WO2021206638A1

WO2021206638A1 - Vaccine and/or antibody for viral infection

Info

Publication number: WO2021206638A1
Application number: PCT/SG2021/050197
Authority: WO
Inventors: Qi Zeng
Original assignee: Intra-Immusg Private Limited
Priority date: 2020-04-09
Filing date: 2021-04-08
Publication date: 2021-10-14
Also published as: WO2022216223A1

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 also includes a nucleic acid molecule encoding the SARS-CoV-2 N-protein and/or an immunogenic fragment thereof; and uses thereof. The SARS-CoV-2 N protein and/or immunogenic fragment thereof may be produced by recombinant DNA technology or may be chemically synthesised. In particular, the SARS-CoV-2 N-protein and/or an immunogenic fragment thereof and/or nucleic acid molecule encoding the SARS-CoV-2 N-protein and immunogenic may be for use as a vaccine. The invention further 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

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 of the invention

Coronaviruses (CoVs) 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.

Summarv 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:

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.

Brief description of the figures

Figure 1 shows the antibody responses to vaccination of nucleocapsid protein in four Balb C mice (A, B, C, D). 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-lgM, -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). (E) 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 antimouse 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 (WT) 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*CD8· T cells in unvaccinated and vaccinated mice. (D) Representative CD62L and CD44 staining on live C D45*C D3*C D335 C D4 C D8* T cells from the blood of Balb/c mice. (E) Change in the percentage of live CD44*CD62I_· 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 IgD* B cells and (I) IgG* class-switched memory B cells. Data representing meant 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- & post-immunization 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-CoVAb (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.

Definitions

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/polypeptide is defined as a fragment of said protein/polypeptide of at least about 4 or 5 amino acids in length, capable of eliciting a specific antibody and/or an immune cell (e.g., a T cell or B cell) 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.

Detailed description of the invention 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 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. 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);

CIAQFAPSASAFFGMSRIGMEVTPSGTWLTY (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 immumogenic 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)]. 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.

The antibody capable of binding to the to the SARS-CoV-2 N-protein may be a monoclonal antibody. The monoclonal antibody may be a chimeric or humanised antibody. It will be appreciated that the monoclonal antibody may be produced by any method, for example hybridoma technology or recombinant DNA technology.

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

MDWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGE TVKISCKASGYTFTNYGMNWVKQA PGKGLKWMGWINTYTGEPTYADDFKGRFAFSLE TSASTAYLQINNLKNEDMAKYFCTRP LYYDYDGHAMDYWGQGTSVTVSS

The regions of the heavy chain are arranged in the following order Signal oeotide-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:

QIQLVQSGPELKKPGETVKISCKASGYT FTNYGMNWVKQAPGKGLKWMGWINTY TGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMAKYFCTRPLYYDYDGHA MDYWGQGTSVTVSS

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:

In SEQ ID NO: 9 above, the nucleotide regions encoding the regions of the heavy chain are shown in the following order, Signal sequence-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).

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.

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.

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)].

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.

Examples

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).

Example 1: Vaccination of mice with SARS-CoV-2 nucleocapsid protein (N- protein) or immunogenic peptides derived from SARS-CoV-2 nucleocapsid protein (N-protein) 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 μg/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-μΜ 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 μL 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 μL 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): CIAQFAPSASAFFGMSRIGMEVTPSGTWLTY (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 antimouse 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 μL 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.

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. 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 antimouse 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-profiling 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 anti-mouse 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 1 D3), 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 1 E). 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 lgG1 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 μL each) was done before each immunization and every 2 weeks after immunization. Similar to N-protein vaccination, serum IgM, IgG and the subclasses lgG1 and lgG2a were measured using ELISA to evaluate the profile of the immune response. Antibody production can be seen after the 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⁺CD62I_- (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⁺CD138lgD⁺lgG^' naive B cells and CD45⁺CD19⁺CD138lgDlgG⁺ 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 al., (2021); Self et al., 2020], Example 2: Cvtokine 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 2m L of provided blocking buffer and incubated for 30m ins at room temperature. 6pL of each serum sample was diluted to a total volume of 500μL 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 500μL 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 500μΙ_ 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 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-α, 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 μL 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 10⁶) 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 x g 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.

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-γ 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 TH1 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 al., (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 al., (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 etal., (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 etal., (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 et al., (2004); Wu et al., (2004); Leung et al., (2004); Zhu etal., (2006); Burbelo etal., (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 et al., (2021); Roltgen et al., (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 et al., (2020); Zhou etal., (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 al., (2020); Pang et al., (2020)]. Wang et al., (2021) reported that volunteers injected with either the Modema (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2 demonstrated high litres of IgM and IgG antibodies against SARS-CoV-2 S protein and RBD [Wang et al., (2021)]. The plasma neutralizing activity and relative numbers of RBD- specific memory B cells of vaccinated individuals is also reported to be similar to patients who recovered from natural infection [Wang et al., (2021); Gaebler et al., (2021); Robbiani et al., (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 et al., (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 post-infection [Marot et al., (2021); Seow et al., (2020); Yamayoshi et al., (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 et al., (2020); Grifoni et al., (2020a); Marra et al., (2003); Drosten et al., (2003); Zhu et al., (2005)]. This data suggests that while anti-S protein antibodies may be key for controlling viral litres 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 et al., (2021); Long et al., (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 et al., (1986); Lecomte et al., (1987)] and influenza A virus [Fujimoto et al., (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 et al., (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 et al, (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 et al., (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 et al., (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 et al., (2011); Fujimoto et al., (2016); Carragher et al., (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

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. Batra, M., et al., Role of IgG against N-protein of SARS-CoV2 in COVID19 clinical outcomes. Sci Rep, 2021. 11(1): p. 3455.

Bolles, M., et al., A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflam matory pulmonary response upon challenge. J Virol, 2011. 85(23): p. 12201-15.

Burbelo, P.D., et al., Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 2019. J Infect Dis, 2020a. 222(2): p. 206- 213.

Burbelo, P.D., et al., Detection of Nucleocapsid Antibody to SARS-CoV-2 is More Sensitive than Antibody to Spike Protein in COVID-19 Patients. medRxiv : the preprint server for health sciences, 2020b: p. 2020.04.20.20071423.

Carragher, D.M., et al., A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus. Journal of immunology (Baltimore, Md. : 1950), 2008. 181(6): p. 4168-4176.

Chen, W.H., et al., The SARS-CoV-2 Vaccine Pipeline: an Overview. Curr Trap Med Rep, 2020: p. 1-4.

Dagan, N., et al., BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting. N Engl J Med, 2021. Dai, L. and G.F. Gao, Viral targets for vaccines against COVID-19. Nat Rev Immunol, 2021. 21(2): p. 73-82.

Deming, D., et al., Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med, 2006. 3(12): p. e525.

Drosten, C., et al., Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med, 2003. 348(20): p. 1967-76.

Dutta, N.K., K. Mazumdar, and J.T. Gordy, The Nucleocapsid Protein of SARS- CoV-2: a Target for Vaccine Development. J Virol, 2020a. 94(13).

Fujimoto, Y., et al., Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection. J Gen Virol, 2016. 97(9): p. 2104-2116.

Gaebler, C., et al., Evolution of antibody immunity to SARS-CoV-2. Nature, 2021.

Gao, W., et al., Effects of a SARS-associated coronavirus vaccine in monkeys. Lancet, 2003. 362(9399): p. 1895-6.

Grifoni, A., et al., A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. Cell Host Microbe, 2020a. 27(4): p. 671-680 e2.

Grifoni, A., et al., Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell, 2020b. 181(7): p. 1489-1501 e15.

Hoffmann, M., et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 2020. 181(2): p. 271-280 e8. Le Bert, N., et al., SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature, 2020. 584(7821): p. 457-462.

Lecomte, J., et al., Protection from mouse hepatitis virus type 3-induced acute disease by an anti-nucleoprotein monoclonal antibody. Brief report. Arch Virol, 1987. 97(1 -2): p. 123-30.

LaMere, M.W., et al., Regulation of Antinucleoprotein IgG by Systemic Vaccination and Its Effect on Influenza Virus Clearance. Journal of Virology, 2011. 85(10): p. 5027.

Leung, D.T., et al., Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid. J Infect Dis, 2004. 190(2): p. 379-86.

Long, Q.X., etal., Antibody responses to SARS-CoV-2 in patients with COVID- 19. Nat Med, 2020. 26(6): p. 845-848.

Marot, S., et al., Rapid decline of neutralizing antibodies against SARS-CoV-2 among infected healthcare workers. Nat Commun, 2021. 12(1): p. 844.

Marra, M.A., et al., The Genome sequence of the SARS-associated coronavirus. Science, 2003. 300(5624): p. 1399-404.

Nakanaga, K., K. Yamanouchi, and K. Fujiwara, Protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice. J Virol, 1986. 59(1): p. 168-71.

Okada, M., et al., The development of vaccines against BARS corona virus in mice and SCID-PBL/hu mice. Vaccine, 2005. 23(17-18): p. 2269-72.

Pang, J., etal., Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review. J Clin Med, 2020. 9(3). Robbiani, D.F., et al., Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature, 2020. 584(7821 ): p. 437-442.

Roltgen, K., et al., Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Sci Immunol, 2020. 5(54).

Sariol, A. and S. Perlman, Lessons for COVID-19 Immunity from Other Coronavirus Infections. Immunity, 2020. 53(2): p. 248-263.

Self, W.H., et al., Decline in SARS-CoV-2 Antibodies After Mild Infection Among Frontline Health Care Personnel in a Multistate Hospital Network - 12 States, April-August 2020. MMWR Morb Mortal Wkly Rep, 2020. 69(47): p. 1762-1766.

Seow, J., et al., Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Nat Microbiol, 2020. 5(12): p. 1598-1607.

Surjit, M. and S.K. Lai, The SARS-CoV nucleocapsid protein: a protein with multifarious activities. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 2008. 8(4): p. 397-405.

Tan, Y.J., et al., Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers. Clin Diagn Lab Immunol, 2004. 11(2): p. 362-71.

Wang, Z., et al., mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature, 2021.

WHO Coronavirus (COVID-19) Dashboard, https://covid19.who.int/ (assessed at 3^rd April 2021). Wu, H.S., et al., Early detection of antibodies against various structural proteins of the SARS-associated coronavirus in SARS patients. J Biomed Sci, 2004. 11(1): p. 117-26.

Yamayoshi, S., et al., Antibody titers against SARS-CoV-2 decline, but do not disappear for several months. EClinicalMedicine, 2021. 32: p. 100734.

Yasui, F., et al., Prior immunization with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV. J Immunol, 2008. 181(9): p. 6337-48.

Zhao, J., et al., Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus. Journal of virology, 2007. 81 (11 ): p. 6079-6088.

Zhou, P., et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020. 579(7798): p. 270-273.

Zhu, Y., et al., Isolation of virus from a SARS patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 SARS- CoV isolates. Virus Genes, 2005. 30(1): p. 93-102.

Zhu, H., et al., Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray. Proc Natl Acad Sci U S A, 2006. 103(11): p. 4011-6.

Claims

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 claim 1, comprising SEQ ID NO: 1.

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

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

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

7. An immunogenic fragment according to claim 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 fragment(s) according to any one of claims 1 to 7.

9. The immunogenic combination and/or immunogenic composition according to claim 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 claim 10.

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

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

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

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

16. The isolated N-protein and/or immunogenic fragment thereof according to any one of claims 1 to 7, the isolated nucleic acid molecule according to claim 10 and/or the vector according to claim 11 , for use as a vaccine.

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

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

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

20. The use according to claim 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 claim 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 claims 1 to 7, the immunogenic combination and/or the immunogenic composition according to claim 8, the isolated nucleic acid molecule according to claim 9 and/or the vector according to claim 10.

23. The method according to claim 22, wherein the viral infection comprises a Coronavirus infection.

24. The method according to claim 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 claim 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 claim 26 wherein the antibody comprises a monoclonal antibody.

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

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

30. The antibody according to claim 26, comprising a heavy chain variable region comprising SEQ ID NO: 6.

31. The antibody according to claim 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 claims 26 to 31.

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

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

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

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

37. The antibody for the use according to claim 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 claim 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 claims 26 to 31 in the preparation of a pharmaceutical composition for treating a viral infection.

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

41. Use according to claim 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 claim 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 claims 26 to 31 to the subject.

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

45. The method according to claim 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 claim 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 claims 26 to 31.

48. The isolated nucleic acid molecule according to claim 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 claims 26 to 31.

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

51. The isolated nucleic acid molecule according to claim 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 claims 26 to 31.

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