WO2022104465A1

WO2022104465A1 - Severe acute respiratory syndrome coronavirus dna vaccines

Info

Publication number: WO2022104465A1
Application number: PCT/CA2021/051633
Authority: WO
Inventors: Gary Kobinger; George BABUADZE; Marc-Antoine DE LA VEGA; Robert Kozak
Original assignee: UNIVERSITé LAVAL; Sunnybrook Health Sciences Centre
Priority date: 2020-11-20
Filing date: 2021-11-18
Publication date: 2022-05-27
Also published as: CA3100236A1

Abstract

DNA vaccine vectors composed of a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen are provided. The DNA vaccines of the present disclosure are able to trigger an immune response towards SARS-CoV-2.

Description

TITLE: SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS DNA

VACCINES

TECHNICAL FIELD

The present disclosure relates to DNA vaccine vectors composed of a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen. The present disclosure more particularly relates to DNA vaccines that induce a humoral and cellular immune response and generate neutralizing antibodies against SARS-CoV-2. The DNA vaccines disclosed herein are used to reduce the risk of infection, the risk of transmission and/or the risk of complications associated with SARS-CoV-2.

BACKGROUND

The first severe acute respiratory syndrome coronavirus (SARS-CoV) now referred to as SARS-CoV- 1 was identified in Asia in 2003. This virus spread rapidly but was eventually contained in a matter of months. Efforts at developing a vaccine were halted for lack of funding. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease pandemic designated COVID-19 was identified in Asia in 2019 and has generated a global health crisis which yet remained to be controlled.

As of November 17, 2020, more than 54 million confirmed cases and more than 1,320,000 deaths have been reported (WHO website). As of November 1^st, 2021, 247 million confirmed cases and more than 5 million deaths have been reported (WHO website).

Unprecedented international efforts are currently aimed at the development of vaccines. To date, more than 160 vaccine candidates are under different stage of development, including mRNA-based vaccines (Moderna/NIAID, BioNTech/Fosun/Pfizer), DNA-based vaccines (Inovio/Intemational Vaccine Institute), pseudo-particles (Medicago), recombinant proteins (Novavax), inactivated virus (Sinovac) and non-replicative or pseudotyped viral particles (Oxford University/AstraZeneca, Johnson & Johnson/Janssen, Merck, Sharpe & Dohme and the International AIDS Vaccine Initiative).

There is an urgent need for the development of vaccine candidates that would confer some level of immunity towards SARS-CoV and especially towards SARS-CoV-2. DNA vaccines have recently attracted high interest. DNA vaccination relies on administration of DNA vectors encoding an antigen, or multiple antigens, for which an immune response is sought into a host. DNA vectors include elements that allow expression of the protein by the host’s cells, and includes a strong promoter, a poly-adenylation signal and sites where the DNA sequence of the transgene is inserted. Vectors also contain elements for their replication and expansion within microorganisms. DNA vectors can be produced in high quantities over a short period of time and as such they represent a valuable approach in response to outbreaks of new pathogens. In comparison with recombinant proteins, whole-pathogen, or subunit vaccines, their methods of manufacturing are relatively cost-effective and they can be supplied without the use of a cold chain system.

DNA vaccines have been tested in animal disease models of infection, cancer, allergy and autoimmune disease. They generate a strong humoral and cellular immune response that has generally been found to protect animals from the disease. Vectors for DNA vaccination have been disclosed in international application No. PCT/CA2019/050686 published on November 21, 2019 under WO2019/218091 Al and in international application No. PCT/CA2019/051592 published on November 26, 2020 under WO2020/232527 Al the entire contents of which are incorporated herein by reference.

SUMMARY

In aspects and embodiments, the present disclosure relates to DNA vaccine vectors composed of at least a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a coronavirus antigen or a fragment thereof.

In exemplary embodiments, the coronavirus antigen may comprise an antigen of a severe acute respiratory syndrome coronavirus (SARS-CoV). The coronavirus antigen may comprise for example, a SARS-CoV-2 antigen or a fragment thereof.

An exemplary embodiment of a coronavirus antigen includes a spike protein or a fragment thereof.

In a first aspect, the present disclosure relates to DNA vaccine vectors composed of at least a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen or a fragment thereof. In other aspects, the present disclosure relates to a DNA vaccine that induces an immune response toward severe acute respiratory syndrome coronavirus (SARS-CoV) antigen. More particularly, the DNA vaccines disclosed herein may trigger a humoral and/or cellular immune response towards SARS-CoV-2. The DNA vaccines disclosed herein may generate neutralizing antibodies against SARS-CoV-2.

In some embodiments, the vector portion may comprise, for example, a sequence from about at least 75% to about 100% identical to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

The present disclosure more specifically relates to a DNA vaccine vector comprising a vector portion and an antigen-coding portion, in which the antigen-coding portion may comprise a nucleic acid sequence encoding a SARS-CoV-2 spike protein or a fragment thereof.

In some embodiments, the DNA vaccine vector may comprise more than one antigencoding portions. For example, the various antigen-coding portions may all encode coronavirus antigens such as SARS-CoV-2 antigens. In other example, the various antigen-coding portions may encode a SARS-CoV-2 antigens and another non-related antigen.

In some embodiments, the vector portion may comprise a sequence at least 80% identical to SEQ ID NO:8, at least 85% identical to SEQ ID NO:8, at least 90% identical to SEQ ID NO:8, at least 95% identical to SEQ ID NO: 8, at least 96% identical to SEQ ID NO: 8, at least 97% identical to SEQ ID NO: 8, at least 98% identical to SEQ ID NO: 8, at least 99% identical to SEQ ID NO: 8 or identical to SEQ ID NO: 8.

The DNA vaccine vector of the present disclosure may further comprise a post- transcriptional regulatory element, such as for example and without limitations, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In some embodiments, the vector portion may comprise a sequence at least 80% identical to SEQ ID NO:9, at least 85% identical to SEQ ID NO:9, at least 90% identical to SEQ ID NO:9, at least 95% identical to SEQ ID NO:9, at least 96% identical to SEQ ID NO:9, at least 97% identical to SEQ ID NO: 9, at least 98% identical to SEQ ID NO: 9, at least 99% identical to SEQ ID NO:9 or identical to SEQ ID NO:9.

In some embodiments, the antigen-coding portion encodes a spike protein.

In other embodiments, the antigen-coding portion encodes a fragment of the spike protein. In some embodiments, the spike protein or fragment thereof may be from SARS-CoV-2.In some embodiments, the spike protein may comprise an amino acid sequence from about at least 95% to about 100% identical to SEQ ID NO:2 or to a fragment thereof.

In some embodiments, the spike protein may comprise an amino acid sequence at least 90% identical to SEQ ID NO:2, at least 91% identical to SEQ ID NO:2, at least 92% identical to SEQ ID NO:2, at least 93% identical to SEQ ID NO:2, at least 94% identical to SEQ ID NO:2, at least 95% identical to SEQ ID NO:2, at least 96% identical to SEQ ID NO:2, at least 97% identical to SEQ ID NO:2, at least 98% identical to SEQ ID NO:2, at least 99% identical to SEQ ID NO:2 or identical to SEQ ID NO:2 or to a fragment thereof.

In exemplary embodiments, the antigen-coding portion may encode the full SARS-CoV-2 spike protein or a fragment of SARS-CoV-2 spike protein.

Accordingly, the antigen-coding portion may encode SEQ ID NO:2 or a fragment of SEQ ID N0:2.

In some embodiments, the spike protein may comprise from 1 to 10 amino acid substitutions or more in comparison with SEQ ID NO:2.

In some embodiments, the spike protein may comprise an amino acid substitution at position A701, P681, D614, A570, N501, E484, S477, Y453, L452, K417 or combinations thereof.

In some embodiments, the spike protein may comprise amino acid substitution P681R, amino acid substitution D614G, amino acid substitution N501 Y, amino acid substitution E484K, amino acid substitution L452R, amino acid substitution Y453F or combination thereof.

The spike protein or fragment thereof may be from the original SARS-CoV-2 Whuan isolate, from the Alpha variant or related lineages (e.g., B. l.1.7 and Q lineages), from the Beta variant or related lineages (e.g., B.1.351 and descendent lineages), from the Gamma variant or related lineages (e.g., P.1 and descendent lineages), from the Delta variant or related lineages (e.g., B.1.617.2 and AY lineages), from the Epsilon variant or related lineages (e.g., B.1.427 and B.1.429), from the Eta variant or related lineages (e.g., B.1.525), from the Iota variant or related lineages (e.g., B.1.526), from the Kappa variant or related lineages (e.g., B.1.617.1), from the 1.617.3 variant or related lineages, from the Mu variant or related lineages (e.g., B.1.621, B.1.621.1), from the Zeta variant or related lineages (e.g., P.2) or from any other variants or lineages.

In some embodiments, the spike protein may comprise an amino acid substitution as described in Long SW et al., mBio Vol. 11(6): e02707-20, November 2020, the entire content of which is incorporated herein by reference.

In some embodiments, the spike protein may carry the so-called “cluster 5” mutations.

In some embodiments, the spike protein may be deleted at its C-terminus. In some embodiments the spike protein comprises an amino acid deletion at its C-terminus.

In some embodiments, the spike protein may be deleted at its N-terminus. In some embodiments the spike protein comprises an amino acid deletion at its N-terminus.

In some embodiments, the spike protein may comprise a deletion of the transmembrane domain or a portion thereof.

In exemplary embodiments, the fragment of the spike protein may be an immunogenic fragment. In another exemplary embodiment, the fragment of the spike protein may be a structural domain.

In some embodiments, the antigen-coding portion may comprise a nucleic acid sequence encoding a peptide adjuvant.

Exemplary and non-limiting examples of peptide adjuvants are provided in US20110305720A1, US20130122031 and US20150306213, the entire content of which is incorporated herein by reference.

In some embodiments, the SARS-CoV antigen and peptide adjuvant are contiguous and are expressed as a single polypeptide chain. As such, in some embodiments, the nucleic acid sequence encoding the SARS-CoV antigen and the nucleic acid sequence encoding the peptide adjuvant are in frame. In some embodiments, the nucleic acid sequence encoding the peptide adjuvant may be contiguous to the nucleic acid sequence encoding the SARS-CoV antigen.

In some embodiments, the antigen-coding portion of the DNA vaccine vector may comprise a nucleic acid sequence encoding a peptide adjuvant in frame with and contiguous to the coronavirus antigen or fragment thereof. In some embodiments, the antigen-coding portion of the DNA vaccine vector may comprise a nucleic acid sequence encoding a peptide adjuvant in frame with and contiguous to the SARS-CoV-2 antigen or fragment thereof.

In some embodiments, the nucleic acid sequence encoding the peptide adjuvant may be at the 3’ end of the nucleic acid sequence encoding the SARS-CoV antigen. In some embodiments, the peptide adjuvant may be at the N-terminal end of the SARS-CoV antigen.

In some embodiments, the nucleic acid sequence encoding the peptide adjuvant may be at the 5’ end of the nucleic acid sequence encoding the SARS-CoV antigen. In some embodiments, the peptide adjuvant may be at the C-terminal end of the SARS-CoV antigen.

In some embodiments, the peptide adjuvant may comprise or consist of the amino acid set forth in any one of SEQ ID NO: 10 to SEQ ID NO: 19.

In some embodiments, the peptide adjuvant may comprise or consist of SEQ ID NO: 10.

In some embodiments, the spike protein may be encoded by a nucleic acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at lease 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein may be encoded by a nucleic acid sequence identical to the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or with a fragment thereof.

In an exemplary embodiment, the spike protein may be encoded by a nucleic acid sequence at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO: 1. Accordingly, the spike protein may be encoded by a nucleic acid sequence at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein may be encoded by a nucleic acid sequence identical to SEQ ID NO: 1. Accordingly, the spike protein may be encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein may be encoded by a nucleic acid sequence identical to SEQ ID NO: 3. Accordingly, the spike protein may be encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 3 over the entire length of SEQ ID NO:3.

In another exemplary embodiment, the spike protein may be encoded by a nucleic acid sequence identical to SEQ ID NO: 5. Accordingly, the spike protein may be encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 5 over the entire length of SEQ ID NO: 5.

In some exemplary embodiments, the DNA vaccine vector may comprise a sequence at least 95% identical, at least 96%, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO:20 or identical to SEQ ID NO:20. Accordingly, in some exemplary embodiments, the DNA vaccine vector may comprise a sequence at least 95% identical, at least 96%, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO:20 or identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In some exemplary embodiments, the DNA vaccine vector may consist of the sequence set forth in SEQ ID NO:20.

In some exemplary embodiments, the DNA vaccine vector may comprise a sequence at least 95% identical, at least 96%, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO:21 or identical to SEQ ID NO:21. Accordingly, in some exemplary embodiments, the DNA vaccine vector may comprise a sequence at least 95% identical, at least 96%, at least 97% identical, at least 98% identical, at least 99% identical to SEQ ID NO:20 or identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some exemplary embodiments, the DNA vaccine vector may consist of the sequence set forth in SEQ ID NO:21.In some embodiments, the nucleic acid sequence may be the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein. In some embodiments, the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein (“S” gene) is as set forth in NCBI Reference sequence No. NC_045512.2:21563-25384 or is a fragment thereof.

In some embodiments, the nucleic acid sequence may be an artificial nucleic acid sequence.

In some embodiments, the nucleic acid sequence encoding the SARS-CoV antigen may be codon-optimized.

In other embodiments, the present disclosure relates to nucleic acid sequences encoding SARS-CoV-2 spike protein or a fragment thereof. The nucleic acid sequence may be cloned into any suitable vector, for example, an expression vector, a DNA vaccine vector and the like. Alternatively, the nucleic acid sequence may be generated as an RNA sequence and used as an RNA-based vaccine.

In some embodiments, the nucleic acid sequence encoding the SARS-CoV-2 spike protein or fragment thereof may be at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or to a fragment thereof, at least 96% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or to a fragment thereof, at least 97% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or to a fragment thereof, at least 98% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or to a fragment thereof, at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or to a fragment thereof.

In some embodiments, the nucleic acid sequence encoding the spike protein or fragment thereof may be at least 95% identical to the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein or to a fragment thereof, at least 96% identical the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein or to a fragment thereof, at least 97% identical to the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein or to a fragment thereof, at least 98% identical to the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein or to a fragment thereof, at least 99% identical to the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein to a fragment thereof.

In some embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 1 or to a fragment thereof. In some embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 3 or to a fragment thereof.

In some embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 5 or to a fragment thereof.

In some embodiments, the nucleic acid sequence may be identical to the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein to a fragment thereof.

In another aspect, the present disclosure relates to a codon-optimized sequence encoding a severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) antigen wherein the nucleic acid molecule may comprise a sequence from about at least 75% to about 100% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or a fragment thereof.

In some embodiments, the codon-optimized sequence may be at least 80% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5, at least 90% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5, at least 95% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5, at least 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5.

In some embodiments, the codon-optimized sequence may be at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to the sequence set forth in SEQ ID NO: 1. Accordingly, the codon- optimized sequence may be at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical to the sequence set forth in SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. In some embodiments, the codon- optimized sequence may be identical to the sequence set forth in SEQ ID NO: 1. Accordingly, the codon-optimized sequence may be 100% identical to the sequence set forth in SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. In some embodiments, the codon-optimized sequence may be identical to the sequence set forth in SEQ ID NO: 3. Accordingly, the codon-optimized sequence may be 100% identical to the sequence set forth in SEQ ID NO: 3 over the entire length of SEQ ID NO:3.

In some embodiments, the codon-optimized sequence may be identical to the sequence set forth in SEQ ID NO.:5. Accordingly, the codon-optimized sequence may be 100% identical to the sequence set forth in SEQ ID NO: 5 over the entire length of SEQ ID NO: 5.

The present disclosure also relates to DNA vaccine vectors comprising the artificial nucleic acid disclosed herein.

The present disclosure also relates to DNA vaccine vectors comprising the codon- optimized sequence disclosed herein.

It is to be understood that the codon-optimized sequence disclosed herein may be the basis of an RNA vaccine. Accordingly, the present disclosure relates to an RNA vaccine comprising a sequence disclosed herein. The RNA vaccine may be composed of ribonucleotides or ribonucleotide analogs. In some embodiments, the DNA vaccine vector may be in a circular form or in a linear form.

In some embodiments, the DNA vaccine vector of the present disclosure may be double stranded or single stranded.

In another aspect, the present disclosure relates to the use of the DNA vaccine vector or the nucleic acid molecule disclosed herein or a fragment thereof for making an immunogenic composition.

In yet another aspect, the present disclosure relates to the use of the DNA vaccine vector or the nucleic acid molecule disclosed herein or a fragment thereof for immunizing a host.

In additional aspects, the present disclosure relates to a DNA vaccine comprising the DNA vaccine vectors disclosed herein.

In a further aspect, the present disclosure relates to a pharmaceutical composition comprising the DNA vaccine vector or nucleic acid molecule disclosed herein and a pharmaceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition may be formulated for vaccination by injection, by electroporation, by inhalation etc.

In some embodiments, the pharmaceutical composition may be formulated as a transdermal patch.

In another aspect, the present disclosure relates to a method of immunizing a host that may comprise administering the pharmaceutical composition of the present disclosure.

In some embodiments, the host may be a human or an animal.

In some embodiments, the delivery of the DNA vaccine is performed by microinjection, biojector, pressure and particle bombardment (gene gun), ultrasound, magnetofection, photoporation (e.g., laser-assisted), hydroporation, droplet-based microfluidic platforms, electrical process (e.g., electroporation), by ultrasound or the like.

In some embodiments, the method may comprise administering the pharmaceutical composition by injection, by electroporation, intradermally, transdermally, intramuscularly, or at a mucosal site. In some embodiments, the method may comprise administering the pharmaceutical composition as a prime and/or boost.

In some embodiments, the method may comprise administering the pharmaceutical composition in combination with another SARS-CoV vaccine such, for example, a SARS-CoV-2 vaccine.

In some embodiments, the other SARS-CoV-2 vaccine may be a mRNA-based vaccine, a DNA vaccine, pseudo-particles, recombinant proteins, inactivated virus or non-replicative pseudotyped viral particles.

In some embodiments, the other SARS-CoV-2 vaccine may be selected from and without limitation, ChAdOxl-S (Covishield or Vaxzevira), Ad5-nCov, rAd26-S+rAd5-S (Sputnik V), Ad26.COV2.S, BNT162b2 (Cominarty), mRNA-1273, NVX-CoV2373 etc.

In some embodiments, the pharmaceutical composition may be administered as a prime and the other SARS-CoV-2 vaccine is administered as a boost. Alternatively, the other SARS- CoV-2 vaccine may be administered as a prime and the pharmaceutical composition is administered as boost. In some embodiments, the DNA vaccine vector may be administered at a dose of 1 nanogram to 10 milligrams, at a dose of 10 nanogram to 2 milligrams, at a dose of 0.5 milligram to 2 milligrams, at a dose of 10 nanogram to 1 milligram, at a dose of 100 nanogram to 500 nanogram, at a dose of 1 microgram to 500 micrograms, at a dose of 10 microgram to 500 micrograms, at a dose of 100 microgram to 500 micrograms etc.

The present disclosure also relates to the use of the DNA vaccine vectors as a DNA vaccine for inducing an immune response against SARS-CoV-2 in an individual in need thereof.

In some embodiments, the DNA vaccines of the present disclosure may induce a humoral immune response.

In some embodiments, the DNA vaccines of the present disclosure may induce a cellular immune response.

In some embodiments, the DNA vaccines of the present disclosure may generate neutralizing antibodies against SARS-CoV-2.

In some embodiments, the DNA vaccine disclosed herein may be used to reduce the risk of infection of SARS-CoV-2, to protect a host against viral dissemination of SARS-CoV-2, to reduce the risk of transmission of SARS-CoV-2 and/or to reduce the risk of complications (e.g., hospitalization, death, pathology, long COVID, acute respiratory distress syndrome etc.) associated with SARS-CoV-2.

The method of the present disclosure involves administering the DNA vaccine by intradermal delivery.

The method of the present disclosure involves administering the DNA vaccine by intramuscular delivery.

In some embodiments, the DNA vaccine may be administered as a single dose.

In other embodiments, the DNA vaccine may be administered in two doses.

In yet other embodiments, the DNA vaccine may be administered in more than two doses.

In accordance with the present disclosure, the doses are administered at different time intervals. The method of the present disclosure comprises, for example, administering the doses at approximately 14 days to 12 weeks, at approximately 6 to 10 weeks interval, at approximately 8 to 10 weeks interval, at approximately 8 to 12 weeks interval.

The method of the present disclosure comprises, for example, administering the doses at approximately 14 days interval, at approximately 28 days interval, at approximately 6 weeks interval, at approximately 8 weeks interval, at approximately 6 months interval, at approximately 1 year interval.

In some embodiments, two doses may be administered at approximately 14 days to 12 weeks interval and a subsequent dose may be administered from approximately 6 months to 1 year interval after the second dose.

Additional doses may be administered every year if required. Accordingly, a subsequent dose is optional but may be recommendable for vulnerable populations (e.g., immunocompromised, elderly, etc.).

In accordance with the present disclosure the vector or DNA vaccines may be used for research applications, for pre-clinical, for clinical, for diagnostic or therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1(A)-(H): sequence alignment of the pIDV-I (SEQ ID NO:8) and pIDV-II (SEQ ID NO: 9) vectors.

Figure 2: histogram representing eGFP expression by fluorescent activated cell sorter (FACS). Vero E6 cells were transfected in triplicate with either pIDV-eGFP, pVAXl-eGFP, or pCAGGS-eGFP using Lipofectamine 2000 (control cells received only Lipofectamine 2000). eGFP expression was analyzed 24 hours after transfection. The average (and standard deviation) eGFP expression of two replicate experiments is presented.

Figure 3: histogram representing eGFP expression by fluorescent activated cell sorter (FACS), 24 hours post-transfection in VeroE6 cells. The graph shows the average and standard deviation of the eGFP expression of 4 different DNA vectors in transfected cells.

Figure 4(A): fFNy ELISpot responses from Balb/c mice immunized with pIDV-II-SARS- CoV2-Spike_Vl (pIDV-II-Vl), pIDV-II-SARS-CoV2-Spike_V3 (pIDV-II-V3) and pIDV-II- SARS-CoV2-Spike_V5 (pIDV-II-V5). T cell response was analyzed by ELISpot 10 days after boost in BALB/c mice. Sham immunized mice were used as control. Splenocytes cell suspension were stimulated with SARS-CoV2 peptide pools partially encompassing the SARS-CoV2 Spike glycoprotein. No significant difference was observed for T-cell response between animals vaccinated with pIDV-II-SARS-CoV2-Spike_Vl and pIDV-ILSARS-CoV2-Spike_V5.

Figure 4(B): Represents Spike-specific antibody responses in vaccinated Balb/c as measured by ELISA. All Balb/c mice were immunized with pIDV-II-SARS-CoV2_Vl (pIDV-II- VI), pIDV-II-SARS-CoV2_V3 (pIDV-II-V3) and pIDV-II-SARS-CoV2_V5 (pIDV-II-V5) or sham vaccination with buffer only (control). All mice received two dose of vaccine one on day 0 and another on day 28. Each dose of vaccine was administered via electroporation (EP) following intramuscular injection of 50 pg/dose (ug DNA by IM+EP route), (sera dilution 1 :400). **** indicates P value = <0.0001, ns= non-significant. Statistical analysis was performed using ANOVA two tailed test and P values < 0.05 were considered significant.

Figure 5(A): IFNy ELISpot responses from C57BL/6 (Black/6) mice immunized with pIDV-II-SARS-CoV2-Spike_Vl (pIDV-II-Vl), and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II- V5) respectively. T cell response was analyzed by ELISpot 10 days after boost in BALB/c mice. Sham immunized mice were used as control. Splenocytes cell suspension were stimulated with SARS-CoV2 peptide pools partially encompassing the SARS-CoV2 Spike glycoprotein. No significant difference was observed for T-cell response between animals vaccinated with pIDV- II-SARS-CoV2-Spike_Vl and pIDV-II-SARS-CoV2-Spike_V5.

Figure 5(B): Represents antibody responses in vaccinated Black/6 mice respectively. All Black/6 mice were immunized with pIDV-II-SARS-CoV2-Spike_Vl (pIDV-II-Vl) or pIDV-II- SARS-CoV2-Spike_V5 (pIDV-II-V5) or sham vaccination with buffer only (control). All mice received two dose of vaccine one on day 0 and another on day 28. Each dose of vaccine was administered via electroporation (EP) following intramuscular injection of 50 pg/dose (ug DNA by IM+EP route), (sera dilution 1 :400). **** indicates P value = <0.0001, ns= non-significant.

Figure 6: Percentage weight changes in vaccinated animals (pIDV-II-SARS-CoV2- Spike Vl (pIDV-ILVl) and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5)) or control animals following virus challenge. Animals were weighed throughout the course of infection and weight change was compared to pre-infection weight (n=8 per group). Error bars represent standard deviation. P value = 0.0001 (****) as determined by two-way ANOVA. Figure 7(A)-(E): Scheme illustrating vaccination with pIDV-II-SARS-CoV2-Spike_Vl (pIDV-II-Vl) and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5) and challenge schedule (A). Sampling timepoints were performed at day 3, day 5 and day 7 post infection (dpi). Groups of animals (n=4) were euthanized at day 4 and day 8 post infection (dpi). Titers of infectious virus from various timepoints were collected (n =4 animals per group) and determined by TCID50 from oral swabs (B), nasal washes (C), nasal turbinates (D), and lungs (E). Error bars represent standard deviation. P value = 0.0001 (****) and 0.0039 (**).

Figure 8(A)-(C): Viral load (mean [SD]) from hamster lung samples in control, pIDV-II- SARS-CoV2-Spike_Vl (pIDV-II-Vl) and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5) groups at day 4 and day 8 post- infection (A). Two-way- ANOVA- Dunnett' s multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4, **p=0.0091, Control vs. pIDV-II-V5 day 4 *p= 0.0457, Control vs. pIDV-II-Vl day 8, * p= 0.0101, Control vs. pIDV-II-V5 day 8 *p= 0.0119). Viral load (mean [SD]) from hamster nasal turbinate samples in control, pIDV-II-Vl & pIDV-II- V5 groups at day 4 and & day 8 post-infection. Two-way- ANOVA- Dunnett's multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4, p=0.1393, Control vs. pIDV-II- V5 day 4 p= 0.5203, Control vs. pIDV-II-Vl day 8, p= 0.3616, Control vs. pIDV-II-V5 day 8 p= 0.4596) (B). Viral load (mean [SD]) from hamster Kidney samples in control, pIDV-II-Vl & pIDV-II-V5 groups at day 4 and & day 8 post-infection. Two-way- ANOVA- Dunnett's multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4, p=0.3591, Control vs. pIDV-II- V5 day 4 p= 0.3768, Control vs. pIDV-II-V5 day 8 p= 0.3432) (C).

Figure 9: Quantification of fibrosis (collagen staining) presented as mean percentage fibrosis of total lung tissue + standard error of the mean. Two-way- ANOVA- Dunnett's multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4,

0.0001, Control vs. pIDV-II-V5 day 4

0.0001, Control vs. pIDV-II-Vl day 8, ****p< 0.0001, Control vs. pIDV-II-V5 day 8 ****p< 0.0001).

Figure 10(A)-(B): Quantification of inflammatory cell infiltration in kidney tissues of control, pIDV-II-SARS-CoV2-Spike_Vl (pIDV-II-Vl) and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5) vaccinated groups. Quantification of cell infiltrates presented as mean percentage of inflammatory cell infiltrates in total kidney tissue + standard error of the mean. Two-way- ANOVA- Dunnett's multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4, **p = 0.0011, Control vs. pIDV-II-V5 day 4 **p = 0.0017, Control vs. pIDV-II-Vl day 8, ***p = 0.0001, Control vs. pIDV-II-V5 day 8 ****p = 0.0404) (A); Quantification of fibrosis (collagen staining) presented as mean percentage fibrosis of total kidney tissue + standard error of the mean. Two-way- ANOVA- Dunnett' s multiple comparisons test was performed (Control vs. pIDV-II-Vl day 4, *p= 0.0241, Control vs. pIDV-II-V5 day 4 **p= 0.0057, Control vs. pIDV-II-Vl day 8, **p= 0.0077, Control vs. pIDV-II-V5 day 8 *p= 0.0120) (B).

Figure 11(A)-(C): Serum neutralizing titers from hamsters vaccinated with TE buffer (control), pIDV-II-Vl, pIDV-II-V5 were analysed against the SB3 isolate (A), Alpha (B.l.1.7) variant isolate (B) and Beta (B.1.351) variant isolate (C). Four hamsters were used per group. Neutralization was performed based on TCID50 assay for neutralization capability and CPE graphed for all groups (4 hamster per group/time point). Stars denote differences (p < 0.0001) as determined by two-way repeated measure ANOVA test.

DETAILED DESCRIPTION

The present disclosure provides amongst other things, DNA vaccine vectors composed of a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a coronavirus antigen. The present disclosure more particularly provides DNA vaccine vectors composed of a vector portion and an antigen-coding portion that comprises a nucleic acid sequence encoding a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen such as SARS- CoV-2 spike protein or a fragment thereof.

Vector portion

The DNA vaccine vectors disclosed herein comprise a vector portion.

In some embodiments, the vector portion may comprise, for example, a sequence from about at least 75% to about 100% identical to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NOV.

In some embodiments the vector portion comprises a sequence at least 80% identical to SEQ ID NO:8. In other embodiments the vector portion comprises a sequence at least 80% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments the vector portion comprises a sequence at least 85% identical to SEQ ID NO:8. In other embodiments the vector portion comprises a sequence at least 85% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8. In some embodiments the vector portion comprises a sequence at least 90% identical to SEQ ID NO:8. In other embodiments the vector portion comprises a sequence at least 90% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments the vector portion comprises a sequence at least 95% identical to SEQ ID NO:8. In other embodiments the vector portion comprises a sequence at least 95% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the vector portion comprises a sequence at least 96% identical to SEQ ID NO:8. In other embodiments, the vector portion comprises a sequence at least 96% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the vector portion comprises a sequence at least 97% identical to SEQ ID NO:8. In other embodiments, the vector portion comprises a sequence at least 97% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the vector portion comprises a sequence at least 98% identical to SEQ ID NO:8. In other embodiments, the vector portion comprises a sequence at least 98% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the vector portion comprises a sequence at least 99% identical to SEQ ID NO:8. In other embodiments, the vector portion comprises a sequence at least 99% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the vector portion comprises a sequence or identical to SEQ ID NO:8. In other embodiments, the vector portion comprises a sequence 100% identical to SEQ ID NO: 8 over the entire length of SEQ ID NO: 8.

In some embodiments, the DNA vaccine vector of the present disclosure comprises a post- transcriptional regulatory element.

In some embodiments, the post-transcriptional regulatory element is woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In some embodiments, the vector portion comprises a sequence at least 80% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 80% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9. In some embodiments, the vector portion comprises a sequence at least 85% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 85% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at least 90% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 90% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at least 95% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 95% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at least 96% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 96% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion v a sequence at least 97% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 97% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at least 98% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 98% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at least 99% identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence at least 99% identical to SEQ ID NO:9 over the entire length of SEQ ID NO:9.

In some embodiments, the vector portion comprises a sequence at identical to SEQ ID NO:9. In other embodiments, the vector portion comprises a sequence 100% identical to SEQ ID NO: 9 over the entire length of SEQ ID NO: 9.

In some embodiments, the vector may consist essentially of the sequence set forth in SEQ ID N0:9.

In some embodiments, the vector may consist in the sequence set forth in SEQ ID NO:9. In some embodiments, the vector comprises one or more regulatory elements such as an initiator, an enhancer, a promoter, cloning site(s), polyadenylation signals, selection markers (e.g., antibiotic resistance genes) or the like.

In some embodiments, the vector comprises an origin of replication.

In some embodiments, the nucleic acid encoding the antigen-binding portion is operably linked to the one or more regulatory elements.

In some embodiments, the vector may also encode an additional antigen, such as for example, a peptide adjuvant, a sequence encoding an antigen from another virus and the like.

In some embodiments, the vector may be in a circular form.

In some embodiments, the vector may be in a linear form.

Antigen-coding portion

The DNA vaccine vectors disclosed herein include an antigen-coding portion that comprises a nucleic acid sequence encoding a coronavirus antigen.

In some embodiments, the DNA vaccine vector may comprise more than one antigencoding portions. For example, the DNA vaccine vector may comprise two antigen coding portions. In other examples, the DNA vaccine vector may comprise more than two antigen coding portions, such as, three antigen coding portions or more.

The various antigen-coding portions may all encode for coronavirus antigens such as SARS-CoV-2 antigens. In other example, the various antigen-coding portions may encode at least one SARS-CoV-2 antigen and other non-related antigen(s). In other examples, the various antigencoding portions may all encode for the same SARS-CoV-2 antigen.

In some embodiments, the coronavirus antigen comprises structural proteins such as spike protein, membrane protein (M), nucleocapside protein (N) or envelope protein (E).

In exemplary embodiments, the DNA vaccine vector comprises various antigen-coding portions, at least one of which encodes for SARS-CoV-2 spike.

In other exemplary embodiments, the DNA vaccine vector comprises various antigencoding portions, at least one of which encodes for SARS-CoV-2 spike and at least one which encodes for another SARS-CoV-2 structural protein. In some embodiments, the coronavirus antigen comprises the spike protein of a fragment thereof. In some embodiments, the DNA vaccine vectors disclosed herein include an antigencoding portion that comprises a nucleic acid sequence encoding a severe acute respiratory syndrome coronavirus (SARS-CoV) antigen.

In some embodiments, the SARS-CoV antigen is a spike protein or a fragment thereof.

In some embodiments, the spike protein or fragment thereof is from SARS-CoV-2.

In some embodiments, the spike protein or fragment thereof is from the original SARS- CoV-2 Whuan isolate.

In some embodiments, the spike protein or fragment thereof is from the Alpha variant or related lineages (e.g., B. l.1.7 and Q lineages).

In some embodiments, the spike protein or fragment thereof is from the Beta variant or related lineages (e.g., B.1.351 and descendent lineages).

In some embodiments, the spike protein or fragment thereof is from the Gamma variant or related lineages (e.g., P. l and descendent lineages).

In some embodiments, the spike protein or fragment thereof is from the Delta variant or related lineages (e.g., B.1.617.2 and AY lineages).

In some embodiments, the spike protein or fragment thereof is from the Epsilon variant or related lineages (e.g., B.1.427 and B.1.429).

In some embodiments, the spike protein or fragment thereof is from the Eta variant or related lineages (e.g., B.1.525).

In some embodiments, the spike protein or fragment thereof is from the Iota variant or related lineages (e.g., B.1.526).

In some embodiments, the spike protein or fragment thereof is from the Kappa variant or related lineages (e.g., B.1.617.1).

In some embodiments, the spike protein or fragment thereof is from the 1.617.3 variant or related lineages. In some embodiments, the spike protein or fragment thereof is from the Mu variant or related lineages (e.g., B.1.621, B.1.621.1).

In some embodiments, the spike protein or fragment thereof is from the Zeta variant or related lineages (e.g., P.2).

In some embodiments, the spike protein comprises an amino acid sequence from about at least 90% to about 100% identical to SEQ ID NO:2 or to a fragment thereof.

In some embodiments, the spike protein comprises an amino acid sequence from about at least 90% to about 100% identical to SEQ ID NO:2.

Alternatively, in some embodiments, the spike protein comprises an amino acid sequence from about at least 95% to about 100% identical to SEQ ID NO:2.

Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO:2. The spike protein or fragment thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least at least 91% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 91% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In other embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least at least 92% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 92% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In yet other embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least at least 93% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 93% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In further embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least at least 94% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 94% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2. In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least 95% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 95% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least 96% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 96% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least 97% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 97% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least 98% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 98% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence at least 99% identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence at least 99% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein or fragment thereof comprises an amino acid sequence identical to SEQ ID NO:2. Accordingly, the spike protein or fragment thereof comprises an amino acid sequence 100% identical to SEQ ID NO:2 over the entire length of SEQ ID NO:2.

In some embodiments, the spike protein consists essentially of the amino acid sequence set forth in SEQ ID NO:2.

In exemplary embodiments, the antigen-coding portion encodes the full SARS-CoV-2 spike protein.

In exemplary embodiments, the antigen-coding portion encodes SEQ ID NO:2. In exemplary embodiments, the antigen-coding portion encodes a fragment of SARS-CoV- 2 spike protein.

In other exemplary embodiments, the antigen-coding portion encodes a fragment of SEQ ID N0:2.

In accordance with the present disclosure, the fragment of SEQ ID NO:2 comprises or consist in the extracellular domain of SARS-CoV-2 spike protein.

In accordance with the present disclosure, the fragment of SEQ ID NO:2 comprises or consist in the receptor binding domain of SARS-CoV-2 spike protein.

In some embodiments, the spike protein comprises from 1 to 10 amino acid substitutions or more in comparison with SEQ ID NO:2.

In exemplary embodiments, the spike protein may comprise 1 amino acid substitution in comparison with SEQ ID NO:2. In other exemplary embodiments, the spike protein may comprise 2 amino acid substitutions in comparison with SEQ ID NO:2. In additional exemplary embodiments, the spike protein may comprise 3 amino acid substitutions in comparison with SEQ ID NO:2. In other exemplary embodiments, the spike protein may comprise 4 amino acid substitutions in comparison with SEQ ID NO:2. In yet other exemplary embodiments, the spike protein may comprise 5 amino acid substitutions in comparison with SEQ ID NO:2. In further exemplary embodiments, the spike protein may comprise 6 amino acid substitutions in comparison with SEQ ID NO:2. In yet further exemplary embodiments, the spike protein may comprise 7 amino acid substitutions in comparison with SEQ ID NO:2. In additional exemplary embodiments, the spike protein may comprise 8 amino acid substitutions in comparison with SEQ ID NO:2. In other exemplary embodiments, the spike protein may comprise 9 amino acid substitutions in comparison with SEQ ID NO:2. In further exemplary embodiments, the spike protein may comprise 10 amino acid substitutions in comparison with SEQ ID NO:2. In yet further exemplary embodiments, the spike protein may comprise 15 amino acid substitutions in comparison with SEQ ID NO:2. In other exemplary embodiments, the spike protein may comprise 20 amino acid substitutions in comparison with SEQ ID NO:2.

In an exemplary embodiment, the spike protein comprises amino acid substitution at position A701. In another example, the spike protein comprises amino acid substitution at position P681.

In yet another example, the spike protein comprises amino acid substitution at position

D614.

In a further example, the spike protein comprises amino acid substitution at position A570.

In another example, the spike protein may comprise amino acid substitution at position N501.

In an additional example, the spike protein comprises amino acid substitution at position E484.

In another example, the spike protein comprises amino acid substitution at position S477.

In yet another example, the spike protein comprises amino acid substitution at position Y453.

In a further example, the spike protein comprises amino acid substitution at position L452.

In yet a further example, the spike protein comprises amino acid substitution at position K417.

Accordingly, the spike protein comprises an amino acid substitution at position A701, P681, D614, A570, N501, E484, S477, Y453, L452, K417 or combinations thereof

In some embodiments, the spike protein comprises amino acid substitution P681R.

In some embodiments, the spike protein comprises amino acid substitution D614G.

In some embodiments, the spike protein comprises amino acid substitution N501 Y.

In some embodiments, the spike protein comprises amino acid substitution E484K.

In some embodiments, the spike protein comprises amino acid substitution L452R.

In some embodiments, the spike protein comprises amino acid substitution Y453F.

In some embodiments, the spike protein may be deleted at its C-terminus, at its N-terminus or at both C- and N-terminus. In some embodiments, the spike protein comprises an amino acid deletion at its C-terminus, at its N-terminus or at both C- and N-terminus. In some embodiments, the deletion may encompass, for example, the transmembrane domain of the spike protein or a portion thereof. In exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 100 amino acid residues. In other exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 90 amino acid residues. In other exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 80 amino acid residues. In further exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 70 amino acid residues. In yet further exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 60 amino acid residues. In additional exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 50 amino acid residues. In yet additional exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 40 amino acid residues. In other exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 30 amino acid residues. In yet other exemplary embodiments, the spike protein may comprise a deletion of from 1 to about 20 amino acid residues. In further exemplary embodiments, the spike protein may comprise at least about 10 amino acid residues. In yet further exemplary embodiments, the spike protein may comprise at least about 20 amino acid residues.

In some embodiments, the antigen is a fragment of the spike protein.

In some embodiments, the fragment of the spike protein comprises at least 10 amino acids. In other embodiments, the fragment comprises at least 20 amino acids. In yet other embodiments, the fragment comprises at least 30 amino acids. In further embodiments, the fragment comprises at least 40 amino acids. In further embodiments, the fragment comprises at least 50 amino acids. In additional embodiments, the fragment comprises at least 60 amino acids. In further embodiments, the fragment comprises at least 70 amino acids. In yet further embodiments, the fragment comprises at least 80 amino acids. In other embodiments, the fragment comprises at least 90 amino acids. In further embodiments, the fragment comprises at least 100 amino acids. In yet further embodiments, the fragment comprises at least 150 amino acids. In additional embodiments, the fragment comprises at least 200 amino acids. In yet additional embodiments, the fragment comprises at least 300 amino acids. In other embodiments, the fragment may comprise at least 500 amino acids.

In some embodiments, the fragment may comprise from at least 20 to at least 1250 amino acid residues of the spike protein including for example, from at least 20 to at least 1000 amino acid residues, from at least 20 to at least 750 amino acid residues, from at least 20 to at least 500 amino acid residues, from at least 20 to at least 250 amino acid residues, from at least 20 to at least 100 amino acid residues, from at least 20 to at least 50 amino acid residues etc.

In some embodiments, the fragment may comprise at least 4 amino acid residues. In other embodiments, the fragment may comprise at least 5 amino acid residues. In yet other embodiments, the fragment may comprise at least 6 amino acid residues. In further embodiments, the fragment may comprise at least 7 amino acid residues. In additional embodiments, the fragment may comprise at least 8 amino acid residues. In yet additional embodiments, the fragment may comprise at least 9 amino acid residues. In further embodiments, the fragment may comprise at least 10 amino acid residues. In yet further embodiments, the fragment may comprise at leastl 1 amino acid residues. In additional embodiments, the fragment may comprise at least 12 amino acid residues. In other embodiments, the fragment may comprise at least 13 amino acid residues. In further embodiments, the fragment may comprise at least 14 amino acid residues. In additional embodiments, the fragment may comprise at least 15 amino acid residues. In other embodiments, the fragment may comprise at least 16 amino acid residues. In other embodiments, the fragment may comprise at least 17 amino acid residues. In additional embodiments, the fragment may comprise at least 18 amino acid residues. In other embodiments, the fragment may comprise at least 19 amino acid residues. In yet other embodiments, the fragment may comprise at least 20 amino acid residues. In other embodiments, the fragment may comprise at least 50 amino acid residues. In yet other embodiments, the fragment may comprise at least 100 amino acid residues. In additional embodiments, the fragment may comprise at least 200 amino acid residues. In yet additional embodiments, the fragment may comprise at least 300 amino acid residues. In further embodiments, the fragment may comprise at least 400 amino acid residues. In yet further embodiments, the fragment may comprise at least 500 amino acid residues. In other embodiments, the fragment may comprise at least 600 amino acid residues. In other embodiments, the fragment may comprise at least 700 amino acid residues. In yet other embodiments, the fragment may comprise at least 800 amino acid residues. In additional embodiments, the fragment may comprise at least 900 amino acid residues. In additional embodiments, the fragment may comprise at least 1000 amino acid residues. In other embodiments, the fragment may comprise at least 1100 amino acid residues.

In exemplary embodiments, the fragment may be an immunogenic fragment. In an exemplary embodiment, the immunogenic fragment may include an amino acid sequence that encompass or is near the ACE2 binding domain. In another exemplary embodiment, the immunogenic fragment includes an amino acid sequence that encompass or is near the fusion peptide(s).

For example, the immunogenic fragment may encompass residues 274-306, 510-586, 587-628, 784-803, or 870-893. In yet other embodiment, the immunogenic fragment may encompass the S14P5 and the S21P2 linear epitopes (see for example, Meng Poh, C, et al., Nature Communications 2020, 11 :2806, the entire content of which is incorporated herein by reference) or other linear epitopes identified by Li, Yang et al., (Linear Epitope Landscape of SARS-Cov-2 Spike Protein Constructed from 1,051 CO VID-19 Patients, 2020).

In another exemplary embodiment, the fragment may be a structural domain of the spike protein. For example, in some embodiments, the structural domain may comprise the receptorbinding domain (amino acid residues 319-541), the receptor binding motif (amino acid residues 437-508), the fusion peptide 1 (amino acid residues 816-837), the fusion peptide 2 (amino acid residues 835 to 855).

In some embodiments, the nucleic acid sequence may be the naturally occurring nucleic acid sequence encoding SARS-CoV-2 spike protein (see NCBI Reference sequence No. NC_045512.2:21563-25384) or a fragment thereof.

In some embodiments, the nucleic acid sequence encoding the antigen is an artificial nucleic acid sequence.

In some embodiments, the nucleic acid sequence encoding the antigen is codon-optimized.

For example, the spike protein may be encoded by a codon-optimized nucleic acid sequence having at least 75% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 80% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof. In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 85% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 90% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 95% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 96% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 97% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 98% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence having at least 99% identity with the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO. :5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a nucleic acid sequence identical to the nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or with a fragment thereof.

In some embodiments, the spike protein is encoded by a naturally occurring sequence.

In some embodiments, the spike protein is encoded by a codon-optimized sequence.

In some embodiments, the codon-optimized sequence is at least 80% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof. In some embodiments, the codon-optimized sequence is at least 90% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In some embodiments, the codon-optimized sequence is at least 95% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In some embodiments, the codon-optimized sequence is at least 96% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In some embodiments, the codon-optimized sequence is at least 97% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In some embodiments, the codon-optimized sequence is at least 98% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In some embodiments, the codon-optimized sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.: 5 or to a fragment thereof.

In an exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 86% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 86% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 87% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 87% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 88% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 88% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 89% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 89% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 90% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 91% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 91% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 92% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 92% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 93% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 93% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 94% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 94% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In yet a further exemplary embodiment, the spike protein is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence identical to SEQ ID NO: 1. Accordingly, the spike protein is encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence identical to SEQ ID NO: 3. Accordingly, the spike protein is encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 3 over the entire length of SEQ ID NO:3.

In another exemplary embodiment, the spike protein is encoded by a nucleic acid sequence identical to SEQ ID NO: 5. Accordingly, the spike protein is encoded by a nucleic acid sequence 100% identical to SEQ ID NO: 5 over the entire length of SEQ ID NO:5.

In some embodiments, the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 1 or to a fragment thereof.

In some embodiments, the codon-optimized sequence consists essentially of the sequence set forth in SEQ ID NO: 1 or of a fragment thereof.

In some embodiments, the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 3 or to a fragment thereof.

In some embodiments, the codon-optimized sequence consists essentially of the sequence set forth in SEQ ID NO: 3 or of a fragment thereof.

In some embodiments, the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO.: 5 or to a fragment thereof. In some embodiments, the codon-optimized sequence consists essentially of the sequence set forth in SEQ ID NO: 5 or of a fragment thereof.

The codon-optimized sequence disclosed herein have other utilities. For example, it may be cloned in other types of vectors and/or it may be used as an RNA vaccine.

In some exemplary embodiments, the codon-optimized nucleic acid sequence disclosed herein may be a DNA. In other exemplary embodiments, the codon-optimized nucleic acid sequence disclosed herein may be a RNA.

The codon-optimized sequence disclosed herein may be cloned into the DNA vector disclosed herein or in other types of vectors including for example, expression vectors, cloning vectors, or DNA vaccine vectors disclosed in the literature.

In some embodiments, DNA vectors used to express the antigen-binding portion or codon- optimized sequence disclosed herein may include pVAXl (see W02019/218091), pVAC™ or pBOOST™ (Invivogen) and the like.

DNA vaccine vectors and DNA vaccines

The DNA vaccine vector of the present disclosure is composed of a vector portion and an antigen-coding portion as described herein.

Therefore, in another aspect, the present disclosure relates to a DNA vaccine vector having a sequence at least 95% identical to SEQ ID NO:20. In some embodiments, the DNA vaccine vector comprises a sequence at least 95% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In some embodiments, the DNA vaccine vector comprises a sequence at least 96% identical to SEQ ID NO:20. Accordingly, the DNA vaccine vector comprises a sequence at least 96% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In some embodiments, the DNA vaccine vector comprises a sequence at least 97% identical to SEQ ID NO:20. Accordingly, the DNA vaccine vector comprises a sequence at least 97% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20. In some embodiments, the DNA vaccine vector comprises a sequence at least 98% identical to SEQ ID NO:20. Accordingly, the DNA vaccine vector comprises a sequence at least 98% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In some embodiments, the DNA vaccine vector comprises a sequence at least 99% identical to SEQ ID NO:20. Accordingly, the DNA vaccine vector comprises a sequence at least 99% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In some embodiments, the DNA vaccine vector comprises a sequence identical to SEQ ID NO:20. Accordingly, the DNA vaccine vector comprises a sequence 100% identical to SEQ ID NO:20 over the entire length of SEQ ID NO:20.

In another aspect, the present disclosure relates to a DNA vaccine vector having a sequence at least 95% identical to SEQ ID N0:21. In some embodiments, the DNA vaccine vector comprises a sequence at least 95% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector comprises a sequence at least 96% identical to SEQ ID NO:21. Accordingly, the DNA vaccine vector comprises a sequence at least 96% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector comprises a sequence at least 97% identical to SEQ ID NO:21. Accordingly, the DNA vaccine vector comprises a sequence at least 97% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector comprises a sequence at least 98% identical to SEQ ID NO:21. Accordingly, the DNA vaccine vector comprises a sequence at least 98% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector comprises a sequence at least 99% identical to SEQ ID NO:21. Accordingly, the DNA vaccine vector comprises a sequence at least 99% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector comprises a sequence identical to SEQ ID NO:21. Accordingly, the DNA vaccine vector comprises a sequence 100% identical to SEQ ID NO:21 over the entire length of SEQ ID NO:21.

In some embodiments, the DNA vaccine vector of the present disclosure is double stranded. In other embodiments, the DNA vaccine vector of the present disclosure is single stranded.

In other embodiments, the DNA vaccine vector of the present disclosure is circular.

In other embodiments, the DNA vaccine vector of the present disclosure is linear.

The DNA vaccine vectors is manufactured and formulated into pharmaceutical compositions to make a DNA vaccine.

Method of manufacturing

Methods for manufacturing DNA vectors for vaccination are known in the art and are based on guidance from the FDA (USA Food and Drug Administration. Guidance for Industry: Considerations for Plasmid DNA Vaccines for Infectious Disease Indications. Rockville, MD, USA: 2007) or the EMA (European Medicines Agency. Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products. London, UK: 2001. CPMP/BWP/3088/99; Presence of the Antibiotic Resistance Marker Gene nptll in GM Plants and Food and Feed Uses. London, UK: 2007. EMEA/CVMP/56937/2007).

Exemplary methods of manufacturing are reviewed in Williams J. A., 2013 (Vaccines, 1(3): 225-249, 2013). Processes for high-scale production and purification are also disclosed in Carnes, A.E. and J. A. Williams, 2007 (Recent Patents on Biotechnology, 1 : 151-66, 2007).

Plasmid DNA production is typically performed in endA (DNA-specific endonuclease I), recA (DNA recombination) deficient A. coli K12 strains such as DH5a, DH5, DH1, XLlBlue, GT115, JM108, DH10B, or endA, recA engineered derivatives of alternative strains such as MG1655, or BL21.

Transformed bacteria are fermented using for example, fed-batch fermentation processes. Clinical grade DNA vector can be obtained by various methods (e.g., HyperGRO™) through service providers such as Aldevron, Eurogentec and VGXI.

DNA vectors are then purified to remove bacterial debris and impurities (RNA, genomic DNA, endotoxins) and formulated with a suitable carrier (for research purposes) or pharmaceutical carrier (for pre-clinical or clinical applications). In some aspects, the present disclosure relates to the use of the DNA vaccine vector or the nucleic acid molecule disclosed herein or a fragment thereof for making an immunogenic composition.

Pharmaceutical compositions

DNA vaccine vectors of the present disclosure may be administered as a pharmaceutical composition, which may comprise for example, the DNA vaccine vector(s) and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition may comprise a single DNA vaccine vector species encoding one or more antigens.

Alternatively, the pharmaceutical composition may comprise a mixture of DNA vaccine vector species (multiple DNA vaccine vector species) each encoding different antigens.

In some embodiments, the pharmaceutical composition of the present disclosure comprises the DNA vaccine vector or nucleic acid molecule disclosed herein and a pharmaceutical acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated for vaccination by injection.

In some embodiments, the pharmaceutical composition is formulated for vaccination by electroporation.

In some embodiments, the pharmaceutical composition is formulated for vaccination by inhalation.

In some embodiments, the pharmaceutical composition is formulated as a transdermal patch.

In some embodiments, the pharmaceutical composition is formulated for intradermal delivery.

In some embodiments, the pharmaceutical composition is formulated for intramuscular delivery.

In some embodiments, the pharmaceutical composition is formulated for intranasal delivery. The pharmaceutical composition may further comprise additional elements for increasing uptake of the DNA vector by the cells, its transport in the nucleic, expression of the transgene, secretion, immune response, etc.

The pharmaceutical composition may comprise for example, adjuvant molecule(s). The adjuvant molecule(s) may be encoded by the DNA vector that encodes the antigen or by another DNA vector. Encoded adjuvant molecule(s) may include DNA- or RNA-based adjuvant (CpG oligonucleotides, immunostimulatory RNA, etc.) or protein-based immunomodulators.

In some embodiments, the adjuvant is a peptide adjuvant encoded by the antigen-coding portion of the DNA vaccine vector.

In some embodiments, the nucleic acid sequence encoding the peptide adjuvant is at the 3’ end of the nucleic acid sequence encoding the SARS-CoV antigen.

In some embodiments, the nucleic acid sequence encoding the peptide adjuvant is contiguous to the nucleic acid sequence encoding the SARS-CoV antigen.

In some embodiments, the nucleic acid sequence encoding the peptide adjuvant is immediately contiguous to the nucleic acid sequence encoding the SARS-CoV antigen.

In some embodiments, the antigen-coding portion of the DNA vaccine vector comprises a nucleic acid sequence encoding a peptide adjuvant in frame with and contiguous to the spike protein or fragment thereof.

In some embodiments, the peptide adjuvant comprises or consists of the amino acid set forth in any one of SEQ ID NO: 10 to SEQ ID NO: 19.

In some embodiments, the peptide adjuvant comprises or consists of SEQ ID NO: 10.

Alternatively, the adjuvant molecule(s) may be co-administered with the DNA vectors.

Adjuvants include, but are not limited to, mineral salts (e.g., A1K(SO4)2, AlNa(SO4)2, A1NH(SO4)2, silica, alum, A1(OH)3, Ca3(PO4)2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs), CpG oligonucleotides, immunostimulatory RNA, poly IC or poly AU acids, saponins such as QS21, QS17, and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod, lipid-polymer matrix (ENABL™ adjuvant), Emulsigen-D™ etc. The DNA vaccines may be formulated for administration by injection (e.g., intramuscular, intradermal, transdermal, subcutaneously) or for mucosal administration (oral, intranasal).

In some embodiments, the DNA vaccine vectors may be incorporated into liposomes.

In accordance with the present disclosure, the pharmaceutical composition may be formulated into nanoparticles.

Treatment modalities

In some aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to prevent or treat an infection or a disease or condition associated with a coronavirus infection.

In other aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to lower the risk of infection, reduce symptoms, and/or lower the risk of complications caused by or associated with a coronavirus.

In particular aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to lower the risk of a host from getting infected with a SARS-CoV and in particular with SARS-CoV-2.

In other particular aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to lower the risk of a host from getting complications related to SARS-CoV infection and in particular to SARS-CoV-2 infection.

In other particular aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to reduce symptoms related with SARS-CoV infection and in particular with SARS-CoV-2 infection. In additional particular aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to prevent infection from SARS-CoV and in particular from SARS- CoV-2.

In yet additional particular aspects and embodiments, the DNA vaccine vectors, nucleic acids or pharmaceutical compositions comprising same may be used to treat an infection caused by SARS-CoV and in particular caused by SARS-CoV-2. In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce the risk of infection of SARS-CoV-2.

In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce the risk of transmission of SARS-CoV-2.

In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce the risk of complications (e.g., hospitalization, death, pathology, etc.) associated with SARS-CoV- 2.

In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce the severity of the coronavirus disease (COVID-19).

In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce the risk of long COVID.

In some exemplary embodiments, the DNA vaccine vectors disclosed herein may be used to protect a host against viral dissemination of SARS-CoV-2.

In some embodiments, the DNA vaccine vectors disclosed herein may be used to reduce acute respiratory distress syndrome.

In other exemplary embodiments, the DNA vaccine vectors disclosed herein may be used to reduce organ pathology associated with SARS-CoV-2.

In yet other exemplary embodiments, the DNA vaccine vectors disclosed herein may be used to lower the risk of hospitalization associated with SARS-CoV-2.

In other exemplary embodiments, the DNA vaccine vectors disclosed herein may be used to lower the risk of death associated with SARS-CoV-2.

In some embodiments, the method of the present disclosure comprises administering a DNA vaccine vector comprising a sequence at least 95% identical, at least 96% identical, at least 97%, at least 98% identical, at least 99% identical to SEQ ID NO:20 or comprising a sequence as set forth in SEQ ID NO:20. In some embodiments, the method of the present disclosure comprises administering a DNA vaccine vector consisting of the sequence set forth in SEQ ID NO: 20.

In some embodiments, the method of the present disclosure comprises administering a DNA vaccine vector comprising a sequence at least 95% identical, at least 96% identical, at least 97%, at least 98% identical, at least 99% identical to SEQ ID NO:21 or comprising a sequence as set forth in SEQ ID NO:21. In some embodiments, the method of the present disclosure comprises administering a DNA vaccine vector consisting of the sequence set forth in SEQ ID NO:21.

In accordance with the present disclosure the DNA vaccine vector may be administered by intradermal delivery.

In accordance with the present disclosure the DNA vaccine vector may be administered by intramuscular delivery.

In some embodiments, the DNA vaccine vector may induce neutralizing antibodies against one or more SARS-CoV-2 isolates.

In some embodiments, the DNA vaccine vector may induce neutralizing antibodies against the Alpha and Beta variants of SARS-CoV-2.

In some embodiments, the DNA vaccine vector may be effective against high dose of SARS-CoV-2.

In some embodiments, the method of the present disclosure comprises administering the DNA vaccine vector as a single dose.

In some embodiments, the method of the present disclosure comprises administering the DNA vaccine vector in two doses.

In some embodiments, the method of the present disclosure comprises administering the DNA vaccine vector in more than two doses, such as for example, three doses, four doses, five doses etc.

In some embodiments, the method of the present disclosure comprises administering the DNA vaccine vector in three doses.

In accordance with the present disclosure, the doses are administered separately.

In accordance with the present disclosure, the doses are administered at different time intervals.

In accordance with the present disclosure, the doses are administered on different days. In some embodiments, the method of the present disclosure comprises administering the doses at approximately 14 days to 12 weeks interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 6 to 10 weeks interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 8 to 10 weeks interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 8 to 12 weeks interval.

The method of the present disclosure comprises, for example, administering the doses at approximately 14 days interval.

The method of the present disclosure comprises, for example, administering the doses at approximately 28 days interval.

The method of the present disclosure comprises, for example, administering the doses at approximately 6 weeks interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 8 weeks interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 6 months interval.

In some embodiments, the method of the present disclosure comprises administering the doses at approximately 1 year interval.

In some embodiments, two doses are administered at approximately 14 days to 12 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 28 days interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose. In some embodiments, two doses are administered at approximately 6 to 12 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 6 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 7 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 8 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 9 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 10 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 11 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at approximately 12 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose.

In some embodiments, two doses are administered at least 12 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval after the second dose. Additional doses may be administered every year if required. Accordingly, a subsequent dose is optional. A subsequent dose may be recommended in vulnerable population (e.g., immunocompromised, elderly, etc.).

The DNA vaccine vectors of the present disclosure may be administered to humans or to animals (non-human primates, cattle, rabbits, mice, rats, sheep, goats, horses, birds, poultry, fish, etc.). The DNA vector may thus be used as a vaccine in order to trigger an immune response against an antigen of interest in a human or animal.

The DNA vaccine vectors may be administered alone (e.g., as a single dose or in multiple doses) or co-administered with a recombinant antigen, with a viral vaccine (live (e.g., replication competent or not), attenuated, inactivated, etc.), with suitable therapy for modulating or boosting the host’s immune response such as for example, adjuvants, immunomodulators (cytokine, chemokines, checkpoint inhibitors, etc.), etc.

The DNA vaccine vectors may also be co-administered with a plasmid encoding molecules that may act as adjuvant (e.g., CpG motifs, cytokine, chemokines, etc.). In accordance with the present disclosure, such adjuvant molecules may also be encoded by the DNA vaccine vectors (e.g., CpG motifs, cytokine, chemokines, etc.).

In some instances, the DNA vaccine vectors may be administered first (for priming) and the recombinant antigen or viral vaccine may be administered subsequently (as a boost), or vice versa.

The DNA vaccine vectors may be administered by injection, intramuscularly, intradermally, transdermally, subcutaneously, to the mucosa (oral, intranasal), etc.

In accordance with the present disclosure, the vaccine may be administered by a physical delivery system including via electroporation, a needleless pressure-based delivery system, particle bombardment, etc.

Following administration, the host’s immune response towards the antigen may be assessed using methods known. In some instances, the level of antibodies against the antigen may be measured by ELISA assay or by other methods known by a person skilled in the art. The cellular immune response towards the antigen may be assessed by ELISPOT or by other methods known by a person skilled in the art. In the case of pre-clinical studies in animals, the level of protection against the pathogen may be determined by challenge experiments where the pathogen is administered to the animal and the animal’s health or survival is assessed. The level of protection conferred by the vaccine expressing a tumor antigen may be determined by tumor shrinkage or inhibition of tumor growth in animal models carrying the tumor.

Method of immunization comprising administering the pharmaceutical composition disclosed herein to a host is encompassed by the present disclosure.

The present disclosure also relates to the use of the DNA vaccine vector or the nucleic acid molecule disclosed herein or a fragment thereof for immunizing a host.In some embodiments, the host is a human.

In some embodiments the human is a child.

In some embodiments, the human is a teenager.

In some embodiments, the human is an adult.

In some embodiments, the human is an elderly.

In some embodiments, the human is healthy.

In some embodiments, the human is immunocompromised.

In some embodiments the child is between about 5-11 years old.

In some embodiments the child is younger than 5 years old.

In some embodiments the child is 6 months old or older.

In some embodiment, the teenager is between about 12-17 years old,

In some embodiments the adult is between about 18-55 years old.

In some embodiments, the adult is between about 56-70 years old.

In some embodiments, the elderly is about 71 years old or older.

In some embodiments, the human has an underlying condition or co-morbidity such as for example and without limitation, heart disease, diabetes, cancer, obesity, chronic kidney disease, chronic obstructive pulmonary disease, immunosuppression (immunocompromised state), liver disease, cystic fibrosis, hypertension, moderate-to-severe asthma, neurologic condition etc. In some embodiments, the host is an animal.

In some embodiments, the method of the present disclosure includes administering the pharmaceutical composition or DNA vaccine vector by injection.

In some embodiments, the method of the present disclosure includes administering the pharmaceutical composition or DNA vaccine vector by electroporation.

In some embodiments, the method of the present disclosure includes administering the pharmaceutical composition or DNA vaccine vector intradermally, transdermally or intramuscularly.

In some embodiments, the method of the present disclosure includes administering the pharmaceutical composition or DNA vaccine vector at a mucosal site.

In some embodiments, the delivery of DNA vaccine is performed with a needle-free pneumatic or jet injectors (Pharmajet™, etc.).

In some embodiments, the delivery of DNA vaccine is performed with a device such as CELLECTRA® 2000 device.

In some embodiments, the delivery of DNA vaccine is performed with an intradermal oscillating needles array injection device.

In some embodiments, the method comprises administering the pharmaceutical composition as a prime.

In some embodiments, the method comprises administering the pharmaceutical composition as a boost.

In some embodiments, the method comprises administering the pharmaceutical composition both as a prime and a boost. In some embodiments, the pharmaceutical composition is administered in combination with another SARS-CoV-2 vaccine.

Exemplary embodiments of other SARS-CoV-2 vaccine include mRNA-based vaccine, DNA vaccine, pseudo-particles, recombinant proteins, inactivated virus or non-replicative and/or pseudotyped viral particles.

In some embodiments, the pharmaceutical composition of the present disclosure is administered as a prime and the other SARS-CoV-2 vaccine is administered as a boost.

In some embodiments, the other SARS-CoV-2 vaccine is administered as a prime and the pharmaceutical composition of the present disclosure is administered as boost.

The dosage of the DNA vaccine may be determined by a clinician. The dose of DNA vaccine vector may vary depending on the weight of the host, his health conditions, route of administration and the like.

It is to be understood herein that one or more doses of DNA vaccines may be administered. For example, two, three or more doses may be administered depending on the patient’s response as measured for example by antibody and/or cellular response against SARS-CoV-2 antigen(s). In some embodiments the doses may be increased at each round of administration.

The doses may be administered at one week-, two weeks-, three weeks-, one month-, two months-, three months-, four months, five months-, six months- intervals, etc. The doses may be administered twice a year, yearly, every two years, etc.

In some embodiments, two doses of the DNA vaccine vector is administered 28 days apart.

In some embodiments, the DNA vaccine vector is administered at a dose of 1 picogram to 10 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 1 picogram to 1 milligram.

In some embodiments, the DNA vaccine vector is administered at a dose of 1 nanogram to 10 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 10 nanograms to 5 milligrams. In some embodiments, the DNA vaccine vector is administered at a dose of 10 nanograms to 2 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 10 nanograms to 1 milligram.

In some embodiments, the DNA vaccine vector is administered at a dose of 100 nanograms to 1 milligram.

In some embodiments, the DNA vaccine vector is administered at a dose of 100 nanograms to 2 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 100 nanograms to 500 nanograms.

In some embodiments, the DNA vaccine vector is administered at a dose of 1 microgram to 500 micrograms.

In some embodiments, the DNA vaccine vector is administered at a dose of 10 micrograms to 500 micrograms.

In some embodiments, the DNA vaccine vector is administered at a dose of 30 micrograms to 500 micrograms.

In some embodiments, the DNA vaccine vector is administered at a dose of 100 micrograms to 500 micrograms.

In some embodiments, the DNA vaccine vector is administered at a dose of 0.5 milligram.

In some embodiments, the DNA vaccine vector is administered at a dose of 1.0 milligram.

In some embodiments, the DNA vaccine vector is administered at a dose of 1.5 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 2.0 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 2.5 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 3.0 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 3.5 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 4.0 milligrams. In some embodiments, the DNA vaccine vector is administered at a dose of 4.5 milligrams.

In some embodiments, the DNA vaccine vector is administered at a dose of 5.0 milligrams. Definitions

As used herein the terms “vector” and “plasmid” are used interchangeably.

The term “transgene” refers to a gene encoding the protein(s) or peptide(s) of interest inserted in the vector of the present disclosure.

As used herein the term “SARS-CoV” is used to identify both SARS-CoV-1 and SARS- CoV-2 and related coronaviruses.

As used herein the term “SARS-CoV2” and “SARS-CoV-2” are used interchangeably.

The term “artificial” with respect to a nucleic acid molecule means that it is not naturally occurring.

The term “naturally occurring” with respect to a sequence means that the sequence is a product of nature.

The term “about” shall generally mean that a given value or range may vary. Variations of 1%- 10% usually represent an acceptable variation range for a given value.

As used herein, the term "regulatory sequences" refers to DNA sequences, such as initiator sequences, enhancer sequences, and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably linked.

As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with the nucleic acid sequence encoding the antigen-coding portion and/or expression control sequences that act in trans or at a distance to control the transcription and expression thereof.

As used herein the term “90% sequence identity”, includes all values contained within and including 90% to 100%, such as 91%, 92%, 92,5%, 95%, 96.8%, 99%, 100%. Likely, the term “at least about 75% identical” includes all values contained within and including 75% to 100%. As used herein the term “adjacent” encompass a sequence that is located near a reference domain either linearly or structurally.

The term “consist(s) essentially of’ or “consisting essentially of’ with respect to a sequence allows for some variation of the sequence (insertions, deletions, changes) that do not substantially affect the ability of the sequence to carry its intended purpose.

Generally, the degree of similarity and identity between two sequences is determined using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247- 250) using default settings, i.e., meagablast program (see NCBI Handout Series | BLAST homepage & search pages | Last Update September 8, 2016).

It is to be understood herein that the nucleic acid sequences encoding protein(s) or peptide(s) of interest may be codon-optimized. The term “codon-optimized” refers to a sequence for which a codon has been changed for another codon encoding the same amino acid but that is preferred or that performs better in a given organism (increases expression, minimize secondary structures in RNA etc.). It is to be understood herein that a “codon-optimized” sequence is an artificial sequence.

As used herein, “pharmaceutical composition” means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), etc.

The term “treatment” for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to slow down (lessen) or reduce infection or pathologic condition or disorder associated with infection, reduce symptoms or disease, reduce transmission of infection, reduce contagion, reduce viral load in a host and the like. All patents, patent applications, and publications referred to herein are incorporated by reference in their entirety.

EXAMPLE 1- Generation of pIDV-II-SARS-CoV-2-Spike vaccines

The pIDV-II vector (SEQ ID NO:9) is a modified version of the pIDV-I vector (SEQ ID NO:8) in which a woodchuck hepatitis virus post-transcriptional regulatory element was inserted at nucleotides 7-595 (see Figure 1(A)-(H)).

Expression experiments indicated that both pIDV-I and pID V-II can drive eGFP expression effectively, with pIDV-II performing better (Figure 2 and Figure 3). The pIDV-II vector was thus selected for generating DNA vaccines against SARS-CoV-2.

We generated DNA vaccine candidates for evaluation using 92 sequences downloaded from NCBI GenBank (accessed February 24, 2020) and from which a consensus sequence was generated using MEGA X. The consensus sequence was aligned to the S-gene from the SARS- CoV-2 isolate Wuhan-Hu- 1, (GenBank accession number MN908947.3) for reference, and the codon optimized gene was cloned into the DNA vector pIDV-II.

Three codon-optimized DNA sequences expressing the SARS-CoV-2 Spike protein or modified versions (Antigen 1 (identified herein as VI or SEQ ID NO: 1), Antigen 2 (identified herein V3 or SEQ ID NO: 3) and Antigen 3 (identified herein as V5 or SEQ ID NO: 5)) were tested.

VI expresses the full-length SARS-CoV-2 Spike protein obtained from NCBI GenBank. V3 expresses a truncated SARS-CoV-2 Spike protein having a 20-amino acid deletion at its C- terminal end and is fused to a short peptide (5Mer). V5 expresses the full-length SARS-CoV2 Spike protein fused with the short peptide (5Mer) at its C-terminal end (Patel et al, PLoS ONE, 2012, US20110305720A1).

Prior to cloning into the pIDV-II vector (SEQ ID NO: 9), the nucleic acid sequence was human codon-optimized (GenScript Biotech Corp.) and fused to the signal sequence of Kozak. The antigen was cloned at the 3’end of the plasmid promoter (see W02019/218091).

In order to exclude introduction of spontaneous mutations in the transgene, the sequence of the vector and insert was confirmed by sequencing. The codon-optimized DNA sequences were cloned into the pIDV-II vector described in international application No. PCT/CA2019/050686 in the name of Kobinger et al., published on November 21, 2019 under WO 2019/218091 and in international application No. PCT/CA2019/051592 in the name of Kobinger et al., published on November 26, 2020 under number WO20201232527 (the entire contents of which are incorporated herein by reference).

The three DNA vaccine vectors thus generated were named pIDV-II-SARS-CoV2- Spike Vl, pIDV-II-SARS-CoV2-Spike_V3 and pIDV-II-SARS-CoV2-Spike_V5.

Protein expression was verified by transient transfection of the DNA vaccine vector in HEK 293T cells, followed by Western blot. Briefly, Lipofectamine™ 2000 transfection of 5 pg pIDV-II-SARS-CoV2-Spike VI, pIDV-II-SARS-CoV2-Spike-V3 or pIDV-II-SARS-CoV2- Spike-V5 was performed in 6 well plates containing 300,000 cells/well. Cell lysis was performed under non-reduced condition. 24 h post-transfection, cell pellets were prepared in Xtractor™ buffer according to manufacturer’s instructions (Clontech Laboratories, Inc. Cat.No 635676). Briefly, cell lysates were centrifuged at 10 000 g for 10 min. The protein content of the supernatant was quantified and 15 ug of each sample was mixed with sample buffer [10 M Tris/HCl (pH 6,8), 2% SDS, 10% glycerol, 5% P-mercaptoethanol, 0,005% bromophenol blue] and incubated at 56 °C for 10 min before electrophoresis in a Criterion Gel. Western blot analysis was performed by using inactivated plasma derived from CoVI19 positive patient and provided by Sunnybrook Science Center (a gift from Dr. Robert Kozak) at a dilution of 1 :200 as primary antibodies and a peroxidase-conjugated secondary antibody, followed by visualization with 4 ml total of substrate (Western blotting detection reagents Bio-Rad).

Western blotting under non-reducing conditions with anti-SARS-CoV2 Spike confirm robust expression of the S protein in vitro with protein of approximately 105, 75 and 45 kDa respectively (Data not shown).

EXAMPLE 2- Animal Study: humoral and cellular responses

Balb/c mouse model

Four groups of 10 mice aged 6-8 weeks (Charles River Company, Canada) were injected intramuscularly by electroporation (Inovio Pharmaceuticals) into the caudal thigh with 50 pg of the SARS-CoV2-Spike DNA vaccines (pIDV-II-SARS-CoV2-Spike_Vl (pIDV-II-Vl), pIDV-II- SARS-CoV2-Spike_V3 (pIDV-II-V3) and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5)) diluted in Endotoxin-free TE buffer or with equivalent volume of Endotoxin-free TE buffer respectively (control group).

A total volume of 100 pl was administered to each animal across two sites; each with 50 pl per limb. All mice received a boost on day 28. Blood was obtained via lateral saphenous vein at days 28 and 56. Serum was separated and kept frozen until analysis. Four mice from each group were euthanized at day 10 after boost for assessment of T-cell response towards SARS-CoV-2 Spike antigen via IFN-y enzyme-linked immunospot (ELISPOT) assay, performed according to the manufacturer's instructions (DB Biosciences). Briefly, splenocytes were collected from immunized animals, cells were seeded in Millipore plate, at 3 x io⁵ splenocytes per well and restimulated with peptide pool containing 158 peptides derived from SARS-CoV2 and spanning complete Spike protein. Peptide pool was applied at a final concentration of 100 pg/ml (JPT Innovative Peptide Solutions). Plates were developed after overnight incubation at 37°C in a humidified incubator supplemented with 5% CO2. Each well was imaged by microscope. Spots were counted and results were expressed as spot forming units (SFU) per IxlO⁶ cells by the CellProfiler™ software (Figure 4(A)).

In Figure 4(A), the SARS-CoV2-Spike DNA vaccines used are pIDV-II-SARS-CoV2- Spike Vl (pIDV-II-Vl), pIDV-II-SARS-CoV2-Spike_V3 (pIDV-II-V3) and pIDV-II-SARS- CoV2-Spike_V5 (pIDV-II-V5). The bar denotes the number of spots against the peptide pool in mice vaccinated with the different DNA vector vaccines. In this assay, animals vaccinated with pIDV-II-SARS-CoV-Spike_V3 shows higher T-cell response pattern at the day 10 after boost compared to groups vaccinated with pIDV-II-SARS-CoV2_Vl and pIDV-II-SARS-CoV2_V5 (Figure 4(A)).

The antibody response at day 28 and 56 was evaluated by ELISA assay.

Briefly, flat bottom ELISA plates were coated overnight at 4°C with a NR-722, a truncated and glycosylated recombinant form of the SARS-CoV spike (S) external envelope glycoprotein (obtained through BEI Resources, NIAID, NIH: SARS-CoV Spike (S) Protein deltaTM, Recombinant from Baculovirus, NR-722) diluted in 1XPBS per 96-well plate.

The following day, plates were washed and then blocked with KPL Milk-Blocking Solution 1/10 diluted in H2O for 90 minutes at 37°C. All washes were done with IX PBS containing 0.05% Tween-20. Plates were washed again, prior to being loaded with mice sera diluted 1 :400 in double replicates. Serum dilution was carried out in blocking buffer. Plates were incubated at 37°C for 90 minutes prior to being washed again, and then incubated with anti-Mouse-HRP secondary antibody diluted 1 :2000 in KPL-Milk Blocking solution and incubated at 37°C in a humified incubator for 90 min. After secondary antibody incubation, plates were washed with 150 pl of PBS-Tween 0.1%, six times. Then 50 pl of freshly prepared KPL two-component ABTS substrate was added into the wells and stabilized at room temperature for 25 minutes at 37°C in a humified incubator. Finally, reaction was stopped by adding of 50 pl/well 1% SDS. Absorbance at 450 nm was determined with a microplate reader.

Individual naive mice sera for each group collected from the same day points were used as an internal control on each assay group. A plate cut-off value was determined based on the average absorbance of the naive control starting dilution plus standard deviation. Only sample dilutions whose average was above this cut-off were registered as positive signal.

All vaccinated mice developed robust IgGl response at day 56 (post prime boost) (Figure 4(B)) After a prime on day 0 with IM+EP route, fast appearance of SARS-COV2 -specific antibodies was detected by ELISA in Balb/c mice vaccinated with pIDV-II-SARS-CoV2-V5. Figure 4(B) shows SARS-COV2 -specific IgG in individual vaccinated mice. All mice received an equal amount of vaccine of lOOpl in total (50 pl/dose). Collected sera from naive mice vaccinated with only Endofree TE buffer (Control group) were tested concurrently and had no detectable background signal.

In vaccinated mice, the SARS-CoV2-specific IgG ELISA titers significantly increased between the first and second vaccinations. Surprisingly all three versions of the DNA vaccines have shown a potent T-cell response in all vaccinated mice with pIDV-II-SARS-CoV2-Spike-Vl or pIDV-II-SARS-CoV2-Spike-V5 vaccines performing the best (Figure 4(B)).

C57BL/6 (Black/6) mice model

The two best candidates were selected for further evaluation in Black/6 mice models. For this purpose three groups of 10 mice -8 weeks (from the Charles River Company, Canada) were injected intramuscular electroporation (Inovio Pharmaceuticals ) per animal into the caudal thigh with 50 pg of optimized pIDV-II-SARS-CoV2-Spike-Vl or pIDV-II-SARS-CoV2-Spike-V5 vaccines diluted in Endotoxin-free TE buffer. An equivalent volume of Endotoxin-free TE buffer respectively was injected into the control group (Figure 5(A)).

In Figure 5(A) the SARS-CoV2-Spike DNA vaccines used are pIDV-II-SARS-CoV2- Spike Vl (pIDV-II-Vl), and pIDV-II-SARS-CoV2-Spike_V5 (pIDV-II-V5). In this assay, animals vaccinated with pIDV-II-SARS-CoV2_Vl and pIDV-II-SARS-CoV2_V5 shows high T- cell response pattern.

The antibody response at day 28 and 56 was evaluated by ELISA assay as indicated above. All mice received an equal amount of vaccine of lOOpl in total (50 pl/dose). Collected sera from naive mice vaccinated with only Endofree TE buffer (Control group) were tested concurrently and had no detectable background signal.

All vaccinated mice developed robust IgGl response at day 56 (post prime boost) (Figure 5(B)) After a prime on day 0 with IM+EP rout fast appearance of SARS-COV2 -specific antibodies was detected by ELISA in Black/6 mice vaccinated with pIDV-II-SARS-CoV2-V5 (Figure 5(B)).

Our study shows that all three vaccines pIDV-II-SARS-CoV2_Vl, pIDV-II-SARS- CoV2_V3 and the pIDV-II-SARS-CoV2_V5 delivered by IM-EP induces a robust T-cell and antibody responses in both mouse models of vaccinated animals with pIDV-II-SARS-CoV2_Vl and pIDV-II-SARS-CoV2_V5 performing the best.

It was noted that mice receiving either pIDV-II-SARS-CoV2_Vl or pIDV-II-SARS- CoV2_V5 had higher antibodies titers than the sham vaccinated group. Interestingly, measurement of the Spike-specific IgG levels at day 56 showed notable increase after animals had received the second vaccine dose, and this was observed in both mouse species. Vaccination also induced a Thl immune response, which was evaluated by an ELISpot assay for IFN-y producing cells 10 days after animals received their second vaccine dose. Notably, vaccinated mice produced more antigen-specific IFN-y cells compared with unvaccinated animals, which is indicative of a strong Thl response. Overall, there were no significant differences in cellular or humoral response between animals that received vaccine pIDV-II-SARS-CoV2_Vl and vaccine pIDV-II-SARS- CoV2_V5. Based on these data, both vaccines were carried forward for challenge studies. Throughout the experiments, no overt adverse clinical events were observed in vaccinated animals.

In summary, this study shows that we generated pIDV-II-SARS-CoV2-Spike DNA vaccines that are capable to provoke robust humoral and cellular immune responses in two different mice models.

EXAMPLE 3- Animal Study: dose-response

A dose-response study is performed by administering varying doses of the one or more DNA vaccines disclosed herein using a vaccination scheme as exemplified in Example 2.

Typically, groups of animals receive a dose ranging from 1 microgram to 2milligrams.

For example, animals may receive 10 micrograms, 30 micrograms, 100 micrograms, 300 micrograms, 500 micrograms, 1 milligram, 2 milligrams or as may deemed necessary.

The immune response is evaluated using the methodology exemplified in Example 2. The optimal dosage is then determined.

EXAMPLE 4- Animal Study: challenge experiments

DNA vaccines are tested in challenge experiments in ferrets, hamsters (e.g., Syrian hamsters) or in any other suitable animal model receptive to SARS-CoV-2 infection.

Briefly, Vero E6 cells are used to grow the SARS-CoV-2 virus (Sunnybrook strain; SARS- CoV-2/SB3-TYAGNC or other suitable strains) in Dulbecco’s modified Eagle medium (DMEM; Fisher Scientific) with supplements (10% fetal bovine serum, 2 mmol/L 1-glutamine, 100 U/mL Pen/Strep).

Viral titer is determined by performing TCID50 assay on monolayers of infected Vero E6 cells in 96-well plates, the first dilution of viral sample typically being 1 : 10 followed by ten-fold serial dilutions. Plates are incubated at 37° C for 4 days followed by cytopathic effect (CPE) examination of infected cells. The back-titration of inoculum is determined. Groups of animals receive the DNA vaccines described herein, whereas control groups typically receive saline. At week 4, all animals are challenged with 100 pl of virus TCID5ol ^x lO⁵/mL SARS-CoV-2 by the intranasal route. Typically, ferrets infected with SARS-CoV-2 are assessed by histopathology on days 7 (n = 12) and 14 (n = 12) whereas challenged hamsters are assessed on days 4 and 8. Nasal washes and oral swabs from ferrets are collected at day 1, 3, 5, 7, 9, 11 and 14 and for hamsters at day 1, 3, 5 and 7. Lungs and nasal turbinates are collected from all euthanized animals. The infectious and/or total viral load is determined.

Protection experiments in pre-clinical studies confirmed, amongst other things, that the DNA vaccine of the present disclosure can substantially lower the levels of infectious SARS-CoV2 virus in the lungs following two doses of the vaccine.

The pIDV-II-SARS-CoV2_Vl, pIDV-II-SARS-CoV2_V5 vaccines were particularly selected for challenge experiments in Syrian hamsters. To this end, three groups of eight hamsters were vaccinated at day 0 and day 28 (prime and boost) with either vaccine pIDV-II-SARS- CoV2_Vl, pIDV-II-SARS-CoV2_V5 or sterile TE buffer (sham vaccination).

For vaccination, 200 pg of DNA in a total volume of 200 pl endotoxin free TE buffer (Thermo Fisher Scientific, Mississauga, Canada) was delivered into the back by intradermal (ID) delivery using an intradermal oscillating needles array injection device (Gomez et al manuscript in revision). Briefly, pressurized vaccine was injected into the skin by a needle array composed of hypodermic needles. The two electromagnetic coils move the needle array up and down at approximately 100 Hz. A valve controls vaccine injection and its opening is synchronized with the needle array oscillation, thereby ensuring that the vaccine is delivered when the needles are inside the skin.

Alternatively, the DNA vaccine is delivered using the CELLECTRA® 2000 device that generates a controlled electric field at the injection site to enhance the cellular uptake and expression of the DNA plasmid. The device delivers a total of four electrical pulses per EP, each pulse of 52 msec in duration, at strengths of 0.2 Amp current and voltage of 40- 200 V per pulse.

Four weeks after the second immunization, hamsters were challenged intranasally with a high dose of SARS-CoV-2 (SARS-CoV-2/SB3-TYAGNC; 8.3 xlO⁵ TCID50). Following infection, animals were monitored for clinical signs of disease and weight loss. Animals in the vaccinated groups showed no weight loss during the experiment. In contrast, hamsters in the sham-vaccinated group showed weight first noted at 3 dpi with a median loss of 8.8% by 5 dpi and maximum percentage weight loss of 16% at 7 dpi (Figure 6). Interestingly, as shown by Tostanoski et al, (Tostanoski, L. H. et al., 2020), infection resulted in partial mortality of animals in the unvaccinated group as one animal died on day 7. No animals succumbed to infection in either vaccinated group. No hierarchy between the two vaccine constructs could be determined based on weight loss post-challenge. Viral shedding was assessed in oral swabs and nasal washes collected from sham and vaccinated hamsters post SARS-CoV-2 challenge to differentiate the two vaccine constructs. Both vaccines reduced the quantity of infectious viral particles as compared to unvaccinated controls at 3, 5 and 7 dpi. Interestingly, no infectious virus was detected in the oral swabs or washes of hamsters that received pIDV-II-SARS-CoV2_Vl (Figures 7(B)-(C)).

EXAMPLE 5- Viral burden in upper and lower respiratory tract and organs

While vaccination has been shown to reduce viral shedding, the quantities of infectious RNA loads were also determined in the lungs and nasal turbinates following euthanization.

The RNA loads from swabs, nasal washes and homogenized tissues were extracted using the QIAamp viral RNA kit (QIAGEN, Toronto, Canada) according to the manufacturer's instructions. Reverse transcription-quantitative PCR (RT-qPCR) was used to determine RNA loads and was performed as described previously (Feld, J. J. et al. 2021).

Examination of the viral burden in lungs and nasal turbinates demonstrated significantly lower quantities of infectious virus at 4 dpi in the pIDV-II-SARS-CoV2_V5 vaccinated group and undetectable levels of infectious virus at 8 dpi compared to the control group. In comparison, in hamsters vaccinated with pIDV-II-SARS-CoV2_Vl, no infectious virus was detected either on 4 or 8 dpi (Figure 7(D) and (E)). Viral RNA was detected in lungs of both vaccinated and unvaccinated groups at both timepoints (Figures 8(A)-(C)). Interestingly, faster viral clearance was observed in the kidneys of hamsters vaccinated with pIDV-II-SARS-CoV2_Vl at 8 dpi, as no viral RNA was detected at this time point.

EXAMPLE 6- Histopathology

Following euthanization of the animals at 4- and 8-dpi, lungs and nasal turbinates were examined and scored to determine the extent of pulmonary disease.

For histopathology assessment, tissues were fixed in 10 % neutral phosphate-buffered formalin, routinely processed, sectioned at 5 p and stained with hematoxylin and eosin (H&E) for histopathologic examination. Sections of nasal turbinates and lung (left and right lobes) were examined and scored by a board-certified veterinary pathologist who was blinded to groups and days of sampling. Nasal turbinates were evaluated for the presence of intraepithelial neutrophils. Lungs were evaluated for the presence of absence of: features of cell or tissue damage (necrosis of bronchiolar epithelial cells (BEC), inflammatory cells and/or cellular debris in bronchi, intraepithelial neutrophils, alveolar emphysema), circulatory changes and vascular lesions (alveolar hemorrhage, significant alveolar edema, vasculitis/vascular endothelialitis), reactive inflammatory patterns (necrosuppurative bronchitis, intralveolar neutrophils and macrophages, mononuclear infiltrates around airways, presence of polymorphonuclear granulocytes, perivascular mononuclear cuffs, and mesothelial reactivity), as well as regeneration and repair (alveolar epithelial hyperplasia/regeneration, BEC hyperplasia/regeneration) (Gruber, A. D. et a!.. 2020). After scoring was complete, the pathologist was unblinded and nasal turbinate and lung pathology scores (“Inflammation Score”) were calculated as the number of lesions present per group for each timepoint. Scores for the control group at 8 dpi were adjusted as only 3 control animals were available for evaluation at this time point.

For kidneys and liver H&E and Masson’ s Trichrome staining were also performed to assess tissue architecture and inflammation and gauge the progression of fibrosis, respectively Images were acquired on a Nikon microscope with NIS Elements AIR 5.02.00 software under lOx objective. Non-overlapping fields of view were taken to image the entire tissue for each section. Inflammation and fibrosis were assessed by a blind observer. The region of aggregation of inflammatory cell infiltrates delineated and represented as percentage of the cell infiltrates area to total tissue area. The regions of collagen content stained in blue were delineated and represented as percentage fibrosis area to total tissue area. Scoring was performed by a clinical evaluator blinded to the identities of the samples.

Results indicate that pulmonary pathology was observed to a greater extent in unvaccinated animals, and disease scores were highest in this group compared to animals that received either vaccine (not shown). The inflammatory response and amount of lung tissue affected at day 4 was most severe in the control group (lung score, though this was only slightly higher than the hamsters vaccinated with pIDV-II-SARS-CoV2-Spike_V5. Hamsters vaccinated with pIDV-II-SARS- CoV2-Spike_Vl had a few lung lesions present at day 4. At day 8, both vaccinated groups had reduced lung scores, with the lesions largely comprised of mildly inflammatory responses. In the control group at day 8, lung scores were also slightly reduced as compared to the day 4 controls. However, there were fewer inflammatory lesions present at day 8 alongside a significant regenerative response was present. Investigation of lung fibrosis was also performed (Puelles, V. G. et al., 2020). A lower percentage of fibrosis was observed in both vaccinated groups compared to the controls, with the lowest percentage observed in the lungs of animals that received the pIDV- II-SARS-CoV2-Spike_Vl vaccine (Figure 9). In the nasal turbinates neutrophilic intraepithelial inflammation was only observed in two control animals at the day 4 timepoint. Overall, the pathology scores and fibrosis percentages suggested more severe disease in the unvaccinated group. Pathology in the kidneys was also evaluated, as renal tropism for SARS-CoV-2 has been described, and kidney injury is a frequent complication of infection (Puelles, V. G. et al., 2020; Pei, G. et al., 2020). Inflammation and an influx of inflammatory cells was observed to a greater degree in the control group compared to vaccinated animals at both timepoints and was suggestive of potential tubular injury or acute kidney disease (Figure 10(A)). Additionally, kidney fibrosis was more severe in unvaccinated than vaccinated animals (Figure 10(B)). Overall, vaccination reduced extrapulmonary pathology.

EXAMPLE 7- Virus neutralization

Neutralizing antibodies are emerging as a potential correlate of protection against SARS- CoV-2 (Khoury, D. S. et al., 2021). In order to assess the ability of our vaccines to elicit neutralizing antibodies against emerging variants of SARS-CoV-2, we evaluated viral neutralizing titers in sera from all groups of hamsters, collected 4- and 8-days post challenge.

For viral neutralization assays, serum was collected at 4 and 8 dpi from hamsters for analysis of neutralizing titers in manner similar to that described by Abe and colleagues (Abe, K. T. etal, 2020). Briefly, Vero E6 cells (2xl0⁵cells/mL) were seeded in 200pL/well DMEM (Gibco, Canada) containing 10% FBS and 1% Penicillium- Streptomycin (P/S) into a 96 plate and incubated for overnight at 37°C. On the following day, each serum (heat inactivated at 56°C for 30 min before use) was twofold serially diluted in DMEM containing 1% P/S and incubated with either SARS-CoV-2 SB3, SARS-CoV-2 Alpha (B.1.1.7) or SARS-CoV-2 Beta (B.1.351) at 37°C for 1 h with shaking every 15 min. The virus-serum mixture (containing SARS-CoV-2 variants at 4* 10² TCID50) was transferred to the Vero E6 cell plate in quadruplicate wells of a 96-well plate. For each serum, the starting dilution was 1/20 with a series of two-fold dilutions to the final dilution of 1/2560. After incubation, all the media from the previously seeded Vero E6 plate was removed and replaced with 50pL/well of infected cells and incubated for Ih. Following the Ih incubation, the inoculum was removed and 200pL DIMEM (2% FBS/1% P/S) containing the same serum dilution per row as the layout was added to all wells. Virus neutralization titers (VNT) were read at the day 3 and 5 following infection, as the reciprocal of the highest dilution of serum where the cytopathic effect (CPE) was recorded.

Viral neutralization against several variants of concern (VOCs) was assessed by mixing serum with viral isolates from different lineages and the assessing CPE on Vero E6 cells at 5 dpi. Hamster sera from the pIDV-II-Vl vaccinated group were able to neutralize three SARS-CoV-2 viruses including SB3 (Bl lineage), the Alpha variant (B. l.1.7) lineage and Beta variant (Bl.351) lineage. Neutralizing antibodies (nAb) were detected in all vaccinated animals at day 4 dpi and at 8 dpi against SB3, B. l.1.7 and Bl.351. Similar to control group, animals vaccinated with pIDV- II-V5 did not have detectable nAb at 4 dpi, while at the 8 dpi, two-fold higher levels of nAb was observed in the same group compared to the control group (Figure 11). Overall, significantly higher titers of nAbs were produced in hamsters vaccinated with pIDV-II-SARS-CoV2-Spike_Vl in comparison to the group vaccinated with pIDV-II-SARS-CoV2-Spike_V5.

In this study we demonstrated differing levels of protection against disease caused by SARS-CoV-2 generated by a DNA vaccine expressing the Spike protein or the same vaccine also expressing a putative immuno-stimulatory peptide. In both cases, a two-dose vaccination resulted in the production of neutralizing antibodies, lower viral loads in the both the upper and lower respiratory tracts, reduced viral shedding, and vaccination protected against weight loss. However, there were differences in the extent of lung and tissue pathology between the two vaccine candidates. Vaccination resulted in decreased inflammation and fibrosis in the kidneys suggesting that our vaccine candidates may limit viral dissemination to other organs. Additionally, differences in the quantity of neutralizing antibody were noted between both vaccines at 4- and 8-days postinfection. Interestingly, despite good immunogenicity, the protection was not uniform for both vaccines. Our DNA vaccine that included the 5mer4 peptide sequence showed similar immunogenicity in mice compared to pIDV-II-SARS-CoV2-Spike_Vl, there was less protection in challenged hamsters. The 5mer4 peptide may affect the tertiary structure of S protein which could influence the antigen presentation and the production of antibodies. Both of our vaccines induced antibodies capable of neutralizing SARS-CoV-2 variants that have been shown to evade immunity derived from natural infection as well as being highly transmissible and pathogenic (Davies, N. G. et al., 2021 (1); Davies, N. G. etal., 2021 (2)). Virus neutralization is a measure of antibody efficacy, and a potential correlate of protection of vaccines (Pollard, A. J. & Bijker, E. M., 2021) and recent vaccination studies with a parainfluenza virus 5 vector expressing the SARS- CoV-2 S-antigen have demonstrated the potential to prevent transmission in the ferret model (An, D. et al., 2021). It is possible that the antibody titers generated by our DNA vaccines limit transmission as well. Gooch etal. have demonstrated a correlation between decrease in viral loads in the throat ofNHPs, and serum neutralizing antibody titers (Gooch, K. E. etal., 2021). We noted a similar association with overall antibody titers and viral shedding. Mucosal antibodies present in the upper respiratory tract induced by our DNA vaccines will be assessed.

Our vaccines have demonstrated the potential to protect against viral dissemination and multi organ pathology as evidenced by reduced viral RNA in the kidneys of both vaccinated groups. While it has been determined that the primary cause of mortality in COVID-19 patients is acute respiratory distress syndrome (ARDS), there is increasing evidence that SARS-CoV-2 is a systemic disease that affects multiple organs, including lungs, kidney, heart and liver (Synowiec, A., et al., 2021; Ruan, Q., et al., 2020). Recent studies in hamsters by Tostanoski et al., demonstrated ongoing inflammation even after viral loads decreased (Tostanoski, L. H. et al.,

2020). Additionally, a recent study investigating intranasal or intramuscular delivery of a SARS- CoV-2 DNA vaccine in hamster showed that it did not protect against lung pathology or pneumonia (Leventhal, S. S., etal., 2021). Interestingly, in contrast, our vaccine both reduced viral load in the lungs correlated with decreased pathology seen in the lungs, indicating that mechanisms other than viral replication may likely be responsible for lung pathology (Leventhal, S. S., et al.,

2021). In addition, the nature of the DNA vaccine and route of administration may have an impact on its efficacy.

Our vaccine has several advantages. It is easy to generate and adapt as needed should vaccine escape variants arise and does not face the challenge of pre-existing immunity that may exist with viral vectored vaccines (Zhu, F. C. et al., 2020). Our dosing regimen is based on previous studies which indicated that two doses were required to generate a robust immune response (Modjarrad, K. et al., 2019; Tebas, P. et al., 2017). Interestingly, we were able to demonstrate protection in our animal study using a smaller quantity of plasmid than a similar vaccine study in non-human primates (Gooch, K. E. et al., 2020). Recent studies on individuals who received a single dose of the currently licensed mRNA COVID-19 vaccine has indicated that significant protection is conferred after one dose (Britton, A. et al., 2021). Thus, it is possible that a single dose of pIDV-II-SARS-CoV2-Spike_Vl is sufficient to protect animals from severe disease.

Overall, our results provide further insights into the immunogenicity of a SARS-CoV2- Spike DNA vaccines and contribute to knowledge and therapies against this devastating pandemic.

The embodiments and examples described herein are illustrative and are not meant to limit the scope of the claims. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Citations listed in the present application are incorporated herein by reference.

REFERENCES

All patents, patent applications and publications referred to throughout the application are incorporated herein by reference.

Patel, A. et al. (2012) PLoS ONE 7(8): e43802.

Long, S.W. et al., mBio Vol. 11(6): e02707-20, November 2020.

Li, Q. et al. N Engl J Med 382, 1199-1207, doi: 10.1056/NEJMoa2001316 (2020).

Zhu, N. et al. N Engl J Med 382, 727-733, doi: 10.1056/NEJMoa2001017 (2020).

Shahhosseini, N. et al., Gene Rep 23, 101045, doi: 10.1016/j. genrep.2021.101045 (2021).

Polack, F. P. et al., N Engl J Med 383, 2603-2615, doi: 10.1056/NEJMoa2034577 (2020).

Jackson, L. A. et al., N Engl J Med 383, 1920-1931, doi: 10.1056/NEJMoa2022483 (2020).

Moghadas, S. M. et al., Clin Infect Dis, doi:10.1093/cid/ciab079 (2021).

Sharma, O. et al., Front Immunol 11, 585354, doi: 10.3389/fimmu.2020.585354 (2020).

Prompetchara, E. et al., Asian Pac J Allerg Immunol 38, 1-9, doi: 10.12932/ AP -200220-0772 (2020).

Johansen, M. D. et al., Mucosal Immunol 13, 877-891, doi: 10.1038/s41385-020-00340-z (2020).

Imai, M. et al., Proc Natl Acad Sci U S A 117, 16587-16595, doi:10.1073/pnas.2009799117 (2020).

Sia, S. F. et al., Nature 583, 834-838, doi:10.1038/s41586-020-2342-5 (2020).

Tostanoski, L. H. et a\., Nat Med 26, 1694-1700, doi: 10.1038/s41591-020-1070-6 (2020).

Leventhal, S. S. et al., Microorganisms 9, doi: 10.3390/microorganisms9051040 (2021).

Chai, K. M. et al. PLoS Negl Trop Dis 15, e0009374, doi:10.1371/joumal.pntd.0009374 (2021).

Kumar, A. et al., Curr Infect Dis Rep 23, 9, doi : 10.1007/s 11908-021-00752-3 (2021).

Modjarrad, K. et al., Lancet Infect Dis 19, 1013-1022, doi: 10.1016/S1473-3099(19)30266-X (2019).

Tebas, P. et al., EClinicalMedicine 31, 100689, doi: 10.1016/j. eclinm.2020.100689 (2021). Tebas, P. et al., N Engl J Med, doi: 10.1056/NEJMoal708120 (2017).

Seo, Y. B. et al., Vaccine doi:10.3390/vaccines9040307 (2021).

Yu, J. et al., Science 369, 806-811, doi: 10.1126/science.abc6284 (2020).

Nasir, J. A. et al., Viruses 12, doi: 10.3390/vl2080895 (2020).

Banerjee, A. et al., Emerg Infect Dis 26, 2054-2063, doi: 10.3201/eid2609.201495 (2020).

Gooch, K. E. et al., Vaccine, doi: 10.1016/j. vaccine.2021.06.057 (2021).

Feld, J. J. et al., Lancet Respir Med 9, 498-510, doi: 10.1016/S2213-2600(20)30566-X (2021).

Abe, K. T. et al., JCI Insight 5, doi : 10.1172/j ci. insight.142362 (2020).

Gruber, A. D. et al., Am J Respir Cell Mol Biol 63, 856-859, doi: 10.1165/rcmb.2020-0280LE (2020).

Chan, J. F. et al., Clin Infect Dis 71, 2428-2446, doi: 10.1093/cid/ciaa325 (2020).

Puelles, V. G. et al., N Engl J Med 383, 590-592, doi: 10.1056/NEJMc2011400 (2020).

Pei, G. et al., Pneumonia. J Am Soc Nephrol 31, 1157-1165, doi: 10.1681/ASN.2020030276 (2020).

Khoury, D. S. et al., Nat Med 27, 1205-1211, doi: 10.1038/s41591-021-01377-8 (2021).

Davies, N. G. et al., Science 372, doi: 10.1126/science.abg3055 (2021 (1)).

Davies, N. G. et al., Nature 593, 270-274, doi: 10.1038/s41586-021-03426-l (2021 (2)).

Pollard, A. J. & Bijker, E. M. Nat Rev Immunol 21, 83-100, doi: 10.1038/s41577-020-00479-7 (2021).

An, D. et al., Sci Adv 7, doi: 10.1126/sciadv.abi5246 (2021).

Synowiec, A. et al., Clin Microbiol Rev 34, doi: 10.1128/CMR.00133-20 (2021).

Ruan, Q. et al., Intensive Care Med 46, 846-848, doi: 10.1007/s00134-020-05991-x (2020).

Zhu, F. C. et al., Lancet 395, 1845-1854, doi: 10.1016/80140-6736(20)31208-3 (2020).

Britton, A. et al., MMWR Morb Mortal Wkly Rep 70, 396-401, doi: 10.15585/mmwr.mm7011e3 (2021). SEQUENCE TABLE

Antigen 1 (VI)- codon-optimized nucleic acid sequence (SEQ ID NO: 1)

ATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTGTCCTCACAGTGCGTCAACCTGACTACCCGAA

CTCAGCTGCCCCCTGCTTATACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTT

TAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGG

TTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTT

TTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGG

CACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATC

AAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATA

AGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGT

GTCCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTC

GTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGC

GCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACAT

CACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGC

GGATGGACCGCAGGAGCAGCAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGA

AGTACAACGAGAATGGCACCATCACAGACGCCGTGGATTGCGCCCTGGATCCTCTGAGCGAGAC

AAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTG

CAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGT

TCAACGCAACCAGGTTCGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGCGTGGC

CGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCC

ACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACG

AGGTGCGCCAGATCGCACCTGGACAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCAGA

CGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAAC

TACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTA

CAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCC

ACTGCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTG

CTGAGCTTTGAGCTGCTGCACGCACCAGCAACAGTGTGCGGACCCAAGAAGTCCACCAATCTGG

TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCGAGTC

CAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTG

CGCGACCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGA

TCACACCAGGAACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGA

GGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCC AACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGT

GCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGAG

AGCACGGAGCGTGGCATCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCT

GTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGA

TCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTAC

CGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACA

GGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACA

AGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAA

GCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGC

TTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCACGGGACCTGATCTGTGCCCAGA

AGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAG

CGCCCTGCTGGCAGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATC

CCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACG

AGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTC

CTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGATATCCTGA

GCAGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTC

TCTGCAGACCTATGTGACACAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTG

GCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGG

GCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTA

CGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCAC

TTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCT

ACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGG

CATCGTGAACAATACCGTGTATGATCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTG

GATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATG

CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGA

GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATC

TGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGA

CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGA

CGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCTGA Antigen 1 (VI)- amino acid sequence of naturally occurring SARS-CoV-2 spike protein (SEQ ID NO: 2)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW

FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWI

KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF

VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS

GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV

QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP

TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN

YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVW

LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV

RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS

VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT

GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG

FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI

PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALN

TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL

AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAH

FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEEL DKYFKNHT SPDVDLGD I SG INAS WN I QKE I DRLNEVAKNLNE SL I DLQELGKYEQ Y I KWPWY I WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

Antigen 2 (V3)- codon-optimized nucleic acid sequence (SEQ ID NO: 3)

ATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTGTCCTCACAGTGCGTCAACCTGACTACCCGAA

CTCAGCTGCCCCCTGCTTATACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTT

TAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGG

TTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTT

TTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGG

CACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATC

AAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATA

AGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGT GTCCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTC

GTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGC

GCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACAT

CACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGC

GGATGGACCGCAGGAGCAGCAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGA

AGTACAACGAGAATGGCACCATCACAGACGCCGTGGATTGCGCCCTGGATCCTCTGAGCGAGAC

AAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTG

CAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGT

TCAACGCAACCAGGTTCGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGCGTGGC

CGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCC

ACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACG

AGGTGCGCCAGATCGCACCTGGACAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCAGA

CGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAAC

TACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTA

CAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCC

ACTGCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTG

CTGAGCTTTGAGCTGCTGCACGCACCAGCAACAGTGTGCGGACCCAAGAAGTCCACCAATCTGG

TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCGAGTC

CAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTG

CGCGACCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGA

TCACACCAGGAACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGA

GGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCC

AACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGT

GCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGAG

AGCACGGAGCGTGGCATCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCT

GTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGA

TCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTAC

CGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACA

GGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACA

AGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAA

GCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGC

TTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCACGGGACCTGATCTGTGCCCAGA AGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAG

CGCCCTGCTGGCAGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATC

CCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACG

AGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTC

CTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGATATCCTGA

GCAGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTC

TCTGCAGACCTATGTGACACAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTG

GCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGG

GCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTA

CGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCAC

TTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCT

ACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGG

CATCGTGAACAATACCGTGTATGATCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTG

GATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATG

CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGA

GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATC

TGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGA

CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCAAGTGGTGCGAATGCTA G

Antigen 2 (V3)- amino acid sequence (SEQ ID NO: 4)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW

FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWI

KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF

VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS

GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV

QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP

TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN

YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVW

LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV

RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT

GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG

FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI

PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALN

TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL

AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAH

FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEEL DKYFKNHT SPDVDLGD I SG INAS WN I QKE I DRLNEVAKNLNE SL I DLQELGKYEQ Y I KWPWY I WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCKWCEC

Antigen 3 (V5)- codon-optimized nucleic acid sequence (SEQ ID NO: 5)

ATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTGTCCTCACAGTGCGTCAACCTGACTACCCGAA

CTCAGCTGCCCCCTGCTTATACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTT

TAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGG

TTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTT

TTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGG

CACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATC

AAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATA

AGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGT

GTCCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTC

GTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGC

GCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACAT

CACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGC

GGATGGACCGCAGGAGCAGCAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTGCTGA

AGTACAACGAGAATGGCACCATCACAGACGCCGTGGATTGCGCCCTGGATCCTCTGAGCGAGAC

AAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTG

CAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCACAAACCTGTGCCCATTTGGCGAGGTGT

TCAACGCAACCAGGTTCGCAAGCGTGTACGCATGGAATAGGAAGCGCATCTCTAACTGCGTGGC

CGACTATAGCGTGCTGTACAACTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCC

ACAAAGCTGAATGACCTGTGCTTTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACG

AGGTGCGCCAGATCGCACCTGGACAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCAGA

CGATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAAC

TACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCTCTA CAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGAGGGCTTTAACTGTTATTTCCC

ACTGCAGTCCTACGGCTTCCAGCCCACAAACGGCGTGGGCTATCAGCCTTACCGCGTGGTGGTG

CTGAGCTTTGAGCTGCTGCACGCACCAGCAACAGTGTGCGGACCCAAGAAGTCCACCAATCTGG

TGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCGAGTC

CAACAAGAAGTTCCTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTG

CGCGACCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGTGA

TCACACCAGGAACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGA

GGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGCGGGTGTACAGCACCGGCTCC

AACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGT

GCGACATCCCAATCGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGAG

AGCACGGAGCGTGGCATCCCAGTCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCT

GTGGCCTACTCTAACAATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGA

TCCTGCCCGTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTAC

CGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACA

GGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACA

AGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCTGATCCATCCAA

GCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGC

TTCATCAAGCAGTATGGCGATTGCCTGGGCGACATCGCAGCACGGGACCTGATCTGTGCCCAGA

AGTTTAATGGCCTGACCGTGCTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAG

CGCCCTGCTGGCAGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATC

CCCTTTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACG

AGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTC

CTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAAT

ACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGATATCCTGA

GCAGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTC

TCTGCAGACCTATGTGACACAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTG

GCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGG

GCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTA

CGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCAC

TTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCT

ACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGG

CATCGTGAACAATACCGTGTATGATCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTG GATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATG

CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGA

GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATC

TGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGA

CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGA

CGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCAAGTGGTGCGAATGCTAG

Antigen 3 (V5)- amino acid sequence (SEQ ID NO: 6)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW

FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWI

KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF

VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS

GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV

QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP

TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN

YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVW

LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV

RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS

NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS

VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT

GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG

FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI

PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALN

TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL

AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAH

FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEEL DKYFKNHT SPDVDLGD I SG INAS WN I QKE I DRLNEVAKNLNE SL I DLQELGKYEQ Y I KWPWY I WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYTKWCEC- pIDV plasmid (SEQ ID NO: 7)

AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCT

GGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG

GAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGC AACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATG

AAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTT

TTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACT

AGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGA

TCCCTCGACCTGCAGCCCAAgctTGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG

CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG

CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT

GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT

TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT

GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC

AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG

TGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG

TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG

TTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC

TGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG

GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAGCACGTGCTATTATTGAAGCATT

TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAG

GGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGTATGCGGTGTGAAATACCGCACAGATGC

GTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATAATTCAGAAGAACTCGTCAAGAA

GGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTC

AGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGG

TCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATAT

TCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAG

CCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACA

AGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGC

AGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGC

AGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTT

CCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATA

GCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAAC

CGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCC

CAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTT

CAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGAT CCTTGGCGGCGAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCC

CCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTAGAAGGCATGCCTGCTAC

TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA

TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTA

TTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT

GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC

CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTT

CTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAA

TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGCCGCGCGCCAGCCGGGGCGGGGCGGGGC

GAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAA

AGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGC

GGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGG

CTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTA

ATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCT

CCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGG

AGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTT

GTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGG

CTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGC

GCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTT

CGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCA

GGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGC

GGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT

CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCG

CCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGC

GGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC

CGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC

CGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGC

AACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCTCATCGATGCATGGT

ACC pIDV- plasmid (SEQ ID NO:8)

AGATCTTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTC

TGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTC

GGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG

CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATAT

GAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTT

TTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTAC

TAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAG

ATCCCTCGACCTGCAGCCCAAgctTGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA

GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG

GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC

TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG

TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC

TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGG

CAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA

GTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA

GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG

GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT

CTGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA

GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAGCACGTGCTATTATTGAAGCAC

ACATTTCCCCGAAAAGTGCCACCTGTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAA

ATACCGCATCAGGAAATTGTAAGCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAG

GCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGC

CGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACC

CAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAG

GCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACA

GTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTC

CATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGA

TCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGT

GAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGT

GACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCC

TCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCT GCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCC

GAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGA

AACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCG

AGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAA

TTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTAGAAGGCATGCCTGCTACTAGTTATTAAT

AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTAC

GGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTAT

GTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAA

CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGA

CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG

TACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTC

TCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC

AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGG

GGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT

TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGC

TGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGA

CTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC

GCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGA

GGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC

GCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGC

TCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGA

GGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCG

GTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTG

CGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGG

GGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCC

CGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCG

AGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCG

CACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG

GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGG

GGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGG

CTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTG

CTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCTCATCGATGCATGGTACC pIDV- (WPRE position 7-595) (SEQ ID NO:9:)

AGATCTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG

CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTAT

GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCC

GTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA

TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA

ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC

GTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTC

TGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGG

CCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCC

CTTTGGGCCGCCTCCCCGCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTG

AGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTT

GTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTG

GTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAG

GTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTG

AGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCC

TTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTC

TTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAgctTGTTGCTGGCGTTTTTCCATAGGCTCC

GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT

ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG

CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT

GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGT

TCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC

TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA

CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGC

TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC

GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG

AAGATCCTTTGATCTGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATG

AGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAGCACGTGCT

ATTATTGAAGCACACATTTCCCCGAAAAGTGCCACCTGTATGCGGTGTGAAATACCGCACAGAT

GCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATAATTCAGAAGAACTCGTCAAG

AAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGG TCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGC

GGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGAT

ATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTG

AGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGA

CAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGG

GCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCG

GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCC

TTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGA

TAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGA

ACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTG

CCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTG

TTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAG

ATCCTTGGCGGCGAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCG

CCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTAGAAGGCATGCCTGCT

ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG

TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC

AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG

TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA

TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTT

TCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACG

TTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTT

AATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCG

GGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTC

CGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC

GGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCC

GCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG

GGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTA

AAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTG

CGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCG

GGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCG

GGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG

TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGC CCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGT

GGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGG

GCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTA

TGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG

GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGA

AATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGG

GGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGC

GTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCT

CCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCTCATCGAT GCATGGTACC

Peptide adjuvant (SEQ ID NO: 10:)

KWCEC

Peptide adjuvant (SEQ ID NO: 11)

KYMCW

Peptide adjuvant (SEQ ID NO: 12)

CYWWW

Peptide adjuvant (SEQ ID NO: 13)

EHWCM

Peptide adjuvant (SEQ ID NO: 14)

FCCWW

Peptide adjuvant (SEQ ID NO: 15)

TCCMW

Peptide adjuvant (SEQ ID NO: 16)

TCWWH

Peptide adjuvant (SEQ ID NO: 17)

TCYWW

Peptide adjuvant (SEQ ID NO: 18)

WMICM Peptide adjuvant (SEQ ID NO: 19)

YWHMW pIDV-II-SARS-CoV2- Spike VI (pIDV-II expressing Antigen 1) (SEQ ID NO:20)

TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC

AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGG

GGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG

CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT

GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGA

GTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCG

CCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCT

CCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGC

GTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGG

GGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGG

CGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCG

AGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAA

CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGT

CGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGG

CTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGG

GGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGG

GCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGC

GAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTT

GTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCG

GGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCT

TCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCG

GGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTG

TGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACA

GCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGA

GCTCATCGATGCATGGTACCGCACCATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTG

TCCTCACAGTGCGTCAACCTGACTACCCGAACTCAGCTGCCCCCTGCTTATACCAAT

TCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGTGCTGCAC

TCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCC ACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAAC

GATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTT

GGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAA

CGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTA

CTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCG

CCAACAATTGCACATTTGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAGGGCA

AGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACT

TCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCT

TCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTC

AGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGA

TGGACCGCAGGAGCAGCAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTG

CTGAAGTACAACGAGAATGGCACCATCACAGACGCCGTGGATTGCGCCCTGGATCC

TCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATC

AGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCA

CAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCAGGTTCGCAAGCGTGTACG

CATGGAATAGGAAGCGCATCTCTAACTGCGTGGCCGACTATAGCGTGCTGTACAACT

CCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACC

TGTGCTTTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCC

AGATCGCACCTGGACAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCAGAC

GATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGC

GGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAG

AGGGACATCTCTACAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGA

GGGCTTTAACTGTTATTTCCCACTGCAGTCCTACGGCTTCCAGCCCACAAACGGCGT

GGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGC

AACAGTGTGCGGACCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACT

TCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCGAGTCCAACAAGAAGTTC

CTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGA

CCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGT

GATCACACCAGGAACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGA

ATTGTACCGAGGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGCGG

GTGTACAGCACCGGCTCCAACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGC AGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTG

CCTCTTACCAGACCCAGACAAACTCTCCAAGGAGAGCACGGAGCGTGGCATCCCAG

TCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCTGTGGCCTACTCTAAC

AATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCC

GTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACC

GAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCC

CTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGT

GAAGCAGATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCA

GATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTT

CAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGG

GCGACATCGCAGCACGGGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTG

CTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGC

AGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCT

TTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGT

ACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAG

GACAGCCTGTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAG

AACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCAT

CTCTAGCGTGCTGAATGATATCCTGAGCAGGCTGGACAAGGTGGAGGCAGAGGTGC

AGATCGACCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTATGTGACACAG

CAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTGGCAGCAACCAAGAT

GTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCA

CCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTAC

GTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAA

GGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGAC

ACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCG

GCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTGCAGC

CCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCC

CCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCA

GAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCG

ATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATCTGG

CTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGT ATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGT

TTGATGAGGACGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCTGA

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT

GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT

CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAG

GAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCA

ACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTT

TCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGA

CAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGT

CCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTG

CTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCT

CTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGG

CCGCCTCCCCGCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTG

AGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA

_{ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATC}

AGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGA

ACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATT

CCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTG

TTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTT

CCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTC

GACCTGCAGCCCAAGCTTGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA

GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA

GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC

GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC

TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG

CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG

TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT

TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT

ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT

TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT

GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA TCCTTTGATCTGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC

ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT

AGCACGTGCTATTATTGAAGCACACATTTCCCCGAAAAGTGCCACCTGTATGCGGTG

TGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGT

TAATAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCG

GGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTC

TTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAG

CCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCA

AGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTG

AGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCC

TGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCT

TGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATC

AGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCC

CCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCA

CAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTT

GCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCC

TGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCA

GTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCAT

CTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCT

GCGCCATCAGATCCTTGGCGGCGAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCC

AACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAAC

CGCCCAGTAGAAGGCATGCCTGCTACTAGTTATTAATAGTAATCAATTACGGGGTCA

TTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCG

CCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCC

ATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAA

ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC

GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC

TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGG pIDV-II-SARS-CoV2- Spike_V5 (pIDV-II expressing Antigen 3) (SEQ ID NO:21)

TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC

AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGG GGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG

CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT

GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGA

GTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCG

CCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCT

CCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGC

GTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGG

GGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGG

CGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCG

AGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAA

CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGT

CGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGG

CTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGG

GGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGG

GCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGC

GAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTT

GTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCG

GGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCT

TCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCG

GGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTG

TGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACA

GCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGA

GCTCATCGATGCATGGTACCGCACCATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTG

TCCTCACAGTGCGTCAACCTGACTACCCGAACTCAGCTGCCCCCTGCTTATACCAAT

TCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTTTAGAAGCTCCGTGCTGCAC

TCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGGTTCCACGCCATCC

ACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTTTTAAC

GATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTT

GGCACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAA

CGTGGTCATCAAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTA

CTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCG CCAACAATTGCACATTTGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAGGGCA

AGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACT

TCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGCGCGACCTGCCTCAGGGCT

TCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACATCACCCGGTTTC

AGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCCGGCGACTCCTCTAGCGGA

TGGACCGCAGGAGCAGCAGCCTACTATGTGGGCTATCTGCAGCCTAGGACCTTCCTG

CTGAAGTACAACGAGAATGGCACCATCACAGACGCCGTGGATTGCGCCCTGGATCC

TCTGAGCGAGACAAAGTGTACACTGAAGTCCTTTACCGTGGAGAAGGGCATCTATC

AGACATCCAATTTCAGGGTGCAGCCAACCGAGTCTATCGTGCGCTTTCCTAATATCA

CAAACCTGTGCCCATTTGGCGAGGTGTTCAACGCAACCAGGTTCGCAAGCGTGTACG

CATGGAATAGGAAGCGCATCTCTAACTGCGTGGCCGACTATAGCGTGCTGTACAACT

CCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACC

TGTGCTTTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCC

AGATCGCACCTGGACAGACAGGCAAGATCGCCGACTACAATTATAAGCTGCCAGAC

GATTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGC

GGCAACTACAATTATCTGTACCGGCTGTTTAGAAAGAGCAATCTGAAGCCCTTCGAG

AGGGACATCTCTACAGAGATCTACCAGGCCGGCAGCACCCCTTGCAATGGCGTGGA

GGGCTTTAACTGTTATTTCCCACTGCAGTCCTACGGCTTCCAGCCCACAAACGGCGT

GGGCTATCAGCCTTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCAGC

AACAGTGTGCGGACCCAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACT

TCAACTTCAACGGCCTGACCGGAACAGGCGTGCTGACCGAGTCCAACAAGAAGTTC

CTGCCATTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGA

CCCACAGACCCTGGAGATCCTGGATATCACACCCTGCTCTTTCGGCGGCGTGAGCGT

GATCACACCAGGAACCAATACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGA

ATTGTACCGAGGTGCCTGTGGCCATCCACGCCGATCAGCTGACCCCAACATGGCGG

GTGTACAGCACCGGCTCCAACGTGTTCCAGACAAGAGCAGGATGCCTGATCGGAGC

AGAGCACGTGAACAATTCCTATGAGTGCGACATCCCAATCGGCGCCGGCATCTGTG

CCTCTTACCAGACCCAGACAAACTCTCCAAGGAGAGCACGGAGCGTGGCATCCCAG

TCTATCATCGCCTATACCATGTCCCTGGGCGCCGAGAATTCTGTGGCCTACTCTAAC

AATAGCATCGCCATCCCAACCAACTTCACAATCTCTGTGACCACAGAGATCCTGCCC

GTGTCCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACC GAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCC

CTGACAGGCATCGCCGTGGAGCAGGATAAGAACACACAGGAGGTGTTCGCCCAGGT

GAAGCAGATCTACAAGACCCCCCCTATCAAGGACTTTGGCGGCTTCAATTTTTCCCA

GATCCTGCCTGATCCATCCAAGCCTTCTAAGCGGAGCTTTATCGAGGACCTGCTGTT

CAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCAGTATGGCGATTGCCTGG

GCGACATCGCAGCACGGGACCTGATCTGTGCCCAGAAGTTTAATGGCCTGACCGTG

CTGCCACCCCTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGGC

AGGAACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCT

TTGCCATGCAGATGGCCTATAGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGT

ACGAGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAG

GACAGCCTGTCCTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAG

AACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCAT

CTCTAGCGTGCTGAATGATATCCTGAGCAGGCTGGACAAGGTGGAGGCAGAGGTGC

AGATCGACCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGACCTATGTGACACAG

CAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTGGCAGCAACCAAGAT

GTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATCA

CCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTAC

GTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAA

GGCCCACTTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGAC

ACAGCGCAATTTCTACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCG

GCAACTGTGACGTGGTCATCGGCATCGTGAACAATACCGTGTATGATCCACTGCAGC

CCGAGCTGGACAGCTTTAAGGAGGAGCTGGATAAGTACTTCAAGAATCACACCTCC

CCTGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCA

GAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCG

ATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATCTGG

CTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGT

ATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGT

TTGATGAGGACGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCAAG

TGGTGCGAATGCTAGAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGT

ATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGT

ATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTG CTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT

GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTT

TCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCC

TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT

CGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC

GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCG

CGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGT

CGGATCTCCCTTTGGGCCGCCTCCCCGCTTTTTCCCTCTGCCAAAAATTATGGGGACA

TCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGC

AATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAA

TCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATAT

GCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGC

CCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTT

TATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTT

ACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTC

TTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGTTGCTGGCGTTTTTCCATAGGCTC

CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCC

GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC

TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA

AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA

ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA

CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG

TGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG

AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC

CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG

ATCTCAAGAAGATCCTTTGATCTGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA

GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA

AAATGAAGTTTTAGCACGTGCTATTATTGAAGCACACATTTCCCCGAAAAGTGCCAC

CTGTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGA

AATTGTAAGCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATG CGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTC

GCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTC

CGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCA

TGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGC

ATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCG

TCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATG

CGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGC

CGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAG

GAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGAC

AACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCG

CTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAA

CCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTC

TGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCG

TGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGC

TTGATCCCCTGCGCCATCAGATCCTTGGCGGCGAGAAAGCCATCCAGTTTACTTTGC

AGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTG

TCCATAAAACCGCCCAGTAGAAGGCATGCCTGCTACTAGTTATTAATAGTAATCAAT

TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGT

AAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA

CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT

ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC

CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA

CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GG

Claims

CLAIMS:

1. A DNA vaccine vector comprising a vector portion and an antigen-coding portion, wherein the vector portion comprises a sequence from about at least 75% to about 100% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 and wherein the antigen-coding portion comprises a nucleic acid sequence encoding a coronavirus antigen or a fragment thereof.

2. The DNA vaccine vector of claim 1, wherein the coronavirus antigen is a spike protein or a fragment thereof.

3. A DNA vaccine vector comprising a vector portion and an antigen-coding portion, wherein the vector portion comprises a sequence at least 95% identical to SEQ ID NO:8 and wherein the antigen-coding portion comprises a nucleic acid sequence encoding a SARS-CoV-2 spike protein or a fragment thereof.

4. The DNA vaccine vector of claim 3, wherein the vector portion comprises a post- transcriptional regulatory element.

5. The DNA vaccine vector of claim 4, wherein the post-transcriptional regulatory element is woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

6. The DNA vaccine vector of any one of claims 3 to 5, wherein the vector portion comprises a sequence as set forth in SEQ ID NO:9.

7. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein comprises an amino acid sequence from about at least 95% to about 100% identical to SEQ ID NO:2 or to a fragment thereof.

8. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence at least 95% identical to SEQ ID NO:2.

9. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence at least 96% identical to SEQ ID NO:2.

10. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence at least 97% identical to SEQ ID NO:2.

11. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence at least 98% identical to SEQ ID NO:2.

89 The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence at least 99% identical to SEQ ID NO:2. The DNA vaccine vector of any one of claims 2 to 6, wherein the spike protein or fragment thereof comprises a sequence identical to SEQ ID NO:2. The DNA vaccine vector of any one of claims 2 to 13, wherein the spike protein comprises from 1 to 10 amino acid substitutions in comparison with SEQ ID NO:2. The DNA vaccine vector of claim 14, wherein the spike protein comprises amino acid substitution at position A701, P681, D614, A570, N501, E484, S477, Y453, L452, K417 or combinations thereof. The DNA vaccine vector of claim 14, wherein the spike protein comprises amino acid substitution P681R, D614G, N501Y, E484K, Y453F, L452R or combination thereof. The DNA vaccine vector of any one of claims 3 to 14, wherein the spike protein or fragment thereof is selected from the SARS-CoV-2 Whuan isolate, the Alpha variant or related lineages, the Beta variant or related lineages, the Gamma variant or related lineages, the Delta variant or related lineages, the Epsilon variant or related lineages, the Eta variant or related lineages, the Iota variant or related lineages, the Kappa variant or related lineages, the 1.617.3 variant or related lineages, the Mu variant or related lineages, the Zeta variant or related lineages. The DNA vaccine vector of any one of claims 2 to 17, wherein the spike protein comprises a deletion of the transmembrane domain or a portion thereof. The DNA vaccine vector of any one of claims 2 to 18, wherein the antigen-coding portion comprises a nucleic acid sequence encoding a peptide adjuvant. The DNA vaccine vector of claim 19, wherein the peptide adjuvant comprises or consists of the amino acid set forth in any one of SEQ ID NO: 10 to SEQ ID NO: 19. The DNA vaccine vector of any one of claims 2 to 20, wherein the spike protein or fragment thereof is encoded by a nucleic acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or identical to the

90 nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or with a fragment thereof. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 86% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 87% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 88% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 89% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 90 % identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 96% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 97% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.

91 The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 98% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence at least 99% identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. The DNA vaccine vector of any one of claims 2 to 21, wherein the spike protein is encoded by a nucleic acid sequence identical to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. A DNA vaccine vector having a sequence at least 95% identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence at least 96% identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence at least 97% identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence at least 98% identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence at least 99% identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence identical to SEQ ID NO:20. The DNA vaccine vector of claim 34, wherein the DNA vaccine vector has a sequence identical to SEQ ID NO:21. The DNA vaccine vector of any one of claims 1 to 40, wherein the DNA vaccine vector is double stranded. The DNA vaccine vector of any one of claims 1 to 40, wherein the DNA vaccine vector is circular. The DNA vaccine vector of any one of claims 1 to 40, wherein the DNA vaccine vector is linear.

92 A pharmaceutical composition comprising the DNA vaccine vector of any one of claims 1 to 43 and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is adapted for intradermal administration. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is adapted for intramuscular administration. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is adapted for subcutaneous administration. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is adapted for mucosal administration. The pharmaceutical composition of any one of claims 44 to 48, wherein the pharmaceutical composition is adapted for electroporation. The pharmaceutical composition of any one of claims 44 to 49, wherein the DNA vaccine vector is at a concentration of about 1 mg/mL to about 25 mg/mL. The pharmaceutical composition of any one of claims 44 to 49, wherein the DNA vaccine vector is at a concentration of about 5 mg/mL to about 10 mg/mL. An artificial nucleic acid molecule comprising a codon-optimized sequence encoding a severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) antigen wherein the nucleic acid molecule comprises a sequence from about at least 75% to about 100% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or a fragment thereof. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 85% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 86% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 87% identical to the sequence set forth in SEQ ID NO: 1.

93 The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 88% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 89% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 90% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is at least 95% identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 1. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 3. The artificial nucleic acid molecule of claim 52, wherein the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 5. A DNA vaccine vector comprising the artificial nucleic acid molecule of any one of claims 52 to 62. A pharmaceutical composition comprising the DNA vaccine vector of claim 63 and a pharmaceutical acceptable carrier or excipient. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition is formulated for vaccination by injection. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition is formulated for vaccination by electroporation. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition is formulated for vaccination by inhalation. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition is formulated as a transdermal patch.

94 Use of the DNA vaccine vector, the artificial nucleic acid molecule or pharmaceutical composition of any of the preceding claims for making an immunogenic composition or medicament. Use of the DNA vaccine vector, the artificial nucleic acid molecule or pharmaceutical composition of any of the preceding claims for treating a host. A method of immunizing a host comprising administering the DNA vaccine vector, the artificial nucleic acid molecule or the pharmaceutical composition of any of the preceding claims to the host. A method of treating a host, the method comprising administering the DNA vaccine vector, the artificial nucleic acid molecule or the pharmaceutical composition of any of the preceding claims to the host. The method or use of any one of claims 70 to 72, wherein the host is a human. The method or use of any one of claims 70 to 72, wherein the host is an animal. The method or use of any one of claims 70 to 74, wherein the pharmaceutical composition is administered by injection. The method or use of any one of claims 70 to 74, wherein the pharmaceutical composition is administered by electroporation. The method or use of any one of claims 70 to 74, wherein the pharmaceutical composition is administered intradermally, transdermally or intramuscularly. The method or use of any one of claims 70 to 74, wherein the pharmaceutical composition is administered at a mucosal site. The method or use of any one of claims 70 to 78, wherein the pharmaceutical composition is administered as a prime. The method or use of any one of claims 70 to 78, wherein the pharmaceutical composition is administered as a boost. The method or use of any one of claims 70 to 80, wherein the pharmaceutical composition is administered in combination with another SARS-CoV-2 vaccine.

95 The method or use of claim 81, wherein the other SARS-CoV-2 vaccine is a mRNA-based vaccine, a DNA vaccine, pseudo-particles, recombinant proteins, inactivated virus or non- replicative pseudotyped viral particles. The method or use of claim 81 or 82, wherein the pharmaceutical composition is administered as a prime and the other SARS-CoV-2 vaccine is administered as a boost. The method or use of claim 81 or 82, wherein the other SARS-CoV-2 vaccine is administered as a prime and the pharmaceutical composition is administered as boost. The method of use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 100 nanograms to 2 milligrams. The method of use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 100 nanograms to 1 milligram. The method or use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 100 nanograms to 500 micrograms. The method or use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 1 microgram to 500 micrograms. The method or use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 10 micrograms to 500 micrograms. The method or use of any one of claims 70 to 84, wherein the DNA vaccine vector is administered at a dose of 100 micrograms to 500 micrograms of the DNA vaccine vector. The method or use of any one of claims 70 to 90, for reducing viral load in a host. The method or use of any one of claims 70 to 90, to reduce symptoms related with SARS- CoV-2 infection. The method or use of any one of claims 70 to 90, to prevent or reduce the risk of infection from SARS-CoV-2. The method or use of any one of claims 70 to 90, to treat an infection caused by SARS- CoV-2.

96 The method or use of any one of claims 70 to 90, to protect against viral dissemination of SARS-CoV-2. The method or use of any one of claims 70 to 90, to reduce organ pathology associated with SARS-CoV-2. The method or use of any one of claims 70 to 90, to lower the risk of transmission of SARS- CoV-2. The method or use of any one of claims 70 to 90, to lower the risk of complications related to SARS-CoV-2 infection. The method or use of any one of claims 70 to 90, to lower the risk of hospitalization associated with SARS-CoV-2. . The method or use of any one of claims 70 to 90, to lower the risk of death associated with SARS-CoV-2. . The method or use of any one of claims 70 to 100, wherein the DNA vaccine vector comprises a sequence at least 95% identical to SEQ ID NO:20. . The method or use of claim 101, wherein the DNA vaccine vector comprises a sequence as set forth in SEQ ID NO:20. . The method or use of claim 101, wherein the DNA vaccine vector comprises a sequence as set forth in SEQ ID NO:21. . The method or use of any one of claims 70 to 103, wherein the DNA vaccine vector is administered by intradermal delivery. . The method or use of any one of claims 70 to 104, wherein the DNA vaccine vector induces neutralizing antibodies against one or more SARS-CoV-2 isolates. . The method or use of any one of claims 70 to 104, wherein the DNA vaccine vector induces neutralizing antibodies against the Alpha and Beta variants. . The method or use of any one of claims 70 to 106, wherein the DNA vaccine vector is effective against high dose of SARS-CoV-2.

. The method or use of any one of claims 70 to 107, wherein the DNA vaccine vector is administered as a single dose. . The method or use of any one of claims 70 to 107, wherein the DNA vaccine vector is administered in two doses. . The method or use of any one of claims 70 to 107, wherein the DNA vaccine vector is administered in more than two doses. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 28 days interval. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 6 weeks interval. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 8 weeks interval. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 8 to 10 weeks interval. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 6 months interval. . The method or use of claim 109 or 110, wherein the doses are administered at approximately 1 year interval. . The method or use of claim 109 or 110, wherein the two doses are administered at approximately 14 days to 12 weeks interval and a subsequent dose is administered from approximately 6 months to 1 year interval. . The method or use of claim 117, wherein the subsequent dose is optional.