WO2023044542A1

WO2023044542A1 - Sars cov-2 vaccine

Info

Publication number: WO2023044542A1
Application number: PCT/AU2022/051148
Authority: WO
Inventors: Branka GRUBOR-BAUK; Makutiro Ghislain MASAVULI; Eric James Gowans
Original assignee: The University Of Adelaide
Priority date: 2021-09-24
Filing date: 2022-09-23
Publication date: 2023-03-30

Abstract

Single and multivalent vaccines against SARS-CoV-2 comprised out of non-structural protein 3 (NSP3), nucleocapsid (N), SI subunit of the Spike protein, the receptor binding domain (RBD) of the Spike protein, or a combination of two or more of them as fusion DNA constructs. Additionally, the vaccines comprise antigens with an oligomerisation domain which can be an IMX313P or Foldon, or additionally encode perforin.

Description

Title of Invention

SARS CoV-2 vaccine

Technical Field

[0001] The present disclosure relates to vaccines for preventing or treating SARS- CoV-2 infection or preventing or treating COVID-19 disease, or SARS-CoV-2 related diseases and complications.

Background of Invention

[0002] The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that has affected hundreds of millions of people worldwide and caused a global pandemic. SARS-CoV-2 infected individuals are characterized by the presence of respiratory symptoms, such as dry cough, shortness of breath and fever.

[0003] In mid-December 2019, a series of pneumonia cases of unknown aetiology emerged in the city of Wuhan, China. Deep sequencing of respiratory aspirates from these patients identified a novel SARS-like corona virus (2019-nCoV). New cases of the virus rapidly increase and began to appear globally. On 11 March 2020, the World Health Organisation declared the novel corona virus, now named SARs-CoV-2, a global pandemic.

[0004] In response, myriad vaccines have been designed and entered clinical trials to try and prevent the infection with SARS-CoV-2 or treat SARS-CoV-2 related condition. As of September 2022, there were more than 960 vaccines in active development or in use (Cochrane vaccine mapping (https://covid- nma.com/vaccines/mapping/).

[0005] As of September 2021 , at least 21 vaccines had been authorized or approved by government agencies globally (www.raps.org - COVID 19 tracker). These vaccines included inactivated virus vaccines, non-replicating viral vectors, protein subunit vaccines, recombinant viral vectors and mRNA nano-lipid vaccines. With the exception of inactivated virus vaccines, the approved vaccines utilise SARS-CoV-2 protein subunits. [0006] However, all of the vaccines to date include limitations. For example: inactivated virus vaccines have shown limited efficacy; mRNA vaccines are complex to manufacture, relatively costly and require cold-chain transport and storage; and recombinant viral vectors induce an immune response to the vector thereby limiting the ability to use the vector for boosting doses of the vaccine.

[0007] The predominate subunit used in the approved SARS-CoV-2 vaccines is the viral Spike protein, or portions of the Spike protein. This protein is targeted because it forms part of the external structure of the virions and is critical for viral entry into cells via the ACE2 receptor. Accordingly, it is both accessible to the immune system and susceptible to being neutralised by antibodies.

[0008] However, the use of a single protein subunit can limit the efficacy of a vaccine by restricting the repertoire of the anti-viral immune response. Further, variants of SARS-CoV-2 containing mutations within the target protein, or protein portion, have evaded the immune response instigated by the vaccine, thereby decreasing the efficacy of the vaccine.

[0009] To this end, the WHO is tracking a range of variants of SARS-CoV-2, including variants of interest and variants of concern. The Omicron variant (B.1.1.529) is currently the most prevalent variant (as of September 2022). Previous variants of concern include the Alpha (B.1.1.7), Beta (B.1.351 ), Gamma (P.1 ) and Delta variant (B.1.617.2). The Omicron variant has shown significant immune evasion. As a result, it is causing significant break-through infection in vaccinated individuals and individuals previously infected with other variants. Analysis of the effective reproduction rate of the Omicron variant showed that it is about 3.8 times that of the Delta variant, which itself was about two times greater than the ancestral Wuhan ancestral variant (see Liu J. and Rockldv (2022). The effective reproductive number of the Omicron variant of SARS- CoV-2 is several times relative to Delta, Travel Medicine May 31 ;29(3):taac037 and Liu J. and Rockldv (2021 ). The reproductive number of the Delta variant of SARS-CoV-2 is far higher compared to the ancestral SARS-CoV-2 virus, Journal of Travel Medicine. Oct 11 ;28(7):Taab124). [0010] Accordingly, there is a need to develop alternative SARS-CoV-2 vaccines which target novel portions of the virus. Further, it is advantageous to develop novel SARS-CoV-2 vaccines which are relatively cheap to produce and stable.

Summary of Invention

[0011 ] The present Inventors have identified various structural and non-structural components of SARS-CoV-2 which are antigenic and induce adaptive immune responses in mammals. Consequently, these components may be useful in vaccination against infection or transmission of SARS-CoV-2 or preventing COVID-19 disease, or related conditions, following infection. Further, the present Inventors have shown that nucleic acid vaccines, such as DNA vector vaccines, can be used to induce an immune response in subjects to the various identified components of the SARS-CoV-2 virus.

[0012] Accordingly, the present invention provides a vaccine comprising an isolated or recombinant immunogenic portion of a SARS-CoV-2 protein selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein. Each of these proteins have been identified as including portions which can induce B-cell and T-cell responses. Accordingly, each of these proteins include at least an immunogenic portion which is MHC-restricted and presented to endogenous T-cells within a subject (as well as inducing a B-cell response).

[0013] Such a T-cell responses may promote an effective immunity to infection as well as promoting strong immune memory.

[0014] For the immunogenic portions of the SARS-CoV-2 proteins to initiate a functional B-cell immune response, it is important for the protein (or portion thereof) to be stabilised in its correct three-dimensional structure. To help facilitate this, particularly when the peptide is not located at its native location - such as a cell membrane - it may be advantageous to oligomerise the immunogenic portion. Therefore, in some embodiments of the vaccine, the immunogenic portion of the protein is linked to an oligomerisation domain. The oligomerisation domain can be any suitable domain, including ferritin, lumazine synthase, [3-annulus peptide, IMX313P or Foldon. Preferably, the oligomerisation domain is IMX313P or Foldon. [0015] Accordingly, in some embodiments the vaccine comprises an immunogenic portion of the S1 subunit of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon. In some embodiments, the vaccine comprises an immunogenic portion of the receptor binding domain of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon. In some embodiments, the vaccine comprises an immunogenic portion of NSP3 linked to IMX313P or Foldon. In some embodiments, the vaccine comprises an immunogenic portion the Nucleocapsid protein linked to IMX313P or Foldon.

[0016] In some embodiments the vaccine comprises immunogenic portions from more than one SARS-CoV-2 protein selected from the group consisting of: Non- Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. These multiple immunogenic portions maybe be collocated (for example is a composition), co-administered or can be provided as a recombinant polyprotein. Therefore, in some embodiments, there is provided a recombinant polyprotein, wherein the polyprotein includes immunogenic portions of two or more SARS-CoV-2 proteins selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0017] In some embodiments, the recombinant polyprotein comprises an immunogenic portion of NSP3 and an immunogenic portion of at least one or more of: Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. In some other embodiments, the recombinant polyprotein comprises an immunogenic portion of Nucleocapsid and an immunogenic portion of at least one or more of: NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0018] It may be advantageous for the recombinant polyprotein to be cleaved in vivo to allow the multiple immunogenic portions to be differentially processed or located. Accordingly, in some embodiments at least two of the multiple immunogenic portions of the SARS-CoV-2 proteins in the polyprotein are separated by a cleavage domain. In some embodiments the vaccine comprises a polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of the proteins are separated by a cleavage domain. Cleavage domains are known in the art; however, in some embodiment the cleavage domain is a self-cleavage domain, preferably a 2A self-cleavage domain such as the foot-and- mouth disease virus (FMDV) 2A peptide.

[0019] Vaccines can be provided in many forms including (but not limited to) subunit protein vaccines, pseudo-virus vaccines, virus-like-particle vaccines, split vaccines or nucleic acid vaccines.

[0020] In a preferred embodiment the vaccine of the present invention comprises a nucleic acid (such as the nucleic acids described below).

[0021 ] Accordingly, the invention provides a nucleic acid encoding an immunogenic portion of a SARS-CoV-2 protein selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein.

[0022] Proteins expressed from nucleic acids will typically remain intracellular unless the protein includes, or is linked to, a signal peptide which directs localisation of the protein. In the context of a vaccine, to elicit an effective immune response, it may be advantageous to secrete a protein from the cell in which it was expressed. Accordingly, the nucleic acid of the present invention includes a signal peptide. Preferably, the signal peptide results in membrane localisation of an expressed protein, or secretion of an expressed protein.

[0023] Accordingly, in some embodiments, there is provided a nucleic acid, comprising an immunogenic portion of the S1 subunit of the Spike protein, and/or an immunogenic portion of the receptor binding domain (RBD) of the Spike protein, linked to a sequence encoding a heterologous signal peptide. In some embodiments, the heterologous signal peptide facilitates secretion of a protein from a eukaryotic cell once expressed. In some preferred embodiments, the heterologous signal peptide is the tissue plasminogen activator (tPA) signal peptide, or a functional portion, or variant, thereof. [0024] As discussed above, it can be advantageous to oligomerise a peptide to allow for an appropriate three-dimensional configuration. Accordingly, in some embodiments, the nucleic acid encodes an immunogenic portion of the SARS-CoV-2 protein linked to an oligomerisation domain, which may be any suitable domain but is preferably IMX313P or Foldon. In some embodiments, the nucleic acid encodes an immunogenic portion of SARS-CoV-2 S1 protein linked to IMX313P or Foldon. In some embodiments, the nucleic acid encodes an immunogenic portion of the receptor binding domain of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon.

[0025] To facilitate the expression of the immunogenic portion of the SARS-CoV-2 peptide from a DNA nucleic acid, the DNA may include a promoter sequence that promotes the expression of the encoded immunogenic portion of the SARS-CoV-2 protein(s). Additionally, in some embodiments, the nucleic acid comprises an expression vector which contains at least one promoter sequence promoting the expression of the encoded immunogenic portion of the SARS-CoV-2 protein(s). In some embodiments, the expression vector is pVax.

[0026] It can also be advantageous to a provide a peptide with an immune stimulant, such as an adjuvant. One possible immune stimulant is perforin, or a functional portion, or variant, thereof. Perforin can induce cell lysis when expressed and therefore promote uptake of cell debris (including expressed proteins) by phagocytic cells which can present them to the immune system. Accordingly, in some embodiments, the nucleic acid further encodes a functional portion, or variant, of a perforin protein.

[0027] In some embodiments, the nucleic acid encodes an immunogenic portion of SARS-CoV-2 Non-Structural Protein 3 (NSP3) and further encodes a functional portion, or variant, of a perforin protein. In some embodiments, the nucleic acid encodes an immunogenic portion of SARS-CoV-2 Nucleocapsid protein and further encodes a functional portion, or variant, of a perforin protein. In some embodiments the perforin protein has at least 80% sequence identity to SEQ ID NO: 28.

[0028] In some embodiments, the expression of the functional portion, or variant, of the perforin protein is under the control of a promoter, preferably a distinct promoter to that linked to the immunogenic portion of the SARS-CoV-2 protein(s). This allows for independent, and possibly differential expression, of the functional portion, or variant, of the perforin protein.

[0029] Nucleic acids of the present invention can be RNA or DNA, including synthetic RNA or DNA.

[0030] In some embodiments, the nucleic acid encodes a recombinant polyprotein, wherein the polyprotein includes immunogenic portions of two or more SARS-CoV-2 proteins selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0031 ] In some embodiments, the encoded polyprotein comprises immunogenic portions of NSP3 and at least one or more of: Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. In some other embodiments, the polyprotein comprises immunogenic portions of Nucleocapsid and at least one or more of: NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0032] In some embodiments, the encoded polyprotein comprises immunogenic portions of the S1 subunit of the Spike protein and at least one or more of: Nucleocapsid; NSP3; and/or the receptor binding domain (RBD) of the Spike protein. In some other embodiments, the polyprotein comprises immunogenic portions of the receptor binding domain (RBD) of the Spike protein and at least one or more of: NSP3; Nucleocapsid; and/or the S1 subunit of the Spike protein.

[0033] In some embodiments there is provided a nucleic acid encoding a polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of proteins are separated by a cleavage domain. In some embodiments, the cleavage domain is a self-cleavage domain, preferably a 2A cleavage domain such as the foot-and-mouth disease virus (FMDV) 2A peptide. [0034] In some embodiments, the encoded polypeptide includes a heterologous signal peptide which is linked to the immunogenic portions of the S1 subunit of the Spike protein and/or the receptor binding domain (RBD) of the Spike protein, and facilitate the secretion of the immunogenic portion(s) from a eukaryotic cell. In some embodiments, the heterologous signal peptide of the polypeptide is the tissue plasminogen activator (tPA) signal peptide.

[0035] The nucleic acids disclosed herein may be used as a vaccine. In such embodiments, the vaccine may comprise at least two distinct nucleic acids each of which encode for different SARS-CoV-2 proteins, or encodes for perforin.

[0036] Further provided is a pharmaceutical composition comprising a nucleic acid or a vaccine as describe herein and a pharmaceutically acceptable carrier, diluent, excipient and/or stabiliser.

[0037] Also provided herein is a method of eliciting an immune response in a subject, the method comprising administering to the subject a nucleic acid, a vaccine or pharmaceutical composition as described herein.

[0038] In some embodiments, the elicited immune response is a B cell immune response and/or T-cell immune response. In some embodiments the method elicits a memory immune response.

[0039] Further provided is a method of preventing or treating a SARS-CoV-2 infection in a subject, preventing or treating COVID-19 disease in a subject or preventing or treating a condition associated with a SARS-CoV-2 infection in a subject, the method comprising administering to a subject a nucleic acid, a peptide, a vaccine or a pharmaceutical composition as described herein.

[0040] Further provided is use of a nucleic acid, or vaccine in the preparation of a medicament for the prevention or treatment of: a SARS-CoV-2 infection, or COVID-19 disease in a subject, or a condition associated with a SARS-CoV-2 infection in a subject.

[0041 ] Also provided is a method for producing an anti-SARS-CoV-2 antibody, the method comprising administering to a subject a nucleic acid, a peptide, a vaccine, or a pharmaceutical composition as described herein, and isolating an anti-SARS-CoV-2 antibody from the subject, identifying and expressing a sequence from the subject encoding an anti-SARS CoV-2 antibody, or isolating a B-cell secreting an anti-SARS- CoV-2 antibody from the subject.

Brief Description of Drawings

[0042] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying drawings.

[0043] Figures 1A to 11 are schematics of the gene construct for DNA vaccines comprising S1 subunit of the Spike protein, the receptor-binding domain (RDB) of the spike protein, Nucleocapsid (Nuc) and Non-structural Protein 3 (NSP3). These may be linked to oligomerization domains such as IMX313P and Foldon, or linked to perforin.

[0044] Figures 2A to 2F are schematics of poly-gene constructs encoding a polypeptide containing immunogenic portions from multiple SARS-CoV-2 genes with, or without, oligomerisation proteins and with, or without, perforin.

[0045] Figure 3 is fluorescent immunohistochemistry images of transduced

HEK293T cells stained with anti-RBD antibodies.

[0046] Figure 4 is fluorescent immunohistochemistry images of transduced HEK293T cells stained with anti-Spike antibodies.

[0047] Figure 5 is fluorescent immunohistochemistry images of transduced

HEK293T cells stained with anti-V5 antibodies.

[0048] Figure 6 is fluorescent immunohistochemistry images of transduced

HEK293T cells stained with anti-Nuc antibodies.

[0049] Figure 7 is fluorescent immunohistochemistry images of polygene transduced HEK293T cells stained with anti-RBD antibodies.

[0050] Figure 8 is fluorescent immunohistochemistry images of polygene transduced HEK293T cells stained with anti-spike antibodies. [0051 ] Figures 9A and B are western immunoblots of cell culture supernatants from HEK293T cell transduced with DNA vectors which express oligomerised RBD.

[0052] Figures 10A and B show anti-RBD antibody titres from mice vaccinated with DNA vectors encoding oligomerized S1 and RBD.

[0053] Figures 11 A and B show anti-Spike antibody titres from mice vaccinated with

DNA vectors encoding oligomerized S1 and RBD.

[0054] Figure 12 shows anti-Nuc antibody titres from mice vaccinated with nucleocapsid-encoding DNA vectors.

[0055] Figures 13A and 13B show anti-RBD antibodies (Figure 13A) and anti-Nuc antibodies (Figure 13B) in mice vaccinated with a DNA vector encoding a polyprotein including immunogenic portions from truncated NSP3, Nucleocapsid protein and RBD.

[0056] Figure 14 shows IFN-y production from T-cells isolated from heptameric S1 and RBD vaccinated mice in response to various pools of peptides spanning the S1 subunit and RBD region of the Spike protein.

[0057] Figure 15 shows IFN-y production from T-cells isolated from trimeric S1 and RBD vaccinated mice in response to various pools of peptides spanning the S1 subunit and RBD region of the Spike protein.

[0058] Figure 16 shows IFN-y production from T-cells isolated from NSP3 vaccinated mice in response to various pools of peptides spanning NSP3.

[0059] Figure 17 shows IFN-y production from T-cells isolated from nucleocapsid vaccinated mice in response to various pools of peptides spanning nucleocapsid protein.

[0060] Figure 18 shows IFN-y production from T-cells isolated from mice vaccinated with poly-gene DNA vector encoding truncated NSP3, nucleocapsid protein and RBD in response to various pools of peptides spanning NSP3.

[0061 ] Figure 19 shows IFN-y production from T-cells isolated from mice vaccinated with a poly-gene DNA vector encoding truncated NSP3, Nucleocapsid protein and RBD in response to various pools of peptides spanning the nucleocapsid protein. [0062] Figure 20 shows IFN-y production from T-cells isolated from mice vaccinated with a poly-gene DNA vector encoding truncated NSP3, Nucleocapsid protein and RBD in response to various pools of peptides spanning the RBD region of the Spike protein.

[0063] Figure 21 shows IFN-y production from T-cells isolated from mice vaccinated with a poly-gene DNA vector encoding truncated NSP3, Nucleocapsid protein and RBD in response to various pools of peptides spanning the S1 subunit of the Spike protein.

[0064] Figure 22 shows specific killing of target cells (pulsed with pools of peptides spanning NSP3) by T-cells from mice vaccinated with a DNA vector encoding NSP3.

[0065] Figure 23 shows T helper cell responses from mice vaccinated with a DNA vector encoding NSP3 in response to target cells pulsed with pools of peptides spanning NSP3.

[0066] Figure 24 shows specific killing of target cells (pulsed with pools of peptides spanning the nucleocapsid proteins) by T-cells from mice vaccinated with a DNA vector encoding the nucleocapsid protein.

[0067] Figure 25 shows T helper cell responses from mice vaccinated with a DNA vector encoding the nucleocapsid protein in response to target cells pulsed with pools of peptides spanning the nucleocapsid protein.

[0068] Figures 26A to 26C show IFN-y, IL-2 and TNF-a cytokine production in CD4+ T-cells isolated from mice vaccinated with DNA vaccines encoding oligomerized S1 or RBD in response to peptides spanning the RBD region or S1 subunit of the Spike protein.

[0069] Figures 27A to 27C show IFN-y, IL-2 and TNF-a cytokine production in CD8+ T-cells isolated from mice vaccinated with DNA vaccines encoding oligomerized S1 or RBD in response to peptides spanning the RBD region or S1 subunit of the Spike protein.

[0070] Figures 28A to 28C show IFN-y, TNF-a and IL-2 cytokine production in CD4+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning truncated NSP3. [0071 ] Figures 29Ato 29C show IFN-y, TNF-a and IL-2 cytokine production in CD8+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning truncated NSP3.

[0072] Figures 30A to 30C show IFN-y, TNF-a and IL-2 cytokine production in CD4+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning the nucleocapsid protein.

[0073] Figures 31 A to 31 C show IFN-y, TNF-a and IL-2 cytokine production in CD8+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning the nucleocapsid protein.

[0074] Figure 32 show IFN-y, TNF-a and IL-2 cytokine production in CD4+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning the RBD region of the Spike protein.

[0075] Figure 33 shows IFN-y, TNF-a and IL-2 cytokine production in CD8+ T-cells isolated from mice vaccinated with DNA vaccines encoding a polyprotein of truncated NSP3, nucleocapsid protein and oligomeric RBD in response to pools of peptides spanning the RBD region of the Spike protein.

[0076] Figure 34 shows fluorescent immunohistochemistry images of transduced HEK293T cells stained with anti-RBD antibodies, anti-spike antibodies, anti-NSP3 antibodies and anti-Nuc antibodies.

[0077] Figure 35 shows fluorescent immunohistochemistry images of transduced HEK293T cells stained with anti-RBD antibodies and anti-spike antibodies.

[0078] Figures 36A to 36C shows a vaccination protocol comprising 2 doses of tPA- RBD(Wuhan)-Foldon and 1 dose of tPA-RBDA-Foldon or 3 doses of tPA-RBDA-Foldon (36A), and the production and function of antibodies generated in response to the vaccination protocol (36B and 36C). [0079] Figure 37 shows the production of IFN-y from splenocytes collected from mice vaccinated with 2 doses of tPA-RBD(Wuhan)-Foldon and 1 dose of tPA-RBDA- Foldon or 3 doses of tPA-RBDA-Foldon following re-stimulation with S1 or RBD peptides from various variants of SARS-CoV-2.

[0080] Figures 38A and 38B show the production of cytokines (IFN-y, TNF-a and IL-2) from T-cells isolated from mice vaccinated with tPA-RBD-Foldon from the Wuhan- Hu-1 isolate or the Delta isolate (tPA-sRBDA-Foldon) following re-stimulation with the SARS-cov-2 RBD (Wuhan-Hu-1 isolate) peptide pool.

[0081 ] Figures 39A to 39D show a vaccination schedule comprising 3 x 75 pg or 3 x 100 ug, doses of a tPA-RBD(Omicron)-Foldon construct (Figure 39A) and the production (Figures 39B and 39C) and function (Figure 39D) of antibodies generated in response to the vaccination schedule.

[0082] Figure 40 shows the production of IFN-y from splenocytes collected from mice vaccinated with 3 doses of the tPA-RBD(Omicron)-Foldon construct (75 pg or 100 pg) following re-stimulation with RBD peptides from various variants of SARS-CoV-2.

[0083] Figures 41 A and 41 B show the production of cytokines (IFN-y, TNF-a and IL-2) from T-cells isolated from mice vaccinated with 3 doses of the tPA-RBD(Omicron)- Foldon construct (75 pg or 100 pg) following re-stimulation with RBD peptides from various variants of SARS-CoV-2.

[0084] Figures 42A to 42D show a vaccination protocol comprising 3 doses of 50 pg tPA-RBD(Delta)-Foldon in combination with: 50 pg of tNSP3-Nuc; or 50 pg of tNSP3-Nuc-Foldon (Figure 42A), and the production (Figures 42B and 42C) and function (Figure 42D) of antibodies generated in response to the vaccination protocol.

[0085] Figures 43A to 43C show the production of IFN-y from splenocytes collected from mice vaccinated with 3 doses of 50 pg tPA-RBD(Delta)-Foldon in combination with: 50 pg of tNSP3-Nuc; or 50 pg of tNSP3-Nuc-Foldon following re-stimulation with NSP3, Nucleocapsid or RBD peptides from SARS-CoV-2.

[0086] Figures 44A to 44F shows specific-killing and T-helper cell function in mice vaccinated with 3 doses of 50 pg tPA-RBD(Delta)-Foldon in combination with: 50 pg of tNSP3-Nuc; or 50 g of tNSP3-Nuc-Foldon, in response to cells pulsed with NSP3, Nucleocapsid or RBD peptides from SARS-CoV-2.

Detailed Description

[0087] The present invention is predicated on the fact that various structural and non-structural components of SARS-CoV-2 are antigenic and can induce an adaptive immune response in mammals. Consequently, these components may be useful in vaccination against infection or transmission of SARS-CoV-2 or preventing COVID-19 disease development following infection. Further, the present inventors have shown that DNA vaccines, and preferably DNA vector vaccines, can be used to induce an immune response in subjects to various components of the SARS-CoV-2 virus.

[0088] SARS-CoV-2 includes a single-stranded RNA genome of approximately 29,000 nucleotides (nt) (RefSeq Gene: NC_045512; BioProject: PRJNA485481/485481 ) which encodes for the structural proteins: Spike (S), Membrane (M), Nucleocapsid (N) and Envelope (E). SARS-CoV-2 has an approximately 21 ,000nt OFRIab region, which encodes 16 non-structural proteins (NSPs), namely NSP1 to NSP16 (Wu A et al. (2020) Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China. Cell Host Microbe Mar 11 ;27(3):325-328). Further encoded are a range of accessory proteins: ORF3a, ORF6, ORF7a, ORF7b, ORF8 and ORF10. A summary of the SARS-CoV-2 proteins and NCBI protein reference sequences is provided in table 1 below.

[0089] Table 1 - SARS-CoV-2 Protein References

[0090] The Spike protein forms a homotrimer that is embedded in the lipid bilayer of the viral envelope and protrudes externally. The Spike protein is a class I viral fusion protein which facilitates entry of the coronavirus into the host cell via the ACE2 receptor. The Spike protein comprises two subunits, termed S1 and S2. The S1 subunit binds to the host cell receptor via a Receptor Binding Domain (RBD) which spans from amino acid 319 to 541 of the spike protein sequence YP_009724390. Notably, different studies report different binding regions for the RBD. For example, Tai et al (2020) identifies the RBD as spanning amino acid positions 331 -524 of the SARS-CoV-2 spike protein based on alignment with SARS-CoV and MERS-CoV proteomes (Tai W. et al. (2020). Characterisation of the receptor binding domain (RBD) of 2019 novel coronavirus: implications for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Molecular Immunology 17: 613-620).

[0091 ] Following binding, the spike protein undergoes a change in configuration allowing S2 interaction with the ACE2 receptor and subsequent fusion of the viral and the host cell membranes and viral entry into the host cell.

[0092] Due to the surface expression and functional role of the spike protein, the majority of COVID-19 vaccines available consist of the Spike protein. The rationale being that antibodies generated against the spike protein can neutralise virus-receptor binding or prevent virus/host cell fusion. For example, Pfizer/BioNTech’s vaccine (BNT162b2), Moderna’s vaccine (mRNA-1273), Johnson and Johnson’s vaccine (JNJ- 78436735) and Novavax’s vaccine (NVX-CoV-2373) all rely on the whole spike protein stabilised in a pre-fusion configuration by the mutation of positions 986 and 987 to introduce two proline residues. Stabilising the Spike protein in the prefusion configuration has the advantage of inducing the production of antibodies which can bind to virions prior to binding with the ACE2 receptor thereby preventing binding and subsequent cell fusion and viral entry. By contrast, the Oxford University/Astra Zeneca vaccine (Ch. Ad. Ox) uses a native (non-modified) spike protein which can alternate between the pre-fusion and post-fusion configuration. None-the-less, Ch. Ad. Ox induces significant levels of neutralising antibodies.

[0093] While targeting the spike protein has obvious benefits, variations in the spike protein, as the result of natural mutation of the virus, may decrease vaccine efficacy and reduce the immunity to such variants. Therefore, it is advantageous to provide a vaccine which includes components of the SARS-CoV-2 virus in addition to, or instead of, the spike protein, or include multiple components of the SARS-CoV-2 virus. Such multi-protein vaccines (comprising multiple subunits) allow for greater redundancy against vaccine escape variants possessing variation in one region of the virus (for example the Spike protein). Further, such multi-protein vaccines may permit protection against other human coronaviruses by utilising regions of the SARS-CoV-2 proteins which are conserved with other human coronaviruses. [0094] The present invention provides a vaccine comprising an immunogenic portion of a SARS-CoV-2 protein selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein.

[0095] In some embodiments, the S1 subunit of the Spike protein has the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof. In some embodiments the RBD of the Spike protein has the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof. In some embodiments, NSP3 has the amino acid sequence set forth SEQ ID NO: 5, or a variant thereof. In some embodiments the Nucleocapsid protein has the amino acid sequence set forth SEQ ID NO: 6, or a variant thereof.

[0096] Variants of SARS-CoV-2 are known in the art based on genome sequencing data. The Pango nomenclature is being used by researchers and public health agencies to denote and track the spread and transmission of SARS-CoV-2 variants (see pango network - www. pango. network). Lineages are assigned based on a series of characteristics including a shared common ancestor, a single evolutionary event (e.g.,a single nucleotide change, insertion/deletion, or recombination event), and at least 5 sequences with high genome coverage (<5% of the genome is represented by IIIPAC ambiguity codes).

[0097] A list of the most common variants as of September 2022 are provided in table 2 (covariants.org).

[0098] Table 2 - SARS-CoV-2 Variants and Reference Sequences

[0099] Accordingly, a person skilled in the art can determine sequence variants of SARS-CoV-2 and it is envisaged that the sequence of the Non-Structural Protein 3 (NSP3), Nucleocapsid protein, S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein have a sequence associated with any identified variant of SARS-CoV-2. In embodiments, the variant has evolutionary homology with the original Wuhan isolate of SARS-CoV-2, or any of the above variants.

[0100] In some specified embodiments, the variant has at least 80% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 85% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 90% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 92% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 94% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 95% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 96% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 97% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 98% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.2% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.4% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.5% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.6% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.7% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.8% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6, or the variant has at least 99.9% sequence identity to any one of SEQ ID NOs: 3, 4, 5 or 6.

[0101] S1 Subunit and RBD Portion of the Spike Protein

[0102] As described above, the Spike protein of SARS-CoV-2 is provided on the envelope of the virus as a homotrimer. Accordingly, it may be advantageous to provide the components of the Spike protein (such as the S1 subunit or the RBD region) in an oligomerised form. Accordingly, in embodiments of the invention comprising the S1 subunit or the RBD of the Spike protein, the immunogenic portions are oligomerised. Oligomerised antigens may be more immunogenic as a result of increased valency and stability of the expressed antigen. Further, stabilising the protein in an oligomerised form may allow emulation of the natural tertiary structure allowing generation of appropriate antibodies.

[0103] Particularly exemplified oligomerisation domains are IMX313P and Foldon. However, other oligomerisation domains, such as ferritin, lumazine synthase, and [3- annulus peptide, can be used for the forming oligomers. For example, see Lainscek, D. et al. (2021 ). A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS- CoV-2 with Minimal Anti-Scaffold Response. Vaccines. Volume 9, issue no. 5: 431 ; and Mayssam A. and Barbara I. (2005). Protein oligomerization: How and why. Bioorganic & Medicinal Chemistry. Volume 13, Issue 17. Pages 5013-5020i.; US2004/0116664A1 ; US2020/0181633A1 ; WO2015/110831 A1 .

[0104] IMX313P is a fusion protein which comprises the oligomerisation domain of the C4 binding protein and has been shown to oligomerise monomer proteins into heptamers. Use of this fusion protein has been demonstrated to act as a potent adjuvant for purified protein antigens in murine models of vaccination (Kask, L. et al., Biochemistry 2002;41 (30): 9349-9357; and Ogun et al., Infect Immun. 2008;76(8):3817-23).

[0105] Foldon is a 27 amino acid domain which constitutes the C-terminal end of the fibriti n protein from bacteriophage T4. This domain is the oligomerisation domain of T4 fibritin and has been shown to oligomerise monomer peptides into trimers (Guthe S, et al. (2004). Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin. Journal of Molecular Biology. 2;337(4):905-15.) [0106] Accordingly, in some embodiments, the immunogenic portion of the S1 subunit is linked to IMX313P. In some embodiments, the immunogenic portion of the S1 subunit is linked to Foldon. In some embodiments, the immunogenic portion of the RBD region is linked to IMX313P. In some embodiments, the immunogenic portion of the RBD region is linked to Foldon. Amino acid sequences for the IMX313P and Foldon are provided in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0107] While it is particularly envisaged that the S1 subunit and the RBD region of the Spike protein are oligomerised, in some embodiments, immunogenic portions of NSP3 or Nucleocapsid proteins may be oligomerized, for example with IMX313P or Foldon.

[0108] NSP3 and Nucleocapsid

[0109] Unlike the S1 subunit and the RBD subunit, Non-Structural Protein 3 and the Nucleocapsid protein are not involved in the fusion of SARS-CoV-2 virions to a host cell. However, the inventors have shown that these proteins include immunogenic portions which can induce an immune response in a mammal.

[0110] SARS-CoV-2 NSP3 is a multifunctional protein and is one of several proteins with protease activity which are expressed from the SARS-CoV-2 genome. It has been hypothesised that NSP3 may inhibit innate immunity as well as being involved in cleavage of viral proteins (Khan, M.T.ef al. (2021 ) SARS-CoV-2 nucleocapsid and Nsp3 binding: an in-silico study. Archives of microbiology vol. 203,1 59-66.)

[0111 ] NSP3, and macrodomains contained therein, have a hydrolytic function which subverts host ADP-ribosylation and helps evade consequential immune responses. Mutations of NSP3 have been shown to render the SARS CoV-2 virus non- pathogenic and therefore it has been touted as a potential antiviral target (Schuller, M et al. (2021 ) Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking. Science advances vol; 7,16).

[0112] Surprisingly, the inventors have identified that Non-structural Protein 3 (NSP3) can induce an immune response in mammals, and can activate both B-cells, resulting in antibody production, and T-cells, thereby offering a target for vaccine development.

[0113] Nucleocapsid protein is the major structural component of the SARS-CoV-2 virion and binds to viral genomic RNA to package the RNA into a ribonucleoprotein (RNP) complex. In addition to viral assembly, the Nucleocapsid protein also has a role in viral mRNA transcription and replication, cytoskeleton organization and immune regulation by binding to double stranded RNA to combat RNAi-mediated antiviral responses in the host (Peng Y, et al. (2020) Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design. EMBO J. 39(20)).

[0114] Like NSP3, the Inventors have identified Nucleocapsid protein as effectively inducing B-cell and T-cell immune responses.

[0115] Therefore, in some embodiments, there is provided a vaccine comprising an immunogenic portion of SARS-CoV-2 Non-Structural Protein 3 (NSP3), or an immunogenic portion of SARS-CoV-2 Nucleocapsid protein. In some embodiments the vaccine comprises Non-Structural Protein 3 (NSP3) or an immunogenic portion of SARS-CoV-2 Nucleocapsid protein, but does not comprise the spike protein, or components thereof. In some embodiments, the vaccine comprises Non-Structural Protein 3 (NSP3) or an immunogenic portion of SARS-CoV-2 Nucleocapsid protein but does not comprise any other structural proteins. In some embodiment, the vaccine comprises Non-Structural Protein 3 (NSP3) or an immunogenic portion of SARS-CoV- 2 Nucleocapsid protein and any one or more proteins expressed from orfl ab. In some embodiment, the vaccine comprises Non-Structural Protein 3 (NSP3) or an immunogenic portion of SARS-CoV-2 Nucleocapsid protein and any one or more of: Nsp1 , Nsp2, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10, Nsp11 , Nsp12, Nsp13, Nsp14, Nsp15, Nsp16, ORF3a, ORF6, ORF7a, ORF7b, ORF8 and/or ORF10

[0116] Multi-Protein and Polyprotein Vaccines

[0117] In some preferred embodiments, the vaccine includes immunogenic portions from at least two of the SARS-CoV-2 proteins. In such a form, the vaccine is a multiprotein vaccine. In some forms, the vaccine includes immunogenic portions from at least two, at least three of the SARS-CoV-2 proteins, or all four of the SARS-CoV-2 proteins, preferably selected from the list of the S1 subunit of the Spike protein, the RBD region of the Spike protein, NSP3 or the Nucleocapsid protein.

[0118] In some embodiments, the vaccine comprises an immunogenic portion of the Nucleocapsid protein. In some embodiments, the vaccine comprises an immunogenic portion of the Non-Structural Protein 3. In some embodiments, the vaccine comprises an immunogenic portion of the Non-Structural Protein 3 and an immunogenic portion of the Nucleocapsid protein.

[0119] In some embodiments, the vaccine includes an immunogenic portion of another SARS-CoV-2 protein in addition to Nucleocapsid and Non-Structural Protein 3. Accordingly, there is provided a vaccine comprising an immunogenic portion of the Nucleocapsid protein and at least one or more immunogenic portions from: NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. In some embodiments, there is provided a vaccine comprising an immunogenic portion from NSP3 and at least one or more immunogenic portions from: the Nucleocapsid protein; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0120] Such a multi-protein vaccine may be provided as immunogenic portions from multiple proteins combined into a single formulation or may be co-administered. Alternatively, the multi-protein vaccine may take the form of a polyprotein, which has the immunogenic portions of the two or more proteins linked together as a single molecule.

[0121 ] In some embodiments, there is provided a vaccine comprising a recombinant polyprotein, wherein the polyprotein includes immunogenic portions from two or more SARS-CoV-2 proteins selected from the group consisting of: Non- Structural Protein 3 (NSP3), Nucleocapsid, S1 protein, and/or the receptor binding domain (RBD) of the Spike protein.

[0122] In some embodiments, there is provided a vaccine comprising a polyprotein wherein the polyprotein includes immunogenic portions from NSP3 and at least one or more immunogenic portions from: Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. [0123] In some embodiments, there is provided a vaccine comprising a polyprotein wherein the polyprotein includes immunogenic portions from Nucleocapsid and at least one or more immunogenic portions from: NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0124] In some embodiments, the multiple proteins in the polyprotein may be linked together by a cleavage domain to allow the multiple proteins to be separated in vivo after administration. Such separation may be desirable as the target location of the various immunogenic portions may differ.

[0125] The immunogenic portion can be provided in any suitable manner. For example, the immunogenic portion may be provided in the vaccine as a protein. Alternatively, the immunogenic portion may be encoded by a nucleic acid and transcribed (in the case of a DNA vaccine) and translated in vivo to produce a protein which induces the desired immune response (such as the nucleic acid vaccines described below). Both of these aforementioned forms are envisaged as embodiments of the present invention.

[0126] When provided as a nucleic acid, the nucleic acid may be a polygene which may encode a polyprotein. Such a polyprotein may include immunogenic portions from two or more different SARS CoV-2 proteins (as described above). It may be desirable for the two different immunogenic portions to be separated, or separable in vivo. Therefore, each of the two or more immunogenic portions can be encoded to provide separated proteins/peptides.

[0127] Alternatively, the multiple immunogenic portions can be encoded to provide as a single polyprotein with an interspacing cleavage domain which allows separation of the components of the polyprotein into the constituent parts either at translation, or post-translation.

[0128] In some embodiments, there is provided a vaccine comprising a polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of proteins are separated by a cleavage domain. In some embodiments, the cleavage domain is a self-cleavage domain, preferably a 2A self-cleavage domain such as the foot-and-mouth disease virus (FMDV) 2A peptide. Alternative cleavage domains are known in the art including many viral associated cleavage domains which can be used with the present invention, including the native furin and TMPRSS2 cleavage domains is SARS CoV-2 Spike protein.

[0129] By allowing the immunogenic portions of the NSP3 or Nucleocapsid to be separated from the immunogenic portions of S1 subunit or the receptor binding domain (RBD) region of the spike protein they can be processed in vivo separately and if desired can be transported or targeted to different cellular regions, for example by way of signal peptides.

[0130] Specifically-exemplified embodiments of the vaccines of the present invention include the proteins set forth in SEQ ID NOs: 35 to 42, 44, 46 or 48. Accordingly, in some embodiments, the vaccine has an amino acid sequence at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to SEQ ID NOs: 35 to 42, 44, 46 or 48.

[0131] Nucleic Acid Vaccines

[0132] In some embodiments of the present invention, the vaccine comprises a nucleic acid encoding the herein described immunogenic portions of SARS-CoV-2 or encoding the vaccines described above.

[0133] Accordingly, in some embodiments, there is provided a nucleic acid encoding an immunogenic portion of one or more of: Non-Structural Protein 3 (NSP3); Nucleocapsid; the S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein.

[0134] Further, the nucleic acid may encode one or more additional proteins as described herein. In particular, the nucleic acid may encode: an oligomerisation domain (such as IMX313P or Foldon), or a perforin protein (or functional portion, or variant, thereof). [0135] As nucleic acid vaccines result in production of proteins intracellularly this presents opportunities and challenges compared to protein-based vaccinations. One of the advantages is cellular trafficking of the translated and/or transcribed proteins. For example, it may be advantageous to express a surface protein (such as the Spike protein) in a manner that ensures that they are transported and embedded in the cellular membrane. This results in them being stabilised in an appropriate three- dimensional configuration and presents them to the immune system. Alternatively, it may be advantageous to express certain proteins (or immunogenic portions) so that they locate intracellularly. This may allow for processing of the protein into peptide fragments which can then be presented to the immune system via MHC molecules. Further, it may be advantageous to secrete certain encoded proteins, or fragments, from the cell.

[0136] In some embodiments of the invention, the nucleic acid encodes an immunogenic portion of one or more of: Non-Structural Protein 3 (NSP3); Nucleocapsid; S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein linked to a signal peptide.

[0137] Some signal peptides will result in secretion of a protein, or immunogenic portion thereof, unless the protein (or portion) includes a transmembrane, or membrane anchoring, region. Some proteins, such as the full-length spike protein, include a membrane anchoring region in the S2 domain. Therefore, nucleic acids which express the entirety of the spike protein will produce a protein which will anchor to the membrane. Even if the protein is linked to a signal peptide that may otherwise drive protein secretion from the cell. By comparison, nucleic acids which only encode the S1 portion of the Spike protein, or the RBD domain (which is contained within the S1 subunit), will not result in a membrane anchored protein when linked to a similar signal peptide.

[0138] The present inventors have shown that linking the S1 subunit, or the RBD region, of the Spike protein to a secretory signal peptide will result in secretion of these proteins. Importantly, these proteins will, when secreted, induce an immune response. Accordingly, in some embodiments of the nucleic acid vaccine, the immunogenic portions of the S1 subunit, or the RBD region, will be linked to a nucleic acid encoding a heterologous signal peptide which will result in the secretion of the linked peptide or protein from a cell, once expressed. Preferably, the cell is a eukaryotic cell.

[0139] The term “signal peptide” or “signal sequence”, or the like, is used in the context of this specification in reference to a peptide which at least directs transport and localisation of a linked product (such as a protein) to specific region within, or from, a host cell. Such peptides may be referred to in the art as leader sequences.

[0140] The signal peptide/sequences may include both co-translational translocation sequences and post-translational translocation sequences. In some embodiments, the signal sequence directs the localisation of the operatively linked portion of SARS-CoV-2 to, or through, a membrane of the host cell. In some embodiments, the membrane is the Endoplasmic Reticulum (ER), which may result in localization of the linked portion of the SARS-CoV-2 protein to the cell surface, or may result in secretion of the portion of the SARS-CoV-2 protein.

[0141 ] In some embodiments, the signal peptide results in transport of the linked immunogenic portion to the surface of the cell in which the nucleic acid is expressed. In some embodiments, the signal peptide results in the secretion of the linked immunogenic portion from the host cell.

[0142] A range of signal peptides are known in the art which are suitable for use in the present invention. These include signal peptides from eukaryotic cells, prokaryotic cells and viral genomes. A range of signalling peptides are available in the literature and are indexed in databases, such as the signal peptide database (http://www.signalpeptide.de/). Further, while signal sequences display little to no sequence conservation, they do display characteristic conserved physiochemical structure. As such, algorithms have been developed which identify signal peptides (for discussion on signal peptides and identification see Nicchitta, CV (2002). Signal Sequence Function in the Mammalian Endoplasmic Reticulum: A Biological Perspective, Current Topics in Membranes. 52, Ch.7:483-499). Further, see https://www.uniprot.org/help/signal in relation to designation of signal peptides within the Uniprot database.

[0143] Signal peptides can be functional in either eukaryotic cells or prokaryotic cells (see Freudl, R. (2018) Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories 17, 52). The signal peptide chosen will be determined by the desired function. In preferred embodiments, the signal peptide is a eukaryotic signal peptide.

[0144] The Spike protein of SARS-CoV-2 includes a 13 amino acid signal peptide at amino acids 1 to 13 (Huang Y, et al. (2020) Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacologica Sinica; 41 :1141-1149). Therefore, in some embodiments, the signal peptide is the spike protein signal peptide. In other embodiments, the signal peptide is heterologous.

[0145] Exemplary signal peptides include, but are not limited to, the tissue plasminogen activator (tPA) signal sequence, Japanese encephalitis virus signal sequence, erythropoietin (epo) signal sequence, VP22 HSV1 signal sequence, Parathyroid hormone-related protein (PTHrP) N-terminal ER signal sequence, Calreticul in (CRT), or Adenovirus E3 signal sequence. In a preferred, and exemplified, embodiment, the signal peptide is a tPA signal peptide. In some embodiments, the tPA signal peptide has the amino acid sequence set forth in SEQ ID NO: 17, or is a functional variant thereof. In some embodiments, the tPA signal peptide is encoded by a sequence set forth in SEQ ID NO: 7, or a variant thereof.

[0146] In some embodiments, the encoded peptide may include a target sequence which targets the encoded product to a desired cell type or cellular subset and may facilitate localisation of the operatively linked immunogenic peptide. In some embodiments, the target sequence targets the operatively linked immunogenic peptide to an immune cell. In some embodiments, the target sequence targets the operatively linked immunogenic peptide to an antigen presenting cell.

[0147] Immune cell targeting sequences include, but are not limited to, heat shock proteins (HSP70, gp96, calreticulin, HSP60), cytokines (FLT-3 ligand, GM-CSF), chemokines (MCP3, MIP1 a, MIP-3a, RANTES, [3-defensin, MC148, vMIP-l), singlechain fragment variable (scFv) antibody fragment, anti-CD40, anti-MHCH, anti-CD21 , anti-DEC205, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), Fc fragment, CD154, fragment of the complement factor C3 (C3d), L-Selectin or fragment C of tetanus toxin (TetC), Immunoglobulin signal sequence (e.g. IgA, IgE or IgG). [0148] In some embodiments, the target sequence targets the operatively linked immunogenic portion of SARS-CoV-2 proteins to the proteasome. Proteasome targeting of expression products can assist in facilitating MHC class I presentation of a linked expression product, such as a peptide or protein. Consequently, proteasome targeting can lead to activation of larger populations of CD8+ Cytotoxic T Lymphocytes, which may generate stronger anti-viral immunity.

[0149] Proteasome target sequences include, but are not limited to, co-translational ubiquitination of an expressed product, thereby fusing the expressed product to ubiquitin, ETA(dll) or y-tubulin, transporters associated with antigen processing (TAP) proteins, or endoplasmic reticulum insertion signal sequences.

[0150] In some embodiments, the target sequence targets the operatively linked portion of a SARS-CoV-2 peptide to the endosome or lysosome of a host cells. Endosome and lysosome targeting sequences may assist in targeting linked antigens to the MHC-II pathways, thereby increasing CD4+ T helper responses. Endosome or lysosome targeting sequences include, but are not limited to, lysosomal-associated membrane protein type 1 (LAMP-1 ), major histocompatibility complex class Il- associated invariant chain (li), melanosome transport sorting signals, or the transferrin receptor (TfR).

[0151 ] As described herein, the expressed immunogenic portion of the SARS-CoV- 2 protein may be linked to an oligomerisation domain such as IMX313P or Foldon. DNA sequences encoding for IMX313P or Foldon are provided in SEQ ID NO: 15 and SEQ ID NO: 16 respectively.

[0152] In some embodiments, there is a provided a nucleic acid vaccine or molecule encoding an immunogenic portion of SARS-CoV-2 S1 protein linked to IMX313P or Foldon. In some embodiments, there is provided a nucleic acid vaccine or molecule encoding an immunogenic portion of the receptor binding domain of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon. While it is particularly envisaged that the encoded S1 subunit or the RBD region of the Spike protein are linked to a nucleic acid encoding and oligomerisation domain, in some embodiments, encoded immunogenic portions of NSP3 or Nucleocapsid proteins maybe linked to oligomerisation domain, for example with IMX313P or Foldon. [0153] The vaccine of the present invention may comprise an immunogenic portion from multiple SARS-CoV-2 proteins (as described above). When provided as a nucleic acid, this may take the form of a poly-gene construct encoding multiple proteins from a single nucleic acid molecule. Such poly-gene constructs, when in the form of a DNA molecule, may have all genes expressed under the control of the same promoter sequence, or may have genes expressed under the control of separate promoter sequences. This can allow optimal expression of each protein or may allow differential expression of the proteins depending on the promoter used. Therefore, in some embodiments the DNA vector comprises multiple promoters. These promoters can be the same or can be different.

[0154] In some other forms, the separate immunogenic portions may be contained on separate nucleic acid molecules and collocated in a mixture or composition. Therefore, in some embodiments, there is provided a nucleic acid vaccine comprising at least two distinct nucleic acids each of which encode for immunogenic portions of different SARS-CoV-2 proteins, preferably selected from the S1 subunit of the Spike protein, the RBD region of the Spike protein, NSP3 or the Nucleocapsid protein. In some embodiments, at least one of the separate nucleic acids encodes for a perforin protein, or a functional portion or variant, thereof. Such an embodiment is particularly envisaged (but not limited to) when the nucleic acid is an RNA. It is to be understood that where there is more than one nucleic acid, the ratio of each nucleic acid (encoding separate proteins) may not be even.

[0155] In some embodiments, there is provided a nucleic acid encoding a polyprotein comprising NSP3 and at least one or more of: Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. In some embodiments, there is provided a nucleic acid encoding a polyprotein comprising Nucleocapsid and at least one or more of: NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0156] In some embodiments, there is provided a nucleic acid encoding a polyprotein comprising the receptor binding domain (RBD) of the Spike protein and at least one or more of: NSP3; Nucleocapsid; and/or the S1 subunit of the Spike protein. In some embodiments, there is provided a nucleic acid encoding a polyprotein comprising the S1 subunit of the Spike protein and at least one or more of: NSP3; Nucleocapsid; and/or the receptor binding domain (RBD) of the Spike protein.

[0157] As explained above, it may be desirable to target immunogenic portions of different SARS-CoV-2 to different cellular regions, such as secreting them from the cell. Therefore, in embodiments of the invention, where multiple immunogenic portions are provided on one nucleic acid (e.g., a poly-gene), one or more of the multiple immunogenic portions (when expressed) may be separated by an interspacing cleavage domain allowing the two adjacent portions to be separated intracellularly. Therefore, in some embodiments, the nucleic acid may include a sequence encoding a cleavage domain. In some embodiments, the nucleic acid includes a sequence encoding for the foot-and-mouth disease virus (FMDV) 2A peptide (i.e,. the “2A” peptide). In some embodiments, the encoded foot-and-mouth disease virus (FMDV) 2A peptide has the sequence set forth in SEQ ID NO: 23, or is a functional variant thereof. In some embodiments, the nucleic acid sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide has the sequence set forth in SEQ ID NO: 13, or is a variant thereof.

[0158] In some embodiments, there is provided a nucleic acid encoding a recombinant polyprotein, wherein the polyprotein includes immunogenic portions of two or more SARS-CoV-2 proteins selected from the group consisting of: Non-Structural Protein 3 (NSP3); Nucleocapsid; S1 protein; and/or the receptor binding domain (RBD) of the Spike protein.

[0159] In some embodiments, there is provided a nucleic acid encoding a recombinant polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of proteins are separated by a cleavage domain. As discussed above, the nucleic acid encoding the first immunogenic portion of a protein and the nucleic acid encoding the second immunogenic portion of a protein are interspaced by a nucleic acid sequence which encodes a cleavage peptide, such as the foot-and-mouth disease virus (FMDV) 2A peptide. In some such embodiments, the S1 subunit and/or the RBD of the Spike protein may be linked to a signal peptide.

[0160] Specifically exemplified embodiments of nucleic acid gene constructs are provided in SEQ ID NOs: 27 to 34, 43, 45 and 47. Accordingly, in some embodiments, the vaccine has a nucleic acid sequence at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to SEQ ID NOs: 27 to 34, 43, 45 and 47. Or a nucleic acid sequence which encodes for a protein having an amino acid sequence at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identical to SEQ ID NOs: 35 to 42, 43, 45 and 47.

[0161 ] Perforin is driven under a separate promoter region and therefore is typically separate from the nucleic acid gene constructs. For example, in embodiments of the invention comprising DNA vectors (see below), perforin may be cloned into a different restriction site in an expression vector and therefore is separate to the gene construct encoding the antigenic portions of SARS-CoV-2.

[0162] DNA vaccines

[0163] Many known forms of vaccines against viruses are used for inducing protection to pathogenic organisms. The most common of which include live-attenuated viruses, inactivated (killed) viruses, subunit or recombinant vaccines that use portions of viral capsule proteins or virus like particles. However, one of the most promising vaccine types are DNA vaccines.

[0164] DNA vaccines, in their simplest form, consist of a DNA plasmid or vector containing a gene (or portion of a gene) encoding an immunogenic protein of a pathogen, and elements needed to transcribe this gene in the target subject. This DNA is administered to a subject during immunization. The encoded immunogen is transcribed and translated, and the antigen is exposed to the immune system to elicit an immune response.

[0165] Unlike protein-based vaccines, DNA vaccines have the advantage of synthesizing the specific antigen in the host’s target organism (as opposed to a model system in vitro). This ensures that the proteins, once translated are processed (e.g. glycosylated and folded) correctly to elicit the appropriate immune response.

[0166] Furthermore, DNA vaccines have the advantage of being simple and cheap to produce and being stable for transportation and storage, when compared to protein- based vaccines. DNA can be readily lyophilised and rehydrated prior to administration without significant loss in functionality.

[0167] Accordingly, the present invention provides a DNA vaccine including a nucleic acid molecule as disclosed herein. In some embodiments, the DNA molecule is a DNA vector. DNA vectors can be administered via intramuscular injection, intradermal injection, gene gun or jet injection. Further, DNA vectors can be formulated to help facilitate uptake of DNA into cells. This can be done by using recombinant viral vectors (see below) including non-human viral vectors, or nano-lipid formulations.

[0168] As will be understood DNA vectors include promotor sequences for driving expression of the encoded peptides. A non-exhaustive list of possible promoters is provided in Table 3.

Table 3 - Promoters

[0169] In some embodiments, the DNA vector includes a promotor which is constitutive in a mammalian cell. In some embodiments, the promoter is selected from the group consisting of CMV, SV40, UBC, EF1A, PGK or CAGG. In some embodiments, the promoter is derived from cytomegalovirus (CMV).

[0170] Any suitable DNA can be used in the present invention, which may include naked DNA. It is therefore not intended that the invention described herein in limited to a specific vector, unless expressly stated.

[0171 ] However, when the term “DNA vector” is used throughout this specification, it is intended to refer to a polynucleotide/nucleic acid, construct designed for transduction/transfection of one or more cell types. These may include, for example; "cloning vectors" which can be stably maintained in a target cell and can facilitate propagation, replication and subsequent isolation of inserted nucleotide molecules; "expression vectors" designed for expression of selected nucleotide sequence in a host cell; a "viral vector" which are designed to facilitate the production of a recombinant DNA containing virus or virus-like particle in a host cell, or "shuttle vectors", which can propagate in host cells from more than one species. [0172] Typically, a vector will include cloning, or restriction, sites for insertion of DNA, a promoter (as discussed above) to induce the production of the inserted DNA, and DNA portion that encodes for proteins which permit selection or identification of vector carrying host cell(s).

[0173] Methods are known in the art for producing, modifying, and optimising suitable vectors for use in the present invention (see, for example, lurescia S. et al. (2014) A Blueprint for DNA Vaccine Design. In: Rinaldi M. et al. (eds); DNA Vaccines. Methods in Molecular Biology (Methods and Protocols), vol 1143. Humana Press, New York, NY).

[0174] DNA vectors suitable for use with the present invention are known in the art, and include vectors approved for therapeutic use by regulatory bodies including the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), the Australian Therapeutic Goods Administration (TGA) and the Chinese Food and Drug Administration (CDFA).

[0175] In some embodiments, the DNA vector is an expression vector such as the gWIZ vector, the pVax 1 vector, the pcDNA3.1 vector (see Gomez L. and Onate A. Plasmid-Based DNA Vaccines, DOI: 10.5772/intechopen.76754), the NTC8385 vector and the NTC9385R vector (see Williams J. Vaccines, 2013, 1 (3): pp.225-249). In some embodiments, the DNA vector is a pVax vector.

[0176] Viral vectors

[0177] In some embodiments the vaccine includes a viral vector, the viral vector including a nucleic acid as described herein.

[0178] Viral vectors suitable for use with the present invention are known in the art, and include vectors approved for therapeutic use by regulatory bodies including the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), the Australian Therapeutic Goods Administration (TGA) and the Chinese Food and Drug Administration (CDFA).

[0179] Any suitable viral vector can be used in the present invention. It is therefore not intended that the invention described herein will be limited to a specific viral vector, unless expressly stated. [0180] Examples of viral vectors suitable for use with the present invention include (but are not limited to): measles virus, adenovirus, Varicella-zoster virus, Human parainfluenza virus 3, Coxsackievirus group B, Retrovirus, Lentivirus, Vaccinia virus Adenovirus, Adeno-associated virus, Cytomegalovirus, Sendai virus and Poxvirus — modified vaccinia Ankara. These include non-human viruses. Methods are known in the art for producing viral vectors comprising a nucleic acid in accordance with the invention. For example, see Lauer KB et al. (2017) Multivalent and Multipathogen Viral Vector Vaccines. Clin Vaccine Immunol. 2017 Jan 5;24(1 ):e00298-16; and Llra T. et al. (2014) Developments in Viral Vector-Based Vaccines; Vaccines, 2(3): pp.624-641.

[0181] RNA vaccines

[0182] RNA vaccines represent an alternative form of nucleic acid vaccine to DNA vaccines. Unlike DNA, RNA is typically considered unstable and readily degraded by abundant RNases. This may result in difficulties formulating and storing the vaccine, as well as low efficacy.

[0183] However, RNA vaccines have the advantage of scalability as RNA transcripts are easily produced, they can be rapidly adaptable, and RNA is not integrated into genomic DNA. Further, RNA is inherently immunogenic and therefore can act to enhance the immune response to the encoded protein once produced.

[0184] Accordingly, in some embodiments, there is provided an RNA molecule including the nucleic acid disclosed herein. In some embodiments, the RNA molecule is a non-replicating mRNA molecule. In some embodiment, the RNA molecule is a selfamplifying RNA molecule.

[0185] Methods are known in the art for producing mRNA transcripts from DNA including T7, T6 or sP6 phage RNA polymerases (see Pardi N et al. (2013) In vitro transcription of long RNA containing modified nucleosides. Methods in Molecular Biology; 969: 29-42).

[0186] Further known in the art are methods for delivering RNA molecules to cells including injection of naked RNA, gene gun delivery of RNA, electroporation and mRNA complexing strategies. Recently, mRNA complexing strategies have become the preferred means for administering mRNA molecules in vivo. These strategies include coupling mRNA with Protamine, Protamine liposomes, Polysaccharide particles, Cationic nanoemulsions, Cationic polymers, Cationic polymer liposomes, Cationic lipid nanoparticles, Cationic lipid-cholesterol nanoparticles, Cationic lipid-cholesterol-PEG nanoparticles and Dendrimer nanoparticles (see Pardi, N et al. (2018). mRNA vaccines - a new era in vaccinology. Nature reviews. Drug discovery; vol. 17,4: 261 -279).

[0187] Adjuvants

[0188] To improve the antigenicity of DNA vaccines, it may be advantageous to coadminister, or include in a pharmaceutical composition, an immune-enhancing agent such as adjuvant.

[0189] Accordingly, in some embodiments, the vaccine or pharmaceutical composition includes an adjuvant. In some embodiments, the nucleic acid encodes an adjuvant.

[0190] Adjuvants for conventional vaccines include, but are not limited to, saponin, non-ionic detergents (such as Tween 80), vegetable oil, aluminium hydroxide, surface active substances (including lysolecithin, pluronic polyols, polyanions) peptides, CpG repeats, oil or hydrocarbon emulsions and keyhole limpet hemocyanins. Further, adjuvants are disclosed in Del G. et al. (2018). Correlates of adjuvanticity: A review on adjuvants in licensed vaccines. Seminars in Immunology. 39:14-21 ; Pulendran, B et al. (2021 ). Emerging concepts in the science of vaccine adjuvants. Nature Review in Drug Discovery 20, 454-475; and Shrestha, A. et al. (2019) Cytolytic Perforin as an Adjuvant to Enhance the Immunogenicity of DNA Vaccines. Vaccines vol. 7(2):38 (the entire contents of which is incorporated herein).

[0191 ] Specific adjuvants for DNA vaccines include plasmids, or other nucleic acid molecules, encoding immunomodulatory proteins - such as cytokines (including IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-18, IL-21 , IFN, TGF[3, and GM-CSF) chemokines (including MIP-1 a, MIP-3a, MIP-3|3, and RANTES) - co-stimulatory and adhesion molecules (including B7-1 , B7-2, LFA-3 and ICAM-1 ), heat shock proteins, viral fusion proteins, molecules that block co-inhibitory molecules (including blocking PD-1 ), free RNA or double-stranded DNA, lipid formulations (such as nanolipids), encoded CpG repeats or free CpG DNA. [0192] Further, vaccines (including nucleic acid vaccine such as those of the present invention) have been shown to be boosted by the use of perforin or other cytolytic proteins (see Shrestha A et al. - see above). Specifically, truncated versions of perforin (PRF), lacking the final 12 amino acid residues of the C terminus, can induce non-apoptotic cell death and enhance immune responses, particularly T-cell mediated immunity (see Wijesundara D et al. (2017). Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by dendritic cells in a time-dependent manner. Scientific Reports. 7:8530; WO 2013/170305 A1 ; Gargett T. et al. (2014). Induction of antigen-positive cell death by the expression of Perforin, but not DTa, from a DNA vaccine enhances the immune response. Immunol. Cell Biol; 92:359-367; Grubor-Bauk B et al. Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs. Gene Then 23:26-37; Chiang SCC et al. (2013). Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production. Blood. 2013;121 :1345. - the entire disclosures of which are incorporated herein).

[0193] Accordingly, in some embodiments, the immunogenic portion of the SARS- CoV-2 peptide is provided in combination with a perforin protein (or a functional portion, or variant, thereof). This perforin protein (or functional portion, or variant, thereof) may be a separate protein or may be linked to an immunogenic portion of SARS-CoV-2. Alternatively, the perforin protein (or functional portion, or variant, thereof) may be encoded by a nucleic acid to be expressed separately or linked to an encoded immunogenic portion of a SARS-CoV-2 protein. When encoded by a separate nucleic acid, the sequence encoding the perforin protein (or functional portion, or variant, thereof) may be linked to, and under the control of, a separated promoter sequence (for example SV40). In the exemplified embodiments of the present invention described in the Examples, perforin (or the truncated version thereof) is cloned separately into the expression vector and is expressed under the control of a separate promoter.

[0194] In some embodiments, the perforin is a non-human protein, such as a mouse perforin. In some embodiments, the functional variant of perforin is mutated or truncated relative to a wild-type form of perforin. In some embodiments, the truncated perforin has the amino acid sequence set forth in SEQ ID NO: 24, or a functional variant thereof. In some embodiments, the functional variant of perforin has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24.

[0195] In some embodiments of the nucleic acid vaccine, the perforin is encoded by the nucleic acid sequence given in SEQ ID NO: 14, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0196] In some embodiment, the invention provides an immunogenic portion of the SARS-CoV-2 protein linked to, co-expressed with, or co-administered with, a portion of the perforin protein. In some preferred embodiments, there is provided an immunogenic portion of NSP3 linked to, co-expressed with, or co-administered with, a perforin protein (or functional portion, or variant, thereof). In some embodiments, the invention provides an immunogenic portion of Nucleocapsid protein linked to, co-expressed with, or coadministered with, a perforin protein (or functional portion, or variant, thereof). In some embodiment, the invention provides an immunogenic portion of Spike protein, or the S1 subunit of the Spike protein, or the RBD region of the spike protein, linked to, coexpressed with, or co-administered with, a perforin protein (or functional portion, or variant, thereof).

[0197] Methods of Use

[0198] The disclosure herein provides methods of immunizing, or vaccinating, an individual for the prevention or treatment of SARS-CoV-2 infection in a subject, the method comprising administering to a subject a vaccine, nucleic acid or pharmaceutical composition as described herein.

[0199] In some embodiments, treatment of a SARS-CoV-2 infection may include: reducing the likelihood of infection with SARS-CoV-2; reducing the seventy of SARS- CoV-2 infection; reducing the duration of SARS-CoV-2 infection; decreasing viral titres during SARS-CoV-2 infection or decreasing viral sheading during SARS-CoV-2 infection.

[0200] In some individuals, a SARS-CoV-2 infection will be asymptomatic or sub- clinical. However, in some subjects, infection with SARS-CoV-2 will become COVID-19 disease and will present with one or more symptoms including: fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting or diarrhea. In severe cases COVID-19 can result in Acute Respiratory Distress Syndrome (ARDS) which can lead to hypoxia and multi-organ failure. Further, severe COVID-19 may result in cytokine storm syndrome.

[0201 ] Accordingly, in some embodiments, there is provided a method of preventing or treating COVID-19 disease in a subject, the method comprising administering a nucleic acid, vaccine or pharmaceutical composition as described herein.

[0202] Further, SARS-CoV-2 infection has been associated with long term symptoms which remain after resolution of the acute infection in a phenomenon known as long-COVID. It has been estimated that approximately 80% of individuals show at least 1 symptom more than 14 days after resolution of acute infection (and two negative PCR tests), the most common of which are: fatigue, headache, attention disorder, anosmia, memory loss, anxiety, depression, fever, sleep disorders, sleep apnea, psychiatric illness, weight loss, pain, sweating, hearing loss, tinnitus, hair loss, dyspnea, reduced pulmonary capacity, ageusia, polypena, chest pain, cough, and joint pain (see Lopez-Leon, S. et al. (2021 ). More than 50 long-term effects of COVID-19: a systematic review and meta-analysis. Scientific Reports; 11 , 16144).

[0203] Accordingly, in some embodiments, there is provided a method of preventing or treating a condition or symptom associated with a SARS-CoV-2 infection in a subject, including a symptom associated with long-COVID, the method comprising administering a nucleic acid, vaccine or pharmaceutical composition as described herein.

[0204] In some embodiments, the above methods are achieved by eliciting an immune response in a subject. As such, in some embodiments, the invention provides a method of eliciting an immune response in a subject, the method including the step of administering to the subject a nucleic acid molecule, a vaccine or a pharmaceutical composition as described herein.

[0205] In some embodiments, the immune response is a T-cell response. In some embodiments, the T-cell response is the generation of anti-SARS-CoV-2 CD8+ T-cells and/or CD4+ T-cells (directed against NSP3, S1 , RBD or nucleocapsid protein). In some embodiments, the T-cell immune response results in memory T-cells.

[0206] In some embodiments, the immune response is a B cell response. In some embodiments, the B cell response results in the generation of antibodies. In some embodiments, the B cell response results in memory B cells.

[0207] Regimens for immunizing a subject are known in the art and may vary depending on the composition of vaccine (e.g., the presence of adjuvants), the route of administration, and the imm uno-competence of the individual. In some embodiments, the method of eliciting an immune response includes a dosage regimen, wherein the vaccine, nucleic acid or pharmaceutical composition is administered in a single dose, in two doses, in three doses, in four doses, or in five doses.

[0208] In some embodiments, the method of eliciting an immune response in a subject includes administering the vaccine, nucleic acid or pharmaceutical composition via a parenteral route. Exemplary parenteral routes include an intravenous route, a subcutaneous route, an intradermal route, or an intramuscular route.

[0209] In some embodiments, the invention provides the use of a vaccine, a nucleic acid or a pharmaceutical composition, as described herein, for eliciting an immune response in a subject or for the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection, COVID-19 disease or a COVID-19 related symptom or condition in a subject.

[0210] Methods are known in the art to evaluate the induction of an immune response. Exemplary methods include (but are not limited to) the induction of an innate immune response, or the induction of an adaptive immune response. In preferred embodiments, the immunogenic portion induces an adaptive immune response. In some embodiments, the immunogenic portion induces an antigen specific immune response. An antigen specific immune response includes (but is not limited to) the expansion of lymphocytes population including T-cells, affinity maturation of B cells, instigation of secretion of macromolecules such as antibodies (including isotype switching to IgA, IgG or IgE isotypes), instigation or enhancement of secretion of chemokines and/or cytokines (such as IL-2, IL-4, IL-5, IL-9, IL-12, IL-13, IL-17, IL-21 , IL-22, IL-26, IFN-y, TNF-a, or TNF-|3), instigation or enhancement of CTL responses, instigation or enhancement of maturation of monocytes and/or dendritic cells, increase in surface expression of co-stimulation molecules, increase in the expression of Fc receptors, increase in the expression of major-histocompatibility (MHC) molecules, recruitment or migration of leukocytes, increase in expression of markers indicative of plasma cells or memory B cells (including CD38, CD21 , CD24, CD19, B220, FcRH4 and CD25), upregulation of markers indicative of memory T-cells (including CD45RO, CCR7, CD62L. CD27 or CD28), and upregulation of activation receptors (including CD69, Ki67 or CD40L).

[0211 ] In some embodiments, the subject is a human. In some embodiments, the subject is a human who is immunologically naive for SARS-CoV-2. In some embodiments, the subject has an active infection or indications of a recent infection. In some embodiments, the subject has a subclinical infection.

[0212] The precise dose of the nucleic acid molecule, vaccine or pharmaceutical composition to be employed in methods of the invention will depend on multiple factors including the route of administration, and the nature of the patient, and should be decided according to the judgment of the practitioner regarding each patient's circumstances, according to standard clinical techniques and clinical evaluation such as clinical trials. In some embodiments a pharmaceutically effective amount will be administered. An "effective amount" is an amount sufficient to achieve a desired biological effect such as to induce enough humoral or cellular immunity, or to provide some degree of protection.

[0213] In some embodiments, there is provided a method for producing an anti- SARS-CoV-2 antibody, the method comprising administering to a subject a nucleic acid, a vaccine or a pharmaceutical composition as described herein and isolating an anti-SARS-CoV-2 antibody from the subject, or isolating a B-cell secreting an anti- SARS-CoV-2 antibody from the subject. In some embodiments, the subject is not a human.

[0214] Examples

[0215] The invention is further described and illustrated in the following examples. The examples are only for the purpose of describing particular embodiments of the invention and are not intended to be limiting with respect to the above description and the scope of the invention as claimed in this application or future applications claiming priority from this application.

[0216] Example 1 - Preparation of DNA Vectors

[0217] DNA vectors encoding various genes of the SARS CoV-2 virus were prepared as described below.

[0218] Single gene DNA vectors (as illustrated in Figure 1 ) encoding single SARS- CoV-2 peptides were prepared as described below.

[0219] Constructs comprising tPA-S1-IMX313P (Figure 1A), tPA-RBD-IMX313P (Figure 1 B), tPA-S1-Foldon (Figure 1 c), and tPA-RBD-Foldon (Figure 1 D) were prepared using the following protocol.

[0220] Codon optimized nucleic acid sequences encoding for the S1 portion of the SARS-CoV-2 Spike protein and receptor binding domain (RBD) portion of the Spike proteins with a C-terminal His-tag (provided by Prof Florian Krammer; see Amanat, F et al. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature Medicine, 26(7), 1033-1036.) were used in series of PCR reactions to introduce the tissue plasminogen activator (tPA) signal peptide upstream of, and operatively linked to, the S1 and RBD gene.

[0221 ] Codon optimised genes for IMX313P and Foldon, were introduced downstream of the S1 and RBD genes to ensure oligomerisation of the antigens after expression, creating the gene constructs tPA-S1 -IMX313P, tPA-RBD-IMX313P, tPA- S1 -Foldon, and tPA-RBD-Foldon.

[0222] Constructs comprising tNSP3-Nuc-2A-tPA-RBD-IMX313P (Figure 2A) tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF (Figure 2B), tNSP3-Nuc-2A-tPA-RBD-Foldon (Figure 2C) and tNSP3-Nuc-2A-tPA-RBD-Foldon-PRF (Figure 2D) were prepared as follows.

[0223] Codon optimised SARS-Cov-2 non-structural protein 3 (NSP3) and Nucleocapsid (Nuc) (GenBank accession number QIK50447.1 ) were linked to a V5 tag. A downstream simian virus 40 (SV40) promoter, linked to a truncated perforin (PRF) gene (see Shrestha AC et al. (2019). Cytolytic Perforin as an Adjuvant to Enhance the Immunogenicity of DNA Vaccines. Vaccines (Basel); 7(2):38), was provided to create the gene inserts, NSP3-PRF (Figure 1 e), and Nuc-PRF (Figure 1f). While gene inserts devoid of perforin - NSP3 (Figure 1g) and Nuc (Figure 1 h) - were also generated to allow the assessment of the contribution of perforin in stimulating an immune response. Finally, a construct comprising the RBD linked to the tPA signal peptide, but without an oligomerisation domain, was generated (Figure 11).

[0224] Poly-gene DNA vectors (as illustrated in Figure 2) encoding a polyprotein comprising multiple linked SARS CoV-2 proteins were prepared as described below.

[0225] Constructs comprising tNSP3-Nuc-2A-tPA-RBD-IMX313P (Figure 2A), tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF (Figure 2B), tNSP3-Nuc-2A-tPA-RBD-Foldon (Figure 2C), tNSP3-Nuc-2A-tPA-RBD-Foldon-PRF (Figure 2D), tNSP3-Nuc (Figure 2E) and tNSP3-Nuc-PRF (Figure 2F) were prepared as follows.

[0226] A nucleic acid encoding a truncated region of the NSP3 protein (tNSP3), which spanned the amino acids from positions 1200 to 1900 of the SARS-CoV-2 NSP3 protein, was linked to the nucleic acid sequence encoding the Nucleocapsid protein to form tNSP3-Nuc. A nucleic acid sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide (the “2A” peptide) was inserted downstream of the Nucleocapsid gene and upstream of a tPA-RBD-IMX313P construct or a tPA-RBD-Foldon construct (as described above) to form tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc-2A- tPA-RBD-Foldon. When expressed, the included 2A peptide facilitates the separation of the downstream portions of the polyprotein (i.e., the tPA-RBD-IMX313P or tPA-RBD- Foldon) at the time of translation. As these are operatively linked to the tPA signal peptide, this facilitates the secretion of the oligomerised forms of the RBD peptide from the cell.

[0227] Finally, three additional versions (tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF, tNSP3-Nuc-2A-tPA-RBD-Foldon-PRF, and tNSP3-Nuc-PRF) of the poly-gene constructs were prepared to include a downstream perforin encoding gene. This was operatively linked to an SV40 promoter sequence to drive its expression independently of the other encoded proteins.

[0228] The gene constructs were cloned into the pVAX DNA Vector (ThermoFisher Catalogue number: V26020) under the control of the CMV promoter. The inserts and vectors were assembled via NEBuilder Hifi DNA assembly (New England Biolabs). Prior to being transformed into DH5-a E. coli cells using heat shock transformation as described below.

[0229] The assembled DNA mix was added to DH5-a E coli cells, incubated on ice for 30 minutes and then heat shocked at 42°C for 45 seconds, followed by a further incubation on ice and the addition of SOC growth media. Transformed DH5-a E coli cells were incubated at 37°C with mixing and then spread on Lura Broth (LB) agar plates supplemented with kanamycin and incubated overnight at 37°C.

[0230] Positive bacterial colonies were picked and suspended in Milli-Q (MQ) water and correct insertion of the constructs was confirmed by PCR and restriction enzyme digestion followed by gel electrophoresis. Positive clones that showed the correct bands were sequenced with the nucleic acid sequence aligned against the known sequence of the gene of interest using BLAST to confirm the correct insertion.

[0231] The above protocols produced DNA vectors including the gene constructs set forth in table 4 below.

Table 4 - List of Prepared Gene Constructs

[0232] Example 2 - Confirmation of Protein Expression

[0233] The DNA vectors were confirmed to be functional, and the proteins expressed in Human Embryonic Kidney 293T (HEK293T), as show in figures 3 to 8.

[0234] To detect protein expression, HEK293T cells were seeded in 96-well flat- bottom plates and transfected with 200ng/well of DNA vector using Lipofectamine LTX

(Life Technologies). Forty-eight hours after transfection (for S1 and RBD vectors) or 36 hours after transfection (for all other vectors), the cells were fixed with 4% paraformaldehyde and permeabilised with methanol. Cells were then incubated with primary antibodies (as set out in table 5) for 24 to 48 hrs at 4°C, washed and then incubated with a suitable secondary antibody (see table 5) for 1 to 2 hrs at 4°C.

Table 5 - Primary and Secondary Antibodies Used

[0235] Fluorescence of the transfected cells was viewed under the Zeiss LSM-700 microscope and the data digitised using the Zen software (Zeiss). [0236] Figures 3 and 4 confirm specific transfection of, and protein expression in,

HEK293T cells by RBD and S1 mono-gene DNA vaccines. Figure 3 illustrates anti- RBD staining in all cells transfected with either the S1 subunit of the Spike protein or the RBD of the Spike protein, while minimal staining was seen in empty pVax transfected cells. Figure 4 illustrates anti-Spike staining in all cells transfected with either the S1 subunit of the Spike protein or the RBD of the Spike protein, while minimal staining was seen in empty pVax transfected cells. As can be seen the anti-Spike antibody binds both the S1 subunit and the RBD region of the S1 subunit indicating that it binds to at least an epitope within the RBD region.

[0237] Figures 5 and 6 confirm specific transfection of, and protein expression in, HEK293T cells by poly-gene DNA vaccines encoding the proteins NSP3 (figure 5) or Nucleocapsid (Figure 6). Figure 5 illustrates anti-V5 staining in all cells transfected with either NSP3, NSP3 linked with truncated perforin (NSP3-PRF), Nucleocapsid or Nucleocapsid linked to truncated perforin (Nuc-PRF), while no staining was seen in empty pVax transfected cells. Because the V5 tag was linked to both NSP3 and the Nucleocapsid genes, all transfected cells should stain positive. Figure 6 illustrates anti- Nucleocapsid staining in all cells transfected with Nucleocapsid expressive pVax vector (i.e., Nuc or Nuc-PRF), while cells transfected with empty pVax vectors showed no staining with anti-Nuc antibodies.

[0238] Figures 7 and 8 confirm specific transfection of, and protein expression in, HEK293T cells by poly-gene DNA vaccines encoding the polyproteins tNSP3-Nuc-2A- tPA-RBD-Foldon, tNSP3-Nuc-2A-tPA-RBD-Foldon-PRF, tNSP3-Nuc-2A-tPA-RBD- IMX313P and tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF. Figure 7 illustrates anti-RBD staining in all cells transfected with the poly-gene vectors. Figure 8 illustrates anti-Spike staining in all cells transfected with the poly-gene vectors.

[0239] Figure 34 confirms specific transfection of, and protein expression in, HEK293T cells of poly-gene DNA vaccines encoding the polyproteins tNSP3-Nuc-2A- tPA-RBD-Foldon, tNSP3-Nuc-2A-tPA-RBD-Foldon-PRF, tNSP3-Nuc and tNSP3-Nuc- PRF, as well as the mono-gene DNA vaccines encoding Nuc, Nuc-PRF and tPA-RBD- Foldon. Empty pVax DNA vectors were used as negative controls. As can be seen, polygenic vectors comprising Nucleocapsid, NSP3 and RBD showed expression of these peptides as confirmed with anti-Nuc, anti-NSP3, anti-RBD and anti-spike antibodies, with the anti-spike antibody binding to epitopes in the RBD (note anti-RBD and anti-spike staining for tNSP3-Nuc-2A- tPA-RBD-Foldon and tNSP3-Nuc-2A-tPA- RBD-Foldon-PRF are duplicates of those in Figures 7 and 8). Further, Nuc and Nuc- PRF were confirmed to successfully transfect and express Nucleocapsid peptide. [0240] To confirm oligomerization and secretion of the expressed RBD protein, western immunoblots were performed on cell supernatants from incubated cells. The supernatants from transfected HEK293T cells were collected 72 hours post transfection and centrifuged at 2,000xg for 5 minutes with the aspirate filtered to remove cell debris. Proteins were concentrated using 30 kDa filters (Merck).

[0241 ] Western immunoblot analysis was performed on 60pg of protein under reducing and non-reducing conditions on a 12% SDSPAGE gels prior to transfer onto PVDF membranes. Membranes were blocked and then incubated with primary anti- RBD antibody (see table 5), washed four times, followed by the addition of the secondary antibody Goat anti-rabbit IgG HRP (Invitrogen). The membranes were washed and visualised using Western Lightning Ultra (Perkin-Elmer) and LAS-4000 western-blot imaging system (Fujifilm).

[0242] As shown in Figure 9, HEK293T transfected with pVAX-tPA-RBD-IMX313P (lane B) and pVax-tPA-RBD-Foldon (lane C) secrete proteins comprising the receptor binding domain of SARS-CoV-2 into culture supernatant. Lane A contained a positive control of SARS-CoV-2 RBD with a C-terminal His-tag (residues 319-541 ; kindly provided by Prof Florian Krammer - see Amanat, F. et al. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature Medicine, 26(7), 1033- 1036), and lane D comprised supernatants from empty pVax transfected cells.

[0243] Proteins isolated from culture supernatants were subjected to SDS-PAGE and immunoblotted under native (figure 9A) and reducing (figure 9B) conditions. The expected molecular masses of RBD is ~37kDa.

[0244] As can be seen in Figure 33 proteins were isolated from culture supernatants indicating protein expression. As shown in figure 9A, under native conditions the molecular weight of the proteins in lanes B and D was higher than the expected weight for the monomer, thereby showing oligomerisation of the secreted proteins. By comparison, when run under reducing conditions (Figure 9B) the molecular weight of the protein in lanes B and C were approximately the expected 37kDa. These figures confirm that the RBD protein was both secreted and oligomerised.

[0245] Example 3 - Immunogenicity of DNA Vectors [0246] Each DNA vector was shown to elicit an immune response in vivo by identifying SARS-CoV-2 -specific antibodies and T-cells in vaccinated mice as described below.

[0247] Vaccination Protocol [0248] Six- to eight-weeks old female BALB/c mice (n = 7 per treatment group) were maintained under PC2 conditions and immunised with the dosage regimen provided in table 6. All DNA vaccines were given via intra-dermal administration.

Table 6 - Murine Vaccination Protocol

[0249] Detection of B-Cell response and Antibody Production

[0250] As shown in figures 9-12, antibodies against S1 protein, RBD or Nuc were identified in mice vaccinated as described above.

[0251 ] Figure 10 shows that mice vaccinated with pVax DNA vectors encoding secreted heptameric S1 protein and secreted heptameric RBD peptide (i.e. , tPA-S1 - IMX313P and tPA-RBD-IMX313P), as well as secreted trimeric S1 protein and secreted trimeric RBD peptide (i.e., tPA-S1 -Foldon and tPA-RBD-Foldon), have circulating anti- RBD antibodies three weeks (Figure 10A) after a single dose. This antibody response is further elevated at week 9, three weeks after the third vaccinations (Figure 10B). By comparison animals administered the empty pVax vector showed no antibodies for the RBD peptide.

[0252] Figure 11 shows that mice vaccinated with pVax DNA vectors encoding secreted heptameric S1 protein and secreted heptameric RBD peptide (i.e., tPA-S1 - IMX313P and tPA-RBD-IMX313P), as well as secreted trimeric S1 protein and secreted trimeric RBD peptide (i.e., tPA-S1 -Foldon and tPA-RBD-Foldon), have anti-Spike antibodies three weeks (Figure 11 A) after a single dose. This antibody response is further elevated at least three weeks after the third vaccinations (Figure 11 B). By comparison animals administered the empty pVax vector showed no antibodies for the Spike protein.

[0253] Figure 12 shows that mice vaccinated with pVax DNA vectors encoding Nucleocapsid protein and Nucleocapsid protein linked to truncated perforin protein (i.e. Nuc and Nuc-PRF) produced elevated anti-Nuc antibodies at least two weeks after the first vaccination and rising with the second (4 week) and third (6 week) vaccination. The antibody response was higher when truncated perforin was expressed together with the Nucleocapsid protein. By comparison animals administered the empty pVax vector showed no antibodies for the Nucleocapsid protein. [0254] Figure 13 shows that mice vaccinated with pVax DNA vectors encoding a polyprotein comprising a truncated Non-Structural Protein 3 linked to the Nucleocapsid protein and including a secreted heptameric RBD peptide (i.e., tNSP3-Nuc-2A-tPA- RBD-IMX313P) produce anti-RBD antibodies (Figure 13A - squares) and anti-Nuc antibodies (Figure 13B - squares). Additionally, vaccination with pVax DNA vectors encoding a polyprotein comprising a truncated Non-Structural Protein 3 linked to the Nucleocapsid protein and including a secreted heptameric RBD peptide and encoding truncated perforin protein (i.e., tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF) produced anti- RBD antibodies (Figure 13A - triangles) and anti-Nuc antibodies (Figure 13B - triangle).

[0255] The data represent mean responses in each group (n =7) ±SEM. *p < 0.05, **p < 0.01 (Mann-Whitney non-parametric t-test) comparison between Nucleocapsid with or without perforin.

[0256] As with the other vaccines these antibodies were present at least two weeks after the first vaccination and rose with the second and third vaccination up to the end point, two weeks after the third vaccination, with the exception of anti-RBD antibodies for the tNSP3

[0257] Nuc-2A-tPA-RBD-IMX313P-PRF vaccine which appeared to peak 1 week after administration of the second dose and then plateaued.

[0258] ELISA Protocol

[0259] The antibody titres in Figures 11 and 12 were determined by ELISA as described below.

[0260] Recombinant SARS-CoV-2 Spike protein and RBD peptide were produced and purified in house. Prefusion SARS-CoV-2 Spike ectodomain (isolate WHLI1 , a. a. residues 1 -1208) with HexaPro mutations (kindly provided by Dr Adam Wheatley - see Hsieh, C.-L., et al. (2020). Structure-based design of prefusion-stabilized SARS-CoV- 2 Spikes. Science, 369 (6510), 1501-1505) and SARS-CoV-2 RBD with a C-terminal His-tag (residues 319-541 ; kindly provided by Prof Florian Krammer - see Amanat, F. et al. (2020). A serological assay to detect SARS- CoV-2 seroconversion in humans. Nature Medicine, 26(7), 1033-1036) were overexpressed in cells and purified. Specifically, HEK293F (Expi293F) cells were cultured in Expi293^TM Expression Medium (ThermoFisher) and transfected with Endofree DNA (SARS-CoV-2 spike or RBD) using ExpiFectamineTM 293 transfection kit (ThermoFisher), according to the manufacturer’s protocol. The encoded SARS-CoV-2 spike protein was the HexaPro variant - see Hsieh C.L., et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science. 2020;369(6510):1501 -1505. doi:10.1126/science.abd0826. Cells were harvested 5 days post-transfection and centrifuged at 4000xg for 20 mins at 4°C. Cell supernatant was collected, filtered and concentrated using a 30kDa Amicon filter. Concentrated supernatant was mixed with NiNTA agarose resin (QIAGEN, cat. no. 166017069) in a ratio of 1 volume of resin to 5 volumes of concentrated supernatant and incubated overnight at 4°C. The mixture was then loaded into 5-mL polypropylene columns (QIAGEN), washed and the recombinant proteins were eluted with the elution buffer (see Stadlbauer, D.et al (2020). SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Current Protocols in Microbiology, 57). The elute was then concentrated using a 30kDa Amicon filter units by centrifugation at 4000xg for 30 mins at 4°C. Protein concentration was determined by Bradford assay.

[0261 ] Nucleocapsid protein was commercially obtained from Sino Biological (Catalogue number 40588-V08B).

[0262] Enzyme-Linked Immunosorbent Assays (ELISAs) were performed by coating MaxiSorp 96-well ELISA plates with 1 pg/mL of recombinant SARS-CoV-2 RBD, Spike protein or Nucleocapsid protein overnight at 4°C. The wells were then blocked with 50pl/well of StartingBlock PBS Blocking Buffer (ThermoFisher) for 5 minutes at room temperature.

[0263] Serial diluted mice serum samples were transferred to ELISA plates and incubated at 37°C for 3 hours (for Spike and RBD) and 4hrs for Nuc. The plates were then washed four times with PBST (1xPBS, 0.05% Tween). Secondary antibody Goat anti-Mouse IgG HRP conjugated (1 :3000 dilution in StartingBlock - Invitrogen), was added to the plates and incubated at 37°C for 2 hours. The plates were then washed 3-6 times with PBST (1xPBS, 0.05% Tween). Plates were then developed with 1 - Step™ Ultra TMB Substrate (ThermoFisher) and the reaction stopped with 2M sulphuric acid. Absorbance (optical density “OD”) was measured at 450nm. The cut-off value for ELISA was determined by average OD reading of negative control (pVax) plus 2 standard deviations. Endpoint titres were determined as the reverse of the highest serum dilution with OD reading above the cut-off.

[0264] Detection of in vitro IFN gamma Production

[0265] Having confirmed that the vaccines of the invention could induce a B-cell response (as indicated by antibody production) the ability of the vaccines to induce a T-cell response and cytokine production to specific portions of SARS-CoV-2 proteins was investigated by an ELIspot assay.

[0266] As can be seen in Figures 14 to 21 all vaccines investigated resulted in the production of IFN-y in response to peptide portion of SARS-CoV-2 proteins.

[0267] An ELISpot assay was used to assess specific IFN-y release by splenocytes form vaccinated mice to peptides spanning the RBD region of the Spike protein, the S1 subunit of the Spike protein, Nucleocapsid protein and Non-Structural Protein 3. ELISpot was performed as previously described (see Masavuli, M.G., et al., (2019). A Hepatitis C Virus DNA Vaccine Encoding a Secreted, Oligomerized Form of Envelope Proteins Is Highly Immunogenic and Elicits Neutralizing Antibodies in Vaccinated Mice. Frontiers in Immunology. 10: p. 1145.)

[0268] Red blood cell-depleted splenocytes from vaccinated mice, at 5x10⁵ cells per well, were stimulated for 36hrs at 37°C with 4 pg/ml of either S1 , RBD, Nuc or NSP3 peptides (SARS-Cov-2 Spike peptides cat. no. NR-3010, Nucleocapsid peptides Cat. No. NR-2670, provided by the National Institutes for Health Biodefense and Emerging Infectious Research Resources Repository (NIAID), Bethesda, MD). Phytohemagglutinin (PHA) was added as a positive control and R10 media alone represented the negative control.

[0269] Secreted IFN-y was detected using anti-mouse IFN-y-biotin (clone R4-6A2; MabTech), streptavidin-AP and SigmaFast BCIP/NBT™ (Sigma). An ELISpot reader (AID Germany) was used to count the spots on the plates. The average number of spot forming units (SFU) in the unstimulated samples was subtracted from the number of spots formed in the stimulated samples and the data adjusted to SFU per 10⁶ splenocytes and presented as mean ± the standard error of the mean (SEM). [0270] RBD and S1 Vaccinated Mice

[0271 ] As can be seen in Figures 14 and 15, mice vaccinated with the vaccine vectors encoding for heptameric and trimeric RBD and S1 proteins had splenocytes reactive with peptides from both RBD and S1 proteins.

[0272] To elucidate the portions of the RBD and S1 proteins which contribute to the T-cell and cytokine response the peptides were separated into pools. The RBD peptide pool consisted of one pool of 30 overlapping 15-19mer peptides spanning the entire RBD protein. The peptides spanning the S1 protein were divided into three pools containing 31 peptides each (referred to as S1 pool 1 , S1 pool 2 and S1 pool 3 - see table 7 ).

[0273] Table 7 - S1 Protein Peptide Pools

[0274] As can be seen in Figure 14, mice vaccinated with heptameric RBD vaccine vectors (i.e., RBD-IMX313P) demonstrated 2040 spot forming units (SFUs) per million cells when exposed to the pooled 15-19mers peptides of the RBD protein. By comparison, less than half the number (917) of SFUs were observed when splenocytes from mice vaccinated with heptameric-S1 (S1 -IMX313P) vaccine vectors were incubated with the same RBD peptides.

[0275] Further, Figure 14 demonstrates the approximately 3500 SFUs were produced when splenocytes from mice vaccinated with both heptameric-RBD (3437 SFUs) and heptameric-S1 (3344) vaccine vectors were incubated with S1 peptides. The relative contribution of each pool is provided in table 8, below.

[0276] Table 8 - Relative Contribution of S1 Peptides in IFN-y Production from Mice Vaccinated with Heptameric RBD and Heptameric S1 Vaccines.

[0277] Figure 15 shows mice vaccinated with trimeric RBD vaccine vectors (RBD- Foldon), demonstrated a relatively lower number (1507) of SFUs per million cells compared to the heptameric RBD vaccines, when splenocytes were exposed to pooled 15-19mers peptides of the RBD protein, with a comparable number (1409) of SFUs also formed when splenocytes from mice vaccinated with the trimeric S1 protein vector (S1 -Foldon) were exposed to peptides from the RBD protein.

[0278] When splenocytes from mice vaccinated with the trimeric RBD vectors were incubated with peptides from the S1 protein 2160 SFUs were generated. Again, and similar to Figure 14, peptides in pool 3 stimulated the largest response (see table 9, below).

[0279] The largest response was seen in splenocytes from mice vaccinated with the trimeric S1 vector and incubated with peptides derived from the S1 protein. As illustrated in Figure 15, 5631 SFUs were generated, with peptides in pool 2 accounting for the greatest number of SFUs (see table 9, below).

[0280] Table 9 - Relative Contribution of S1 Peptides in IFN-y Production from Mice Vaccinated with Trimeric RBD and Trimeric S1 Vaccines.

[0281] Non-Structural Protein 3 Vaccinated Mice [0282] As can be seen in Figure 16, mice vaccinated with the vaccine vectors encoding for NSP3 or NSP3 linked to truncated perforin (NSP3-PRF) had splenocytes reactive with peptides from the NSP3 protein.

[0283] To elucidate the portions of the NSP3 protein which contribute to T-cell and cytokine responses, peptides, which covered the length of the NSP3 protein, were separated into ordered pools. Specifically, the NSP3 peptides were formed into 22 pools each comprising 22 overlapping 15-19mer peptides as set out in table 10.

[0284] Table 10 - Non-Structural Protein 3 Peptide Pools

0285] Pool and peptide numbers refer to table 14: P1_01 - Pool 1 peptide 1 . [0286] As illustrated in Figure 16 isolated splenocytes were particularly reactive to

NSP3 peptide in pool 8, 16 and 19-22 with cells from mice vaccinated with both NSP3 alone and NSP3 together with truncated perforin showing comparable reactivity.

[0287] Nucleocapsid Vaccinated Mice [0288] As can be seen in Figure 17, mice vaccinated with vectors encoding for Nucleocapsid or Nucleocapsid linked to truncated perforin (Nuc-PRF) had splenocytes reactive with peptides from the Nucleocapsid protein.

[0289] To elucidate the portions of the Nucleocapsid protein which contributed to T- cell and cytokine responses, peptides, which covered the length of the Nucleocapsid protein, were separated into eight ordered pools consisting of 8 overlapping 15-19mer peptides (with the exception of pool 4 which consisted of 4 overlapping peptides) as set out in table 11 .

[0290] Table 11 - Nucleocapsid Peptides Pools

[0291 ] As illustrated in figure 17, peptides in pools 2 to 5 stimulated the most IFN-y release from splenocytes from vaccinated mice. Notably, for these pools, the IFN-y release from mice vaccinated with the Nucleocapsid protein alone was similar to that released from splenocytes isolated from mice vaccinated with vectors encoding both the Nucleocapsid and truncated perforin. However, pools 1 and 6-8 only induced IFN- y release in mice vaccinated with Nuc-PRF.

[0292] Polyprotein Vaccinated Mice

[0293] As can be seen in Figures 18 to 21 , mice vaccinated with the poly-gene vectors encoding three SARS CoV-2 proteins had splenocytes which were reactive with peptides derived from the S1 portion of the SARS CoV-2 Spike protein, the RBD portion of the Spike protein, the Nucleocapsid protein and NSP3. Specifically, the mice were vaccinated with poly-gene vectors encoding a polyprotein comprising truncated Non- Structural Protein 3, Nucleocapsid protein and the heptameric receptor binding domain portion of the Spike protein with or without truncated perforin (i.e. tNSP3-Nuc-2A-tPA- RBD-IMX313P and tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF). [0294] Identification of T-Cell Restricted Peptide Portions

[0295] Truncated NSP3 Peptides

[0296] To elucidate the portions of the truncated NSP3 protein that contribute to T- cell and cytokine responses in polyprotein vaccinated mice, peptides, which covered the length of the truncated NSP3 protein, were separated into ordered pools. Specifically, the NSP3 peptides were formed into seven peptide pools of 30 or 31 overlapping peptides which covered the truncated portion of the NSP3 protein spanning from amino acids 1200 to 1946, each pool comprising 22 overlapping 15-19mer peptides as set out in table 12. The pools of peptides were then incubated with splenocytes from vaccinated mice. [0297] Table 12 - truncated NSP3 Peptide Pool

[0298] Pool and peptide numbers refer to table 14: P1_01 - Pool 1 peptide 1 .

[0299] Figure 18 illustrates that splenocytes from mice vaccinated with the polygene vector encoding a polyprotein produced significant IFN-y when incubated with peptides from pools 14 and 16, with only modest IFN-y produced when incubated with the remaining pools. The presence of truncated perforin in the vaccine did not make a notable difference to the IFN-y release from splenocytes incubated with any of the seven pools of peptides.

[0300] Nucleocapsid Peptides

[0301 ] To elucidate the portions of the Nucleocapsid protein that contribute to T-cell and cytokine responses in polyprotein vaccinated mice, peptides, which covered the length of the Nucleocapsid protein, were separated into two pools containing 30 and 29 overlapping 15-19mer peptides (as set out in table 13). The pools of peptides were then incubated with splenocytes from vaccinated mice.

[0302] Table 13 - Nucleocapsid Protein Peptide Pools

[0303] Figure 19 shows that peptides in both pool 1 and pool 2 induced similar IFN- y release from splenocytes isolated from vaccinated mice. Further, mice vaccinated with the poly-gene vector including truncated perforin produced similar levels of IFN-y to mice vaccinated in the absence of truncated perforin.

[0304] Receptor Binding Domain Peptides

[0305] To elucidate the portions of the RBD region of the Spike protein that contribute to T-cell and cytokine responses in polyprotein vaccinated mice, peptides, which covered the length of the RBD region were incubated with splenocytes from vaccinated mice.

[0306] As illustrated in Figure 20, approximately 300 SFUs were formed when splenocytes from vaccinated mice were incubated with RBD peptides, with a comparable number of SFUs being generated in the absence and presence of truncated perforin.

[0307] S1 Portion of Spike Protein Peptides [0308] To elucidate the portions of the S1 subunit of the Spike protein that contribute to T-cell and cytokine responses in vaccinated mice, peptides, which covered the length of the S1 subunit were separated into three pools (see table 7). The pools of peptides were then incubated with splenocytes from vaccinated mice.

[0309] As illustrated in Figure 21 over 500 SFUs were formed when splenocytes from vaccinated mice were incubated with S1 peptide pools. The majority of the SFUs were produced in response to the peptides in pool 3. Again, comparable numbers of SFUs were generated from the vaccine in the absence and presence of truncated perforin.

[0310] Detection of T-cell Activation and Killing

[0311 ] To determine the portion of the proteins used in the vaccines which are targets for T-cell responses, a fluorescent target assay (FTA) was performed.

[0312] As can be seen from Figures 22 and 23, T-cells are activated during vaccination which target the NSP3 peptides in pools 6, 10, 11 and 14-16. Furthermore, as shown in Figures 24 and 25, T-cells are activated during vaccination which primarily target peptides in pool 1 (i.e., the first half) of the Nucleocapsid peptide.

[0313] To perform the FTA, mice were immunised as described above and 13 days after the final vaccination FTA was conducted. Briefly, naive autologous splenocytes collected from age and sex matched BALB/c mice were serially labelled with various concentrations of cell proliferation dye (CPD), cell trace violet (CTV), and carboxyfluorescein succinimidyl ester (CFSE) at RT for 5 min to produce distinct populations of cell with discernible CFSE, CPP and CTV profiles (as previously described in Mekonnen, Z.A., et al., (2019) Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T-cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus. Journal of Virology, 93, 19). The dye-labelled cells were un-pulsed (nil) or pulsed with 10 pg/ml of peptide pools spanning Nuc (see table 13, above) or NSP3 proteins (see table 14, below) for 4 h at 37°C with 5% CO2. Following labelling, 1 .5 million Nil or peptide pulsed targets were resuspended in PBS and injected into the lateral vein of mice vaccinated with vectors encoding NSP3, NSP3-PRF, Nuc or Nuc-PRF. [0314] Fifteen hours after the FTA challenge, red blood cell-depleted splenocytes were isolated and stained with PE-Cy7-conjugated anti-mouse CD69 (clone H1.2F3; BD Biosciences) and Alexa Fluor 700-conjugated anti-mouse B220 (RA3-6B2; Invitrogen) for flow cytometric analysis as previously described (Mekonnen et al. - see above).

[0315] The percentage of specific killing of target cells was calculated based on the percentage of FTA cells recovered using the formula [(nil target value % - peptide- pulsed target value %)/nil target value %] x 100. The plotted values for Geometric Mean Fluorescent Intensity (GMFI) of CD69 on B220+ FTA targets was calculated using the formula B220+ peptide-pulsed target value (GMFI of CD69) - B220+ nil target value

(GMFI of CD69).

[0316] Table 14 - NSP3 Peptide Pools Used for FTA

[0317] Figure 22 illustrates that target cells pulsed with peptides in pools 6, 10, 11 and 14-16 were specifically killed (as demonstrated by increase killing in vaccinated mice compared to sham (pVax) vaccinated mice). This indicates that cytotoxic T- lymphocytes (CTLs) are activated by peptide sequences in these pools as a result of vaccination with NSP3 encoding vectors. [0318] Upon recognition of cognate antigens, T helper (Th) cells co-stimulate B cells, which leads to upregulation of CD69 on mature (B220+) B cells. Consequently, in vivo T helper responses from the sham vaccinated mice and the mice vaccinated with NSP3, NSP3-PRF, Nuc or Nuc-PRF were analysed by assessing the activation of B220+ B cell populations within each distinct population of peptide pulsed cells (i.e. each population having a distinct CFSE, CPD and CTV profile). Specifically, the expression (as determined by the GMFI) of the activation marker CD69 was assessed on each of distinct populations of cells which had been pulsed with pooled peptides.

[0319] Figure 23 illustrates that T-helper cells in NSP3 vaccinated mice were activated by peptides in pools 7, 10, 11 and 14-16 as indicated by an elevated expression of CD69 on B220+ cells isolated from vaccinated mice compared to sham vaccinated mice.

[0320] Figure 24 illustrates that target cells pulsed with peptides in pool 1 were specifically killed by cytotoxic T-lymphocytes (CTLs) in mice vaccinated with vectors encoding Nucleocapsid protein. By comparison, peptides in pool 2 did not induce any demonstrable killing.

[0321 ] Figure 25 illustrates that T-helper cells in Nuc vaccinated mice were primarily activated by peptides in pool 1 , with peptides in pool 2 inducing a lower expression of CD69 on B220+. However, peptides in both pool 1 and pool 2 induced elevated CD69 expression on B220+ cells in mice vaccinated with vectors encoding Nuc compared to sham vaccinated mice.

[0322] T-Cell Cytokine Production

[0323] To further characterise vaccine induced T-cell responses, intracellular cytokine staining assays were used to measure the numbers of CD4⁺ and CD8⁺ T-cells reactive with SARS-CoV-2 peptides.

[0324] As illustrated in Figures 25 to 32, mice vaccinated with various DNA vaccine vectors showed elevated production of IFN-y, TNF-a and IL-2 in both CD8+ and CD4+ T-cells [0325] Cells were isolated from vaccinated mice and stimulated with various SARS- CoV-2 peptides before the frequency of cells secreting IFN-y, TNF-a and IL-2 was assessed via flow cytometry (FACS).

[0326] Specifically, mice were vaccinated with one of tPA-S1 -IMX313P, tPA-S1- Foldon, tPA-RBD-IMX313P, tPA-RBD-Foldon, tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF, as described above. Three-weeks post final vaccination splenocytes were harvested and stimulated for 12 hours with peptides spanning SARS-CoV-2 RBD, the S1 subunit of the Spike protein, NSP3 (pools 10-16, see table 12) or Nucleocapsid (pools 1 and 2, see table 13). Stimulated cells were further incubated for 4 hours after the addition of BFA and stained for extracellular cell markers (CD3, CD44, CD8 and CD4) and intracellular cytokine markers (IFN-y, IL-2, and TNF-a).

[0327] FACS analysis was performed on splenocytes which were gated to select lymphocytes using forward and side scatter followed by gating on live cell population.

[0328] Figure 26A illustrates that the number of IFN-y producing CD3⁺CD4⁺ T-cells was greater in mice that were vaccinated with tPA-S1-Foldon irrespective of the peptides used to stimulate these cells (i.e., RBD or S1 peptides). As shown in Figure 26B and C The absolute numbers of IL-2 or TNF-a secreting CD3⁺CD4⁺ T-cells were comparable between mice vaccinated with vectors encoding tPA-S1-Foldon and mice vaccinated with vectors encoding tPA-RBD-Foldon.

[0329] Figure 27 shows CD8⁺ T-cells from mice vaccinated with vectors encoding for tPA-S1 -Foldon showed greater production of IFN-y (Figures 27A), TNF-a (Figure 27B) or IL-2 (Figure 27C) when cells were stimulated with S1 peptide in pool 1 (P1 ) and pool 2 (P2). Likewise, CD8⁺ T-cells from mice vaccinated with vectors encoding tPA- RBD-Foldon also showed elevated IFN-y, TNF-a and IL-2 when stimulated with RBD or S1 pool 3 (P3) peptides.

[0330] As illustrated in Figures 28 and 29, isolated CD4+ and CD8+ T-cells from mice vaccinated with vectors encoding for the polyprotein vaccines tNSP3-Nuc-2A-tPA- RBD-IMX313P and tNSP3-Nuc-2A-tPA-RBD-IMX313P-PRF showed increased production of IFN-y, TNF-a and IL-2 when exposed to pooled peptides from NSP3 (pools 10 to 16 - see table 14). [0331 ] As illustrated in Figure 28 CD4+ T-cells showed an increase in the production of IFN-y (Figure 28A), TNF-a (Figure 28B) and IL-2 (Figure 28C) indicating significant activation of T-helper cells following vaccination with vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P and tNSP3-Nuc-2A-tPA-RBD- IMX313P-PRF.

[0332] As illustrated in Figure 29, CD8+ T-cells showed an increase in the production of IFN-y (Figure 29A), TNF-a (Figure 29B) and IL-2 (Figure 29C) indicating significant activation of cytotoxic T-cells following vaccination with vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P and tNSP3-Nuc-2A-tPA-RBD- IMX313P-PRF, respectively.

[0333] Figure 30 illustrates that CD4+ T-cells from mice vaccinated with DNA vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc- 2A-tPA-RBD-IMX313P-PRF produced elevated levels of IFN-y (Figure 30A), TNF-a (Figure 30B) and IL-2 (Figure 30C) in response to Nuc peptide pools 1 and 2 (see table 13).

[0334] Figure 31 illustrates that CD8+ T-cells from mice vaccinated with DNA vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc- 2A-tPA-RBD-IMX313P-PRF produced elevated levels of IFN-y (Figure 31 A), TNF-a (Figure 31 B) and IL-2 (Figure 31 C) in response to Nuc peptide pools 1 and 2 (see table 13).

[0335] The results in Figures 29 and 30 show that vaccination with DNA vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc-2A-tPA- RBD-IMX313P-PRF results in activating T-cell populations reactive with Nucleocapsid protein peptides. Notably, peptides in both pool 1 and pool 2 re-stimulated primed T- cells to a comparative level indicating that T-cell restricted peptides exist in both pools.

[0336] Figures 32 and 33 illustrates expression of IFN-y, TNF-a and IL-2 in CD4+ T-cells (Figures 32) and CD8+ T-cells (Figures 33) from mice vaccinated with DNA vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc- 2A-tPA-RBD-IMX313P-PRF which were re-stimulated with peptides spanning the RBD of the Spike protein. Consequently, vaccination with the DNA vectors encoding the polyproteins tNSP3-Nuc-2A-tPA-RBD-IMX313P or tNSP3-Nuc-2A-tPA-RBD-IMX313P- PRF results in activation of RBD specific T-cells.

[0337] SARS-CoV-2 Peptide Pools

[0338] Table 15 - S1 peptides

[0339] Bo d sequence = Peptides in RBD region

[0340] Table 16 - truncated NSP3 peptides

[0341] Table 17 - Nucleocapsid Peptides

[0342] Example 4 - SARS CoV-2 Variants Constructs

[0343] Preparation of RBD Variant Constructs

[0344] DNA vectors encoding secreted receptor binding domain (sRBD - i.e. , tPA signal peptide linked to RBD as per Figure 1 i) was generated for the ancestral Wuhan strain, the Delta strain and the Omicron strain. Versions of this construct, including the Foldon oligomerization protein (as per Figure 1 d), were also generated - as described above.

[0345] HEK293T cells were transduced with these vectors prior to staining with anti- RBD and anti-Spike antibodies (in accordance with the methods of Example 2, above).

[0346] Figure 35 shows in intracellular expression of the RBD portion of the S1 protein from these constructs.

[0347] Vaccination of Mice with Constructs Including Variants of SARS CoV- 2 Induces Neutralising Antibodies

[0348] Delta Variant.

[0349] To identify if RBD variants of SARS CoV-2 showed comparable immunogenicity to the original Wuhan variant, variant versions of RBD vaccines were produced.

[0350] Mice were administered with one of two vaccination schedules: Group (1 ) two doses of 75 pg of tPA-RBD-Foldon vector (black syringes in Figure 36A) on day 0 and 14, and one dose of 75 pg tPA-RBDA-Foldon vector (delta variant of RBD - grey syringes) on day 28; or group (2) three 75 pg doses of tPA-RBDA-Foldon vector (grey syringes) at days 0, 14 and 28 (see schedule Figure 36A).

[0351 ] Anti-RBD (Wuhan-Hu-1 ) serum antibodies titers were measured by ELISA at the days 7, 14, 21 , 28, 35 and 42, as describe in Example 3.

[0352] Neutralising antibodies were measured in according with the protocol disclosed in Tea, F., et al., SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants. PLoS Med, 2021. 18(7): p. e1003656. Briefly, HEK-ACE2/TMPRSS cells (Clone 24) were seeded in 384-well plates at a concentration of 5 x 10³ cells/well in the presence of the live cell nuclear stain Hoechst- 33342 dye (NucBlue, Invitrogen) at a concentration of 5% v/v. Two-fold dilutions of mouse serum samples from vaccinated mice were mixed with an equal volume of SARS-CoV-2 virus solution (1.25 x 10⁴ TCID50/ml) and incubated at 37°C for 1 h before adding 40 pl, in duplicate, to the cells (final MOI = 0.05). Viral variants used included the key variants of concern; Beta (B.1 .351 ), Delta (B.1 .617.2) and Omicron (B.1 .1 .529), as well as control virus (A.2.2) from clade A and presenting no aa mutations in Spike (denoted as “Wuhan”). Plates were incubated for 24 h post-infection and entire wells were imaged by high-content fluorescence microscopy, cell counts obtained with automated image analysis software, and the percentage of virus neutralization was calculated with the formula: %N = (D-(1 -Q)) x 100/D. An average %N > 50% was defined as having neutralizing activity.

[0353] Figures 36B shows antibody titers (expressed as the reciprocal of the dilution factor of the serum and plotted as Log10). Statistical significance tested between group vaccinated with 3 x tPA-RBDA-Foldon and the group vaccinated with 2 x tPA-RBD-Foldon + 1 x tPA-RBDA-Foldon.

[0354] Figure 36C shows serum neutralization end-point cut-off titers (highest dilution factor that yields >50% inhibition of cell death after live-virus infection) against Wuhan, Beta, Delta and Omicron live-virus particles. Neutralization activity was considered negative, value of zero, when neutralization of initial serum dilution was <50%. The data represent mean responses in each group (n =7) ±SEM. *p < 0.05, **p < 0.01 (Mann-Whitney non-parametric t-test). [0355] As can be seen in figure 36B, both the vaccination regimens set out in figure 36A resulted in anti-RBD antibodies, with the dosage regimen of group 2 (squares on figure 36b) showing higher levels of antibody titres across all time points measured.

[0356] In figure 36C it is shown that the dosing schedule of group 2 (3 x delta RBD- Foldon) resulted in significantly higher neutralising antibody titres for delta infection and comparable neutralising antibody titres for infection with the ancestral Wuhan virus.

[0357] Vaccination of Mice with Constructs Including Variants of SARS CoV- 2 Induces Cytokine Secretion from Splenocytes

[0358] To identify if RBD variants of SARS CoV-2 showed comparable T-cell immunogenicity to the original Wuhan variant, splenocytes from mice vaccinated with variant versions of RBD construct were analysed.

[0359] Mice were vaccinated three times at 2-week intervals with DNA constructs encoding secreted RBD (Wuhan-Hu-1 and Delta isolate) fused to Foldon as shown in figure 36A. On day 28, splenocytes were harvested before being re-stimulated in duplicate with overlapping SARS-CoV-2 peptide pools representing the S1 subunit of the Spike protein (pools 1 to 3 as per Table 7) or RBD proteins from the Wuhan-Hu-1 , Alpha, Beta, Epsilon, Delta and Gamma isolates. IFN-y secretion was measured by ELISpot assay as set out above in Example 3. The data are expressed as mean (n = 7) SFUs per 10⁶ cells responses to different peptide pools and presented as the mean ± SEM per group. The number of SFUs in unstimulated cells was subtracted from the number in cells stimulated with peptides to generate the net RBD responses. *p < 0.05, ** P < 0.01 , ***p < 0.001 (Mann-Whitney test non-parametric t-test).

[0360] As can be seen in figure 37A, vaccination with 3 doses of vector expressing Wuhan RBD and Delta RB induced RBD-specific splenocytes which produce IFN-y in response to Wuhan spike protein peptides.

[0361 ] Figure 27B shows that splenocytes from mice vaccinated with 3 doses of the delta RBD-foldon construct had a significantly higher IFN-y secretion in response to RBD peptides from epsilon and delta variants, compared to mice who received 2 x Wuhan RBD and 1 x Delta doses of vaccine. Responses, to the Alpha, Beta and Gamma variants were comparable between the two vaccination groups. [0362] The frequency of cytokine producing T-cells in response to SARS-CoV-2 RBD peptides was assessed in mice vaccinated with RBD-Foldon (Wuhan-Hu-1 isolate) or RBDA-Foldon (Delta isolate) vaccine. Splenocytes from mice vaccinated as per figure 36A were harvested 14 days post-vaccination, before being re-stimulated with RBD pool peptides. CD8+ (38A) and CD4+ (38B) T-cells were permeabilised and stained for intracellular cytokines interferon gamma, interleukin 2, and/or tumor necrosis factor alpha, as described above in Example 3. Cells were analysed by flow cytometry.

[0363] CD8+ cells primarily produced either IFN-y or TNF-a or IFN-y and IL-2, with almost no notable expression of IL-2 alone (Figure 38A). A portion of CD4+ cells expressed one of IFN-y, TNF-a or IL-2, with a small portion of cells simultaneously expressing more than one cytokine (Figure 38B).

[0364] Omicron Variant

[0365] Female BALB/C mice (n = 7/group) were vaccinated at 2 weeks intervals (days 0, 14 and 28) via the ID route with three doses of either 75pg or 100pg of tPA- RBD-Foldon comprising the RBD sequence from the Omicron variant (Figure 39A). Antibody titres and neutralisation antibodies were assessed as previously discussed.

[0366] As shown in figures 39B and 39C, mice vaccinated with 3 x 75 pg showed comparable anti-RBD antibody titres to both the Wuhan and Delta variants, compared to mice vaccinated with 3 x 100 pg.

[0367] Further, the antibodies demonstrated neutralisation of infection from each of the Wuhan, Beta, Delta and Omicron viral variants, with the greatest neutralisation seen in serum derived from mice administered 3 x 75 pg doses against infection by the Delta variant virus (Figure 39D).

[0368] Interferon gamma production, as assessed via ELISpot (Figure 40), was seen in splenocytes of mice vaccinated as per figure 39A. Splenocytes were harvested 21 days after the final vaccination, re-stimulated in duplicate with overlapping SARS- CoV-2 peptide pools representing the RBD proteins from the Wuhan-Hu-1 , Delta and Omicron isolates, prior to ELISpot assessment. Specifically, cells were re-stimulated with RBD Delta mutant pooled peptides (RBD Delta mut pool - Miltenyi Biotec™ Cat# 130-129-568) which cover the mutated regions in the spike protein of the SARS-CoV- 2 Delta AY.1 lineage (a subvariant of B.1 .617.2 Delta variant) and the RBD Wuhan Ref Pool (Miltenyi Biotec™, Cat# 130-129-564) which consists of the 44 homologous peptides of the Wuhan sequence and serves as a control. Also used were pooled peptides corresponding to the RBD of the omicron variant.

[0369] As can be seen in Figure 40, vaccination of the RBD of the Omicron variant induces T-cells which are responsive to RBD peptides from the Wuhan and Delta variant strains, in addition to the Omicron RBD. Re-stimulation with the variant regions of the Wuhan and Delta RBD also resulted in IFN-y production, indicating a degree of immunogenic redundancy in the epitope regions of the RBD.

[0370] Additional analysis (by intracellular cytokine staining as described above) showed that IFN-y, TNFa and IL-2 were secreted by both CD8+ T-cells (Figures 41A) and CD4+ T-cells (Figure 41 B) in response to restimulation with peptides from the RBD of all three variants.

[0371] Response to Variants of SARS CoV-2 in Nucleocapsid and Non- Structural Protein 3 Vaccinated Mice

[0372] To determine the immune response to variants of SARs CoV-2 in mice vaccinated with polygenic DNA vaccines encoding truncated NSP3 (tNSP3) and Nucleocapsid protein, vaccinated mice were analysed for antibody production and T- cell responses to portions of the SARs CoV-2 genome of different variants.

[0373] Antibody production

[0374] Groups of mice (n = 7) were vaccinated with one of two vaccine schedules: (1 ) 50 pg of a construct encoding the polyprotein truncated NSP3 and Nucleocapsid (tNSP3-Nuc - Figure 2E) as well as 50 pg of a separate construct encoding the secreted form of the Delta variant RBD and Foldon (sRBDA-Foldon - Delta variant of the constructs of Figure 2D); or (2) 50 pg of a construct encoding the polyprotein truncated NSP3 and Nucleocapsid with Perforin (tNSP3-Nuc-PRF - Figure 2F) as well as 50 pg of the separate construct encoding the secreted form of the Delta variant RBD and Foldon. [0375] Mice were vaccinated at three-week intervals (days 0, 21 and 42 - see Figure 42A) before being sacrificed at 9 weeks. Anti-RBD- and anti-Nuc-specific serum antibodies titers (Figures 42B and 42C) were measured by ELISA (as described above) at the indicated time points. Titers are expressed as the reciprocal of the dilution factor of the serum and plotted as Log10. Figure 42D illustrates serum neutralization endpoint cut-off titers (highest dilution factor that yields >50% inhibition of cell death after live-virus infection) against Wuhan, Beta, Delta and Omicron live-virus particles (performed using the method discussed above). Twenty or 40 was the initial dilution for all serum samples. Neutralization activity was considered negative, value of zero, when neutralization of initial serum dilution was <50%. The data represent mean responses in each group (n =7) ±SEM. *p < 0.05, ** P < 0.01 , ***p < 0.001 (Mann-Whitney test non-parametric t-test).

[0376] As can be seen in figures 42B and 42C, both vaccination schedules resulted in anti-RBD antibodies (Figure 42B) and anti-Nucleocapsid antibodies (Figure 42C). Further, as illustrated in Figure 42D, both schedules resulted in significant neutralisation of virus infection with the Delta variant, and showed some neutralisation of infection with other variants including the Wuhan variant.

[0377] Cytokine production

[0378] Further groups of mice (n = 7/group) were vaccinated three times at 2-week intervals (days 0, 14, and 28) with one of the two schedules above (i.e. , tNSP3-Nuc + sRBDA-Foldon or tNSP3-Nuc-PRF + sRBDA-Foldon) before the mice were sacrificed on week 6 and splenocytes were collected. The splenocytes were re-stimulated in duplicate with overlapping peptide pools representing the SARS-Cov-2 NSP3 (Figure 43A), Nucleocapsid (Figure 43B), or RBD proteins (Figure 43C). Interferon gamma secretion was measured by ELISpot assay as previously described. The data are expressed as mean (n = 7) SFU per 10⁶ cells and presented as the mean ± SEM per group. The number of SFU in unstimulated cells was subtracted from the number in cells stimulated with peptides to generate the net SARS-CoV-2 response.

[0379] Interferon gamma was produced by splenocytes re-stimulated with peptides spanning from amino acids 1081 to 1945 of NSP3 (Figure 43A), with the peptides spanning positions 1565 to 1689 and 1813 to 1945 stimulating the most IFN-y production.

[0380] Further, as shown in Figure 43B, peptides in both pool 1 and pool 2 of the Nucleocapsid pools (see Table 13) induced IFN-y. And peptides from the Wuhan RBD, Delta mutant (Mut) pool and RBD Wuhan Ref pool (see discussion above), stimulated IFN-y production in cells from mice in both vaccination schedules (see Figure 43C).

[0381 ] Cell killing

[0382] Cells from mice vaccinated with vaccine constructs expressing NSP3, Nucleocapsid and RBD were shown to selectively kill cells pulsed with peptides from NSP3, Nucleocapsid and RBD of the Omicron and Wuhan variants.

[0383] Mice (n = 7/group) were vaccinated three times at 3-week intervals (days 0, 21 , and 42) with one of two schedules: (1 ) tNSP3-Nuc + sRBDA-Foldon or (2) tNSP3- Nuc-PRF + sRBDA-Foldon (as per Figure 42A). An FTA cell killing assay was performed as described in Example 3 with mice being challenged with I.V. injected naive peptide-pulsed or un-pulsed autologous splenocytes labelled with cell tracking dyes (CTV, CFSE, and CPD). Data is expressed as mean (n = 7) + SEM of the specific killing responses detected against each target cell population (in the FTA stained cells recovered from the spleen from immunized mice - Figure 44A to 44C). In Figures 44E to 44F, the graphs show the mean (n = 7) + SEM of the specific (beyond nil) expression (GMFI) of CD69 on gated B220+ cells within the FTA cells pulsed with different SARS- cov-2 peptides recovered from the spleen of vaccinated mice.

[0384] As can be seen cells pulsed with various NSP3 peptides were targeted for killing indicating anti-NSP3 cytotoxic cells (see Figure 44A). Further, target cells pulsed with Nucleocapsid peptides (particularly peptides in pool 1 ) and peptides of the Wuhan RBD were killed by cytolytic cells present in vaccinated mice (see Figures 44B and 44C, respectively). Helper cells were also present and responded to presented peptides in the NSP3 peptide pools (see Figure 44D), Nucleocapsid pool 1 (see Figure 44E) and Wuhan RBD peptides (see Figure 44F).

[0385] Definitions and Qualifications

[0386] Reference to an Electronic Sequence Listing [0387] The contents of the electronic sequence listing (SARS_CoV-2_ Vaccine. xml; Size: 152,352 bytes; and Date of Creation: 22 September 2022) is herein incorporated by this reference in its entirety.

[0388] The nucleotide and protein sequences referred to herein are represented by a sequence identifier number (SEQ ID NO.). A summary of the sequence identifiers is provided in Table 4. A sequence listing is also provided as part of the specification.

[0389] Table 18 - Sequence Listings

accordance with RefSeq protocols.

[0390] As discussed herein, the sequences listed above include functional variants which may have modifications and mutations in the sequence. As would be understood to those skilled in the art a “functional variant” still maintains a portion of, or all, of the function of the original protein or nucleic acid of the sequence. As such, the functional variant may, for example and in relation to a protein, have one or more amino acid insertions, deletions or substitutions relative to one of the SEQ ID NOs. provided above. Or, in the case of a nucleic acid, a function variant may include one or more synonymous mutation(s) thereby still encoding the same amino acid sequence, or may include one or more non-synonymous mutation(s) so long as the encoded protein is a functional variant of the originally encoded protein.

[0391 ] The term "encoding", as used herein, refers to the property of specific sequences of nucleotides, in a nucleic acid molecule such as a genomic DNA, cDNA, or mRNA, to serve as a template for the synthesis of polymers or macromolecules. The polymers or macromolecules in themselves may have a defined sequence, being a sequence of nucleotides (i.e. , rRNA, tRNA and mRNA) or a defined sequence of amino acids (i.e. peptides such as proteins). Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (with the exception of uracil “U” in place of thymine “T”), and the complementary “non-coding” or “template” strand are to be considered as encoding the protein or other product of the specified sequence. Unless otherwise specified, a "nucleotide sequence encoding” an amino acid sequence, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Degenerative codons are known in the art and are provided in the table 19. Table 19 - Degenerate Codons

[0392] Sequences described herein may have one or more deletions, substitutions or insertions without departing from the present invention. Where a functional requirement is described with regard to the sequence, it is to be understood that the deletions, substitutions or insertions will not abrogate the function of the specified sequence. However, the function may be diminished without departing from the invention.

[0393] In some embodiments, a functional variant, or variant, may comprise at least 50% amino acid sequence identity, at least 55% amino acid sequence identity, at least

60% amino acid sequence identity, at least 65% amino acid sequence identity, at least

70% amino acid sequence identity, at least 75% amino acid sequence identity, at least

80% amino acid sequence identity, at least 85% amino acid sequence identity, at least

90% amino acid sequence identity, at least 91 % amino acid sequence identity, at least

92% amino acid sequence identity, at least 93% amino acid sequence identity, at least

94% amino acid sequence identity, at least 95% amino acid sequence identity, at least

96% amino acid sequence identity, at least 97% amino acid sequence identity, at least

98% amino acid sequence identity, at least 99% amino acid sequence identity, or at least 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% amino acid sequence identity to any one of SEQ ID Nos. listed and recited herein. In some embodiments, the function variant maintains 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% of the function of the original peptide/protein.

[0394] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0395] It is to be further understood that terminology such as “comprise”, or variations such as “comprises” or “comprising” inherently include within their scope (without being limited to) versions of the invention that excludes other elements directly related to the invention. Accordingly, terminology such as “consisting of” or “consisting essentially of’ can be substituted for terminology such as “comprise”, “comprises” or “comprising” with the effect of limiting the scope of the invention to the specifically recited elements. Notably, where it is explicitly intended for the invention to be considered in an exhaustive manner, such limitations should be considered to relate only to the inventive concept disclosed herein and other features can be added which fall outside of the scope of the inventive concept. Such features or elements may include, but are not limited to, excipients, formulations, additives, diluents, packaging, adjuvants and collocated features which are not to be excluded by terminology such as “consisting of” or “consisting essentially of”.

[0396] When comparing nucleic acid sequences, the sequences should be compared over a comparison window which is determined by the length of the nucleic acid or is otherwise specified. For example, a comparison window of at least 20 residues, at least 50 residues, at least 75 residues, at least 100 residues, at least 200 residues, at least 300 residues, at least residues, at least 500 residues, at least 600 residues, or over the full length of any one of the sequences listed in table 18 is envisaged. The comparison window may comprise additions or deletions of about 20%, about 18%, about 16%, about 14% about 12%, about 9%, about 8%, about 6%, about 4% or about 2% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al., (1997) (Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402). Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al. (2000). EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16(6):276-7), and the GGSEARCH program (available at fasta.bioch. Virginia. edu/fasta_www2/fasta_www.cgi?rm=compare&pgm=gnw) which is part of the FASTA package (Pearson Wand Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman- Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al Eds. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, Chapter 19, 2003).

[0397] The term "substitution", in reference to a peptide or protein, refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (e.g., by changing the amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping; e.g. substituting a hydrophilic amino acid with a hydrophobic amino acid) or in a conservative manner (e.g., by changing the amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping; e.g. substituting a hydrophilic amino acid with a hydrophilic amino acid). Such a conservative change generally leads to a reduction in conformational and functional changes in the modified peptide/protein. The following are examples of various groupings of amino acids: 1 ) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine. [0398] A person skilled in the art will recognise that any amino acid can be substituted with a chemically (functionally) similar amino acid and retain function of the protein. Such conservative amino acid substitutions are well known in the art. The following groups in Table 5 and 6 provide some conservative amino acids.

Table 20 - Exemplary amino acid conservative substitutions

Table 21 - Conservative amino acid groups

[0399] The term “insertion”, in reference to a peptide or protein, refers to addition of amino acids within the interior of the sequence. “Addition” refers to addition of amino acids to the terminal ends of the sequence. “Deletion” refers to removal of amino acids from the sequence.

[0400] As will be understood the term “modification” or “mutation” includes any addition, deletion, insertion or substitution to an amino acids sequence, or a nucleic acid sequence.

[0401 ] The term “immunogenic portion”, as used herein relates to the capacity of the portion of the larger protein to induce an immune response within a subject administered the vaccine. What constitutes an immune response will be understood in the art. However, for clarity, an immune response may include (but is not limited to) the induction of an innate immune response, or the induction of an adaptive immune response. In preferred embodiments, the immunogenic portion induces an adaptive immune response. In some embodiments, the immunogenic portion induces an antigen specific immune response. An antigen specific immune response includes (but is not limited to) an; expansion of a lymphocyte population including T-cells, affinity maturation of B cells, instigation of secretion of macromolecules such as antibodies (including isotype switching to IgA, IgG or IgE isotypes), instigation or enhancement of secretion of chemokines and/or cytokines (such as IL-2, IL-4, IL-5, IL-9, IL-12, IL-13, IL-17, IL-21 , IL-22, IL-26, IFN-y, TNF-a, or TNF-[3), instigation or enhancement of CTL responses, instigation or enhancement of maturation of monocytes and/or dendritic cells, increase in surface expression of co-stimulation molecules, increase in the expression of Fc receptors, increase in the expression of major-histocompatibility (MHC) molecules, recruitment or migration of leukocytes, increase in expression of markers indicative of plasma cells or memory B cells (including CD38, CD21 , CD24, CD19, B220, FcRH4 and CD25), upregulation of markers indicative of memory T-cells (including CD45RO, CCR7, CD62L. CD27 or CD28), and upregulation of activation receptors (including CD69, Ki67 or CD40L).

[0402] In some embodiments, an “immunogenic portion” induces a T-cell response. In some embodiments the T-cell response is a T-helper response and/or a CTL response. In some embodiment the immune response includes a B cell response. Preferably, an immunogenic portion induces a T-helper response, a CTL response and a B cell response.

[0403] The term “immunogenic portion” may be relative to the desired or measured immune response. As will be understood to a person skilled in the art, a protective antibody response typically requires an antigenic region to be provided in a three- dimensional configuration similar, or identical, to the native protein. Therefore, this may require the entirety of, or a significant portion of, a protein that is stabilised in its three- dimensional configuration. A T-cell response, however, may only require an epitope portion of a protein. MHC (or HLA) class-l epitopes can be from 8 to 15 amino acids in length, or more preferably from 8 to 13 amino acids in length or from 8 to 11 amino acids in length, or from 8 to 10 amino acids in length or most preferably 9 amino acids in length (see Trolle T et al. (2016). The Length Distribution of Class l-Restricted T-cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference. Journal of Immunology.196:4). Class II MHC epitopes are typically 13 to 17 amino acids in length (Stewart C. T. et al. (2006). Peptide length-based prediction of peptide-MHC class II binding, Bioinformatics, 22:22, pp 2761-2767). Accordingly, an immunogenic portion may be as small as 8 amino acids in length if an MHC class I mediated response (e.g. a CD8+ T-cell response) is desired. However, as will be understood, processing pathways exist in vivo, particularly in antigen-presenting cells, to digest and fragment longer peptides, or proteins, into epitope fragments which can be presented by MHC molecules. Therefore, in some embodiments, the immunogenic portion may be a full-length protein (such as NSP3 or nucleocapsid), or a full-length region (such as the RBD region, S1 region) of a protein, or fragment of a full protein or region (such as truncated NSP3). In some embodiments, the immunogenic portion is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% of the full-length of a specified protein or region. In some embodiments, the immunogenic portion is up to 95%, up to 90%, up to 85%, up to 80%, up to 75%, up to 70%, up to 65%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10% of the full-length of a specified protein or region.

[0404] Techniques, such as those provided in the Examples, and those described above, can be used for identifying immunogenic portions of a SARS-CoV-2 protein, or protein region.

[0405] A, "vaccine" composition refers to a composition comprising at least a vector as described herein, which is useful to establish immunity to the SARS-CoV-2 in the subject. It is contemplated that the vaccine comprises a pharmaceutically acceptable carrier, solvent, excipient and/or an adjuvant.

[0406] A vaccine composition envisages a prophylactic or therapeutic treatment. A "prophylactic" treatment is a treatment administered to a subject, who does not exhibit signs of an infection, for the purpose of reducing the likelihood of a SARS-CoV-2 infection, decreasing the risk of developing pathology from a SARS-CoV-2 infection, decreasing the severity of a Covid-19 as a result of a SARS-CoV-2 infection, or decreasing the risk of transmitting a SARS-CoV-2 infection to another subject. [0407] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, for the purpose of reducing the severity of infection, shortening the duration of infection, reducing or eliminating signs or symptoms of an infection, reducing viral shedding of the infection, or reducing the likelihood of transmitting the infection. A reference to therapeutic treatment of a SARS- CoV-2 infection should be considered equivalent to treatment of COVID-19 disease. However, it may be that treatment of Covid-19 disease is not equivocal to treating a SARS-CoV-2 infection. For example, and depending on the context, prevention of Covid-19 may relate only to the prevention of the onset of symptoms, not prevention of infection.

[0408] A "DNA Vaccine" as used throughout the specification refers to a synthetic DNA structure that can be administered to a subject and transfected into one or more host cells whereby it is transcribed. A DNA vaccine can comprise any suitable DNA molecule including linear nucleic acid, such as a purified DNA molecule, a plasmid incorporating a DNA molecule, or a DNA molecule incorporated into another suitable vector for introduction (transfection, transduction, transformation etc.) of the DNA molecule into the cell of a treated subject. Accordingly, in some embodiments, the DNA vaccine can be naked DNA, a DNA vector or a viral vector vaccine (live, attenuated, inactivated, recombinant, modified or killed).

[0409] As used herein, "pharmaceutical composition" refers to a composition suitable for administration to a subject animal, including humans. In the present context, a pharmaceutical composition comprises a pharmacologically effective amount of a nucleic acid molecule or peptide of the present invention and also a pharmaceutically acceptable carrier, solvent or excipient. Accordingly, pharmaceutical compositions of the present invention encompass any composition made by admixing a nucleic acid molecule, vector, or protein in accordance with the present invention and a pharmaceutically acceptable carrier.

[0410] As used herein, a "pharmaceutically acceptable carrier" includes any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and excipients, such as a phosphate buffered saline solution (PBS), aqueous solutions of dextrose or mannitol, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21 st Ed. (Mack Publishing Co., Easton, 2006). Pharmaceutical carriers useful for the composition depend upon the intended mode of administration of the active agent. Typical modes of administration include parenteral administration, including subcutaneous, intramuscular, intravenous or intraperitoneal injection; transdermal; or transmucosal administration.

[0411 ] Reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention. See, for example, Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0412] Referenced documents, publications and patents are to be included in their entirety by way of reference. The teachings and disclosures in such documents, publications and patents are therefore considered to form part of the disclosure of this specification.

[0413] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context or the understanding of a skilled addressee. The use of any and all examples, or exemplary language (e.g., "such as", “i.e.”, “for example”), is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention, unless otherwise claimed or stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0414] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0415] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations. [0416] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0417] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0418] Future patent applications may be filed on the basis of, or claiming priority from, the present application. It is to be understood that the following claims are not intended to limit the scope of what may be claimed in any such future application(S). Features may be added to or omitted from the claims at a later date so as to further define or re-define the claimed invention or inventions.

[0419] It will be apparent to the person skilled in the art that while the invention is described herein in detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0420] List of Abbreviations

Claims

94 The claims defining the invention are as follows:

1 . A nucleic acid encoding an immunogenic portion of a SARS-CoV-2 protein selected from the group consisting of:

Non-Structural Protein 3 (NSP3);

Nucleocapsid;

S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein.

2. A nucleic acid according to claim 1 , comprising an immunogenic portion of the S1 subunit of the Spike protein, and/or an immunogenic portion of the receptor binding domain (RBD) of the Spike protein, wherein the immunogenic portion(s) is linked to a sequence encoding a heterologous signal peptide.

3. A nucleic acid according to claim 2, wherein the heterologous signal peptide facilitates secretion of a protein from a eukaryotic cell.

4. A nucleic acid according to claim 2 or claim 3, wherein the heterologous signal peptide is the tissue plasminogen activator (tPA) signal peptide, or a functional portion, or variant, thereof.

5. A nucleic acid according to any one of claims 1 to 4, wherein the immunogenic portion of the SARS-CoV-2 protein is linked to an oligomerisation domain.

6. A nucleic acid according to claim 5, wherein the oligomerisation domain is IMX313P or Foldon.

7. A nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid further encodes a functional portion, or variant, of a perforin protein.

8. A nucleic acid encoding an immunogenic portion of the S1 subunit of the SARS-CoV-2 Spike protein, linked to IMX313P or Foldon.

9. A nucleic acid encoding an immunogenic portion of the receptor binding domain of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon. 95 A nucleic acid encoding an immunogenic portion of SARS-CoV-2 Non- Structural Protein 3 (NSP3), wherein the nucleic acid further encodes a functional portion, or variant, of a perforin protein. A nucleic acid encoding an immunogenic portion of SARS-CoV-2 Nucleocapsid protein, wherein the nucleic acid further encodes a functional portion, or variant, of a perforin protein. A nucleic acid according to any one of claims 7, 10 or 11 , wherein the perforin protein has at least 80% sequence identity to SEQ ID NO: 24., or wherein the sequence encoding the perforin protein has at least 80% sequence identity to SEQ ID NO: 14, or the reverse compliment thereof. A nucleic acid according to any one of claims 7 or 10 to 12, wherein the sequence encoding the functional portion, or variant, of perforin is under the control of a promoter sequence. A nucleic acid according to any one of claims 1 to 13, wherein the nucleic acid sequence includes a promoter sequence that promotes the expression of the encoded immunogenic portion of the SARS-CoV-2 protein(s). A nucleic acid according to any one of claims 1 to 14, comprising an expression vector which contains at least one promoter sequence promoting the expression of the encoded immunogenic portion of the SARS-CoV-2 protein(s). A nucleic acid according to claim 15, wherein the expression vector is pVax. A nucleic acid according to any one of claims 1 to 12 consisting of RNA. A nucleic acid according to any one of claims 1 to 16 consisting of DNA. A nucleic acid encoding a recombinant polyprotein, wherein the polyprotein includes immunogenic portions of two or more SARS-CoV-2 proteins selected from the group consisting of:

Non-Structural Protein 3 (NSP3);

Nucleocapsid; the S1 subunit of the Spike protein; and/or 96 the receptor binding domain (RBD) of the Spike protein. A nucleic acid according to claim 19, further encoding a heterologous signal peptide which is linked to the immunogenic portions of the S1 subunit of the Spike protein and/or the receptor binding domain (RBD) of the Spike protein, and facilitates the secretion of the immunogenic portions from a eukaryotic cell. A nucleic acid according to claim 20, wherein the heterologous signal peptide is the tissue plasminogen activator (tPA) signal peptide, or a functional portion, or variant, thereof. A nucleic acid according to claim 19, wherein the polyprotein comprises immunogenic portions of NSP3 and at least one or more of:

Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. A nucleic acid according to claim 19, wherein the polyprotein comprises immunogenic portions of Nucleocapsid and at least one or more of:

NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. A nucleic acid according to any one of claims 19 to 23 encoding a polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of proteins are separated by a cleavage domain. A nucleic acid according to claim 24, wherein the cleavage domain is the foot- and-mouth disease virus (FMDV) 2A peptide. A vaccine comprising a nucleic acid according to any one of claims 1 to 25. 97 A vaccine according to claim 26 comprising at least two distinct nucleic acids each of which encode for different SARS-CoV-2 proteins. A vaccine according to claim 26 or claim 27, wherein at least one of the nucleic acids encodes for a perforin protein, or a functional portion or variant, of a perforin protein. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 25, and a pharmaceutically acceptable carrier, diluent, excipient and/or stabiliser. A pharmaceutical composition according to claim 29, comprising at least two distinct nucleic acids, each of which encode for different SARS-CoV-2 proteins, or wherein at least one of the two distinct nucleic acids encodes for a perforin protein, or a functional portion, or variant, of a perforin protein. A vaccine comprising an isolated or recombinant immunogenic portion of a SARS-CoV-2 protein selected from the group consisting of:

Non-Structural Protein 3 (NSP3);

Nucleocapsid; the S1 subunit of the Spike protein; or the receptor binding domain (RBD) of the Spike protein. A vaccine according to claim 31 , wherein the immunogenic portion of the protein is linked to an oligomerisation domain. A vaccine according to claim 32, wherein the oligomerisation domain is IMX313P or Foldon. A vaccine comprising an immunogenic portion of the S1 subunit of the SARS- CoV-2 Spike protein linked to IMX313P or Foldon. A vaccine comprising an immunogenic portion of the receptor binding domain of the SARS-CoV-2 Spike protein linked to IMX313P or Foldon. A vaccine comprising a recombinant polyprotein, wherein the polyprotein includes immunogenic portions of two or more SARS-CoV-2 proteins selected from the group consisting of: 98

Non-Structural Protein 3 (NSP3);

Nucleocapsid;

S1 protein; and/or the receptor binding domain (RBD) of the Spike protein. A vaccine according to claim 36, wherein the polyprotein comprises an immunogenic portion of NSP3 and an immunogenic portion of at least one or more of:

Nucleocapsid; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. A vaccine according to claim 36, wherein the polyprotein comprises an immunogenic portion of Nucleocapsid and an immunogenic portion of at least one or more of:

NSP3; the S1 subunit of the Spike protein; and/or the receptor binding domain (RBD) of the Spike protein. A vaccine comprising a polyprotein comprising: at least a first immunogenic portion of a protein selected from the group consisting of: Non-Structural Protein 3 (NSP3) or Nucleocapsid; and at least a second immunogenic portion of a protein selected from the group consisting of: the S1 subunit of the Spike protein or the receptor binding domain (RBD) of the Spike protein, wherein the first and second immunogenic portions of the proteins are separated by a cleavage domain. A vaccine according to claim 39, wherein the cleavage domain is the foot-and- mouth disease virus (FMDV) 2A peptide. A method of eliciting an immune response in a subject, the method comprising administering to the subject a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or 30. 99 A method according to claim 41 , wherein the immune response is a B cell immune response and/or T-cell immune response. A method according to claim 41 or claim 42, wherein the immune response is a memory immune response. A method of preventing or treating a SARS-CoV-2 infection in a subject or preventing or treating COVID-19 disease in a subject, the method comprising administering to a subject a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30. A method of preventing or treating a condition associated with a SARS-CoV-2 infection in a subject, the method comprising administering to a subject a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30. Use of a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30 in the preparation of a medicament for the prevention or treatment of a SARS-CoV-2 infection, or COVID-19 disease in a subject. Use of a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30 in the preparation of a medicament for preventing or treating a condition associated with a SARS- CoV-2 infection in a subject. A method for producing an anti-SARS-CoV-2 antibody, the method comprising administering to a subject a nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30, and isolating an anti-SARS-CoV-2 antibody from the subject, identify a sequence of an anti- SARS CoV-2 antibody from the subject, identifying and expressing a sequence 100 encoding an anti-SARS CoV-2 antibody from the subject and or isolating a 13- cell secreting an anti-SARS-CoV-2 antibody from the subject. A nucleic acid according to any one of claims 1 to 25, a vaccine according to any one of claims 26 to 28 or 31 to 40, or a pharmaceutical composition according to claim 29 or claim 30 for use in eliciting an immune response in a subject, preventing or treating a SARS-CoV-2 infection in a subject, preventing or treating COVID-19 disease in a subject, or preventing or treating a condition associated with a SARS-CoV-2 infection in a subject.