorder can be measured, without implying any limitation, by a biomarker or by a clinical parameter. The “effective amount of a DNA vaccine” refers to an amount of DNA vaccine or compositions thereof sufficient to prevent ameliorate, reverse, or mitigate infection and / or spread of a SARS-CoV-2 virus. It should be noted that the terms “effective amount” and “therapeutically effective amount” can be used interchangeably. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue. A non-limiting example of “enhancing” refers to mutation of a ubiquitin protein to enhance stability of said protein. A “virus” is a microscopic infectious agent that replicates only inside the living cells of an organism. Viruses can infect all life forms, including mammalian and non-mammalian animals, plants, and other microorganisms. A complete virus, also known as a virion, consists of nucleic acid genetic material surrounded by a protective coat of protein called a capsid. Virus can have a lipid envelope derived from the infected host cell membrane. In general, there are five morphological virus types including helical, icosahedral, prolate, enveloped, and complex virus. A virus can either have a DNA or RNA genome. Irrespective of the type of nucleic acid genome, a viral genome can be either a single-stranded genome or a double-stranded genome. A “vaccine” refers to a biological preparation that provides active acquired immunity to a particular infectious diseases caused by a virus, bacteria, parasite, or any other microorganisms. Vaccines typically comprise an agent or several agents, also referred to as antigens, resembling the disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins, or its surface proteins / peptides. Vaccines are also made to comprise additional components, such as adjuvants, preservatives, and / or stabilizers to boost the immune response, improve safety, and improve vaccine storage. An “antigen” refers to a molecule, moiety, foreign particulate matter, or an allergen that can bind to a specific antibody or T cell receptor. The presence of antigens within a host can illicit an immune response against said molecule, moiety, foreign particulate matter, or allergen. An “epitope” or “antigenic determinant” refer to the part of an antigen, a molecular structure, or foreign particulate that can bind to a specific antibody or T-cell receptor. The presence of antigens or epitopes of antigens within a host can illicit an immune response. An “adjuvant” refers to a drug, molecule, substance, or a combination thereof that is used to increase the efficacy or potency of certain therapeutic agents, such as for example vaccines and / or antibodies. “Adjuvant(s)” are often at least one ingredient used in some vaccines that help create a stronger immune response in the host receiving said vaccine. A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule. A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material. A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full- length coding sequences of the gene with which they are associated. As used herein, a “mutation” refers to changing the structure of a gene, resulting in a variant form that may be transmitted to later generations. A mutation is caused by the alteration of single nucleotides in DNA, or the deletion, insertion, or rearrangement of larger sections of genes. A mutation can led to the expression of a protein that has been changed physically or functionally leading to lethality, non-lethal dysfunction effects, or no effects. A non-limiting example of a mutation refers to changes in a nucleic acid sequence resulting in replacing one amino acid for another amino acid within a polypeptide sequence. A “protein”, "polypeptide", or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like. The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino- propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2- Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3- Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4- Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid. Fusion proteins and fusion polynucleotides are also contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N-terminus, the C- terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein. A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3′ end of a first polynucleotide to a 5′ end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter). The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra- joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Vaccine Compositions The present disclosure provides DNA vaccines, vectors, and compositions thereof for preventing SARS-CoV-2 infection. The present disclosure also provides methods of inducing T cell-based immune responses using the DNA vaccines, vectors, and compositions thereof. The present disclosure also provides methods of preventing a SARS-CoV-2 infection using the DNA vaccines, vectors, and compositions thereof. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) comprises four structural proteins, the S protein, the N protein, the M protein, and the E protein. The S protein, or surface spike protein, comprises two subunits, S1 and S2. Further, the S1 subunit comprises a receptor- binding domain (RBD), which binds to host receptors to initiate viral entry. The S protein is a non- limiting example of a key target for developments of SARS-CoV-2 vaccines. A “T cell” refers to a type of lymphocyte that is one of the most important white blood cells of the immune system. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. The immune-mediated cell death function of T cells is carried by two major subtypes: CD8+“killer” T cells and CD4+“helper T cells. Thus, the present disclosure provides a DNA vaccine that induces a T cell-based immune response to prevent or inhibit infections from SARS-CoV-2 strains and substrains. In one aspect, disclosed herein is a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein. In some embodiments, the vaccine of any preceding aspect encodes a whole SARS-CoV- 2 Spike protein. In one embodiment, the vaccine of any preceding aspect encodes a fragment of the SARS-CoV-2 Spike protein. In one embodiment, the Spike protein is split and rearranged into three parts. In one embodiment, the vaccine of any preceding aspect encodes 30% or more of a whole SARS-CoV-2 Spike protein. In one embodiment, the vaccine of any preceding aspect encodes 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a whole SARS-CoV-2 spike protein. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Spike protein comprising at least 60% sequence identity to SEQ ID NO: 53. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike protein comprising at least 80% sequence identity to SEQ ID NO: 53. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike protein comprising at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike protein comprising at least 95% sequence identity to SEQ ID NO: 53. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike protein comprising at least 99% sequence identity to SEQ ID NO: 53. Specifically, the SARS-CoV-2 Spike protein can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 53, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike protein comprising SEQ ID NO: 53. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Spike nucleotide comprising at least 60% sequence identity to SEQ ID NO: 52. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike nucleotide comprising at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike nucleotide comprising at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike nucleotide comprising at least 95% sequence identity to SEQ ID NO: 52. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike nucleotide comprising at least 99% sequence identity to SEQ ID NO: 52. Specifically, the SARS-CoV-2 Spike nucleotide can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 52, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a SARS-CoV-2 Spike nucleotide comprising SEQ ID NO: 52. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub- Spike construct. In some embodiments, the vaccine of any preceding aspect encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising at least 60% sequence identity to SEQ ID NO: 42. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising at least 99% sequence identity to SEQ ID NO: 42. Specifically, the SARS-CoV-2 Ub-Spike construct can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 42, or any amount in- between, below, or above these ranges. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct comprising SEQ ID NO: 42. In some embodiments, the vaccine of any preceding aspect encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct. In some embodiments, the vaccine of any preceding aspect encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising at least 60% sequence identity to SEQ ID NO: 43. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising at least 99% sequence identity to SEQ ID NO: 43. Specifically, the SARS-CoV-2 Ub-Spike construct can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 43, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct comprising SEQ ID NO: 43. In one embodiment, the vaccine of any preceding aspect encodes an S-a fragment of the SARS-CoV-2 Spike protein. In some embodiments, the vaccine of any preceding aspect encodes an S-a fragment of the SARS-CoV-2 Spike protein comprising at least 60% sequence identity to SEQ ID NO: 49. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV- 2 Spike protein comprising at least 80% sequence identity to SEQ ID NO: 49. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike protein comprising at least 90% sequence identity to SEQ ID NO: 49. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike protein comprising at least 95% sequence identity to SEQ ID NO: 49. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike protein comprising at least 99% sequence identity to SEQ ID NO: 49. Specifically, the SARS- CoV-2 Spike protein can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 49, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike protein comprising SEQ ID NO: 49. In some embodiments, the vaccine of any preceding aspect encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising at least 60% sequence identity to SEQ ID NO: 48. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising at least 80% sequence identity to SEQ ID NO: 48. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising at least 90% sequence identity to SEQ ID NO: 48. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising at least 95% sequence identity to SEQ ID NO: 48. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising at least 99% sequence identity to SEQ ID NO: 48. Specifically, the SARS-CoV-2 Spike nucleotide can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 48, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-a fragment of the SARS-CoV-2 Spike nucleotide comprising SEQ ID NO: 48. In one embodiment, the vaccine of any preceding aspect encodes an S-b fragment of the SARS-CoV-2 Spike protein. In some embodiments, the vaccine of any preceding aspect encodes an S-b fragment of the SARS-CoV-2 Spike protein comprising at least 60% sequence identity to SEQ ID NO: 47. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV- 2 Spike protein comprising at least 80% sequence identity to SEQ ID NO: 47. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike protein comprising at least 90% sequence identity to SEQ ID NO: 47. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike protein comprising at least 95% sequence identity to SEQ ID NO: 47. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike protein comprising at least 99% sequence identity to SEQ ID NO: 47. Specifically, the SARS- CoV-2 Spike protein can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 47, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike protein comprising SEQ ID NO: 47. In some embodiments, the vaccine of any preceding aspect encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising at least 60% sequence identity to SEQ ID NO: 46. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising at least 80% sequence identity to SEQ ID NO: 46. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising at least 90% sequence identity to SEQ ID NO: 46. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising at least 95% sequence identity to SEQ ID NO: 46. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising at least 99% sequence identity to SEQ ID NO: 46. Specifically, the SARS-CoV-2 Spike nucleotide can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 46, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-b fragment of the SARS-CoV-2 Spike nucleotide comprising SEQ ID NO: 46. In one embodiment, the vaccine of any preceding aspect encodes an S-c fragment of the SARS-CoV-2 Spike protein. In some embodiments, the vaccine of any preceding aspect encodes an S-c fragment of the SARS-CoV-2 Spike protein comprising at least 60% sequence identity to SEQ ID NO: 45. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV- 2 Spike protein comprising at least 80% sequence identity to SEQ ID NO: 45. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike protein comprising at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike protein comprising at least 95% sequence identity to SEQ ID NO: 45. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike protein comprising at least 99% sequence identity to SEQ ID NO: 45. Specifically, the SARS- CoV-2 Spike protein can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 45, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike protein comprising SEQ ID NO: 45. In some embodiments, the vaccine of any preceding aspect encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising at least 60% sequence identity to SEQ ID NO: 44. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising at least 80% sequence identity to SEQ ID NO: 44. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising at least 90% sequence identity to SEQ ID NO: 44. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising at least 95% sequence identity to SEQ ID NO: 44. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising at least 99% sequence identity to SEQ ID NO: 44. Specifically, the SARS-CoV-2 Spike nucleotide can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 44, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an S-c fragment of the SARS-CoV-2 Spike nucleotide comprising SEQ ID NO: 44. In one embodiment, the vaccine of any preceding aspect encodes a ubiquitin protein. In one embodiment, the vaccine of any preceding aspect encodes a ubiquitin protein fused to SARS- CoV-2 Spike protein or a fragment thereof. In some embodiments, the vaccine of any preceding aspect encodes a ubiquitin protein comprising at least 60% sequence identity to SEQ ID NO: 51. In some embodiments, the vaccine encodes a ubiquitin protein comprising at least 80% sequence identity to SEQ ID NO: 51. In some embodiments, the vaccine encodes a ubiquitin protein comprising at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, the vaccine encodes a ubiquitin protein comprising at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the vaccine encodes a ubiquitin protein comprising at least 99% sequence identity to SEQ ID NO: 51. Specifically, the SARS-CoV-2 Spike protein can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 51, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a ubiquitin protein comprising SEQ ID NO: 51. In some embodiments, the vaccine of any preceding aspect encodes a ubiquitin nucleotide comprising at least 60% sequence identity to SEQ ID NO: 50. In some embodiments, the vaccine encodes a ubiquitin nucleotide comprising at least 80% sequence identity to SEQ ID NO: 50. In some embodiments, the vaccine encodes a ubiquitin nucleotide comprising at least 90% sequence identity to SEQ ID NO: 50. In some embodiments, the vaccine encodes a ubiquitin nucleotide comprising at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the vaccine encodes a ubiquitin nucleotide comprising at least 99% sequence identity to SEQ ID NO: 50. Specifically, the SARS-CoV-2 Spike nucleotide can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 50, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a ubiquitin nucleotide comprising SEQ ID NO: 50. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub- Spike construct comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the S protein is split and rearranged into three parts (S-a, S-b and S-c), and wherein the three parts of the S Protein are rearranged with overlap regions in between the S- a, S-b and S-c, wherein the overlap regions are overlap region 1 and overlap region 2. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub-Spike construct overlap region 1. In some embodiments, the vaccine of any preceding aspect encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 60% sequence identity to SEQ ID NO: 54. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 80% sequence identity to SEQ ID NO: 54. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV- 2 Ub-Spike construct overlap region 1 comprising at least 90% sequence identity to SEQ ID NO: 54. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 95% sequence identity to SEQ ID NO: 54. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 99% sequence identity to SEQ ID NO: 54. Specifically, the SARS-CoV-2 Ub-Spike construct overlap region 1 can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 54, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a nucleotide sequence of SARS- CoV-2 Ub-Spike construct overlap region 1 comprising SEQ ID NO: 54. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub- Spike construct overlap region 1. In some embodiments, the vaccine of any preceding aspect encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 60% sequence identity to SEQ ID NO: 55. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 80% sequence identity to SEQ ID NO: 55. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 90% sequence identity to SEQ ID NO: 55. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 95% sequence identity to SEQ ID NO: 55. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising at least 99% sequence identity to SEQ ID NO: 55. Specifically, the SARS-CoV-2 Ub-Spike construct overlap region 1 can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 55, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 1 comprising SEQ ID NO: 55. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub- Spike construct overlap region 2. In some embodiments, the vaccine of any preceding aspect encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 60% sequence identity to SEQ ID NO: 56. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 80% sequence identity to SEQ ID NO: 56. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 90% sequence identity to SEQ ID NO: 56. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 95% sequence identity to SEQ ID NO: 56. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV- 2 Ub-Spike construct overlap region 2 comprising at least 99% sequence identity to SEQ ID NO: 56. Specifically, the SARS-CoV-2 Ub-Spike construct overlap region 2 can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 56, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes a nucleotide sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising SEQ ID NO: 56. In some embodiments, the vaccine of any preceding aspect encodes a SARS-CoV-2 Ub- Spike construct overlap region 2. In some embodiments, the vaccine of any preceding aspect encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 60% sequence identity to SEQ ID NO: 57. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising at least 99% sequence identity to SEQ ID NO: 57. Specifically, the SARS-CoV-2 Ub-Spike construct overlap region 2 can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 57, or any amount in-between, below, or above these ranges. In some embodiments, the vaccine encodes an amino acid sequence of SARS-CoV-2 Ub-Spike construct overlap region 2 comprising SEQ ID NO: 57. Selective protein degradation uses ubiquitin protein as a marker that targets proteins for rapid degradation. Ubiquitin is a highly conserved 76 amino acid polypeptide that attaches to one or more amino acid residues of the target protein. It should be noted that multiple ubiquitin proteins can be added to the target protein as termed “polyubiquitination”. Once ubiquitinated, the target protein is subsequently degraded by a proteasome complex. Herein, the degraded SARS-CoV-2 spike (S) protein activated and enhanced the functions of T lymphocytes. In one aspect, disclosed herein is a composition comprising the DNA vaccine of any preceding aspect, an adjuvant, and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion), various types of wetting agents, any excipient, diluent, filler, salt, buffer, stabilizer, 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, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; 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 TWEENTM(ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM(BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent. In some embodiments, the adjuvant comprises Imiquimod, Alum, monophosphoryl lipid A (MPL), MF59, Poly(I:C), Montanide ISA51, AddaVax, or a combination thereof. In some embodiments, the DNA vaccine of any preceding aspect further encodes at least one cytotoxic T lymphocyte (CTL) epitope. In some embodiments, the protein of any preceding aspect comprises a mutation that can improve protein degradation. As used herein, a “protein degradation-enhancing mutation” or a “proteolysis-enhancing mutation” refers to an alteration in a genetic sequence to optimize, increase, improve, and / or enhance the functions, activity, stability, and / or conformation of the expressed protein, such as, for example ubiquitin protein. Herein, the ubiquitin protein comprises a protein degradation-enhancing mutation to ensure stability while attached to the target protein. In some embodiments, the protein degradation-enhancing mutation comprises a glycine (G) residue at amino acid position 76 of the protein replaced by an alanine (A) residue. In some embodiments, the protein degradation-enhancing mutation comprises a G76A mutation to the ubiquitin protein. It should be noted that “protein degradation-enhancing mutation” and “proteolysis-enhancing mutation” can be used interchangeably throughout the disclosure. Viruses, including SARS-CoV-2, are constantly mutating to adapt to their environment. These genetic mutations can lead to emergence of a new variant of the virus that comprises different or improved functions, including but not limited to easier transmission (spreading from organism to organism) or better evasion mechanisms from the host immune system, not seen in the original strain. Virus variants are further divided into subvariants, when the one or more distinctive mutants of a variant emerges. Thus, an original virus can mutate into a variant, and a variant virus can further mutate into a subvariant. In some embodiments, the DNA vaccine of any preceding aspect induces a T cell-based response against an original SARS-CoV-2 (WT-S), Alpha, Beta, Gamma, Delta, or Omicron SARS-CoV-2 variants, or subvariants thereof. In some embodiments, the SARS-CoV-2 spike protein is from an Omicron variant of SARS-CoV-2. In some embodiments, the Omicron variant is a BA1 variant. In some embodiments, the Omicron variant is BA1-S. In some embodiments, the Omicron variant is BA2. In some embodiments, the Omicron variant is BA2.12.1. In some embodiments, the Omicron variant is BA4. In some embodiments, the Omicron variant is BA5. In some embodiments, the Omicron variant is XBB1.5. In some embodiments, the Omicron variant is HV.1. In some embodiments, the Omicron variant is EG.5. In some embodiments, the Omicron variant is BA.2.86. In some embodiments, the Omicron variant is JN.1. In some embodiments, the SARS-CoV-2 spike (S) protein is from an Alpha variant. In some embodiments, the Alpha variant is B.1.1.7. In some embodiments, the SARS-CoV-2 (S) protein is from a Beta variant. In some embodiments, the Beta variant is B.1.351. In some embodiments, the SARS-CoV-2 S protein is from a Gamma variant. In some embodiments, the Gamma variant is P.1. In some embodiments, the SARS-CoV-2 S protein is from a Delta variant. In some embodiments, the Delta variant is B.1.1.529. In some embodiments, the T cell-based response comprises a response from CD8+T cells, CD4+T cells, or a combination thereof. In some embodiments, the vaccine of any preceding aspect provides protection without inducing an antibody response against the spike protein. In one aspect, disclosed herein is a vector comprising the DNA vaccine of any preceding aspect. In some embodiments, the vector comprises a plasmid or a virus or viral vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.). Methods of inducing a T cell-based response to prevent and / or inhibit SARS-CoV-2 infections In one aspect, disclosed herein is a method of inducing a T cell-based response to a SARS- CoV-2 virus in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein. In one aspect, disclosed herein is a method of preventing or inhibiting a SARS-CoV-2 infection in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein. In some embodiments, the method of any preceding aspect comprises the DNA vaccine compositions of any preceding aspect. In some embodiments, the method of any preceding aspect further comprises inducing the T cell-based response against an original SARS-CoV-2 (WT-S), Alpha, Beta, Gamma, Delta, or Omicron SARS-CoV-2 variants, or subvariants thereof. In some embodiments, the method of any preceding aspect induces a T cell-based response from CD8+T cells, CD4+T cells, or a combination thereof. In some embodiments, the method of any preceding aspect comprises the vaccine compositions of any preceding aspect. In some embodiments, the method of any preceding aspect further comprises testing splenocytes from the immunized subject for T cell-based responses. The splenocytes are stimulated with peptide fragments from the SARS-CoV-2 S protein, wherein the peptide fragments from the SARS-CoV-2 S protein comprises CTL and T helper cell epitopes. In some embodiments, the peptide fragments from the SARS-CoV-2 S protein of any preceding aspect encode an amino acid sequence selected from a group comprising SEQ ID NOS: 1-41. In some embodiments, the peptide fragments from the SARS-CoV-2 S protein of any preceding aspect encode an amino acid sequence comprising at least 60% sequence identity to any one of SEQ ID NOS: 1-41. In some embodiments, the peptide fragments encode an amino acid sequence comprising at least 80% sequence identity to any one of SEQ ID NOS: 1-41. In some embodiments, the peptide fragments encode an amino acid sequence comprising at least 90% sequence identity to SEQ ID NOS: 1-41. In some embodiments, the peptide fragments encode an amino acid sequence comprising at least 95% sequence identity to any one of SEQ ID NOS: 1-41. In some embodiments, the peptide fragments encode an amino acid sequence comprising at least 99% sequence identity to any one of SEQ ID NOS: 1-41. Specifically, the peptide fragments can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOS: 1-41, or any amount in-between, below, or above these ranges. In some embodiments, the peptide fragments encode an amino acid sequence comprising any one of SEQ ID NOS: 1-41. In some embodiments, the method of any preceding aspect provides protection without inducing an antibody response against the spike protein. In some embodiments, the induced T cell response prevents and / or inhibits a SARS-CoV-2 infection. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human. Administration of vaccine compositions The vaccine composition of any preceding aspect may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the vaccine composition of any preceding aspect will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like. The vaccine composition of any preceding aspect is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the patient; route of administration, and rate of excretion of the specific vaccine composition employed; and like factors well known in the medical arts. The vaccine composition of any preceding aspect may be administered by any route. In some embodiments, the composition is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the vaccine composition (e.g., its stability in the environment of the respiratory system), the condition of the subject (e.g., whether the subject is able to tolerate administration), etc. The exact amount of a vaccine composition of any preceding aspect required to achieve a therapeutically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of any side effects, mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art. Example 1. A T-cell based SARS-CoV-2 spike protein vaccine provides protection without antibodies. SARS-CoV-2 spike (S)-based vaccines are used to control the COVID-19 pandemic. However, emerging variants became resistant to antibody neutralization and further mutations can lead to full resistance. It was tested whether T cells alone could provide protection without antibodies. Herein, a T cell-based vaccine was designed in which SARS-CoV-2 S sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies but strong specific T cell responses. Mice were challenged with SARS-CoV-2 wild- type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by SARS- CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with SARS-CoV-2 wild-type or the Omicron variant were significantly reduced. Importantly, depletion of CD4+or CD8+T cells led to significant loss of the protection. The analyses of S protein sequences of the variants indicated that fewer than 1 / 3 presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in subunit 1 region. The S protein has two subunits, S1 and S2, which are responsible for receptor angiotensin- converting enzyme 2 (ACE2) binding and membrane fusion, respectively. As the neutralizing antibodies are mostly against receptor-binding domain (RBD) of the S1 subunit, the mutations in the RBD lead to reduction of neutralizing activity of immune sera from individuals vaccinated with SARS-CoV-2 vaccines or from convalescent COVID-19 patients. This raises a burning question whether the current vaccines will still protect against emerging variant strains without significant amounts of neutralizing antibodies or boost immunizations should be provided using mutated S proteins as the current bivalent vaccines from Moderna and Pfizer-BioNTech. Although it is believed that neutralizing antibodies are the primary immune effector to provide viral protection, cellular immunity (CD4+T cells and CD8+T cells) also plays important roles in controlling viruses. It has been shown that cytotoxic T lymphocytes (CTLs) alone are sufficient to provide full protection against Zika virus-induced fetal damage. Thus, the question of whether the current vaccines targeting the S protein will still provide protection in the absence of an optimal SARS-CoV-2 antibody response was addressed herein. To this end, a T cell-based S protein vaccine was designed that induces only T cell immunity but no antibodies, and it was tested if it protects against SARS-CoV-2 challenge. Generation of virus-specific CD8 T cells depends on the presentation of epitopes (~9 amino acids) in the context of MHC class I. Usually, intracellular proteins subject to continuous turnover are degraded at different rates into short peptides in the proteasome. Peptides generated in the proteasome are transported by peptide transporters to the endoplasmic reticulum for loading onto MHC class I. The intracellular proteins targeted for proteolysis often have ubiquitin attached to them. Ubiquitin-protein conjugates are degraded by the proteasome. It is shown that ubiquitination of a viral protein greatly enhanced degradation of the viral protein and consequently caused an enhanced induction of specific CTLs. Accordingly, the T cell-based SARS-CoV-2 vaccine was made by rearranging S gene to disrupt S protein conformation and adding ubiquitin gene to enhance S protein degradation in the proteosome for effective CTL generation. This vaccine induced only specific T cell responses without specific antibodies and provided protection in mouse models. Results Analysis of T cell epitope mutations in S protein The conservation of human T cell epitopes was analyzed in S proteins including MHC class I- and class II-restricted epitopes in the variants of concern strains. Initially, the epitopes presented by the most prevalent MHC class I and class II alleles were identified and analyzed. The top six most frequently occurring alleles were selected (Tables 1 and 2). TepiTool, a T cell epitope prediction resource from the Immune Epitope Database (IEDB), was used to find the top ten highest scoring epitopes of S protein from original strain (GenBank accession number QHR63250.2) for each allele based on the 50% inhibitory concentration (IC50) value, then the number of mutated epitopes in the variants of concern were determined. The T cell epitopes presented by MHC Class I alleles were more conserved than those presented by MHC class II alleles in these variants; on average, the 3.7% of epitopes presented by HLA-A alleles, 6.7% of those by HLA-B alleles, 5.7% of those by HLA-C alleles, 15.7% of those by HLA-DR alleles, 9.7% of those by HLA-DQ alleles and 24% of those by HLA-DP alleles are mutated (Figures 7A, 7B, 7C, 7D, 7E, and 7F; and Figures 8A, 8B, 8C, 8D, 8E, and 8F). Then, a list of confirmed T cell epitopes presented by all MHC alleles were analyzed and a similar trend was found: The MHC class I epitope mutation rate is about 6.4% and the MHC class II epitope mutation rate is about 11.5% (Figures 7G and 7H; and Figures 8G and 8H). Most of the mutations are located in the epitopes in S1 subunit. Thus, the data shows that T cell responses to S protein may survive the mutations. Construction of a T cell-based SARS-CoV-2 Ub-S DNA vaccine Because gene rearrangement usually changes original folding of a protein and prevents induction of antibodies, in particular those to conformational epitopes, the SARS-CoV-2 S gene was split into three parts (segment a,b,c) and rearranged them (segment c:b:a ratio is 1539:1425:1290) (Figure 1A). Thirty nucleotides before and after cleaved sites were added back in order to preserve CTL epitopes that were disrupted. To enhance degradation of the rearranged S protein, a ubiquitin gene was added to the construct. Ubiquitin gene can be mutated to encode ubiquitin with an alanine at residue 76 to enhance ubiquitin-protein complex stability. Because human and mouse ubiquitin genes share the same sequences, the gene encoding a monomer of human mutated ubiquitin was fused to the 5’ end of the rearranged S DNA sequence, with its glycine (G) at the 76thresidue replaced by alanine (A) to enhance stability of the Ub-S complex (Ub-S DNA) (Figure 1A). The rearranged SARS-CoV-2 Ub-S DNA sequence was inserted into pVAX1 vector, and digested for the correct size (Figure 1B). Two additional constructs expressing either full-length original S protein without ubiquitin (S DNA) or full-length original S protein fused to ubiquitin (Ub-S Unmodified) were also made as the controls of rearranged Ub-S DNA. 293T cells were transfected with the plasmids and cultured the cells in the presence and absence of proteasome inhibitor MG312 and performed Western blot analyses. As shown in Figure 1C, the ubiquitinated and rearranged S protein (Ub-S DNA) and ubiquitinated original S protein (Ub-S Unmodified) are reduced as compared to the original S protein without ubiquitin. The expression of these two ubiquitinated proteins are enhanced in the presence of MG 312, showing that ubiquitinated proteins are degraded in the proteosome. There is no significant difference at the protein level between Ub-S DNA and Ub-S Unmodified, indicating that gene arrangement has no impact on the protein degradation. SARS-CoV-2 Ub-S DNA vaccine induced strong specific T cell responses without antibodies In the next sets of experiments, the short-term and long-term immune responses induced by the Ub-S DNA vaccine expressing the ubiquitinated S protein were tested separately, wherein the S protein was rearranged in different mouse strains and with various adjuvants. First, young adult BALB / c mice (6-8-week-old) were immunized with this DNA vaccine in the presence of TLR7 agonist Imiquimod adjuvant, or SARS-CoV-2 full-length S protein (S protein) in the presence of Alum plus monophosphoryl lipid A (MPL) adjuvants, and boosted twice at a three-week interval. Here, different adjuvants were used as they have been optimized for DNA and proteins, respectively. Mice were sacrificed at 10 days after the 3rdimmunization, and spleen and sera were collected to determine the T cell responses to SARS-CoV-2 S protein and antibody responses to the S protein or different S protein fragments. The results indicated that full-length S protein induced both CD8+and CD4+T cells that produced IFN-γ and TNF-α in response to stimulation with S peptides (Figures 2A, 2B, 2C, and 2D; Table 3) and IgG antibodies specific to SARS-CoV-2 S protein and its fragments, including N-terminal domain (NTD), receptor-binding domain (RBD), S1, and S2 (Figures 2E, 2F, 2G, 2H, and 2I). In contrast, the Ub-S DNA vaccine expressing the rearranged and ubiquitinated S protein induced higher CD8+and CD4+T cell responses than the full-length S protein control vaccine, but did not induce S, NTD, RBD, S1, or S2-specific antibody unlike the full-length S protein control vaccine (Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I). PBS control only elicited no or background level of antibody and T cell responses (Figure 2). These data indicate that this DNA vaccine strategy induced CTLs but also T helper cells, due to leaking of the rearranged protein into MHC class II processing pathway. As Figure 1 showed that transfection of 293T cells with the plasmid expressing the rearranged and ubiquitinated S protein had a thin band without proteasome inhibitor MG312, showing that the protein is not fully degraded in proteasome and then leaked to MHC class II processing pathway. Because no antibody responses were induced in BALB / c mice, no rearranged and ubiquitinated S protein is secreted. Second, to verify these data in a different mouse strain, to identify the immunogenicity of Ub-S DNA in the presence of other adjuvants, and to investigate booster effect after 6 months, young adult C57BL / 6 (B6) mice (6-8-week-old) were immunized with the DNA vaccine expressing ubiquitinated and rearranged S protein (Ub-S DNA) or full-length S protein (S protein) control vaccine in the presence of Imiquimod adjuvant and Alum + MPL adjuvants, respectively, boosted twice at a three-week interval and once at 6 months, and then tested for specific antibody responses and T cell responses 10 days after last immunization. In addition, a DNA containing original SARS-CoV-2 S sequence without ubiquitin (S DNA) plus Imiquimod adjuvant was included as a control for Ub-S DNA. Mice immunized with PBS with or without respective adjuvants were included as background controls. As shown in Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H, full-length S protein adjuvanted with either Imiquimod or Alum + MPL elicited IFN- γ and TNF-α-secreting CD8+(Figure 3A, 3B, 3E, and 3F) and CD4+(Figure 3C, 3D, 3G, and 3H) T cells, in response to stimulation with S peptides for B6 mice (Table 4), particularly high-titer IgG antibodies specific to the SARS-CoV-2 S protein, NTD, RBD, S1, and S2 fragments, respectively (Figure 3I, 3J, 3K, 3L 3M, 3N, 3O, 3P, 3Q, and 3R). Imiquimod or Alum + MPL- adjuvanted Ub-S DNA vaccine expressing the rearranged and ubiquitinated S protein elicited significantly higher and durable CD8+and CD4+T cell responses than the full-length S protein (Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H), but did not elicit any S, RBD, NTD, S1, or S2- specific antibody responses (Figure 3I, 3J, 3K, 3L 3M, 3N, 3O, 3P, 3Q, and 3R). Unlike Ub-S DNA, S DNA expressing original SARS-CoV-2 S protein without ubiquitin induced both S- specific T cell and antibody responses (Figure 3A, 3B, 3C, 3D, 3I, 3J, 3K, 3L, and 3M), but the T cell responses were significantly lower, or lower than those induced by Ub-S DNA (Figure 3A, 3B, 3C, and 3D). Interestingly, S DNA induced antibody responses only in the presence of Imiquimod adjuvant, and the antibodies were undetectable in the presence of Alum + MPL adjuvants (Figure 3I, 3J, 3K, 3L 3M, 3N, 3O, 3P, 3Q, and 3R). In contrast, PBS control with or without adjuvants elicited no or background level of antibody and T cell responses (Figure 3), and only background level of T cell responses were elicited in vaccinated and control mice in response to stimulation with unrelated peptides of non-structural protein 3 (NS3) of Zika virus (ZIKV) (data not shown). These data confirm the ability of Ub-S DNA conjugated with different adjuvants in eliciting strong and specific T cell immune responses without antibodies in B6 mice. SARS-CoV-2 Ub-S DNA vaccine induced protection against SARS-CoV-2 infection To evaluate the protection of SARS-CoV-2 Ub-S DNA vaccine in mature-adult mice, 4-6- month-old mice expressing SARS-CoV-2 receptor human ACE2 (hACE2-Tg) were first immunized with this vaccine in the presence of Imiquimod adjuvant. The mice were also immunized separately with the SARS-CoV-2 full-length S protein with Alum plus MPL adjuvants, or PBS. Here, different adjuvants were used based on previously optimized protocols (Shi, J., et al., Effective vaccination strategy using SARS-CoV-2 spike cocktail against Omicron and other variants of concern. NPJ Vaccines, 2022.7(1): p.169)( Gambino, F., Jr., et al., A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage. Cell Rep, 2021. 35(6): p.109107). The mice were boosted twice at 3 weeks. The immunized mice were challenged with a prototypic wild-type SARS-CoV-2 (2019n-CoV / USA-WA1 / 2020) at a high lethal dose (5,000 PFU / mouse) 2 weeks post-booster, and mouse survival and weight loss were monitored for 14 days post-infection (p.i.). All mice immunized with full-length S protein survived without weight loss during the monitoring period, whereas 86% of the mice immunized with SARS-CoV- 2 Ub-S DNA vaccine survived with minimal weight loss. In contrast, 100% of the mice in the PBS control group died on 10 days p.i. with severe and significant weight loss (Figures 4A and 4B). These data demonstrate that, similar to the full-length S protein, the Ub-S DNA vaccine was able to protect mature adult transgenic mice expressing SARS-CoV-2 receptor ACE2 from high-dose SARS-CoV-2 infection. The protection of SARS-CoV-2 Ub-S DNA vaccine was then evaluated in middle-aged mice. As such, wild-type B6 mice at 8-9-month-old were immunized with the Ub-S DNA, full- length S protein, and PBS as described above, challenged with a mouse-adapted SARS-CoV-2 (N501YMA30, 5,000 PFU / mouse, not lethal for the mice) 2 weeks after the last boost, and examined for viral titers 2 days p.i. Notably, the Ub-S DNA significantly reduced SARS-CoV-2 titers in the lung than the PBS control, although it was not as effective as the full-length S protein vaccine (Figure 9A). Ub-S DNA vaccine did not induce specific antibody responses (as shown below) as it can reduce but not provide sterilizing immunity. Different from the full-length S protein, which induced a high-titer, specific IgG antibodies, the Ub-S DNA only elicited a background-level antibody response similar to that induced by PBS control in these mice (Figure 9B). The above data indicate that SARS-CoV-2 Ub-S DNA vaccine significantly reduced viral infection in the challenged middle-aged mice, and that no vaccine-induced antibodies play a role in this protection. The cross-protective efficacy of SARS-CoV-2 Ub-S DNA vaccine against a recent SARS- CoV-2 Omicron variant was further evaluated and the protection with that provided by the DNA containing original S sequence (S DNA) and the original S protein (S protein) in the presence of same adjuvant was compared. Specifically, young adult BALB / c mice (10-week-old) were immunized with Ub-S DNA (with ubiquitin), S DNA (without ubiquitin), or full-length S protein, all of which were mixed with Imiquimod adjuvant, and challenged with SARS-CoV-2 Omicron- BA5 variant (50,000 PFU / mouse) 4 weeks post-booster. Here, Omicron-BA5 variant was used because it is a prevalent variant at the time of experiment. BALB / c mice were utilized due to their susceptibility to the Omicron variant, and use of a different viral titer and time point was based on the optimal protocols for Omicron challenge. Sera were collected before challenge for evaluation of IgG antibody responses, and the challenged mice were observed for viral titers in the lungs 2 days p.i. The full-length S protein induced high-titer IgG antibodies, and protected mice against Omicron-BA5 challenge, with significantly reduced viral titers than other groups (Figures 5A, 5B, 5C, 5D, 5E, and 5F). The Ub-S DNA also provided effective protection despite the lower efficacy than the full-length protein vaccine (Figure 5F), because the Ub-S DNA did not induce antibodies (Figures 5A, 5B, 5C, 5D, and 5E). Nevertheless, the wild-type S DNA induced low-titer IgG antibodies and provided less protection than the Ub-S DNA, resulting in significantly higher viral titers in the lungs of immunized mice than those in the lungs of mice immunized with Ub-S DNA (Figures 5A, 5B, 5C, 5D, 5E, and 5F). These data show that T cell-based Ub-S DNA vaccine induced effective cross-protective efficacy against recent SARS-CoV-2 Omicron variant, and that the protection did not depend on the antibody responses, which is different from the S protein. SARS-CoV-2 Ub-S DNA vaccine-induced T cells played a critical role in protection Since SARS-CoV-2 Ub-S DNA did not induce antibody for preventing SARS-CoV-2 infection, next the contribution of CD8+and CD4+T cells induced by this vaccine to protection against SARS-CoV-2 was determined. To do this, mature adult hACE2-Tg mice (4-6-month-old) were immunized and challenged with a lethal dose of SARS-CoV-2 (2019n-CoV / USA- WA1 / 2020, 2,000 PFU / mouse) with or without depletion of CD8+or CD4+T cells 8 weeks post- booster, and mouse survival and weight loss were monitored for 14 days p.i. as described above. The reason for using 2,000 PFU / mouse, instead of a high lethal dose (5,000 PFU / mouse), was to evaluate whether Ub-S DNA-induced T cells can provide complete protection against SARS-CoV- 2 infection at a lower lethal dose. Also, the reason for challenging the immunized mice at 8 weeks after the last immunization was to evaluate whether the induced T cells may provide a longer protection against SARS-CoV-2 infection. Indeed, depletion of CD8+or CD4+T cells significantly reduced protection against SARS-CoV-2, with increased weight loss (Figures 6A and 6B); 80% or 100% of mice died after CD8+or CD4+T cell depletion, respectively (Figures 6C and 6D). In contrast, all the mice immunized with SARS-CoV-2 Ub-S DNA vaccine and treated with antibody isotype control survived without weight loss at the lethal dose tested (Figure 6E and 6F). As expected, mice receiving PBS and treated with antibody isotype Control had constant weight loss, leading to death, after challenge (Figure 6G and 6H). These data demonstrate that SARS-CoV-2 Ub-S DNA vaccine-induced T cells substantially and durably prevent SARS-CoV-2-induced clinical disease, particularly death, and that both CD4+and CD8+T cells play a role in this protection, since depletion of either CD4+or CD8+T cells led to the complete, or almost complete, loss of protection. Discussion SARS-CoV-2 continues to infect humans with high human-to-human transmissibility, and it keeps mutating to generate new variants, calling for the consistent effort to design and develop effective vaccines to prevent infection of the variants of concern and diseases. SARS-CoV-2 S protein-induced neutralizing antibodies are considered as key effectors in protecting against SARS-CoV-2 infection. The role of T cell-induced protection is demonstrated in animals. Importantly, patients with X-linked agammaglobulinemia without B cells can recover from SARS- CoV-2 infection, providing evidence that T cell immunity alone may provide protection against SARS-CoV-2 in humans. However, the patients infected with SARS-CoV-2 may develop broader T cell responses than the vaccines targeting only S protein, which may result in different outcomes. Herein, it is demonstrated that different from the DNA without rearrangement and ubiquitination which elicited low-titer antibody and low-level T cell responses, or the full-length S protein which induced high-titer antibody but low-level T cell responses, the rearranged and ubiquitinated DNA vaccine targeting original SARS-CoV-2 S protein induced robust specific T cell responses without antibody responses, which provided protection against SARS-CoV-2 (original strain), extending survival, and reducing weight loss in B6-backgrounded hACE2-Tg mice. It also cross-protected mice against Omicron variant with significantly reduced viral titers in the lungs of BALB / c mice. Notably, the adjuvants alone without DNA did not show protective efficacy from SARS-CoV-2 infection (data not shown). It was also found that both SARS-CoV-2 Ub-S DNA vaccine-induced CD4+and CD8+T cells play a critical role in the protection against SARS-CoV-2 infection, as depletion of either one resulted in significant loss of vaccine-mediated protection. One important function of CD4+T cells is to provide help for the generation of CD8+CTLs, in particular for memory CTLs. It is contemplated that CD4+T cell effector functions are also important for direct protection because the depletion of CD4+T cells was done just before the viral challenge. Most likely, CD4+T cells mediated their effects via cytokines such as IFN-γ, which directly inhibit SARS-CoV-2 replication as shown by other research groups. CD8+T cells provide protection via cytolytic activity. CD4+T cells provide better protection than CD8+T cells, due to IFN-γ. In this study, a ubiquitination strategy was used to promote degradation of SARS-CoV-2 in the proteosome. Although the degradation of SARS-CoV-2 S protein was clearly shown in Figure 1, the degradation was not complete (the S protein band can still be seen without the proteosome inhibitor). It is contemplated that the S protein was not degraded in the proteosome because of leakage into MHC class II processing pathway, resulting in generation of specific CD4+T cells for protection. Neutralizing activity against newer SARS-CoV-2 variants are reduced in the sera from individuals vaccinated with the SARS-CoV-2 vaccines targeting the original S protein (Wuhan clone). However, mRNA vaccine-induced T cells responded similarly to different SARS-CoV-2 variants, e.g. people vaccinated with mRNA vaccines targeting the original S are equally reactive to the Omicron strain. Analyses were performed on the mutations of MHC class I- and II-restricted epitopes of the S proteins and most of the epitopes were found to be preserved. Furthermore, Convalescent COVID-19 patients presented dominant CTL responses to S, M and N proteins; T cell memory responses to SARS-CoV-2 infection or vaccination are shown to last. These data support that the current vaccines should provide protection against future variants. However, it is unclear if T cell responses alone prevent breakthrough infections. Notably, mRNA-vaccinated individuals with induction of both T cells and neutralizing antibodies against SARS-CoV-2 can get infected. Most likely, insufficient amount or lack of upper respiratory tract mucosal IgA and tissue-resident T cells against SARS-CoV-2 lead to the breakthrough infections. Nonetheless, the T cells are capable of preventing severe symptoms as shown in the mouse models. Materials and Methods Construction of the vaccine and control plasmids The ubiquitinated and rearranged S gene construct (Ub-S DNA) and ubiquitinated original (unmodified) S gene construct (Ub-S Unmodified) for SARS-CoV-2 S DNAs were made as previously described with some modifications. To construct Ub-S DNA, SARS-CoV-2 S protein sequence from SARS-CoV-2 original strain (GenBank accession number QHR63250.2) was rearranged, and ubiquitin sequence was added to the front of the rearranged S (see Results section). A Kozak sequence was placed ahead of the Ub / S open reading frame to ensure efficient transcription of the plasmid. Upstream the Kozak sequence, linker DNA as well as an EcoRI restriction site was placed to facilitate proper cloning of the gene segment into the vector of interest. Downstream the rearranged Ub / S gene sequence, a stop codon was placed along with linker DNA and a NotI restriction site. This nucleotide sequence was ordered from GenScript. The S gene was delivered in a vector in a lyophilized form. Plasmid DNA was resuspended in Molecular Biology Grade water (Fisher Scientific) to a concentration of approximately 200 ng / uL. DNA was digested with FastDigest (FD) EcoRI and NotI for 15 min in a 37oC heat block. The digested DNA was then loaded directly onto a 1% Agarose gel and allowed to run for 40 min at 80V. The Ub-S DNA with correct size (4170 bp) was exposed to 440 nm UV light in the imaging room and excised. The gel slice was placed into a clear, 1.5 mL microcentrifuge tube and the purified DNA was extracted using a QIAquick Gel Extraction Kit (Qiagen). This gene fragment was stored in a -20°C freezer. The pVAX1 vector (Thermo Fisher Scientific) was digested with FD EcoRI and FD NotI, run on a gel, and extracted using the same kit. The Ub-S gene fragment was ligated using a 3:1 ratio of insert DNA (Ub-S) to vector DNA (pVAX1) at room temperature in the presence of T4 DNA ligase. To construct Ub-S unmodified DNA, ubiquitin sequence was added to the front of the original S sequence and cloned into pVAX1 as above. To construct S gene without ubiquitin, a sequence encoding an N-terminal CD5 signal peptide was added to the front of the original S sequence and cloned into pVAX1 as above. Bacterial transformation Previously made Chemically Competent DH5α E. coli was removed from -80°C freezer and thawed on ice for 25 min. A plasmid was introduced to the E. coli and remained on ice for 30 min. Heat shock transformation occurred by placing the tube into a 42°C heat block for 45 sec and back on ice for 2 min.1 mL of SOC media was added to the E. coli and was grown in a shaking incubator at 37°C for 45 min. The E. coli was then spun down at 9,000 rpm for 2 min, resuspended in 200 μL SOC media, and streaked on an LB Kanamycin (Kan+) plate. Once dry, the plate was inverted and incubated overnight at 37°C. Colonies from the plate were inoculated into 2 mL LB Kan+media, and a Miniprep was conducted using a QIAprep Spin Miniprep Kit according to the manufacturer’s instructions (Qiagen). The plasmid was subsequently re-digested with FD EcoRI and FD NotI. Upon observance of correct band digestion, the plasmid was sent out for sequencing (ACGT, Inc) for confirmation of correct insertion. Plasmid transfection 293T cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% Penicillin / Streptomycin. Cells were split upon reaching 90% confluence (every 2-3 days). Upon sufficient generation of 293T cells, 10 μg of the plasmid was transfected using PEI transfection reagent overnight. Media was removed the next morning and cells were cultured in DMEM. For subsequent experiments using proteasome inhibitor, MG132 (Sigma) was added to cell culture medium at 50 μM overnight at either 12, 36, or 60 h after transfection. Western blot 293T cells (ATCC) were cultured, transfected, and treated with proteasome inhibitor as described above. After overnight treatment with 50 μM MG132, 293T cells were treated with 0.25% trypsin for 5 min at 37°C in 5% CO2.293T cells were resuspended in DMEM, centrifuged at 1,200 rpm for 5 min, and cell pellets were resuspended in RIPA buffer with freshly added proteinase inhibitor. Total protein concentration was calculated via Bradford Assay (Bio-Rad), and 6x SDS Loading Buffer was added to 20 μg of total protein. Protein was denatured at 100°C for 10 min and then placed on ice for 2-3 min. Proteins were run on a 12% polyacrylamide gel for 20 min at 80V and for 40 min at 120V, or until the dye line reached the bottom of the gel. Bands from the polyacrylamide gel were transferred to a nitrocellulose membrane using an iBlot Gel Transfer Device (Thermo Fisher Scientific). Membrane was blocked in 5% blocking buffer (5% non-fat milk in PBS) for 1 h. Membrane was washed in PBS-Tween 20 (PBS-T) and blocked in 5% blocking buffer containing a 1:1,000 dilution of anti-SARS-CoV-2 S antisera overnight. The next day, the membrane was washed with PBS-T for 20 min, and the membrane was blocked in 5% blocking buffer with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (BioLegend) for 1 h. Membrane was washed in PBS-T for 20 min, and then exposed to 10 mL Chemiluminescent substrate for 3 min devoid of light. Proteins were visualized using a FluorChem E system (ProteinSimple). Preparation of recombinant proteins SARS-CoV-2 full-length S protein and its fragments were prepared. Briefly, DNA sequences of SARS-CoV-2 S or its fragments (RBD, NTD, or S2) were amplified by PCR using a plasmid encoding codon-optimized S protein of SARS-CoV-2 original strain (GenBank accession number QHR63250.2). The recombinant plasmids were constructed by inserting respective PCR fragments (S containing a C-terminal folden trimeric sequence and His6 tag; RBD, NTD, or S2 containing a C-terminal His6tag with or without a foldon tag) into a pLenti expression vector. This was followed by transfecting each recombinant plasmid into 293T cells, and purifying each protein from cell culture supernatants using Ni-NTA Superflow (Qiagen). Ethical statement BALB / c, C57BL / 6 (B6), and hACE2-Tg mice were used in the study. The animal protocols were approved by Institutional Animal Care and Use Committees (IACUC) of NYBC, GSU, and University of Iowa. The mouse-related experiments were carried out according to the guidelines of the approved IACUC protocols. Immunization of mice with vaccines and sample collection SARS-CoV-2 vaccines were used to immune mice using the following three immunization protocols (32, 33). First, female BALB / c mice (6-8-week-old) were intramuscularly (I.M.) vaccinated with Ub-S DNA (10 μg / mouse) plus Imiquimod adjuvant (20 μg / mouse, InvivoGen). Mice immunized (I.M.) with a mammalian cell-expressed SARS-CoV-2 full-length S protein (10 μg / mouse) plus Alhydrogel (Alum) (500 μg / mouse) and Monophosphoryl lipid A (MPL, 10 μg / mouse) adjuvants (InvivoGen) were used as a vaccine control, and mice injected with PBS were included as a background control. The immunized mice were boosted twice with the same immunogens and adjuvants at 3-week intervals. 10 days after the 3rdimmunization, sera and splenocytes were collected for detection of SARS-CoV-2 S, NTD, RBD, S1, or S2-specific IgG antibodies and S-specific T cell responses, respectively. Second, female B6 mice (6-8-week-old) were immunized (I.M.) with Ub-S DNA, original S DNA control, full-length S protein control (10 μg / mouse), or PBS control in the presence of above Imiquimod adjuvant and Alum + MPL adjuvants, respectively, and boosted with the same immunogens and adjuvants twice at 3 weeks and once at 6 months. Sera were collected 10 days after the 3rdimmunization as above to test for SARS-CoV-2 S, NTD, RBD, S1, or S2-specific IgG antibodies, and splenocytes were collected 10 days after last immunization to detect S-specific T cell responses. Third, female BALB / c mice (10- week-old) were immunized with Imiquimod-adjuvanted Ub-S DNA, original S DNA control, full- length S protein control (10 μg / mouse), or PBS background control, and boosted twice at 3-week intervals. Sera collected before SARS-CoV-2 challenge were tested for IgG antibodies specific to SARS-CoV-2 S and its fragments (NTD, RBD, S1 or S2) as described above. ELISA SARS-CoV-2-specific IgG antibodies were detected by ELISA using the collected mouse sera. Briefly, ELISA plates were pre-coated, respectively, with above purified SARS-CoV-2 NTD, RBD, S2, or full-length S, as well as S1 (BEI Resources), protein (1 μg / ml) at 4°C overnight, followed by being blocked with PBS-T containing 2% fat-free milk at 37°C for 2 h. The plates were washed for three washes with PBS-T, and then sequentially incubated with serially diluted mouse sera and HRP-conjugated goat-anti-mouse IgG antibody (Fab-specific: 1:5,000, Sigma) at 37°C for 1 h. The plates were further incubated with substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (Sigma), and then H2SO4(1N) to stop the reaction. Absorbance at 450 nm (A450) was measured using Cytation 7 Microplate Multi-Mode Reader (BioTek Instruments). Flow cytometry Flow cytometry analysis was performed to detect SARS-CoV-2 S-specific CD4+and CD8+T cell responses in the above collected mouse splenocytes. Briefly, splenocytes (1×106cells / well) were incubated with a mixture of peptides predicted to be mouse CTL and T helper cell epitopes in SARS-CoV-2 S protein (final concentration 5 μg / ml / peptide; Tables 3 and 4), and cultured at 37°C.42 h later, the cells were restimulated as above in the presence of mouse IL-2 (1 μg; R&D Systems) and Brefeldin A (5 μg / ml; Sigma).6 h later, the cells were washed with PBS, and stained for surface markers using anti-mouse-CD45-AF700, anti-mouse-CD8-PerCP / Cy5.5 (Biolegend), and anti-mouse-CD4-FITC (BD Pharmingen) antibodies. After fixation and permeabilization, the cells were further stained for intracellular markers using anti-mouse-IFN-γ-PE (BD Pharmingen) and anti-mouse-TNF-α-BV421 (Biolegend) antibodies, followed by analysis using flow cytometry (CytoFLEX flow cytometer: Beckman Coulter Life Sciences). Evaluation of vaccine efficacy in the immunized mice The hACE2-Tg mice (6-8-week-old female or 4-6-month-old male and female), BALB / c mice (10-week-old female), and C57BL / 6 (B6) mice (8-9-month-old male and female) were respectively immunized with SARS-CoV-2 Ub-S DNA, original S DNA, full-length S protein, and / or PBS as described above. Four separate experiments were performed to evaluate the efficacy of SARS-CoV-2 vaccines in the immunized mice. First, two weeks post-last dose, hACE2-Tg mice respectively immunized with Ub-S DNA (Imiquimod adjuvant), full-length S protein control (Alum + MPL adjuvants), or PBS, were intranasally (I.N.) challenged with SARS-CoV-2 (human strain 2019n-CoV / USA-WA1 / 2020, 5,000 PFU / mouse, 50 μl / mouse), and observed for survival and weight changes daily for 14 days. Second, two weeks post-last dose, B6 mice respectively immunized with Ub-S DNA, full-length S protein control, or PBS, as described above, were challenged (I.N.) with SARS2-CoV-2 (mouse-adapted N501YMA30, 5,000 PFU / mouse, 50 μl / mouse). The challenged mice were sacrificed 2 days post-infection, and lung tissues were detected for viral titers as described below. Third, four weeks post-last dose, BALB / c mice, which were respectively immunized with Imiquimod-adjuvanted Ub-S DNA, original S DNA control, or full-length S protein control, as well as PBS background control (as described above), were challenged (I.N.) with SARS-CoV-2 Omicron-BA5 (50,000 PFU / mouse, 50 μl / mouse), and evaluated for viral titers in the lungs 2 days post-challenge. Fourth, eight weeks post-last dose of Imiquimod-adjuvanted Ub-S DNA vaccine and PBS, immunized hACE2-Tg mice were intraperitoneally injected with anti-mouse-CD4 (IgG2b, for depleting CD4+T cells), anti-mouse- CD8a (IgG2b, for depleting CD8+T cells) (200 μg / mouse) or IgG2b isotype control (without depleting CD4+and CD8+T cells) (Bio X Cell) antibody, respectively, at -2, -1, and 1 days post- SARS-CoV-2 challenge, and the mice were challenged (I.N.) with SARS-CoV-2 (2019n- CoV / USA-WA1 / 2020, 2,000 PFU / mouse, 50 μl / mouse). The challenged mice were observed for survival and weight changes daily for 14 days. The challenged mice with greater than 30% weight loss and significant clinical symptoms were humanely euthanized. SARS-CoV-2 plaque assay SARS-CoV-2 titers in the challenged mouse lung were detected by plaque assay. Specifically, supernatants from the homogenized lung tissues were serially diluted in DMEM, and incubated with Vero E6 cells pre-plated in 12-well plates at 37°C for 1 h in 5% CO2 with gentle rocking every 15 min. After removing the inoculum, the plates were overlaid with 1.2% agarose containing 4% FBS. After further incubation for 2 days, overlays were removed, and plaques were visualized by staining with 0.1% crystal violet. Statistical analysis Statistical significance among different groups was performed using GraphPad Prism 9 software. Statistical significance of vaccine-induced T cell responses was calculated using Ordinary one-way ANOVA Multiple comparison test. Statistical significance of viral titers and weight loss was performed using unpaired student t test. P < 0.05 was considered significant. *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively. It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0002]

[0003] SEQUENCES 1) SEQ ID NO: 1 – 2019CoV-19 TVLPPLTDEMIAQYTSALL 2) SEQ ID NO: 2 – 2019CoV-20 LQIPFAMQMAYRFNGIGVTQ 3) SEQ ID NO: 3 – 2019CoV-21 NVLYENQKLIANQFNSAIGK 4) SEQ ID NO: 4 – 2019CoV-22 IQDSLSSTASALGKLQDVVN 5) SEQ ID NO: 5 – 2019CoV-23 LQDVVNQNAQALNTLVKQLS 6) SEQ ID NO: 6 – 2019CoV-24 KQLSSNFGAISSVLNDILSR 7) SEQ ID NO: 7 – 2019CoV-25 MSFPQSAPHGVVFLHVTYVP 8) SEQ ID NO: 8 – 2019CoV-26 CGPKKSTNLVKNKCVNFNFN 9) SEQ ID NO: 9 – 2019CoV-27 VNFNFNGLTGTGVLTESNKK 10) SEQ ID NO: 10 – 2019CoV-28 NSPRRARSVASQSIIAYTMSL 11) SEQ ID NO: 11 – 2019CoV-29 SIIAYTMSLGAENSVAYSNNSI 12) SEQ ID NO: 12 – 2019CoV-30 LPPAYTNSFTRGVYYPDKVF 13) SEQ ID NO: 13 – 2019CoV-31 STQDLFLPFFSNVTWFHAIHVS 14) SEQ ID NO: 14 – 2019CoV-32 SGTNGTKRFDNPVLPFNDGVYF 15) SEQ ID NO: 15 – 2019CoV-33 YSSANNCTFEYVSQPFLMDL 16) SEQ ID NO: 16 – 2019CoV-34 WTAGAAAYYVGYLQPRTFLL 17) SEQ ID NO: 17 – 2019CoV-35 IYQTSNFRVQPTESIVRFPN 18) SEQ ID NO: 18 – 7 of 181 FRSSVLHSTQDLFLPFF 19) SEQ ID NO: 19 – 8 of 181 STQDLFLPFFSNVTWFH 20) SEQ ID NO: 20 – 11 of 181 SGTNGTKRFDNPVLPFN 21) SEQ ID NO: 21 – 15 of 181 NIIRGWIFGTTLDSKTQ 22) SEQ ID NO: 22 – 31 of 181 NLVRDLPQGFSALEPLV 23) SEQ ID NO: 23 – 32 of 181 QGFSALEPLVDLPIGIN 24) SEQ ID NO: 24 – 33 of 181 PLVDLPIGINITRFQTL 25) SEQ ID NO: 25 – 38 of 181 AGAAAYYVGYLQPRTFL 26) SEQ ID NO: 26 – 57 of 181 TNVYADSFVIRGDEVRQ 27) SEQ ID NO: 27 – 67 of 181 PFERDISTEIYQAGSTP 28) SEQ ID NO: 28 – 68 of 181 TEIYQAGSTPCNGVEGF 29) SEQ ID NO: 29 – 69 of 181 STPCNGVEGFNCYFPLQ 30) SEQ ID NO: 30 – 73 of 181 YQPYRVVVLSFELLHAP 31) SEQ ID NO: 31 – 74 of 181 VLSFELLHAPATVCGPK 32) SEQ ID NO: 32 – 77 of 181 LVKNKCVNFNFNGLTGT 33) SEQ ID NO: 33 – 94 of 181 GAEHVNNSYECDIPIGA 34) SEQ ID NO: 34 – 99 of 181 VASQSIIAYTMSLGAEN 35) SEQ ID NO: 35 – 117 of 181 SKRSFIEDLLFNKVTLA 36) SEQ ID NO: 36 – 118 of 181 DLLFNKVTLADAGFIKQ 37) SEQ ID NO: 37 – 150 of 181 GKGYHLMSFPQSAPHGV 38) SEQ ID NO: 38 – 153 of 181 VTYVPAQEKNFTTAPAI 39) SEQ ID NO: 39 – 154 of 181 EKNFTTAPAICHDGKAH 40) SEQ ID NO: 40 – 172 of 181 IDLQELGKYEQYIKWPW 41) SEQ ID NO: 41 – 173 of 181 KYEQYIKWPWYIWLGFI 42) SEQ ID NO: 42 – SARS-CoV-2 Ub-S Construct- Nucleotide sequence CTAGAATTCGTTCTTGCCACCATGCAGATCTTCGTGAAGACTCTGACTGGTAAGACCATC ACCCTCGAGGTTGAGCCCAGTGACACCATCGAGAATGTCAAGGCAAAGATCCAAGATAAG GAAGGCATCCCTCCTGACCAGCAGAGGCTGATCTTTGCTGGAAAACAGCTGGAAGATGGG CGCACCCTGTCTGACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTGCTCCGTCTC AGAGGTGCCATCGCCGCCAGAGACCTGATCTGTGCCCAGAAGTTTAATGGCCTG ACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCC TGCTGGCCGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAG ATCCCCTTTGCCATGCAGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAAT GTGCTGTACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAA GATCCAGGACAGCCTGTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGT GAATCAGAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCG GCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCA GAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTACGT GACACAGCAGCTGATCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAA CCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAG GGCTATCACCTGATGTCCTTCCCACAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACG TGACCTACGTGCCAGCCCAGGAGAAGAACTTCACCACAGCACCAGCCATCTGCCAC GATGGCAAGGCACACTTTCCTAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTG GTTTGTGACACAGCGCAATTTCTACGAGCCACAGATCATCACCACAGACAATACATT CGTGTCTGGCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCC TCTGCAGCCAGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATC ACACCTCCCCCGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGA ACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGTCC CTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTA TATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCT GTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGC TGTAAGTTTGATGAGGACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTA CACCAAGCTGCCTGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACA ATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAG AGCAATCTGAAGCCATTCGAGAGGGACATCTCTACAGAAATCTACCAGGCAGGCAG CACCCCATGCAATGGAGTGGAGGGCTTTAACTGTTATTTCCCTCTGCAGTCCTACGG CTTCCAGCCAACAAACGGCGTGGGCTATCAGCCCTACCGCGTGGTGGTGCTGAGCTT TGAGCTGCTGCACGCACCTGCAACAGTGTGCGGACCAAAGAAGTCCACCAATCTGG TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTG ACCGAGTCCAACAAGAAGTTCCTGCCTTTTCAGCAGTTCGGCAGGGACATCGCAGAT ACCACAGACGCCGTGCGCGACCCTCAGACCCTGGAGATCCTGGACATCACACCATG CTCTTTCGGCGGCGTGAGCGTGATCACACCAGGCACCAATACAAGCAACCAGGTGG CCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCAATCCACGCAGATC AGCTGACCCCTACATGGCGGGTGTACAGCACCGGCTCCAACGTGTTCCAGACAAGA GCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCC TATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCACGGAGAGC CCGGAGCGTGGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAA CAGCGTGGCCTACTCTAACAATAGCATCGCCATCCCTACCAACTTCACAATCTCTGT GACCACAGAGATCCTGCCAGTGTCCATGACCAAGACATCTGTGGACTGCACAATGT ATATCTGTGGCGATTCTACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTT GTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGACAAGAACACA CAGGAGGTGTTCGCCCAGGTGAAGCAAATCTACAAGACCCCACCCATCAAGGACTT TGGCGGCTTCAACTTCAGCCAGATCCTGCCCGATCCTTCCAAGCCATCTAAGCGGAG CTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAA GCAGTATGGCGATTGCCTGGGCGACATCGCCGCCAGAGACCTGATCTGTGCCCA GAAGTTTAATGGCCTGACCGTGCTGCCTCCACAGTGCGTGAACCTGACCACAAG GACCCAGCTGCCCCCTGCCTATACCAATTCCTTCACACGGGGCGTGTACTATCCCGA CAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCTTTCTTT AGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCG GTTCGACAATCCAGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCTACCGAGAA GAGCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGT CTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATCAAGGTGTGCGAGTTCCAGT TTTGTAATGATCCATTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGG AGTCCGAGTTTCGCGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGTGTCCC AGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAG TTCGTGTTTAAGAATATCGATGGCTACTTCAAAATCTACTCTAAGCACACCCCAATC AACCTGGTGCGCGACCTGCCACAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCT GCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTA CCTGACACCAGGCGACTCCTCTAGCGGATGGACCGCCGGCGCTGCCGCCTACTATGT GGGCTATCTGCAGCCCAGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCA CAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGCGAGACCAAGTGTACACTGAAG TCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCTACC GAGTCTATCGTGCGCTTTCCCAATATCACAAACCTGTGCCCTTTTGGCGAGGTGTTCA ACGCAACCAGGTTCGCCAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGC GTGGCCGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTAT GGCGTGTCCCCTACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCCGATTCT TTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCCCCTGGCCAGACAGGCAAGAT CGCCGACTACAATTATAAGCTGCCTGACGATTTCACCGGCTGCGTGATCGCCTG GAACAGCAACAATCTGGATTCCtagGTTCTTGCGGCCGCCCTA Bold underline = Kozak sequence Italics underline = Linker DNA Bold italics= Restriction site (EcoRI and NotI restriction sites) Bold = Overlap regions lowercase = Stop codon Italics = Ubiquitin sequence Underline= SARS-CoV-2 Spike Sequence S-c, S-b, and S-a, respectively 43) SEQ ID NO: 43- SARS-CoV-2 Ub-S Construct Amino Acid Sequence LEFVLATMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLS DYNIQKESTLHLVLRLRGAIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASA LGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSL QTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVF LHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTF VSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQK EIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCC SCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYTKLPDDFTGCVIAWNSNNLDSKVGGNY NYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR VVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRD IADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQ LTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVA SQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPS KPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPQCVNLTT RTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFL GVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDG YFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN NLDS Italics: Ubiquitin sequence Bold and underline “A” is modified glycine to alanine residue 44) SEQ ID NO: 44 - SARS-CoV-2 Spike Sequence S-c- Nucleotide Sequence CTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCAT CACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCCATGCA GATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACC AGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTG TCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCA GGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGCGT GCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACC GGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTACGTGACACAGCAGCTGATC AGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAGTG CGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATGTC CTTCCCACAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCAGC CCAGGAGAAGAACTTCACCACAGCACCAGCCATCTGCCACGATGGCAAGGCACACT TTCCTAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCA ATTTCTACGAGCCACAGATCATCACCACAGACAATACATTCGTGTCTGGCAACTGTG ACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCTCTGCAGCCAGAGCTGG ACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCCCCCGACGTG GATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGAT CGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGTCCCTGATCGATCTGCAGG AGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCA TCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCT GCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGG ACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACC 45) SEQ ID NO: 45- SARS-CoV-2 Spike Sequence S-c-Amino Acid Sequence LLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQK LIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILS RLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVD FCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNG THWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNH TSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLG FIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 46) SEQ ID NO: 46 - SARS-CoV-2 Spike Sequence S-b-Nucleotide Sequence AAAGTGGGCGGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAA GCCATTCGAGAGGGACATCTCTACAGAAATCTACCAGGCAGGCAGCACCCCATGCA ATGGAGTGGAGGGCTTTAACTGTTATTTCCCTCTGCAGTCCTACGGCTTCCAGCCAA CAAACGGCGTGGGCTATCAGCCCTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGC ACGCACCTGCAACAGTGTGCGGACCAAAGAAGTCCACCAATCTGGTGAAGAACAAG TGCGTGAACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGACCGAGTCCAA CAAGAAGTTCCTGCCTTTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACG CCGTGCGCGACCCTCAGACCCTGGAGATCCTGGACATCACACCATGCTCTTTCGGCG GCGTGAGCGTGATCACACCAGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTAT CAGGACGTGAATTGTACCGAGGTGCCCGTGGCAATCCACGCAGATCAGCTGACCCC TACATGGCGGGTGTACAGCACCGGCTCCAACGTGTTCCAGACAAGAGCCGGATGCC TGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCTATCGGCGCC GGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCACGGAGAGCCCGGAGCGT GGCCTCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGC CTACTCTAACAATAGCATCGCCATCCCTACCAACTTCACAATCTCTGTGACCACAGA GATCCTGCCAGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGG CGATTCTACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCT GAATAGAGCCCTGACAGGCATCGCCGTGGAGCAGGACAAGAACACACAGGAGGTG TTCGCCCAGGTGAAGCAAATCTACAAGACCCCACCCATCAAGGACTTTGGCGGCTTC AACTTCAGCCAGATCCTGCCCGATCCTTCCAAGCCATCTAAGCGGAGCTTTATCGAG GACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGC GATTGCCTGGGCGAC 47) SEQ ID NO: 47- SARS-CoV-2 Spike Sequence S-b-Amino Acid Sequence KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNG VGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV PVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS PRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYI CGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNF SQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD 48) SEQ ID NO: 48 - SARS-CoV-2 Spike Sequence S-a- Nucleotide Sequence CAGTGCGTGAACCTGACCACAAGGACCCAGCTGCCCCCTGCCTATACCAATTCCTTC ACACGGGGCGTGTACTATCCCGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACA CAGGATCTGTTTCTGCCTTTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGA GCGGCACCAATGGCACAAAGCGGTTCGACAATCCAGTGCTGCCCTTTAACGATGGC GTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGGCACC ACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTC ATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCATTCCTGGGCGTGTACTATCAC AAGAACAATAAGAGCTGGATGGAGTCCGAGTTTCGCGTGTATTCTAGCGCCAACAA TTGCACATTTGAGTACGTGTCCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGG CAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAAAT CTACTCTAAGCACACCCCAATCAACCTGGTGCGCGACCTGCCACAGGGCTTCAGCGC CCTGGAGCCACTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACT GCTGGCCCTGCACAGAAGCTACCTGACACCAGGCGACTCCTCTAGCGGATGGACCG CCGGCGCTGCCGCCTACTATGTGGGCTATCTGCAGCCCAGGACCTTCCTGCTGAAGT ACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGC GAGACCAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATC CAATTTCAGGGTGCAGCCTACCGAGTCTATCGTGCGCTTTCCCAATATCACAAACCT GTGCCCTTTTGGCGAGGTGTTCAACGCAACCAGGTTCGCCAGCGTGTACGCATGGAA TAGGAAGCGCATCTCTAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTC TTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCTACAAAGCTGAATGACCTGTGCTTT ACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGC CCCTGGCCAGACAGGCAAGATCGCCGACTACAATTAT 49) SEQ ID NO: 49 - SARS-CoV-2 Spike Sequence S-a-Amino Acid Sequence QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGT NGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEF QFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLRE FVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGD SSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY 50) SEQ ID NO: 50 – Ubiquitin-Nucleotide Sequence CAGATCTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGGTTGAGCCCAGT GACACCATCGAGAATGTCAAGGCAAAGATCCAAGATAAGGAAGGCATCCCTCCTGA CCAGCAGAGGCTGATCTTTGCTGGAAAACAGCTGGAAGATGGGCGCACCCTGTCTG ACTACAACATCCAGAAAGAGTCCACCCTGCACCTGGTGCTCCGTCTCAGAGGTGCC 51) SEQ ID NO: 51 – Ubiquitin-Amino Acid Sequence QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQK ESTLHLVLRLRGA 52) SEQ ID NO: 52 – SARS-CoV-2 Spike-Nucleotide Sequence CAGTGCGTGAACCTGACCACAAGGACCCAGCTGCCCCCTGCCTATACCAATTCCTTC ACACGGGGCGTGTACTATCCCGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACA CAGGATCTGTTTCTGCCTTTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGA GCGGCACCAATGGCACAAAGCGGTTCGACAATCCAGTGCTGCCCTTTAACGATGGC GTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGGCACC ACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTC ATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCATTCCTGGGCGTGTACTATCAC AAGAACAATAAGAGCTGGATGGAGTCCGAGTTTCGCGTGTATTCTAGCGCCAACAA TTGCACATTTGAGTACGTGTCCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGG CAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTTCAAAAT CTACTCTAAGCACACCCCAATCAACCTGGTGCGCGACCTGCCACAGGGCTTCAGCGC CCTGGAGCCACTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACT GCTGGCCCTGCACAGAAGCTACCTGACACCAGGCGACTCCTCTAGCGGATGGACCG CCGGCGCTGCCGCCTACTATGTGGGCTATCTGCAGCCCAGGACCTTCCTGCTGAAGT ACAACGAGAATGGCACCATCACAGACGCAGTGGATTGCGCCCTGGACCCCCTGAGC GAGACCAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATC CAATTTCAGGGTGCAGCCTACCGAGTCTATCGTGCGCTTTCCCAATATCACAAACCT GTGCCCTTTTGGCGAGGTGTTCAACGCAACCAGGTTCGCCAGCGTGTACGCATGGAA TAGGAAGCGCATCTCTAACTGCGTGGCCGACTATAGCGTGCTGTACAACTCCGCCTC TTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCTACAAAGCTGAATGACCTGTGCTTT ACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGC CCCTGGCCAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCTGACGATTTCAC CGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACT ACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCATTCGAGAGGGAC ATCTCTACAGAAATCTACCAGGCAGGCAGCACCCCATGCAATGGAGTGGAGGGCTT TAACTGTTATTTCCCTCTGCAGTCCTACGGCTTCCAGCCAACAAACGGCGTGGGCTA TCAGCCCTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCTGCAACAGT GTGCGGACCAAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACT TCAACGGACTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGCCT TTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCTCA GACCCTGGAGATCCTGGACATCACACCATGCTCTTTCGGCGGCGTGAGCGTGATCAC ACCAGGCACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTA CCGAGGTGCCCGTGGCAATCCACGCAGATCAGCTGACCCCTACATGGCGGGTGTAC AGCACCGGCTCCAACGTGTTCCAGACAAGAGCCGGATGCCTGATCGGAGCAGAGCA CGTGAACAATTCCTATGAGTGCGACATCCCTATCGGCGCCGGCATCTGTGCCTCTTA CCAGACCCAGACAAACTCTCCACGGAGAGCCCGGAGCGTGGCCTCCCAGTCTATCA TCGCCTATACCATGTCCCTGGGCGCCGAGAACAGCGTGGCCTACTCTAACAATAGCA TCGCCATCCCTACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCAGTGTCCA TGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCA GCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAG GCATCGCCGTGGAGCAGGACAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCA AATCTACAAGACCCCACCCATCAAGGACTTTGGCGGCTTCAACTTCAGCCAGATCCT GCCCGATCCTTCCAAGCCATCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAA GGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGGGCGACAT CGCCGCCAGAGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCTCC ACTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGCCGGCACCA TCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTTGCCATGC AGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAAC CAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCT GTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCC AGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAACTTCGGCGCCATCTCTAGC GTGCTGAATGACATCCTGAGCCGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGA CCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTACGTGACACAGCAGCTGA TCAGGGCCGCCGAGATCAGGGCCAGCGCCAATCTGGCAGCAACCAAGATGTCCGAG TGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCACCTGATG TCCTTCCCACAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACCTACGTGCCA GCCCAGGAGAAGAACTTCACCACAGCACCAGCCATCTGCCACGATGGCAAGGCACA CTTTCCTAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCG CAATTTCTACGAGCCACAGATCATCACCACAGACAATACATTCGTGTCTGGCAACTG TGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCTCTGCAGCCAGAGCT GGACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCCCCCGACG TGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAG ATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGTCCCTGATCGATCTGCA GGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATATCTGGCTGGGCTT CATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGACATC CTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAG GACGATAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACCTAG 53) SEQ ID NO: 53 – SARS-CoV-2 Spike- Amino Acid Sequence QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGT NGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEF QFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLRE FVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGD SSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFP LQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGT GVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAV LYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGI CASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMT KTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKT PPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQ KFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVT QNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFP REGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKE ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYI KWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVK LHYT 54) SEQ ID NO: 54 – SARS-CoV-2 Ub-S DNA Construct overlap region 1-Nucleotide Sequence ATCGCCGCCAGAGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTGCTGCCT CCA 55) SEQ ID NO: 55 – SARS-CoV-2 Ub-S DNA Construct overlap region 1-Amino Acid Sequence IAARDLICAQKFNGLTVLPP 56) SEQ ID NO: 56 – SARS-CoV-2 Ub-S DNA Construct overlap region 2-Nucleotide Sequence AAGCTGCCTGACGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGAT TCC 57) SEQ ID NO: 57 – SARS-CoV-2 Ub-S DNA Construct overlap region 2-Amino Acid Sequence KLPDDFTGCVIAWNSNNLDS

Claims

CLAIMS What is claimed is:

1. A DNA vaccine encoding a SARS-CoV-2 spike (S) protein, or a fragment thereof, and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein.

2. The DNA vaccine of claim 1, wherein said vaccine further encodes at least one cytotoxic T lymphocyte (CTL) epitope.

3. The DNA vaccine of claim 1 or 2, wherein the ubiquitin protein comprises a protein degradation-enhancing mutation.

4. The DNA vaccine of claim 3, wherein the protein degradation-enhancing mutation comprises a glycine (G) residue at amino acid position 76 of the protein replaced by an alanine (A) residue.

5. A vector comprising the DNA vaccine of any one of claims 1-4.

6. A composition comprising the DNA vaccine of any one of claims 1-4, an adjuvant, and pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the adjuvant comprises Imiquimod, Alum, monophosphoryl lipid A (MPL), MF59, Poly(I:C), Montanide ISA51, AddaVax, or a combination thereof.

8. A method of inducing a T cell-based response to a SARS-CoV-2 virus in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein, or a fragment thereof, and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein.

9. The method of claim 8, wherein said vaccine further encodes at least one cytotoxic T lymphocyte (CTL) epitope.

10. The method of claim 8 or 9, wherein the ubiquitin protein comprises a protein degradation- enhancing mutation.

11. The method of claim 10, wherein the protein degradation-enhancing mutation comprises a glycine (G) residue at amino acid position 76 of the protein replaced by an alanine (A) residue.

12. The method of any one of claims 8-11, wherein the composition further comprises an adjuvant and a pharmaceutically acceptable carrier.

13. The method of claim 12, wherein the adjuvant comprises Imiquimod, Alum, monophosphoryl lipid A (MPL), MF59, Poly(I:C), Montanide ISA51, AddaVax, or a combination thereof.

14. The method of any one of claims 8-13, wherein further comprising inducing the T cell- based response against an original SARS-CoV-2 (WT-S), Alpha, Beta, Gamma, Delta, or Omicron SARS-CoV-2 variants, or subvariants thereof.

15. The method of any one of claims 8-14, wherein the T cell-based response comprises a response from CD8+T cells, CD4+T cells, or a combination thereof.

16. A method of preventing a SARS-CoV-2 infection in a subject, comprising administering to the subject a composition comprising a DNA vaccine encoding a SARS-CoV-2 spike (S) protein, or a fragment thereof, and a ubiquitin protein, wherein the S protein is split and rearranged into three parts, and wherein the S protein is fused to the ubiquitin protein.

17. The method of claim 16, wherein said vaccine further encodes at least one cytotoxic T lymphocyte (CTL) epitope.

18. The method of claim 16 or 17, wherein the ubiquitin protein comprises a protein degradation-enhancing mutation.

19. The method of claim 18, wherein the protein degradation-enhancing mutation comprises a glycine (G) residue at amino acid position 76 of the protein replaced by an alanine (A) residue.

20. The method of any one of claims 16-19, wherein the composition further comprises an adjuvant and a pharmaceutically acceptable carrier.

21. The method of claim 20, wherein the adjuvant comprises Imiquimod, Alum, monophosphoryl lipid A (MPL), MF59, Poly(I:C), Montanide ISA51, AddaVax, or a combination thereof.

22. The method of any one of claims 16-21, wherein the DNA vaccine induces a T cell-based immune response against an original SARS-CoV-2 (WT-S), Alpha, Beta, Gamma, Delta, or Omicron SARS-CoV-2 variants, or subvariants thereof.

23. The method of claim 22, wherein the T cell-based response comprises a response from CD8+T cells, CD4+T cells, or a combination thereof.

24. The method of any one of claims 16-23, wherein the vaccine provides protection without inducing an antibody response against the spike (S) protein.

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