CA3100236A1 - Severe acute respiratory syndrome coronavirus dna vaccines - Google Patents

Severe acute respiratory syndrome coronavirus dna vaccines Download PDF

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CA3100236A1
CA3100236A1 CA3100236A CA3100236A CA3100236A1 CA 3100236 A1 CA3100236 A1 CA 3100236A1 CA 3100236 A CA3100236 A CA 3100236A CA 3100236 A CA3100236 A CA 3100236A CA 3100236 A1 CA3100236 A1 CA 3100236A1
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seq
dna vaccine
nucleic acid
vaccine vector
sars
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Gary Kobinger
George BABUADZE
Marc-Antoine DE LA VEGA
Robert Kozak
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Universite Laval
Sunnybrook Health Sciences Centre
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Babuadze George
de la Vega Marc Antoine
Kozak Robert
Universite Laval
Sunnybrook Health Sciences Centre
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Application filed by Babuadze George, de la Vega Marc Antoine, Kozak Robert, Universite Laval, Sunnybrook Health Sciences Centre filed Critical Babuadze George
Priority to CA3100236A priority Critical patent/CA3100236A1/en
Priority to PCT/CA2021/051633 priority patent/WO2022104465A1/en
Publication of CA3100236A1 publication Critical patent/CA3100236A1/en
Pending legal-status Critical Current

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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. DNA vaccines that are able to trigger an immune response towards SARS-CoV-2.

Description

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 towards 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).
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/International 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 Date Recue/Date Received 2020-11-20 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 W02019/218091 and in international application No. PCT/CA2019/051592 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 a spike protein. In other exemplary embodiments, the coronavirus antigen may comprise an antigen of a severe acute respiratory syndrome coronavirus (SARS-CoV).
In a first aspect, the present disclosure relates to DNA vaccine vectors composed at least 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 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.
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.

Date Recue/Date Received 2020-11-20 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 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 amino acid substitution D614G.
In some embodiments, the spike protein may comprise amino acid substitution Y453F.
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 may be deleted at its N-terminus.
3 Date Recue/Date Received 2020-11-20 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 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 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
4 Date Recue/Date Received 2020-11-20 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 some embodiments, the nucleic acid sequence encoding the 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 Date Recue/Date Received 2020-11-20 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 identical to the sequence set forth in SEQ ID NO: 1.
In some embodiments, the codon-optimized sequence may be identical to the sequence set forth in SEQ ID NO: 3.
In some embodiments, the codon-optimized sequence may be identical to the sequence set forth in SEQ ID NO.:5.
In some embodiments, the DNA vaccine vector may be in a circular form or in a linear form.
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 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.
6 Date Recue/Date Received 2020-11-20 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 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 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 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.
In accordance with the present disclosure the vector may be used for research applications, for pre-clinical, for clinical, for diagnostic or therapeutic applications.
7 Date Recue/Date Received 2020-11-20 BRIEF DESCRIPTION OF THE DRAWINGS
Figure la-f: sequence alignment of the pIDV-I and pIDV-II vectors.
Figure 2: histogram representing eGFP expression by fluorescent activated cell sorter (FACS). Vero E6 cells were transfected in triplicate with either pIDV-eGFP, pVAX1-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 4a: IFNy ELISpot responses from Balb/c mice immunized with pIDV-II-SARS-CoV2-Spike V1 (G1), pIDV-II-SARS-CoV2-Spike V3 (G2) and pIDV-II-SARS-CoV2-Spike V5 (G3) respectively.
Figure 4b. Represents antibody responses in vaccinated Balb/c. All Balb/c mice were immunized with pIDV-II-SARS-CoV2 V1, pIDV-II-SARS-CoV2 V3 and pIDV-II-SARS-CoV2 V5, designated on the figure as Group 1, Group 2 and Group 3 respectively.
Figure 5a: IFNy ELISpot responses from Black/6 mice immunized with pIDV-II-SARS-CoV2-Spike V1 (G1), and pIDV-II-SARS-CoV2-Spike V5 (G2) respectively.
Figure 5b. Represents antibody responses in vaccinated Black/6 mice respectively. All Black/6 mice were immunized with only two selected vaccine versions V1 and V5 corresponding to Group 1 and Group 2 respectively.
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.
Vector portion
8 Date Recue/Date Received 2020-11-20 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 NO:9.
In some embodiments, the vector portion may comprise a sequence at least 80%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 85%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 90%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 95%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 96%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 97%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 98%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at least 99%
identical to SEQ ID NO:9.
In some embodiments, the vector portion may comprise a sequence at identical to SEQ ID
NO:9.
In some embodiments, the vector may consist essentially of the sequence set forth in SEQ
ID NO: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.
9 Date Recue/Date Received 2020-11-20 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 coronavirus antigen comprises the spike protein of a portion thereof.
In some embodiments, the DNA vaccine vectors disclosed herein include an antigen-coding 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 portion thereof.
In some embodiments, the spike protein or portion thereof is from SARS-CoV-2.
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.
In some embodiments, the spike protein comprises an amino acid sequence at least 95%
identical to SEQ ID NO:2.
In some embodiments, the spike protein comprises an amino acid sequence at least 96%
identical to SEQ ID NO:2.
In some embodiments, the spike protein comprises an amino acid sequence at least 97%
identical to SEQ ID NO:2.
In some embodiments, the spike protein comprises an amino acid sequence at least 98%
identical to SEQ ID NO:2.
Date Recue/Date Received 2020-11-20 In some embodiments, the spike protein comprises an amino acid sequence at least 99%
identical to SEQ ID NO:2.
In some embodiments, the spike protein comprises an amino acid sequence identical to 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 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 some embodiments, the spike protein may comprise amino acid substitution D614G.
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 deletion may encompass, for example, the transmembrane domain of the spike protein or a portion thereof.

Date Recue/Date Received 2020-11-20 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 may be a fragment of the spike protein.
In some embodiments, the fragment of the spike protein may comprise at least
10 amino acids. In other embodiments, the fragment may comprise at least 20 amino acids. In yet other embodiments, the fragment may comprise at least 30 amino acids. In further embodiments, the fragment may comprise at least 40 amino acids. In further embodiments, the fragment may comprise at least 50 amino acids. In additional embodiments, the fragment may comprise at least 60 amino acids. In further embodiments, the fragment may comprise at least 70 amino acids. In yet further embodiments, the fragment may comprise at least 80 amino acids. In other embodiments, the fragment may comprise at least 90 amino acids. In further embodiments, the fragment may comprise at least 100 amino acids. In yet further embodiments, the fragment may comprise at least 150 amino acids. In additional embodiments, the fragment may comprise at least 200 amino acids. In yet additional embodiments, the fragment may comprise 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 Date Recue/Date Received 2020-11-20 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 embodiments, the fragment may comprise at least 20 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 at., (Linear Epitope Landscape of SARS-Cov-2 Spike Protein Constructed from 1,051 COV1D-19 Patients, 2020).

Date Recue/Date Received 2020-11-20 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 receptor-binding 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 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 Date Recue/Date Received 2020-11-20 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 pVAX1 (see W02019/218091), pVACTM or pBOOSTTm (Invivogen) and the like.
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, Date Recue/Date Received 2020-11-20 Preclinical and Clinical Aspects of Gene Transfer Medicinal Products. London, UK: 2001.
CPMP/BWP/3088/99; Presence of the Antibiotic Resistance Marker Gene nptII 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 E. coil K12 strains such as DH5a, DH5, DH1, XL1Blue, 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., HyperGROTM) 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 vectors of the present disclosure may be administered as a pharmaceutical composition, which may comprise for example, the DNA vector(s) and a pharmaceutically acceptable carrier.
The pharmaceutical composition may comprise a single DNA vector species encoding one or more antigens.
Alternatively, the pharmaceutical composition may comprise a mixture of DNA
vector species (multiple DNA vector species) each encoding different antigens.

Date Recue/Date Received 2020-11-20 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 inj ecti on.
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.
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 peptide adjuvant comprises or consists of the amino acid set forth in any one of SEQ ID NO:10 to SEQ ID NO:19.

Date Recue/Date Received 2020-11-20 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(504)2, AlNa(504)2, A1NH(504)2, silica, alum, Al(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 Q521, Q517, and Q57 (U.S. Pat. Nos.
5,057,540; 5,650,398;
6,524,584; 6,645,495), monophosphoryl lipid A, such as 3-de-0-acylated monophosphoryl lipid A (3D-MPL), imiquimod, lipid-polymer matrix (ENABLTM adjuvant), Emulsigen-DTM
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.

Date Recue/Date Received 2020-11-20 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.
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. 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.

Date Recue/Date Received 2020-11-20 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 adult is between about 18-55 years of age.
In some embodiments, the human adult is between about 56-70 years of age.
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.
Date Recue/Date Received 2020-11-20 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 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.

Date Recue/Date Received 2020-11-20 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 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 1 milligram.
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.

Date Recue/Date Received 2020-11-20 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.

Date Recue/Date Received 2020-11-20 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 1Last 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-HC1., 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), anti-oxidants (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.

Date Recue/Date Received 2020-11-20 EXAMPLE 1- Generation of pIDV-II-SARS-CoV-2-Spike vaccines We generated three codon-optimized DNA sequences expressing the SARS-CoV-2 Spike protein or modified versions (Antigen 1 (identified herein as V1 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)).
V1 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 at, PLoS ONE, 2012, U520110305720A1).
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 that spontaneous mutations have been introduced in the transgene, the sequence of the vector and insert was confirmed by the sequencing.
The codon-optimized DNA sequences were cloned into the plDV-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 (the entire contents of which are incorporated herein by reference).
The three DNA vaccine vectors thus generated were named plDV-II-SARS-CoV2-Spike V1, 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, LipofectamineTM 2000 transfection of 5 [tg pIDV-II-SARS-CoV2-Spike V1, pIDV-II-SARS-CoV2-Spike-V3 or pIDV-II-SARS-CoV2-Spike-VS 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 XtractorTM
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 Date Recue/Date Received 2020-11-20 was quantified and 15 ug of each sample was mixed with sample buffer [10 M
Tris/HC1 (pH 6,8), 2% SDS, 10% glycerol, 5% 13-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 pIDV-II-SARS-CoV2-Spike DNA vaccines (pIDV-II-SARS-CoV2-Spike V1 (G1), pIDV-II-SARS-CoV2-Spike V3 (G2) and plDV-II-SARS-CoV2-Spike V5 (G3)) diluted in Endotoxin-free TE buffer or with equivalent volume of Endotoxin-free TE buffer respectively (control group).
A total volume of 100 pi was administered to each animal across two sites;
each with 50 Ill 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 105 splenocytes per well and re-stimulated 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 [tg/m1 (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 1x106 cells by the CellProfilerTM software (Figures 4a).

Date Recue/Date Received 2020-11-20 In Figure 4a, the plDV-II-SARS-CoV2-Spike DNA vaccines used are pIDV-II-SARS-CoV2-Spike V1 for group 1 (G1 -Pp 1), pIDV-II-SARS-CoV2-Spike V3 for group 2 (G2-Pp 1) and plDV-II-SARS-CoV2-Spike V5 for group 3 (G3-Pp 1) respectively. The black light bar denotes the number of spots against the peptide pool in mice vaccinated with pIDV-II-SARS-CoV2 V1 while gray non-patterned bar reflects number of spots in mice vaccinated with plDV-II-SARS-CoV2 V3 and the gray patterned bar shows the number of spots in mice samples immunized with pIDV-II-SARS-CoV2 V5. In this assay, animals vaccinated with plDV-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 V1 and pIDV-II-SARS-CoV2 V5.
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 BET Resources, NIAID, NIH: SARS-CoV Spike (S) Protein deltaTM, Recombinant from Baculovirus, NR-722) diluted in 1XPB S per 96-well plate.
The following day, plates were washed and then blocked with KPL Milk-Blocking Solution 1/10 diluted in H20 for 90 minutes at 37 C. All washes were done with 1X 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 .1 of PBS-Tween 0.1%, six times. Then 50 .1 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 I/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.

Date Recue/Date Received 2020-11-20 All vaccinated mice developed robust IgG1 response at day 56 (post prime boost)(Figure 4a). After a prime on day 0 with IM+EP route, fast appearance of SARS-COV2 -specific antibodies was detected by ELISA in Black/6 mice Group 2 vaccinated with pIDV-II-SARS-CoV2-V5. Figures 4b shows SARS-COV2 -specific IgG in individual vaccinated mice. All mice received an equal amount of vaccine of 100 1 in total (50 1/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-V1 or pIDV-II-SARS-CoV2-Spike-V5 vaccines performing the best.
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 electoporation (Inovio Pharmaceuticals ) per animal into the caudal thigh with 50 pg of optimized pIDV-II-SARS-CoV2-Spike-V1 or pIDV-II-SARS-CoV2-Spike-vaccines diluted in Endotoxin-free TE buffer. An equivalent volume of Endotoxin-free TE buffer respectively was injected into the control group (Figure 5a).
In Figure 5a the plDV-II-SARS-CoV2-Spike DNA vaccines used are plDV-II-SARS-CoV2-Spike V1 for group 1 (G1 -Pp 1), and pIDV-II-SARS-CoV2-Spike V5 for group 2 (G2-Pp1) respectively. In this assay, animals vaccinated with pIDV-II-SARS-CoV2 V1 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 100 1 in total (50 1/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 IgG1 response at day 56 (post prime boost) (Figure 5b). After a prime on day 0 with IM+EP rout fast appearance of SARS-COV2 -specific antibodies Date Recue/Date Received 2020-11-20 was detected by ELISA in Black/6 mice Group 2 vaccinated with plDV-II-SARS-CoV2-V5 (Figure 5b).
Our study shows that all three vaccines pIDV-II-SARS-CoV2 V1, plDV-II-SARS-CoV2 V3 and the plDV-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 V1 and plDV-II-SARS-CoV2 V5 performing the best.
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 1 milligram.
For example, animals may receive 10 micrograms, 30 micrograms, 100 micrograms, micrograms, 500 micrograms, 1 milligram, 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 SARS-CoV-2 (Sunnybrook strain) 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 Date Recue/Date Received 2020-11-20 week 4, all animals are challenged with 100 1 of virus TOD501x105/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 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.
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, Dong JC, Trost B, Richardson JS, Tohme S, et al. (2012) Pentamers Not Found in the Universal Proteome Can Enhance Antigen Specific Immune Responses and Adjuvant Vaccines.
PLoS ONE 7(8): e43802.
Long SW et at., Molecular Architecture of Early Dissemination and Massive Second Wave of th SARS-CoV-2 Virus in a Major Metropolitan Area, mBio Vol. 11(6): e02707-20, November Date Recue/Date Received 2020-11-20 SEQUENCE TABLE
SEQ ID NO: 1 Antigen 1 (V1)- codon-optimized nucleic acid sequence 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

Date Recue/Date Received 2020-11-20 AGAACCAGAAGCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTC
CTCTACAGCCTCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAAT
ACCCTGGTGAAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGATATCCTGA
GCAGGCTGGACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTC
TCTGCAGACCTATGTGACACAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTG
GCAGCAACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGG
GCTATCACCTGATGTCCTTCCCTCAGTCTGCCCCACACGGCGTGGTGTTTCTGCACGTGACCTA
CGTGCCCGCCCAGGAGAAGAACTTCACCACAGCCCCTGCCATCTGCCACGATGGCAAGGCCCAC
TTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCT
ACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGG
CATCGTGAACAATACCGTGTATGATCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTG
GATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATG
CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGA
GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATC
TGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGA
CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGA
CGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCTGA
SEQ ID NO: 2 Antigen 1 (VI)- amino acid sequence of naturally occurring SARS-CoV-2 spike protein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW
FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVI
KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF
VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS
GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV
QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP
TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN
YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV
LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV
RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS
NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS
VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT
GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG
FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI
PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN
TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL
AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL
DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI
WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
SEQ ID NO: 3 Antigen 2 (V3)- codon-optimized nucleic acid sequence ATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTGTCCTCACAGTGCGTCAACCTGACTACCCGAA
CTCAGCTGCCCCCTGCTTATACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTT
TAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGG

Date Recue/Date Received 2020-11-20 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

Date Recue/Date Received 2020-11-20 TTTCCAAGGGAGGGCGTGTTCGTGTCCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCT
ACGAGCCCCAGATCATCACCACAGACAATACCTTCGTGAGCGGCAACTGTGACGTGGTCATCGG
CATCGTGAACAATACCGTGTATGATCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTG
GATAAGTACTTCAAGAATCACACCTCCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAATG
CCTCCGTGGTGAACATCCAGAAGGAGATCGACCGCCTGAACGAGGTGGCCAAGAATCTGAACGA
GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCATGGTACATC
TGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGTATGA
CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCAAGTGGTGCGAATGCTA
G
SEQ ID NO: 4 Antigen 2 (V3)- amino acid sequence MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW
FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVI
KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF
VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS
GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV
QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP
TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN
YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV
LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV
RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS
NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS
VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT
GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG
FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI
PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN
TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL
AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL
DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI
WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCKWCEC
SEQ ID NO:5 Antigen 3 (V5)- codon-optimized nucleic acid sequence ATGTTTGTCTTCCTGGTCCTGCTGCCTCTGGTGTCCTCACAGTGCGTCAACCTGACTACCCGAA
CTCAGCTGCCCCCTGCTTATACCAATTCCTTCACCCGGGGCGTGTACTATCCTGACAAGGTGTT
TAGAAGCTCCGTGCTGCACTCTACACAGGATCTGTTTCTGCCATTCTTTAGCAACGTGACCTGG
TTCCACGCCATCCACGTGAGCGGCACCAATGGCACAAAGCGGTTCGACAATCCCGTGCTGCCTT
TTAACGATGGCGTGTACTTCGCCTCTACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGG
CACCACACTGGACTCCAAGACACAGTCTCTGCTGATCGTGAACAATGCCACCAACGTGGTCATC
AAGGTGTGCGAGTTCCAGTTTTGTAATGATCCCTTCCTGGGCGTGTACTATCACAAGAACAATA
AGAGCTGGATGGAGTCCGAGTTTAGAGTGTATTCTAGCGCCAACAATTGCACATTTGAGTACGT
GTCCCAGCCTTTCCTGATGGACCTGGAGGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTC
GTGTTTAAGAATATCGATGGCTACTTCAAGATCTACTCTAAGCACACCCCCATCAACCTGGTGC
GCGACCTGCCTCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGATCTGCCTATCGGCATCAACAT

Date Recue/Date Received 2020-11-20 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
Date Recue/Date Received 2020-11-20 CATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGA
CGACTCCGAGCCAGTGCTGAAAGGCGTGAAGCTGCATTACACCAAGTGGTGCGAATGCTAG
SEQ ID NO:6 Antigen 3 (V5)- amino acid sequence MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTW
FHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVI
KVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREF
VFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS
GWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV
QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP
TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN
YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV
LSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAV
RDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS
NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS
VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALT
GIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAG
FIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI
PFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN
TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANL
AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL
DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI
WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYTKWCEC-SEQ ID NO: 7 pIDV plasmid AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCT
GGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG
GAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGC
AACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATG
AAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTT
TTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACT
AGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGA
TCCCTCGACCTGCAGCCCAAgctTGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG
CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT
TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT
GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC
AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG
TGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG
TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG

Date Recue/Date Received 2020-11-20 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

Date Recue/Date Received 2020-11-20 CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCG
CCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGC
GGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC
CGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGAC
CGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGC
AACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCTCATCGATGCATGGT
ACC
SEQ ID NO:8 pIDV-I plasmid 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

Date Recue/Date Received 2020-11-20 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
SEQ ID NO:9:
pIDV-II (WPRE position 7-595) AGATCTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTG
CTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTAT
GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCC
GTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA
TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA
ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC
GTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTC
TGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGG

Date Recue/Date Received 2020-11-20 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
Date Recue/Date Received 2020-11-20 TCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACG
TTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTT
AATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCG
GGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTC
CGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC
GGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCC
GCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG
GGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTA
AAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTG
CGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCG
GGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCG
GGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG
TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGC
CCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGT
GGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGG
GCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTA
TGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG
GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGA
AATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGG
GGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGC
GTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCT
CCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCTCATCGAT
GCATGGTACC
SEQ ID NO:10 Peptide adjuvant KWCEC
SEQ ID NO:!!
Peptide adjuvant KYMCW
SEQ ID NO:12 Peptide adjuvant CYWWW
SEQ ID NO:13 Peptide adjuvant EHWCM
SEQ ID NO:14 Peptide adjuvant Date Recue/Date Received 2020-11-20 FCCWW
SEQ ID NO:15 Peptide adjuvant TCCMW
SEQ ID NO:16 Peptide adjuvant TCWWH
SEQ ID NO:17 Peptide adjuvant TCYWW
SEQ ID NO:18 Peptide adjuvant WMICM
SEQ ID NO:19 Peptide adjuvant YWHMW

Date Recue/Date Received 2020-11-20

Claims (80)

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 portion thereof.
3. 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 severe acute respiratory syndrome coronavirus (SARS-CoV) antigen or a fragment thereof
4. The DNA vaccine vector of any one of claims 1 to 3, wherein the vector portion comprises a sequence at least 80% identical to SEQ ID NO:9.
5. The DNA vaccine vector of any one of claims 1 to 3, wherein the vector portion comprises a sequence at least 90% identical to SEQ ID NO:9.
6. The DNA vaccine vector of any one of claims 1 to 3, wherein the vector portion comprises a sequence at least 95% identical to SEQ ID NO:9.
7. The DNA vaccine vector of any one of claims 1 to 3, wherein the vector portion comprises a sequence at least 99% identical to SEQ ID NO:9.
8. The DNA vaccine vector of any one of claims 1 to 3, wherein the vector portion comprises a sequence at identical to SEQ ID NO:9.
9. The DNA vaccine vector of any one of claims 3 to 8, wherein the SARS-CoV
antigen is a spike protein or a portion thereof
10. The DNA vaccine vector of any one of claims 2 to 9, wherein the spike protein or portion thereof is a spike protein from SARS-CoV-2 or a portion thereof.

Date Recue/Date Received 2020-11-20
11. The DNA vaccine vector of claim 10, 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
12. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence at least 95% identical to SEQ ID NO:2 or to a fragment thereof.
13. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence at least 96% identical to SEQ ID NO:2 or to a fragment thereof.
14. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence at least 97% identical to SEQ ID NO:2 or to a fragment thereof.
15. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence at least 98% identical to SEQ ID NO:2 or to a fragment thereof.
16. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence at least 99% identical to SEQ ID NO:2 or to a fragment thereof.
17. The DNA vaccine vector of claim 11, wherein the spike protein comprises a sequence identical to SEQ ID NO:2 or to a fragment thereof
18. The DNA vaccine vector of any one of claims 2 to 17, wherein the spike protein comprises from 1 to 10 amino acid substitutions in comparison with SEQ ID NO:2.
19. The DNA vaccine vector of claim 18, wherein the spike protein comprises amino acid sub stituti on D614G.
20. The DNA vaccine vector of any one of claims 2 to 19, wherein the spike protein is deleted at a C-terminus.
21. The DNA vaccine vector of any one of claims 2 to 19, wherein the spike protein comprises a deletion of the transmembrane domain or a portion thereof
22. The DNA vaccine vector of any one of claims 1 to 21, wherein the antigen-coding portion comprises a nucleic acid sequence encoding a peptide adjuvant.
23. The DNA vaccine vector of claim 22, wherein the nucleic acid sequence encoding the SARS-CoV antigen and the nucleic acid sequence encoding the peptide adjuvant are in frame.

Date Recue/Date Received 2020-11-20
24. The DNA vaccine vector of claim 23, wherein the nucleic acid sequence encoding the peptide adjuvant is at the 3' end of the nucleic acid sequence encoding the SARS-CoV
antigen.
25. The DNA vaccine vector of claim 23, wherein the nucleic acid sequence encoding the peptide adjuvant is immediately contiguous to the nucleic acid sequence encoding the SARS-CoV antigen.
26. The DNA vaccine vector of any one of claims 22 to 25, 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.
27. The DNA vaccine vector of claim 26, wherein the peptide adjuvant comprises or consists of SEQ NO:10.
28. The DNA vaccine vector of any one of claims 2 to 27, wherein the spike protein is encoded by a 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
29. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is at least 95%
identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or to a fragment thereof
30. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is at least 96%
identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or to a fragment thereof
31. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is at least 97%
identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or to a fragment thereof
32. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is at least 98%
identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or to a fragment thereof
33. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is at least 99%
identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO.:5 or to a fragment thereof
34. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is identical to SEQ
lD NO: 1 or to a fragment thereof.
35. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is identical to SEQ
lD NO: 3 or to a fragment thereof.
Date Recue/Date Received 2020-11-20
36. The DNA vaccine vector of claim 28, wherein nucleic acid sequence is identical to SEQ
ID NO: 5 or to a fragment thereof.
37. The DNA vaccine vector of any one of claims 1 to 36, wherein the nucleic acid sequence encoding the antigen-coding portion is an artificial nucleic acid sequence.
38. The DNA vaccine vector of any one of claims 1 to 36, wherein the nucleic acid sequence encoding the antigen-coding portion is codon-optimized.
39. 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
40. The artificial nucleic acid molecule of claim 39, wherein 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.
41. The artificial nucleic acid molecule of claim 39, wherein 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.
42. The artificial nucleic acid molecule of claim 39, wherein 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.
43. The artificial nucleic acid molecule of claim 39, wherein 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.
44. The artificial nucleic acid molecule of claim 39, wherein the codon-optimized sequence is identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID
NO.:5.
45. A DNA vaccine vector comprising the artificial nucleic acid molecule of any one of claims 39 to 44.

Date Recue/Date Received 2020-11-20
46. A pharmaceutical composition comprising the DNA vaccine vector of any one of claims 1 to 38 or 45 or artificial nucleic acid molecule of any one of claims 39 to 44 and a pharmaceutical acceptable carrier.
47. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated for vaccination by injection.
48. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated for vaccination by electroporation.
49. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated for vaccination by inhalation.
50. The pharmaceutical composition of claim 46, wherein the pharmaceutical composition is formulated as a transdermal patch.
51. 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.
52. Use of the DNA vaccine vector, the artificial nucleic acid molecule or pharmaceutical composition of any of the preceding claims for making a medicament.
53. Use of the DNA vaccine vector, the artificial nucleic acid molecule or pharmaceutical composition of any of the preceding claims for treating a host.
54. 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.
55. 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.
56. The method or use of any one of claims 53 to 55, wherein the host is a human.
57. The method or use of any one of claims 53 to 55, wherein the host is an animal.
58. The method or use of any one of claims 53 to 57, wherein the pharmaceutical composition is administered by injection.

Date Recue/Date Received 2020-11-20
59. The method or use of any one of claims 53 to 57, wherein the pharmaceutical composition is administered by electroporation.
60. The method or use of any one of claims 53 to 57, wherein the pharmaceutical composition is administered intradermally, transdermally or intramuscularly.
61. The method or use of any one of claims 53 to 57, wherein the pharmaceutical composition is administered at a mucosal site.
62. The method or use of any one of claims 53 to 61, wherein the pharmaceutical composition is administered as a prime.
63. The method or use of any one of claims 53 to 61, wherein the pharmaceutical composition is administered as a boost.
64. The method or use of any one of claims 53 to 63, wherein the pharmaceutical composition is administered in combination with another SARS-CoV-2 vaccine.
65. The method or use of claim 64, 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.
66. The method or use of claim 64 or 65, wherein the pharmaceutical composition is administered as a prime and the other SARS-CoV-2 vaccine is administered as a boost.
67. The method or use of claim 64 or 65, wherein the other SARS-CoV-2 vaccine is administered as a prime and the pharmaceutical composition is administered as boost.
68. The method of use of any one of claims 53 to 67, wherein the DNA vaccine vector is administered at a dose of 100 nanograms to 1 milligram.
69. The method or use of any one of claims 53 to 67, wherein the DNA vaccine vector is administered at a dose of 100 nanograms to 500 micrograms.
70. The method or use of any one of claims 53 to 67, wherein the DNA vaccine vector is administered at a dose of 1 microgram to 500 micrograms.
71. The method or use of any one of claims 53 to 67, wherein the DNA vaccine vector is administered at a dose of 10 micrograms to 500 micrograms.

Date Recue/Date Received 2020-11-20
72. The method or use of any one of claims 53 to 67, wherein the DNA vaccine vector is administered at a dose of 100 micrograms to 500 micrograms of the DNA vaccine vector.
73. The method or use of any one of claims 53 to 72, for treating infection caused by the coronavirus.
74. The method or use of any one of claims 53 to 72, for preventing infection from the coronavirus.
75. The method or use of any one of claims 53 to 72, for reducing viral load in a host.
76. The method or use of any one of claims 53 to 72, to lower the risk of a host from getting infected with a SARS-CoV.
77. The method or use of any one of claims 53 to 72, to lower the risk of a host from getting complications related to SARS-CoV infection.
78. The method or use of any one of claims 53 to 72, to reduce symptoms related with SARS-CoV infection.
79. The method or use of any one of claims 53 to 72, to prevent infection from SARS-CoV.
80. The method or use of any one of claims 53 to 72, to treat an infection caused by SARS-CoV.

Date Recue/Date Received 2020-11-20
CA3100236A 2020-11-20 2020-11-20 Severe acute respiratory syndrome coronavirus dna vaccines Pending CA3100236A1 (en)

Priority Applications (2)

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PCT/CA2021/051633 WO2022104465A1 (en) 2020-11-20 2021-11-18 Severe acute respiratory syndrome coronavirus dna vaccines

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Application Number Priority Date Filing Date Title
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Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5656857B2 (en) * 2008-11-28 2015-01-21 ハー マジェスティ ザ クイーン イン ライト オブ カナダ アズ リプリゼンテド バイ ザ ミニスター オブ ヘルスＨＥＲ ＭＡＪＥＳＴＹ ＴＨＥ ＱＵＥＥＮ ＩＮ ＲＩＧＨＴＯＦ ＣＡＮＡＤＡ ａｓ ｒｅｐｒｅｓｅｎｔｅｄ ｂｙ ＴＨＥ ＭＩＮＩＳＴＥＲ ＯＦ ＨＥＡＬＴＨ Peptide adjuvant

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