WO2021207420A1 - An innovative dna vaccine for sars-cov, sars-cov-2, and mers-cov - Google Patents

Abstract

Provided herein are DNA vaccine constructs comprising linking polynucleotides encoding human albumin to polynucleotides comprising one or more of the receptor binding domains (RBD) of the spike protein of SARS-CoV-2 and/or MERS-CoV and/or SARS-CoV. Methods for making the vaccine constructs and their use in prophylaxis and treatment of SARS coronavirus infections are also provided.

Description

AN INNOVATIVE DNA VACCINE FOR SARS-CoV, SARS-CoV-2, and MERS-CoV

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. Nos. 63/007,608, filed April 9, 2020 and 63/042,260, filed June 22, 2020, the disclosures of which are incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 7, 2021, is named 0184_0148-PCT_(P16311-03)_SL.txt and is 97,416 bytes in size.

BACKGROUND OF THE INVENTION

[0003] SARS-CoV-2, the novel coronavirus that causes COVID-19, has been rapidly spreading since the World Health Organization’ s first citation of the disease in December 2019. Since then the virus has spread throughout 196 countries (The World Health Organization, www.who.int/emergencies/diseases/novel-coronavirus-2019), and as of Monday, March 30, Johns Hopkins reported more than 700,000 worldwide-confirmed cases and cited the disease as causing over 35,000 deaths (Johns Hopkins University, coronavirus.jhu.edu). In the United States, all 50 states, Washington DC, and four territories have reported cases. As of March 29, there have been over 120,000 confirmed cases in the US; some cases apparently due to community transmission (Center for Disease Control, www.cdc.gov/coronavims/2019-ncov/index.html). Since the World Health Organization has declared COVID-19 a pandemic, the virus has spread, showing its potential to negatively impact economies, overfill hospitals, and cause the death of many individuals.

[0004] The elderly and people with underlying conditions or compromised immune systems appear to be the most at risk. Although mortality rates vary from <1% to 27% depending on age class³, about 1 in 5 patients require hospitalization due to serious illness³. The number of new infections appears to be accelerating, highlighting the urgent need for a treatment or vaccine to prevent unnecessary death and economic damage. [0005] While SARS-CoV-2 is not the first coronavirus found to infect humans, several distinct features make the virus particularly complicated to halt the outbreak. COVID-19 is often compared to the 2003 Sudden Acute Respiratory Syndrome (SARS) and its respective virus, SARS-CoV. The two viruses cause similar respiratory tract infections and are from the same viral family; however, SARS and COVID-19 differ on biological and epidemiological levels.

[0006] While SARS-CoV-2 and SARS-CoV share roughly 79% of genetic information, the structure and genetic coding for the binding spike protein are dissimilar (Wang, Y.,

Wang, Y., Chen, Y., and Qin, Q. (2020). Unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (COVID-19) implicate special control measures. J Med Virol). Both viruses use these proteins to infect human cells via human angiotensin-converting enzyme 2 (ACE2), but the different forms make the viruses distinct despite a majority of similar RNA.

[0007] Epidemiologically, COVID-19 is harder to contain. This pattern is already reflected in the numbers of cases: SARS was able to be contained and infected approximately 8000 people in total (Id.), whereas 800,000 people have COVID-19 as of March 31². This is due to several factors. First, the reproductive value, Ro , of COVID-19 is higher, around 2- 3.5. SARS had an Ro closer to 2, meaning the average infected person infects fewer other people, slowing exponential spread (Id.). Second, as many as 80% of cases of SARS-CoV-2 are asymptomatic or mild despite high levels of viral shedding and viral load (Id.), so seemingly-healthy carriers are apt to unwittingly spread the disease. Third, there is a major epidemiological difference in the fatality rates. SARS had a global fatality rate of 9.6%, whereas COVID-19’s rate hovers at about 3% (Id.). The lower mortality enables the vims to spread to relatively more uninfected individuals than if the mortality rate was higher, resulting in more gross deaths despite the lower rate. Since SARS-CoV-2 spreads quickly, is likely to have asymptomatic carriers, and has a relatively low death rate, COVID-19 is difficult to contain. These characteristics make eradication unlikely.

[0008] Furthermore, it is also not practical to let SARS-CoV-2 infections continue to spread with the intent of generating herd immunity. Although the herd immunity approach is similar to what we do for seasonal flu, the case mortality rate is far too high for SARS-CoV-2 infections compared to seasonal flu (3% versus 0.1%). If we let SARS CoV-2 continue to spread without intervention, it will result in too many deaths and complete exhaustion of our medical systems. The current approach to manage the spread COVID-19 is to practice social distancing; however, this technique is not sustainable long-term.

[0009] Middle East Respiratory Syndrome (MERS) is an illness also caused by a coronavirus called Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Most MERS patients developed severe respiratory illness with symptoms of fever, cough and shortness of breath. According to the CDC, about 3 or 4 out of every 10 patients reported with MERS have died. Health officials first reported the disease in Saudi Arabia in September 2012. Through retrospective (backward-looking) investigations, they later identified that the first known cases of MERS occurred in Jordan in April 2012. So far, all cases of MERS have been linked through travel to, or residence in, countries in and near the Arabian Peninsula. The largest known outbreak of MERS outside the Arabian Peninsula occurred in the Republic of Korea in 2015. The outbreak was associated with a traveler returning from the Arabian Peninsula.

[0010] In May 2014, the Centers for Disease Control and Prevention (CDC) confirmed two cases of MERS. Both patients were healthcare providers who recently traveled from Saudi Arabia, where they are believed to have been infected. Both were hospitalized in the U.S. and later discharged after fully recovering. CDC and other public health partners continue to look for and test people who may have MERS; more than 750 people in the United States have tested negative.

[0011] As such, there exists an unmet need for continued vaccine development to curb these coronavirus pandemics and prevent future infections.

SUMMARY OF THE INVENTION

[0012] In accordance with one embodiment of the present invention, the inventors now provide a vaccine to prevent new infections and to slow the spread of COVID-19. The preventive vaccine is designed to produce neutralizing antibodies that would bind the SARS- CoV-2 spike protein receptor-binding domain (RBD). The SARS-CoV-2 vims’ RBD binds ACE2 in order to infect human cells. A vaccine that could rapidly produce neutralizing antibody to bind the SARS-CoV-2 RBD should render the pathogen unable to bind ACE2, resulting in the inability of SARS-CoV-2 to cause COVID-19. Therefore, the RBD of the spike protein serves as an important target for the development of a preventive vaccine against COVID-19.

[0013] Furthermore, in an effort to prevent new infections and slow the spread of other disease causing coronaviruses, the present invention provides a number of embodiments of a DNA vaccine which is directed to RBDs of SARS-CoV2 RBD, MERS-CoV RBD, and SARS-CoV RBD.

[0014] In accordance with an embodiment, the present invention provides a DNA vaccine composition comprising a synthetic polynucleotide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, or a functional portion or fragment or variant thereof.

[0015] In accordance with an embodiment, the present invention provides a DNA vaccine composition comprising a synthetic polynucleotide encoding human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, MERS- CoV RBD, and SARS-CoV RBD, or a functional portion or fragment or variant thereof. [0016] In accordance with an embodiment, the present invention provides a composition comprising a synthetic polypeptide consisting of albumin protein, or a functional portion, or fragment or variant thereof, conjugated to SARS-CoV2 RBD, or a functional portion or fragment or variant thereof.

[0017] In accordance with an embodiment, the present invention provides a composition comprising a synthetic polypeptide consisting of SARS-CoV2 RBD, MERS-CoV RBD, and SARS-CoV RBD human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to, or a functional portion or fragment or variant thereof.

[0018] In accordance with an embodiment, the present invention provides a recombinant vector encoding the DNA vaccine compositions described herein.

[0019] In accordance with an embodiment, the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions described herein. [0020] In accordance with an embodiment, the present invention provides a cell or population of cells expressing the synthetic polypeptide compositions described herein.

[0021] In accordance with an embodiment, the present invention provides a method for treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein. [0022] In accordance with an embodiment, the present invention provides a method for treating a SARS-CoV-2 infection, and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.

[0023] In accordance with an embodiment, the present invention provides a method for treating COVID-19 in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.

[0024] In accordance with an embodiment, the present invention provides a method for treating COVID-19 and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.

[0025] In accordance with an embodiment, the present invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.

[0026] In accordance with an embodiment, the present invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection and/or a MERS-CoV infection, and/or a SARS-CoV infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Amino acid sequence of SARS-CoV-2 spike protein receptor binding domain linked to human albumin. Linked RBD and human albumin amino acid sequence. EF is the EcoRI cloning site between albumin and RBD within the pcDNA3 vector.

[0028] Figure 2. Genetic sequence of SARS-CoV-2 spike protein receptor binding domain linked to human albumin. Genetic Sequence of albumin-RBD protein inserted into pcDNA3 sequence. GAATTC is the EcoRI cloning site between albumin and RBD.

[0029] Figures 3A-3B. pcDNA3-Hal-RBD expresses higher levels of RBD-albumin in a western blot. (3 A) Schematic of plasmids used to cell transfection and mice vaccination. RBD, SARS-CoV-2 Spike receptor binding domain. Hal, Human albumin. CMV, cytomegalovirus (CMV) immediate early promoter. (3B) Western blot of RBD in HEK 293 cells transfected with pcDNA3-RBD or pcDNA3-Hal-RBD. To identify the protein expression level of RBD and Hal-RBD plasmid. HEK 293 cells were transfected with lOug of each plasmid. After 48 hours transfection, cell lysates were collected for Western blot analysis. GAPDH as loading control.

[0030] Figures 4A-4B. pcDNA3 -Hal-RBD promotes higher levels of RBD antibody in vivo. Characterization of Hal-RBD DNA vaccination by ELISA. (4A) Schematic illustration of vaccination. 6-8 week old female C57BL/6 mice (5 mice/group) were vaccinated with 20 μg/mouse of pcDNA3-RBD or pcDNA3 -Hal-RBD DNA by intramuscular injection. The mice were boosted with the same regimen twice with a 2-week interval. One week after last vaccination, serum was collected for ELISA. (4B) Antibody specific for the human SARS-CoV-2 Spike protein were analyzed by ELISA. Briefly, lug/ml of human SARS-CoV-2 Spike protein in PBS were coated on BRANDplates® microplates for overnight. The next day wells were washed, blocked with eBioscience™ ELISA/ELISPOT Diluent, and added serial dilutions of serum (all ranging from 1:100- 1:10240) for two hours at room temperature. Non- vaccinated mice serum as control. Goat anti-mouse IgG-HRP secondary antibody was added for one hour at 1:5000 dilution, followed by TMB substrate. The OD at 450 nm was determined by an ELISA reader.

[0031] Figures 5A-5B. pcDNA3 -Hal-RBD vaccination generate the highest levels of RBD-specific antibody in vaccinated mice among all of the various DNA vaccine methods tested. Characterization of Hal-RBD DNA vaccination by ELISA. (5A) is a graphic depiction of the dosing regimen for various vaccine compositions. (5B) Antibodies specific for the human SARS-CoV-2 Spike protein were analyzed by ELISA. Briefly, 1 μg/ml of human SARS-CoV-2 Spike protein in PBS were coated on BRANDplates® microplates for overnight. The next day wells were washed, blocked with eBioscience™ ELISA/ELISPOT Diluent, and added serial dilutions of serum (all ranging from 1:100-1:10,240) for two hours at room temperature. Non- vaccinated mice serum as control. Goat anti-mouse IgG-HRP secondary antibody was added for one hour at 1:5000 dilution, followed by TMB substrate. The OD at 450 nm was determined by an ELISA reader. Legend: sig=RBD conjugated to signal peptide; Ig = RBD conjugated to Fc portion of IgG. [0032] Figure 6 (Panels A and B). Flow cytometry analysis to characterize the binding of the spike protein of SARS-CoV-2 by the antibody generated by vaccination with pcDNA3- Hal-RBD DNA. Flow cytometry assay of the binding of the spike protein of SARS-CoV-2 on the cell surface of 293 cells by antibodies generated by vaccination with the various DNA constructs. HEK 293 cells were transfected with DNA encoding the spike protein of SARS- CoV-2. At 48 h post-transfection, binding to the spike protein at the cell surface was analyzed by incubation with sera from mice vaccinated pcDNA3-RBD DNA (light trace) (Panel A), pcDNA3-Hal-RBD DNA (light trace) (Panel B) or PBS (dark distribution curves in both Panels A and B) control followed by staining with PE-conjugated anti-mouse IgG. Representative figures of flow cytometry analysis are shown.

[0033] Figures 7A-7B. Genetic sequence of alternative embodiment of SARS-CoV-2 spike protein receptor binding domain linked to human albumin showing nucleotide (7 A) and amino acid (7B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD. [0034] Figures 8A-8B. Genetic sequence of another embodiment of SARS-CoV-2 spike protein receptor binding domain, the MERS-CoV RBD, and the SARS-CoV RBD linked to human albumin showing nucleotide (8A) and amino acid (8B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD.

[0035] Figures 9A-9B. Genetic sequence of another embodiment of SARS-CoV-2 spike protein receptor binding domain, the MERS-CoV RBD, and the SARS-CoV RBD linked to human albumin showing nucleotide (9 A) and amino acid (9B) sequences. Genetic Sequence of albumin-RBD protein inserted into pNGVL4a vector sequence. GAATTC is the EcoRI cloning site between albumin and RBD. Figure 9A discloses SEQ ID NO: 21.

[0036] Figure 10. pcDNA3-Hal-RBD and pNGVL4a-Hal RBD expresses higher levels of secreted protein in a western blot. Western blot of RBD in HEK 293 cells transfected with pcDNA3-Hal-RBD or pNGVL4a -Hal-RBD. To identify the protein expression level of Hal- RBD HEK 293 cells were transfected with 10 μg of each plasmid. After 48 hours transfection, cell lysates and supernatant were collected for Western blot analysis. Lane 1: cell pellet from pcDNA3 -Hal-RBD transfected cells. Lane 2: cell pellet from pNGVL4a-Hal- RBD transfected cells. Lane 3: supernatant from pcDNA3-Hal-RBD transfected cells. Lane 4: supernatant from pNGVL4a -Hal-RBD transfected cells.

[0037] Figure 11. Hal-RBD protein generates higher levels of RBD antibody in vivo. ELISA assays using sera derived from mice vaccinated with RBD, RBD-FC or Hal-RBD protein. C57BL/6 mice (5 per group) were vaccinated subcutaneously with 10 μg of RBD, 20 μg RBD-FC or 40 μg of Hal-RBD protein vaccine with 5 μg MPL/mouse three times at 1 week interval. Sera from each group of mice were collected through tail vein 1 week after the last vaccination. Serial dilution of the sera was used for ELISA using 96 well plate coated with RBD protein.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Among different forms of preventive vaccines, naked DNA vaccines have emerged as a potentially promising strategy due to their simplicity, stability, and safety profiles. However, because naked DNA vaccine does not amplify in the transfected cells and spread throughout the body, there is limited potency. Thus, DNA vaccine strategies require techniques to improve potency.

[0039] A common approach to increasing potency is to administer the vaccine via electroporation. However, electroporation is uncomfortable and difficult, making it unlikely to become a widely adopted vaccine. Instead of modifying the delivery mechanism, the present inventors modified the DNA vaccine itself to increase potency.

[0040] The inventors provide herein an innovative strategy to improve naked DNA vaccine potency by conjugating albumin protein with the target antigen: RBD of the spike protein of SARS-CoV-2. The inventors now show that the linkage of albumin with the target antigen leads to the enrichment of targeted antigen protein, specifically within the draining lymph nodes. These elevated target antigen protein levels within the lymph nodes leads to high titers of antibody against the linked antigen in vaccinated mice. The linking of albumin therefore increases potency of a DNA vaccine strategy while avoiding cumbersome electroporation, making the vaccine a more feasible approach.

[0041] In addition, the inventors now also provide a broader spectrum coronavirus vaccine that can potentially protect subject from all three know disease-causing coronaviruses, by linking albumin with the SARS-CoV-2 RBD antigen, MERS-CoV RBD antigen, and SARS-CoV RBD antigen.

[0042] Therefore, in accordance with an embodiment, the present invention provides a DNA vaccine composition comprising a synthetic polynucleotide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.

[0043] It will be understood by those of skill in the art that the DNA vaccine compositions of the present invention can be used for other SARS coronavirus infections having the same or similar RBD.

[0044] In some embodiments, the DNA vaccine composition comprises a synthetic polynucleotide encoding human albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof. [0045] As used herein, the term “albumin” means the most abundant circulating protein in the plasma of most mammals. Human albumin (Hal) is present in the plasma of healthy individuals at a concentration of (3.5-5 g/dL) and it represents approximately 50% of the total protein content. HA is a small globular protein (molecular weight: 66.5 kDa), consisting of a single chain of 585 amino acids organized in three repeated homologue domains (sites I, II, and III), each of which comprises two separate sub-domains (A and B).

[0046] It will be understood that the term “albumin protein” means the full-length expressed polypeptide of a nucleic acid encoding the albumin gene, or a functional portion or fragment, or variant thereof.

[0047] It will be understood by those of ordinary skill in the art that many different isoforms of both human and murine albumin protein exist, and can be used in the compositions disclosed herein. Examples of albumin isoforms include, but are not limited to, human albumin isoforms (NM_000477, AAH41789.1, and AAH35969), for example and mouse albumin ligand isoform (AAH49971), for example.

[0048] In some embodiments, the DNA sequence of Hal used in the present invention is detailed in Fig. 2 and has the following sequence:

ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAG

GGGTGTGTTTCGTCGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGAT

TTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTC AGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGC

AAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACC

_{CTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAA}

TGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAAC

ACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGA

TGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGA

AATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAA

GGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCT

GTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACA

GAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATG

GGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCC

AAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTG

CTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAG

ATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATC

CCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTA

GCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGG

ATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCT

GTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCT

GTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACC

TCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAG

CTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTAC

CCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGG

GCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACT

ATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAG

TGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTT

TCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACGT

TCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGA

AACAAACTGCACTTGTTGAGCTTGTGAAACACAAGCCCAAGGCAACAAAAGAGC

AACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGC

TGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAG

TCAAGCTGCCTTAGGCTTA (SEQ ID NO: 1). [0049] The term “functional portion or fragment thereof,” with respect to the albumin protein, means that the portion or fragment of the albumin polypeptide retains its ability to bind to the neonatal Fc receptor and traffic through the lymphatic system.

[0050] In some embodiments, the albumin protein is mammalian. In certain embodiments, the albumin protein can be murine, porcine, ovine, bovine, human, or combinations thereof.

[0051] In some embodiments, the amino acid sequence of Hal used in the present invention is detailed in Fig. 1 and has the following sequence:

MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ CPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARR HPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS S AKQRLKCA SLQKFGER AFKAW A V ARLS QRFPKAEFAE V S KLVTDLTKVHTECCHGDLLEC ADDR ADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNY AEAKD VFLGMFLYEY ARRHPD Y S VVLLLRLAKTYETTLEKCCAAADPHECY AKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVS RNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVN RRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 2). [0052] As used herein, the term “SARS coronavirus infection” means a subject who is infected with one or more coronaviruses (CoVs). CoVs are enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease chickens to potentially lethal human respiratory infections.

[0053] The initial attachment of the CoV virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of receptor binding domains (RBD) within the SI region of a coronavirus S protein vary depending on the virus, with some having the RBD at the N-terminus of SI (MHV) while others (SARS-CoV) have the RBD at the C- terminus of SI. The S -protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. It is unclear why peptidases are used, as entry occurs even in the absence of the enzymatic domain of these proteins. Many a- coronaviruses utilize aminopeptidase N (APN) as their receptor, SARS-CoV, SARS-CoV-2, and HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) as their receptor, MHV enters through CEACAM1, and the recently identified MERS-CoV binds to dipeptidyl- peptidase 4 (DPP4) to gain entry into human cells.

[0054] In some embodiments, the subject is infected with SARS-CoV-2, which causes the COVID-19 disease.

[0055] SARS-CoV-2 is an enveloped virus carrying a positive-sense, single- stranded RNA genome and a nucleocapsid with helical symmetry. The pathogenesis of COVID-19 is complex but begins with the binding of the spike (S) protein to the Angiotensin Converting Enzyme 2 (ACE2) receptor on Type I and Type II pneumocytes in the lower respiratory tract. This binding event facilitates viral entry into respiratory epithelial cells, whereupon the viral genome replicates inside the cell and newly assembled virions bud from the plasma membrane to infect other cells.

[0056] As used herein, the term “SARS-CoV-2 RBD” means the receptor-binding domain of the spike protein of a SARS corona vims, and in some embodiments, it is the RBD of the SARS-CoV-2 virus, or a functional portion or fragment thereof. In one embodiment, the RBD has the following nucleotide sequence:

ATGAGGGTCCAACCAACAGAGAGCATTGTGAGGTTTCCAAACATCACCAACCTG

TGTCCATTTGGAGAGGTGTTCAATGCCACCAGGTTTGCCTCTGTCTATGCCTGGA

ACAGGAAGAGGATTAGCAACTGTGTGGCTGACTACTCTGTGCTCTACAACTCTGC

CTCCTTCAGCACCTTCAAGTGTTATGGAGTGAGCCCAACCAAACTGAATGACCTG

TGTTTCACCAATGTCTATGCTGACTCCTTTGTGATTAGGGGAGATGAGGTGAGAC

AGATTGCCCCTGGACAAACAGGCAAGATTGCTGACTACAACTACAAACTGCCTG

ATGACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAGG

TGGGAGGCAACTACAACTACCTCTACAGACTGTTCAGGAAGAGCAACCTGAAAC

CATTTGAGAGGGACATCAGCACAGAGATTTACCAGGCTGGCAGCACACCATGTA

ATGGAGTGGAGGGCTTCAACTGTTACTTTCCACTCCAATCCTATGGCTTCCAACC

AACCAATGGAGTGGGCTACCAACCATACAGGGTGGTGGTGCTGTCCTTTGAACTG CTCCATGCCCCTGCCACAGTGTGTGGACCAAAGAAGAGCACCAACCTGGTGAAG AACAAGTGTGTGAACTTCTGA (SEQ ID NO: 3).

[0057] In some embodiments, the amino acid sequence of RBD used in the present invention is detailed in Fig. 1 and has the following sequence:

MRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKFNDFCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKFPDDFTGC VIAWNSNNFDSKVGGNYNYFYRFFRKSNFKPFERDISTEIYQAGSTPCNGVEGFNCY FPLQS Y GFQPTN G V GY QPYR V V VLS FELLH APAT V CGPKKS TNL VKNKC VNF (SEQ ID NO: 4).

[0058] As used herein, the term “MERS-CoV RBD” means the receptor-binding domain of the spike protein of a MERS-CoV corona virus, and in some embodiments, it is the RBD of the MERS-CoV virus, or a functional portion or fragment thereof. In one embodiment, the

RBD is shown in Fig. 8A, and has the following nucleotide sequence:

GAGGCTAAGCCCAGCGGCTCTGTGGTTGAACAAGCCGAAGGCGTGGAATGCGAC

TTCTCTCCACTGCTGTCTGGCACCCCTCCACAGGTGTACAACTTCAAGCGGCTGG

TGTTCACCAACTGCAATTACAACCTGACAAAGCTGCTGAGCCTGTTCAGCGTGAA

CGACTTTACCTGCAGCCAGATCTCTCCTGCCGCCATTGCCAGCAACTGTTACAGC

TCCCTGATCCTGGACTACTTCAGCTACCCTCTGAGCATGAAGTCCGACCTGTCTGT

GTCTAGCGCCGGACCTATCAGCCAGTTCAATTACAAGCAGTCCTTCAGCAACCCC

ACCTGTCTGATTCTGGCCACCGTGCCTCACAATCTGACCACCATCACCAAGCCAC

TGAAGTACAGCTACATCAACAAGTGCAGCCGGTTCCTGAGCGACGACAGAACAG

AAGTGCCACAGCTCGTCAACGCCAACCAGTACAGCCCCTGTGTGTCTATCGTGCC

TAGCACAGTGTGGGAGGACGGCGACTACTACAGAAAGCAGCTGTCTCCACTCGA

AGGCGGAGGATGGCTGGTGGCTTCTGGAAGCACAGTGGCTATGACAGAGCAGCT

GCAGATGGGCTTTGGCATCACCGTGCAGTACGGCACCGATACCAATAGCGTGTG

CCCCAAGCTGGAATTCGCCAACGACACCAAGATTGCCAGCCAGCTGGGCAATTG

CGTCGAGTACAGA (SEQ ID NO: 15).

[0059] In some embodiments, the amino acid sequence of MERS-CoV RBD used in the present invention is detailed in Fig. 8B and has the following sequence: FEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDF TCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATV PHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYR KQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIAS QLGNCVEYR (SEQ ID NO: 16) or a functional portion or fragment thereof.

[0060] As used herein, the term “SARS-CoV RBD” means the receptor-binding domain of the spike protein of a SARS-CoV corona vims, and in some embodiments, it is the RBD of the SARS-CoV virus, or a functional portion or fragment thereof. In one embodiment, the

RBD is shown in Fig. 8A, and has the following nucleotide sequence:

GTGGTGCCTAGCGGCGACGTTGTGCGCTTTCCTAATATCACAAACCTGTGTCCAT

TCGGGGAAGTGTTTAACGCCACAAAGTTCCCTTCCGTGTATGCCTGGGAGCGCAA

GAAAATCTCCAACTGTGTGGCTGATTACTCCGTCCTGTACAACAGCACCTTTTTCT

CCACGTTCAAATGTTATGGGGTGTCCGCCACCAAACTCAATGACCTCTGTTTTAG

CAACGTCTACGCCGACTCCTTCGTCGTGAAAGGGGATGATGTTCGCCAGATCGCC

CCAGGACAAACCGGCGTTATCGCCGACTATAATTACAAACTCCCCGATGATTTCA

TGGGCTGTGTGCTGGCCTGGAACACCAGAAATATCGATGCCACCTCCACCGGGA

ACTATAACTACAAGTACAGATACCTGCGGCACGGCAAGCTGAGGCCCTTTGAGA

GGGATATCTCCAACGTGCCATTCAGCCCCGACGGCAAGCCTTGTACACCACCAGC

TCTGAATTGCTACTGGCCCCTGAACGATTACGGCTTCTACACCACAACCGGCATC

GGCTACCAACCATACAGGGTCGTCGTGCTGAGCTTCGAATTGCTGAACGCCCCAG

CCACAGTGTGTGGCCCAAAGCTGAGCACCGACCTGATTAAGAACCAGTGCGTCA

ACTTCAAC (SEQ ID NO: 17).

[0061] In some embodiments, the amino acid sequence of SARS-CoV RBD used in the present invention is detailed in Fig. 8B and has the following sequence: VVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTF KCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGC VLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYW PLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFN (SEQ ID NO: 18) or a functional portion or fragment thereof.

[0062] It will be understood by those of skill in the art that in one embodiment, the DNA vaccine composition comprises a first polynucleotide sequence encoding a synthetic polypeptide encoding albumin, or a functional portion, or fragment or variant thereof, linked via one or more linking polynucleotides, to polynucleotide encoding a synthetic polypeptide encoding RBD, or a functional portion, or fragment or variant thereof. As shown in Figs. 1, 2, 8, and 9, the conjugate composition has the albumin amino acid sequence domain at the N- terminal side of the polypeptide followed by a linker, and then the RBD domain at the C- terminal end of the polypeptide. The corresponding DNA polynucleotide of the present invention is analogous with the polynucleotide domain encoding the albumin protein at the 5 ’ end of the polynucleotide, followed by a linker, and then the RBD polynucleotide domain is at the 3’ end of the polynucleotide. It will be understood that this orientation can be reversed. [0063] In some embodiments, the linker comprises a polynucleotide which encodes one or more amino acids. The linker can be a polynucleotide encoding 1 to 50 amino acids, including, for example, 2, 3, 4, 5, 10, 15, 20, 30, 40, up to 50 amino acids. In an embodiment, the linker is a dipeptide. In another embodiment, the linker is the dipeptide Glu-Phe.

[0064] In some embodiments, the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 5. In some embodiments, the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 5, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 5.

[0065] In some other embodiments, the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 11 (Albumin-RBD (lowercase single underlined sequence is albumin, italicized sequence is linker, lowercase double underlined sequence RBD, and remainder of the sequence is vector)). In some embodiments, the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 11, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 11:

GGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGT

CCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAA

TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC

GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA

ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG

TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC

CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCAT CGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT

TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATT

GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCG

TTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT

AGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACG

CGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACA

CCCCTTTGGCTCTT ATGC ATGCT AT ACTGTTTTTGGCTTGGGGCCT AT AC ACCCCC

GCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGA

CCATTATTGACCACTCCAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTG

CTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTC

CATGGGTCTTTTCTGCAGTCACCGTCGTCGACGGTATCGATAAGCTTGATATCGA

ATTCACGTGGGCCCGGTACCGTATACTCTAGAGCCACCatgaagtgggtcaccttcatcagcctgc tgtttctgttcagcagcgcctacagcagaggcgtgttcagaagagatgcccacaagagcgaggtggcccacagattcaaggacctgg gcgaagagaacttcaaggccctggtgctgatcgccttcgctcagtatctgcagcagtgccccttcgaggatcacgtgaagctggtcaa cgaagtgaccgagttcgccaagacctgtgtggccgatgagagcgccgagaactgtgataagagcctgcacaccctgttcggcgaca agctgtgtacagtggccacactgagagaaacctacggcgagatggccgactgctgtgccaagcaagagcccgagagaaacgagtg cttcctgcagcacaaggacgacaaccccaacctgcctagactcgtgcgacccgaagtggatgtgatgtgcaccgccttccacgacaa cgaggaaaccttcctgaagaagtacctgtacgagatcgccagacggcacccctacttttatgcccctgagctgctgttcttcgccaagc ggtataaggccgccttcaccgaatgttgccaggccgctgataaggctgcctgtctgctgcctaagctggacgagctgagagatgagg gcaaagccagctctgccaagcagagactgaagtgcgccagcctgcagaagttcggcgagagagcctttaaagcctgggccgttgcc agactgagccagagatttcctaaggccgagtttgccgaggtgtccaagctcgtgaccgatctgacaaaggtgcacaccgagtgctgtc acggcgatctgctggaatgtgccgacgatagagccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagct gaaagagtgctgcgagaagcccctgctggaaaagtctcactgtatcgccgaggtggaaaacgacgagatgcctgccgatctgccta gcctggctgccgatttcgtggaaagcaaggacgtgtgcaagaactacgccgaggccaaggatgtgttcctgggcatgtttctgtatga gtacgcccgcagacaccccgactattctgtggttctgctgctgcggctggccaaaacctacgagacaaccctggaaaaatgctgcgc cgctgccgatcctcacgagtgttatgccaaggtgttcgacgagttcaagcctctggtggaagaaccccagaacctgatcaagcagaac tgcgagctgttcgagcagctgggcgagtacaagttccagaatgccctgctcgtgcggtacaccaagaaagtgcctcaggtgtccaca cctacactggttgaggtgtcccggaatctgggcaaagtgggcagcaagtgttgcaagcaccctgaggccaagagaatgccttgcgcc gaggattacctgagcgtggtgctgaatcagctgtgcgtgctgcacgagaaaacccctgtgtccgacagagtgaccaagtgctgtacc gagagcctcgtgaacagaaggccttgctttagcgccctggaagtggacgagacatacgtgcccaaagagttcaacgccgagacattc accttccacgccgacatctgcaccctgtccgagaaagagcggcagatcaagaagcagacagccctggtcgagctggttaagcacaa gcccaaggccaccaaagaacagctgaaggccgtgatggacgacttcgccgcctttgtcgagaagtgctgcaaggccgacgacaaa gagacatgcttcgccgaagagggcaagaaactggtggctgcctctcaggctgccctgggccttGAGTYTatgagagtgcagcct accgagtccatcgtgcggttccccaacatcaccaatctgtgcccctttggcgaggtgttcaatgccaccagatttgccagcgtgtacgc ctggaaccggaagagaatcagcaactgcgtggccgactacagcgtgctgtacaatagcgccagcttcagcaccttcaagtgctacgg cgtgtcccctaccaagctgaacgacctgtgcttcaccaatgtgtacgccgacagcttcgtgatcagaggcgacgaagtgcggcagatt gctcctggacagaccggcaagatcgccgattacaactacaagctgcccgacgacttcaccggctgcgtgatcgcctggaatagcaac aacctggacagcaaagtcggcggcaactacaactacctgtaccggctgttccggaagtccaacctgaagcctttcgagcgggacatc agaagtctaccaacctggtcaagaacaaatgcgtgaacttcTAATAAGGATCCAGATCTTTTTCCCTCTGC

CAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAA

AGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGG

AAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTA

GAGTTTGGCAACATATGCCCATTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCT

CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT

ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC

AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC

GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC

CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC

TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAA

GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT

CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT

TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT

GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC

AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGT

ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT

CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT

TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCT

GACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA

AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT AAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC

ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGG

GGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGGCA

ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT

TCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT

GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC

CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC

CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGA

ATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC

GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC

GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCG

TGCACCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATG

AGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGG

AACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGT

TCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGT

GTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAA

ACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTC

TGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGG

TATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTC

GTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGG

TGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCA

TTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATT

GCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG

GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCAC

CTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGT

GGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAG

AGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTG

GCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCAT

ACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATA

CCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTT

TCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACA GTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTT

TGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTT

ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG

GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATT

ATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGC

GTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA

CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAG

CGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGT

ACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAA

AT ACCGC ATC AG ATT GGCT ATT (SEQ ID NO: 11).

[0066] In yet another embodiment the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 13 (Albumin- COVID19 RBD- Mers RBD- Sars

RBD (single underlined polynucleotide sequence is albumin, italicized polynucleotide sequence is COVID19 RBD, bold polynucleotide sequence is Mers RBD, double underlined polynucleotide sequence is Sars RBD, and the remainder of the polynucleotide sequence is the vector pNGVL4a)). In some embodiments, the synthetic polynucleotide encoding the

DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 13, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 13:

GGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGT

CCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAA

TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC

GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA

ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGG

TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC

CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCAT

CGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT

TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATT

GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCG

TTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT AGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACG

CGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACA

CCCCTTTGGCTCTT ATGC ATGCT AT ACTGTTTTTGGCTTGGGGCCT AT AC ACCCCC

GCTTCCTTATGCTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGA

CCATTATTGACCACTCCAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTG

CTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTC

CATGGGTCTTTTCTGCAGTCACCGTCGTCGACGGTATCGATAAGCTTGATATCGA

ATTCACGTGGGCCCGGTACCGTATACTCTAGAGCCACCatgaagtgggtcaccttcatcagcctgc tgtttctgttcagcagcgcctacagcagaggcgtgttcagaagagatgcccacaagagcgaggtggcccacagattcaaggacctgg gcgaagagaacttcaaggccctggtgctgatcgccttcgctcagtatctgcagcagtgccccttcgaggatcacgtgaagctggtcaa cgaagtgaccgagttcgccaagacctgtgtggccgatgagagcgccgagaactgtgataagagcctgcacaccctgttcggcgaca agctgtgtacagtggccacactgagagaaacctacggcgagatggccgactgctgtgccaagcaagagcccgagagaaacgagtg cttcctgcagcacaaggacgacaaccccaacctgcctagactcgtgcgacccgaagtggatgtgatgtgcaccgccttccacgacaa cgaggaaaccttcctgaagaagtacctgtacgagatcgccagacggcacccctacttttatgcccctgagctgctgttcttcgccaagc ggtataaggccgccttcaccgaatgttgccaggccgctgataaggctgcctgtctgctgcctaagctggacgagctgagagatgagg gcaaagccagctctgccaagcagagactgaagtgcgccagcctgcagaagttcggcgagagagcctttaaagcctgggccgttgcc agactgagccagagatttcctaaggccgagtttgccgaggtgtccaagctcgtgaccgatctgacaaaggtgcacaccgagtgctgtc acggcgatctgctggaatgtgccgacgatagagccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagct gaaagagtgctgcgagaagcccctgctggaaaagtctcactgtatcgccgaggtggaaaacgacgagatgcctgccgatctgccta gcctggctgccgatttcgtggaaagcaaggacgtgtgcaagaactacgccgaggccaaggatgtgttcctgggcatgtttctgtatga gtacgcccgcagacaccccgactattctgtggttctgctgctgcggctggccaaaacctacgagacaaccctggaaaaatgctgcgc cgctgccgatcctcacgagtgttatgccaaggtgttcgacgagttcaagcctctggtggaagaaccccagaacctgatcaagcagaac tgcgagctgttcgagcagctgggcgagtacaagttccagaatgccctgctcgtgcggtacaccaagaaagtgcctcaggtgtccaca cctacactggttgaggtgtcccggaatctgggcaaagtgggcagcaagtgttgcaagcaccctgaggccaagagaatgccttgcgcc gaggattacctgagcgtggtgctgaatcagctgtgcgtgctgcacgagaaaacccctgtgtccgacagagtgaccaagtgctgtacc gagagcctcgtgaacagaaggccttgctttagcgccctggaagtggacgagacatacgtgcccaaagagttcaacgccgagacattc accttccacgccgacatctgcaccctgtccgagaaagagcggcagatcaagaagcagacagccctggtcgagctggttaagcacaa gcccaaggccaccaaagaacagctgaaggccgtgatggacgacttcgccgcctttgtcgagaagtgctgcaaggccgacgacaaa gagacatgcttcgccgaagagggcaagaaactggtggctgcctctcaggctgctctgggacttAGAGTGCAGCCTACAG

AGTCCATCGTGCGGTTCCCCAACATCACCAATCTGTGCCCCTTTGGCGAGGTGTTCAA

TGCCACCAGATTTGCCAGCGTGTACGCCTGGAACCGGAAGAGAATCAGCAACTGCGT GGCCGACTACAGCGTGCTGTACAATAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGC GTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAATGTGTACGCCGACAGCTTCG TGATCAGAGGCGACGAAGTGCGGCAGATTGCTCCTGGACAGACCGGCAAGATCGCCG ATTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAATAGCAA CAACCTGGACAGCAAAGTCGGCGGCAACTACAACTACCTGTACCGGCTGTTCCGGAAG TCCAACCTGAAGCCTTTCGAGCGGGACATCAGCACCGAGATCTATCAGGCCGGCAGC ACCCCTTGTAATGGCGTCGAGGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCT TCCAGCCTACCAATGGCGTGGGCTACCAGCCTTATAGAGTGGTGGTGCTGTCCTTCGA ACTGCTGCATGCCCCTGCTACCGTGTGCGGCCCTAAGAAGTCTACCAACCTGGTCAAG AACAAATGCGTGAACTTCgaggctaagcccagcggctctgtggttgaacaagccgaaggcgtggaatgcgacttc tctccactgctgtctggcacccctccacaggtgtacaacttcaagcggctggtgttcaccaactgcaattacaacctgacaaag ctgctgagcctgttcagcgtgaacgactttacctgcagccagatctctcctgccgccattgccagcaactgttacagctccctga tcctggactacttcagctaccctctgagcatgaagtccgacctgtctgtgtctagcgccggacctatcagccagttcaattacaa gcagtccttcagcaaccccacctgtctgattctggccaccgtgcctcacaatctgaccaccatcaccaagccactgaagtacag ctacatcaacaagtgcagccggttcctgagcgacgacagaacagaagtgccacagctcgtcaacgccaaccagtacagccc ctgtgtgtctatcgtgcctagcacagtgtgggaggacggcgactactacagaaagcagctgtctccactcgaaggcggaggat ggctggtggcttctggaagcacagtggctatgacagagcagctgcagatgggctttggcatcaccgtgcagtacggcaccgat accaatagcgtgtgccccaagctggaattcgccaacgacaccaagattgccagccagctgggcaattgcgtcgagtacaga GTGGTGCCTAGCGGCGACGTTGTGCGCTTTCCTAATATCACAAACCTGTGTCCAT TCGGGGAAGTGTTTAACGCCACAAAGTTCCCTTCCGTGTATGCCTGGGAGCGCAA GAAAATCTCCAACTGTGTGGCTGATTACTCCGTCCTGTACAACAGCACCTTTTTCT CCACGTTCAAATGTTATGGGGTGTCCGCCACCAAACTCAATGACCTCTGTTTTAG CAACGTCTACGCCGACTCCTTCGTCGTGAAAGGGGATGATGTTCGCCAGATCGCC CCAGGACAAACCGGCGTTATCGCCGACTATAATTACAAACTCCCCGATGATTTCA TGGGCTGTGTGCTGGCCTGGAACACCAGAAATATCGATGCCACCTCCACCGGGA ACTATAACTACAAGTACAGATACCTGCGGCACGGCAAGCTGAGGCCCTTTGAGA GGGATATCTCCAACGTGCCATTCAGCCCCGACGGCAAGCCTTGTACACCACCAGC TCTGAATTGCTACTGGCCCCTGAACGATTACGGCTTCTACACCACAACCGGCATC GGCTACCAACCATACAGGGTCGTCGTGCTGAGCTTCGAATTGCTGAACGCCCCAG CCACAGTGTGTGGCCCAAAGCTGAGCACCGACCTGATTAAGAACCAGTGCGTCA ACTTCAACtaataaggatccagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggc taataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatatgggagggcaaatcatttaaaac atcagaatgagtatttggtttagagtttggcaacatatgcccattcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgc ggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaa ggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaat cgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctca gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgt cttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggt gctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcg gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga aaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagt taccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctga ggtctgcctcgtgaagaaggtgttgctgactcataccagggcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgttt ggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggt cctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatac gggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg ctgttgagatccagttcgatgtaacccactcgtgcacctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgag agctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatcctt caactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaatt ctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaa tgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaa cctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttat gcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcg cctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagc gcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatc atcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattg gcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacat tatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctc ataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgag acacaacgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa ataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa ataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcac agcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaact atgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatca gattggctatt (SEQ ID NO: 13).

[0067] In a further embodiment, the DNA vaccine composition comprises at least the polynucleotide sequence of SEQ ID NO: 19 (Albumin- COVID19 RBD- Mers RBD- Sars RBD (single underline is albumin, uppercase bold and italicized is EcoRI cloning site, lowercase italicized is COVID19 RBD, uppercase bold is Mers RBD, double underline is Sars RBD, and remainder is vector pNGVL4a)). In some embodiments, the synthetic polynucleotide encoding the DNA vaccine composition has the nucleic acid sequence of SEQ ID NO: 19, or a polynucleotide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 19:

GGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGT

CCAACATTACCGCCATGTTGACATTGATTATTGactagtTATTAATAGTAATCAATTA

CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGT

AAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG

ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

ACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAG

TACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG

TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC

TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT

GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGAC

GCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTG

GCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTAT

AGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGActcgagGCCACCatga agtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcacacaagagtgaggttgctcat cggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctcagtatcttcagcagtgtccatttgaagatcatgta aaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttgga gacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatg cttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatga agagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaa gctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcg tctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccaga gatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttga atgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaac ctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagt aaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgt cgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaag tgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattcc agaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtg ggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttg catgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaag tcgatgaaacatacgttcccaaagagtttaatgctgaaacgttcaccttccatgcagatatatgcacactttctgagaaggagagacaaat caagaaacaaactgcacttgttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgca gcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc cttaggctta GAATTCatscscstscascccactsastccatastsasstttcctaacataactaacctctscccattcssssa agtgtttaacgccacccggttcgctagcgtgtacgcctggaaccgtaagaggattagtaactgcgtagctgactatagcgtactgta taatagcgctagttttagcacctttaagtgctacggggtcagccccactaagctgaatgatttgtgcttcactaacgtgtacgctgata gcttcgtaattaggggggatgaggtgagacagatagcccccggacagaccggcaagatcgctgattacaactacaagctgcctg atgacttcacaggctgcgtgatcgcctggaactctaacaacttggactctaaggtcggcggaaactacaattacctttaccgcctgtt tagaaagtccaacctgaaacccttcgagcgggatatcagcacagagatctaccaggccgggagcaccccctgcaacggggtg gagggcttcaactgctacttccccctgcagtcctacgggttccagccaaccaacggcgtgggctaccagccctacagagtcgtggt gctgtcctttgagctgctgcacgcccccgctaccgtctgcggccccaagaagtccacaaacctcgtgaagaacaagtgcgtgaac ii/GAGGCCAAGCCATCCGGGAGCGTCGTGGAGCAGGCCGAAGGGGTCGAGT

GCGACTTCAGCCCACTGCTGAGCGGCACCCCCCCACAGGTGTACAACTTTA

AGAGGCTGGTGTTCACTAACTGCAACTACAACCTCACCAAGCTCCTGTCCCT

GTTCTCCGTGAACGACTTCACATGCAGCCAGATCAGCCCAGCCGCCATCGC

CTCCAACTGCTACAGCAGCCTGATCCTCGACTACTTCTCCTACCCCCTCAGC

ATGAAGTCCGACCTCTCCGTGTCCAGCGCCGGCCCCATTAGCCAGTTTAACT ACAAGCAGAGCTTTTCCAACCCCACCTGCCTGATCCTGGCCACAGTGCCCCA

TAACCTGACCACAATTACCAAGCCCCTGAAGTACAGCTACATTAACAAGTGC

TCCAGGTTCCTCAGCGATGATCGGACCGAGGTGCCCCAGCTCGTCAACGCC

AACCAGTACAGCCCTTGCGTGAGCATTGTCCCCAGCACCGTGTGGGAGGAC

GGCGACTACTACAGAAAGCAGCTGAGCCCTCTGGAGGGCGGCGGGTGGCTG

GTGGCCTCCGGGAGCACAGTGGCCATGACAGAGCAGCTGCAGATGGGGTTC

GGCATTACTGTGCAGTACGGAACAGATACAAACAGCGTGTGCCCTAAGCTG

GAGTTCGCCAACGACACTAAGATCGCCTCCCAGCTGGGCAACTGCGTCGAG tccacattcaagtgctacggcgtgagcgccacaaagctgaacgacctctgcttcagcaacgtgtacgccgacagcttcgtggtcaagg gcgatgatgtgcggcagatcgcccccggccagaccggcgtgatcgccgattacaactataagctgcccgacgacttcatggggtgc gtgctggcctggaacacaaggaacattgatgccaccagcacaggcaactacaactacaagtacaggtacctgaggcacgggaagct gcggcccttcgagcgggacatctccaacgtgcccttcagccccgacggcaagccctgcaccccccccgccctgaactgctactggc ccctgaacgattacggcttctacacaaccaccggcattggctaccagccttaccgggtcgtggtgctgagctttgagctgctgaacgcc cccgccaccgtgtgcgggcctaagctgagcactgacctgattaagaaccagtgcgtgaactttaacTGATGAGGATCCA

GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATC

TGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTT

TTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGA

ATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATTCTTCCGCTTCCTCGCTC

ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA

AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT

GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG

TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTC

AGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA

GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC

TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG

TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAG

CCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC

ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT

ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG AAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA

GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG

TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA

TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT

GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAAT

GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT

GCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCT

GACTCATACCAGGGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCT

CGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTAC

ATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT

GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA

ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA

ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGT

CAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG

GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCTGAATCGCCCCATCATCCAGCCAGAAAGTGA

GGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAA

CTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCC

TTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAG

CGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCA

TCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATT

TTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA

GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACA

ACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGA

GTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTT

GTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACC

GTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA

GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA

TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTT

CCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATG CTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCA

TCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCG

CATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATC

GCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGC

CTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTT

TATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGT

AACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCATTATTGAAG

CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA

AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT

AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGC

CCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCT

CCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG

TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGC

ATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAG

ATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATT (SEQ ID NO: 19).

[0068] The DNA vaccine composition can be comprised within an expression cassette. [0069] The term "expression cassette" or "expression vector" as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. "Operably linked', refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant "naked DNA" vector, and the like.

[0070] The term "immunogen" or "immunogenic composition" is synonymous with “antigen or antigenic” and refers to a compound or composition comprising a peptide, polypeptide or protein which is "immunogenic," i.e., capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance. An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a fragment 15 amino acids in length, a fragment 20 amino acids in length or greater; smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen's coding sequence operably linked to a promoter, e.g., an expression cassette.

The immunogen includes one or more antigenic determinants or epitopes, which may vary in size from about 3 to about 15 amino acids.

[0071] In one or more embodiments, the immunogen or antigen is a synthetic polypeptide encoding RBD or portion or fragment thereof.

[0072] In some embodiments, the RBD antigen is from one or more different viruses or can contain more than one copy of the RBD antigen. In some embodiments, the RBD antigen is from three different coronaviruses, such as SARS-Cov2, MERS-CoV, and SARS-CoV. [0073] In accordance with an embodiment, the present invention provides a recombinant vector encoding the DNA vaccine compositions described herein.

[0074] By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.

[0075] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.

For purposes herein, the replication can be in vitro replication or in vivo replication.

[0076] The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine- substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3- (3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0077] In some embodiments, the substituted nucleic acid sequence may be optimized. Without being bound to a particular theory, it is believed that optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0078] The invention also provides an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein. [0079] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally occurring as a whole. However, parts of the vectors can be naturally occurring. The inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages does not hinder the transcription or replication of the vector.

[0080] In some embodiments, the DNA vaccine composition or expression cassette will be inserted into a DNA vector or plasmid.

[0081] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGT10, λGT11, λλZap I I (Stratagene), /.EMBL4, and lNM1149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). [0082] In some embodiments, the plasmid can be one of the pcDNA3 family of plasmids known in the art. In another embodiment, the plasmid can be the pNGVL4a expression vector. [0083] As the research progresses towards clinical trials, we should carefully consider the expression vector used. Any vector used needs to be non -harmful to human body. The pcDNA3 contains the ampicillin resistant gene, rendering it inappropriate for clinical usage. The inventors have previously used a mammalian cell expression vector, pNGVL4a DNA construct for our clinical studies. The pNGVL4a DNA construct does not contain ampicillin resistant gene.

[0084] In accordance with another embodiment Applicants’ have cloned various constructs including the albumin-SARS-CoV2 RBD fusion construct, and two albumin- SARS-CoV2 RBD, MERS-CoV RBD and SARS-CoV fusion constructs into the pNGVL4a vector and are analyzing the expression and immunogenicity of these DNA constructs.

[0085] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 m plasmid, l, SV40, bovine papilloma virus, and the like.

[0086] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0087] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0088] The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the fusion proteins, polypeptide, or protein. The selection of promoters, e.g., strong, weak, inducible, tissue- specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.

[0089] In accordance with an embodiment, the present invention provides a composition comprising a synthetic polypeptide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.

[0090] It will be understood by those of skill in the art that the synthetic polypeptide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof is a fusion polypeptide which acts as an immunogen to the immune system and is expressed in the cells of the subject that have taken up the DNA vaccine of the present invention.

[0091] In some embodiments, the synthetic polypeptide encoding human albumin protein has the amino acid sequence of SEQ ID NO: 2, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 2.

[0092] In some embodiments, the synthetic polypeptide encoding RBD has the amino acid sequence of SEQ ID NO: 4, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 4.

[0093] In some embodiments, the synthetic polypeptide encoding RBD, or a functional portion, or fragment or variant thereof, conjugated to human albumin protein, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 6, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 6.

[0094] In accordance with another embodiment, the synthetic polypeptide molecules described herein, such as SEQ ID NO: 6, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 6, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 6, can also be used as a vaccine.

[0095] In accordance with an embodiment, the synthetic polypeptide molecules described herein, such as SEQ ID NO: 12, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 12, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 12, can also be used as a vaccine. [0096] In accordance with another embodiment, the synthetic polypeptide molecules described herein, such as SEQ ID NO: 14, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 14, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 14, can also be used as a vaccine.

[0097] In accordance with another embodiment, the synthetic polypeptide molecules described herein, such as SEQ ID NO: 20, or a functional portion or fragment or variant thereof comprises the amino acid sequence of at least SEQ ID NO: 20, or a polypeptide having at least 80%, 85%, 90%, 95%, 99% identity with SEQ ID NO: 20, can also be used as a vaccine.

[0098] The term "functional portion" when used in reference to the antigenic epitope or immunogen refers to any part or fragment, which part or fragment retains the biological activity of which it is a part (the parent molecule, antibody, or antigen). Functional portions encompass, for example, those parts that retain the ability to specifically bind to the antigen (e.g., in an MHC-independent manner), or detect, treat, or prevent the disease, to a similar extent, the same extent, or to a higher extent, as the parent molecule. In reference to the parent molecule, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent molecule.

[0099] In this regard, the invention also provides a synthetic polypeptide molecule comprising at least one of the polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. See, for instance, Choi et al., Mol. Biotechnol. 31: 193-202 (2005).

[00100] Included in the scope of the invention are functional variants of the inventive fusion proteins, and polypeptides, and proteins described herein. The term "functional variant" as used herein refers to fusion proteins, polypeptides, or proteins having substantial or significant sequence identity or similarity to a parent fusion proteins, polypeptides, or proteins, which functional variant retains the biological activity of the fusion proteins, polypeptides, or proteins of which it is a variant. In reference to the parent fusion proteins, polypeptides, or proteins, the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent fusion proteins, polypeptide, or protein.

[0100] The functional variant can, for example, comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, lie, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

[0101] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent fusion proteins, polypeptide, or protein with at least one non conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent fusion proteins, polypeptide, or protein.

[0102] The fusion polypeptides, and/or proteins of the invention (including functional portions and functional variants thereof) can be obtained by methods known in the art. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the fusion proteins, polypeptides, and proteins of the invention (including functional portions and functional variants thereof) can be isolated and/or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art. Alternatively, the fusion proteins, polypeptides, and/or proteins described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies, such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this respect, the inventive fusion proteins, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.

[0103] In accordance with an embodiment, the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions described herein. [0104] In accordance with a further embodiment, the present invention provides a pharmaceutical composition comprising the synthetic polypeptide compositions described herein for use as a vaccine.

[0105] Thus, in further embodiments, the present invention provides the use of a pharmaceutical composition comprising vaccine, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of a SARS coronavirus infection in a subject.

[0106] In another embodiment, the present invention provides the use of a pharmaceutical composition comprising the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of COVID 19-related disease or SARS-CoV2 infection in a subject.

[0107] In a further embodiment, the present invention provides a method for treating a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.

[0108] In some embodiments, the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV-2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.

[0109] In some alternative embodiments the present invention provides the use of a pharmaceutical composition comprising vaccine, and a pharmaceutically acceptable carrier, as a medicament, preferably as a medicament for the treatment of a SARS-CoV, MERS-CoV or SARS-CoV2 coronavirus infection in a subject.

[0110] In further embodiments, the present invention provides a method for treating a a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.

[0111] In some embodiments, the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV, MERS-CoV or SARS-CoV2infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein.

[0112] In some embodiments, the term "administering" means that the compositions of the present invention are introduced into a subject, preferably a subject receiving treatment for an infectious disease, and the compounds are allowed to come in contact with the one or more disease related cells or population of cells in vivo.

[0113] It will be understood to those of ordinary skill in the art that the DNA vaccine compositions described herein can be administered in a regimen where there is a first or priming dose of vaccine composition administered to the subject, then after a period of 5 to 50 days, a second, third or more boost dose of vaccine is then administered to the subject. In some embodiments, the boost dose is administered 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 up to 50 days apart.

[0114] Thus, in accordance with an embodiment, the present invention provides a method for treating a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising: a) administering to the subject an effective amount of a first or priming dose of the DNA vaccine compositions and/or the synthetic polypeptide compositions; b) administering to the subject after an interval of between about 5 to 180 days an effective amount of a second or boost dose of the DNA vaccine compositions and/or the synthetic polypeptide compositions; and c) optionally repeating step b).

[0115] The carrier is preferably a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration· The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0116] The choice of carrier will be determined in part by the chemical properties of the vaccines as well as by the particular method used to administer the vaccines. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and intraperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the first and second vaccine, and in certain instances, a particular route can provide an immediate and more effective response than another route. In a preferred embodiment, the vaccine is administered intra-muscularly. In another embodiment, and the vaccine is administered intra-dermally. [0117] Injectable formulations are in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well- known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 14th ed., (2007)).

[0118] In accordance with some embodiments, the vaccines of the present invention can be administered other ways known in the art. For example, the vaccines can be administered via use of electroporation techniques. Suitable electroporation techniques are disclosed in U.S. Pat. Nos. 6,010,613, 6,603,998, and 6,713,291, all of which are incorporated herein by reference. Other physical approaches can include gene gun, biojector, ultrasound, and hydrodynamic delivery, all of which employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer. Chemical vaccination approaches typically use synthetic or naturally occurring compounds (e.g. cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to deliver the nucleic acid into the cells. [0119] In some other embodiments, intramuscular administration of the vaccines of the present invention may be achieved by the use of a needless injection device to administer a virus or plasmid DNA suspension (using, e.g., Biojector™) or a freeze-dried powder containing the vaccine (e.g., in accordance with techniques and products of Powderject). [0120] For purposes of the invention, the amount or dose of the vaccine administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the first and second vaccine and the condition of a human, as well as the body weight of a human to be treated. [0121] Typically, the attending physician will decide the dosage of first and second vaccine with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the vaccine is about 1 to 10,000 μg of vaccine to the subject being treated. In some embodiments, the dosage range of the vaccine is about 500 μg-6,000 μg of vaccine. In a preferred embodiment, the dosage of the vaccine is about 3,000 Pg·

[0122] In accordance with some embodiments, the present invention provides a pharmaceutical composition comprising the DNA vaccine compositions and/or the synthetic polypeptide compositions described herein in combination with at least one additional biologically active agent. [0123] An “active agent” and a “biologically active agent” are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0124] The biologically active agent may vary widely with the intended purpose for the composition. The term active is art-recognized and refers to any moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of biologically active agents, that may be referred to as “drugs”, are described in well-known literature references such as the Merck Index, the Physicians’ Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0125] Non- limiting examples of biologically active agents include following: anti inflammatory agents such as steroids, non-steroidal anti-inflammatory agents, anti-pyretic and analgesic agents, antigenic materials, and anti-viral drugs.

[0126] Various forms of the biologically active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms and the like, which are biologically activated when implanted, injected or otherwise placed into a subject.

[0127] Thus, in a further embodiment, the present invention provides a method for treating a SARS-CoV, MERS-CoV or SARS-CoV2infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions in combination with one or more additional biologically active agents.

[0128] In some embodiments the invention provides a method for post-exposure prophylaxis or treatment of a SARS-CoV, MERS-CoV or SARS-CoV2 infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions and/or the synthetic polypeptide compositions in combination with an effective amount of one or more additional biologically active agents.

[0129] It will be understood that the methods of treatment using an effective amount of the DNA vaccine compositions in combination with an effective amount of one or more additional biologically active agents, the combination can occur either simultaneously or serially with at least one other.

[0130] The dosing regimens of the above methods can also comprise a first dose of vaccine an additional biologically active agent, followed by a second or more dose of vaccine and optionally an additional biologically active agent as needed.

[0131] As used herein, the term "subject" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0132] In accordance with an embodiment, the present invention provides a cell or population of cells expressing the synthetic polypeptide compositions described herein. It will be understood that the cells or population of cells expressing the synthetic polypeptide compositions were in contact with the DNA vaccine compositions and/or the synthetic polypeptide compositions in vitro or in vivo.

[0133] As defined herein, in another embodiment, the term "contacting" means that the one or more compounds of the present invention are introduced into a sample having at least one cancer cell and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding and uptake of the at least one compound to the cancer cell. Methods for contacting the samples with the compounds, and other specific binding components are known to those skilled in the art, and may be selected depending on the type of assay protocol to be ran. Incubation methods are also standard and are known to those skilled in the art.

[0134] In accordance with an embodiment, the present invention provides kits that contain the compositions or pharmaceutical compositions used with the inventive methods, as described above, to practice the methods of the invention. The kits can contain various combinations of vaccines and the like. The kit can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the invention, means to inject or infect cells, patients or animals with vaccines of the invention, means to monitor the resultant immune response and assess the reaction of the individual to which the compositions have been administered, and the like.

[0135] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

[0136] Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0137] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0138] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1 , and the like) and any range within that range.

[0139] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

EXAMPLES

[0140] To generate pcDNA3-albumin, human albumin (Hal) was first amplified via PCR using the cDNA template of human albumin (GenBank: AAA98797.1, TransOMIC Technologies, Huntsville, AL) and the following set of primers: 5'- A A ACTCG AGGCC ACC ATGA AGTGGGT A ACCTTT- 3 ' (SEQ ID NO: 7) and 5'- TTTGAATTCTAAGCCTAAGGCAGCTTG-3 ' (SEQ ID NO: 8). The amplified human albumin product was then cloned into the Xho/EcoRI sites of a pcDNA3 vector (Invitrogen Corp., Carlsbad, California). Next, for the generation of pcDNA3-Albumin-RBD, RBD was amplified via PCR using the cDNA template of RBD (GenBank QHD43416.1) from Sino Biological (Wayne, PA 19087) and the following primers: 5'- AAAGAATTCATGAGGGTCCAACCAACAGAG-3' (SEQ ID NO: 9) and 5'- TTTGGATCCTC AGAAGTTC ACAC ACTTGTT-3 ' (SEQ ID NO: 10). The amplified product was then cloned into the EcoRI/BamHI sites of pcDNA3 -Albumin creating pcDNA3- Hal-RBD. For the generation of pcDNA3-RBD, the amplified cDNA RBD product was cloned into the EcoRI/BamHI sites of pcDNA3 with no albumin product.

[0141] To generate pNGVL4a-Albumin-RBD DNA vaccine vector, albumin-RBD cDNA was synthesized by Thermofisher and cloned into Xbal/BamHI of pNGVL4a vector.

[0142] To generate the pNGVL4a-Albumin- SARS-CoV-MERS-CoVSARS-CoV2 vaccine vector, albumin- SARS-CoV-MERS-CoVSARS-CoV2 RBD cDNA was synthesized by Thermofisher and cloned into Xbal/BamHI of pNGVL4a vector.

[0143] Western blot

[0144] To characterize the SARS-CoV2 RBD protein expression levels in cells, HEK 293 cells were transfected with 10 μg of either pcDNA3-RBD or pcDNA3-Hal-RBD plasmid. After 48 hours of transfection, cell lysates were collected to complete a Western blot analysis. Blots were blocked with PBS/0.05%, Tween 20 (PBST) containing 5% nonfat milk for one hour at room temperature. Membranes were probed with anti-SARS-CoV Spike Antibody (Cat: 40150-T62-COV2, Sino Biological) at 1:3000 dilution in PBST containing 5% nonfat milk overnight at 4 °C, washed three times with PBST, and then incubated with goat antimouse IgG conjugated to alkaline phosphatase (Amersham, Piscataway, NJ) at 1:5000 dilution in PBST containing 5% nonfat milk. Membranes were washed three times with PBST and developed using enhanced Hyperfilm- enhanced chemiluminescence (Amersham, Piscataway, NJ). GAPDH (Cat: 60004- 1-Ig, Proteintech) was loaded as a control.

[0145] Animal Vaccination

[0146] To test the plasmid and generate sera, 6-8 week old female C57BL/6 mice (5 mice per group) were vaccinated. Each mouse was vaccinated with 20 μg/mouse of pcDNA3- SARS-CoV2 RBD, pcDNA3-Hal- SARS-CoV2 RBD, pNGVL4a-Hal-SARS-CoV3 RBD, and the variant pNGVL4a-Hal-SARS-CoV3 RBD-MERS-CoV RBD-SARS-CoV RBD plasmids via intramuscular injection in the right and left hind leg (10 μg/leg) at day 0. The mice were boosted with the same regimen twice, once at day 7 and once at day 14. One week after the final vaccination (day 21), sera was collected.

[0147] Characterization of Antibody Response

[0148] To characterize the SARS-CoV-2 MERS-CoV RBD-SARS-CoV RBD Spike Protein RBD antibody response, mouse serum was analyzed by ELISA lug/ml of RBD protein (from Sino Biological) in PBS was coated on BRANDplates ® microplates overnight at 4 °C. The next morning, the plates were washed, blocked with eBioscience™ ELISA/ELISPOT Diluent (Thermo Fisher Scientific), and added serial dilution of serum for two hours at room temperature. All serum used ranged from 1:100 - 1:10,240, and non- vaccinated mice serum was used as control. Goat anti-mouse IgG-HRP secondary antibody was added at 1:5000 dilution for 1 hour, followed by TMB substrate. The OD at 450 nm was determined by 800™ TS Absorbance Reader (BioTek Instruments, Inc).

EXAMPLE 1

[0149] Generation and characterization of an exemplary DNA vaccine construct encoding human albumin (Albumin) linked to the receptor binding domain (RBD) of the spike protein of the SARS-CoV-2, MERS-CoV RBD, and SARS-CoV RBD.

[0150] In one embodiment, pcDNA3 was first generated encoding human albumin (pcDNA3- Albumin). The DNA sequence encoding human albumin was amplified with PCR using the cDNA template of human albumin and a set of primers framing the albumin gene. The amplified product was then cloned into the Xho/EcoRI sites of the pcDNA3 vector to form pcDNA3 -Albumin. Next, to generate DNA encoding human albumin linked to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2, RBD gene was amplified via PCR using the cDNA template of RBD and a set of primers framing the RBD gene. The amplified gene product was then cloned into the EcoR I/Bam HI sites of pcDNA3- Albumin to create pcDNA3-Albumin-RBD (See Figures 1 and 2). We also generated DNA encoding RBD alone (pcDNA3-RBD) as control. The accuracy of the DNA constructs was confirmed by DNA sequencing.

[0151] In some other embodiments, pNGVL4a plasmids were substituted for the pcDNA3 plasmid and the fusion constructs for Hal-SARS-CoV-2, MERS-CoV RBD, and SARS-CoV RBD were constructed in the same manner. EXAMPLE 2

[0152] The linkage of albumin to SARS-CoV2 RBD enhances the expression of the chimeric RBD protein in cells transfected the DNA constructs.

[0153] The resultant pcDNA3-albumin-RBD or pcDNA3-RBD DNA constructs were transfected into HEK 293 cells in order to probe for resultant expressed protein. HEK 293 cells were transfected with either pcDNA3-RBD or pcDNA3-albumin-RBD DNA constructs for 48 hours. A western blot was then completed to gauge protein expression, using GAPDH as a control. As shown in Figure 3, the HEK 293 cells transfected with pcDNA3-albumin- RBD DNA construct produced the novel chimeric albumin-RBD protein with a molecular weight of around 50-75 kD. In comparison, HEK 293 cells transfected with the pcDNA3- RBD DNA construct (without the albumin) produced the SARS-CoV2 RBD protein with a molecular size of around 25 kD (Fig 3B). Further, the amount of the chimeric albumin-RBD protein is significantly larger compared to that of RBD protein. Taken together, our data indicated that the linkage of albumin to RBD enhances the protein expression of the chimeric albumin-RBD fusion protein in cells transfected with the DNA construct.

EXAMPLE 3

[0154] The linkage of albumin to SARS-CoV2 RBD drastically enhanced the RBD- specific antibody responses in mice vaccinated with the DNA construct.

[0155] 6-8 week old female C57BL/6 mice (5 per group) were vaccinated with 20 μg/mouse of either pcDNA3-RBD or pcDNA3-Hal-RBD three times at 1-week intervals (Fig 4A). One week after final vaccination, mouse sera was harvested to analyze via ELISA for SARS-CoV2 RBD-specific antibody responses. As shown in Figure 4B, the mice vaccinated with pcDNA3 -Albumin-RBD had significantly higher levels of RBD-specific antibody responses compared to mice vaccinated with pcDNA3-RBD. The mice vaccinated with pcDNA3-RBD DNA vaccine showed no significant difference in SARS-CoV2 RBD-specific antibody levels when compared to the control group receiving no RBD DNA injection at all (Fig 4B). Thus, our data indicated that the linkage of Albumin to RBD drastically enhanced the RBD-specific antibody responses in the DNA vaccinated mice.

EXAMPLE 4 [0156] pcDNA3-Hal-RBD vaccination generate the highest levels of SARS-CoV2 RBD- specific antibody in vaccinated mice among all of the DNA vaccines tested. Characterization of Hal-RBD DNA vaccination by ELISA. (Fig. 5A) 6-8 week old female C57BL/6 mice (5 mice/group) were vaccinated with 20 μg/mouse of pcDNA3-Sig/RBD, pcDNA3-Sig-IgG- RBD or pcDNA3-Hal-RBD DNA by intramuscular injection. The mice were boosted with the same regimen once with a 1-week interval. One week after last vaccination, serum were collected for ELISA. (Fig. 5B) Antibodies specific for the human SARS-CoV-2 Spike protein were analyzed by ELISA. Briefly, 1 μg/ml of human SARS-CoV-2 Spike protein in PBS were coated on BRANDplates® microplates for overnight. The next day wells were washed, blocked with eBioscience™ ELISA/ELISPOT Diluent, and added serial dilutions of serum (all ranging from 1:100-1:10,240) for two hours at room temperature. Non-vaccinated mice serum as control. Goat anti-mouse IgG-HRP secondary antibody was added for one hour at 1:5000 dilution, followed by TMB substrate. The results demonstrate that the inventive vaccine compositions are far superior to other technologies demonstrated in the past, such as making the protein secreted out of cells by adding the signal peptide (sig) to SARS-CoV2 RBD or linking the SARS-CoV2 RBD to the Fc portion of IgG to enhance humoral immune responses etc.

EXAMPLE 5

[0157] Vaccination with DNA encoding albumin-RBD elicits antibody responses recognizing the spike protein of SARS-CoV-2 expressed on the cell surface.

[0158] In order to determine whether the RBD-specific antibody responses generated by vaccination with pcDNA3-Hal-RBD is able to bind with the spike protein of SARS-CoV-2 in the right conformation, we used HEK 293 cells transfected with DNA encoding the full length of the spike protein of the SARS-CoV-2. The binding of the spike protein of SARS- CoV-2 by the RBD-specific antibody from vaccinated mice were characterized using flow cytometry analysis. As shown in Figure 6, only sera from mice vaccinated with pcDNA3- Hal-RBD DNA, but not pcDNA3-RBD DNA, were able to bind with the 293 cells transfected with DNA encoding the full length of the spike protein of the SARS-CoV-2. These data suggest the RBD-specific antibody generated by vaccination with pcDNA3-Hal-RBD DNA were able to bind with the spike protein of SARS-CoV-2 in the right conformation. [0159] The inventors have successfully generated a pcDNA3-Albumin-RBD DNA construct to generate antibodies for the SARS-CoV-2 spike protein RBD. Our study has shown that pcDNA3-Albumin-RBD generates a higher amount of RBD-Alb protein than pcDNA3-RBD generates its respective RBD protein in DNA transfected cells, suggesting pcDNA3-Albumin-RBD may be more immunogenic. Additionally, the inventors have shown that pcDNA3-Albumin-RBD DNA vaccine generates a significantly higher level of SARS- CoV-2 spike protein RBD specific antibody responses compared to pcDNA3-RBD DNA vaccine in vaccinated mice. The stronger immune response elicited by pcDNA3-Albumin- RBD indicates potential opportunity of the DNA vaccine in preventing COVID-19.

EXAMPLE 6

[0160] pcDNA3-Hal-RBD and pNGVL4a-Hal RBD expresses higher levels of secreted protein in a western blot.

[0161] To identify the protein expression level of Hal-RBD, HEK 293 cells were transfected with 10 μg of each DNA construct. After 48 hours transfection, cell lysates and supernatant were collected for Western blot analysis. Lane 1: cell pellet from pcDNA3-Hal- RBD transfected cells. Lane 2: cell pellet from pNGVL4a-Hal-RBD transfected cells. Lane 3: supernatant from pcDNA3-Hal-RBD transfected cells. Lane 4: supernatant from pNGVL4a - Hal-RBD transfected cells. The blot shows that the secreted protein in the supernatant of cell culture media had a higher concentration of expressed fusion protein than the transfected cells themselves (Lig. 7).

EXAMPLE 7

[0162] Mice immunized with the synthetic fusion protein constructs generated antibodies to RBD. C57BL/6 mice (5 per group) were vaccinated subcutaneously with 10 μg of RBD,

20 μg RBD-LC or 40 μg of Hal-RBD protein vaccine with 5 μg MPL/mouse three times at a 1 week interval. Sera from each group of mice were collected through tail vein 1 week after the last vaccination. Serial dilution of the sera was used for ELISA using 96 well plate coated with RBD protein. Figure 8 shows that the mice vaccinated with the Hal-RBD protein generated a higher titer of RBD antibody than RBD or RBD-LC alone.

[0163] There are several reasons that may account for the observed enhancement of RBD-specific immune responses when albumin gene is linked to RBD gene in the DNA construct. First, linkage to albumin can extend the life of the protein in vivo (Makrides, S. C., Nygren, P. A., Andrews, B., Ford, P. J., Evans, K. S., Hayman, E. G., Adari, H., Uhlen, M., and Toth, C. A. (1996). Extended in vivo half-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor. J Pharmacol Exp Ther 277, 534-542). By extending the time the body is exposed to the RBD antigen, the immune system is provided with a longer span of time to properly mount an immune response and produce antibody when compared to a shorter period of antigen exposure. Second, linking albumin to RBD also promotes drainage of the RBD protein to the lymph nodes, as albumin is noted in its ability to target the lymph nodes, which is important for triggering immune responses (Wang, Y.,

Lang, L., Huang, P., Wang, Z., Jacobson, O., Kiesewetter, D. O., Ali, I. U., Teng, G., Niu, G., and Chen, X. (2015). In vivo albumin labeling and lymphatic imaging. Proc Natl Acad Sci U S A 112, 208-213). The lymph nodes contain high levels of immune cells, meaning that when RBD is targeted to the lymph nodes, the immune system can better mount an immune response. Without albumin, less RBD is drained to the lymph nodes, which results in a less pronounced antibody response. Finally, as described above, linking albumin to RBD resulted in increased expression the chimeric protein (Fig 3). Higher expression levels make the vaccine more potent, as immune cells get more exposure and opportunity to respond to the foreign antigen. Although other factors may also contribute to the higher levels of antibody responses produced in response to the pcDNA3-Albumin-RBD, linkage to albumin appears to increase the volume, life span, and lymph node targeting of RBD, which provides a strong rationale for why antibody levels were notably higher compared to pcDNA-RBD alone, and it is expected that the same results will be seen in vaccinations with the pNGVL4a Hal- SARS- CoV2 RBD and SARS-CoV-MERS-CoVSARS-CoV2 RBD vaccine compositions.

[0164] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0165] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0166] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. A DNA vaccine composition comprising a synthetic polynucleotide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to one or more coronavirus receptor binding domains (RBD), or a functional portion or fragment or variant thereof.

2. The DNA vaccine composition of claim 1, wherein the vaccine has the sequence of SEQ ID. NO: 5 and/or SEQ ID NO: 11 and/or SEQ ID NO: 13 and/or SEQ ID NO: 19.

3. The DNA vaccine composition of claim 1, wherein the albumin is human.

4. The DNA vaccine composition of claim 3, wherein the human albumin has the polynucleotide sequence of SEQ ID NO: 1.

5. The DNA vaccine composition of claim 3, wherein the RBD has the sequence of SEQ ID NO: 3, and/or SEQ ID NO: 15, and/or SEQ ID NO: 17.

6. A recombinant vector encoding the DNA vaccine compositions of any of claims 1- 5.

7. The vector of claim 6, wherein the vector is pcDNA3 or pNGVL4a.

8. A pharmaceutical composition comprising the recombinant vector of claim 6 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, and at least one additional biologically active agent.

10. A method for treating a SARS coronavirus infection in a subject in need thereof comprising administering to the subject an effective amount of the DNA vaccine compositions of any of claims 6-9.

11. The method of claim 10, wherein the SARS coronavirus is SARS-CoV-2 and/or MERS-CoV and/or SARS-CoV.

12. The method of either of claims 10 or 11, wherein the treatment is for post exposure prophylaxis or treatment of a SARS-CoV-2 and/or MERS-CoV and/or SARS-CoV infection.

13. The method of any of claims 10 to 12, wherein the vaccine composition is administered as a boost dose at least one or more additional times at least 5 or more days after the initial administration.

14. A vaccine composition comprising a synthetic polypeptide encoding albumin protein, or a functional portion, or fragment or variant thereof, conjugated to RBD, or a functional portion or fragment or variant thereof.

15. The vaccine composition of claim 14, wherein the synthetic polypeptide has the amino acid sequence of SEQ ID NO: 6 and/or SEQ ID NO: 12 and/or SEQ ID NO: 14 and/or SEQ ID NO: 20.

16. The synthetic polypeptide vaccine composition of either of claims 14 or 15, wherein the albumin is human.

17. The synthetic polypeptide vaccine composition of any of claims 14 to 16, wherein the human albumin has the sequence of SEQ ID NO: 2.

18. The synthetic polypeptide vaccine composition of any of claims 14 to 17, wherein the RBD has the sequence of SEQ ID NO: 4 and/or SEQ ID NO: 16 and/or SEQ ID NO: 18.

19. The synthetic polypeptide vaccine compositions of any of claims 14 to 18, wherein the compositions are expressed in a cell or population of cells which have taken up the DNA vaccine compositions of any of claims 6-9.

20. A pharmaceutical composition comprising the synthetic polypeptide vaccine composition of claim 13, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, and at least one additional biologically active agent.

22. A method for treating a SARS coronavirus infection in a subject in need thereof comprising administering to the subject an effective amount of the synthetic polypeptide vaccine compositions of any of claims 14-21.

23. The method of claim 22, wherein the SARS coronavirus is SARS-CoV-2 and/or MERS-CoV and/or SARS-CoV.

24. The method of either of claims 22 or 23, wherein the treatment is for postexposure prophylaxis or treatment of a SARS-CoV-2 and/or MERS-CoV and/or SARS-CoV infection.

25. The method of any of claims 22 to 24, wherein the vaccine composition is administered as a boost dose at least one or more additional times at least 5 or more days after the initial administration.