WO2022216223A1

WO2022216223A1 - Vaccine and/or antibody for viral infection

Info

Publication number: WO2022216223A1
Application number: PCT/SG2021/050497
Authority: WO
Inventors: Qi Zeng
Original assignee: Intra Immusg Private Ltd Sg
Current assignee: Intra Immusg Private Ltd Sg
Priority date: 2020-04-09
Filing date: 2021-08-25
Publication date: 2022-10-13
Anticipated expiration: 2023-10-08
Also published as: WO2021206638A1

Abstract

The present invention relates to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein and/or an immunogenic fragment thereof and uses thereof. The invention includes an antibody capable of binding to the SARS-CoV-2 N-protein or antigen-binding fragment thereof and uses thereof

Description

Vaccine and/or antibody for viral infection

This application claims priority from PCT/SG2021/050197 filed 8 April 2021 , the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to prophylaxis and treatment of viral infection. In particular, the invention relates to immune therapies such as novel vaccines for prophylaxis and antibodies for treatment of viral infection, for example Coronavirus infection.

Background

Coronaviruses (Co Vs) are enveloped, positive-sense, single-stranded RNA viruses of the family Coronaviridae. While most viruses cause mild illnesses such as the common cold, a few viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) resulted in the severe acute respiratory syndrome (SARS) public health crises in 2003, Middle East respiratory syndrome coronavirus (MERS-CoV) caused Middle East respiratory syndrome (MERS) in 2009. In addition, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused Coronavirus disease 2019 (COVID-19) from late 2019. The outbreaks for SARS-CoV and MERS-CoV were regional, while that of SARS-CoV-2 is global. The World Health Organisation (WHO) declared COVID-19 as a pandemic on 11th March 2020 and SARS-CoV-2 has infected almost 128 million people and caused over 2.8 million deaths worldwide as of 3rd April 2021 , with severe outbreaks occurring in first in China, then Europe and in the USA (WHO Coronavirus (COVID-19) Dashboard). While infections are generally self-resolving in healthy subjects, it can also lead to severe pneumonia, multi-organ failure, and death in significant portions of infected patients, especially those with pre-existing comorbidities. Along with drastic social distancing measures in an attempt to slow the spread of the virus, the current COVID-19 pandemic has caused widespread medical, social, political, and financial repercussions. There are predictions that COVID-19, like flu, could become seasonal and may recur in the future even after recovery. The global pandemic of COVID-19 has prompted the current interest in the pursuit of immune therapies against SARS-CoV-2. It is desirable to develop novel and effective immune therapies such as vaccines and antibody therapeutics for coronavirus infections.

The present invention has been devised in light of the above considerations.

Summary of the Invention

According to a first aspect, the present invention relates to an isolated nucleocapsid protein (N-protein) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or an immunogenic fragment thereof. The sequence of the SARS-CoV-2 nucleocapsid protein (N-protein) comprises or consists of:

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTN SSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANN AAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLE SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFA PSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQR QKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 1)

Another aspect of the present invention includes an isolated nucleic acid molecule encoding the SARS- CoV-2 nucleocapsid protein (N-protein) and/or an immunogenic fragment thereof.

The SARS-CoV-2 N-protein and/or immunogenic fragment thereof and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment may be for use as a medicament.

The SARS-CoV-2 N-protein and/or immunogenic fragment thereof and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment may be for use as a vaccine.

According to a further aspect, the invention includes an antibody capable of binding to the isolated N- protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or an immunogenic fragment thereof. The immunogenic fragment thereof is also capable of binding to SARS-CoV-2 N- protein. In particular, the antibody binds to SARS-CoV-2 N-protein. The antigenic-binding fragment also binds to the SARS-CoV-2 N-protein.

The antibody may be for use as a medicament.

In one aspect, the present disclosure provides an antibody. The amino acid sequence of the antibody may comprise the amino acid sequences i) to (iii), or the amino acid sequences (iv) to vi), or preferably the amino acid sequences i) to vi): i) NYGMN (SEQ ID NO: 7) ii) WINTYTGEPTYADDFKG (SEQ ID NO: 8) iii) PLYYDYDGHAMDY (SEQ ID NO: 9) iv) RSSKSLLHSNGITY (SEQ ID NO: 11) v) QMSNLAS (SEQ ID NO: 12) vi) QNLELMWT (SEQ ID NO: 13) or a variant thereof in which one or two or three amino acids in one or more of the sequences (i) to (vi) are replaced with another amino acid.

The antibody may comprise at least one light chain variable region incorporating the following CDRs: LC-CDR1 : RSSKSLLHSNGITY (SEQ ID NO: 11);

LC-CDR2: QMSNLAS (SEQ ID NO: 12); and

LC-CDR3: QNLELMWT (SEQ ID NO: 13).

The antibody may comprise at least one heavy chain variable region incorporating the following CDRs:

HC-CDR1 : NYGMN (SEQ ID NO: 7); HC-CDR2: INTYTGEPTYADDFKG (SEQ ID NO: 8); and

HC-CDR3: PLYYDYDGHA DY (SEQ ID NO: 9).

The antibody may comprise at least one light chain variable region incorporating the CDRs shown in Figure 6. The antibody may comprise at least one heavy chain variable region incorporating the CDRs shown in Figure 6.

The antibody may comprise at least one light chain variable region comprising the amino acid sequence shown in Figure 6 or an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VL chain amino acid sequence of SEQ ID NO: 14 shown in Figure 6.

The antibody may comprise at least one heavy chain variable region comprising the amino acid sequence shown in Figure 6 or an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VH chain amino acid sequence of SEQ ID NO: 6 shown in Figure 6.

The antibody may comprise at least one light chain variable region comprising the amino acid sequence as shown in Figure 6 (or an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VL chain amino acid sequence of SEQ ID NO:14 shown in Figure 6) and at least one heavy chain variable region comprising the amino acid sequence as shown in Figure 6 (or an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VH chain amino acid sequence of SEQ ID NO: 6 shown in Figure 6).

The antibody may optionally bind N protein. The antibody may optionally bind SARS-Cov-2 N protein.

The antibody may optionally bind SARS-Cov N-protein. The antibody may optionally bind SARS-Cov-2 N protein and SARS-Cov N-protein. The antibody may bind a protein having the amino acid sequence set out in SEQ ID NO: 1 . The antibody may optionally have amino acid sequence components as described above. In some cases, the antibody does not bind a peptide comprising or consisting of the amino acid of SEQ ID NO: 3, SEQ ID NO: 3 and/or SEQ ID NO:4.

The antibody may be a humanized antibody. It may be a murine or chimeric antibody.

In some embodiments the antibody may be antibody 6H3.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which: Figure 1 shows the amino acid sequences of Nucleocapsid proteins from 4 different coronaviruses (CoVs) and antibody responses to vaccination of nucleocapsid protein in four Balb C mice. (A) The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), 2003 Pandemic severe acute respiratory syndrome coronavirus (SARS-CoV), BAT Coronavirus (BAT-CoV) and BAT Severe Acute Respiratory virus (BAT-SARS), were aligned, and there is 90 % similarity of amino acid sequences (Conserved domains are highlighted). (B, C, D, E) The whole N protein vaccination was repeated 4 times (2-week interval). Red arrow indicates the time points for vaccination. Blood samples were taken before vaccination followed by every 2 weeks until 22^nd week. Serum antibodies were detected by using anti- IgM, -lgG1 , -lgG2 and anti IgG Fc horseradish peroxidase (HRP) conjugated antibodies. Antibody responses can be detected after 2^nd vaccination and sustained till last sample collection in mouse#1 (A), #2 (B), #3 (C) and #4 (D). (F) Mean data of antibody productions in the BALB/c mice (n=4) Mean antibody production of N protein vaccination in FVB mice (n=3). Data represent Mean ±S.D. (F) shows the same trend in FVB mice vaccinated with N protein. Figure 1 shows that N protein is an excellent immunogen for vaccination.

Figure 2: (A) shows the sequences of peptides which were selected based on N-protein sequence. (B) anti-N polyclonal Abs (at 1 : 1000 & 1 :2000 dilutions) were tested by Elisa for the binding affinity to three individual N-peptides, whole N protein as controls, which were coated respectively with 5ng & 20ng/well, detected by anti-mouse IgG Fc (HRP). The Optical Density (OD) was measured. anti-N polyclonal Abs bind not only whole N protein, but also enriched binding to Peptide#3, the highest OD compared to Peptide#1 and #2. (C) Peptide#3 was used to vaccine BALB/c mice in 2-week interval, 3 repeats. Red arrow indicates each vaccine time point. (D) Anti-Peptide#3 Ab serum were classified by using anti-lgM, - lgG1 , -lgG2a and -IgG Fc horseradish peroxidase (HRP) conjugated antibodies. Data represent Mean ±S.D, n=3. (E) Anti-Peptide#3 Ab serum were tested by Elisa for the binding capacity. Anti-Peptide#3 Ab serum binds to Peptide#3 and whole N protein, detected by anti-mouse IgG Fc (HRP). The Optical Density (OD) was measured. Figure 2 shows that Peptide#3 is a good immunogen.

Figure 3. Vaccination results in an increased frequency of CD4⁺ & CD8⁺ memory T cells and a decreased frequency of memory T cells. (A) Representative CD62L and CD44 staining on live CD45⁺CD3⁺CD335^_ CD4⁺CD8 T cells from the blood of Balb/c mice. Mice were either unvaccinated (W T) or vaccinated with Freund’s adjuvant and N protein (vaccinated mice). (B) Change in the percentage of live CD44⁺CD62L^_ memory T cells as a proportion of total live CD45⁺CD3⁺CD335 CD4⁺CD8 T cells in unvaccinated and vaccinated mice. (C) Change in the percentage of live CD44 CD62L* naive T cells as a proportion of total live CD45⁺CD3⁺CD335 CD4⁺CD8T cells in unvaccinated and vaccinated mice. (D) Representative CD62L and CD44 staining on live CD45⁺CD3⁺CD335 CD4 CD8⁺ T cells from the blood of Balb/c mice. (E) Change in the percentage of live CD44⁺CD62L^_ memory T cells as a proportion of total live CD45⁺CD3⁺CD335 CD4 CD8⁺ T cells in unvaccinated and vaccinated mice. (F) Change in the percentage of live CD44 CD62L⁺ naive T cells as a proportion of total live CD45⁺CD3⁺CD335 CD4 CD8⁺ T cells in unvaccinated and vaccinated mice. (G) Representative IgD and IgG staining on live CD45⁺CD19⁺CD138 B cells from the blood of unvaccinated and vaccinated Balb/c mice. Change in the percentage of (H) naive lgD⁺ B cells and (I) lgG⁺ class-switched memory B cells. Data representing mean± SEM. n=4 in BALB/c mice & n=3 in FVB mice.

Figure 4. Vaccination with whole N protein in complete Freund’s adjuvant (CFA) can induce the secretion of pro-inflammatory memory cell and TH1 associated cytokines. (A) Cytokine array blot of pre- & postimmunization mouse sera. The orange box indicates the cytokines which increased more than 2 folds than pre-immunization sample. (B) Map of cytokine array. (C) The table indicating fold increases in cytokine level based on pre-immunization sample. (D) Bar graph of cytokines with more than 2 folds increase compared to pre-immunization sample. Cytokine array performed for wild type(pre), 4 weeks treated (N4), and 12 weeks treated (N12) mice sera.

Figure 5. Clone 6H3 mouse monoclonal antibody binds to SARS-CoV-2 N-protein with good affinity. ELISA was done to analyzed the binding affinity of peptides & N-protein (SARS-CoV2) to in house produced mouse SARS-CoV Ab (clone 6H3). ELISA plate was coated with 5ng & 20ng/ well of different peptides & N-protein (SARS-CoV2). mouse 6H3 antibodies were diluted at 1 :1000 & 1 :5000 dilution. The binding of antibody was detected by anti-mouse IgG (HRP). The Optical Density (OD) was measured.

Figure 6. Sequences of an antibody 6H3.

Detailed Description of the Invention

As used herein, the term “adjuvant” refers to any substance or combination of substances which non- specifically enhances the immune response to an antigen.

As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of’. The variations of the word “comprising”, such as

“comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.

An immunogenic fragment is defined as a part of an antigen which is capable of inducing/eliciting an immune response in a host. An immunogenic fragment of a protein/polypeptide preferably comprises one or more epitopes of said protein/polypeptide. An epitope of a protein/poiypeptide is defined as a fragment of said protein/polypeptide of at ieast about 4 or 5 amino acids in length, capable of eiiciting a specific antibody and/or an immune ceil (e.g,, a T cell or B ceil) bearing a receptor capable of specifically binding said epitope. Two different kinds of epitopes exist: linear epitopes and conformational epitopes. A linear epitope comprises a stretch of consecutive amino acids. A conformational epitope is typically formed by several stretches of consecutive amino acids that are folded in position and together form an epitope in a properly folded protein. An immunogenic fragment as used herein refers to either one, or both, of said types of epitopes. As used herein, the term “vaccine” refers to a composition comprising an antigen capable of stimulating an immune response specifically against the antigen and preferably to engender immunological memory that leads to mounting of a protective immune response should the subject encounter that antigen at some future time.

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

According to a first aspect, the present invention relates to an isolated nucleocapsid protein (N-protein) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or an immunogenic fragment thereof.

The sequence of the SARS-CoV-2 N-protein comprises or consists of:

Another aspect of the present invention includes an isolated nucleic acid molecule encoding the SARS- CoV2 N-protein and/or an immunogenic fragment thereof.

The SARS-CoV-2 N-protein and/or immunogenic fragment thereof may be prepared by recombinant DNA technology or chemically synthesised. The nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment may also be prepared by recombinant DNA technology or chemically synthesised.

A further aspect of the invention includes a vector comprising a nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof. The invention further includes a host cell comprising a nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof. The invention also includes a host cell comprising a vector comprising a nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof.

The immunogenic fragment of the SARS-CoV-2 N-protein comprises or consists of a sequence selected from the group consisting of:

CIRQGTDYKHWPQIAQFAPSASAFFGMSRIG (SEQ ID NO: 2);

ClAQFAPSASAFFGMSRIGMEVTPSGTWLTY (SEQ ID NO: 3);

CVILLNKHIDAYKTFPPTEPKKDKKKKADET (SEQ ID NO: 4). In particular, the immunogenic fragment of the SARS-CoV-2 N-protein comprises SEQ ID NO: 4. More in particular, the immunogenic fragment of the SARS-CoV-2 N-protein consists of SEQ ID NO: 4.

The invention includes an immunogenic combination and/or immunogenic composition comprising two or more components as described herein according to any aspect of the invention. It will be appreciated that the components of an immunogenic combination are administered in combination, for example, they may be combined together before administration or may be administered simultaneously or sequentially.

For example, the immunogenic combination and/or immunogenic composition may comprise any two or more components selected from the group consisting of:

(i) an isolated nucleocapsid protein (N-protein) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2);

(ii) the immunogenic fragment(s) as described herein.

In particular, the immunogenic combination and/or immunogenic composition may comprise two or more immunogenic fragments as described herein.

Accordingly, the invention includes the use of SARS-CoV-2 N-protein and/or immunogenic fragment thereof and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof in the preparation of a vaccine.

The invention includes a pharmaceutical composition comprising a SARS-CoV-2 N-protein and/or immunogenic fragment thereof and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof. The pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient.

The invention further includes a vaccine comprising a SARS-CoV-2 N-protein and/or immunogenic fragment thereof and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment thereof. The vaccine may also comprise at least one pharmaceutically acceptable excipient. The vaccine may further comprise at least one adjuvant.

The vaccine may be for immunizing a subject against a viral infection.

The invention includes a method for immunizing a subject against a viral infection, comprising administering to the subject the isolated SARS-CoV-2 N-protein and/or immunogenic fragment thereof, the immunogenic combination and/or the immunogenic composition and/or the nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or immunogenic fragment and/or the vector; as described herein.

The viral infection may be a Coronavirus infection. For example, the vaccine may be for immunising against severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) infection [Coronavirus disease 2019 (COVID-19)]. In particular, the vaccine is for immunising against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)]. Antibodies

According to a further aspect, the invention includes an antibody capable of binding to the SARS-CoV-2 N-protein and/or an immunogenic fragment thereof or an antigen-binding fragment of the antibody. The immunogenic fragment thereof is also capable of binding to SARS-CoV-2 N-protein. In particular, the antibody binds to SARS-CoV-2 N-protein. The antigenic-binding fragment also binds to the SARS-CoV-2 N-protein. In some aspects, the antibody is capable of binding to SARS-CoV-2 N-protein and SARS-CoV (also known as SARS-Cov-1) N-Protein. The antigenic-binding fragment is also capable of binding to SARS-CoV-2 N-protein and SARS-CoV N-Protein. In other words, the antibody and antigenic-binding fragment are capable of binding to both SARS-CoV-2 N-protein and SARS-CoV N-Protein.

The antibody capable of binding to the to the SARS-CoV-2 N-protein may be a monoclonal antibody. Monoclonal antibodies (mAbs) are a homogenous population of antibodies specifically targeting a single epitope on an antigen.

The antibody capable of binding to the to the SARS-CoV-2 N-protein may be a polyclonal antibody. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

The monoclonal antibody may be a chimeric or humanised antibody. The antibody may be a murine antibody. Preferably, the antibody is a humanised monoclonal antibody.

Antibodies according to the present invention may be provided in isolated form.

By “antibody” we include a fragment or derivative thereof, or a synthetic antibody or synthetic antibody fragment, preferably an antigenic binding fragment.

In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in ''Monoclonal Antibodies: A manual of techniques ", FI Zola (CRC Press, 1988) and in "Monoclonal Flybridoma Antibodies: Techniques and Applications ", J G R FHurrell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Fragments of antibodies, such as Fab and Fab2 fragments may also be provided as can genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855). That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341 , 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293- 299.

By "ScFv molecules" we mean molecules wherein the VH and VL partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies, and F(ab')2 fragments are "bivalent". By "bivalent" we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site. Synthetic antibodies which bind to SEQ ID NO:

4 may also be made using phage display technology as is well known in the art. It will be appreciated that the monoclonal antibody may be produced by any method, for example hybridoma technology or recombinant DNA technology.

In certain methods, the antibody is 6H3, or a variant of 6H3. 6H3 comprises the following CDR sequences:

(i) NYG N (SEQ ID NO: 7)

(ii) WINTYTGEPTYADDFKG (SEQ ID NO: 8)

(iii) PLYYDYDGHAMDY (SEQ ID NO: 9)

(iv) RSSKSLLHSNGITY (SEQ ID NO: 11)

(v) QMSNLAS (SEQ ID NO: 12)

(vi) QNLELMWT (SEQ ID NO: 13)

CDR sequences determined by Kabat definition.

Antibodies according to the present invention may comprise the CDRs of 6H3. In an antibody according to the present invention one or two or three or four of the sequences (i) to (vi) may vary. A variant may have one or two amino acid substitutions in one or two of the sequences (i) to (vi).

The amino acid sequence (and encoding polynucleotide sequence) of the V_H and V_L chains of 6H3 have been determined as shown in Figure 6.

The light and heavy chain CDRs 1-3 of 6H3 may also be particularly useful in conjunction with a number of different framework regions. Accordingly, light and/or heavy chains having CDRs 1-3 of 6H3 may possess an alternative framework region. Suitable framework regions are well known in the art and are described for example in M. Lefranc & G. Le:franc (2001) "The Immunoglobulin FactsBook", Academic Press, incorporated herein by reference.

In this specification, antibodies may have V_H and/or VL chains comprising an amino acid sequence that has a high percentage sequence identity to the V_H and/or V_L amino acid sequences of Figure 6.

For example, antibodies according to the present invention include antibodies that bind severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have a V_H chain that comprises an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the V_H chain amino acid sequence of 6H3 shown in Figure 6.

Some antibodies according to the present invention include antibodies that bind severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have a V_L chain that comprises an amino acid sequence having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the V_L chain amino acid sequence of 6H3 shown in Figure 6.

Antibodies according to the present invention may be detectably labelled or, at least, capable of detection. For example, the antibody may be labelled with a radioactive atom or a coloured molecule or a fluorescent molecule or a molecule which can be readily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radiolabels. The binding moiety may be directly labelled with a detectable label or it may be indirectly labelled. For example, the binding moiety may be an unlabelled antibody which can be detected by another antibody which is itself labelled. Alternatively, the second antibody may have bound to it biotin and binding of labelled streptavidin to the biotin is used to indirectly label the first antibody.

According to one embodiment, the antibody comprises a heavy chain comprising SEQ ID NO: 5:

MPWLWNI_JIJFLMAAAQSAQAQIQIIVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEP

TUARREK6HEAE3IiET3A3TAU1¾INNIiKNERMAKUE0TKRRUURUR6HAMRUΐG6¾0T3ntn33

The regions of the heavy chain are arranged in the following order Signal peptide-FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4, with the framework regions FR1 , FR2 and FR3 in bold and the complementary regions CDR1 , CDR2 and CDR3 underlined.

According to one embodiment, the antibody comprises a heavy chain comprising a variable region comprising SEQ ID NO: 6:

QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYAPPFKGRFAFSLETSAS TAYLQINNLKNEPMAKYFCTRPLYYPYPGHAMPYWGQGTSVTVSS

The complementary determining CDR regions are underlined.

According to a further embodiment, the antibody comprises a heavy chain comprising a CDR region 1 (CDR1) comprising NYGMN (SEQ ID NO: 7), a CDR region 2 (CDR2) comprising WINTYTGEPTYADDFKG (SEQ ID NO: 8), and a CDR region 3 (CDR3) comprising PLYYDYDGHAMDY (SEQ ID NO: 9). The invention includes an isolated nucleic acid encoding the heavy chain of an antibody as described herein.

The invention includes an isolated nucleic acid molecule encoding the heavy chain of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

In particular, the isolated nucleic acid molecule encoding the heavy chain of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprises SEQ ID NO: 10:

ATGGATTGGCTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGTGCAGTC

TGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATG GAAT GAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCT GGATAAACACCTACACT GGAGAGCCA ACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGTACTGCCTATTTGCAGATCAA CAACCTCAAAAATGAGGACATGGCTAAATATTTCTGTACAAGACCCCTCTACTATGATTACGACGGCCATGCTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

In SEQ ID NO: 9 above, the nucleotide regions encoding the regions of the heavy chain are shown in the following order, Signal seguence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 with framework regions FR1 , FR2 and FR3 in bold and the complementary regions CDR1 , CDR2 and CDR3 underlined.

The invention also includes an isolated nucleic acid molecule encoding the heavy chain variable region of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The invention includes an isolated nucleic acid molecule encoding a heavy chain antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a variable region comprising SEQ ID NO: 6.

The invention includes an isolated nucleic acid molecule encoding a heavy chain antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a CDR region 1 (CDR1) comprising NYGMN (SEQ ID NO: 7), a CDR region 2 (CDR2) comprising WINTYTGEPTYADDFKG (SEQ ID NO: 8), and a CDR region 3 (CDR3) comprising PLYYDYDGHAMDY (SEQ ID NO: 9).

The invention includes an isolated nucleic acid encoding the light chain of an antibody as described herein.

The invention includes an isolated nucleic acid molecule encoding the light chain of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The invention also includes an isolated nucleic acid molecule encoding the light chain variable region of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

According to one embodiment, the antibody comprises a light chain comprising SEQ ID NO: 14: DIVMTQAAFSNPVTITSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLIIYQMSNLASGVPDRFSSSGSTDFTLR ISRVEAEDVGVYYCAQNLELMWTFGGGTKLEIK

The complementary determining CDR regions are underlined.

According to a further embodiment, the antibody comprises a light chain comprising a CDR region 1 (CDR1) comprising RSSKSLLHSNGITY (SEQ ID NO: 11), a CDR region 2 (CDR2) comprising QMSNLAS (SEQ ID NO: 12), and a CDR region 3 (CDR3) comprising QNLELMWT (SEQ ID NO: 13).

The invention also includes an isolated nucleic acid molecule encoding the light chain variable region of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The invention also includes an isolated nucleic acid molecule encoding the light chain variable region of an antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV).

The invention includes an isolated nucleic acid molecule encoding a light chain antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a variable region comprising SEQ ID NO: 14. The invention also includes an isolated nucleic acid molecule encoding the light chain variable region of an antibody capable of binding to the isolated N- protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV) comprising a variable region comprising SEQ ID NO:

14.

The invention includes an isolated nucleic acid molecule encoding a light chain antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a CDR region 1 (CDR1) comprising RSSKSLLHSNGITY (SEQ ID NO: 11), a CDR region 2 (CDR2) comprising QMSNLAS (SEQ ID NO: 12), and a CDR region 3 (CDR3) comprising QNLELMWT (SEQ ID NO: 13). The invention includes an isolated nucleic acid molecule encoding a light chain antibody capable of binding to the isolated N-protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV) comprising a CDR region 1 (CDR1) comprising RSSKSLLHSNGITY (SEQ ID NO: 11), a CDR region 2 (CDR2) comprising QMSNLAS (SEQ ID NO: 12), and a CDR region 3 (CDR3) comprising QNLELMWT (SEQ ID NO: 13).

The antibody and/or antigenic fragment thereof may be for use as a medicament. The antibody may be formulated into a pharmaceutical composition. The pharmaceutical composition may comprise at least one pharmaceutically acceptable excipient. Formulating pharmaceutically useful compositions and medicaments

Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.

The term “carrier” refers to diluents, binders, lubricants and d is integrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.

The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability, bioavailability, safety, and/or efficacy of the active ingredients, i.e. an antibody disclosed herein used in the composition. Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

Therapeutic uses

Accordingly, the invention includes the antibody and/or antigen-binding fragment thereof for use in treating a viral infection.

The invention also includes the use of the antibody and/or antigen-binding fragment thereof as described herein in the preparation of a pharmaceutical composition for treating a viral infection.

The invention further includes a method for treating a viral infection in a subject comprising administering the antibody and/or antigenic fragment as described herein thereof to the subject.

Also disclosed herein is an antibody for use in a method of treating a viral infection and the use of an antibody in the manufacture of a medicament for use in a method of treating a viral infection. Such methods may involve administering the antibody and/or antigenic fragment described herein thereof to a subject.

A subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in human or animals (veterinary use). The subject may have been determined to have a coronavirus infection. The subject may have been determined to have a SARS-CoV-2 infection. The subject may have been determined to have a SARS-CoV-1 infection. The subject may have been determined to have an infection via a PCR test. The subject may be suspected of having a coronavirus infection wherein the specific strain of coronavirus has not yet been determined or is indeterminate.

The viral infection may be a Coronavirus infection. For example, the viral infection may be severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural, oral and nasal. The medicaments and compositions may be formulated for injection.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Sequence Identity

Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (referred to by the SEQ ID NO) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated overthe entire length of the respective sequences.

Where the aligned sequences are of different length, sequence identity of the shorter comparison sequence may be determined overthe entire length of the longer given sequence or, where the comparison sequence is longer than the given sequence, sequence identity of the comparison sequence may be determined overthe entire length of the shorter given sequence.

Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1 , Protein/DNA GAPDIST = 4. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Statements relating to particular embodiments of the invention

1 . An isolated nucleocapsid protein (N-protein) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or an immunogenic fragment thereof.

2. The isolated N-protein according to statement 1 , comprising SEQ ID NO: 1.

3. The isolated N-protein according to statement 1 , consisting of SEQ ID NO: 1 .

4. An immunogenic fragment according to statement 1 , comprising a sequence selected from the group consisting of:

SEQ ID NO: 2;

SEQ ID NO: 3;

SEQ ID NO: 4.

5. An immunogenic fragment according to statement 1 , consisting of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

6. An immunogenic fragment according to statement 1 , comprising SEQ ID NO: 4.

7. An immunogenic fragment according to statement 1 , consisting of SEQ ID NO: 4.

8. An immunogenic combination and/or or immunogenic composition comprising any two or more components selected from the group consisting of:

(i) the isolated nucleocapsid protein (N-protein) according to claim 1 ;

(ii) immunogenic fragments) according to any one of claims 1 to 7. 9. The immunogenic combination and/or immunogenic composition according to statement 5 comprising two or more immunogenic fragments according to any one of claims 1 to 7.

10. An isolated nucleic acid molecule encoding the SARS-CoV2 nucleocapsid (N-protein) and/or an immunogenic fragment thereof.

11. A vector comprising the isolated nucleic acid according to statement 10.

12. A host cell comprising the isolated nucleic acid molecule according to claim 10 or the vector according to statement 11 .

13 A vaccine comprising the isolated N-protein and/or immunogenic fragment thereof according to any one of statements 1 to 7, the isolated nucleic acid molecule according to statement 10 and/or the vector according to statement 11.

14. The vaccine according to statement 13, further comprising a pharmaceutically acceptable excipient.

15. The vaccine according to statement 13 or 14, further comprising an adjuvant.

16. The isolated N-protein and/or immunogenic fragment thereof according to any one of statements

1 to 7, the isolated nucleic acid molecule according to statement 10 and/or the vector according to statement 11 , for use as a vaccine.

17. Use of the isolated N-protein and/or immunogenic fragment thereof according to any one of statements 1 to 7, the isolated nucleic acid molecule according to statement 10 and/or the vector according to claim 10; in the preparation of a vaccine.

18. The use according to statement 17, in the preparation of a vaccine for immunizing against a viral infection.

19. The use according to statement 18, wherein the viral infection comprises a Coronavirus infection.

20. The use according to statement 19, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

21. The use according to statement 17, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

22. A method of immunising a subject against a virus infection comprising administering to the subject the isolated N-protein and/or immunogenic fragment thereof according to any one of statements 1 to 7, the immunogenic combination and/or the immunogenic composition according to statement 8, the isolated nucleic acid molecule according to statement 9 and/or the vector according to statement 10.

23. The method according to statement 22, wherein the viral infection comprises a Coronavirus infection. 24. The method according to statement 23, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

25. The method according to statement 24, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID- 19)].

26. An antibody capable of binding to the SARS-CoV-2 nucleocapsid protein (N-protein) and/or an immunogenic fragment thereof or an antigen-binding fragment of the antibody.

27. The antibody according to statement 26 wherein the antibody comprises a monoclonal antibody.

28 The monoclonal antibody according to statement 27, wherein the antibody comprises a chimeric or humanised antibody.

29. The antibody according to statement 26, comprising a heavy chain comprising SEQ ID NO: 5

30. The antibody according to statement 26, comprising a heavy chain variable region comprising

SEQ ID NO: 6.

31. The antibody according to statement 26, comprising a heavy chain comprising a CDR region 1

(CDR1) comprising SEQ ID NO: 7, a CDR region 2 (CDR2) comprising SEQ ID NO: 8, and a CDR region 3 (CDR3) comprising SEQ ID NO: 9.

32. A pharmaceutical composition comprising the antibody according to any one of statements 26 to 31.

33. The pharmaceutical composition according to statement 32, further comprising a pharmaceutically acceptable excipient.

34. The antibody and/or antigen-binding fragment according to any one of statements 26 to 31 , for use as a medicament.

35. The antibody according to any one of statements 26 to 31 , for use in treating a viral infection.

36. The antibody for the use according to statement 35, wherein the viral infection comprises a

Coronavirus infection.

37. The antibody for the use according to statement 36, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

38. The antibody for the use according to statement 37, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)]. 39. Use of the antibody and/or antigen-binding fragment according to any one of statements 26 to 31 in the preparation of a pharmaceutical composition fortreating a viral infection.

40. Use according to statement 39, wherein the viral infection comprises a Coronavirus infection.

41. Use according to statement 40, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

42. Use according to statement 41 , wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

43. A method of treating a viral infection in a subject comprising administering the antibody and/or antigenic fragment according to any one of statements 26 to 31 to the subject.

44. The method according to statement 43, wherein the viral infection comprises a Coronavirus infection.

45. The method according to statement 44, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID-19)].

46. The method according to statement 45, wherein the Coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Coronavirus disease 2019 (COVID- 19)].

47. An isolated nucleic acid molecule encoding the heavy chain of an antibody according to any one of statements 26 to 31.

48. The isolated nucleic acid molecule according to statement 47, comprising SEQ ID NO: 10.

49. An isolated nucleic acid molecule encoding the heavy chain variable region of an antibody according to any one of statements 26 to 31 .

50. The isolated nucleic acid molecule according to statement 49, wherein the variable region of the antibody comprises SEQ ID NO: 6.

51. The isolated nucleic acid molecule according to statement 47, wherein the heavy chain of the antibody comprising a CDR region 1 (CDR1) comprises SEQ ID NO: 7, the CDR region 2 (CDR2) comprising SEQ ID NO: 8, and a CDR region 3 (CDR3) comprising SEQ ID NO: 9.

52. An isolated nucleic acid molecule encoding the light chain of an antibody according to any one of statements 26 to 31.

53. An isolated nucleic acid molecule encoding the light chain variable region of an antibody according to any one of statements 26 to 31 . Examples

Example 1: Vaccination of mice with SARS-CoV-2 nucleocaosid protein ( N-orotein ) or immunogenic peptides derived from SARS-CoV-2 nucleocaosid protein ( N-orotein )

Materials and methods Preparation of GST-N protein:

SARS-CoV-2 GST-N protein bacterial clone was kindly supplied by Dr Yee Joo TAN (Monoclonal Antibody Unit, IMCB, A*STAR, Singapore). Bacterial clone was inoculated in 5 mL of Luria-Bertani (LB media) with 100 pg/mL ampicillin and cultured overnight, added to 500 mL of Luria-Bertani/ampicillin and grown until its OD reached 0.6 to 0.8 at A600 nm. Isopropyl-L-thio-h-D-galactopyranoside was added to the culture at 0.5 mmol/L/mL and the culture was shaken overnight at room temperature. The culture was then centrifuged at 5,000 rpm for 10 minutes. The pellet was in 25 mL GST extraction buffer [1 mg/mL lysozyme, 5 mmol/L DTT and 0.5 mmol/L phenylmethylsulfonyl fluoride in GST buffer: PBS, 50 mmol/L Tris (pH 8) and 0.5 mmol/L MgCI2] The lysate was incubated on ice for 15 minutes and sonicated for 5 minutes followed by centrifugation at 15,000 rpm for 30 minutes at 4°C. The supernatant was passed through a 0.45-pM filter. One milliliter of glutathione slurry (Pharmacia, Piscataway, NJ) was packed into a column, which was washed several times with PBS. The extract was incubated with the column at 4°C for 1 hour. The unbound extract was drained out and the column was washed with GST buffer for 3 times. The GST fusion proteins were eluted with elution buffer [20 mmol/L of reduced glutathione, 100 mmol/L Tris-HCI (pH 8.0), and 120 mmol/L NaCI] and the fractions were collected and then analyzed by SDS- PAGE.

It will be appreciated that the SARS-CoV-2 N-protein from another source may be used for the present invention.

Immunization:

Animal: All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of A*STAR. 8 weeks old female BALB/c mice were purchased from In Vivos, Singapore.

Vaccine preparation & injection:

GST-Nucleocapsid protein (GST-N- protein) was purified from GST-N protein bacterial clone. Nucleocapsid peptide with or without KLH tagged, Peptide#1 , Peptide#2, Peptide#3 were synthesized by Genemed Synthesis, Inc (USA). GST-N-protein (75ug), KLH-Peptide#3 (50ug) and Peptide#3 vaccine (20ug) in 100 ul of PBS were mixed thoroughly with 100 pL of Freund’s adjuvant (complete adjuvant for 1^st immunization and incomplete adjuvant for subsequent immunization, Pierce). The mice were immunized by intraperitoneal injection of each vaccine in 2 week intervals for 3 or 4 times. The vaccine without any protein or peptide (Adjuvant only) was injected in a group of mice as control. Blood samples (20 pL each time) were taken by tail bleed in Eppendorf tube, and serum was prepared. The antibody titer was measured by ELISA.

The peptides are:

Peptide#1 (P1): CIRQGTDYKHWPQIAQFAPSASAFFGMSRIG (SEQ ID NO: 2)

Peptide#2 (P2): CIAQFAPSASAFFGMSRIG EVTPSGTWLTY (SEQ ID NO: 3)

Peptide#3 (P3): CVILLNKHIDAYKTFPPTEPKKDKKKKADET (SEQ ID NO: 4)

Preparation of serum samples for Elisa:

Collected blood at different time points were centrifuged at 5000 rpm for 15 minutes. The supernatant serum was collected and stored at -70°C. 2-fold serial dilution of serum was done in PBS starting from dilution of 2 pL serum in 1024 pL of PBS (which will be the same as 2 fold per steps in 10 steps).

Classified specific antibody subtypes induced in mice vaccinated with whole N Protein or Peptide#3:

Ninety-six-well plates (IWAKI, Japan) were coated with 100 pL of solution containing 50 ng of GST-N- protein, KLH-peptides or 20ng of peptides in PBS overnight at 4°C. Coated plates were blocked with 3% bovine serum albumin (BSA) for 1 hr room temperature and washed with PBS-t (PBS with 0.05% Tween- 20). 0.1 ml of diluted mouse serum (2-fold serial dilution) was added to each well, and incubated for 1.5 hours at 37°C. After extensive washing, different subtypes of bound antibody were detected with horseradish peroxidase (HRP)-conjugated different antibodies by incubating for 1 hour at 37°C. Anti-N Protein IgM antibody was detected by goat anti-mouse IgM-HRP Antibody (Invitrogen 626820). Subtype of anti N Protein IgG, lgG2a & lgG1 , were detected by goat anti-mouse lgG2a-HRP (Invitrogen A10685)

& goat anti-mouse lgG1-HRP (Invitrogen A10551) secondary antibodies. Anti N-protein IgG (whole IgG) were detected by goat anti-mouse IgG-HRP (H+L) secondary antibody (Invitrogen 31430). The plates were washed with PBST subsequently and 100 pL of tetramethy!benzidine (TMB) peroxidase substrate (Pierce) was added. The reaction was stopped by adding 100 pL of 2 M H2S04. Optical Density (OD) was measured at 450 nm using a plate reader (Tecan). Normal mouse serum at 10 steps dilution is used as control. OD > 3 times of normal mouse serum was considered as positive signal. The positive signal at specific steps of dilution was considered as the titer of that sample.

Elisa Assays:

Ninety-six-well plates (IWAKI, Japan) were coated with 100 pL of solution containing 50 ng of GST-N- protein, KLH-peptides or 20ng of peptides in PBS overnight at 4°C. Coated plates were blocked with 3% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20 and washed with PBS. 0.1 ml of diluted mouse serum (2-fold serial dilution) was added to each well, and incubated for 1 .5 hours at 37°C. After extensive washing, bound antibody was detected using horseradish peroxidase-conjugated anti-mouse antibody IgM, lgG2a, lgG1 , IgG-Fc by incubating for 1 hour at 37°C. Development was done using Turbo- TMB substrate (Pierce) and stopped by adding 100 pL of 2 M H2S04. Optical Density (OD) was measured at 450 nm using a plate reader (Tecan). Normal mouse serum at 10 steps dilution is used as control. OD > 3 times of normal mouse serum was considered as positive signal. The positive signal at specific steps of dilution was considered as the titer of that sample.

& goat anti-mouse lgG1-HRP (Invitrogen A10551) secondary antibodies. Anti N-protein IgG (whole IgG) were detected by goat anti-mouse IgG-HRP (H+L) secondary antibody (Invitrogen 31430). The plates were washed with PBST subsequently and 100 pL of tetramethylbenzidine (TMB) peroxidase substrate (Pierce) was added. The reaction was stopped by adding 100 pL of 2 M H2S04. Optical Density (OD) was measured at 450 nm using a plate reader (Tecan). Normal mouse serum at 10 steps dilution is used as control. OD > 3 times of normal mouse serum was considered as positive signal. The positive signal at specific steps of dilution was considered as the titer of that sample.

Immuno-profilina of blood from unvaccinated and vaccinated mice:

Whole blood from mice was lysed with ACK Lysing Buffer (Gibco, A1049201) for 10 min at RT to remove RBCs. The remaining single-cell suspensions were then stained with Zombie UV Fixable Viability dye (BioLegend) for 30 min at 4 °C, approximately 300,000 - 500,000 cells were used per stain. Non-specific labelling was blocked with anti-CD16/32 (clone 2.4G2; BD Biosciences) for 30 min at 4 °C before multiplex labelling for 30 min at 4 °C with the following antibodies from BioLegend: Brilliant Violet 711 antimouse CD3e (clone 145-2C11), PE-Cy7 anti-mouse CD4 (clone RM4-5), Brilliant Violet 786 anti-mouse CD8a (clone 53-6.7), PE/Dazzle 594 anti-mouse CD11b (clone M1/70), APC-Cy7 anti-mouse CD19 (clone 6D5), Brilliant Violet 510™ anti-mouse CD25 (clone PC61), AF488 anti-mouse CD45 (clone 30- F11), APC anti-mouse CD69 (clone H1.2F3) PE/Dazzle 594 anti-mouse CD127 (clone A7R34), Brilliant Violet 421 anti-mouse CD335 (clone 29A1 .4), Brilliant Violet 421 anti-mouse IgG (clone Poly4053). And the following antibodies from BD Biosciences: BV711 Anti-Mouse CD3e (clone 145-2C11), APC-Cy7 Rat Anti-Mouse CD19 (clone 1D3), PE Anti-Mouse CD44 (clone IM7), FITC Anti-Mouse CD45 (clone 30-F11), BV650 Anti-Mouse CD62L (clone MEL-14), PE-CF594 Anti-Mouse CD80 (clone 16-10A1), BV786 Anti- Mouse CD138 (clone 281-2), BV510 Anti-Mouse CD273 (clone TY25), APC Anti-Mouse IgD (clone 11 - 26c.2a). All samples were run on a BD LSR II flow cytometer and analysed using the FlowJo software 10.5.3 (FlowJo).

Results

N-Protein vaccination can produce high and sustainable anti N-protein different antibody subtypes

Immunization of N-protein vaccine was done in BALB/c mice and the antibody response at different time intervals was analyzed. Serum IgM, IgG and the subclasses lgG1 and lgG2a were measured using ELISA to evaluate the profile of the immune response. lgG1 indicates a humoral immune response, whereas lgG2a indicates a cellular immune response. Antibody production was not detected after 1^st dose of vaccination. IgM antibody was detected after 2^nd vaccination, but it stays at the plateau phase, and at a sustainable level throughout the time point. IgG and its subclasses lgG1 & lgG2a antibodies can also be detected after the 2^nd dose of vaccination. These antibodies gradually increase upon subsequent vaccination and remain in the plateau phase throughout the time points. Peak antibody responses were detected after the 4^th dose of vaccination in each BALB/c mouse (Figure 1A, B, C, D). The second dose of vaccine significantly boosted the concentrations of all antibody subtypes, IgM (p-value < 0.001), lgG2a (p-value <0.001), lgG1 (p-value <0.0001) and IgG (p-value <0.0001) comparison between 2 weeks (2weeks after 1^st vaccination) and 4 weeks (2 weeks after 2^nd vaccination) time point (Figure 1E). All types of antibodies were not detected in the mice vaccinated with adjuvant only. For all mice, the second dose of vaccine elicited a greater increase in the IgG 1 antibody concentration than in the lgG2a antibody concentration, which resulted in a lower lgG2a/lgG1 ratio compared to the ratio observed after the first vaccination.

Next, to confirm the above antibody production, we did immunization of N-protein in another mouse species, FVB mice. The same trend of antibody production was detected in FVB mice also (Figure 1 F).

By ELISA assays, we showed that mice vaccinated with whole N protein produced high titer of specific anti-N antibody subtypes: IgM (p-value <0.045), IgG (p-value <0.0037), lgG1 (p-value <0.045), lgG2a (p- value <0.029), suggesting that N-protein is an excellent antigen with high immunogenicity to evoke a protective immunity and produce anti-N specific antibodies at high titers.

Peptides selected on N-protein sequence could bind specifically to anti-N protein antibody

N protein vaccination resulted in a high and sustained production of different IgG subtypes in mice, indicating that N-protein is a good vaccine candidate. Based on N-protein sequences (419 amino acid sequence), we considered the development of a peptide vaccine of a specific epitope which possesses a higher accuracy in targeting N protein. We selected 3 different peptides: peptide 1 , peptide 2, and peptide 3 that are derived from N-protein, and 3 N-protein peptides were synthesized, 30 amino acids in the length of each peptide (Figure 2A) with or without KLH carrier.

To test which synthetic peptides (Peptide #1 , #2, and #3) induces the host to produce the highest titer of anti-N-protein specific antibodies, we performed ELISA assays by coating the ELISA plate with different concentration, 5 ng or 50 ng per well of each peptide, incubated with serum sample taken after the 4^th immunization of N-protein vaccine at 2-fold serial dilution at 10, 11 and 13 steps. N-protein coating was used as positive control. By the appearance of the ELISA assay, peptide #3 showed the best binding activity compared to other peptides. Quantitating the reaction by measuring Optical Density (OD) showed similar results (Figure 2B). The OD of Peptide #3 (with or without KLH carrier) were nearly 2/3 of N- protein OD at dilution step 10. The OD of other peptides, peptide #1 and Peptide #2 were nearly 10 times lower than peptide #3, indicating that Peptide 3 is the best candidate to represent N protein epitope to produce antibody which is specific to N-protein, and the peptide#3 could potentially be combined with the cocktail of traditional influenza vaccine to be a general safe vaccine.

Peptide vaccination could induce high and sustainable antibody production similar to N protein vaccine.

Among the 3 peptides synthesized, most polyclonal anti-N antibodies react highly with peptide#3, but not peptide 1 and peptide 2, suggesting that peptide 3 alone could be a good immunogen for vaccination to evoke host immune system to produce antibody specific to N protein. By mixing with Freund adjuvant, the immunization was done on BALB/c mice for 3 times in a 2-week interval. The blood collection (20 pL each) was done before each immunization and every 2 weeks after immunization. Similar to N-protein vaccination, serum Ig , IgG and the subclasses lgG1 and lgG2a were measured using ELISA to evaluate the profile of the immune response.

Antibody production can be seen afterthe 2^nd vaccine dose. The pattern and quantity of rise in antibody titer is similar to N-protein vaccine (Figure 2C, D).

To test the binding of anti-Peptide#3 antibody in mouse serum and N-protein, ELISA assay was performed by coating the ELISA plate with different concentration, 5ng or 50 ng per well, of N protein, incubated with serum sample taken after the 3^rd immunization of Peptide#3 vaccine at 2-fold serial dilution at 10, 11 and 13 steps. By the appearance of the ELISA reaction, anti-Peptide#3 antibody bound to N- protein with good affinity (Figure 2E). Quantitating the reaction by measuring Optical Density (OD) showed similar results (Figure 2F).

N protein vaccination results in the accumulation of memory T cells

To investigate if N protein vaccination can result in the accumulation of memory immune cells, 8-week-old BALB/c mice were vaccinated once weekly with a combination of Freund’s adjuvant and N protein (Vaccinated mice) for four weeks. Vaccinated mice were then bled and sacrificed eight weeks after the last vaccination to determine if they had elevated levels of memory cells compared to unvaccinated mice.

Circulating live CD45⁺CD3⁺CD335 CD4⁺CD8 CD44⁺CD62L· (Figure 3A, 3B) and CD45⁺CD3⁺CD335 CD4- CD8⁺CD44⁺CD62L^_ (Figure 3D, 3E) memory T cell frequencies were significantly (CD4⁺, p-value = 0.0000129; CD8⁺, p-value = 0.000306, one way-ANOVA) increased in vaccinated BALB/c mice compared with unvaccinated WT controls, suggesting that our vaccination protocol can successfully induce a robust and lasting memory CD4⁺ and CD8⁺ T cell population. Additional phenotypic analysis of T cell subpopulations reveal a corresponding decrease in the proportion of circulating live CD45⁺CD3⁺CD335 CD4⁺CD8 CD44 CD62L⁺ (Figure 3A, 3C) and CD45⁺CD3⁺CD335- CD4 CD8⁺CD44 CD62L⁺ (Figure 3D, 3F) naive T cell levels (CD4⁺, p-value = 0.00817; CD8⁺ p-value = 0.0160) in vaccinated BALB/c mice compared with unvaccinated WT controls, supporting our observation that our vaccination protocol results in a decrease in antigen naive T cells and an elevated frequency of antigen experienced memory CD4⁺ and CD8⁺ T cells.

In contrast, the change in live CD45⁺CD19⁺CD138 lgD⁺lgG^_ naive B cells and CD45⁺CD19⁺CD138 lgD lgG⁺ class-switched memory B cells is less distinct. The frequency of both IgG class-switched memory B cells in vaccinated mice is similar to their unvaccinated counterparts, while the frequency of naive B cells is elevated in vaccinated BALB/c mice (Figure 3G-I). The lack of a permanent large increase in the frequency of memory B cells in our vaccinated mice may indicate that T cells may be more important for mediating long term immunity against SARS-CoV-2 and may explain why some former SARS-CoV-2 patients experience a decline in antibodies several months after recovery [Marot et at, (2021); Self et at, 2020]

Example 2: Cytokine production in vaccinated mice

Materials and methods

Cytokine Array:

Mouse serum cytokines from unvaccinated and vaccinated mice were analysed with the RayBiotech mouse cytokine array C1 (Cat: #AMM-CYT-1-8) using the provided experimental protocol unless otherwise indicated. In brief, blots were blocked with 2mL of provided blocking buffer and incubated for 30mins at room temperature. 6pL of each serum sample was diluted to a total volume of 500pL with blocking buffer and arrays were incubated overnight at 4°C with dilute serum samples. Arrays were then washed with provided washing buffers according to the standard protocol. Next arrays were incubated with 500pL of pre-diluted biotinylated antibody cocktail for 4hrs at room temperature. Arrays were then washed with provided washing buffers according to the standard protocol. Arrays were then incubated with 500pL of x1 HRP-Streptavidin for 2hrs at room temperature. Arrays were washed and incubated with detection buffers for chemiluminescence detection.

Chemiluminescence Detection:

The fold change of N4 and N12 vaccinated mice was calculated for all the cytokine proteins against untreated mice (see Figure 5). A significant cut-off fold change > 2 is used and highlighted in red. Seven proteins (IFN gamma, CCL2, GCSF, IL-10, CCL5, TNFR1 and TNFalpha) highlighted in yellow exhibits both significant and steady rise in the fold change in both groups. Results

Vaccination N protein with complete Freund’s adjuvant (CFA) can induce the secretion of pro- inflammatory memory cell and TH1 associated cytokines.

Cytokine array studies of mouse serum suggests that our vaccinated mice have elevated levels of pro- inflammatory cytokines and chemokines such as CCL2, CCL5, IFN-y, TNF-a, TNF-RI, GCSF, IL-4, and IL-10 compared to unvaccinated mice (Figure 4A-4D). A subsequent cycle of vaccination (4^th immunization) also results in an increased level of these cytokines compared to a prior cycle (2^nd immunization), suggesting that repeated vaccinations with N protein can result in progressively elevated cytokine levels in mice (Figure 4C-4D) and likely enhanced immune responses during subsequent vaccinations due to the accumulation of memory immune cells.

Example 3: Generation of Anti N-protein antibody against Nucleocapsid N protein for therapeutic Materials and methods

Generation of mouse anti-N protein monoclonal antibody:

N-protein hybridoma clone generated by fusion of splenocytes from N-protein immunized BALB/c mice and BALB/c parental myeloma SP2/0 cells was kindly provided by Dr Yee Joo TAN (Monoclonal Antibody Unit, IMCB, A*STAR, Singapore). Hybridoma cells (5 x 10⁵) were suspended in 200 pL of Phosphate Buffered Saline (PBS) and injected into the peritoneal cavity and wait until the mouse developed a large quantity of ascitic fluid. The mouse was sacrificed and ascitic fluid was collected, centrifuged and frozen at -70°C until further use.

Total RNA was isolated from the hybridoma cells following the technical manual of RNeasy Plus Micro Kit. Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase. Antibody fragments of heavy chain procedure (SOP) of rapid amplification of cDNA ends (RACE). Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes.

Generation of Ascetic Fluids:

Hybridoma cells (5 x 10⁸) were suspended in 200 L of serum-free DMEM medium and injected with a 26- gauge needle into the peritoneal cavity to BALB/c mice. After 10 days, the mouse developed a large quantity of ascitic fluid, and the abdomen was greatly distended. The mouse was sacrificed and a small shallow was cut to open the abdominal cavity. The ascitic fluid was drawn with a 10-mL syringe fitted with an 18-gauge needle. The fluid was centrifuged at 200 xg for 10 minutes at 4°C. The supernatant fluid was collected and frozen at -70°C until further use. Results

In 2003, we generated a monoclonal antibody (clone 6H3) against SARS-CoV. This SARS-CoV antibody binds to SAR-CoV2 N-protein with good affinity (Figure 5). We further demonstrated 6H3 cross-reacting with SAR-CoV-2, using ELISA assay to access the binding affinity. ELISA plate was coated with 5 & 20 ng/well of Peptide #1 , Peptide #2, Peptide #3 & N-protein (SARS-CoV2). Anti-SARS-CoV-N protein antibody clone (6H3) was diluted at 1 :1000 & 1 :5000, followed by goat anti-mouse IgG-HRP secondary antibody. The measurement of Optical Density showed Mouse SAR-CoV-N-protein antibody could not bind well to Peptide #1 , Peptide #2, and Peptide #3 but bind strongly to SARS-CoV2 N-protein in a concentration dependent manner (Figure 5), suggesting clone 6H3 epitope presents in the whole N protein but not in all 3 peptides. The mouse antibody (6H3) can be developed for the First in Class humanized antibody to treat patients infected with coronavirus. The sequences for 6H3 are provided in Figure 6.

Example 4: Discussion

Severe Acute Respiratory Syndrome Coronavirus 2 (SAR-CoV-2) caused the global pandemic of the Coronavirus disease in late 2019 (COVID-19). Vaccine development efforts have predominantly been aimed at ‘Extra-viral’ Spike (S) mRNA as vaccine vehicles but there are concerns regarding ‘viral immune escape’ since multiple mutations may enable the mutated virus strains to escape from immunity against S protein. The ‘Intra-viral’ Nucleocapsid (N-protein) is relatively conserved among mutant strains of coronaviruses during spread and evolution. Herein, we demonstrate novel vaccine candidates against SARS-CoV-2 by using the whole conserved N-protein or its fragment/peptides. Using ELISA assay, we showed that high titers of specific anti-N antibodies (IgG, lgG1 , lgG2a, IgM) were maintained > 5 months, suggesting that N-protein is an excellent immunogen to stimulate host immune system and robust B cell activation. We synthesized 3 peptides located at the conserved regions of N-protein among CoVs. One peptide showed as a good immunogen for vaccination as well. Cytokine arrays on post-immunization mouse sera showed progressive upregulation of various cytokines such as IFN-y and CCL5, suggesting that TH1 associated responses are also stimulated. Furthermore, vaccinated mice exhibited an elevated memory T cells population. Here, we propose an unconventional vaccine strategy targeting the conserved N-protein as an alternative ‘Universal vaccine’ for coronaviruses. Moreover, we generated a mouse monoclonal antibody specifically against an epitope shared between SAR-CoV and SAR-CoV-2, and we are currently developing the First-in-Class humanized anti-N-protein antibody to potentially treat patients infected by various CoVs in the future.

In this study, we have demonstrated that mice vaccinated with N protein/or N protein fragment/peptides had sustainably high titers of anti-N antibodies (IgG, lgG1 , lgG2a, IgM). Interestingly, vaccination with peptide #3 gave similar results as that of the whole N protein, suggesting that peptide #3 is not only the major epitope in the N-protein but also sufficient to elicit protective immunity in the host. We also observed a robust induction of CD4⁺ and CD8⁺ memory T cells along with the induction of pro- inflammatory and T_H1 associated cytokines. A major challenge in the early development of SARS coronavirus vaccines has been the discovery that double-inactivated SARS-CoV whole viral vaccines have low efficacy and resulted in enhanced immune pathology especially in aged animal model [Bolles et ai, (2011)]. Interestingly, a further study demonstrated that while vaccination with Venezuelan equine encephalitis virus replicon particles (VRP) containing the SARS-CoV strain spike (S) glycoprotein could provide protection against viral challenges, vaccination with nucleocapsid (N) protein resulted in enhanced immunopathology with increased eosinophilic lung infiltrates in challenged mice [Deming et ai, (2006)]. Another study also reported that SARS-CoV N protein vaccination in mice resulted in severe pneumonia upon viral challenge, suggesting that excessive host immune response against N protein may cause the severe acute lung injury observed in SARS-CoV infection [Yasui etai, (2008)]. In addition, only mice immunized with S protein exhibited reduced viral titers after vaccination, in contrast to vaccination with other SARS-CoV structural proteins, such as the N, membrane (M), and envelop (E) proteins [Yasui et ai, (2008)].

Clinically, patients with both SARS-CoV and SARS-CoV-2 first exhibit antibodies against N protein and antibodies against N protein are the most sensitive for serologic diagnosis [Tan etai. , (2004); Wu etai, (2004); Leung et ai, (2004); Zhu et ai, (2006); Burbelo etai, (2020a)]. Interestingly patients with elevated levels of antibodies against N protein and lower levels of anti-S protein antibodies have a higher risk of admission to the intensive care unit and longer hospitalization stays [Batra etai, (2021); Roltgen et ai, (2020)]. This may suggest that N protein antibodies may also favor a stronger inflammatory response in human patients.

Similar to the SARS coronavirus (SARS-CoV), SARS-CoV-2 infects target cells via spike protein receptor binding domain (RBD) and ACE2 receptor interactions [Hoffmann etai, (2020); Zhou etai, (2020)]. To generate effective neutralizing antibodies to block SARS-CoV-2 viral entry, the SARS-CoV-2 spike protein and its RBD were selected as the leading target antigens in vaccine development [Chen et ai, (2020); Pang et ai, (2020)]. Wang etai, (2021) reported that volunteers injected with either the Moderna (mRNA- 1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2 demonstrated high titres of IgM and IgG antibodies against SARS-CoV-2 S protein and RBD [Wang et ai, (2021)]. The plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated individuals is also reported to be similarto patients who recovered from natural infection [Wang et ai, (2021); Gaebler ef ai, (2021); Robbiani etai, (2020)]. A study group involving approximately 600,000 individuals in Israel also demonstrated that the BNT162b2 vaccine has an 92% effectiveness of preventing SARS-CoV-2 infection [Dagan etai, (2021)].

However, the presence of neutralizing antibodies against SARS-CoV-2 S protein and its RBD does not confer complete protection against SARS-CoV-2 infection in all vaccinated individuals, even in recently vaccinated individuals. Surprisingly, a subset of recently vaccinated individuals still contract SARS-CoV-2 despite multiple vaccinations which should have induced robust levels of neutralizing antibodies. In addition, it has been reported that the titer of SARS-CoV-2 neutralizing antibodies decline fairly rapidly, with some individuals reporting close to baseline neutralizing antibody levels as soon as two months postinfection [Marot etai, (2021); Seow et ai, (2020); Yamayoshi etai, (2021)]. The SARS-CoV-2 S gene also has a relatively lower amino acid similarity (76%) compared to the SARS-CoV S gene with a higher rate of mutation compared to the more conserved (90)% N gene [Dutta etal., (2020); Grifoni et ai, (2020a); Marra et al., (2003); Drosten etal., (2003); Zhu et al., (2005)]. This data suggests that while anti- S protein antibodies may be key for controlling viral titres during an ongoing infection, other immune mediators may be responsible for conferring long-term immunity to SARS-CoV-2.

The N protein is highly immunogenic and is the most abundant viral protein during coronavirus infections [Dai etal., (2021); Long etal., (2020)]. It is also a major target for antibody and T cell responses [Sariol and Perlman (2020)]. Importantly, non-neutralizing antibodies against N protein can protect mice against some other viruses, such as the mouse hepatitis virus [Nakanaga etal., (1986); Lecomte etal., (1987)] and influenza A virus [Fujimoto etal., (2016)]. N protein is also commonly externalized on the cell surface membrane of infected cells, and can act as a potential target for both antibody and T cell responses [Fujimoto etal., (2016)].

Furthermore, memory T cells may play a critical role in conferring long-term immunity to SARS-CoV-2. Grifoni et al (2020b) reports the induction of robust CD4⁺ and CD8⁺ T cells in convalescent SARS-CoV-2 patients. Surprisingly even some non-exposed individuals demonstrate T cell reactivity against SARS- CoV-2 epitopes, suggesting that prior infections in these individuals could also enhance immunity against SARS-CoV-2 [Grifoni et al (2020b)]. Le Bert et al also demonstrates that former SARS-CoV patients possess long lasting memory T cells which are reactive to N protein over 17 years after the SARS epidemic in 2003 [Le Bert etal, (2020)]. These memory T cells were also highly cross-reactive to the SARS-CoV-2 N protein, suggesting that these individuals may be less susceptible to SARS-CoV-2 infection and other similar coronavirus [Le Bert et al, (2020)]. Other animal model studies involving vaccination with SARS-CoV N protein have also demonstrated robust SARS-specific T cell proliferation and cytotoxic responses [Gao etal., (2003); Okada et al., (2003)]. N protein specific CD8⁺ T cells also protect against infectious bronchitis virus model in chickens. This data suggests that T cells are essential for mediating long-term immunity.

In short, our findings have shown that N protein vaccination provides sustainably long protective immunity. Much emphasis has been placed on the extra-viral spike (S) protein in vaccine development. This is due to its importance in the detection by host immune system and viral entry into host cells. However, the glycosylation and mutation of the S protein have posed challenges in vaccine development. Intra-viral nucleocapsid (N) protein, on the other hand, is more conserved [Surjit etal., (2008)] in sequence. Importantly, N protein can be detected by the host immune system as there is a presence of anti-N protein antibodies in the sera of SARS-CoV-2 infected patients [Burbelo et al., (2020b)]. On the potential of anti-N protein antibodies in the prevention of infection, dominant helper T-cell epitopes in the N protein of SARS-CoV have been identified to assist in antiviral neutralizing antibody production [Zhao et al., (2007). The anti-N protein antibodies have been previously shown to confer protection against several types of lethal influenza A viruses [La Mere etal., (2011); Fujimoto et al., (2016); Carragher etal., (2008)]. A combination of neutralizing antibodies targeting S protein and its RBD, anti-N protein antibodies, and memory T cells against N protein epitopes may be essential to confer long-term protection against SARS- CoV-2.

Our data showed that monoclonal anti-N protein antibody raised against N protein of SARS-CoV can recognize/bind to the SARS-CoV-2 N protein with good affinity. From this, it is hypothesized that a humanized anti-N protein antibody could potentially be used as a therapy in the eradication of infected host cells.

Similar strategies of Intra-viral protein unconventional immunotherapies could apply to other viral infections, such as HBV or HIV.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that such document is part of the state of the art or is common general knowledge.

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Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

Claims

Claims:

1 . An antibody, or antigen binding fragment, optionally isolated, that binds SARS-Cov-2 N-protein.

2. The antibody, or antigen binding fragment, of claim 1 , having the amino acid sequences i) to iii), or the amino acid sequences iv) to vi), or preferably the amino acid sequences i) to vi): vii) NYGMN (SEQ ID NO: 7) viii) WINTYTGEPTYADDFKG (SEQ ID NO: 8) ix) PLYYDYDGHAMDY (SEQ ID NO: 9) x) RSSKSLLHSNGITY (SEQ ID NO: 11) xi) QMSNLAS (SEQ ID NO: 12) xii) QNLELMWT (SEQ ID NO: 13) or a variant thereof in which one or two or three amino acids in one or more of the sequences (i) to (vi) are replaced with another amino acid.

3. The antibody, or antigen binding fragment, of any one of claims 1 to 3, having at least one light chain variable region incorporating the following CDRs:

LC-CDR1 : RSSKSLLHSNGITY (SEQ ID NO: 11);

LC-CDR2: QMSNLAS (SEQ ID NO: 12); and

LC-CDR3: QNLELMWT (SEQ ID NO: 13).

4. The antibody, or antigen binding fragment, of any one of claims 1 to 4, having at least one heavy chain variable region incorporating the following CDRs:

HC-CDR1 : NYGMN (SEQ ID NO: 7);

HC-CDR2: INTYTGEPTYADDFKG (SEQ ID NO: 8); and

HC-CDR3: PLYYDYDGHAMDY (SEQ ID NO: 9).

5. An antibody, or antigen binding fragment, optionally isolated, having the amino acid sequences i) to iii), or the amino acid sequences iv) to vi), or preferably the amino acid sequences i) to vi): i) NYGMN (SEQ ID NO: 7) ii) WINTYTGEPTYADDFKG (SEQ ID NO: 8) iii) PLYYDYDGHAMDY (SEQ ID NO: 9) iv) RSSKSLLHSNGITY (SEQ ID NO: 11) v) QMSNLAS (SEQ ID NO: 12) vi) QNLELMWT (SEQ ID NO: 13) or a variant thereof in which one or two or three amino acids in one or more of the sequences (i) to (vi) are replaced with another amino acid.

6. The antibody, or antigen binding fragment, of claim 6, having at least one light chain variable region incorporating the following CDRs: LC-CDR1 : RSSKSLLHSNGITY (SEQ ID NO: 11);

LC-CDR2: QMSNLAS (SEQ ID NO: 12); and

LC-CDR3: QNLELMWT (SEQ ID NO: 13).

7. The antibody, or antigen binding fragment, of claim 6 or 7, having at least one heavy chain variable region incorporating the following CDRs:

HC-CDR1 : NYGMN (SEQ ID NO: 7);

HC-CDR2: INTYTGEPTYADDFKG (SEQ ID NO: 8); and

HC-CDR3: PLYYDYDGHAMDY (SEQ ID NO: 9).

8. An in vitro complex, optionally isolated, comprising an antibody, or antigen binding fragment, according to any one of claims 1 to 9 bound to SARS-Cov-2 N-protein.

9. An isolated light chain variable region polypeptide comprising the following CDRs:

LC-CDR1 : RSSKSLLHSNGITY (SEQ ID NO: 11);

LC-CDR2: QMSNLAS (SEQ ID NO: 12); and

LC-CDR3: QNLELMWT (SEQ ID NO: 13).

10. An isolated heavy chain variable region polypeptide comprising the following CDRs:

HC-CDR1 : NYGMN (SEQ ID NO: 7);

HC-CDR2: INTYTGEPTYADDFKG (SEQ ID NO: 8); and

HC-CDR3: PLYYDYDGHAMDY (SEQ ID NO: 9).

11. An antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence, wherein: the heavy chain comprises a HC-CDR1 , HC-CDR2, HC-CDR3, having at least 85% overall sequence identity to NYGMN (SEQ ID NO: 7), INTYTGEPTYADDFKG (SEQ ID NO: 8) and PLYYDYDGHAMDY (SEQ ID NO: 9) respectively, and the light chain comprises a LC-CDR1 , LC-CDR2, LC-CDR3,, having at least 85% overall sequence identity to RSSKSLLHSNGITY (SEQ ID NO: 11), QMSNLAS (SEQ ID NO: 12) and QNLELMWT (SEQ ID NO: 13) respectively.

12. An antibody or antigen binding fragment, optionally isolated, comprising a heavy chain and a light chain variable region sequence, wherein: the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRF AFSLETSASTAYLQINNLKNEDMAKYFCTRPLYYDYDGHAMDYWGQGTSVTVSS (SEQ ID NO:6), and the light chain sequence has at least 85% sequence identity to the light chain sequence:

DIVMTQAAFSNPVTLTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGS TDFTLRISRVEAEDVGVYYCAQNLELMWTFGGGTKLEIK (SEQ ID NO:14).

13. A composition comprising the antibody, or antigen binding fragment, or polypeptide of any one of claims 1 to 7 and at least one pharmaceutically acceptable carrier.

14. An isolated nucleic acid encoding the antibody, or antigen binding fragment or polypeptide of any of one of claims 1 to 7.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell comprising the vector of claim 14.

17. A method for making an antibody, or antigen binding fragment or polypeptide of any of one of claims 1 to 12 comprising culturing the host cell of claim 16 under conditions suitable for the expression of a vector encoding the antibody, or antigen binding fragment or polypeptide, and recovering the antibody, or antigen binding fragment or polypeptide.

18. An antibody or antigen binding fragment according to any of one of claims 1 to 7 for use in therapy, or in a method of medical treatment.

19. An antibody or antigen binding fragment according to any of one of claims 1 to 7 for use in the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

20. Use of an antibody or antigen binding fragment according to any of one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

21. A method of treating a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the method comprising administering an antibody or antigen binding fragment according to any one of claims 1 to 7 to a patient determined to have a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.