Patent Publication Number: US-2023143228-A1

Title: Coronavirus vaccines, compositions, and methods related thereto

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 63/012,920 filed Apr. 21, 2020, U.S. Provisional Application No. 63/044,711 filed Jun. 26, 2020, U.S. Provisional Application No. 63/084,065 filed Sep. 28, 2020, and U.S. Provisional Application No. 63/143,191 filed Jan. 29, 2021. The entirety of each of these applications is hereby incorporated by reference for all purposes. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under AI148378 awarded by the National Institutes of Health. The government has certain rights in this invention. 
    
    
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB) 
     The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 20121PCT_ST25.txt. The text file is 240 KB, was created on April 20, 2021, and is being submitted electronically via EFS-Web. 
     BACKGROUND 
     About 10% of common colds are due to certain coronavirus (CoV) strains associated with mild symptoms. More dangerous human strains such as severe acute respiratory syndrome associated coronavirus (SARS-CoV-1) and SARS-CoV-2 (also referred to as COVID-19) are believed to result from coronavirus strains jumping to humans by secondary zoonotic transfers, e.g., from bats to cats and cats to humans. In humans, SARS-CoV-2 can be transferred from individuals who have mild symptoms or are asymptomatic and has caused numerous deaths worldwide. Thus, there is a need to find an effective vaccine. 
     The SARS-CoV-2 genome has about 30 kb that can be directly read by ribosomes with host cells. The RNA forms a ribonucleoprotein complex within virus particles having a viral lipid envelope membrane made up of membrane (M) glycoproteins, trimeric spike (S) glycoproteins and envelope (E) proteins. The trimeric units of the spike protein contain a receptor binding domain and a fusion domain that anchors it into lipid membrane. 
     Walls et al. report that the SARS-CoV-2 spike protein is involved in viral cell entry by recognizing human ACE2. Cell, 2020, 180, 1-12. 
     Andersen et al. report six receptor binding domain amino acids L455, F486, Q493, S494, N501 and Y505 are involved in binding to ACE2 receptors in SARS-CoV-2. Nat Med, 2020. 
     Altenburg et al. report modified vaccinia virus Ankara (MVA) as a production platform for vaccines against influenza and other viral respiratory diseases. Viruses, 2014, 6(7):2735-61. 
     Graham et al. report prefusion coronavirus spike proteins and uses. See WO 2018/081318. 
     References cited herein are not an admission of prior art. 
     SUMMARY 
     This disclosure relates to methods of promoting immune responses against coronavirus, such as SARS-CoV-2, and compositions related thereto. In certain embodiments, this disclosure relates to methods of vaccinating for coronavirus comprising administering to the subject a composition disclosed herein. In certain embodiments, the composition comprises a recombinant virus such as recombinant MVA that encodes a coronavirus spike protein. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 986. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 987. 
     In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject. 
     In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein in combination with a nucleic acid encoding a T cell stimulating chimeric protein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject. 
     In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising virus-like particles having a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. 
     In certain embodiments, this disclosure contemplates nucleic acids, recombinant vectors, viral vectors, and bacterial plasmids encoding a coronavirus spike protein or segment thereof as disclosed herein which forms trimeric protein complexes and uses in vaccination methods disclosed herein. 
     In certain embodiments, this disclosure relates to cells and other expression vectors and expression systems for use in producing a coronavirus spike protein or segment thereof as disclosed herein and trimeric coronavirus spike proteins or segment thereof as disclosed herein, or variants thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG.  1 A  illustrates a recombinant nucleic acid that encodes the spike protein of SARS-CoV-2 as a full-length protein displayed on VLPs (S-VLP) expressed within an MVA construct. In the s2 domain, the amino acids at positions 986 and 987 are mutated to proline. This expresses the full-length coronavirus S protein (aa 1 to 1273) followed by membrane protein (M; aa 1 to 222) and envelope glycoprotein (E; aa 1 to 75). The S, M and E are expresses as a single transcript from a single mH5 promoter using Porcine 2A sequences between S and M, and M and E. This construct is designed to produce VLPs that will display S, M and E proteins on the VLP membrane. Two point-mutations were introduced at positions 986 (K986P) and 987 (V987P) to introduce prolines. These mutations stabilize the protein in pre-fusion confirmation. The spike protein of SARS-CoV-2 is expressed as a trimer on the surface of the virion. Multimeric expression of the antigen in the form of virus-like particles (VLPs) generates antibodies by focusing the response away from unwanted epitopes. NTD—N terminal domain; CTD—C terminal domain; FP—Fusion peptide; HR-N—Heptad repeat N; HR-C—Heptad repeat C; TM—Transmembrane anchor; IC—intracellular tail, M—membrane protein; E—Envelope protein; P2A—Porcine 2A; aa—amino acid. 
         FIG.  1 B  illustrates a recombinant nucleic acid that encodes the spike protein of SARS-CoV-2 with a truncated spike ACE2 receptor domain (S) lacking the fusion domain. GM-CSFss refers to a GM-CSF signal sequence. This expresses the amino acids 14-780 of the S protein (contains S1 and part of S2). The first 13 amino acids of the spike protein were replaced with the GM-CSF secretory signal sequence to enhance protein secretion. The protein is expected to be secreted out of the cell as a monomer. 
         FIG.  1 C  illustrates a recombinant nucleic acid that encodes the spike protein of SARS-CoV-2 for forming a trimeric complex (S-Tri) in a lipid membrane for coating on cells. This expresses the full-length S protein (aa 1 to 1273) under the control of mH5 promoter. This leads to expression of a trimeric S protein that will be anchored on a membrane but will not make VLPs. 
         FIG.  1 D  illustrates a recombinant nucleic acid that encodes the spike protein of SARS-CoV-2 with a self-folding sequence on the C-terminus for forming a trimeric complex (S-Tri-sec). This expresses the amino acids 14-1208 of the S protein (contains most of S without transmembrane and cytoplasmic tail regions). The first 13 amino acids of the spike protein were replaced with the GM-C SF secretory signal sequence to enhance protein secretion. A Fold-on trimerization sequence downstream of S at position 1208 was added. This protein is secreted out of the cell as a stabilized trimeric protein. GM-CSFss refers to a GM-CSF signal sequence. 
         FIG.  2 A  shows flow cytometry data indicating the expression of full-length spike protein. DF-1 cells were infected with wild type MVA and transfected with pLW-73-MVA/S-Tri or MVA/S-VLP DNA expression plasmids. Polyclonal rabbit serum was used to detect spike protein. 
         FIG.  2 B  shows a Western blot on spike protein expression by MVA recombinants. Bacterially expressed and purified spike protein (deltaTM) (spike control) was used as a positive control and total lysate from MVA infected DF-1 cells were used as a negative control. The arrow indicates that the spike protein VLP moves higher than the soluble spike protein. 
         FIG.  3    shows flow cytometry data on spike protein expression by DNA recombinants where 293T cells were transfected with DNA/S-VLP or DNA/S1-Mono plasmids and the expression of spike protein was confirmed by a flow cytometry using rabbit polyclonal serum generated against SRAS-CoV. 
         FIG.  4 A  illustrates two MVA recombinants one expressing full length Spike with stabilizing mutations and the other expressing only the S1 region of Spike. Both constructs expressed the proteins at the correlated size. MVA/S and MVA/S1, spike protein inserts of SARS-CoV-2 were cloned in between essential regions in plasmid pLW72 (18R and G1L), under mH5 promoter. 
         FIG.  4 B  shows data when mice were immunized with two doses of the vaccines described in  FIG.  4 A . Both vaccines induced comparable binding antibody responses to RBD and S proteins. The MVA/S1 mice induced a stronger binding antibody response to S1 protein. Antibody responses were induced by MVA/S or MVA/S1 vaccinated Balb/c mice. BALB/c mice were immunized on week 0 and 3 with recombinant MVA expressing either S (MVA/S) (n=5) or S1 (MVA/S1) (n=5) in a prime-boost strategy through intramuscular (i.m.) route. Unvaccinated (naïve) animals served as controls (n=5). Endpoint IgG titers against SARS-CoV-2 RBD, S1 and S measured at week 2 after immunization. Titers are presented as the reciprocal of the serum dilution and plotted as log10. 
         FIG.  4 C  shows data indicating inducible bronchus associated lymphoid tissues (iBALT) formation upon MVA/S vaccination. Frozen lung sections from vaccinated mice were either stained for H&amp;E to analyze tissue structure and formation of iBALT aggregates, or immunofluorescence stained to visualize B cell and T cell (B) forming B cell follicle like structure (iBALT) induced by MVA/S vaccination given via i.m. route and compared with unvaccinated control mice. Total number of iBALT like structures visualized in each section per mice was quantified and compared between the groups. 
         FIG.  4 D  shows data on neutralizing antibody responses. Six to 8-week-old Balb/c mice were immunized via i.m. twice on week 0 and 3 with different SARS-CoV-2 MVA-based vaccines candidates MVA/S or MVA/S1. Serum was collected two weeks post-boost and performed SARS-CoV-2 virus (expressing GFP-mNG50) neutralization assay in Vero cells in serial dilutions. Serum collected from the naïve animals used as negative controls. The SARS-COV-2 FRNT-mNG50 titers in naïve, MVA/S and MVA/S1 immunized animals were quantified. 
         FIG.  4 E  shows data which compares binding and neutralizing data indicating MVA/S vaccine is highly immunogenic and can induce strong neutralizing antibody responses. Correlations analysis was performed to compare the relation between SARS-CoV-2 proteins (RBD, S 1  and S)-binding IgG antibody endpoint titers analyzed by ELISA assay with neutralization titers induced by MVA/S and MVA/S1, respectively. Strong neutralizing antibody response was observed only in mice immunized with MVA/S but not in MVA/S1. This difference was unexpected considering the binding antibodies are comparable or higher in the MVA/S1 group. This data suggests that the MVA/S vaccine has high potential to protect against SARS-CoV-2 infection. 
         FIG.  5 A  illustrates experimental timeline for evaluation of immunogenicity and protective efficacy of MVA-SARS-2 Spike (prefusion stabilized). 
         FIG.  5 B  shows data indicating strong binding antibody response against SARS-2 S1 and S 1+S2 in MVA/S vaccinated Rhesus Macaques, performed by ELISA. 
         FIG.  5 C  shows data indicating neutralization and correlation between binding ab response and functional neutralization titer. 
         FIG.  5 D  shows data indicating intracellular cytokine stimulation (ICS), IFNg+ CD8 response against S1 peptide pool of SARS-2 spike. 
         FIG.  5 E  shows data indicating Efficacy of MVA-S against upper and lower respiratory viral replication, estimated sub genomic viral RNA copies by quantitative real time PCR. 
         FIG.  6 A  illustrates an experimental schedule for assaying Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine candidates&#39; (MVA/S-tri and MVA/S-tri-dFCS) in BALB/c mice. Female mice were brought to the experimental room and adapted for 1 week prior to study initiation. Approximately, 6-8-week-old female BALB/c mice intramuscularly (i.m.), immunized on wk0 and wk4 with either MVA/S-tri (107 PFU) or MVA/S-tri-dFCS (10 7  PFU). Control group received no treatment served as controls. 
         FIG.  6 B  illustrates of Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine candidates (MVA-S-tri and MVA-S-tri-dFCS). Recombinant inserts were cloned in the essential region in between 18R and G1L under mH5 promoter. Spike protein (S) based vaccines. NTD—N terminal domain; CTD—C terminal domain; FP—Fusion peptide; HR-N—Heptad repeat N; HR-C—Heptad repeat C; TM—Transmembrane anchor; IC—intracellular tail; Active FCS (FCS—Furin cleavage site—RRAR (SEQ ID NO: 21)); Inactive FCS (FCS mutation—SRAG (SEQ ID NO: 22)). Arrows represents amino acid number and protease cleavage sites. 
         FIG.  6 C  shows data of experiments. Right shows data on measured RBD binding IgG antibody using ELISA and presented Endpoint IgG titers of serum from 3-weeks post-prime and 2-weeks post-boost immunization. Left shows data on neutralization titer against live mNeonGreen SARS-CoV-2 virus in serum collected at week 2 post-boost immunizations. 
         FIG.  7    illustrates chimeric constructs wherein CMV mH5 is is the promoter in DNA, S(delta)RBD is the spike protein of SARS-CoV-2 with the RBD deleted, N is the SARS-CoV-2 Nucleocapsid, M is the SARS-CoV-2 Membrane protein, NSPs are SARS-CoV-2 Non-structural proteins (e.g., nsp3, nsp4, and nsp6). Chimeric antigens with and without transmembrane regions (TM) are used for inducing T cells to DNA and MVA immunogens. 
         FIG.  8    shows data indicating MVA/S vaccine protects from SARS-CoV-2 infection in rhesus macaques. 
         FIG.  9    shows data on the pathology score of lungs of MVA/S vaccinated and MVA/Wt immunized rhesus macaques 10 days post infection. 
         FIGS.  10 A-C  shows data indicating MVA-based vaccines (MVA-S-tri and MVA-S-tri-dFCS) induces a robust neutralizing antibody response and provides protection against SARS-CoV-2 challenge in mice. 
         FIG.  10 A  shows data where six-week-old female BALB/c mice were immunized either with MVA-S-tri (circles) or MVA-S-tri-dFCS (upward triangle) vaccines via intramuscular route at weeks 0 and 4. Immunized mice were infected with 10{circumflex over ( )}5 PFU SARS-CoV-2 MA10. Endpoint IgG titers against SARS-CoV-2 RBD measured in serum collected at week 2 post-boost immunizations. 
         FIG.  10 B  shows data on neutralization titer against live mNeonGreen SARS-CoV-2 virus. The dotted line represents the limit of detection. 
         FIG.  10 C  shows data on lung SARS-CoV-2 (MA10) viral titers of vaccinated animals compared to unvaccinated. 
     
    
    
     DETAILED DISCUSSION 
     Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. 
     All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. 
     As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. 
     Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. 
     It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably. It is further noted that the claims may be drafted to exclude any optional element. 
     “Subject” refers to any animal, preferably a human patient, livestock, rodent, monkey or domestic pet. The term is used herein to encompasses apparently healthy, non-infected individuals or a patient who is known to be infected with, diagnosed with, a pathogen. 
     As used herein, the term “combination with” when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof. 
     The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of in reference to a peptide having an amino acid sequence refers a peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide may consist of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the C-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide may consist of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a rage of amino acids specified in the claim however the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. 
     The terms “protein” and “peptide” refer to polymers comprising amino acids joined via peptide bonds and are used interchangeably. Amino acids may be naturally or non-naturally occurring. A “chimeric protein” or “fusion protein” is a molecule in which different portions of the protein are derived from different origins such that the entire molecule is not naturally occurring. A chimeric protein may contain amino acid sequences from the same species of different species as long as they are not arranged together in the same way that they exist in a natural state. Examples of a chimeric protein include sequences disclosed herein that are contain one, two or more amino acids attached to the C-terminal or N-terminal end that are not identical to any naturally occurring protein, such as in the case of adding an amino acid containing an amine side chain group, e.g., lysine, an amino acid containing a carboxylic acid side chain group such as aspartic acid or glutamic acid, a polyhistidine tag, e.g. typically four or more histidine amino acids. Contemplated chimeric proteins include those with self-cleaving peptides such as P2A-GSG. See Wang. Scientific Reports 5, Article number: 16273 (2015). 
     In certain embodiments, the disclosure relates to recombinant polypeptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule. 
     A “label” refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as  35 S or  131 I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. 
     In certain embodiments, this disclosure contemplates that chimeric proteins disclosed herein may be variants. Variants may include 1 or 2 amino acid substitutions or conserved substitutions. Variants may include 3 or 4 amino acid substitutions or conserved substitutions. Variants may include 5 or 6 or more amino acid substitutions or conserved substitutions. Variant include those with not more than 1% or 2% of the amino acids are substituted. Variant include those with not more than 3% or 4% of the amino acids are substituted. Variants include proteins with greater than 80%, 89%, 90%, 95%, 98%, or 99% identity or similarity. 
     Variant peptides can be produced by mutating a vector to produce appropriate codon alternatives for polypeptide translation. Active variants and fragments can be identified with a high probability using computer modeling. Shihab et al. report an online genome tolerance browser. BMC Bioinformatics, 2017, 18(1):20. Ng et al. report methods of predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet, 2006, 7:61-80. Teng et al. Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions. Curr Pharm Biotechnol, 2008, 9(2): 123-33. 
     Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See also Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3):e1004775; Marks et al. Protein structure from sequence variation, Nat Biotechnol. 2012, 30(11):1072-80; Mackenzie et al. Curr Opin Struct Biol, 2017, 44:161-167 Mackenzie et al. Proc Natl Acad Sci U S A. 113(47):E7438-E7447 (2016); Joseph et al. J R Soc Interface, 2014, 11(95):20131147, Wei et al. Int. J. Mol. Sci. 2016, 17(12), 2118. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). 
     Sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. For example, the polypeptides GGGGGG (SEQ ID NO: 32) and GGGGT (SEQ ID NO: 33) have a sequence identity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP (SEQ ID NO: 34) and GGGAPPP (SEQ ID NO: 35) have a sequence identity of 6 out of 7 or 85%. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic—F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative—D E; Polar—S T N Q. The amino acid groups are also considered conserved substitutions. 
     Percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. 
     The term “recombinant vector” when made in reference to vectors and nucleic acids refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term recombinant nucleic acid is distinguished from the natural recombinants that result from crossing-over between homologous chromosomes. Recombinant nucleic acids as used herein are an unnatural union of nucleic acids from nonhomologous sources, usually from different organisms. 
     The terms “expression vector ” refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. 
     In certain embodiments, a vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col E1 origin of replication, f1 origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides. 
     Protein “expression systems” refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfected with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vector, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labelling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara &amp; Chong, Nucleic Acids Research, 2010, 38(13): e141, both hereby incorporated by reference in their entirety. 
     A “selectable marker” is a nucleic acid introduced into a vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5′ phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (at1D), UDP-glucose:galactose-1-phosphate uridyltransferasel (galT), feedback-insensitive α subunit of anthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR), benzyladenine-N-3-glucuronide,  E. coli  threonine deaminase, glutamate 1-semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSB1), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA). 
     Coronavirus Vaccines 
     In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of a coronavirus spike protein, a trimeric spike protein complex, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that spike protein, trimeric complex, and/or virus-like particles with spike protein are formed in the subject. 
     In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a coronavirus spike protein, trimeric complex, or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising virus-like particles having a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. In certain embodiments, this disclosure contemplates pharmaceutical compositions comprising a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein and variants thereof. 
     In certain embodiments, this disclosure contemplates nucleic acids, recombinant vectors, viral vectors, and bacterial plasmids encoding a coronavirus spike protein or segment thereof as disclosed herein which form trimeric protein complexes and uses in vaccination methods disclosed herein. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, further comprises a proline mutation at position 987. In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 1) 
     MF VFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKT SVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNF SQILPDP SKP SKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. In certain embodiments, the amino acid position of a coronavirus protein is in relation to SEQ ID NO: 1. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein comprises a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, further comprises a proline mutation at position 987. In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 23) 
     MFVFLVLLPLVS SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a lysine mutation at position 484, an asparagine mutation at position 417, a tyrosine mutation at position 501, or combinations thereof and a mutation in the furin cleavage site at position 682, 683, 684 or 685, a serine mutation at position 682, a proline mutation at position 986, a proline mutation at position 987, or combinations thereof. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 484. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a lysine mutation at position 484 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685. In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987. 
     In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 28) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF SALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 417. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising an asparagine mutation at position 417 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987. 
     In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 29) 
     MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS SVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GNIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNF SQILPDP SKP SKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. 
     In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation at position 501. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a tyrosine mutation at position 501 optionally in combination with other mutations below. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a mutation in the furin cleavage site at position 682, 683, 684 or 685, In certain embodiments, the mutation in the furin cleavage site is at position 682. In certain embodiments, the coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a serine mutation at position 682. In certain embodiments, this disclosure contemplates non-naturally occurring coronavirus spike protein, or nucleic acid having a sequence encoding a mutation, comprising a proline mutation at position 986. In certain embodiments, the coronavirus spike protein further comprises a proline mutation at position 987. 
     In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 30) 
     MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPSRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPP (spike sequence amino acids 1 to 987) or variants thereof. 
     In certain embodiments, the coronavirus spike protein further comprises a heterologous N-terminal signal sequence. 
     In certain embodiments, the coronavirus spike protein further comprises a C-terminal trimerization sequence. 
     In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 2) 
     MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIR AAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKN LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCC SCLKGCCSCGS CCKFDEDDSEPVLKGVKLHYT (S-Tri) or variants thereof. 
     In certain embodiments, the coronavirus spike protein comprises a coronavirus M protein sequence downstream from the C-terminal end of the coronavirus spike protein sequence, and wherein the M protein sequence and the coronavirus spike sequence are separated by a self-cleaving sequence. In certain embodiments, the coronavirus spike protein comprises a coronavirus E protein sequence downstream from the C-terminal end of the M protein sequence, and wherein the E protein sequence and the coronavirus M protein sequence are separated by a self-cleaving sequence. 
     In certain embodiments, the coronavirus spike protein comprises amino acid sequence (SEQ ID NO: 3) 
     MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPF LMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQ SYGFQPTNGVGYQPYRVVVL SFELLHAPATVCGPKKSTNL VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFG GVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIG AEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIA IPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ DKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIK QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAAL QIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLS STASALGKLQDVVN QNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIR AAEIRASANLAATKMSECVLGQ SKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGI VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKN LNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCC SCLKGCCSCGS CCKFDEDDSEPVLKGVKLHYTGSGATNFSLLKQAGDVEENPGPMADSNGTITVEELKKL LEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWI TGGIAIAMACLVGLMWLSYFIASFRLFARTRSMW SFNPETNILLNVPLHGTILTRPLLESE LVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYS RYRIGNYKLNTDHSSSSDNIALLVQGSGATNFSLLKQAGDVEENPGPMYSFVSEETGTLI VNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKP SFYVYSRVKNLNS SRVPDL LV (S-VLP) or variants thereof. 
     With specific regard to coronavirus proteins disclosed herein, any amino acid substitution is permissible so long as the activity of the protein is not significantly affected. In this regard, it is appreciated in the art that amino acids can be classified into groups based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants that contain substitutions are those in which an amino acid is substituted with an amino acid from the same group. Such substitutions are referred to as conservative substitutions. 
     In certain embodiments, this disclosure relates to virus-like particles comprising a coronavirus spike protein disclosed herein. 
     In certain embodiments, this disclosure relates nucleic acids comprising a sequence encoding a coronavirus spike protein disclosed herein in operable combination with a heterologous promotor. 
     In certain embodiments, the nucleic acid the sequence encoding a coronavirus spike protein comprises (SEQ ID NO: 4) 
     ATGTGGTTACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAATTTCACA ATGTGTAAATCTAACTACAAGAACTCAATTACCGCCTGCCTATACTAATTCTTTTACA AGAGGAGTATATTATCCTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAG ATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGA ACTAATGGAACAAAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTAT TTTGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTA GATTCTAAAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAA GTATGTGAATTTCAATTTTGTAATGATC CATTTTTGGGAGTTTATTATCATAAAAATA ATAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTACATT TGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAACTTTAA AAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATCTATTCTAA ACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACC ACTAGTAGATTTGC CTATAGGAATTAATATTACTAGATTTCAAACATTATTAGC TTTA CATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGATGGACTGCAGGAGCTGCA GCTTATTATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAATATAATGAAAAT GGAACTATAACAGATGCAGTTGATTGTGCTTTAGATCCTCTATCTGAAACTAAATGT ACTTTAAAATCTTTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTAGAGTA CAACCAACTGAATCTATTGTTAGATTTCCAAATATAACAAATCTATGTCCTTTTGGA GAAGTTTTTAATGCAACTAGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATA TCTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGCATCTTTTTCTACTTTTAA ATGTTATGGAGTATCTCCAACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCA GATTCTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGG AAAAATAGCAGATTATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGC TTGGAATTCTAATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAG ATTGTTTAGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTA TCAAGCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTAGTT GTATTATC TTTTGAATTATTACATGCACCAGCTACAGTATGTGGACC TAAAAAATCT ACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTAACTGGAACA GGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAATTTGGAAGAGAT ATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTGGATATT ACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCAGGAACTAATACATCTAAT CAAGTTGCTGTATTATATCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCATG CTGATCAATTAACTCCAACATGGAGAGTATATTCTACTGGATCTAATGTTTTTCAAA CAAGAGCTGGATGTCTAATTGGAGCAGAACATGTAAATAATTCTTATGAATGTGATA TTCCTATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAAATTCTCCAAGAA GAGCTAGATCTGTTGCATCTCAATCTATAATTGCTTATACAATGTCTTTAGGAGCTGA AAATTCTGTAGCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTACTATTTCT GTAACTACAGAAATATTGCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATG TATATATGTGGAGATTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTT GTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACA CAAGAAGTTTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTT GGAGGTTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTT TTATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAACA ATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGC TCAAAAGTT TAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCACAATATAC ATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGAGCTGGAGCAGC TTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAATGGAATTGGAGTTAC TCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAATCAATTCAATTCTGCAAT TGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCTGCTTTAGGAAAACTACAAGA TGTTGTAAATCAAAATGCACAAGCTTTAAATACTCTAGTTAAACAACTATCTTCTAA TTTTGGAGCTATTTCTTCTGTTTTAAATGATATATTGTCTAGACTAGATCCACCT (encoding spike sequence amino acids 1 to 987) or variants with great than 85% identity thereto. In certain embodiments, variants are synonymous or non-synonymous codons. 
     Recombinant Nucleic Acids and Viral Vectors 
     In certain embodiments, the disclosure relates to recombinant viral vectors, recombinant vectors, and recombinant plasmids comprising nucleic acids encoding coronavirus spike proteins disclosed herein. In certain embodiments, this disclosure relates to expression systems comprising nucleic acids and vectors disclosed herein. 
     Nucleic acids, vectors, and expression constructs can be introduced in vivo via lipofection (DNA transfection via liposomes prepared from synthetic cationic lipids). Synthetic cationic lipids can be used to prepare liposomes to encapsulate a nucleic acid, vector, or expression construct of the disclosure. A nucleic acid, vector, or expression construct can also be introduced as naked DNA or RNA using methods known in the art, such as transfection, microinjection, electroporation, calcium phosphate precipitation, and by biolistic methods. 
     If a recombinant virus vector of this disclosure is constructed starting with a vaccinia virus, the majority of the nucleic acid molecules and proteins in the recombinant virus vector will come from vaccinia virus and thus the final recombinant virus vector can be referred to, for example, as a recombinant vaccinia virus vector or a vaccinia-based recombinant virus vector. In certain embodiments, the recombinant virus vector is selected from the group consisting of a recombinant poxvirus vector, a recombinant vaccinia virus vector, a recombinant chordopoxvirus vector, a recombinant iridovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant SV40 virus vector, a recombinant Epstein-Barr virus vector, a recombinant herpes virus vector, and a recombinant JC virus vector. 
     In certain embodiments, this disclosure contemplates that methods disclosed herein are used with recombinant virus, preferably recombinant modified vaccinia virus Ankara (MVA). MVA is an attenuated strain of vaccinia virus originally developed as a vaccine for smallpox. The ability of MVA to infect mammalian, including human host cells, is restricted due to known deletions in the virus genome. In addition to the safe use in human vaccinations, Wyatt et al. report mice with severe combined immunodeficiency disease remained healthy when inoculated with MVA. Proc Natl Acad Sci USA. 2004, 101(13):4590-5. 
     MVA can be engineered in deleted regions to express heterologous genes to induce protective immunity to other viruses. Combined DNA and recombinant modified vaccinia Ankara (MVA62B) vaccines can produce virus-like particles that display membrane-bound trimeric forms of envelope proteins. As a result of extensive passage in cell culture, the MVA virus genome contains six major deletions, referred to as Del I, II, III, IV, V and VI. Historically, the region around Del II and Del III has been used for insertion of heterologous nucleic acid sequences. 
     As used herein, the term heterologous is a comparative term, and refers to a molecule that is from an organism different from that to which it is being referenced or that is made synthetically. The molecule can be a protein or a nucleic acid sequence (i.e., RNA or DNA). For example, a heterologous nucleic acid sequence in a recombinant virus vector refers to the fact that the heterologous nucleic acid sequence is or may be from an organism other than the base virus used to construct the recombinant virus vector. As a further example, a heterologous nucleic acid sequence in a recombinant vaccinia virus vector refers to the fact that the heterologous nucleic acid sequence is from an organism other than vaccinia virus or that was made synthetically. 
     A heterologous nucleic acid sequence can be inserted at any location in a recombinant virus vector genome, as long as such insertion does not unintentionally alter the functioning of the resulting recombinant virus vector. For example, a nucleic acid sequence can be inserted into a non-essential region. Such non-essential regions include, but are not limited to, naturally occurring deletions within the viral genome (e.g., Del I, II, III, etc. of modified vaccinia virus Ankara (MVA)), intergenic regions or non-essential genes. A non-essential region is a genomic region, the alteration of which has no, or almost no, discernible effect on viral replication and the production of progeny virus. One example of a non-essential region is a non-essential gene such as, for example, the vaccinia virus hemagglutinin gene. 
     Alternatively, a nucleic acid sequences can be inserted into an essential region of the genome (e.g., an essential gene). It will be appreciated that interruption of an essential region will result in a recombinant virus vector unable to complete the virus life cycle and produce progeny virus. However, such recombinant virus vectors can produce progeny virus when grown in cells that provide the missing function. Such a cell can be referred to as a complementing cell because it provides the function usually provided by the essential gene. That is, it “complements” the recombinant virus vector. Conversely, a cell that is unable to provide the missing viral function can be referred to as a non-commenting cell. Such culture systems are contemplated herein. At least one heterologous nucleic acid sequence may be inserted into the gene required for expression of post-replicative viral genes. 
     Methods of Use 
     This disclosure relates to methods of promoting immune responses against coronavirus, such as SARS-CoV-2, and compositions related thereto. In certain embodiments, this disclosure relates to methods of vaccinating for coronavirus, such as SARS-CoV-2, comprising administering to the subject a composition disclosed herein. In certain embodiments, the composition comprises a coronavirus spike protein, VLP containing the same, or a recombinant virus such as recombinant MVA that encodes a coronavirus, such as SARS-CoV-2 spike protein. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 986. In certain embodiments, the coronavirus spike protein comprises a proline mutation at position 987. 
     In certain embodiments, this disclosure relates to methods of vaccinating or immunizing comprising administering to a human subject an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein under conditions such that spike protein and/or virus-like particles with spike protein are formed in the subject. 
     In certain embodiments, the methods are conducted in combination with an adjuvant. In certain embodiments, methods include using a coronavirus spike protein, trimeric complex or virus-like particle or nucleic acid encoding the same in combination with an adjuvant. 
     In certain embodiments, administering is to the skin, muscle, or buccal cavity. In certain embodiments, administration is by syringe, microneedle, topically, or using pressurized devices, e.g., device comprising a nozzle to push a solution into tissue by means of pressure, e.g., spring-powered without the use of a needle (needle-free devices). 
     DNA-based vaccines typically use bacterial plasmids to express protein immunogens in vaccinated hosts. Recombinant DNA technology is used to clone cDNAs encoding immunogens of interest into eukaryotic expression plasmids. Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated. DNA typically is inoculated by a needle injection of DNA in saline, or by a gene gun device that delivers DNA-coated gold beads into skin. The plasmid DNA is taken up by host cells, the vaccine protein is expressed, processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules, and an immune response against the DNA-encoded immunogen is generated. 
     In certain embodiments the present disclosure is a method to generate an immune response against coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein. Such a response can be a CD8+T cell immune response or an antibody response. More particularly, the present disclosure relates to “prime and boost” immunization regimes in which the immune response induced by administration of a priming composition is boosted by administration of a boosting composition. The present disclosure is based on experimental demonstration that effective priming can be achieved using modified vaccinia Ankara (MVA) vectors, following boosting with coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein. 
     A major protective component of the immune response against a number of pathogens is mediated by T lymphocytes of the CD8+type, also known as cytotoxic T lymphocytes (CTL). An important function of CD8+cells is secretion of gamma interferon (IFNγ), and this provides a measure of CD8+T cell immune response. A second component of the immune response is antibody directed to the proteins of the pathogen. 
     It is contemplated that a vaccination regime using needle-free, intradermal, intramuscular, or mucosal immunization for both prime and boost can be employed, constituting a general immunization regime suitable for inducing CD8+T cells and also eliciting an antibody response, e.g., in humans. An immune response to coronavirus spike protein, trimeric complex or virus-like particle thereof may be primed by immunization with plasmid DNA, recombinant virus, or by infection with an infectious agent. 
     A further aspect of this disclosure provides a method of inducing a CD8+T cell immune response to a coronavirus spike protein, trimeric complex or virus-like particle thereof in an individual, and also eliciting an antibody response. 
     A further aspect provides for use of coronavirus spike protein, trimeric complex or virus-like particle thereof as disclosed herein, in the manufacture of a medicament for administration to a mammal to boost a CD8+T cell immune response and also eliciting an antibody response. Such a medicament is generally for administration following prior administration of a priming composition comprising nucleic acid and/or recombinant virus encoding the antigen. 
     The priming composition may comprise DNA encoding a coronavirus spike protein, trimeric complex or virus-like particle thereof, such DNA being in the form of a circular plasmid that is not capable of replicating in mammalian cells. Any selectable marker should preferably not be resistance to an antibiotic used clinically, so for example Kanamycin resistance is preferred to Ampicillin resistance. Antigen expression should be driven by a promoter which is active in mammalian cells, for instance the cytomegalovirus immediate early (CMV IE) promoter. 
     In particular embodiments of the various aspects of the present disclosure, administration of a priming composition is followed by boosting with a boosting composition, or first and second boosting compositions, the first and second boosting compositions being the same or different from one another. 
     In certain embodiments, the subject is a human subject. In certain embodiments, the human subject is of advanced age or elderly e.g., more than 45, 55, or 65 years old. 
     In certain embodiments, an “effective amount” in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a viral infection, disease or symptom associated therewith; (ii) reduce the duration of a viral infection, disease or symptom associated therewith; (iii) prevent the progression of a viral infection, disease or symptom associated therewith; (iv) cause regression of a viral infection, disease or symptom associated therewith; (v) prevent the development or onset of a viral infection, disease or symptom associated therewith; (vi) prevent the recurrence of a viral infection, disease or symptom associated therewith; (vii) reduce or prevent the spread of a viral from one cell to another cell, one tissue to another tissue, or one organ to another organ; (viii) prevent or reduce the spread of a viral from one subject to another subject; (ix) reduce organ failure associated with a viral infection; (x) reduce hospitalization of a subject; (xi) reduce hospitalization length; (xii) increase the survival of a subject with a viral infection or disease associated therewith; (xiii) eliminate a viral infection or disease associated therewith; (xiv) inhibit or reduce viral replication; (xv) inhibit or reduce the entry of an virus into a host cell(s); (xvi) inhibit or reduce replication of the virus genome; (xvii) inhibit or reduce synthesis of virus proteins; (xviii) inhibit or reduce assembly of virus particles; (xix) inhibit or reduce release of virus particles from a host cell(s); (xx) reduce virus titer; and/or (xxi) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. 
     In certain embodiments, the effective amount does not result in complete protection from a coronavirus infection but results in a lower titer or reduced number of viruses compared to an untreated subject with a viral infection. In certain embodiments, the effective amount results in a 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reduction in titer of virus relative to an untreated subject with a viral infection. Benefits of a reduction in the titer, number or total burden of virus include, but are not limited to, less severe symptoms of the infection, fewer symptoms of the infection and a reduction in the length of the disease associated with the infection. 
     Compositions described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, conjunctival and subcutaneous routes. In some embodiments, a composition is formulated for topical administration, for example, for application to the skin. In specific embodiments, the route of administration is nasal, e.g., as part of a nasal spray. In certain embodiments, a composition is formulated for intramuscular administration. In some embodiments, a composition is formulated for subcutaneous administration. In certain embodiments, a composition is not formulated for administration by injection. 
     In certain embodiments, immunogenic compositions disclosed herein are administered intradermally. In certain embodiments, this disclosure contemplates administration using a transdermal patch for diffusion of the drug across the skin or by microneedle injection. In certain embodiments, it may be desirable to introduce the pharmaceutical compositions into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. 
     In certain embodiments, this disclosure contemplates a combination vaccine that is designed to induce a strong neutralizing antibody response and broad cytotoxic CD8 T cell response against the SARS-CoV-2 providing long-lasting protection against SARS-CoV-2 and other SARS corona viruses. To achieve one combine DNA and modified vaccinia Ankara (MVA) vaccines such that both neutralizing antibodies and CD8 T cells are induce. The DNA and MVA immunogens express nucleocapsid, membrane and envelope proteins and a string of conserved epitopes from other proteins of SARS-CoV-2. In certain embodiments, priming with a DNA or MVA construct disclosed herein plus chimeric construction disclosed herein and boosting with DNA or MVA constructs disclosed herein. The DNA or MVA constructs for the priming and boosting may be the same or different. T cell epitopes in DNA and MVA vaccines promotes T cells against SARS corona viruses that could potentially provide protection even when the virus escapes from antibody responses providing induction of high levels of neutralizing antibodies and CD8 T cells with fewer immunizations. 
     In certain embodiments, this disclosure relates to vaccination methods using nucleic acids encoding T cell stimulating chimeric proteins. In certain embodiments, this disclosure relates to methods of vaccinating or immunizing a human subject comprising administering an effective amount of coronavirus spike protein, a virus-like particle comprising a coronavirus spike protein, a nucleic acid and/or recombinant virus that encodes a coronavirus spike protein or segment thereof as disclosed herein in combination with a nucleic acid encoding a T cell stimulating chimeric protein under conditions such that a spike protein and/or virus-like particles with a spike protein are formed in the subject. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26) NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26), NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses; and the boost is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein. In certain embodiments the prime is a DNA coronavirus construct, and the boost is an MVA coronavirus construct. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M_dTM (SEQ ID NO: 25) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding NSP3-4-6 (SEQ ID NO: 26) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses, and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA coronavirus construct disclosed herein in combination with chimeric sequences such as a nucleic acid encoding NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses; and the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein and the boost is a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein. 
     In certain embodiments, this disclosure relates to a vaccination method comprising administering a prime and boost, wherein the prime is any coronavirus vaccine, e.g., mRNA vaccines viral vector coronavirus vaccines conventionally known by their supplier/brand name such as the Pfizer-BioNTech COVID-19 vaccine, Moderna COVID-19 vaccine, Johnson &amp; Johnson&#39;s Janssen COVID-19 vaccine, AstraZeneca COVID-19 vaccine, and Novavax COVID-19 vaccine and the boost is a corona virus vaccine such as a DNA or RNA vaccine encoding a corona virus spike protein of fragment thereof and preferably a DNA or MVA or combination of a DNA and MVA coronavirus construct disclosed herein. 
     In certain embodiments, the boost is in combination with chimeric sequences such as a nucleic acid encoding SdRBD-N-M (SEQ ID NO: 24), N-M, SdRBD-N-M_dTM (SEQ ID NO: 25), N-M_dTM, NSP3-4-6 (SEQ ID NO: 26), NSP3-4-6_dTM (SEQ ID NO: 27) or variants or combinations thereof (wherein d/delta is deleted) for the purpose of stimulating T cell responses. 
     In certain embodiments, the boost is vaccine comprises mutation E484K, K417N, N501Y, or combinations thereof. In certain embodiments, the boost is MVA/S-tri-dFCS or variants optionally comprising mutations E484K, K417N, N501Y, or combinations thereof. 
     In certain embodiments, the boost is administered more than one or two weeks after the prime. In certain embodiments, the boost is administered more than one or two months after the prime. In certain embodiments, the boost is administered more than six months after the prime. In certain embodiments, the boost is administered more than one year after the prime. 
     Spike Protein of SARS-CoV-2 
     Four forms ( FIG.  1 A-D ) of SARS-CoV-2 spike protein are disclosed, i.e., full-length protein displayed on VLPs like in the virus (S-VLP), soluble monomeric S1 (S1-Mono), trimeric S protein displayed on the membrane but does not produce VLPs (S-Tri) and stabilized soluble S Trimer (S-Tri-sec). See  FIGS.  1 A-D . 
     Construction and Characterization of rMVAs: 
     Full-length consensus spike protein sequences of SARS-CoV-2 was modified recombinant methods. DNA sequences encoding the proteins were codon-optimized for vaccinia virus codon usage, synthesized, and subcloned in between Xma1 and BamH1 restriction sites of the plasmid transfer vector pLW-73 (see Patent EP2402451). Inserts are transfer in between two essential genes I8R and G1L of MVA, under the control of an independent early/late vaccinia virus promoter (modified H5 [mH5]) to generate stable MVAs. Recombinant MVAs are characterized for protein expression using Western blotting and flow cytometry, grown in large-scale in chicken embryo fibroblasts, purified, quality tested, and titrated. Expression data for two of the MVA recombinants MVA/S-Tri and MVA/S-VLP are shown in  FIG.  2 A and  2 B . 
     MVA Construct 1: MVA/S-VLP 
       
                                     Name   Range   Description                  Flank1 I8R    1-537   Essential gene region on                MVA for recombination       P11   545-573   promoter       GFP    574-1293   GFP       DR   1294-1528    Direct Repeats       mH5   1553-1619    Promoter       Spike   1634-5452    Spike protein sequence       Aminoacid changed   4589-4591    K986P 4589AAA4591                changed to 4589CCA4591       Aminoacid changed   4592-4594    V987P 4591GTT4594                changed to 4591CCT4594       P2A   5453-5518    Porcine 2A sequence       Membrane   5519-6184    Membrane protein sequence       P2A   6185-6250    Porcine 2A sequence       Envelope   6251-6475    Envelope protein sequence       Flank 2 G1L   6513-7214    Essential gene region on                MVA for recombination       Ampicillin resistance    8500-9290    Confers resistance to Ampicillin       gene                    
Plasmid Sequence (SEQ ID NO: 5) and Sequence encoding spike protein fusion (bold, SEQ ID NO: 6)
 
     
       
         
           
               
               
            
               
                 GAATTCCCTGGGACATACGTATATTTCTATGATCTGTCTTATATGAAGTCTATACAGC 
                   
               
               
                   
               
               
                 GAATAGATTCAGAATTTCTACATAATTATATATTGTACGCTAATAAGTTTAATCTAA 
               
               
                   
               
               
                 CACTCCCCGAAGATTTGTTTATAATCCCTACAAATTTGGATATTCTATGGCGTACAA 
               
               
                   
               
               
                 AGGAATATATAGACTCGTTCGATATTAGTACAGAAACATGGAATAAATTATTATCCA 
               
               
                   
               
               
                 ATTATTATATGAAGATGATAGAGTATGCTAAACTTTATGTACTAAGTCCTATTCTCGC 
               
               
                   
               
               
                 TGAGGAGTTGGATAATTTTGAGAGGACGGGAGAATTAACTAGTATTGTACAAGAAG 
               
               
                   
               
               
                 CCATTTTATCTCTAAATTTACGAATTAAGATTTTAAATTTTAAACATAAAGATGATGA 
               
               
                   
               
               
                 TACGTATATACACTTTTGTAAAATATTATTCGGTGTCTATAACGGAACAAACGCTAC 
               
               
                   
               
               
                 TATATATTATCATAGACCTCTAACGGGATATATGAATATGATTTCAGATACTATATTT 
               
               
                   
               
               
                 GTTCCTGTAGATAATAACTAAGGCGCGCCTTTCATTTTGTTTTTTTCTATGCTATAAA 
               
               
                   
               
               
                 TGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG 
               
               
                   
               
               
                 GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC 
               
               
                   
               
               
                 CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC 
               
               
                   
               
               
                 CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC 
               
               
                   
               
               
                 CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC 
               
               
                   
               
               
                 AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG 
               
               
                   
               
               
                 AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA 
               
               
                   
               
               
                 GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC 
               
               
                   
               
               
                 GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG 
               
               
                   
               
               
                 CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC 
               
               
                   
               
               
                 CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCG 
               
               
                   
               
               
                 CCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG 
               
               
                   
               
               
                 ACCGCCGCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGAGCTCGAGGA 
               
               
                   
               
               
                 CGGGAGAATTAACTAGTATTGTACAAGAAGCCATTTTATCTCTAAATTTACGAATTA 
               
               
                   
               
               
                 AGATTTTAAATTTTAAACATAAAGATGATGATACGTATATACACTTTTGTAAAATAT 
               
               
                   
               
               
                 TATTCGGTGTCTATAACGGAACAAACGCTACTATATATTATCATAGACCTCTAACGG 
               
               
                   
               
               
                 GATATATGAATATGATTTCAGATACTATATTTGTTCCTGTAGATAATAACTAACTCG 
               
               
                   
               
               
                 AGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTT 
               
               
                   
               
               
                 GTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCGGGACC ATGTTTGTTTT   
               
               
                   
               
               
                 
                   TCTAGTTTTGCTACCGTTGGTTTCAAGTCAATGTGTAAATCTAACTACAAGAAC 
                 
               
               
                   
               
               
                 
                   TCAATTACCGCCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATCCTGA 
                 
               
               
                   
               
               
                 
                   TAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAGATTTGTTTTTACCATTT 
                 
               
               
                   
               
               
                 
                   TTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGAACTAATGGAACA 
                 
               
               
                   
               
               
                 
                   AAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTTTGCTTCTA 
                 
               
               
                   
               
               
                 
                   CTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTAGATTCTA 
                 
               
               
                   
               
               
                 
                   AAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAAGTAT 
                 
               
               
                   
               
               
                 
                   GTGAATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTATCATAAAAATAA 
                 
               
               
                   
               
               
                 
                   TAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTAC 
                 
               
               
                   
               
               
                 
                   ATTTGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAA 
                 
               
               
                   
               
               
                 
                   CTTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATC 
                 
               
               
                   
               
               
                 
                   TATTCTAAACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCT 
                 
               
               
                   
               
               
                 
                   GCTTTAGAACCACTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTCAA 
                 
               
               
                   
               
               
                 
                   ACATTATTAGCTTTACATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGAT 
                 
               
               
                   
               
               
                 
                   GGACTGCAGGAGCTGCAGCTTATTATGTTGGATATTTGCAACCAAGAACATTTT 
                 
               
               
                   
               
               
                 
                   TGTTAAAATATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGCTTTAG 
                 
               
               
                   
               
               
                 
                   ATCCTCTATCTGAAACTAAATGTACTTTAAAATCTTTTACTGTAGAAAAAGGAA 
                 
               
               
                   
               
               
                 
                   TCTATCAAACATCTAACTTTAGAGTACAACCAACTGAATCTATTGTTAGATTTCC 
                 
               
               
                   
               
               
                 
                   AAATATAACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACTAGATTTGC 
                 
               
               
                   
               
               
                 
                   TTCTGTATATGCATGGAATAGAAAAAGAATATCTAATTGCGTAGCTGATTATTC 
                 
               
               
                   
               
               
                 
                   TGTATTATATAATTCTGCATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCA 
                 
               
               
                   
               
               
                 
                   ACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATTCTTTTGTAATAA 
                 
               
               
                   
               
               
                 
                   GAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGGAAAAATAGCAGAT 
                 
               
               
                   
               
               
                 
                   TATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCT 
                 
               
               
                   
               
               
                 
                   AATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAGATTGTTT 
                 
               
               
                   
               
               
                 
                   AGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTATCAA 
                 
               
               
                   
               
               
                 
                   GCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA 
                 
               
               
                   
               
               
                 
                   CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTA 
                 
               
               
                   
               
               
                 
                   GTTGTATTATCTTTTGAATTATTACATGCACCAGCTACAGTATGTGGACCTAAA 
                 
               
               
                   
               
               
                 
                   AAATCTACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTA 
                 
               
               
                   
               
               
                 
                   ACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAA 
                 
               
               
                   
               
               
                 
                   TTTGGAAGAGATATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTA 
                 
               
               
                   
               
               
                 
                   GAAATATTGGATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCA 
                 
               
               
                   
               
               
                 
                   GGAACTAATACATCTAATCAAGTTGCTGTATTATATCAAGATGTTAATTGTACT 
                 
               
               
                   
               
               
                 
                   GAAGTTCCTGTAGCAATTCATGCTGATCAATTAACTCCAACATGGAGAGTATAT 
                 
               
               
                   
               
               
                 
                   TCTACTGGATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGGAGCAGAA 
                 
               
               
                   
               
               
                 
                   CATGTAAATAATTCTTATGAATGTGATATTCCTATAGGAGCTGGAATATGTGCA 
                 
               
               
                   
               
               
                 
                   TCTTATCAAACTCAAACAAATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAA 
                 
               
               
                   
               
               
                 
                   TCTATAATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTAGCATATTCTA 
                 
               
               
                   
               
               
                 
                   ATAATTCTATTGCAATTCCTACTAACTTTACTATTTCTGTAACTACAGAAATATT 
                 
               
               
                   
               
               
                 
                   GCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATGTATATATGTGGAGA 
                 
               
               
                   
               
               
                 
                   TTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTTGTACTCAATTG 
                 
               
               
                   
               
               
                 
                   AATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACACAAGAAGT 
                 
               
               
                   
               
               
                 
                   TTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAGG 
                 
               
               
                   
               
               
                 
                   TTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTTTT 
                 
               
               
                   
               
               
                 
                   ATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAA 
                 
               
               
                   
               
               
                 
                   CAATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGCTCAA 
                 
               
               
                   
               
               
                 
                   AAGTTTAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCA 
                 
               
               
                   
               
               
                 
                   CAATATACATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGA 
                 
               
               
                   
               
               
                 
                   GCTGGAGCAGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAAT 
                 
               
               
                   
               
               
                 
                   GGAATTGGAGTTACTCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAAT 
                 
               
               
                   
               
               
                 
                   CAATTCAATTCTGCAATTGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCT 
                 
               
               
                   
               
               
                 
                   GCTTTAGGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTTTAAATACT 
                 
               
               
                   
               
               
                 
                   CTAGTTAAACAACTATCTTCTAATTTTGGAGCTATTTCTTCTGTTTTAAATGATA 
                 
               
               
                   
               
               
                 
                   TATTGTCTAGACTAGATCCACCTGAAGCAGAAGTACAAATTGATAGACTAATTA 
                 
               
               
                   
               
               
                 
                   CAGGAAGATTACAATCTCTACAAACTTATGTAACACAACAACTAATTAGAGCAG 
                 
               
               
                   
               
               
                 
                   CTGAAATAAGAGCATCTGCTAATTTGGCAGCTACTAAAATGTCTGAATGCGTAT 
                 
               
               
                   
               
               
                 
                   TAGGACAATCTAAAAGAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTT 
                 
               
               
                   
               
               
                 
                   TTCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTTACATATGTACCTG 
                 
               
               
                   
               
               
                 
                   CACAAGAAAAGAACTTTACTACAGCACCAGCTATATGTCATGATGGAAAAGCTC 
                 
               
               
                   
               
               
                 
                   ATTTTCCTAGAGAAGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACAC 
                 
               
               
                   
               
               
                 
                   AAAGAAACTTTTATGAACCACAAATTATAACTACAGATAATACATTTGTATCTG 
                 
               
               
                   
               
               
                 
                   GAAATTGTGATGTTGTAATTGGAATTGTTAATAATACTGTATATGATCCACTAC 
                 
               
               
                   
               
               
                 
                   AACCTGAACTAGATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATCATA 
                 
               
               
                   
               
               
                 
                   CTTCTCCTGATGTTGATTTGGGAGATATATCTGGAATTAATGCTTCTGTTGTAA 
                 
               
               
                   
               
               
                 
                   ATATTCAAAAAGAAATAGATAGATTGAATGAAGTAGCAAAAAATTTGAATGAAT 
                 
               
               
                   
               
               
                 
                   CTCTAATTGATTTGCAAGAATTAGGAAAATATGAACAATATATCAAATGGCCAT 
                 
               
               
                   
               
               
                 GGTATATTTGGCTAGGTTTTATAGCTGGATTAATAGCAATTGTTATGGTAACTA 
               
               
                   
               
               
                 
                   TTATGTTATGTTGTATGACATCTTGTTGTTCTTGTCTAAAAGGATGTTGTTCTTG 
                 
               
               
                   
               
               
                 
                   TGGATCTTGTTGTAAATTTGATGAAGATGATTCTGAACCTGTTTTGAAAGGTGT 
                 
               
               
                   
               
               
                 
                   TAAACTACATTATACTGGATCTGGAGCAACTAATTTTTCTTTGTTAAAACAAGC 
                 
               
               
                   
               
               
                 
                   TGGAGATGTAGAAGAAAATCCAGGACCTATGGCTGATTCTAATGGAACTATAA 
                 
               
               
                   
               
               
                 
                   CAGTTGAAGAATTGAAAAAACTATTAGAACAATGGAATTTGGTAATAGGATTTT 
                 
               
               
                   
               
               
                 
                   TGTTTTTAACATGGATTTGTTTATTACAATTTGCATATGCTAATAGAAATAGATT 
                 
               
               
                   
               
               
                 
                   TTTGTATATCATAAAACTAATATTTTTGTGGTTATTATGGCCAGTTACTTTAGCA 
                 
               
               
                   
               
               
                 
                   TGTTTTGTTTTAGCAGCTGTATATAGAATTAATTGGATTACAGGAGGAATTGCA 
                 
               
               
                   
               
               
                 
                   ATAGCTATGGCATGTCTAGTAGGATTAATGTGGCTATCTTACTTTATAGCATCT 
                 
               
               
                   
               
               
                 
                   TTTAGACTATTTGCTAGAACTAGATCTATGTGGTCTTTTAATCCTGAAACAAAT 
                 
               
               
                   
               
               
                 
                   ATATTGTTAAATGTACCATTACATGGAACTATATTGACAAGACCTCTACTAGAA 
                 
               
               
                   
               
               
                 
                   TCTGAATTAGTTATTGGAGCAGTAATATTAAGAGGACATTTGAGAATTGCTGGA 
                 
               
               
                   
               
               
                 
                   CATCATTTGGGAAGATGTGATATCAAAGATTTGCCTAAAGAAATTACTGTTGCT 
                 
               
               
                   
               
               
                 
                   ACATCTAGAACTTTATCTTATTATAAACTAGGAGCATCTCAAAGAGTAGCTGGA 
                 
               
               
                   
               
               
                 
                   GATTCTGGATTTGCAGCTTATTCTAGATATAGAATTGGAAATTATAAATTGAAT 
                 
               
               
                   
               
               
                 
                   ACTGATCATTCTTCTTCTTCTGATAATATTGCATTATTAGTACAAGGATCTGGA 
                 
               
               
                   
               
               
                 
                   GCTACAAATTTTTCTTTGTTAAAACAGGCAGGAGATGTTGAAGAAAATCCAGGA 
                 
               
               
                   
               
               
                 
                   CCAATGTATTCTTTTGTATCTGAAGAAACTGGAACATTAATTGTTAATTCTGTAT 
                 
               
               
                   
               
               
                 
                   TATTGTTTTTAGCTTTTGTAGTATTTTTGCTAGTTACATTAGCAATATTGACTGC 
                 
               
               
                   
               
               
                 
                   TTTAAGATTATGTGCATATTGTTGTAATATTGTTAATGTATCTTTAGTAAAACCA 
                 
               
               
                   
               
               
                 
                   TCTTTTTATGTATATTCAAGAGTTAAAAATCTAAATTCATCAAGAGTTCCTGATC 
                 
               
               
                   
               
               
                   TATTGGTATAATAA TTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAAACTCTAAT 
               
               
                   
               
               
                 GACCACATCTTTTTTTAGAGATGAAAAATTTTCCACATCTCCTTTTGTAGACACGACT 
               
               
                   
               
               
                 AAACATTTTGCAGAAAAAAGTTTATTAGTGTTTAGATAATCGTATACTTCATCAGTG 
               
               
                   
               
               
                 TAGATAGTAAATGTGAACAGATAAAAGGTATTCTTGCTCAATAGATTGGTAAATTCC 
               
               
                   
               
               
                 ATAGAATATATTAATCCTTTCTTCTTGAGATCCCACATCATTTCAACCAGAGACGTTT 
               
               
                   
               
               
                 TATCCAATGATTTACCTCGTACTATACCACATACAAAACTAGATTTTGCAGTGACGT 
               
               
                   
               
               
                 CGTATCTGGTATTCCTACCAAACAAAATTTTACTTTTAGTTCTTTTAGAAAATTCTAA 
               
               
                   
               
               
                 GGTAGAATCTCTATTTGCCAATATGTCATCTATGGAATTACCACTAGCAAAAAATGA 
               
               
                   
               
               
                 TAGAAATATATATTGATACATCGCAGCTGGTTTTGATCTACTATACTTTAAAAACGA 
               
               
                   
               
               
                 ATCAGATTCCATAATTGCCTGTATATCATCAGCTGAAAAACTATGTTTTACACGTATT 
               
               
                   
               
               
                 CCTTCGGCATTTCTTTTTAATGATATATCTTGTTTAGACAATGATAAAGTTATCATGT 
               
               
                   
               
               
                 CCATGAGAGACGCGTCTCCGTATCGTATAAATATTTCATTAGATGTTAGACGCTTCA 
               
               
                   
               
               
                 TTAGGGGTATACTTCTATAAGGTTTCTTAATCAGTCCATCATTGGTTGCGTCAAGAAC 
               
               
                   
               
               
                 AAGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATAGCT 
               
               
                   
               
               
                 GTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAG 
               
               
                   
               
               
                 CATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT 
               
               
                   
               
               
                 GCGCTCACTGCCCGCTTTCGAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAAT 
               
               
                   
               
               
                 CGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCT 
               
               
                   
               
               
                 CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA 
               
               
                   
               
               
                 GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG 
               
               
                   
               
               
                 CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC 
               
               
                   
               
               
                 GATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG 
               
               
                   
               
               
                 GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG 
               
               
                   
               
               
                 TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC 
               
               
                   
               
               
                 GGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT 
               
               
                   
               
               
                 CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC 
               
               
                   
               
               
                 CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT 
               
               
                   
               
               
                 GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG 
               
               
                   
               
               
                 AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT 
               
               
                   
               
               
                 GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA 
               
               
                   
               
               
                 AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA 
               
               
                   
               
               
                 GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT 
               
               
                   
               
               
                 GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC 
               
               
                   
               
               
                 ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG 
               
               
                   
               
               
                 TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC 
               
               
                   
               
               
                 TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC 
               
               
                   
               
               
                 GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA 
               
               
                   
               
               
                 CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG 
               
               
                   
               
               
                 TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA 
               
               
                   
               
               
                 GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCTACAGGCATC 
               
               
                   
               
               
                 GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA 
               
               
                   
               
               
                 GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTC 
               
               
                   
               
               
                 CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCAC 
               
               
                   
               
               
                 TGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTA 
               
               
                   
               
               
                 CTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC 
               
               
                   
               
               
                 GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGG 
               
               
                   
               
               
                 AAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC 
               
               
                   
               
               
                 GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT 
               
               
                   
               
               
                 TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA 
               
               
                   
               
               
                 CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCA 
               
               
                   
               
               
                 GGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT 
               
               
                   
               
               
                 AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTAT 
               
               
                   
               
               
                 TATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCG 
               
               
                   
               
               
                 TTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAG 
               
               
                   
               
               
                 CTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGT 
               
               
                   
               
               
                 GTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAG 
               
               
                   
               
               
                 AGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCA 
               
               
                   
               
               
                 TCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGG 
               
               
                   
               
               
                 GCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGT 
               
               
                   
               
               
                 TGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTG 
               
               
                   
               
               
                 GATTTAGGTGACACTATA 
               
            
           
         
       
     
     MVA Construct 2: MVA/S-Tri 
       
     
       
         
           
               
               
               
             
               
                   
               
               
                 Name 
                 Range 
                 Description 
               
               
                   
               
             
            
               
                 Flank1 I8R 
                  1-537 
                 Essential gene region on  
               
               
                   
                   
                 MVA for recombination 
               
               
                 P11 
                 545-573 
                 promoter 
               
               
                 GFP 
                  574-1293 
                 GFP 
               
               
                 DR 
                 1294-1528 
                 Direct Repeats 
               
               
                 mHS 
                 1553-1619 
                 Promoter 
               
               
                 Spike 
                 1634-5452 
                 Spike Protein sequence 
               
               
                 Aminoacid changed 
                 4589-4591 
                 K986P 4589AAA4591  
               
               
                   
                   
                 changed to 4589CCA4591 
               
               
                 Aminoacid changed 
                 4592-4594 
                 V987P 4591GTT4594  
               
               
                   
                   
                 changed to 4591CCT4594 
               
               
                 Flank 2 G1L 
                 5490-6191 
                 Essential gene region on  
               
               
                   
                   
                 MVA for recombination 
               
               
                 Ampicillin resistance  
                 7477-8267 
                 Confers resistance to Ampicillin 
               
               
                 gene 
               
               
                   
               
            
           
         
       
     
     Plasmid Sequence (SEQ ID NO: 7) and Sequence encoding spike protein (bold, SEQ ID NO: 8) 
     
       
         
           
               
               
            
               
                 GAATTCCCTGGGACATACGTATATTTCTATGATCTGTCTTATATGAAGTCTATACAGC 
                   
               
               
                   
               
               
                 GAATAGATTCAGAATTTCTACATAATTATATATTGTACGCTAATAAGTTTAATCTAA 
               
               
                   
               
               
                 CACTCCCCGAAGATTTGTTTATAATCCCTACAAATTTGGATATTCTATGGCGTACAA 
               
               
                   
               
               
                 AGGAATATATAGACTCGTTCGATATTAGTACAGAAACATGGAATAAATTATTATCCA 
               
               
                   
               
               
                 ATTATTATATGAAGATGATAGAGTATGCTAAACTTTATGTACTAAGTCCTATTCTCGC 
               
               
                   
               
               
                 TGAGGAGTTGGATAATTTTGAGAGGACGGGAGAATTAACTAGTATTGTACAAGAAG 
               
               
                   
               
               
                 CCATTTTATCTCTAAATTTACGAATTAAGATTTTAAATTTTAAACATAAAGATGATGA 
               
               
                   
               
               
                 TACGTATATACACTTTTGTAAAATATTATTCGGTGTCTATAACGGAACAAACGCTAC 
               
               
                   
               
               
                 TATATATTATCATAGACCTCTAACGGGATATATGAATATGATTTCAGATACTATATTT 
               
               
                   
               
               
                 GTTCCTGTAGATAATAACTAAGGCGCGCCTTTCATTTTGTTTTTTTCTATGCTATAAA 
               
               
                   
               
               
                 TGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG 
               
               
                   
               
               
                 GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC 
               
               
                   
               
               
                 CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC 
               
               
                   
               
               
                 CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC 
               
               
                   
               
               
                 CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC 
               
               
                   
               
               
                 AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG 
               
               
                   
               
               
                 AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA 
               
               
                   
               
               
                 GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC 
               
               
                   
               
               
                 GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG 
               
               
                   
               
               
                 CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC 
               
               
                   
               
               
                 CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCG 
               
               
                   
               
               
                 CCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG 
               
               
                   
               
               
                 ACCGCCGCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGAGCTCGAGGA 
               
               
                   
               
               
                 CGGGAGAATTAACTAGTATTGTACAAGAAGCCATTTTATCTCTAAATTTACGAATTA 
               
               
                   
               
               
                 AGATTTTAAATTTTAAACATAAAGATGATGATACGTATATACACTTTTGTAAAATAT 
               
               
                   
               
               
                 TATTCGGTGTCTATAACGGAACAAACGCTACTATATATTATCATAGACCTCTAACGG 
               
               
                   
               
               
                 GATATATGAATATGATTTCAGATACTATATTTGTTCCTGTAGATAATAACTAACTCG 
               
               
                   
               
               
                 AGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTT 
               
               
                   
               
               
                 GTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCGGGACC ATGTTTGTTTT   
               
               
                   
               
               
                 
                   TCTAGTTTTGCTACCGTTGGTTTCAAGTCAATGTGTAAATCTAACTACAAGAAC 
                 
               
               
                   
               
               
                 
                   TCAATTACCGCCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATCCTGA 
                 
               
               
                   
               
               
                 
                   TAAAGTTTTTAGATCTTCTGTATTACATTCTACACAAGATTTGTTTTTACCATTT 
                 
               
               
                   
               
               
                 
                   TTCTCTAATGTTACTTGGTTTCATGCAATACATGTATCTGGAACTAATGGAACA 
                 
               
               
                   
               
               
                 
                   AAAAGATTTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTTTGCTTCTA 
                 
               
               
                   
               
               
                 
                   CTGAAAAATCTAATATAATTAGAGGATGGATATTTGGAACTACATTAGATTCTA 
                 
               
               
                   
               
               
                 
                   AAACACAATCTCTACTAATTGTTAATAATGCAACTAATGTAGTTATAAAAGTAT 
                 
               
               
                   
               
               
                 
                   GTGAATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTATCATAAAAATAA 
                 
               
               
                   
               
               
                 
                   TAAGTCTTGGATGGAATCTGAATTCAGAGTATATTCTTCTGCTAATAATTGTAC 
                 
               
               
                   
               
               
                 
                   ATTTGAATATGTATCTCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAA 
                 
               
               
                   
               
               
                 
                   CTTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATGGATACTTTAAAATC 
                 
               
               
                   
               
               
                 
                   TATTCTAAACATACTCCAATTAATCTAGTAAGAGATTTGCCTCAAGGATTTTCT 
                 
               
               
                   
               
               
                 
                   GCTTTAGAACCACTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTCAA 
                 
               
               
                   
               
               
                 
                   ACATTATTAGCTTTACATAGATCTTATTTGACACCTGGAGATTCTTCTTCTGGAT 
                 
               
               
                   
               
               
                 
                   GGACTGCAGGAGCTGCAGCTTATTATGTTGGATATTTGCAACCAAGAACATTTT 
                 
               
               
                   
               
               
                 
                   TGTTAAAATATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGCTTTAG 
                 
               
               
                   
               
               
                 
                   ATCCTCTATCTGAAACTAAATGTACTTTAAAATCTTTTACTGTAGAAAAAGGAA 
                 
               
               
                   
               
               
                 
                   TCTATCAAACATCTAACTTTAGAGTACAACCAACTGAATCTATTGTTAGATTTCC 
                 
               
               
                   
               
               
                 
                   AAATATAACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACTAGATTTGC 
                 
               
               
                   
               
               
                 
                   TTCTGTATATGCATGGAATAGAAAAAGAATATCTAATTGCGTAGCTGATTATTC 
                 
               
               
                   
               
               
                 
                   TGTATTATATAATTCTGCATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCA 
                 
               
               
                   
               
               
                 
                   ACAAAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATTCTTTTGTAATAA 
                 
               
               
                   
               
               
                 
                   GAGGAGATGAAGTTAGACAAATAGCTCCTGGACAAACAGGAAAAATAGCAGAT 
                 
               
               
                   
               
               
                 
                   TATAATTATAAATTACCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCT 
                 
               
               
                   
               
               
                 
                   AATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTTGTATAGATTGTTT 
                 
               
               
                   
               
               
                 
                   AGAAAATCTAATTTGAAACCTTTTGAAAGAGATATTTCTACAGAAATCTATCAA 
                 
               
               
                   
               
               
                 
                   GCAGGATCTACTCCATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACTA 
                 
               
               
                   
               
               
                 
                   CAATCTTATGGATTTCAACCTACAAATGGAGTAGGATATCAACCATATAGAGTA 
                 
               
               
                   
               
               
                 
                   GTTGTATTATCTTTTGAATTATTACATGCACCAGCTACAGTATGTGGACCTAAA 
                 
               
               
                   
               
               
                 
                   AAATCTACTAATTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGATTA 
                 
               
               
                   
               
               
                 
                   ACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAATTTTTGCCTTTTCAACAA 
                 
               
               
                   
               
               
                 
                   TTTGGAAGAGATATTGCTGATACTACAGATGCAGTAAGAGATCCTCAAACTTTA 
                 
               
               
                   
               
               
                 
                   GAAATATTGGATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAACACCA 
                 
               
               
                   
               
               
                 
                   GGAACTAATACATCTAATCAAGTTGCTGTATTATATCAAGATGTTAATTGTACT 
                 
               
               
                   
               
               
                 
                   GAAGTTCCTGTAGCAATTCATGCTGATCAATTAACTCCAACATGGAGAGTATAT 
                 
               
               
                   
               
               
                 
                   TCTACTGGATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGGAGCAGAA 
                 
               
               
                   
               
               
                 
                   CATGTAAATAATTCTTATGAATGTGATATTCCTATAGGAGCTGGAATATGTGCA 
                 
               
               
                   
               
               
                 
                   TCTTATCAAACTCAAACAAATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAA 
                 
               
               
                   
               
               
                 
                   TCTATAATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTAGCATATTCTA 
                 
               
               
                   
               
               
                 
                   ATAATTCTATTGCAATTCCTACTAACTTTACTATTTCTGTAACTACAGAAATATT 
                 
               
               
                   
               
               
                 
                   GCCAGTTTCTATGACTAAAACATCTGTAGATTGTACAATGTATATATGTGGAGA 
                 
               
               
                   
               
               
                 
                   TTCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTTTTTGTACTCAATTG 
                 
               
               
                   
               
               
                 
                   AATAGAGCTTTAACAGGAATAGCAGTAGAACAAGATAAAAATACACAAGAAGT 
                 
               
               
                   
               
               
                 
                   TTTTGCTCAAGTAAAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAGG 
                 
               
               
                   
               
               
                 
                   TTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACCTTCTAAAAGATCTTTT 
                 
               
               
                   
               
               
                 
                   ATTGAAGATTTGTTGTTTAATAAGGTTACATTAGCAGATGCTGGTTTTATAAAA 
                 
               
               
                   
               
               
                 
                   CAATATGGAGATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGCTCAA 
                 
               
               
                   
               
               
                 
                   AAGTTTAATGGATTAACTGTATTACCACCTCTACTAACAGATGAAATGATAGCA 
                 
               
               
                   
               
               
                 
                   CAATATACATCTGCATTATTAGCTGGAACTATTACATCTGGATGGACTTTTGGA 
                 
               
               
                   
               
               
                 
                   GCTGGAGCAGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGATTCAAT 
                 
               
               
                   
               
               
                 
                   GGAATTGGAGTTACTCAAAATGTATTATATGAAAATCAAAAACTAATTGCTAAT 
                 
               
               
                   
               
               
                 
                   CAATTCAATTCTGCAATTGGAAAAATTCAAGATTCTCTATCTTCTACAGCATCT 
                 
               
               
                   
               
               
                 
                   GCTTTAGGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTTTAAATACT 
                 
               
               
                   
               
               
                 
                   CTAGTTAAACAACTATCTTCTAATTTTGGAGCTATTTCTTCTGTTTTAAATGATA 
                 
               
               
                   
               
               
                 
                   TATTGTCTAGACTAGATCCACCTGAAGCAGAAGTACAAATTGATAGACTAATTA 
                 
               
               
                   
               
               
                 
                   CAGGAAGATTACAATCTCTACAAACTTATGTAACACAACAACTAATTAGAGCAG 
                 
               
               
                   
               
               
                 
                   CTGAAATAAGAGCATCTGCTAATTTGGCAGCTACTAAAATGTCTGAATGCGTAT 
                 
               
               
                   
               
               
                 
                   TAGGACAATCTAAAAGAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTT 
                 
               
               
                   
               
               
                 
                   TTCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTTACATATGTACCTG 
                 
               
               
                   
               
               
                 
                   CACAAGAAAAGAACTTTACTACAGCACCAGCTATATGTCATGATGGAAAAGCTC 
                 
               
               
                   
               
               
                 
                   ATTTTCCTAGAGAAGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACAC 
                 
               
               
                   
               
               
                 
                   AAAGAAACTTTTATGAACCACAAATTATAACTACAGATAATACATTTGTATCTG 
                 
               
               
                   
               
               
                 
                   GAAATTGTGATGTTGTAATTGGAATTGTTAATAATACTGTATATGATCCACTAC 
                 
               
               
                   
               
               
                 
                   AACCTGAACTAGATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATCATA 
                 
               
               
                   
               
               
                 
                   CTTCTCCTGATGTTGATTTGGGAGATATATCTGGAATTAATGCTTCTGTTGTAA 
                 
               
               
                   
               
               
                 
                   ATATTCAAAAAGAAATAGATAGATTGAATGAAGTAGCAAAAAATTTGAATGAAT 
                 
               
               
                   
               
               
                 
                   CTCTAATTGATTTGCAAGAATTAGGAAAATATGAACAATATATCAAATGGCCAT 
                 
               
               
                   
               
               
                 
                   GGTATATTTGGCTAGGTTTTATAGCTGGATTAATAGCAATTGTTATGGTAACTA 
                 
               
               
                   
               
               
                 
                   TTATGTTATGTTGTATGACATCTTGTTGTTCTTGTCTAAAAGGATGTTGTTCTTG 
                 
               
               
                   
               
               
                 
                   TGGATCTTGTTGTAAATTTGATGAAGATGATTCTGAACCTGTTTTGAAAGGTGT 
                 
               
               
                   
               
               
                   TAAACTACATTATACTTAATAA TTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAA 
               
               
                   
               
               
                 ACTCTAATGACCACATCTTTTTTTAGAGATGAAAAATTTTCCACATCTCCTTTTGTAG 
               
               
                   
               
               
                 ACACGACTAAACATTTTGCAGAAAAAAGTTTATTAGTGTTTAGATAATCGTATACTT 
               
               
                   
               
               
                 CATCAGTGTAGATAGTAAATGTGAACAGATAAAAGGTATTCTTGCTCAATAGATTGG 
               
               
                   
               
               
                 TAAATTCCATAGAATATATTAATCCTTTCTTCTTGAGATCCCACATCATTTCAACCAG 
               
               
                   
               
               
                 AGACGTTTTATCCAATGATTTACCTCGTACTATACCACATACAAAACTAGATTTTGC 
               
               
                   
               
               
                 AGTGACGTCGTATCTGGTATTCCTACCAAACAAAATTTTACTTTTAGTTCTTTTAGAA 
               
               
                   
               
               
                 AATTCTAAGGTAGAATCTCTATTTGCCAATATGTCATCTATGGAATTACCACTAGCA 
               
               
                   
               
               
                 AAAAATGATAGAAATATATATTGATACATCGCAGCTGGTTTTGATCTACTATACTTT 
               
               
                   
               
               
                 AAAAACGAATCAGATTCCATAATTGCCTGTATATCATCAGCTGAAAAACTATGTTTT 
               
               
                   
               
               
                 ACACGTATTCCTTCGGCATTTCTTTTTAATGATATATCTTGTTTAGACAATGATAAAG 
               
               
                   
               
               
                 TTATCATGTCCATGAGAGACGCGTCTCCGTATCGTATAAATATTTCATTAGATGTTAG 
               
               
                   
               
               
                 ACGCTTCATTAGGGGTATACTTCTATAAGGTTTCTTAATCAGTCCATCATTGGTTGCG 
               
               
                   
               
               
                 TCAAGAACAAGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGG 
               
               
                   
               
               
                 TCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA 
               
               
                   
               
               
                 GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT 
               
               
                   
               
               
                 AATTGCGTTGCGCTCACTGCCCGCTTTCGAGTCGGGAAACCTGTCGTGCCAGCTGCA 
               
               
                   
               
               
                 TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC 
               
               
                   
               
               
                 TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC 
               
               
                   
               
               
                 TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA 
               
               
                   
               
               
                 ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT 
               
               
                   
               
               
                 GGCGTTTTTCGATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG 
               
               
                   
               
               
                 TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA 
               
               
                   
               
               
                 GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT 
               
               
                   
               
               
                 TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG 
               
               
                   
               
               
                 GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC 
               
               
                   
               
               
                 CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA 
               
               
                   
               
               
                 TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG 
               
               
                   
               
               
                 TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT 
               
               
                   
               
               
                 TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG 
               
               
                   
               
               
                 ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT 
               
               
                   
               
               
                 TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA 
               
               
                   
               
               
                 CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA 
               
               
                   
               
               
                 GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA 
               
               
                   
               
               
                 TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT 
               
               
                   
               
               
                 CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAAC 
               
               
                   
               
               
                 TACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC 
               
               
                   
               
               
                 CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG 
               
               
                   
               
               
                 CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG 
               
               
                   
               
               
                 GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCT 
               
               
                   
               
               
                 ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC 
               
               
                   
               
               
                 AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT 
               
               
                   
               
               
                 TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA 
               
               
                   
               
               
                 TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC 
               
               
                   
               
               
                 TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC 
               
               
                   
               
               
                 TTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT 
               
               
                   
               
               
                 CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAG 
               
               
                   
               
               
                 ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC 
               
               
                   
               
               
                 ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA 
               
               
                   
               
               
                 TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAA 
               
               
                   
               
               
                 GCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA 
               
               
                   
               
               
                 ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAA 
               
               
                   
               
               
                 GAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT 
               
               
                   
               
               
                 CGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAG 
               
               
                   
               
               
                 ACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC 
               
               
                   
               
               
                 GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGA 
               
               
                   
               
               
                 TTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGA 
               
               
                   
               
               
                 AAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCG 
               
               
                   
               
               
                 ATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA 
               
               
                   
               
               
                 GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACG 
               
               
                   
               
               
                 GCCAGTGAATTGGATTTAGGTGACACTATA 
               
            
           
         
       
     
     MVA Construct 3: MVA/S-Tri-Sec 
       
                                     Name   Range   Description                  Flank1 I8R    1-537   Essential gene region on                MVA for recombination       P11   545-573   promoter       GFP    574-1293   GFP       DR   1294-1528   Direct Repeats       mH5   1553-1619   Promoter       GmCSE signal peptide   1634-1684   Secretory signal sequence       Spike   1685-5269   Spike protein sequence and                transmembrane domain deleted       Aminoacid changed   4601-4603   K986P 4601AAA4603                changed to 4601CCA4603       Aminoacid changed   4592-4594   V987P 4604GTT4606                changed to 4604CCT4606       Fold on sequence   5270-5359   Fold on sequence added       Flank 2 G1L   5397-6098   Essential gene region on                MVA for recombination       Ampicillin resistance    7384-8174   Confers resistance to Ampicillin       gene                    
Plasmid Sequence (SEQ ID NO: 9) and Sequence encoding spike protein (bold, SEQ ID NO: 10)
 
     
       
         
           
               
               
            
               
                   
                 GAATTCCCTGGGACATACGTATATTTCTATGATCTGTCTT 
               
               
                   
               
               
                   
                 ATATGAAGTCTATACAGCGAATAGATTCAGAATTTCTACA 
               
               
                   
               
               
                   
                 TAATTATATATTGTACGCTAATAAGTTTAATCTAACACTC 
               
               
                   
               
               
                   
                 CCCGAAGATTTGTTTATAATCCCTACAAATTTGGATATTC 
               
               
                   
               
               
                   
                 TATGGCGTACAAAGGAATATATAGACTCGTTCGATATTAG 
               
               
                   
               
               
                   
                 TACAGAAACATGGAATAAATTATTATCCAATTATTATATG 
               
               
                   
               
               
                   
                 AAGATGATAGAGTATGCTAAACTTTATGTACTAAGTCCTA 
               
               
                   
               
               
                   
                 TTCTCGCTGAGGAGTTGGATAATTTTGAGAGGACGGGAGA 
               
               
                   
               
               
                   
                 ATTAACTAGTATTGTACAAGAAGCCATTTTATCTCTAAAT 
               
               
                   
               
               
                   
                 TTACGAATTAAGATTTTAAATTTTAAACATAAAGATGATG 
               
               
                   
               
               
                   
                 ATACGTATATACACTTTTGTAAAATATTATTCGGTGTCTA 
               
               
                   
               
               
                   
                 TAACGGAACAAACGCTACTATATATTATCATAGACCTCTA 
               
               
                   
               
               
                   
                 ACGGGATATATGAATATGATTTCAGATACTATATTTGTTC 
               
               
                   
               
               
                   
                 CTGTAGATAATAACTAAGGCGCGCCTTTCATTTTGTTTTT 
               
               
                   
               
               
                   
                 TTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTC 
               
               
                   
               
               
                   
                 ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG 
               
               
                   
               
               
                   
                 TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGG 
               
               
                   
               
               
                   
                 CGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC 
               
               
                   
               
               
                   
                 ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA 
               
               
                   
               
               
                   
                 CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC 
               
               
                   
               
               
                   
                 CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATG 
               
               
                   
               
               
                   
                 CCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG 
               
               
                   
               
               
                   
                 ACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGA 
               
               
                   
               
               
                   
                 GGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC 
               
               
                   
               
               
                   
                 GACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGG 
               
               
                   
               
               
                   
                 AGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA 
               
               
                   
               
               
                   
                 CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGC 
               
               
                   
               
               
                   
                 CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT 
               
               
                   
               
               
                   
                 ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCT 
               
               
                   
               
               
                   
                 GCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGC 
               
               
                   
               
               
                   
                 AAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGG 
               
               
                   
               
               
                   
                 AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGCACGA 
               
               
                   
               
               
                   
                 GCTGTACAAGTAAGAGCTCGAGGACGGGAGAATTAACTAG 
               
               
                   
               
               
                   
                 TATTGTACAAGAAGCCATTTTATCTCTAAATTTACGAATT 
               
               
                   
               
               
                   
                 AAGATTTTAAATTTTAAACATAAAGATGATGATACGTATA 
               
               
                   
               
               
                   
                 TACACTTTTGTAAAATATTATTCGGTGTCTATAACGGAAC 
               
               
                   
               
               
                   
                 AAACGCTACTATATATTATCATAGACCTCTAACGGGATAT 
               
               
                   
               
               
                   
                 ATGAATATGATTTCAGATACTATATTTGTTCCTGTAGATA 
               
               
                   
               
               
                   
                 ATAACTAACTCGAGGCCGCTGGTACCCAACCTAAAAATTG 
               
               
                   
               
               
                   
                 AAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGA 
               
               
                   
               
               
                   
                 AAGCGAGAAATAATCATAAATAAGCCCGGGACC ATGTGGT   
               
               
                   
               
               
                   
                 
                   TACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAAT 
                 
               
               
                   
               
               
                   
                 
                   TTCACAATGTGTAAATCTAACTACAAGAACTCAATTACCG 
                 
               
               
                   
               
               
                   
                 
                   CCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATC 
                 
               
               
                   
               
               
                   
                 
                   CTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACA 
                 
               
               
                   
               
               
                   
                 
                   AGATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTT 
                 
               
               
                   
               
               
                   
                 
                   CATGCAATACATGTATCTGGAACTAATGGAACAAAAAGAT 
                 
               
               
                   
               
               
                   
                 
                   TTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTT 
                 
               
               
                   
               
               
                   
                 
                   TGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATA 
                 
               
               
                   
               
               
                   
                 
                   TTTGGAACTACATTAGATTCTAAAACACAATCTCTACTAA 
                 
               
               
                   
               
               
                   
                 
                   TTGTTAATAATGCAACTAATGTAGTTATAAAAGTATGTGA 
                 
               
               
                   
               
               
                   
                 
                   ATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTAT 
                 
               
               
                   
               
               
                   
                 
                   CATAAAAATAATAAGTCTTGGATGGAATCTGAATTCAGAG 
                 
               
               
                   
               
               
                   
                 
                   TATATTCTTCTGCTAATAATTGTACATTTGAATATGTATC 
                 
               
               
                   
               
               
                   
                 
                   TCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAAC 
                 
               
               
                   
               
               
                   
                 
                   TTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATG 
                 
               
               
                   
               
               
                   
                 
                   GATACTTTAAAATCTATTCTAAACATACTCCAATTAATCT 
                 
               
               
                   
               
               
                   
                 
                   AGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACCA 
                 
               
               
                   
               
               
                   
                 
                   CTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTC 
                 
               
               
                   
               
               
                   
                 
                   AAACATTATTAGCTTTACATAGATCTTATTTGACACCTGG 
                 
               
               
                   
               
               
                   
                 
                   AGATTCTTCTTCTGGATGGACTGCAGGAGCTGCAGCTTAT 
                 
               
               
                   
               
               
                   
                 
                   TATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAAT 
                 
               
               
                   
               
               
                   
                 
                   ATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGC 
                 
               
               
                   
               
               
                   
                 
                   TTTAGATCCTCTATCTGAAACTAAATGTACTTTAAAATCT 
                 
               
               
                   
               
               
                   
                 
                   TTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTA 
                 
               
               
                   
               
               
                   
                 
                   GAGTACAACCAACTGAATCTATTGTTAGATTTCCAAATAT 
                 
               
               
                   
               
               
                   
                 
                   AACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACT 
                 
               
               
                   
               
               
                   
                 
                   AGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATAT 
                 
               
               
                   
               
               
                   
                 
                   CTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGC 
                 
               
               
                   
               
               
                   
                 
                   ATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCAACA 
                 
               
               
                   
               
               
                   
                 
                   AAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATT 
                 
               
               
                   
               
               
                   
                 
                   CTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCC 
                 
               
               
                   
               
               
                   
                 
                   TGGACAAACAGGAAAAATAGCAGATTATAATTATAAATTA 
                 
               
               
                   
               
               
                   
                 
                   CCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCTA 
                 
               
               
                   
               
               
                   
                 
                   ATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTT 
                 
               
               
                   
               
               
                   
                 
                   GTATAGATTGTTTAGAAAATCTAATTTGAAACCTTTTGAA 
                 
               
               
                   
               
               
                   
                 
                   AGAGATATTTCTACAGAAATCTATCAAGCAGGATCTACTC 
                 
               
               
                   
               
               
                   
                 
                   CATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACT 
                 
               
               
                   
               
               
                   
                 
                   ACAATCTTATGGATTTCAACCTACAAATGGAGTAGGATAT 
                 
               
               
                   
               
               
                   
                 
                   CAACCATATAGAGTAGTTGTATTATCTTTTGAATTATTAC 
                 
               
               
                   
               
               
                   
                 
                   ATGCACCAGCTACAGTATGTGGACCTAAAAAATCTACTAA 
                 
               
               
                   
               
               
                   
                 
                   TTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGA 
                 
               
               
                   
               
               
                   
                 
                   TTAACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAAT 
                 
               
               
                   
               
               
                   
                 
                   TTTTGCCTTTTCAACAATTTGGAAGAGATATTGCTGATAC 
                 
               
               
                   
               
               
                   
                 
                   TACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTG 
                 
               
               
                   
               
               
                   
                 
                   GATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAA 
                 
               
               
                   
               
               
                   
                 
                   CACCAGGAACTAATACATCTAATCAAGTTGCTGTATTATA 
                 
               
               
                   
               
               
                   
                 
                   TCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCAT 
                 
               
               
                   
               
               
                   
                 
                   GCTGATCAATTAACTCCAACATGGAGAGTATATTCTACTG 
                 
               
               
                   
               
               
                   
                 
                   GATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGG 
                 
               
               
                   
               
               
                   
                 
                   AGCAGAACATGTAAATAATTCTTATGAATGTGATATTCCT 
                 
               
               
                   
               
               
                   
                 
                   ATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAA 
                 
               
               
                   
               
               
                   
                 
                   ATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAATCTAT 
                 
               
               
                   
               
               
                   
                 
                   AATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTA 
                 
               
               
                   
               
               
                   
                 
                   GCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTA 
                 
               
               
                   
               
               
                   
                 
                   CTATTTCTGTAACTACAGAAATATTGCCAGTTTCTATGAC 
                 
               
               
                   
               
               
                   
                 
                   TAAAACATCTGTAGATTGTACAATGTATATATGTGGAGAT 
                 
               
               
                   
               
               
                   
                 
                   TCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTT 
                 
               
               
                   
               
               
                   
                 
                   TTTGTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGT 
                 
               
               
                   
               
               
                   
                 
                   AGAACAAGATAAAAATACACAAGAAGTTTTTGCTCAAGTA 
                 
               
               
                   
               
               
                   
                 
                   AAACAAATCTATAAAACTCCACCTATAAAAGATTTTGGAG 
                 
               
               
                   
               
               
                   
                 
                   GTTTTAATTTTTCTCAAATATTGCCAGATCCTTCTAAACC 
                 
               
               
                   
               
               
                   
                 
                   TTCTAAAAGATCTTTTATTGAAGATTTGTTGTTTAATAAG 
                 
               
               
                   
               
               
                   
                 
                   GTTACATTAGCAGATGCTGGTTTTATAAAACAATATGGAG 
                 
               
               
                   
               
               
                   
                 
                   ATTGTTTAGGAGATATTGCAGCTAGAGATTTGATTTGTGC 
                 
               
               
                   
               
               
                   
                 
                   TCAAAAGTTTAATGGATTAACTGTATTACCACCTCTACTA 
                 
               
               
                   
               
               
                   
                 
                   ACAGATGAAATGATAGCACAATATACATCTGCATTATTAG 
                 
               
               
                   
               
               
                   
                 
                   CTGGAACTATTACATCTGGATGGACTTTTGGAGCTGGAGC 
                 
               
               
                   
               
               
                   
                 
                   AGCTTTACAAATACCATTTGCTATGCAAATGGCATATAGA 
                 
               
               
                   
               
               
                   
                 
                   TTCAATGGAATTGGAGTTACTCAAAATGTATTATATGAAA 
                 
               
               
                   
               
               
                   
                 
                   ATCAAAAACTAATTGCTAATCAATTCAATTCTGCAATTGG 
                 
               
               
                   
               
               
                   
                 
                   AAAAATTCAAGATTCTCTATCTTCTACAGCATCTGCTTTA 
                 
               
               
                   
               
               
                   
                 
                   GGAAAACTACAAGATGTTGTAAATCAAAATGCACAAGCTT 
                 
               
               
                   
               
               
                   
                 
                   TAAATACTCTAGTTAAACAACTATCTTCTAATTTTGGAGC 
                 
               
               
                   
               
               
                   
                 
                   TATTTCTTCTGTTTTAAATGATATATTGTCTAGACTAGAT 
                 
               
               
                   
               
               
                   
                 
                   CCACCTGAAGCAGAAGTACAAATTGATAGACTAATTACAG 
                 
               
               
                   
               
               
                   
                 
                   GAAGATTACAATCTCTACAAACTTATGTAACACAACAACT 
                 
               
               
                   
               
               
                   
                 
                   AATTAGAGCAGCTGAAATAAGAGCATCTGCTAATTTGGCA 
                 
               
               
                   
               
               
                   
                 
                   GCTACTAAAATGTCTGAATGCGTATTAGGACAATCTAAAA 
                 
               
               
                   
               
               
                   
                 
                   GAGTAGATTTTTGTGGAAAAGGATATCATTTGATGTCTTT 
                 
               
               
                   
               
               
                   
                 
                   TCCACAATCTGCTCCTCATGGAGTAGTATTTTTGCATGTT 
                 
               
               
                   
               
               
                   
                 
                   ACATATGTACCTGCACAAGAAAAGAACTTTACTACAGCAC 
                 
               
               
                   
               
               
                   
                 
                   CAGCTATATGTCATGATGGAAAAGCTCATTTTCCTAGAGA 
                 
               
               
                   
               
               
                   
                 
                   AGGAGTTTTTGTATCTAATGGAACTCATTGGTTTGTTACA 
                 
               
               
                   
               
               
                   
                 
                   CAAAGAAACTTTTATGAACCACAAATTATAACTACAGATA 
                 
               
               
                   
               
               
                   
                 
                   ATACATTTGTATCTGGAAATTGTGATGTTGTAATTGGAAT 
                 
               
               
                   
               
               
                   
                 
                   TGTTAATAATACTGTATATGATCCACTACAACCTGAACTA 
                 
               
               
                   
               
               
                   
                 
                   GATTCTTTTAAAGAAGAACTAGATAAATACTTTAAAAATC 
                 
               
               
                   
               
               
                   
                 
                   ATACTTCTCCTGATGTTGATTTGGGAGATATATCTGGAAT 
                 
               
               
                   
               
               
                   
                 
                   TAATGCTTCTGTTGTAAATATTCAAAAAGAAATAGATAGA 
                 
               
               
                   
               
               
                   
                 
                   TTGAATGAAGTAGCAAAAAATTTGAATGAATCTCTAATTG 
                 
               
               
                   
               
               
                   
                 
                   ATTTGCAAGAATTAGGAAAATATGAACAAGGATCTGCTGG 
                 
               
               
                   
               
               
                   
                 
                   ATATATTCCAGAAGCACCTAGAGATGGACAAGCGTATGTT 
                 
               
               
                   
               
               
                   
                 
                   AGAAAAGATGGTGAATGGGTATTATTGAGTACATTTTTGT 
                 
               
               
                   
               
               
                   
                   AATAA TTTTTATGGATCCTCTAGAGTCGACCTGCAGTCAA 
               
               
                   
               
               
                   
                 ACTCTAATGACCACATCTTTTTTTAGAGATGAAAAATTTT 
               
               
                   
               
               
                   
                 CCACATCTCCTTTTGTAGACACGACTAAACATTTTGCAGA 
               
               
                   
               
               
                   
                 AAAAAGTTTATTAGTGTTTAGATAATCGTATACTTCATCA 
               
               
                   
               
               
                   
                 GTGTAGATAGTAAATGTGAACAGATAAAAGGTATTCTTGC 
               
               
                   
               
               
                   
                 TCAATAGATTGGTAAATTCCATAGAATATATTAATCCTTT 
               
               
                   
               
               
                   
                 CTTCTTGAGATCCCACATCATTTCAACCAGAGACGTTTTA 
               
               
                   
               
               
                   
                 TCCAATGATTTACCTCGTACTATACCACATACAAAACTAG 
               
               
                   
               
               
                   
                 ATTTTGCAGTGACGTCGTATCTGGTATTCCTACCAAACAA 
               
               
                   
               
               
                   
                 AATTTTACTTTTAGTTCTTTTAGAAAATTCTAAGGTAGAA 
               
               
                   
               
               
                   
                 TCTCTATTTGCCAATATGTCATCTATGGAATTACCACTAG 
               
               
                   
               
               
                   
                 CAAAAAATGATAGAAATATATATTGATACATCGCAGCTGG 
               
               
                   
               
               
                   
                 TTTTGATCTACTATACTTTAAAAACGAATCAGATTCCATA 
               
               
                   
               
               
                   
                 ATTGCCTGTATATCATCAGCTGAAAAACTATGTTTTACAC 
               
               
                   
               
               
                   
                 GTATTCCTTCGGCATTTCTTTTTAATGATATATCTTGTTT 
               
               
                   
               
               
                   
                 AGACAATGATAAAGTTATCATGTCCATGAGAGACGCGTCT 
               
               
                   
               
               
                   
                 CCGTATCGTATAAATATTTCATTAGATGTTAGACGCTTCA 
               
               
                   
               
               
                   
                 TTAGGGGTATACTTCTATAAGGTTTCTTAATCAGTCCATC 
               
               
                   
               
               
                   
                 ATTGGTTGCGTCAAGAACAAGCTTGTCTCCCTATAGTGAG 
               
               
                   
               
               
                   
                 TCGTATTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTC 
               
               
                   
               
               
                   
                 CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACAT 
               
               
                   
               
               
                   
                 ACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAA 
               
               
                   
               
               
                   
                 TGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC 
               
               
                   
               
               
                   
                 CCGCTTTCGAGTCGGGAAACCTGTCGTGCCAGCTGCATTA 
               
               
                   
               
               
                   
                 ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATT 
               
               
                   
               
               
                   
                 GGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCT 
               
               
                   
               
               
                   
                 CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA 
               
               
                   
               
               
                   
                 GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCA 
               
               
                   
               
               
                   
                 GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA 
               
               
                   
               
               
                   
                 ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCGATAGGCT 
               
               
                   
               
               
                   
                 CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT 
               
               
                   
               
               
                   
                 CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG 
               
               
                   
               
               
                   
                 CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC 
               
               
                   
               
               
                   
                 GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT 
               
               
                   
               
               
                   
                 TCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT 
               
               
                   
               
               
                   
                 ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTG 
               
               
                   
               
               
                   
                 TGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA 
               
               
                   
               
               
                   
                 TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG 
               
               
                   
               
               
                   
                 ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG 
               
               
                   
               
               
                   
                 CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG 
               
               
                   
               
               
                   
                 TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTG 
               
               
                   
               
               
                   
                 GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAG 
               
               
                   
               
               
                   
                 AGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT 
               
               
                   
               
               
                   
                 AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA 
               
               
                   
               
               
                   
                 GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC 
               
               
                   
               
               
                   
                 GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG 
               
               
                   
               
               
                   
                 ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGA 
               
               
                   
               
               
                   
                 TCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAG 
               
               
                   
               
               
                   
                 TATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA 
               
               
                   
               
               
                   
                 ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT 
               
               
                   
               
               
                   
                 CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC 
               
               
                   
               
               
                   
                 GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG 
               
               
                   
               
               
                   
                 ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG 
               
               
                   
               
               
                   
                 CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGG 
               
               
                   
               
               
                   
                 TCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT 
               
               
                   
               
               
                   
                 TGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT 
               
               
                   
               
               
                   
                 TGCGCAACGTTGTTGGCATTGCTACAGGCATCGTGGTGTC 
               
               
                   
               
               
                   
                 ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC 
               
               
                   
               
               
                   
                 CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA 
               
               
                   
               
               
                   
                 AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG 
               
               
                   
               
               
                   
                 AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA 
               
               
                   
               
               
                   
                 GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT 
               
               
                   
               
               
                   
                 GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG 
               
               
                   
               
               
                   
                 AGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG 
               
               
                   
               
               
                   
                 TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA 
               
               
                   
               
               
                   
                 AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACT 
               
               
                   
               
               
                   
                 CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA 
               
               
                   
               
               
                   
                 CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTT 
               
               
                   
               
               
                   
                 TCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAA 
               
               
                   
               
               
                   
                 TGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA 
               
               
                   
               
               
                   
                 ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT 
               
               
                   
               
               
                   
                 ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG 
               
               
                   
               
               
                   
                 TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT 
               
               
                   
               
               
                   
                 CCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTA 
               
               
                   
               
               
                   
                 TCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCC 
               
               
                   
               
               
                   
                 CTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTC 
               
               
                   
               
               
                   
                 TGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGT 
               
               
                   
               
               
                   
                 AAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTC 
               
               
                   
               
               
                   
                 AGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCG 
               
               
                   
               
               
                   
                 GCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGT 
               
               
                   
               
               
                   
                 GTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCAT 
               
               
                   
               
               
                   
                 CAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAA 
               
               
                   
               
               
                   
                 GGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGG 
               
               
                   
               
               
                   
                 CGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAAC 
               
               
                   
               
               
                   
                 GCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCC 
               
               
                   
               
               
                   
                 AGTGAATTGGATTTAGGTGACACTATA 
               
            
           
         
       
     
     MVA Construct 4: MVA/S1-Mono 
       
                                     Name   Range   Description                  Flank1 I8R    1-537   Essential gene region on                MVA for recombination       P11   545-573   promoter       GFP    574-1293   GFP       DR   1294-1528   Direct Repeats       mH5   1553 1619   Promoter       GmCSF signal peptide   1634-1684   Secretory signal sequence       Spike   1685-3985   Secreted spike protein sequence       Flank 2 G1L   4623-4724   Essential gene region on MVA                for recombination       Ampicillin resistance    6010-6800   Confers resistance to Ampicillin       gene                    
Plasmid Sequence (SEQ ID NO: 11) and Sequence encoding spike protein (bold, SEQ ID NO: 12)
 
     
       
         
           
               
               
            
               
                   
                 GAATTCCCTGGGACATACGTATATTTCTATGATCTGTCTT 
               
               
                   
               
               
                   
                 ATATGAAGTCTATACAGCGAATAGATTCAGAATTTCTACA 
               
               
                   
               
               
                   
                 TAATTATATATTGTACGCTAATAAGTTTAATCTAACACTC 
               
               
                   
               
               
                   
                 CCCGAAGATTTGTTTATAATCCCTACAAATTTGGATATTC 
               
               
                   
               
               
                   
                 TATGGCGTACAAAGGAATATATAGACTCGTTCGATATTAG 
               
               
                   
               
               
                   
                 TACAGAAACATGGAATAAATTATTATCCAATTATTATATG 
               
               
                   
               
               
                   
                 AAGATGATAGAGTATGCTAAACTTTATGTACTAAGTCCTA 
               
               
                   
               
               
                   
                 TTCTCGCTGAGGAGTTGGATAATTTTGAGAGGACGGGAGA 
               
               
                   
               
               
                   
                 ATTAACTAGTATTGTACAAGAAGCCATTTTATCTCTAAAT 
               
               
                   
               
               
                   
                 TTACGAATTAAGATTTTAAATTTTAAACATAAAGATGATG 
               
               
                   
               
               
                   
                 ATACGTATATACACTTTTGTAAAATATTATTCGGTGTCTA 
               
               
                   
               
               
                   
                 TAACGGAACAAACGCTACTATATATTATCATAGACCTCTA 
               
               
                   
               
               
                   
                 ACGGGATATATGAATATGATTTCAGATACTATATTTGTTC 
               
               
                   
               
               
                   
                 CTGTAGATAATAACTAAGGCGCGCCTTTCATTTTGTTTTT 
               
               
                   
               
               
                   
                 TTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTC 
               
               
                   
               
               
                   
                 ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG 
               
               
                   
               
               
                   
                 TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGG 
               
               
                   
               
               
                   
                 CGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGC 
               
               
                   
               
               
                   
                 ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA 
               
               
                   
               
               
                   
                 CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC 
               
               
                   
               
               
                   
                 CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATG 
               
               
                   
               
               
                   
                 CCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG 
               
               
                   
               
               
                   
                 ACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGA 
               
               
                   
               
               
                   
                 GGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC 
               
               
                   
               
               
                   
                 GACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGG 
               
               
                   
               
               
                   
                 AGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA 
               
               
                   
               
               
                   
                 CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGC 
               
               
                   
               
               
                   
                 CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT 
               
               
                   
               
               
                   
                 ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCT 
               
               
                   
               
               
                   
                 GCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGC 
               
               
                   
               
               
                   
                 AAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGG 
               
               
                   
               
               
                   
                 AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGCACGA 
               
               
                   
               
               
                   
                 GCTGTACAAGTAAGAGCTCGAGGACGGGAGAATTAACTAG 
               
               
                   
               
               
                   
                 TATTGTACAAGAAGCCATTTTATCTCTAAATTTACGAATT 
               
               
                   
               
               
                   
                 AAGATTTTAAATTTTAAACATAAAGATGATGATACGTATA 
               
               
                   
               
               
                   
                 TACACTTTTGTAAAATATTATTCGGTGTCTATAACGGAAC 
               
               
                   
               
               
                   
                 AAACGCTACTATATATTATCATAGACCTCTAACGGGATAT 
               
               
                   
               
               
                   
                 ATGAATATGATTTCAGATACTATATTTGTTCCTGTAGATA 
               
               
                   
               
               
                   
                 ATAACTAACTCGAGGCCGCTGGTACCCAACCTAAAAATTG 
               
               
                   
               
               
                   
                 AAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGA 
               
               
                   
               
               
                   
                 AAGCGAGAAATAATCATAAATAAGCCCGGGACC ATGTGGT   
               
               
                   
               
               
                   
                 
                   TACAAGGACTACTATTACTAGGTACTGTTGCCTGTTCAAT 
                 
               
               
                   
               
               
                   
                 
                   TTCACAATGTGTAAATCTAACTACAAGAACTCAATTACCG 
                 
               
               
                   
               
               
                   
                 
                   CCTGCCTATACTAATTCTTTTACAAGAGGAGTATATTATC 
                 
               
               
                   
               
               
                   
                 
                   CTGATAAAGTTTTTAGATCTTCTGTATTACATTCTACACA 
                 
               
               
                   
               
               
                   
                 
                   AGATTTGTTTTTACCATTTTTCTCTAATGTTACTTGGTTT 
                 
               
               
                   
               
               
                   
                 
                   CATGCAATACATGTATCTGGAACTAATGGAACAAAAAGAT 
                 
               
               
                   
               
               
                   
                 
                   TTGATAATCCAGTATTACCTTTTAATGATGGAGTTTATTT 
                 
               
               
                   
               
               
                   
                 
                   TGCTTCTACTGAAAAATCTAATATAATTAGAGGATGGATA 
                 
               
               
                   
               
               
                   
                 
                   TTTGGAACTACATTAGATTCTAAAACACAATCTCTACTAA 
                 
               
               
                   
               
               
                   
                 
                   TTGTTAATAATGCAACTAATGTAGTTATAAAAGTATGTGA 
                 
               
               
                   
               
               
                   
                 
                   ATTTCAATTTTGTAATGATCCATTTTTGGGAGTTTATTAT 
                 
               
               
                   
               
               
                   
                 
                   CATAAAAATAATAAGTCTTGGATGGAATCTGAATTCAGAG 
                 
               
               
                   
               
               
                   
                 
                   TATATTCTTCTGCTAATAATTGTACATTTGAATATGTATC 
                 
               
               
                   
               
               
                   
                 
                   TCAACCATTTTTGATGGATTTGGAAGGAAAACAAGGAAAC 
                 
               
               
                   
               
               
                   
                 
                   TTTAAAAATTTGAGAGAATTTGTTTTTAAAAATATTGATG 
                 
               
               
                   
               
               
                   
                 
                   GATACTTTAAAATCTATTCTAAACATACTCCAATTAATCT 
                 
               
               
                   
               
               
                   
                 
                   AGTAAGAGATTTGCCTCAAGGATTTTCTGCTTTAGAACCA 
                 
               
               
                   
               
               
                   
                 
                   CTAGTAGATTTGCCTATAGGAATTAATATTACTAGATTTC 
                 
               
               
                   
               
               
                   
                 
                   AAACATTATTAGCTTTACATAGATCTTATTTGACACCTGG 
                 
               
               
                   
               
               
                   
                 
                   AGATTCTTCTTCTGGATGGACTGCAGGAGCTGCAGCTTAT 
                 
               
               
                   
               
               
                   
                 
                   TATGTTGGATATTTGCAACCAAGAACATTTTTGTTAAAAT 
                 
               
               
                   
               
               
                   
                 
                   ATAATGAAAATGGAACTATAACAGATGCAGTTGATTGTGC 
                 
               
               
                   
               
               
                   
                 
                   TTTAGATCCTCTATCTGAAACTAAATGTACTTTAAAATCT 
                 
               
               
                   
               
               
                   
                 
                   TTTACTGTAGAAAAAGGAATCTATCAAACATCTAACTTTA 
                 
               
               
                   
               
               
                   
                 
                   GAGTACAACCAACTGAATCTATTGTTAGATTTCCAAATAT 
                 
               
               
                   
               
               
                   
                 
                   AACAAATCTATGTCCTTTTGGAGAAGTTTTTAATGCAACT 
                 
               
               
                   
               
               
                   
                 
                   AGATTTGCTTCTGTATATGCATGGAATAGAAAAAGAATAT 
                 
               
               
                   
               
               
                   
                 
                   CTAATTGCGTAGCTGATTATTCTGTATTATATAATTCTGC 
                 
               
               
                   
               
               
                   
                 
                   ATCTTTTTCTACTTTTAAATGTTATGGAGTATCTCCAACA 
                 
               
               
                   
               
               
                   
                 
                   AAATTGAATGATCTATGTTTTACTAATGTTTATGCAGATT 
                 
               
               
                   
               
               
                   
                 
                   CTTTTGTAATAAGAGGAGATGAAGTTAGACAAATAGCTCC 
                 
               
               
                   
               
               
                   
                 
                   TGGACAAACAGGAAAAATAGCAGATTATAATTATAAATTA 
                 
               
               
                   
               
               
                   
                 
                   CCAGATGATTTCACTGGATGCGTAATTGCTTGGAATTCTA 
                 
               
               
                   
               
               
                   
                 
                   ATAATTTGGATTCTAAAGTAGGAGGAAATTATAATTATTT 
                 
               
               
                   
               
               
                   
                 
                   GTATAGATTGTTTAGAAAATCTAATTTGAAACCTTTTGAA 
                 
               
               
                   
               
               
                   
                 
                   AGAGATATTTCTACAGAAATCTATCAAGCAGGATCTACTC 
                 
               
               
                   
               
               
                   
                 
                   CATGTAATGGAGTTGAAGGTTTTAATTGTTATTTTCCACT 
                 
               
               
                   
               
               
                   
                 
                   ACAATCTTATGGATTTCAACCTACAAATGGAGTAGGATAT 
                 
               
               
                   
               
               
                   
                 
                   CAACCATATAGAGTAGTTGTATTATCTTTTGAATTATTAC 
                 
               
               
                   
               
               
                   
                 
                   ATGCACCAGCTACAGTATGTGGACCTAAAAAATCTACTAA 
                 
               
               
                   
               
               
                   
                 
                   TTTGGTTAAAAATAAGTGCGTAAACTTTAACTTTAATGGA 
                 
               
               
                   
               
               
                   
                 
                   TTAACTGGAACAGGAGTTTTAACTGAATCTAATAAGAAAT 
                 
               
               
                   
               
               
                   
                 
                   TTTTGCCTTTTCAACAATTTGGAAGAGATATTGCTGATAC 
                 
               
               
                   
               
               
                   
                 
                   TACAGATGCAGTAAGAGATCCTCAAACTTTAGAAATATTG 
                 
               
               
                   
               
               
                   
                 
                   GATATTACACCATGTTCTTTTGGAGGAGTTTCTGTAATAA 
                 
               
               
                   
               
               
                   
                 
                   CACCAGGAACTAATACATCTAATCAAGTTGCTGTATTATA 
                 
               
               
                   
               
               
                   
                 
                   TCAAGATGTTAATTGTACTGAAGTTCCTGTAGCAATTCAT 
                 
               
               
                   
               
               
                   
                 
                   GCTGATCAATTAACTCCAACATGGAGAGTATATTCTACTG 
                 
               
               
                   
               
               
                   
                 
                   GATCTAATGTTTTTCAAACAAGAGCTGGATGTCTAATTGG 
                 
               
               
                   
               
               
                   
                 
                   AGCAGAACATGTAAATAATTCTTATGAATGTGATATTCCT 
                 
               
               
                   
               
               
                   
                 
                   ATAGGAGCTGGAATATGTGCATCTTATCAAACTCAAACAA 
                 
               
               
                   
               
               
                   
                 
                   ATTCTCCAAGAAGAGCTAGATCTGTTGCATCTCAATCTAT 
                 
               
               
                   
               
               
                   
                 
                   AATTGCTTATACAATGTCTTTAGGAGCTGAAAATTCTGTA 
                 
               
               
                   
               
               
                   
                 
                   GCATATTCTAATAATTCTATTGCAATTCCTACTAACTTTA 
                 
               
               
                   
               
               
                   
                 
                   CTATTTCTGTAACTACAGAAATATTGCCAGTTTCTATGAC 
                 
               
               
                   
               
               
                   
                 
                   TAAAACATCTGTAGATTGTACAATGTATATATGTGGAGAT 
                 
               
               
                   
               
               
                   
                 
                   TCTACTGAATGTTCTAATTTGCTACTACAATATGGATCTT 
                 
               
               
                   
               
               
                   
                 
                   TTTGTACTCAATTGAATAGAGCTTTAACAGGAATAGCAGT 
                 
               
               
                   
               
               
                   
                   AGAACAAGATAAAAATACACAAGAATAATAA TTTTTATGG 
               
               
                   
               
               
                   
                 ATCCTCTAGAGTCGACCTGCAGTCAAACTCTAATGACCAC 
               
               
                   
               
               
                   
                 ATCTTTTTTTAGAGATGAAAAATTTTCCACATCTCCTTTT 
               
               
                   
               
               
                   
                 GTAGACACGACTAAACATTTTGCAGAAAAAAGTTTATTAG 
               
               
                   
               
               
                   
                 TGTTTAGATAATCGTATACTTCATCAGTGTAGATAGTAAA 
               
               
                   
               
               
                   
                 TGTGAACAGATAAAAGGTATTCTTGCTCAATAGATTGGTA 
               
               
                   
               
               
                   
                 AATTCCATAGAATATATTAATCCTTTCTTCTTGAGATCCC 
               
               
                   
               
               
                   
                 ACATCATTTCAACCAGAGACGTTTTATCCAATGATTTACC 
               
               
                   
               
               
                   
                 TCGTACTATACCACATACAAAACTAGATTTTGCAGTGACG 
               
               
                   
               
               
                   
                 TCGTATCTGGTATTCCTACCAAACAAAATTTTACTTTTAG 
               
               
                   
               
               
                   
                 TTCTTTTAGAAAATTCTAAGGTAGAATCTCTATTTGCCAA 
               
               
                   
               
               
                   
                 TATGTCATCTATGGAATTACCACTAGCAAAAAATGATAGA 
               
               
                   
               
               
                   
                 AATATATATTGATACATCGCAGCTGGTTTTGATCTACTAT 
               
               
                   
               
               
                   
                 ACTTTAAAAACGAATCAGATTCCATAATTGCCTGTATATC 
               
               
                   
               
               
                   
                 ATCAGCTGAAAAACTATGTTTTACACGTATTCCTTCGGCA 
               
               
                   
               
               
                   
                 TTTCTTTTTAATGATATATCTTGTTTAGACAATGATAAAG 
               
               
                   
               
               
                   
                 TTATCATGTCCATGAGAGACGCGTCTCCGTATCGTATAAA 
               
               
                   
               
               
                   
                 TATTTCATTAGATGTTAGACGCTTCATTAGGGGTATACTT 
               
               
                   
               
               
                   
                 CTATAAGGTTTCTTAATCAGTCCATCATTGGTTGCGTCAA 
               
               
                   
               
               
                   
                 GAACAAGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTT 
               
               
                   
               
               
                   
                 GGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT 
               
               
                   
               
               
                   
                 TATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA 
               
               
                   
               
               
                   
                 TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACT 
               
               
                   
               
               
                   
                 CACATTAATTGCGTTGCGCTCACTGCCCGCTTTCGAGTCG 
               
               
                   
               
               
                   
                 GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAAC 
               
               
                   
               
               
                   
                 GCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC 
               
               
                   
               
               
                   
                 TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG 
               
               
                   
               
               
                   
                 CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGT 
               
               
                   
               
               
                   
                 TATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG 
               
               
                   
               
               
                   
                 AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC 
               
               
                   
               
               
                   
                 GCGTTGCTGGCGTTTTTCGATAGGCTCCGCCCCCCTGACG 
               
               
                   
               
               
                   
                 AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA 
               
               
                   
               
               
                   
                 CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA 
               
               
                   
               
               
                   
                 AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTA 
               
               
                   
               
               
                   
                 CCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC 
               
               
                   
               
               
                   
                 GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG 
               
               
                   
               
               
                   
                 TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC 
               
               
                   
               
               
                   
                 CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG 
               
               
                   
               
               
                   
                 TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG 
               
               
                   
               
               
                   
                 GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG 
               
               
                   
               
               
                   
                 TAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA 
               
               
                   
               
               
                   
                 CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTG 
               
               
                   
               
               
                   
                 CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTT 
               
               
                   
               
               
                   
                 GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT 
               
               
                   
               
               
                   
                 TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT 
               
               
                   
               
               
                   
                 CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC 
               
               
                   
               
               
                   
                 AGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG 
               
               
                   
               
               
                   
                 ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA 
               
               
                   
               
               
                   
                 AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAA 
               
               
                   
               
               
                   
                 CTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC 
               
               
                   
               
               
                   
                 TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC 
               
               
                   
               
               
                   
                 TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT 
               
               
                   
               
               
                   
                 TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCC 
               
               
                   
               
               
                   
                 ACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA 
               
               
                   
               
               
                   
                 GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT 
               
               
                   
               
               
                   
                 CCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG 
               
               
                   
               
               
                   
                 AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT 
               
               
                   
               
               
                   
                 GGCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG 
               
               
                   
               
               
                   
                 GTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG 
               
               
                   
               
               
                   
                 AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGC 
               
               
                   
               
               
                   
                 TCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG 
               
               
                   
               
               
                   
                 CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC 
               
               
                   
               
               
                   
                 TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACT 
               
               
                   
               
               
                   
                 GGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC 
               
               
                   
               
               
                   
                 GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAA 
               
               
                   
               
               
                   
                 TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT 
               
               
                   
               
               
                   
                 GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC 
               
               
                   
               
               
                   
                 CGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC 
               
               
                   
               
               
                   
                 CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT 
               
               
                   
               
               
                   
                 GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG 
               
               
                   
               
               
                   
                 GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT 
               
               
                   
               
               
                   
                 CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT 
               
               
                   
               
               
                   
                 CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA 
               
               
                   
               
               
                   
                 AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC 
               
               
                   
               
               
                   
                 ACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC 
               
               
                   
               
               
                   
                 TATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGC 
               
               
                   
               
               
                   
                 GTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT 
               
               
                   
               
               
                   
                 CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGG 
               
               
                   
               
               
                   
                 AGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCG 
               
               
                   
               
               
                   
                 GGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGAT 
               
               
                   
               
               
                   
                 TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCAC 
               
               
                   
               
               
                   
                 AGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGC 
               
               
                   
               
               
                   
                 CATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCG 
               
               
                   
               
               
                   
                 GGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGT 
               
               
                   
               
               
                   
                 GCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCC 
               
               
                   
               
               
                   
                 AGTCACGACGTTGTAAAACGACGGCCAGTGAATTGGATTT 
               
               
                   
               
               
                   
                 AGGTGACACTATA 
               
            
           
         
       
     
     DNA Vaccines 
     Four DNA vaccines are made using the coronavirus spike protein as shown in  FIG.  1 A-D . The DNA inserts are codon-optimized for human codon usage and expressed under the human CMV promoter with intron A in pGA1 vector. 
     DNA Construct 1: DNA/S-VLP 
       
                                     Name   Range   Description                  CMV Promoter   476 to 675   Human cytomegalovirus (CMV)                immediate early promoter       intron A    702 to 1645   Intron A positively regulate                expression from the hCMV                immediate-early enhancer/promoter       ORF   1651 to 6495   Insert nCoV VIP (Spike protein                (1273 aa)-P2A (22 aa)-Matrix                (222 aa)-P2A (22 aa)-Envelope (75 aa)       bGH poly(A)    6535 to 6742   Bovine growth hormone        signal       polyadenylation signal       Kanamycin    6775 to 756S   Confers resistance to neomycin,        resistance gene       kanamycin, and               G418 (Geneticin(R))       Replication    7981 to 8570   High-copy-number                ColE1/pMB1/pBR322/pUC        Origin       origin of replication                    
pGA8-nCoV S-VLP Plasmid sequences (SEQ ID NO: 13) and Sequence encoding spike protein (bold, SEQ ID NO: 14)
 
     
       
         
           
               
               
            
               
                 CGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATT 
                   
               
               
                   
               
               
                 TATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATT 
               
               
                   
               
               
                 AATAGTAATCAATTACGGGTTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA 
               
               
                   
               
               
                 CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGA 
               
               
                   
               
               
                 CGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC 
               
               
                   
               
               
                 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA 
               
               
                   
               
               
                 TGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG 
               
               
                   
               
               
                 CCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGGTATTAGTC 
               
               
                   
               
               
                 ATCGGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCG 
               
               
                   
               
               
                 GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT 
               
               
                   
               
               
                 TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACG 
               
               
                   
               
               
                 CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT 
               
               
                   
               
               
                 GAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACA 
               
               
                   
               
               
                 CCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCC 
               
               
                   
               
               
                 GTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGCTC 
               
               
                   
               
               
                 TTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA 
               
               
                   
               
               
                 TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTC 
               
               
                   
               
               
                 CCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACT 
               
               
                   
               
               
                 ATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTAT 
               
               
                   
               
               
                 TTTTACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGT 
               
               
                   
               
               
                 CCCCCGTGCCCGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGG 
               
               
                   
               
               
                 TACCGTGTTCCGGACATGGGYTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGC 
               
               
                   
               
               
                 CCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAG 
               
               
                   
               
               
                 TGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAGTGTGCCGCACAAG 
               
               
                   
               
               
                 GCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCGCACCGC 
               
               
                   
               
               
                 TGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTT 
               
               
                   
               
               
                 GTTGTATTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAG 
               
               
                   
               
               
                 GGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAG 
               
               
                   
               
               
                 CTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCATCGAT AT   
               
               
                   
               
               
                 
                   GTTTGTCTTCCTGGTCCTGCTGCCTCTGGTCTCCTCACAGTGCGTCAATCTGAC 
                 
               
               
                   
               
               
                 
                   TACCCGAACTCAGCTGCCCCCCGCCTACACCAACTCCTTCACCCGGGGCGTGT 
                 
               
               
                   
               
               
                 
                   ACTATCCAGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCCACCCAGGATCTGT 
                 
               
               
                   
               
               
                 
                   TTCTGCCCTTCTTTTCTAATGTGACATGGTTCCACGCCATCCACGTGAGCGGCA 
                 
               
               
                   
               
               
                 
                   CCAACGGCACAAAGAGGTTCGACAACCCTGTGCTGCCATTCAATGATGGCGTG 
                 
               
               
                   
               
               
                 
                   TACTTTGCCTCCACCGAGAAGTCTAACATCATCCGCGGCTGGATCTTTGGCACC 
                 
               
               
                   
               
               
                 
                   ACACTGGACTCCAAGACCCAGTCCCTGCTGATCGTGAACAATGCCACAAACGT 
                 
               
               
                   
               
               
                 
                   GGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAACGATCCTTTCCTGGGCGTGTA 
                 
               
               
                   
               
               
                 
                   CTATCACAAGAACAATAAGTCTTGGATGGAGAGCGAGTTTAGGGTGTATTCTA 
                 
               
               
                   
               
               
                 
                   GCGCCAACAATTGCACCTTCGAGTACGTGTCCCAGCCATTTCTGATGGACCTG 
                 
               
               
                   
               
               
                 
                   GAGGGCAAGCAGGGCAATTTCAAGAACCTGCGGGAGTTCGTGTTTAAGAACAT 
                 
               
               
                   
               
               
                 
                   CGACGGCTACTTCAAGATCTACTCCAAGCACACCCCCATCAACCTGGTGCGGG 
                 
               
               
                   
               
               
                 
                   ACCTGCCACAGGGCTTCTCTGCCCTGGAGCCTCTGGTGGATCTGCCAATCGGC 
                 
               
               
                   
               
               
                 
                   ATCAACATCACACGGTTTCAGACCCTGCTGGCCCTGCACAGAAGCTACCTGAC 
                 
               
               
                   
               
               
                 
                   CCCTGGCGACTCCTCTAGCGGATGGACAGCAGGAGCAGCAGCATACTATGTGG 
                 
               
               
                   
               
               
                 
                   GCTATCTGCAGCCACGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATC 
                 
               
               
                   
               
               
                 
                   ACAGACGCCGTGGATTGCGCCCTGGATCCACTGTCTGAGACAAAGTGTACACT 
                 
               
               
                   
               
               
                 
                   GAAGAGCTTTACAGTGGAGAAGGGCATCTATCAGACCAGCAACTTCAGGGTGC 
                 
               
               
                   
               
               
                 
                   AGCCCACAGAGTCCATCGTGCGCTTTCCAAATATCACCAACCTGTGCCCCTTCG 
                 
               
               
                   
               
               
                 
                   GCGAGGTGTTTAATGCCACAAGATTCGCCAGCGTGTACGCCTGGAACAGGAAG 
                 
               
               
                   
               
               
                 
                   CGCATCTCCAATTGCGTGGCCGACTATTCTGTGCTGTACAACTCTGCCAGCTTC 
                 
               
               
                   
               
               
                 
                   TCCACCTTTAAGTGCTATGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTC 
                 
               
               
                   
               
               
                 
                   ACAAACGTGTACGCCGATTCCTTTGTGATCAGGGGCGACGAGGTGCGCCAGAT 
                 
               
               
                   
               
               
                 
                   CGCACCAGGACAGACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGACG 
                 
               
               
                   
               
               
                 
                   ATTTCACAGGCTGCGTGATCGCCTGGAATTCCAACAATCTGGATTCTAAAGTGG 
                 
               
               
                   
               
               
                 
                   GCGGCAACTACAATTATCTGTACAGGCTGTTCCGCAAGTCTAACCTGAAGCCTT 
                 
               
               
                   
               
               
                 
                   TTGAGCGGGACATCTCCACCGAGATCTACCAGGCCGGCTCTACACCATGCAAC 
                 
               
               
                   
               
               
                 
                   GGCGTGGAGGGCTTCAATTGTTATTTTCCCCTGCAGAGCTACGGCTTCCAGCCT 
                 
               
               
                   
               
               
                 
                   ACCAATGGCGTGGGCTATCAGCCATACAGAGTGGTGGTGCTGTCTTTTGAGCT 
                 
               
               
                   
               
               
                 
                   GCTGCACGCACCAGCAACCGTGTGCGGACCTAAGAAGAGCACAAATCTGGTGA 
                 
               
               
                   
               
               
                 
                   AGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTG 
                 
               
               
                   
               
               
                 
                   ACCGAGTCCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGGCAGGGACATCGC 
                 
               
               
                   
               
               
                 
                   AGATACCACAGACGCCGTGCGCGACCCCCAGACACTGGAGATCCTGGATATCA 
                 
               
               
                   
               
               
                 CCCCTTGCAGCTTCGGCGGCGTGTCCGTGATCACCCCTGGAACCAATACAAGC 
               
               
                   
               
               
                 
                   AACCAGGTGGCCGTGCTGTATCAGGACGTGAACTGTACAGAGGTGCCAGTGGC 
                 
               
               
                   
               
               
                 
                   CATCCACGCCGATCAGCTGACCCCCACATGGCGGGTGTACTCCACAGGCTCTA 
                 
               
               
                   
               
               
                 
                   ACGTGTTCCAGACCAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAAT 
                 
               
               
                   
               
               
                 
                   AGCTATGAGTGCGACATCCCCATCGGCGCCGGCATCTGTGCCTCCTACCAGAC 
                 
               
               
                   
               
               
                 
                   CCAGACAAACTCCCCTCGGAGAGCCAGGTCTGTGGCCTCTCAGAGCATCATCG 
                 
               
               
                   
               
               
                 
                   CCTATACCATGAGCCTGGGCGCCGAGAACTCCGTGGCCTACAGCAACAATTCC 
                 
               
               
                   
               
               
                 
                   ATCGCCATCCCCACCAATTTCACAATCTCCGTGACCACAGAGATCCTGCCCGTG 
                 
               
               
                   
               
               
                 
                   AGCATGACCAAGACAAGCGTGGACTGCACCATGTATATCTGTGGCGATTCCAC 
                 
               
               
                   
               
               
                 
                   AGAGTGCTCTAATCTGCTGCTGCAGTACGGCTCTTTTTGTACACAGCTGAACCG 
                 
               
               
                   
               
               
                 
                   CGCCCTGACCGGAATCGCAGTGGAGCAGGACAAGAATACCCAGGAGGTGTTCG 
                 
               
               
                   
               
               
                 
                   CCCAGGTGAAGCAGATCTACAAGACACCCCCTATCAAGGACTTTGGCGGCTTC 
                 
               
               
                   
               
               
                 
                   AACTTTAGCCAGATCCTGCCTGATCCATCTAAGCCTAGCAAGAGGTCCTTCATC 
                 
               
               
                   
               
               
                 
                   GAGGACCTGCTGTTTAATAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCA 
                 
               
               
                   
               
               
                 
                   GTATGGCGATTGCCTGGGCGACATCGCAGCACGCGACCTGATCTGTGCCCAGA 
                 
               
               
                   
               
               
                 
                   AGTTTAACGGCCTGACAGTGCTGCCACCCCTGCTGACCGATGAGATGATCGCA 
                 
               
               
                   
               
               
                 
                   CAGTACACCTCTGCCCTGCTGGCCGGAACCATCACAAGCGGATGGACATTCGG 
                 
               
               
                   
               
               
                 
                   CGCAGGAGCCGCCCTGCAGATCCCATTCGCCATGCAGATGGCCTATCGGTTTA 
                 
               
               
                   
               
               
                 
                   ATGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAATCAGAAGCTGATCGCC 
                 
               
               
                   
               
               
                 
                   AATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACTCTCTGTCCTCTACCGCC 
                 
               
               
                   
               
               
                 
                   AGCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAA 
                 
               
               
                   
               
               
                 
                   CACACTGGTGAAGCAGCTGAGCTCCAATTTCGGCGCCATCTCTAGCGTGCTGA 
                 
               
               
                   
               
               
                 
                   ACGATATCCTGAGCAGGCTGGACAAGGTGGAGGCCGAGGTGCAGATCGACCG 
                 
               
               
                   
               
               
                 
                   GCTGATCACCGGCAGACTGCAGTCCCTGCAGACCTACGTGACACAGCAGCTGA 
                 
               
               
                   
               
               
                 
                   TCAGGGCAGCAGAGATCAGGGCCTCTGCCAACCTGGCAGCAACAAAGATGAGC 
                 
               
               
                   
               
               
                 
                   GAGTGCGTGCTGGGACAGTCCAAGAGGGTGGACTTTTGTGGCAAGGGCTATCA 
                 
               
               
                   
               
               
                 
                   CCTGATGAGCTTCCCACAGTCCGCCCCACACGGAGTGGTGTTTCTGCACGTGA 
                 
               
               
                   
               
               
                 
                   CCTACGTGCCTGCCCAGGAGAAGAATTTCACCACAGCCCCAGCCATCTGCCAC 
                 
               
               
                   
               
               
                 
                   GATGGCAAGGCACACTTCCCAAGGGAGGGCGTGTTTGTGAGCAATGGCACACA 
                 
               
               
                   
               
               
                 
                   CTGGTTCGTGACCCAGAGAAACTTTTACGAGCCTCAGATCATCACCACAGACAA 
                 
               
               
                   
               
               
                 
                   CACCTTCGTGAGCGGCAATTGTGACGTGGTCATCGGCATCGTGAACAATACAG 
                 
               
               
                   
               
               
                 
                   TGTATGATCCCCTGCAGCCTGAGCTGGACTCTTTCAAGGAGGAGCTGGATAAG 
                 
               
               
                   
               
               
                 
                   TACTTTAAGAACCACACCAGCCCCGACGTGGATCTGGGCGACATCTCCGGCAT 
                 
               
               
                   
               
               
                 
                   CAACGCCTCTGTGGTGAATATCCAGAAGGAGATCGACAGACTGAATGAGGTGG 
                 
               
               
                   
               
               
                 
                   CCAAGAATCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTATGAG 
                 
               
               
                   
               
               
                 
                   CAGTACATCAAGTGGCCATGGTATATCTGGCTGGGCTTCATCGCCGGCCTGAT 
                 
               
               
                   
               
               
                 
                   CGCCATCGTGATGGTGACAATCATGCTGTGCTGTATGACCTCTTGCTGTAGCTG 
                 
               
               
                   
               
               
                 
                   CCTGAAGGGCTGCTGTTCCTGTGGCTCTTGCTGTAAGTTCGATGAGGACGATT 
                 
               
               
                   
               
               
                 
                   CCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACAGGCTCCGGCGCCACC 
                 
               
               
                   
               
               
                 
                   AACTTTTCTCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAACCCAGGACCTAT 
                 
               
               
                   
               
               
                 
                   GGCCGATAGCAATGGCACCATCACAGTGGAGGAGCTGAAGAAGCTGCTGGAGC 
                 
               
               
                   
               
               
                 
                   AGTGGAACCTGGTCATCGGCTTCCTGTTTCTGACCTGGATCTGCCTGCTGCAGT 
                 
               
               
                   
               
               
                 
                   TCGCCTATGCCAATCGGAACAGATTTCTGTACATCATCAAGCTGATCTTCCTGT 
                 
               
               
                   
               
               
                 
                   GGCTGCTGTGGCCAGTGACCCTGGCCTGCTTCGTGCTGGCCGCCGTGTATCGG 
                 
               
               
                   
               
               
                 
                   ATCAACTGGATCACAGGCGGCATCGCCATCGCCATGGCCTGTCTGGTGGGCCT 
                 
               
               
                   
               
               
                 
                   GATGTGGCTGAGCTACTTCATCGCCTCCTTTAGACTGTTCGCCAGGACCCGCA 
                 
               
               
                   
               
               
                 
                   GCATGTGGTCCTTTAATCCCGAGACAAACATCCTGCTGAATGTGCCTCTGCACG 
                 
               
               
                   
               
               
                 
                   GCACCATCCTGACAAGGCCACTGCTGGAGTCCGAGCTGGTCATCGGAGCCGTG 
                 
               
               
                   
               
               
                 
                   ATCCTGAGGGGACACCTGAGAATCGCAGGACACCACCTGGGCCGCTGCGATAT 
                 
               
               
                   
               
               
                 
                   CAAGGACCTGCCTAAGGAGATCACCGTGGCCACATCTAGGACCCTGAGCTACT 
                 
               
               
                   
               
               
                 
                   ATAAGCTGGGAGCCAGCCAGAGGGTGGCAGGCGACAGCGGATTCGCAGCATA 
                 
               
               
                   
               
               
                 
                   TTCCCGGTACAGAATCGGCAACTACAAGCTGAATACCGATCACTCCTCTAGCTC 
                 
               
               
                   
               
               
                 
                   CGACAATATCGCCCTGCTGGTGCAGGGATCTGGAGCAACAAACTTCAGCCTGC 
                 
               
               
                   
               
               
                 
                   TGAAGCAGGCCGGCGATGTGGAAGAAAACCCAGGACCCATGTATTCTTTTGTG 
                 
               
               
                   
               
               
                 
                   AGCGAGGAGACAGGCACACTGATCGTGAATAGCGTGCTGCTGTTTCTGGCCTT 
                 
               
               
                   
               
               
                 
                   CGTGGTGTTTCTGCTGGTGACACTGGCCATCCTGACCGCCCTGAGACTGTGCG 
                 
               
               
                   
               
               
                 
                   CCTACTGCTGTAATATCGTGAACGTGTCTCTGGTGAAGCCCAGCTTTTACGTGT 
                 
               
               
                   
               
               
                 
                   ATAGTAGGGTGAAGAATCTGAACTCAAGTAGGGTGCCCGATCTGCTGGTCTAA 
                 
               
               
                   
               
               
                 GCTAGCCCCGGGTGATAAACGGACCGCGCAATCCCTAGGCTGTGCCTTCTAGTTGCC 
               
               
                   
               
               
                 AGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCC 
               
               
                   
               
               
                 CACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA 
               
               
                   
               
               
                 TTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA 
               
               
                   
               
               
                 ATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATATAAAAAACGCCCGGCGGCAAC 
               
               
                   
               
               
                 CGAGCGTTCTGAACGCTAGAGTCGACAAATTCAGAAGAACTCGTCAAGAAGGCGAT 
               
               
                   
               
               
                 AGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCG 
               
               
                   
               
               
                 GTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTC 
               
               
                   
               
               
                 CTGATAGCGGTCTGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGC 
               
               
                   
               
               
                 CATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCT 
               
               
                   
               
               
                 CGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCT 
               
               
                   
               
               
                 GATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTG 
               
               
                   
               
               
                 CTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCG 
               
               
                   
               
               
                 TATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGT 
               
               
                   
               
               
                 GAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCG 
               
               
                   
               
               
                 CTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCAC 
               
               
                   
               
               
                 GATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTG 
               
               
                   
               
               
                 ACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGC 
               
               
                   
               
               
                 AGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCG 
               
               
                   
               
               
                 GAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCT 
               
               
                   
               
               
                 CTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCC 
               
               
                   
               
               
                 AGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCG 
               
               
                   
               
               
                 GTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGC 
               
               
                   
               
               
                 AAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCT 
               
               
                   
               
               
                 GACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGAAAAGGAT 
               
               
                   
               
               
                 CTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG 
               
               
                   
               
               
                 TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT 
               
               
                   
               
               
                 TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT 
               
               
                   
               
               
                 TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA 
               
               
                   
               
               
                 GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG 
               
               
                   
               
               
                 AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT 
               
               
                   
               
               
                 GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT 
               
               
                   
               
               
                 AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG 
               
               
                   
               
               
                 AACGACCTACACCCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG 
               
               
                   
               
               
                 CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG 
               
               
                   
               
               
                 GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC 
               
               
                   
               
               
                 GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG 
               
               
                   
               
               
                 AGCCTATGGAAAAACGCCAGCAACGCGGCCCTTTTACGGTTCCTGGCCTTTTGCTGG 
               
               
                   
               
               
                 CCTTTTGCTCACATGTTGT 
               
            
           
         
       
     
     DNA Construct 2: DNA/S-Tri 
       
                                     Name   Range   Description                  CMV Promoter   476 to 675   Human cytomegalovirus (CMV)                immediate early promoter       Intron A    702 to 1645   Intron A positively regulate                expression from the hCMV                immediate-early enhancer/promoter       ORF   1651 to 5472   nCoV S-Tri Spike protein ss                14 to 1273       bGH poly(A)    5512 to 5719   Bovine growth hormone        signal       polyadenylation signal       Kanamycin    5752 to 6546   Confers resistance to neomycin,        resistance gene       kanamycin, and               G418 (Geneticin(R))       Replication    6958 to 7547   High-copy-number        Origin       ColE1/pMB1/PBR322/pUC                origin of replication                    
pGA8-nCoV-S-Tri Plasmid sequences (SEQ ID NO: 15) and Sequence encoding spike protein (bold, SEQ ID NO: 16)
 
     
       
         
           
               
               
            
               
                 CGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATT 
                   
               
               
                   
               
               
                 TATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATT 
               
               
                   
               
               
                 AATAGTAATCAATTACGGGTTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA 
               
               
                   
               
               
                 CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGA 
               
               
                   
               
               
                 CGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC 
               
               
                   
               
               
                 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA 
               
               
                   
               
               
                 TGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG 
               
               
                   
               
               
                 CCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGGTATTAGTC 
               
               
                   
               
               
                 ATCGGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCG 
               
               
                   
               
               
                 GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT 
               
               
                   
               
               
                 TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACG 
               
               
                   
               
               
                 CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT 
               
               
                   
               
               
                 GAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACA 
               
               
                   
               
               
                 CCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCC 
               
               
                   
               
               
                 GTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGCTC 
               
               
                   
               
               
                 TTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA 
               
               
                   
               
               
                 TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTC 
               
               
                   
               
               
                 CCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACT 
               
               
                   
               
               
                 ATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTAT 
               
               
                   
               
               
                 TTTTACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGT 
               
               
                   
               
               
                 CCCCCGTGCCCGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGG 
               
               
                   
               
               
                 TACCGTGTTCCGGACATGGGYTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGC 
               
               
                   
               
               
                 CCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAG 
               
               
                   
               
               
                 TGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAGTGTGCCGCACAAG 
               
               
                   
               
               
                 GCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCGCACCGC 
               
               
                   
               
               
                 TGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTT 
               
               
                   
               
               
                 GTTGTATTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAG 
               
               
                   
               
               
                 GGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAG 
               
               
                   
               
               
                 CTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCATCGAT AT   
               
               
                   
               
               
                 
                   GTTCGTGTTTCTGGTCTTGCTGCCCCTGGTGTCCAGCCAGTGCGTCAACCTGAC 
                 
               
               
                   
               
               
                 
                   AACCAGAACCCAACTGCCCCCAGCCTACACCAACTCCTTCACAAGAGGCGTGT 
                 
               
               
                   
               
               
                 
                   ATTACCCTGACAAGGTGTTTCGGAGCAGCGTGCTGCACTCCACCCAGGACTTG 
                 
               
               
                   
               
               
                 
                   TTTCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGC 
                 
               
               
                   
               
               
                 
                   ACCAATGGAACCAAGAGATTCGACAATCCTGTGCTCCCCTTCAACGACGGCGT 
                 
               
               
                   
               
               
                 
                   CTACTTCGCCAGCACCGAAAAGTCTAACATCATCAGGGGCTGGATCTTCGGCA 
                 
               
               
                   
               
               
                 
                   CAACACTGGACAGCAAGACCCAGTCCCTGCTGATTGTGAACAACGCCACAAAT 
                 
               
               
                   
               
               
                 
                   GTGGTGATCAAGGTGTGCGAATTCCAGTTTTGCAACGATCCCTTTTTGGGCGTG 
                 
               
               
                   
               
               
                 
                   TATTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAATTCCGGGTGTACAG 
                 
               
               
                   
               
               
                 
                   CAGCGCCAACAACTGTACCTTCGAATACGTGAGCCAGCCTTTCCTGATGGACCT 
                 
               
               
                   
               
               
                 
                   GGAAGGCAAACAGGGCAACTTCAAGAACCTGCGGGAATTCGTGTTCAAGAACA 
                 
               
               
                   
               
               
                 
                   TCGACGGGTACTTCAAGATCTACTCTAAGCACACCCCTATCAACCTGGTCAGAG 
                 
               
               
                   
               
               
                 
                   ACCTGCCTCAAGGCTTTAGCGCCCTGGAACCTCTGGTGGACCTGCCGATCGGC 
                 
               
               
                   
               
               
                 
                   ATTAACATCACCAGATTCCAGACACTGCTGGCTCTGCACAGATCCTACCTGACC 
                 
               
               
                   
               
               
                 
                   CCTGGCGATAGCTCCAGCGGCTGGACCGCCGGAGCTGCTGCTTACTACGTGGG 
                 
               
               
                   
               
               
                 
                   CTACCTGCAACCAAGAACCTTTCTGCTGAAGTACAACGAAAACGGCACCATCAC 
                 
               
               
                   
               
               
                 
                   AGACGCCGTGGACTGCGCCCTGGATCCTCTCAGCGAGACAAAGTGTACCCTCA 
                 
               
               
                   
               
               
                 
                   AGTCGTTCACCGTGGAAAAGGGCATATACCAGACCTCTAACTTCAGAGTGCAG 
                 
               
               
                   
               
               
                 
                   CCTACAGAGAGCATCGTAAGATTCCCTAACATCACCAACCTCTGTCCCTTTGGC 
                 
               
               
                   
               
               
                 
                   GAGGTTTTCAACGCCACCAGATTCGCCAGCGTATACGCCTGGAACAGAAAGAG 
                 
               
               
                   
               
               
                 
                   AATCTCCAATTGCGTGGCCGACTACAGCGTGCTGTACAATTCTGCATCTTTTAG 
                 
               
               
                   
               
               
                 
                   CACATTCAAATGCTACGGCGTGTCCCCAACCAAGCTAAACGACCTGTGCTTCAC 
                 
               
               
                   
               
               
                 
                   CAACGTCTACGCCGACTCATTTGTGATTCGGGGCGACGAAGTGCGCCAGATCG 
                 
               
               
                   
               
               
                 
                   CCCCTGGCCAGACCGGCAAAATCGCCGATTACAACTACAAGCTGCCAGATGAC 
                 
               
               
                   
               
               
                 
                   TTCACCGGCTGTGTGATCGCCTGGAACAGCAATAATCTGGACAGCAAGGTTGG 
                 
               
               
                   
               
               
                 
                   AGGAAACTACAACTACCTGTATCGGCTGTTCAGAAAGAGCAACCTGAAGCCTTT 
                 
               
               
                   
               
               
                 
                   CGAGCGGGACATCAGTACAGAGATCTACCAGGCTGGCTCCACGCCATGCAATG 
                 
               
               
                   
               
               
                 
                   GCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGAGCTATGGCTTCCAGCCC 
                 
               
               
                   
               
               
                 
                   ACAAACGGCGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCT 
                 
               
               
                   
               
               
                 
                   GCTTCATGCCCCTGCTACAGTCTGCGGCCCTAAGAAAAGCACCAATCTGGTGA 
                 
               
               
                   
               
               
                 
                   AAAATAAATGCGTGAACTTCAACTTTAACGGCCTGACCGGAACTGGAGTCCTTA 
                 
               
               
                   
               
               
                 
                   CCGAGAGCAACAAGAAGTTCCTGCCTTTTCAGCAGTTCGGAAGAGATATCGCC 
                 
               
               
                   
               
               
                 
                   GACACCACCGATGCCGTGCGGGATCCCCAGACCCTGGAGATCCTGGATATCAC 
                 
               
               
                   
               
               
                 
                   CCCCTGCAGCTTCGGCGGCGTGTCTGTGATCACGCCCGGCACCAACACCAGCA 
                 
               
               
                   
               
               
                 
                   ACCAGGTGGCCGTTCTGTACCAGGATGTGAATTGCACCGAGGTGCCTGTGGCC 
                 
               
               
                   
               
               
                 
                   ATCCACGCCGATCAGCTGACACCCACCTGGCGGGTGTATAGCACCGGATCTAA 
                 
               
               
                   
               
               
                 
                   TGTGTTCCAGACAAGAGCCGGATGTCTGATCGGAGCCGAACACGTGAACAATA 
                 
               
               
                   
               
               
                 
                   GCTACGAGTGTGACATCCCTATCGGCGCCGGAATCTGCGCCAGCTACCAAACA 
                 
               
               
                   
               
               
                 
                   CAGACTAACAGCCCTCGGAGAGCCAGAAGCGTGGCCTCTCAGTCAATCATCGC 
                 
               
               
                   
               
               
                 
                   CTACACCATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCA 
                 
               
               
                   
               
               
                 
                   TCGCGATTCCTACCAACTTTACCATCAGCGTTACGACAGAGATCCTGCCTGTGA 
                 
               
               
                   
               
               
                 
                   GCATGACCAAAACCTCCGTGGACTGCACAATGTACATCTGCGGCGACAGCACC 
                 
               
               
                   
               
               
                 
                   GAGTGCAGCAACCTGCTGCTGCAATACGGAAGCTTCTGCACCCAGCTGAATCG 
                 
               
               
                   
               
               
                 
                   GGCCCTGACCGGCATCGCCGTTGAACAGGACAAGAACACTCAGGAGGTGTTTG 
                 
               
               
                   
               
               
                 
                   CCCAGGTCAAGCAGATATACAAGACCCCTCCTATCAAGGACTTCGGCGGATTT 
                 
               
               
                   
               
               
                 
                   AACTTTTCTCAGATCCTGCCTGACCCCAGCAAACCTTCCAAAAGAAGCTTCATC 
                 
               
               
                   
               
               
                 
                   GAAGACCTGCTGTTCAACAAGGTGACACTCGCCGACGCCGGATTTATCAAGCA 
                 
               
               
                   
               
               
                 
                   GTACGGCGATTGCCTGGGAGACATCGCCGCTAGAGATCTGATCTGCGCCCAAA 
                 
               
               
                   
               
               
                 
                   AATTCAACGGCCTGACAGTGCTGCCTCCACTGCTGACAGACGAGATGATCGCC 
                 
               
               
                   
               
               
                 
                   CAATACACCTCTGCCCTGCTGGCCGGAACCATCACAAGCGGCTGGACCTTCGG 
                 
               
               
                   
               
               
                 
                   CGCCGGCGCAGCCCTGCAAATCCCCTTCGCCATGCAGATGGCTTATAGATTCA 
                 
               
               
                   
               
               
                 
                   ATGGCATCGGCGTCACACAGAACGTGCTGTACGAGAATCAGAAGCTGATCGCC 
                 
               
               
                   
               
               
                 
                   AACCAGTTCAACTCTGCTATCGGCAAAATCCAGGATTCACTAAGCAGCACCGCC 
                 
               
               
                   
               
               
                 
                   TCAGCCCTGGGCAAACTGCAGGATGTGGTTAATCAGAATGCCCAGGCCCTGAA 
                 
               
               
                   
               
               
                 
                   CACACTGGTGAAGCAACTGTCCAGCAATTTCGGGGCTATTAGCAGTGTGCTGA 
                 
               
               
                   
               
               
                 
                   ACGATATCCTGAGTAGGCTGGATCCACCTGAGGCCGAAGTGCAGATCGACCGG 
                 
               
               
                   
               
               
                 
                   CTCATCACAGGGAGACTGCAGTCCCTGCAGACCTACGTGACCCAGCAGCTCAT 
                 
               
               
                   
               
               
                 
                   CAGAGCTGCTGAGATACGGGCCTCTGCTAATCTGGCCGCTACCAAAATGAGCG 
                 
               
               
                   
               
               
                 
                   AGTGCGTGCTGGGCCAGTCTAAGCGGGTAGATTTCTGCGGCAAGGGCTATCAC 
                 
               
               
                   
               
               
                 
                   CTGATGAGCTTCCCACAGAGCGCTCCGCACGGCGTAGTGTTCTTACATGTGAC 
                 
               
               
                   
               
               
                 
                   ATACGTCCCTGCCCAGGAGAAGAACTTCACCACAGCTCCTGCCATCTGTCACG 
                 
               
               
                   
               
               
                 
                   ATGGCAAGGCCCACTTCCCCAGAGAGGGCGTGTTCGTGTCCAACGGCACCCAC 
                 
               
               
                   
               
               
                 
                   TGGTTCGTGACGCAGCGGAACTTCTACGAGCCTCAGATTATCACAACAGACAA 
                 
               
               
                   
               
               
                 
                   CACCTTCGTGTCTGGAAATTGCGACGTTGTCATCGGCATCGTCAACAACACCGT 
                 
               
               
                   
               
               
                 
                   GTACGACCCACTGCAGCCTGAGCTGGACAGCTTCAAGGAAGAGCTGGACAAGT 
                 
               
               
                   
               
               
                 
                   ACTTCAAGAACCACACCAGCCCCGATGTGGACCTGGGCGACATCAGCGGAATC 
                 
               
               
                   
               
               
                 
                   AACGCCTCTGTGGTGAACATCCAGAAGGAAATCGACAGACTGAACGAGGTGGC 
                 
               
               
                   
               
               
                 
                   CAAGAACCTGAATGAGTCACTTATTGACCTGCAGGAACTGGGCAAATACGAAC 
                 
               
               
                   
               
               
                 
                   AGTACATCAAATGGCCCTGGTACATCTGGCTGGGATTCATCGCTGGCCTGATC 
                 
               
               
                   
               
               
                 
                   GCCATCGTGATGGTGACAATCATGCTGTGTTGCATGACATCTTGTTGTAGCTGC 
                 
               
               
                   
               
               
                 
                   CTGAAGGGCTGCTGTAGCTGTGGCTCTTGTTGCAAGTTCGACGAGGACGACAG 
                 
               
               
                   
               
               
                   CGAGCCCGTGCTCAAGGGAGTGAAGCTGCACTATACCTAA ACCATGATATTCGG 
               
               
                   
               
               
                 CAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTT 
               
               
                   
               
               
                 GAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATC 
               
               
                   
               
               
                 CTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGC 
               
               
                   
               
               
                 TTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCAT 
               
               
                   
               
               
                 CAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGC 
               
               
                   
               
               
                 CCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGC 
               
               
                   
               
               
                 ACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTC 
               
               
                   
               
               
                 TTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCC 
               
               
                   
               
               
                 CCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCC 
               
               
                   
               
               
                 CAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCC 
               
               
                   
               
               
                 ATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATCTTGATCCC 
               
               
                   
               
               
                 CTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTC 
               
               
                   
               
               
                 CCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAA 
               
               
                   
               
               
                 ACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTG 
               
               
                   
               
               
                 CGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGC 
               
               
                   
               
               
                 ACCGTTTCTGCGGACTGGCTTTCTACGTGAAAAGGATCTAGGTGAAGATCCTTTTTG 
               
               
                   
               
               
                 ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC 
               
               
                   
               
               
                 CCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG 
               
               
                   
               
               
                 CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT 
               
               
                   
               
               
                 ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT 
               
               
                   
               
               
                 TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC 
               
               
                   
               
               
                 ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG 
               
               
                   
               
               
                 TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT 
               
               
                   
               
               
                 GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCCGAACT 
               
               
                   
               
               
                 GAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG 
               
               
                   
               
               
                 CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT 
               
               
                   
               
               
                 TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTT 
               
               
                   
               
               
                 GAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAG 
               
               
                   
               
               
                 CAACGCGGCCCTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTGT 
               
            
           
         
       
     
     DNA Construct 3: DNA/S-Tri-Sec 
       
                                     Name   Range   Description                  CMV Promoter   476 to 675   Human cytomegalovirus (CMV)                immediate early promoter       intron A    702 to 1645   Intron A positively regulate                expression from the hCMV                immediate-early enhancer/promoter       ORF   1651 to 5379   Insert nCoV S-Tri-Sec (GMCSF                ss (17 aa)-Spike aa 14 to 1208                (1195 aa)-FOLD ON (30 aa))       bGH poly(A)    5419 to 5626   Bovine growth hormone        signal       polyadenylation signal       Kanamycin    6775 to 7569   Confers resistance to neomycin,        resistance gene       kanamycin, and               G418 (Geneticin(R))       Replication    6865 to 7454   High-copy-number        Origin       ColE1/pMB1/pBR322/pUC                origin of replication                    
pGA8-nCoV S-Tri-sec Plasmid sequences (SEQ ID NO: 17) and Sequence encoding spike protein (bold, SEQ ID NO: 18)
 
     
       
         
           
               
               
            
               
                 CGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATT 
                   
               
               
                   
               
               
                 TATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATT 
               
               
                   
               
               
                 AATAGTAATCAATTACGGGTTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA 
               
               
                   
               
               
                 CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGA 
               
               
                   
               
               
                 CGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC 
               
               
                   
               
               
                 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA 
               
               
                   
               
               
                 TGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG 
               
               
                   
               
               
                 CCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGGTATTAGTC 
               
               
                   
               
               
                 ATCGGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCG 
               
               
                   
               
               
                 GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT 
               
               
                   
               
               
                 TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACG 
               
               
                   
               
               
                 CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT 
               
               
                   
               
               
                 GAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACA 
               
               
                   
               
               
                 CCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCC 
               
               
                   
               
               
                 GTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGCTC 
               
               
                   
               
               
                 TTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA 
               
               
                   
               
               
                 TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTC 
               
               
                   
               
               
                 CCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACT 
               
               
                   
               
               
                 ATCTCTATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTAT 
               
               
                   
               
               
                 TTTTACAGGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGT 
               
               
                   
               
               
                 CCCCCGTGCCCGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGG 
               
               
                   
               
               
                 TACCGTGTTCCGGACATGGGYTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGC 
               
               
                   
               
               
                 CCTGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAG 
               
               
                   
               
               
                 TGGAGGCCAGACTTAGGCACAGCACAATGCCCACCACCACCAGTGTGCCGCACAAG 
               
               
                   
               
               
                 GCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGAGATTGGGCTCGCACCGC 
               
               
                   
               
               
                 TGACGCAGATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTT 
               
               
                   
               
               
                 GTTGTATTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAG 
               
               
                   
               
               
                 GGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAG 
               
               
                   
               
               
                 CTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCATCGAT AT   
               
               
                   
               
               
                 
                   GTGGCTGCAGGGCCTGCTGCTGCTGGGCACCGTGGCATGCAGTATCAGCCAAT 
                 
               
               
                   
               
               
                 
                   GTGTGAACCTGACCACCAGAACCCAACTGCCTCCTGCCTACACCAACTCTTTCA 
                 
               
               
                   
               
               
                 
                   CCAGAGGCGTGTACTACCCTGACAAGGTGTTCAGAAGCAGCGTGCTGCATTCT 
                 
               
               
                   
               
               
                 
                   ACCCAGGACCTGTTTCTGCCATTCTTCAGCAACGTCACCTGGTTCCACGCCATC 
                 
               
               
                   
               
               
                 
                   CACGTGTCTGGCACCAATGGCACTAAGAGATTCGACAACCCCGTGCTGCCTTT 
                 
               
               
                   
               
               
                 
                   CAACGACGGCGTGTACTTTGCCTCAACTGAGAAGAGCAACATCATCAGAGGAT 
                 
               
               
                   
               
               
                 
                   GGATCTTCGGCACCACACTTGACTCAAAGACACAGTCACTGCTGATCGTGAAC 
                 
               
               
                   
               
               
                 
                   AATGCTACCAATGTGGTGATCAAGGTGTGTGAATTCCAGTTTTGCAACGATCCT 
                 
               
               
                   
               
               
                 
                   TTCCTGGGTGTATACTACCACAAGAACAACAAGTCTTGGATGGAGAGCGAGTT 
                 
               
               
                   
               
               
                 
                   CCGGGTGTATAGTAGCGCCAACAACTGCACCTTCGAATACGTGAGCCAGCCTT 
                 
               
               
                   
               
               
                 
                   TCCTCATGGACCTGGAAGGCAAGCAAGGCAACTTCAAGAACCTGAGAGAGTTC 
                 
               
               
                   
               
               
                 
                   GTGTTTAAGAACATTGATGGCTACTTCAAGATCTACAGCAAGCACACCCCCATC 
                 
               
               
                   
               
               
                 
                   AACCTGGTGCGGGACCTCCCTCAGGGCTTCAGCGCCCTGGAACCCTTGGTTGA 
                 
               
               
                   
               
               
                 
                   TCTGCCAATTGGCATCAATATCACTCGGTTCCAAACCCTGCTGGCCCTGCACAG 
                 
               
               
                   
               
               
                 
                   AAGCTATCTGACACCTGGAGACAGCAGCAGCGGCTGGACCGCCGGAGCCGCC 
                 
               
               
                   
               
               
                 
                   GCCTACTACGTGGGCTACCTGCAGCCCCGGACCTTCCTGCTGAAGTACAACGA 
                 
               
               
                   
               
               
                 
                   GAACGGGACCATTACCGACGCCGTCGACTGCGCCCTGGATCCTCTGAGCGAAA 
                 
               
               
                   
               
               
                 
                   CCAAGTGCACACTTAAAAGCTTCACAGTGGAGAAGGGCATCTACCAAACCTCC 
                 
               
               
                   
               
               
                 
                   AATTTTCGGGTCCAGCCAACCGAGAGCATCGTTAGATTCCCCAACATCACCAAC 
                 
               
               
                   
               
               
                 
                   TTGTGCCCCTTCGGAGAAGTGTTCAACGCCACAAGATTCGCCAGCGTCTACGC 
                 
               
               
                   
               
               
                 
                   CTGGAACAGAAAGAGAATTTCCAATTGCGTCGCAGACTACTCTGTGCTGTACAA 
                 
               
               
                   
               
               
                 
                   CAGCGCCAGCTTTTCTACATTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGA 
                 
               
               
                   
               
               
                 
                   ACGACCTATGCTTCACAAACGTGTACGCCGACAGCTTTGTGATCCGGGGCGAC 
                 
               
               
                   
               
               
                 
                   GAGGTGCGCCAGATCGCGCCAGGACAGACCGGTAAGATCGCCGATTACAATTA 
                 
               
               
                   
               
               
                 
                   CAAACTGCCTGACGACTTCACCGGCTGCGTCATCGCTTGGAACAGCAACAACC 
                 
               
               
                   
               
               
                 
                   TGGACTCTAAGGTGGGCGGAAACTACAACTACCTGTACCGGCTGTTTAGAAAG 
                 
               
               
                   
               
               
                 
                   AGCAACCTGAAGCCTTTTGAACGGGACATCTCTACAGAGATCTACCAGGCCGG 
                 
               
               
                   
               
               
                 
                   ATCTACCCCTTGTAATGGCGTGGAGGGCTTTAATTGCTACTTCCCCCTGCAATC 
                 
               
               
                   
               
               
                 
                   GTACGGCTTCCAGCCGACAAACGGCGTCGGCTACCAGCCTTACAGAGTGGTGG 
                 
               
               
                   
               
               
                 
                   TCCTGTCCTTCGAGCTGCTGCATGCCCCTGCTACAGTGTGCGGCCCTAAGAAA 
                 
               
               
                   
               
               
                 
                   AGCACCAACCTGGTGAAGAACAAGTGTGTGAACTTCAATTTCAATGGCCTGACT 
                 
               
               
                   
               
               
                 
                   GGCACCGGAGTGCTGACCGAATCCAACAAGAAGTTCCTGCCCTTCCAGCAGTT 
                 
               
               
                   
               
               
                 
                   CGGCAGAGACATCGCAGACACTACCGATGCTGTGCGGGATCCTCAGACACTGG 
                 
               
               
                   
               
               
                 
                   AGATCCTGGATATCACCCCCTGCAGCTTCGGAGGCGTGAGCGTGATCACACCT 
                 
               
               
                   
               
               
                 
                   GGCACAAACACATCCAACCAGGTGGCCGTGCTGTACCAGGATGTGAACTGCAC 
                 
               
               
                   
               
               
                 AGAAGTGCCGGTTGCCATCCACGCCGATCAGCTCACACCTACTTGGCGGGTGT 
               
               
                   
               
               
                 
                   ACTCCACAGGCAGCAACGTGTTCCAAACCAGAGCTGGCTGTCTGATCGGCGCT 
                 
               
               
                   
               
               
                 
                   GAACACGTGAACAATAGCTATGAGTGCGACATCCCAATCGGCGCCGGTATCTG 
                 
               
               
                   
               
               
                 
                   CGCCTCCTATCAGACGCAGACGAACAGCCCTAGGCGGGCTAGAAGCGTGGCCA 
                 
               
               
                   
               
               
                 
                   GCCAGAGCATCATCGCATATACAATGAGCCTGGGCGCCGAAAACTCTGTCGCC 
                 
               
               
                   
               
               
                 
                   TACAGCAACAACAGCATCGCTATCCCTACCAACTTCACCATAAGCGTAACAACC 
                 
               
               
                   
               
               
                 
                   GAGATCCTGCCTGTGTCCATGACAAAGACCAGCGTGGACTGTACAATGTACAT 
                 
               
               
                   
               
               
                 
                   CTGTGGCGACTCCACCGAGTGCAGCAACCTGCTCCTGCAATACGGCTCTTTCT 
                 
               
               
                   
               
               
                 
                   GCACCCAGCTGAATCGCGCCTTAACAGGCATTGCCGTGGAACAGGATAAGAAC 
                 
               
               
                   
               
               
                 
                   ACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTATAAGACCCCACCCATCAA 
                 
               
               
                   
               
               
                 
                   GGACTTCGGCGGATTCAATTTCAGTCAAATCCTGCCCGATCCTAGCAAGCCCA 
                 
               
               
                   
               
               
                 
                   GTAAGAGATCTTTCATCGAGGACCTGCTTTTCAACAAAGTGACCCTGGCGGAC 
                 
               
               
                   
               
               
                 
                   GCCGGATTTATCAAACAGTACGGCGACTGTCTGGGCGACATCGCCGCTAGAGA 
                 
               
               
                   
               
               
                 
                   TCTGATCTGCGCCCAGAAATTCAACGGCCTGACGGTGCTGCCTCCTCTGCTGA 
                 
               
               
                   
               
               
                 
                   CAGATGAGATGATCGCCCAGTATACCAGCGCCCTGCTGGCTGGAACCATCACC 
                 
               
               
                   
               
               
                 
                   TCTGGCTGGACATTTGGCGCCGGTGCCGCTCTCCAGATCCCCTTTGCCATGCA 
                 
               
               
                   
               
               
                 
                   GATGGCCTACAGATTCAATGGAATCGGCGTGACCCAGAACGTGCTGTACGAGA 
                 
               
               
                   
               
               
                 
                   ACCAGAAGCTGATCGCTAATCAGTTCAACTCTGCCATTGGCAAAATCCAGGACA 
                 
               
               
                   
               
               
                 
                   GCCTGTCTTCCACCGCCAGCGCCCTGGGCAAACTGCAAGACGTGGTGAATCAA 
                 
               
               
                   
               
               
                 
                   AACGCCCAGGCCCTGAACACTCTGGTGAAGCAGCTGTCCAGCAACTTCGGAGC 
                 
               
               
                   
               
               
                 
                   CATCAGCAGCGTGCTGAACGACATACTGAGCAGACTGGACCCTCCGGAGGCCG 
                 
               
               
                   
               
               
                 
                   AGGTGCAGATCGACAGGCTGATCACAGGCAGACTGCAGAGCCTGCAGACCTAC 
                 
               
               
                   
               
               
                 
                   GTCACACAGCAGCTGATCAGAGCCGCTGAGATCCGAGCTTCTGCCAATCTCGC 
                 
               
               
                   
               
               
                 
                   CGCGACAAAGATGTCTGAGTGCGTGCTCGGCCAGAGCAAAAGAGTGGATTTCT 
                 
               
               
                   
               
               
                 
                   GCGGAAAAGGCTATCACCTGATGAGCTTCCCTCAGTCTGCCCCACACGGCGTC 
                 
               
               
                   
               
               
                 
                   GTGTTCCTGCACGTGACCTACGTGCCTGCCCAGGAAAAAAACTTTACCACCGC 
                 
               
               
                   
               
               
                 
                   CCCGGCCATCTGCCACGACGGCAAGGCCCACTTCCCTAGAGAAGGCGTGTTCG 
                 
               
               
                   
               
               
                 
                   TGAGCAATGGCACCCACTGGTTCGTGACACAAAGAAACTTCTACGAGCCTCAA 
                 
               
               
                   
               
               
                 
                   ATCATCACAACAGATAACACCTTCGTGTCAGGCAACTGTGACGTGGTCATCGG 
                 
               
               
                   
               
               
                 
                   AATCGTGAATAATACCGTGTACGACCCCCTGCAGCCTGAACTGGACAGCTTTAA 
                 
               
               
                   
               
               
                 
                   GGAGGAACTGGACAAGTACTTCAAAAACCACACATCTCCTGATGTGGACCTGG 
                 
               
               
                   
               
               
                 
                   GGGATATCAGCGGCATCAACGCTTCTGTGGTGAACATCCAGAAGGAAATCGAC 
                 
               
               
                   
               
               
                 
                   AGACTGAACGAGGTGGCCAAGAATCTCAACGAAAGCCTCATTGACCTTCAGGA 
                 
               
               
                   
               
               
                 
                   GCTGGGGAAGTACGAGCAGGGCTCTGCCGGCTACATCCCTGAGGCTCCTAGGG 
                 
               
               
                   
               
               
                 
                   ACGGCCAGGCCTACGTGCGGAAGGACGGGGAGTGGGTGCTGCTGAGCACATT 
                 
               
               
                   
               
               
                   CCTGTAA ACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCT 
               
               
                   
               
               
                 CGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCT 
               
               
                   
               
               
                 GATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTG 
               
               
                   
               
               
                 CTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCG 
               
               
                   
               
               
                 TATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGT 
               
               
                   
               
               
                 GAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCG 
               
               
                   
               
               
                 CTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCAC 
               
               
                   
               
               
                 GATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTG 
               
               
                   
               
               
                 ACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGC 
               
               
                   
               
               
                 AGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCG 
               
               
                   
               
               
                 GAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCT 
               
               
                   
               
               
                 CTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCC 
               
               
                   
               
               
                 AGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCG 
               
               
                   
               
               
                 GTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGC 
               
               
                   
               
               
                 AAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCT 
               
               
                   
               
               
                 GACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGAAAAGGAT 
               
               
                   
               
               
                 CTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG 
               
               
                   
               
               
                 TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT 
               
               
                   
               
               
                 TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT 
               
               
                   
               
               
                 TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA 
               
               
                   
               
               
                 GCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG 
               
               
                   
               
               
                 AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT 
               
               
                   
               
               
                 GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT 
               
               
                   
               
               
                 AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG 
               
               
                   
               
               
                 AACGACCTACACCCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG 
               
               
                   
               
               
                 CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG 
               
               
                   
               
               
                 GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC 
               
               
                   
               
               
                 GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG 
               
               
                   
               
               
                 AGCCTATGGAAAAACGCCAGCAACGCGGCCCTTTTACGGTTCCTGGCCTTTTGCTGG 
               
               
                   
               
               
                 CCTTTTGCTCACATGTTGT 
               
            
           
         
       
     
     DNA Construct 4: DNA/S1-Mono 
       
                                     Name   Range   Description                  CMV Promoter   476 to 675   Human cytomegalovirus (CMV)               immediate early promoter       Intron A    702 to 1645   Intron A positively regulate               expression from the hCMV               immediate-early               enhancer/promoter       GM-CSF signal    1651 to 1761   Secreted, signal peptide       sequences               ORF   1651 to 4005   Insert nCoV GM-CSF S1 soluble       bGH poly(A)    4045 to 4252   Bovine growth hormone       signal       polyadenylation signal       Kanamycin    4285 to 5079   Confers resistance to neomycin,       resistance gene       kanamycin, and G418               (Geneticin(R))       Replication    5491 to 6080   High-copy-number       Origin       ColE1/pMB1/pBR322/pUC                origin of replication                    
pGA8-nCoV GMCSF-S1 Plasmid sequences (SEQ ID NO: 19) and Sequence encoding spike protein (bold, SEQ ID NO: 20)
 
     
       
         
           
               
               
            
               
                   
                 CGACAATATTGGCTATTGGCCATTGCATACGTTGT 
               
               
                   
               
               
                   
                 ATCTATATCATAATATGTACATTTATATTGGCTCA 
               
               
                   
               
               
                   
                 TGTCCAATATGACCGCCATGTTGACATTGATTATT 
               
               
                   
               
               
                   
                 GACTAGTTATTAATAGTAATCAATTACGGGTTCAT 
               
               
                   
               
               
                   
                 TAGTTCATAGCCCATATATGGAGTTCCGCGTTACA 
               
               
                   
               
               
                   
                 TAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC 
               
               
                   
               
               
                   
                 CAACGACCCCCGCCCATTGACGTCAATAATGACGT 
               
               
                   
               
               
                   
                 ATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT 
               
               
                   
               
               
                   
                 TGACGTCAATGGGTGGAGTATTTACGGTAAACTGC 
               
               
                   
               
               
                   
                 CCACTTGGCAGTACATCAAGTGTATCATATGCCAA 
               
               
                   
               
               
                   
                 GTCCGCCCCCTATTGACGTCAATGACGGTAAATGG 
               
               
                   
               
               
                   
                 CCCGCCTGGCATTATGCCCAGTACATGACCTTACG 
               
               
                   
               
               
                   
                 GGACTTTCCTACTTGGCAGTACATCTACGGTATTA 
               
               
                   
               
               
                   
                 GTCATCGGCTATTACCATGGTGATGCGGTTTTGGC 
               
               
                   
               
               
                   
                 AGTACACCAATGGGCGTGGATAGCGGTTTGACTCA 
               
               
                   
               
               
                   
                 CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA 
               
               
                   
               
               
                   
                 TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT 
               
               
                   
               
               
                   
                 TTCCAAAATGTCGTAATAACCCCGCCCCGTTGACG 
               
               
                   
               
               
                   
                 CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTA 
               
               
                   
               
               
                   
                 TATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG 
               
               
                   
               
               
                   
                 CCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT 
               
               
                   
               
               
                   
                 AGAAGACACCGGGACCGATCCAGCCTCCGCGGCCG 
               
               
                   
               
               
                   
                 GGAACGGTGCATTGGAACGCGGATTCCCCGTGCCA 
               
               
                   
               
               
                   
                 AGAGTGACGTAAGTACCGCCTATAGACTCTATAGG 
               
               
                   
               
               
                   
                 CACACCCCTTTGGCTCTTATGCATGCTATACTGTT 
               
               
                   
               
               
                   
                 TTTGGCTTGGGGCCTATACACCCCCGCTTCCTTAT 
               
               
                   
               
               
                   
                 GCTATAGGTGATGGTATAGCTTAGCCTATAGGTGT 
               
               
                   
               
               
                   
                 GGGTTATTGACCATTATTGACCACTCCCCTATTGG 
               
               
                   
               
               
                   
                 TGACGATACTTTCCATTACTAATCCATAACATGGC 
               
               
                   
               
               
                   
                 TCTTTGCCACAACTATCTCTATTGGCTATATGCCA 
               
               
                   
               
               
                   
                 ATACTCTGTCCTTCAGAGACTGACACGGACTCTGT 
               
               
                   
               
               
                   
                 ATTTTTACAGGATGGGGTCCCATTTATTATTTACA 
               
               
                   
               
               
                   
                 AATTCACATATACAACAACGCCGTCCCCCGTGCCC 
               
               
                   
               
               
                   
                 GCAGTTTTTATTAAACATAGCGTGGGATCTCCACG 
               
               
                   
               
               
                   
                 CGAATCTCGGGTACCGTGTTCCGGACATGGGYTCT 
               
               
                   
               
               
                   
                 TCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCC 
               
               
                   
               
               
                   
                 TGGTCCCATGCCTCCAGCGGCTCATGGTCGCTCGG 
               
               
                   
               
               
                   
                 CAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTA 
               
               
                   
               
               
                   
                 GGCACAGCACAATGCCCACCACCACCAGTGTGCCG 
               
               
                   
               
               
                   
                 CACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAA 
               
               
                   
               
               
                   
                 TGAGCTCGGAGATTGGGCTCGCACCGCTGACGCAG 
               
               
                   
               
               
                   
                 ATGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCA 
               
               
                   
               
               
                   
                 GGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGA 
               
               
                   
               
               
                   
                 GGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGG 
               
               
                   
               
               
                   
                 GCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG 
               
               
                   
               
               
                   
                 CGCGCCACCAGACATAATAGCTGACAGACTAACAG 
               
               
                   
               
               
                   
                 ACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACC 
               
               
                   
               
               
                   
                 ATCGAT ATGTGGCTGCAGGGGCTGCTGCTGCTGGG   
               
               
                   
               
               
                   
                 
                   AACCGTGGCTTGCTCCATTTCTCAGTGCGTCAATC 
                 
               
               
                   
               
               
                   
                 
                   TGACTACCCGAACTCAGCTGCCCCCCGCCTACACC 
                 
               
               
                   
               
               
                   
                 
                   AACTCCTTCACCCGGGGCGTGTACTATCCAGACAA 
                 
               
               
                   
               
               
                   
                 
                   GGTGTTTAGAAGCTCCGTGCTGCACTCCACCCAGG 
                 
               
               
                   
               
               
                   
                 
                   ATCTGTTTCTGCCCTTCTTTTCTAATGTGACATGG 
                 
               
               
                   
               
               
                   
                 
                   TTCCACGCCATCCACGTGAGCGGCACCAACGGCAC 
                 
               
               
                   
               
               
                   
                 
                   AAAGAGGTTCGACAACCCTGTGCTGCCATTCAATG 
                 
               
               
                   
               
               
                   
                 
                   ATGGCGTGTACTTTGCCTCCACCGAGAAGTCTAAC 
                 
               
               
                   
               
               
                   
                 
                   ATCATCCGCGGCTGGATCTTTGGCACCACACTGGA 
                 
               
               
                   
               
               
                   
                 
                   CTCCAAGACCCAGTCCCTGCTGATCGTGAACAATG 
                 
               
               
                   
               
               
                   
                 
                   CCACAAACGTGGTCATCAAGGTGTGCGAGTTCCAG 
                 
               
               
                   
               
               
                   
                 
                   TTTTGTAACGATCCTTTCCTGGGCGTGTACTATCA 
                 
               
               
                   
               
               
                   
                 
                   CAAGAACAATAAGTCTTGGATGGAGAGCGAGTTTA 
                 
               
               
                   
               
               
                   
                 
                   GGGTGTATTCTAGCGCCAACAATTGCACCTTCGAG 
                 
               
               
                   
               
               
                   
                 
                   TACGTGTCCCAGCCATTTCTGATGGACCTGGAGGG 
                 
               
               
                   
               
               
                   
                 
                   CAAGCAGGGCAATTTCAAGAACCTGCGGGAGTTCG 
                 
               
               
                   
               
               
                   
                 
                   TGTTTAAGAACATCGACGGCTACTTCAAGATCTAC 
                 
               
               
                   
               
               
                   
                 
                   TCCAAGCACACCCCCATCAACCTGGTGCGGGACCT 
                 
               
               
                   
               
               
                   
                 
                   GCCACAGGGCTTCTCTGCCCTGGAGCCTCTGGTGG 
                 
               
               
                   
               
               
                   
                 
                   ATCTGCCAATCGGCATCAACATCACACGGTTTCAG 
                 
               
               
                   
               
               
                   
                 
                   ACCCTGCTGGCCCTGCACAGAAGCTACCTGACCCC 
                 
               
               
                   
               
               
                   
                 
                   TGGCGACTCCTCTAGCGGATGGACAGCAGGAGCAG 
                 
               
               
                   
               
               
                   
                 
                   CAGCATACTATGTGGGCTATCTGCAGCCACGGACC 
                 
               
               
                   
               
               
                   
                 
                   TTCCTGCTGAAGTACAACGAGAATGGCACCATCAC 
                 
               
               
                   
               
               
                   
                 
                   AGACGCCGTGGATTGCGCCCTGGATCCACTGTCTG 
                 
               
               
                   
               
               
                   
                 
                   AGACAAAGTGTACACTGAAGAGCTTTACAGTGGAG 
                 
               
               
                   
               
               
                   
                 
                   AAGGGCATCTATCAGACCAGCAACTTCAGGGTGCA 
                 
               
               
                   
               
               
                   
                 
                   GCCCACAGAGTCCATCGTGCGCTTTCCAAATATCA 
                 
               
               
                   
               
               
                   
                 
                   CCAACCTGTGCCCCTTCGGCGAGGTGTTTAATGCC 
                 
               
               
                   
               
               
                   
                 
                   ACAAGATTCGCCAGCGTGTACGCCTGGAACAGGAA 
                 
               
               
                   
               
               
                   
                 
                   GCGCATCTCCAATTGCGTGGCCGACTATTCTGTGC 
                 
               
               
                   
               
               
                   
                 
                   TGTACAACTCTGCCAGCTTCTCCACCTTTAAGTGC 
                 
               
               
                   
               
               
                   
                 
                   TATGGCGTGAGCCCCACCAAGCTGAACGACCTGTG 
                 
               
               
                   
               
               
                   
                 
                   CTTCACAAACGTGTACGCCGATTCCTTTGTGATCA 
                 
               
               
                   
               
               
                   
                 
                   GGGGCGACGAGGTGCGCCAGATCGCACCAGGACAG 
                 
               
               
                   
               
               
                   
                 
                   ACCGGCAAGATCGCAGACTACAACTATAAGCTGCC 
                 
               
               
                   
               
               
                   
                 
                   CGACGATTTCACAGGCTGCGTGATCGCCTGGAATT 
                 
               
               
                   
               
               
                   
                 
                   CCAACAATCTGGATTCTAAAGTGGGCGGCAACTAC 
                 
               
               
                   
               
               
                   
                 
                   AATTATCTGTACAGGCTGTTCCGCAAGTCTAACCT 
                 
               
               
                   
               
               
                   
                 
                   GAAGCCTTTTGAGCGGGACATCTCCACCGAGATCT 
                 
               
               
                   
               
               
                   
                 
                   ACCAGGCCGGCTCTACACCATGCAACGGCGTGGAG 
                 
               
               
                   
               
               
                   
                 
                   GGCTTCAATTGTTATTTTCCCCTGCAGAGCTACGG 
                 
               
               
                   
               
               
                   
                 
                   CTTCCAGCCTACCAATGGCGTGGGCTATCAGCCAT 
                 
               
               
                   
               
               
                   
                 
                   ACAGAGTGGTGGTGCTGTCTTTTGAGCTGCTGCAC 
                 
               
               
                   
               
               
                   
                 
                   GCACCAGCAACCGTGTGCGGACCTAAGAAGAGCAC 
                 
               
               
                   
               
               
                   
                 
                   AAATCTGGTGAAGAACAAGTGCGTGAACTTCAACT 
                 
               
               
                   
               
               
                   
                 
                   TCAACGGCCTGACCGGAACAGGCGTGCTGACCGAG 
                 
               
               
                   
               
               
                   
                 
                   TCCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGG 
                 
               
               
                   
               
               
                   
                 
                   CAGGGACATCGCAGATACCACAGACGCCGTGCGCG 
                 
               
               
                   
               
               
                   
                 
                   ACCCCCAGACACTGGAGATCCTGGATATCACCCCT 
                 
               
               
                   
               
               
                   
                 
                   TGCAGCTTCGGCGGCGTGTCCGTGATCACCCCTGG 
                 
               
               
                   
               
               
                   
                 
                   AACCAATACAAGCAACCAGGTGGCCGTGCTGTATC 
                 
               
               
                   
               
               
                   
                 
                   AGGACGTGAACTGTACAGAGGTGCCAGTGGCCATC 
                 
               
               
                   
               
               
                   
                 
                   CACGCCGATCAGCTGACCCCCACATGGCGGGTGTA 
                 
               
               
                   
               
               
                   
                 
                   CTCCACAGGCTCTAACGTGTTCCAGACCAGAGCAG 
                 
               
               
                   
               
               
                   
                 
                   GATGCCTGATCGGAGCAGAGCACGTGAACAATAGC 
                 
               
               
                   
               
               
                   
                 
                   TATGAGTGCGACATCCCCATCGGCGCCGGCATCTG 
                 
               
               
                   
               
               
                   
                 
                   TGCCTCCTACCAGACCCAGACAAACTCCCCTCGGA 
                 
               
               
                   
               
               
                   
                 
                   GAGCCAGGTCTGTGGCCTCTCAGAGCATCATCGCC 
                 
               
               
                   
               
               
                   
                 
                   TATACCATGAGCCTGGGCGCCGAGAACTCCGTGGC 
                 
               
               
                   
               
               
                   
                 
                   CTACAGCAACAATTCCATCGCCATCCCCACCAATT 
                 
               
               
                   
               
               
                   
                 
                   TCACAATCTCCGTGACCACAGAGATCCTGCCCGTG 
                 
               
               
                   
               
               
                   
                 
                   AGCATGACCAAGACAAGCGTGGACTGCACCATGTA 
                 
               
               
                   
               
               
                   
                 
                   TATCTGTGGCGATTCCACAGAGTGCTCTAATCTGC 
                 
               
               
                   
               
               
                   
                 
                   TGCTGCAGTACGGCTCTTTTTGTACACAGCTGAAC 
                 
               
               
                   
               
               
                   
                 
                   CGCGCCCTGACCGGAATCGCAGTGGAGCAGGACAA 
                 
               
               
                   
               
               
                   
                   GAATACCCAGGAGTAA ACCATGATATTCGGCAAGC 
               
               
                   
               
               
                   
                 AGGCATCGCCATGGGTCACGACGAGATCCTCGCCG 
               
               
                   
               
               
                   
                 TCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTC 
               
               
                   
               
               
                   
                 GGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGAT 
               
               
                   
               
               
                   
                 CATCCTGATCGACAAGACCGGCTTCCATCCGAGTA 
               
               
                   
               
               
                   
                 CGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTC 
               
               
                   
               
               
                   
                 GAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCC 
               
               
                   
               
               
                   
                 GCCGCATTGCATCAGCCATGATGGATACTTTCTCG 
               
               
                   
               
               
                   
                 GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCC 
               
               
                   
               
               
                   
                 CGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCG 
               
               
                   
               
               
                   
                 CTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGA 
               
               
                   
               
               
                   
                 ACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGC 
               
               
                   
               
               
                   
                 CTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGT 
               
               
                   
               
               
                   
                 CGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCT 
               
               
                   
               
               
                   
                 GACAGCCGGAACACGGCGGCATCAGAGCAGCCGAT 
               
               
                   
               
               
                   
                 TGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCT 
               
               
                   
               
               
                   
                 CCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCA 
               
               
                   
               
               
                   
                 TCTTGTTCAATCATGCGAAACGATCCTCATCCTGT 
               
               
                   
               
               
                   
                 CTCTTGATCAGATCTTGATCCCCTGCGCCATCAGA 
               
               
                   
               
               
                   
                 TCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTG 
               
               
                   
               
               
                   
                 CAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGC 
               
               
                   
               
               
                   
                 TGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCG 
               
               
                   
               
               
                   
                 CCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAA 
               
               
                   
               
               
                   
                 GCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCT 
               
               
                   
               
               
                   
                 TGTCCAGATAGCCCAGTAGCTGACATTCATCCGGG 
               
               
                   
               
               
                   
                 GTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTG 
               
               
                   
               
               
                   
                 AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT 
               
               
                   
               
               
                   
                 CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC 
               
               
                   
               
               
                   
                 ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA 
               
               
                   
               
               
                   
                 TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTG 
               
               
                   
               
               
                   
                 CTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG 
               
               
                   
               
               
                   
                 TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT 
               
               
                   
               
               
                   
                 TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT 
               
               
                   
               
               
                   
                 ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAG 
               
               
                   
               
               
                   
                 GCCACCACTTCAAGAACTCTGTAGCACCGCCTACA 
               
               
                   
               
               
                   
                 TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC 
               
               
                   
               
               
                   
                 TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG 
               
               
                   
               
               
                   
                 ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGG 
               
               
                   
               
               
                   
                 TCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAG 
               
               
                   
               
               
                   
                 CTTGGAGCGAACGACCTACACCCGAACTGAGATAC 
               
               
                   
               
               
                   
                 CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC 
               
               
                   
               
               
                   
                 CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG 
               
               
                   
               
               
                   
                 GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT 
               
               
                   
               
               
                   
                 CCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT 
               
               
                   
               
               
                   
                 CGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT 
               
               
                   
               
               
                   
                 TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA 
               
               
                   
               
               
                   
                 AACGCCAGCAACGCGGCCCTTTTACGGTTCCTGGC 
               
               
                   
               
               
                   
                 CTTTTGCTGGCCTTTTGCTCACATGTTGT 
               
            
           
         
       
     
     Chimera T Cell Immunogens and Vaccination Approach to Generate Broadly Cross-Reactive T Cells 
     DNA based T cell chimera antigens we have designed encoding proteins derived from 6 genes of SARS-CoV-2 i.e., Spike, N, M, NSP3, NSP4 and NSP6. These proteins have been chosen because: 1) they show strong conservation between multiple human betacoronaviruses; and 2) they account for greater than 90% of the CoV2 specific T cell response observed in SARS-CoV-2 infected individuals. Chimera 1 carry immunodominant T cell epitopes of spike glycoprotein (S), nucleocapsid (N) and membrane (M) proteins. The other construct has regions derived from non-structural regions, expressed during virus active replication and translation. This includes non-structural protein 3 (NSP3), NSP4, and NSP6. about 66% of the N terminal region of the NSP3 protein was deleted since this region contains peptide sequences and functional domains which can disrupt the process of epitope processing and presentation. These regions include nucleic acid binding domains, viral proteinase activity domains and autophagy modulating domains. However, the remaining C-terminal region contains three immunodominant CD8 T cell epitopes that are conserved in SARS-CoV and CoV2. Versions of each construct lacking transmembrane (TM) regions were developed providing the four chimera constructs (d/delta is deleted): Chimera 1 (SdRBD-N-M) and 3 (NSP3-4-6), and the chimeras lacking TM, Chimera 2 (SdRBD-N-M_dTM) and 4 (NSP3-4-6_dTM), respectively. 
     The RBD region (major target of neutralization) was deleted from the S protein to avoid antibody response to this region induced by improperly folded chimeric protein which could interfere with the neutralizing antibody responses induced by properly folded RBD protein immunogen. Two versions for each construct are provided, one with and the other without the transmembrane regions from S, M, Nsp3, Nsp4 and Nsp6 proteins in order to compare their ability to induce T cell responses. The chimeric proteins without transmembrane regions are expected to be localized to the cytoplasm and will be susceptible to degradation by proteasomes. This could potentially promote class I HLA epitope presentation to generate CD8 T cell response. In addition, these chimeric proteins expressed as fusion proteins and do not have secretory signals facilitating the priming of T cell response as opposed to antibody response with the idea that the expressed chimeric proteins may not retain the proper conformation to generate a neutralizing antibody response. These chimeric immunogens that are designed to induce a broad CD4 and CD8 T cell response with cross-reactivity to other coronaviruses by use in combination with the other DNA prime/MVA boost strategies reported herein providing an improved T cell response. 
     Chimera 1: SdRBD-N-M 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 24) 
               
               
                   
                 MCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQD 
               
               
                   
                   
               
               
                   
                 LFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFA 
               
               
                   
                   
               
               
                   
                 STEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEF 
               
               
                   
                   
               
               
                   
                 QFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQ 
               
               
                   
                   
               
               
                   
                 PFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLV 
               
               
                   
                   
               
               
                   
                 RDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGD 
               
               
                   
                   
               
               
                   
                 SSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL 
               
               
                   
                   
               
               
                   
                 DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFATVC 
               
               
                   
                   
               
               
                   
                 GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQF 
               
               
                   
                   
               
               
                   
                 GRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS 
               
               
                   
                   
               
               
                   
                 NQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQT 
               
               
                   
                   
               
               
                   
                 RAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAG 
               
               
                   
                   
               
               
                   
                 SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE 
               
               
                   
                   
               
               
                   
                 ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR 
               
               
                   
                   
               
               
                   
                 ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQI 
               
               
                   
                   
               
               
                   
                 LPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA 
               
               
                   
                   
               
               
                   
                 ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG 
               
               
                   
                   
               
               
                   
                 WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIAN 
               
               
                   
                   
               
               
                   
                 QFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ 
               
               
                   
                   
               
               
                   
                 LSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQ 
               
               
                   
                   
               
               
                   
                 TYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGK 
               
               
                   
                   
               
               
                   
                 GYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG 
               
               
                   
                   
               
               
                   
                 KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN 
               
               
                   
                   
               
               
                   
                 CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD 
               
               
                   
                   
               
               
                   
                 LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK 
               
               
                   
                   
               
               
                   
                 YEQSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSK 
               
               
                   
                   
               
               
                   
                 QRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNS 
               
               
                   
                   
               
               
                   
                 SPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPE 
               
               
                   
                   
               
               
                   
                 AGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIV 
               
               
                   
                   
               
               
                   
                 LQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNST 
               
               
                   
                   
               
               
                   
                 PGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKG 
               
               
                   
                   
               
               
                   
                 QQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRG 
               
               
                   
                   
               
               
                   
                 PEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMS 
               
               
                   
                   
               
               
                   
                 RIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHID 
               
               
                   
                   
               
               
                   
                 AYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAA 
               
               
                   
                   
               
               
                   
                 DLDDFSKQLQQSMSSADSTQAADSNGTITVEELKKLLEQW 
               
               
                   
                   
               
               
                   
                 NLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPV 
               
               
                   
                   
               
               
                   
                 TLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASF 
               
               
                   
                   
               
               
                   
                 RLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVI 
               
               
                   
                   
               
               
                   
                 GAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYK 
               
               
                   
                   
               
               
                   
                 LGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALL 
               
               
                   
                   
               
               
                   
                 VQ 
               
            
           
         
       
     
     Chimera 2: SdRBD-N-M_dTM 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 25) 
               
               
                   
                 MCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQD 
               
               
                   
                   
               
               
                   
                 LFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFA 
               
               
                   
                   
               
               
                   
                 STEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEF 
               
               
                   
                   
               
               
                   
                 QFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQ 
               
               
                   
                   
               
               
                   
                 PFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLV 
               
               
                   
                   
               
               
                   
                 RDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGD 
               
               
                   
                   
               
               
                   
                 SSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL 
               
               
                   
                   
               
               
                   
                 DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFATVC 
               
               
                   
                   
               
               
                   
                 GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQF 
               
               
                   
                   
               
               
                   
                 GRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS 
               
               
                   
                   
               
               
                   
                 NQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQT 
               
               
                   
                   
               
               
                   
                 RAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAG 
               
               
                   
                   
               
               
                   
                 SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE 
               
               
                   
                   
               
               
                   
                 ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR 
               
               
                   
                   
               
               
                   
                 ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQI 
               
               
                   
                   
               
               
                   
                 LPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA 
               
               
                   
                   
               
               
                   
                 ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSG 
               
               
                   
                   
               
               
                   
                 WTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIAN 
               
               
                   
                   
               
               
                   
                 QFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ 
               
               
                   
                   
               
               
                   
                 LSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQ 
               
               
                   
                   
               
               
                   
                 TYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGK 
               
               
                   
                   
               
               
                   
                 GYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG 
               
               
                   
                   
               
               
                   
                 KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN 
               
               
                   
                   
               
               
                   
                 CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD 
               
               
                   
                   
               
               
                   
                 LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK 
               
               
                   
                   
               
               
                   
                 YEQSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSK 
               
               
                   
                   
               
               
                   
                 QRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNS 
               
               
                   
                   
               
               
                   
                 SPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPE 
               
               
                   
                   
               
               
                   
                 AGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIV 
               
               
                   
                   
               
               
                   
                 LQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNST 
               
               
                   
                   
               
               
                   
                 PGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKG 
               
               
                   
                   
               
               
                   
                 QQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRG 
               
               
                   
                   
               
               
                   
                 PEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMS 
               
               
                   
                   
               
               
                   
                 RIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHID 
               
               
                   
                   
               
               
                   
                 AYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAA 
               
               
                   
                   
               
               
                   
                 DLDDFSKQLQQSMSSADSTQAADSNGTITVEELKKLLEQN 
               
               
                   
                   
               
               
                   
                 RNRFLYIIKLTLACFVLAAVNWITGGLMWLSYFIARTRSM 
               
               
                   
                   
               
               
                   
                 WSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHL 
               
               
                   
                   
               
               
                   
                 RIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAG 
               
               
                   
                   
               
               
                   
                 DSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ 
               
            
           
         
       
     
     Chimera 3: NSP3-4-6 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 26) 
               
               
                   
                 MTNSRIKASMPTTIAKNTVKSVGKFCLEASFNYLKSPNFS 
               
               
                   
                   
               
               
                   
                 KLINIIIWFLLLSVCLGSLIYSTAALGVLMSNLGMPSYCT 
               
               
                   
                   
               
               
                   
                 GYREGYLNSTNVTIATYCTGSIPCSVCLSGLDSLDTYPSL 
               
               
                   
                   
               
               
                   
                 ETIQITISSFKWDLTAFGLVAEWFLAYILFTRFFYVLGLA 
               
               
                   
                   
               
               
                   
                 AIMQLFFSYFAVHFISNSWLMWLIINLVQMAPISAMVRMY 
               
               
                   
                   
               
               
                   
                 IFFASFYYVWKSYVHVVDGCNSSTCMMCYKRNRATRVECT 
               
               
                   
                   
               
               
                   
                 TIVNGVRRSFYVYANGGKGFCKLHNWNCVNCDTFCAGSTF 
               
               
                   
                   
               
               
                   
                 ISDEVARDLSLQFKRPINPTDQSSYIVDSVTVKNGSIHLY 
               
               
                   
                   
               
               
                   
                 FDKAGQKTYERHSLSHFVNLDNLRANNTKGSLPINVIVFD 
               
               
                   
                   
               
               
                   
                 GKSKCEESSAKSASVYYSQLMCQPILLLDQALVSDVGDSA 
               
               
                   
                   
               
               
                   
                 EVAVKMFDAYVNTFSSTFNVPMEKLKTLVATAEAELAKNV 
               
               
                   
                   
               
               
                   
                 SLDNVLSTFISAARQGFVDSDVETKDVVECLKLSHQSDIE 
               
               
                   
                   
               
               
                   
                 VTGDSCNNYMLTYNKVENMTPRDLGACIDCSARHINAQVA 
               
               
                   
                   
               
               
                   
                 KSHNIALIWNVKDFMSLSEQLRKQIRSAAKKNNLPFKLTC 
               
               
                   
                   
               
               
                   
                 ATTRQVVNVVTTKIALKGGKIVNNWLKQLIKVTLVFLFVA 
               
               
                   
                   
               
               
                   
                 AIFYLITPVHVMSKHTDFSSEIIGYKAIDGGVTRDIASTD 
               
               
                   
                   
               
               
                   
                 TCFANKHADFDTWFSQRGGSYTNDKACPLIAAVITREVGF 
               
               
                   
                   
               
               
                   
                 VVPGLPGTILRTTNGDFLHFLPRVFSAVGNICYTPSKLIE 
               
               
                   
                   
               
               
                   
                 YTDFATSACVLAAECTIFKDASGKPVPYCYDTNVLEGSVA 
               
               
                   
                   
               
               
                   
                 YESLRPDTRYVLMDGSIIQFPNTYLEGSVRVVTTFDSEYC 
               
               
                   
                   
               
               
                   
                 RHGTCERSEAGVCVSTSGRWVLNNDYYRSLPGVFCGVDAV 
               
               
                   
                   
               
               
                   
                 NLLTNMFTPLIQPIGALDISASIVAGGIVAIVVTCLAYYF 
               
               
                   
                   
               
               
                   
                 MRFRRAFGEYSHVVAFNTLLFLMSFTVLCLTPVYSFLPGV 
               
               
                   
                   
               
               
                   
                 YSVIYLYLTFYLTNDVSFLAHIQWMVMFTPLVPFWITIAY 
               
               
                   
                   
               
               
                   
                 IICISTKHFYWFFSNYLKRRVVFNGVSFSTFEEAALCTFL 
               
               
                   
                   
               
               
                   
                 LNKEMYLKLRSDVLLPLTQYNRYLALYNKYKYFSGAMDTT 
               
               
                   
                   
               
               
                   
                 SYREAACCHLAKALNDFSNSGSDVLYQPPQTSITSAVLQS 
               
               
                   
                   
               
               
                   
                 AVKRTIKGTHHWLLLTILTSLLVLVQSTQWSLFFFLYENA 
               
               
                   
                   
               
               
                   
                 FLPFAMGIIAMSAFAMMFVKHKHAFLCLFLLPSLATVAYF 
               
               
                   
                   
               
               
                   
                 NMVYMPASWVMRIMTWLDMVDTSLSGFKLKDCVMYASAVV 
               
               
                   
                   
               
               
                   
                 LLILMTARTVYDDGARRVWTLMNVLTLVYKVYYGNALDQA 
               
               
                   
                   
               
               
                   
                 ISMWALIISVTSNYSGVVTTVMFLARGIVFMCVEYCPIFF 
               
               
                   
                   
               
               
                   
                 ITGNTLQCIMLVYCFLGYFCTCYFGLFCLLNRYFRLTLGV 
               
               
                   
                   
               
               
                   
                 YDYLVSTQEFRYMNSQGLLPPKNSIDAFKLNIKLLGVGGK 
               
               
                   
                   
               
               
                   
                 PCIKVATVQ 
               
            
           
         
       
     
     Chimera 4: NSP3-4-6_dTM 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 27) 
               
               
                   
                 MTNSRIKASMPTTIAKNTVKSVGKFCLEASFNYLKSPNFS 
               
               
                   
                   
               
               
                   
                 KLINLMSNLGMPSYCTGYREGYLNSTNVTIATYCTGSIPC 
               
               
                   
                   
               
               
                   
                 SVCLSGLDSLDTYPSLETIQITISSFKWDLTAFGLVAEWS 
               
               
                   
                   
               
               
                   
                 YFAVHFISNSWLMWLIINLKSYVHVVDGCNSSTCMMCYKR 
               
               
                   
                   
               
               
                   
                 NRATRVECTTIVNGVRRSFYVYANGGKGFCKLHNWNCVNC 
               
               
                   
                   
               
               
                   
                 DTFCAGSTFISDEVARDLSLQFKRPINPTDQSSYIVDSVT 
               
               
                   
                   
               
               
                   
                 VKNGSIHLYFDKAGQKTYERHSLSHFVNLDNLRANNTKGS 
               
               
                   
                   
               
               
                   
                 LPINVIVFDGKSKCEESSAKSASVYYSQLMCQPILLLDQA 
               
               
                   
                   
               
               
                   
                 LVSDVGDSAEVAVKMFDAYVNTFSSTFNVPMEKLKTLVAT 
               
               
                   
                   
               
               
                   
                 AEAELAKNVSLDNVLSTFISAARQGFVDSDVETKDVVECL 
               
               
                   
                   
               
               
                   
                 KLSHQSDIEVTGDSCNNYMLTYNKVENMTPRDLGACIDCS 
               
               
                   
                   
               
               
                   
                 ARHINAQVAKSHNIALIWNVKDFMSLSEQLRKQIRSAAKK 
               
               
                   
                   
               
               
                   
                 NNLPFKLTCATTRQVVNVVTTKIALKGGKIVNNWSKHTDF 
               
               
                   
                   
               
               
                   
                 SSEIIGYKAIDGGVTRDIASTDTCFANKHADFDTWFSQRG 
               
               
                   
                   
               
               
                   
                 GSYTNDKACPLIAAVITREVGFVVPGLPGTILRTTNGDFL 
               
               
                   
                   
               
               
                   
                 HFLPRVFSAVGNICYTPSKLIEYTDFATSACVLAAECTIF 
               
               
                   
                   
               
               
                   
                 KDASGKPVPYCYDTNVLEGSVAYESLRPDTRYVLMDGSII 
               
               
                   
                   
               
               
                   
                 QFPNTYLEGSVRVVTTFDSEYCRHGTCERSEAGVCVSTSG 
               
               
                   
                   
               
               
                   
                 RWVLNNDYYRSLPGVFCGVPLIQPIGALDRFRRAFGEYSH 
               
               
                   
                   
               
               
                   
                 SFLPGHIQWMVMFTPLWFFSNYLKRRVNKEMYLKLRSDVL 
               
               
                   
                   
               
               
                   
                 LPLTQYNRYLALYNKYKYFSGAMDTTSYREAACCHLAKAL 
               
               
                   
                   
               
               
                   
                 NDFSNSGSDVLYQPPQTSITSAVLQSAVKRTIKGTHLYEN 
               
               
                   
                   
               
               
                   
                 AKHKHAFSWVMRIMTWLDMVDTSLSGFKLKDCDDGARRVW 
               
               
                   
                   
               
               
                   
                 TLMNVLTLVALDQAISMWALIISVRGIVFMCVEYCCTCYF 
               
               
                   
                   
               
               
                   
                 GLFCLLNRYFRLTLGVYDYLVSTQEFRYMNSQGLLPPKNS 
               
               
                   
                   
               
               
                   
                 IDAFKLNIKLLGVGGKPCIKVATVQ 
               
            
           
         
       
     
     Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full Spike but not S1 Induces Strong Neutralizing Antibody Response 
     Modified vaccinia Ankara (MVA) based vaccines were developed one expressing the full-length spike protein (MVA/S) that is designed to be stabilized in prefusion state and anchored on the membrane of MVA infected cells, and the other expressing the S1 region of the spike (MVA/S1) that forms trimers and is secreted ( FIG.  4 A ). Both immunogens contained the receptor binding domain (RBD) that is the primary target for neutralization. Following immunization of mice, both recombinants induced strong binding antibody to S protein but differed in their specificity. The MVA/S induced strong antibody to RBD, S1 and S2, whereas the MVA/S1 induced strong antibody to S1 but regions other than RBD. Both vaccines induced antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. Sera from MVA/S mice but not MVA/S1 mice showed a strong neutralizing activity against SARS-CoV-2 virus that correlated with RBD binding titer. Binding to ACE-2 revealed that S1 presents RBD in the proper confirmation but this interaction is less stable at room temperature with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection. 
     Modified vaccinia Ankara (MVA) is a highly attenuated strain of vaccinia virus. There are several advantages to MVA based vaccines. MVA can accommodate large inserts (&gt;10 kb) that will allow expression of multiple antigens in a single vector. MVA recombinants are quite stable and can be produced at high titer that makes vaccine manufacture feasible. MVA vaccines also induce strong CD4 and CD8 T cell responses that will be important for protection against viral infections. MVA vaccination can provide protection against multiple virus infections including SARS-CoV, MERS, Zika and Ebola viruses. 
     MVA recombinants, one expressing the full-length spike protein (MVA/S) that is anchored on the membrane of MVA infected cells and the other expressing the S1 portion of the spike (MVA/S1) that is secreted were developed. Both constructs contained the RBD that is the prime target for neutralizing antibody response. The MVA/S also incorporated two mutations that have been shown to keep spike in a prefusion confirmation. These two recombinants were tested in mice for their ability to generate neutralizing antibody response. 
     MVA vaccines expressing either the full length prefusion stabilized spike or secreted Si demonstrated that while both immunogens induce strong binding antibody response to spike only the former induces a strong neutralizing antibody response against the SARS-CoV-2. The failure of MVA/S1 immunogen to induce neutralizing activity was associated with its failure to induce antibody to RBD. This was surprising given the fact that RBD is part of S1. Binding to ACE-2 revealed that S1 presents RBD in the proper confirmation at cold temperature however the stability of RBD confirmation seems to change markedly at the room temperature. This instability of S1 protein seems to contribute to induction of strong binding antibody to other regions in S1 other than RBD following immunization. Systemic MVA vaccination also induced T cell and antibody responses in the lung that will be critical for protection against respiratory infections such as SARS-CoV-2. A dose of about 10 8  pfu (between 10 7  and 10 9 ) is contemplated for human vaccination. Collectively these results demonstrate that MVA/S is a promising vaccine for SARS-CoV-2. 
     Recombinant MVA Vaccines 
     The full-length spike protein sequences of the SARS-CoV-2 strain was obtained from GenBank (Accession number QHD43416.1) and generated various forms of antigens for the improved immunization responses in our vaccination studies. These antigens were expressed using Modified Vaccinia Ankara (MVA) vectors. SARS-CoV-2 full-length spike (S) (aa 1 to 1273) has site-specific mutations introduced at K986P, and V987P for better stabilization and whereas, S1-mono, aa 14 to 780 of spike protein were fused at N-terminus with 16 aa long granulocyte-macrophage colony-stimulating factor (GM-C SF) signal sequences for better secretions. Inserts of rMVA were subcloned in between Xmal and BamHI restriction sites of the pLW-73 transfer vectors, to transfer the inserts into deletion III site. These inserts express under the control of an independent early/late vaccinia virus promoter (modified H5 [mH5]). 
     For MVA/S, the 3821-nt ORF (GenBank accession #MN996527.1_30-Dec-2019 China: Wuhan) encoding the SARS-nCoV Spike gene was codon optimized for vaccinia virus expression, and cloned into pLW-73 using the Xmal and BamH1 sites under the control of the vaccinia virus modified H5 early late promoter and adjacent to the gene encoding enhanced GFP regulated by the vaccinia virus P11 late promoter. Similarly, to develop MVA/S1, spike secreted monomeric form, GMCSF signal sequence followed with Spike DNA sequence of 14-780 AA was synthesized and cloned between Xmal and BamH1 sites of pLW-73 vector as described above. These plasmid DNAs were subsequently used to generate recombinant MVAs by transfecting transfer plasmids into DF-1 cells that were infected with 0.05 plaque forming units (pfu) of MVA per cell into the essential region of MVA 1974 strain between genes I8R and G1L. Recombinant MVA (rMVA) was isolated using standard methods, but sorting was used during the first round of selection using green fluorescent protein (GFP). Each round GFP plaque picked were characterized for the expression using anti SARS-CoV-2 spike antibody to detect cell surface spike protein expression of MVA/S. For MVA/S1, anti SARS-CoV-2 RBD antibody was used to stain intracellularly. Plaques were picked after 7 rounds to obtain GFP-negative rMVA/S, rMVA/S1 and spike DNA sequences were confirmed. The recombinants were characterized for spike expression by flow cytometry and Western blotting. Viral stocks were purified from lysates of infected DF1cells using a 36% sucrose cushion and titrated using DF-1 cells by counting pfu/ml. Absence of wildtype MVA was confirmed by PCR using recombinant specific primers of flanking sequences with rMVA/S and rMVA/S1 infected cellular DNA isolated from DF-1 cells. Absence of 542 bp (essential region) band indicates there is no wild type reverted MVAs in the preps. 
     The MVA Vaccines Express High Levels of Full-Length Stabilized Spike and Trimeric Soluble S1 Proteins 
     To develop the MVA recombinants the full-length spike gene (amino acids 1-1273) was synthesized with stabilizing mutations (K986P, V987P) or just the S1 region with a small portion of S2 region (amino acids 14 to 780). To promote active secretion of the S 1, the first 14 amino acids of the spike sequence were replaced with the signal sequence from GM-CSF ( FIG.  4 A ). Both sequences were optimized for MVA codon usage, corrected for poxvirus transcription termination sequences and cloned into pLW73 vector that will allow us to insert the recombinant sequences under mH5 promoter in the essential region of MVA. The recombinants were selected and characterized for protein expression by flow cytometry and Western blotting. The MVA/S expressed high levels of spike on the cell surface and the expressed protein had a molecular moss of about 180 kDa. Similarly, the MVA/S1 expressed at high levels intracellularly, a protein with a molecular moss of about 100 kDA that was also secreted into the supernatants of the MVA infected cells. The spike protein expressed by MVA/S on the surface seemed folded correctly based on strong binding to ACE2. Interestingly, the S1 protein was found to form trimers based on gel filtration profile and native-PAGE analysis. 
     Both MVA/S and MVA/S1 Vaccines Induce a Strong Binding Antibody Response but with Different Specificities 
     Balb/c mice were immunized with MVA/S or MVA/S1 on weeks 0 and 4, and measured binding antibody responses to total and different parts of spike i.e. RBD, S1, and S (S) using ELISA at 2 weeks post prime and boost. While both vaccines induced a strong binding antibody response to S, they differentially targeted binding to RBD and S1. The MVA/S sera showed strong binding to RBD whereas MVA/S1 sera showed strong binding to S1. This was interesting considering that S1 protein includes complete RBD and suggested that binding activity in MVA/S1 sera may be targeting regions other than RBD in S1. Luminex assay were performed using sera obtained from 3 weeks post boost to measure binding to different parts of S including S2, and to determine the antibody subclass and their ability to bind different soluble FcgRs. These analyses revealed that antibody responses in MVA/S group binding equally to RBD, S1 and S2 whereas in MVA/S1 group the antibody bound primarily to S1 but not to RBD and S2. While the lack of binding to S2 is expected, poor binding to RBD was not expected. Analysis of IgG subclass and FcgR binding of RBD-specific antibody showed strong IgG2a response (Th1 biased) and binding to all three FcRs tested with strongest binding to FcR2 and FcR4 in the MVA/S group. In contrast, poor binding of RBD-specific antibody was observed in general with MVA/S1 sera. However, the S1-specific antibody showed similar results in both groups. These results demonstrated differential targeting of spike specific antibody with Th1 profile induced by MVA/S and MVA/S1 vaccines. 
     MVA Vaccination Induces Strong Bronchus-Associated Lymphoid Tissue and Antibody Responses in the Lung 
     Experiments were performed to determine if vaccination induced immune responses in the lung, a primary site of SARS-CoV-2 virus exposure. The formation of bronchus-associated lymphoid tissue (BALT) was measure using the immunohistochemistry at 3 weeks after the MVA boost by staining for B and T cells. The naïve mice showed very little or no BALT, however, the MVA vaccinated mice showed significant induction of BALT indicating the generation of local lymphoid tissue ( FIG.  4 C ). While we do not know the longevity of persistence of these BALT, they are hoped to help with rapid expansion of immunity in the lung following exposure to SARS-CoV-2 infection. Consistent with BALT, the induction of spike specific IgG and IgA responses in the BAL was observed. These results demonstrate strong induction of antibody responses in the lung following MVA vaccination. 
     MVA/S but not MVA/S1 Induces Strong Neutralizing Antibody Response 
     Neutralization against the SARS-CoV-2 virus was tested using the FRNT-GFP assay using sera from 2 weeks post boost. Impressively, a strong neutralizing activity was observed with sera from mice vaccinated with MVA/S that ranged from 20-900 with a median of 200 ( FIG.  4 D ). In contrast, detectable neutralization was not observed in sera from mice immunized with MVA/S1. This was despite the fact that MVA/S1 mice showed comparable or higher binding antibody response to RBD, S1 and S proteins. There was an indication of higher infection at lower dilutions of MVA/S1 sera. The neutralization titer correlated directly with the RBD binding titer ( FIG.  4 E ) whereas correlated inversely with S1 binding titer. These results demonstrated that MVA/S immunogen can induce a strong neutralizing antibody response against SARS-CoV-2 and could serve as a potential vaccine for SARS-CoV-2. Importantly, they also reveal that MVA/S1 is not a good vaccine as it fails to induce antibody with neutralizing activity. 
     SARS-CoV-2 S1 Exhibits Lower Affinity to ACE2 than RBD, which Further Weakens upon Incubation at 25° C. 
     To further understand the failure of MVA/S1 vaccine to induce strong RBD binding antibody and neutralizing antibody, we purified the S1 trimer protein expressed by MVA/S1 vaccine and determined its ability to bind to human ACE-2 using biolayer interferometry (BLI). Purified RBD protein was used as a benchmark. SARS-CoV-2 S1 bound to hu-ACE2 quite strongly but at 2-fold lower affinity than RBD (KD=70.1 nM and 36 nM respectively). S1 exhibited 10-fold lower association rate than RBD (k on (1/Ms) 1.1E+04 and 1.3E+05 respectively). However, the affinity of S1-ACE2 further decreased by 5-fold when the protein was incubated at 25° C. for 60min. In contrary, RBD was stable and retained its affinity (KD=24 nM). The data indicated the receptor binding domain of S1 to be unstable, thereby loosing association with ACE2 protein upon prolonged incubation at room temperature, unlike RBD. A 10-fold reduction in the association rate for S1-ACE2 was observed, compared to RBD which was meagerly affected. 
     RBD-Binding IgG Antibody Titers for Assaying Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Candidate having a Mutant Furin Cleavage Site (MVA/S-Tri-dFCS) in BALB/c Mice. 
     A mutation of the furin cleavage site was introduced in order to stabilize the expressed proteins of the MVA vaccines, i.e., sequence encoding RRAR was altered to produce FCS mutation—SRAG. MVA/S-tri and MVA/S-tri-dFCS recombinants were expressed as membrane anchored spike protein variants (MVA/S-tri and MVA/S-tri-dFCS) on the surface which was confirmed by flow cytometry and western blot analysis bind studies of hACE2 to MVA/S-tri and MVA/S-tri-dFCS expressing infected cells. 
     Female BALB/c mice were intramuscularly (i.m.) immunized on wk0 and wk4 with either MVA/S-tri (107 PFU) or MVA/S-tri-dFCS (107 PFU) ( FIG.  6 A-B ). Control group received no treatment served as controls. Serum from 3-weeks post-prime and 2-weeks post-boost immunization was used to measure RBD binding IgG antibody using ELISA and presented Endpoint IgG titers. Neutralization titer against live mNeonGreen SARS-CoV-2 virus was performed in serum collected at week 2 post-boost immunizations ( FIG.  6 C ). 
     Vaccination of Rhesus Macaques 
     Two MVA based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the soluble secreted trimeric S1 of the spike (MVA/S1). Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization in SARS-CoV-2 infected individuals. The MVA/S also incorporated two mutations that maintain the spike protein in a prefusion confirmation. 
     
       
         
           
               
            
               
                   
               
               
                 MVA/S-Tri-dFCS 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Length 
                   
               
               
                 Name 
                 Range 
                 (bp) 
                 Description 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Flank1 I8R 
                  1-537 
                 537 
                 Essential gene region on  
               
               
                   
                   
                   
                 MVA for recombination 
               
               
                 P11 
                 545-573 
                 29 
                 promoter 
               
               
                 GFP 
                  574-1293 
                 720 
                 GFP 
               
               
                 DR 
                 1294-1528 
                 235 
                 Direct Repeats 
               
               
                 mH5 
                 1553-1619 
                 67 
                 Promoter 
               
               
                 Spike 
                 1634-5452 
                 3819 
                 Spike Protein sequence 
               
               
                 Furin cleavage site 
                 3677-3679 
                 3 
                 R682S AGA changed to  
               
               
                 change 
                   
                   
                 TCT 
               
               
                 Furin cleavage site 
                 3686-3688 
                 3 
                 R685G AGA changed to  
               
               
                 change 
                   
                   
                 GGA 
               
               
                 Proline mutation 1 
                 4589-4591 
                 3 
                 K986P AAA changed to  
               
               
                   
                   
                   
                 CCA 
               
               
                 Proline mutation 2 
                 4592-4594 
                 3 
                 V987P GTT changed to  
               
               
                   
                   
                   
                 CCT 
               
               
                 Flank 2 G1L 
                 5490-6191 
                 702 
                 Essential gene region on  
               
               
                   
                   
                   
                 MVA for recombination 
               
               
                 Ampicillin  
                 7477-8267 
                 791 
                 Confers resistance to  
               
               
                 resistance gene 
                   
                   
                 Ampicillin 
               
               
                   
               
            
           
         
       
     
     Vaccination of rhesus macaques followed by SARS-CoV-2 challenge demonstrated MVA/S vaccine induces neutralizing antibodies and CD8 T cells and protects from SARS-CoV-2 infection and replication in the lung. 
     The MVA recombinants expressing the full-length spike (amino acids 1-1273) carrying the prefusion-stabilized mutations (MVA/S) or only S1 portion of spike (amino acids 14-780)(MVA-S1) were generated and confirmed by standard methods. SARS-CoV-2 (MN996527.1; Wuhan strain) S ORF was codon optimized for vaccinia virus expression, synthesized, and cloned into pLW-73 between the Xmal and BamHI sites under the control of the vaccinia virus modified H5 (mH5) early late promoter and adjacent to the gene encoding enhanced GFP (green fluorescent protein). To promote active secretion of the S1, amino acids 1-14 of the spike sequence were replaced with the signal sequence from GMCSF, SEQ ID NO: 31 (WLQGLLLLGTVACSIS). Plaques were picked for 7 rounds to obtain GFP-negative recombinants and DNA sequenced to confirm lack of any mutations. Viral stocks were purified from lysates of infected DF-1 cells using a 36% sucrose cushion and titrated using DF-1 cells by counting pfu/ml. Absence of the wildtype MVA was confirmed by PCR using recombinant specific primers, flanking the inserts. 
     Ten adult male rhesus macaques (Macaca mulatta), 4-5 years old, were randomly allocated into two groups; one group (n=5) received MVA empty vector (MVA-wt) and the second group (n=5) received MVA-expressing prefusion stabilized (with proline mutations) SARS-CoV-2 full-length spike protein (MVA-S). Animals received 1×10 8  pfu in 1 ml vaccines at week 0 and week 4 by the intramuscular (IM) route. 
     In addition to the neutralizing activity, the vaccine induced sera showed strong antibody dependent complement deposition (ADCD) activity and low antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities. The MVA/S vaccine also generated a strong spike-specific IFNγ+CD8 T cell response that was evident as early as one week post priming immunization. The frequency of CD8 T cell response was not further boosted following the 2nd MVA/S immunization. The vaccine-induced CD8 T cells were also positive for TNFα and IL-2 and negative for IL-17. The MVA/S vaccine induced very low frequencies of IFNγ+CD4 T cells. These data demonstrated that MVA/S vaccinations induced a poly-functional CD8 T cell response capable of producing IFNγ, IL-2 and TNFα in macaques. 
     Following vaccination, all macaques were challenged with SARS-CoV-2 at week 8 by intratracheal (IT) and intranasal (IN) route. MVA/S vaccinated animals rapidly controlled SARS-CoV-2 replication in the lung at Day 2 (p&lt;0.05) and Day 4 (p&lt;0.05) compared to controls with 4 of the 5 vaccinated animals being negative in BAL. However, in the throat, all vaccinated animals tested negative at Day 2 (p&lt;0.01) but low titer of virus replication was evident in one or two vaccinated animals on Days 4 and 7. Similarly, in nasopharynx one or two animals showed virus replication on Days 2, 4 and 7 and the virus replication was not significantly different between controls and vaccinated animals at all time points. By Day 10 all control and vaccinated animals were negative in all compartments. These results demonstrated that MVA/S vaccination provides protection from SARS-CoV2 infection or replication in the lower respiratory tract. Virus replication, lung pathology, binding and neutralizing antibody titer and T cell responses were measured. Data indicates MVA/S vaccine protects from SARS-CoV-2 infection and replication and reduces lung pathology in rhesus macaques. 
     In order to define protection offered by MVA vaccine expressing Spike and nucleocapsid (NC) against SARS-CoV-2 South African variant (B.1.351) one can immunize rhesus macaques with a double recombinant MVA/S-tri-dFCS-NC on weeks 0 and 4, and challenge with B.1.351. One can assess the protective immune responses generated by the vaccine by measuring antibody and T cell responses in blood and mucosal secretions following vaccination. Animals can be challenged with SARS-CoV-2 virus intranasally and intratracheally to determine vaccine protection. One can collect blood, bone marrow, LN biopsies, BAL, rectal biopsies, rectal swabs, nasal and salivary/oral swabs at multiple times during vaccination and challenge. 
     Evaluation of MVA Based Vaccination Induced Neutralizing Antibody Responses Against SARS-CoV-2 Variants of Concern in Macaques and Mouse Models. 
     Vaccines were injected via intramuscular (IM) route at weeks 0 and 4. Serum collected at week 6 (peak) were used to asses neutralizing antibody titers against live Washington SARS-CoV-2, and variants of concern—UK variants, 501Y.V1, VOC 202012/01 (B.1.1.7) and South African variants (B.1.351), and Fold-Change in neutralization titers between WA virus to the variants of concern are presented. Each sample was analyzed in duplicates and repeated twice and repeated twice and GMT values for each vaccination groups were presented in table. 
                                                         UK                       SARS-   variants,                       CoV-2   501Y.V1,   South                   Wuhan   VOC   African   WA/UK   WA/RSA       Vaccine   Wild-type   202012/01   variants   Fold-   Fold-       Type   strain   (B.1.1.7)   (B.1.351)   Change   Change                  MVA/Stri    63.8   51.3    18.0   1.2   3.6       (macaque)                           MVA-   489.1   NT   134.0   NT   3.7       Stri-dFCS                           (mouse)                    
MVA/S study, n=5 rhesus macaques were immunized with 10{circumflex over ( )}8 pfu/macaques MVA/S-tri vaccine. MVA/S-tri-dFCS study, n=5 BALB/c mice were immunized with 10{circumflex over ( )}7 pfu/mice MVA/S-tri-dFCS vaccine. NT, not tested
 
     Heterologous Vector (DNA/MVA)-Based Vaccine Induce Greater Magnitude of CD8 T Cells Compared to MVA-Only Vaccination in Mice. 
     BALB/c mice were primed with DNA (50 ug/mice) and boosted with 10{circumflex over ( )}7 pfu/mice with spike expressing vaccine. All the vaccines were injected via intramuscular (IM) route at weeks 0 and 4. Blood collected at week 5 (peak) was used to assess % spike-specific tetramer positive CD8 T cells analyzed using flow cytometry. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 S (539-541)-specific 
               
               
                   
                 Vaccine Type 
                 tetramer positive CD8 T 
               
               
                   
                 (Prime/Boost) 
                 cells (%) 
               
               
                   
                   
               
             
            
               
                   
                 DNA- 
                 21 
               
               
                   
                 SdFCS/MVA- 
                   
               
               
                   
                 SdFCS 
                   
               
               
                   
                 MVA- 
                  3 
               
               
                   
                 SdFCS/MVA- 
                   
               
               
                   
                 SdFCS