CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. § 119 (e), of US provisional application No. 60/564/984 filed April 26, 2004 and US provisional application No. 60/643,574 filed January 14, 2005. Both applications are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to peptides and corresponding nucleic acids and uses thereof for prevention, treatment and diagnosis of SARS, and particularly relates to peptides and corresponding nucleic acids derived from the SARS coronavirus nucleocapsid protein, and uses thereof for prevention, treatment and diagnosis of SARS.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome (SARS) is a pneumonia-like disease of a previously unknown etiology that emerged and spread globally affecting countries in Asia, Europe, and North America in early 2003. According to the World Health Organization, the fatality rate of SARS is 14-15% overall and >50% in persons over the age of 65 (•www.who.int/csr/sars/en/). Simultaneously, two groups identified a novel coronavirus as the etiological agent responsible for the cause of SARS designating it the SARS coronavirus (SARS-CoV) . Subsequent infection of non-human primates with SARS-CoV led to similar pathological and clinical features of severe acute respiratory syndrome, confirming that the SARS-CoV was the etiological agent l. As

i with other members of the coronavirus family, the SARS-CoV genome encodes 5 proteins of predicted function; a membrane protein (M protein) , envelope protein (small E protein) , surface glycoprotein (Spike) and nucleocapsid (N protein) , as well as open reading frames of unknown predicted function, however it does lack a haemagluttinin protein (HA) 2'3. Coronaviruses have a crown-like morphology attributed to the surface-expressed Spike protein. The SARS- CoV Spike protein is a glycoprotein of approximately 180 kDa that is composed of two subunits Sl and S2 4. The function of the Spike protein in coronavirus infection is to act as the fusogen aiding in viral attachment and entry into host cells 5. The cellular receptor for the Spike protein of the SARS-CoV has been identified as angiotensin-converting enzyme-2 6, with the binding domain localized to the Sl subunit 7. Studies have been published regarding the ability of Spike to elicit neutralizing antibody responses and provide protection during challenge experiments in animal models of coronavirus infection 8~10. These studies show that there is a strong ' antibody response elicited from inoculation with the Spike protein, and this antibody response is able to provide neutralizing activity against viral infection. The course of SARS-CoV infection is marked by a significant SARS specific IgM response within the first 8-14 days of onset of symptoms with the generation of an IgG response later on in the infection 1X. Inoculation of mice with a plasmid encoding the Spike gene from the SARS-CoV into mice was capable of eliciting an antibody response against the protein and prevented pulmonary viral replication in the absence of T lymphocytes 12. The N protein of the SARS-CoV consists of 422 amino acids, of which there is a unique basic sequence located near the carboxy terminus 3. There are no known post-translational modifications of the protein. Functionally, the N protein is predicted to associate with the genomic DNA and contains an RNA-binding domain. Moreover, the N protein has been shown to interact with the M protein playing a role in viral assembly 13. The nucleoprotein has been shown to localize to the host cell nucleolus where it is postulated to promote translation of viral mRNA 14. Certain viral proteins of the types noted above have been shown to be involved in eliciting immune responses. For example, inoculation of chickens with plasmid DNA encoding' the carboxyl terminus of the infection bronchitis virus (IBV) N protein led to the induction of a cytotoxic T lymphocyte population capable of recognizing two distinct IBV strains. Additionally, the adoptive transfer of T cells from animals inoculated with IBV to naϊve chicks provided CD8+ αβ specific protection 15. Rhesus macaques immunized with a cocktail of adenoviral vectors encoding Sl fragment of Spike, M protein and N protein of the SARS-CoV led to the generation of antibody responses against the Sl fragment of Spike protein i and T-cell responses against the N protein 16. The injection of a T helper type 1 (ThI) T cell line resistant to MHV into susceptible mice led to a marked increase in IFNγ expression with a decrease in IL-4 production concomitant with complete protection against infection 17. Demonstration that the immunity against MHV is a CD8+, perforin, IFNγ dependent process in the absence of CD4+ T cells 18 and the generation of IgG2a antibodies following DNA immunization of N protein 19 suggests that there is a significant ThI type response in coronavirus infection. Further, after surveying the sera of over 400 probable' SARS patients, Liu et al found that more than 90% of these patients generated an antibody response against the N protein 14 days after the onset of symptoms 20, indicative of an adaptive immune response to foreign antigen. The reemergence of SARS in early January 2004 indicates that there is a reservoir for the virus and the reentry of the virus into a large population is probable. There is therefore a need to develop methods and agents for the prevention and treatment of SARS.

SUMMARY OF THE INVENTION

The invention relates to epitopes derived from the SARS-CoV nucleocapsid protein (N protein) and uses thereof. Accordingly, the invention provides an isolated immunogenic polypeptide derived from the SARS-CoV nucleocapsid protein. In an embodiment, the polypeptide comprises an epitope of at least 8, in a further embodiment at least 9, contiguous amino acids of SEQ ID NO: 496, wherein said polypeptide is not the full length SARS CoV N protein set forth in SEQ ID NO: 496. In embodiments, the epitope is selected from the epitopes defined by the start and end positions within SEQ ID NO: 496 set forth in Table 5. In a further embodiment, the polypeptide comprises an epitope of at least 8, in a further embodiment at least 9, contiguous amino acids of an amino acid sequence selected from SEQ ID NOs: 499-505. In an embodiment, the isolated polypeptide is selected from: (a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43-46, 52-54, 61, 62, 64, 66, 67, 69-75, 78, 79, 85, 87, 90, 93, 111, 117, 119, 122, 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181-186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245-248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352, 366, 372, 374, 376, 377, 380-382, 384, 386-390, 392-397 , 400, 405, 409, 410 , 411 , 412 , 419, 426, 432-434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505; and (b) a functional variant or fragment of (a) , wherein said functional variant or fragment has an immune system-related activity.

In an embodiment, the immune response related activity is selected from: induction of T-cell activation or proliferation; an induction in T-cell lytic activity; binding to an MHC class I molecule; an alteration in cytokine or chemokine expression or production; and an alteration in expression of immunoregulatory cell surface molecules. In an embodiment, the isolated polypeptide is 50 amino acids or less in length. In an embodiment, the T-cell is selected from a CD8+ T-cell and a CD4+ T-cell. In an embodiment, the cytokine is selected from IFNγ, IL-2,4, 5, 6, 10, 12, 13, TGF-β, and TNF-α. In embodiments, the functional variant comprises 1- 6, 1-15 or 1-30 amino acid additions to an amino acid sequence selected from SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43- 46, 52-54, 61, 62, 64, 66, 61, 69-75, 78, 79, 85, 87, 90, 93, 111, 117, 119, 122, 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181-186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245-248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352, 366, 372, 374, 376, 377, 380-382, 384, 386-390, 392-397, 400, 405, 409, 410, 411, 412, 419, 426, 432-434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505. In embodiments, the polypeptide consists essentially of an amino acid sequence selected from SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43-46, 52-54, 61, 62, 64, 66, 67, 69- 75, 78, 79, 85, 87, 90, 93, 111, 117, 119, 122, 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181-186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245-248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352,' 366, 372, 374, 376, 377, 380-382, 384, 386-390, 392-397, 400, 405, 409, 410, 411, 412, 419, 426, 432-434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505. In embodiments, the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 44, 66, 70, 186, 303, 412, 426 and 432. The invention further provides an isolated SARS-CoV N protein peptide epitope, wherein said peptide epitope has an immune—related activity, with the proviso that said peptide epitope is not the full length SARS-CoV N protein set forth in SEQ ID NO: 496. In embodiments, the epitope is selected from the epitopes defined by the start and end positions within SEQ ID NO: 496 set forth in Table 5. In an embodiment, the immune-related activity is selected from: induction of T-cell activation or proliferation; an induction in T-cell lytic activity; binding to an MHC class I molecule; an alteration in cytokine or chemokine expression or production; and an alteration in expression of immunoregulatory cell surface molecules. In an embodiment, the peptide epitope comprises of 8 to about 50, 8 to about 12, 8, 9, 10 or 11 contiguous amino acids of the SARS-CoV N protein set forth in SEQ ID NO: 496. In an embodiment, the peptide epitope consists essentially of 8 to about 50, 8 to about 12, 8, 9, 10 or 11 contiguous amino acids of the SARS-CoV N protein set forth in SEQ ID NO: 496. The invention further provides a pharmaceutical composition comprising the above-mentioned isolated polypeptide and a pharmaceutically acceptable carrier. In an embodiment, the composition further comprises an adjuvant. In an embodiment, the composition further comprises an MHC molecule. The invention further provides a composition comprising a multimer of two or more MHC peptide complex monomers, each of said monomers comprising a polypeptide of the invention and an MHC molecule (e.g. an MHC class I heavy- chain or fusion protein thereof, a β2 microglobulin or fusion protein thereof) . In an embodiment, the monomers are joined together into said multimer by virtue of a multivalent entity. In embodiments, monomers are associated with said multivalent entity by virtue of an interaction chosen from biotin-avidin interactions, biotin-streptavidin interactions, coiled-coil domain interactions, and liposome-monomer cross-linking. In an embodiment, the above-mentioned pharmaceutical composition further comprises a second polypeptide (e.g. an additional SARS-CoV polypeptide) different from said isolated polypeptide, wherein said second polypeptide is capable of inducing a SARS-CoV immune response. The invention further provides an antibody (e.g. monoclonal, polyclonal, recombinant, and fragments thereof) capable of specifically binding to the above mentioned polypeptide. The invention further provides an isolated nucleic acid encoding the above-mentioned polypeptide, wherein said nucleic acid does not encode the full length SARS-CoV protein set forth in SEQ ID NO: 496. In an embodiment, the nucleic acid comprises a fragment of a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 496. In an embodiment, the nucleic acid comprises a fragment of the nucleotide sequence of SEQ ID NO: 495. The invention further provides a vector comprising the above-noted nucleic acid operably-linked to a transcriptional regulatory sequence. The invention further provides a host cell transformed or transfected with the above-mentioned vector. The invention further provides a method of producing the above-mentioned polypeptide, said method comprising culturing the above-mentioned host cell under conditions permitting expression of said polypeptide. The invention further provides a method of preventing or treating SARS71 or for inducing an immunological or protective immune response against SARS-CoV, in an animal, said method comprising administering to said animal an agent selected from the above mentioned polypeptide, pharmaceutical composition and vector. In an embodiment, the animal is a mammal, in a further embodiment, a human. The invention further provides a use of the above- mentioned polypeptide for the preparation of a medicament. The invention further provides a use of an agent selected from the above-mentioned polypeptide, pharmaceutical composition, and vector, for preventing or treating SARS, or for inducing an immunological or protective immune response against SARS-CoV. The invention further provides a kit or commercial package comprising an agent selected from the above-mentioned polypeptide, pharmaceutical composition, and vector, together with instructions for preventing or treating SARS, or for inducing an immunological or protective immune response against SARS-CoV. The invention further provides a method of detecting or diagnosing SARS or SARS-CoV infection in an animal, said method comprising assaying a biological sample of said animal with the above-mentioned polypeptide, antibody, and/or composition comprising a multimer. The invention further provides a method of detecting or diagnosing SARS or SARS-CoV infection in an animal, said method comprising: contacting a biological sample of said animal with the above-mentioned polypeptide, antibody, and/or composition comprising a multimer; and determining the binding of a constituent of the biological sample to said the above-mentioned polypeptide, antibody, and/or composition comprising a multimer; wherein said binding is indicative of SARS or SARS-CoV infection. In an embodiment, the biological sample is a tissue or body fluid (e.g. blood, plasma, lymphocytes, etc.) of said animal. The invention further provides a use of a complex comprising the above-mentioned polypeptide and an MHC molecule for labelling, detecting or isolating T-cells; or for detecting, selecting, sorting, or identifying T cell epitopes and/or amino acid sequences. In an embodiment, the above-mentioned composition is an immunogenic or vaccine composition. The invention further provides a polypeptide microarray comprising an isolated polypeptide of the invention bound to a substrate. In an embodiment, the microarray further comprises an MHC molecule bound to said substrate. The invention further provides a polypeptide microarray comprising the above-mentioned antibody bound to a substrate. The invention further provides a polypeptide microarray comprising an MHC complex bound to a substrate, said complex comprising the above-mentioned polypeptide and an MHC molecule. The invention further provides a method of detecting or diagnosing SARS or SARS-CoV infection in an animal, said method comprising: contacting a biological sample of said animal with the above-mentioned polypeptide microarray; and determining the binding of a constituent of the biological sample to said polypeptide microarray; wherein said binding is indicative of SARS or SARS-CoV infection. The invention further provides a kit or commercial package comprising a component selected from the above- mentioned polypeptide, antibody and/or polypeptide microarray, together with instructions for detecting or diagnosing SARS or SARS-CoV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Activation of immunological responses of a convalescent SARS patient from stimulation with peptides of N protein of SARS-CoV. Representative responses of CD4+ and CD8+ peripheral blood leukocytes to sequential overlapping 15 amino acid peptides (see Figure 5) . Fold proliferation (relative to nonstimulated patient cells) of leukocytes pools of ten 15 amino acid peptides A) or individual 15 amino acid peptides B) were incubated with IxIO6 peripheral blood leukocytes for 7 days and analysed for expression of CD4+ and CD8+ cell surface receptors. B), left panel: Proliferation of blood leukocytes from SARS convalescent patient 6 and healthy donors stimulated with nucleocapsid protein peptides; B), right panel: Proliferation of blood leukocytes from SARS convalescent patient 6 stimulated with nucleocapsid protein peptides. The inflammatory cytokine, IFNγ production was measured from culture media of patient (patient 6) cells stimulated with N peptides C) , left panel. Representative dot plot displaying proliferative responses of CD4+ or CD8+ peripheral blood leukocytes C) , right panel.

Figure 2: Activation of immunological responses of a convalescent SARS patient from stimulation with peptides of N protein of SARS-CoV. Representative responses of CD4+ and CD8+ peripheral blood leukocytes to sequential overlapping 15 amino acid peptides (see Figure 5) . Fold proliferation (relative to nonstimulated patient cells) of leukocytes pools of ten 15 amino acid peptides A) or individual 15 amino acid peptides B) were incubated with IxIO6 peripheral blood leukocytes for 7 days and analysed for expression of CD4+ and CD8+ cell surface receptors. The inflammatory cytokine, IFNγ production was measured from culture media of patient cells stimulated with N peptides {C, left panel) . Representative dot plot displaying proliferative responses of CD4+ or CD8+ peripheral blood leukocytes (C, right panel). A) Proliferation of CD4+/CD8+ peripheral blood leukocytes from SARS convalescent patient 8 stimulated with N protein peptides; B) Proliferation of peripheral blood leukocytes from SARS convalescent patient 8 stimulated with N protein peptides; C) IFNγ production from SARS convalescent patient stimulated with N protein peptides.

Figure 3: Binding Affinity and off rate of overlapping 9 amino acid peptides of the SARS-CoV,N protein to various major histocompatibility complex class I alleles. Nonameric peptides (see Figure 6) were incubated for the binding affinity and rate of dissociation to HLA alleles A2 A), A3 B), All C), and A24 D). Binding affinity (left panels of subfigures) of the 9 amino acid peptides that displayed higher affinity as compared to positive control for the alleles had an effective dose (ED50) less than 5xlO~6 M following incubation of allele complexes with sequentially diluted peptides. Rate of dissociation (right panels of subfigures) of 9 amino acid peptides from the allele complexes was assayed by incubating peptides with a specific allele complex over an 8-hour period.

Figure 4: Frequency of SARS convalescent patients exhibiting 2 fold or greater proliferation to immunogenic 15 amino acid peptides of the SARS-CoV N protein. Percent of patients responding to stimulation with identified target N protein polypeptides. CD4+ and CD8+ proliferation induced by identified individual overlapping 15 amino acid peptides (Figure 5) were assessed against 12 patients. Those patients demonstrating 2 fold proliferation (relative to nonstimulated patient cells) were classified as positive responders.

Figure 5: List in tabular format of sequential overlapping 15 amino acid peptides derived from SARS-CoV N protein (SEQ ID NOs: 1-80) which were analyzed according to the Examples below.

Figure 6: List in tabular format of sequential overlapping 9 amino acid peptides derived from SARS-CoV N protein (SEQ ID NOs: 81-494) which were analyzed according to the Examples below.

Figure 7: DNA (SEQ ID NO: 495) and amino acid (SEQ ID NO: 496) sequences of SARS-CoV N protein (Genbank Accession No. NC_004718; nucleotides 28120-29388 and corresponding translated sequence) .

Figure 8: Schematic overview of iTopia™ assay system (Beckman Coulter, Inc. Fullerton, CA) . Figures 9 to 11: Results of SARS N-protein HLA-A*0101 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 8 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 12: Results of SARS N-protein HLA-A*0101 - peptide binding studies. Cluster effect of peptides - HLA-A*0101 is examined. Peptide numbers indicated in X-axes correspond to 9- mer peptide numbers listed in Figure 6.

Figure 13: Results of affinity ED50 studies of SARS N-protein peptide - HLA-A*0101 binding. Peptide numbers indicated in X- axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 14: Results of off-rate half life {tH or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*0101 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 15: Summary of results of SARS N-protein peptide - HLA-A*0101 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6. BIMAS rank determined via BIMAS (Biolnformatics & Molecular Analysis Section) analysis (for reference, see e.g., http://bimas.cit.nih.gov/) . SYFPEITHI rank determined via SYFPEITHI analysis (for reference, see, e.g., http://www.syfpeithi.de/scripts/MHCServer.dll/home.htm) .

Figure 16: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-A*0101. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 17: Summary of results of SARS N-protein peptide - HLA-A*0101 studies. Six epitopes (listed in ^table) were identified in respect of A*0101. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 18 to 20: Results of SARS N-protein HLA-A*0201 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 31 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 21: Results of SARS N-protein HLA-A*0201 - peptide binding studies. Cluster effect of peptides - HLA-A*0201 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figures 22 to 25: Results of affinity ED50 studies of SARS N- protein peptide - HLA-A*0201 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 26 and 27: Results of off-rate half life (tH or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*0201 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 28: Summary of results of SARS N-protein peptide - HLA-A*0201 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6. Figure 29: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-A*0201. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 30: Summary of results of SARS N-protein peptide - HLA-A*0201 studies. 30 epitopes {listed in table) were identified in respect of A*0201. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 31 to 33: Results of SARS N-protein HLA-A*0301 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 21 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 34: Results of SARS N-protein HLA-A*0301 - peptide binding studies. Cluster effect of peptides - HLA-A*0301 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figures 35 to 37: Results of affinity ED50 studies of SARS N- protein peptide - HLA-A*0301 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 38 to 40: Results of off-rate half life (t^ or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*0301 binding. For Figure 38, Tl/2 of positive control is 17.3 hrs. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6. Figure 41: Summary of results of SARS N-protein peptide - HLA-A*0301 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 42: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-A*0301. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 43: Summary of results of SARS N-protein peptide - HLA-A*0301 studies. 18 epitopes (listed in table) were identified in respect of A*0301. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 44 to 46: Results of SARS N-protein HLA-A*1101 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 28 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 47: Results of SARS N-protein HLA-A*1101 - peptide binding studies. Cluster effect of peptides - HLA-A*1101 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figure 48: Comparison of cluster effect of SARS N-protein peptides - HLA-A*0301 and HLA-A*1101. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figures 49 to 51: Results of affinity ED50 studies of SARS N- protein peptide - HLA-A*1101 binding. Peptide numbers 11 indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 52 to 54: Results of off-rate half life {tH or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*1101 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 55: Summary of results of SARS N-protein peptide - HLA-A*1101 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 56: Rank comparison of iTopia vs. BIMAS for SARS N- protein peptide - HLA-A*1101. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 57: Summary of results of SARS N-protein peptide - HLA-A*1101 studies. 28 epitopes (listed in table) were identified in respect of A*1101. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 58 to 60: Results of SARS N-protein HLA-A*2402 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. For this analysis, cutoff was adjusted to' >40%, and a total of 55 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 61 to 66: Results of affinity ED50 studies of SARS N- protein peptide - HLA-A*2402 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6. Figures 67 to 72: Results of off-rate half life (t4 or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*2402 binding. For Figure 67, Tl/2 of positive control is 3.3 hrs. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 73 and 74: Summary of results of SARS N-protein peptide - HLA-A*2402 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 75 and 76: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-A*2402. Peptide numbers indicated, correspond to 9-mer peptide numbers listed in Figure 6.

Figure 77: Summary of results of SARS N-protein peptide - HLA-A*2402 studies. 37 epitopes (listed in table) were identified in respect of A*2402. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 78 to 80: Results of SARS N-protein HLA-B*0702 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 55 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 81: Results of SARS N-protein HLA-B*0702 - peptide binding studies. Cluster effect of peptides - HLA-B*0702 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figures 82 to 87: Results of affinity ED50 studies of SARS N- protein peptide - HLA-B*0702 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 88 to 93: Results of off-rate half life {tH or tl/2; ' hrs) studies of SARS N-protein peptide - HLA-B*0702 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 94 and 95: Summary of results of SARS N-protein peptide - HLA-B*0702 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 96 and 97: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-B*0702. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6. «

Figure 98: Summary of results of SARS N-protein peptide - HLA-B*0702 studies. 18 epitopes (listed in table) were identified in respect of B*0702. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 99 to 101: Results of SARS N-protein HLA-B*0801 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 8 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 102: Results of SARS N-protein HLA-B*0801 - peptide binding studies. Cluster effect of peptides - HLA-B*0801 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6. Figure 103: Comparison of cluster effect of SARS N-protein peptides - HLA-B*0801 and HLA-A*0101. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed in Figure 6.

Figure 104: Results of affinity ED50 studies of SARS N- protein peptide - HLA-B*0801 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 105: Results of off-rate half life (t*s or tl/2; hrs) studies of SARS N-protein peptide - HLA-B*0801 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 106: Summary of results of SARS N-protein peptide - HLA-B*0801 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 107: Rank comparison of iTopia vs. BIMAS vs. SYFPEITHI for SARS N-protein peptide - HLA-B*0801. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 108: Summary of results of SARS N-protein peptide - HLA-B*0801 studies. 7 epitopes (listed in table) were identified in respect of B*0801. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figures 109 to 111: Results of SARS N-protein HLA-B*1501 - peptide binding studies. Peptide numbers indicated in X-axes and table correspond to 9-mer peptide numbers listed in Figure 6. Total of 35 binding peptides were identified, which were further analyzed for ED50 and off-rate half life.

Figure 112: Results of SARS N-protein HLA-B*1501 - peptide binding studies. Cluster effect of peptides - HLA-B*1501 is examined. Peptide numbers indicated in X-axes correspond to 9-mer peptide numbers listed In Figure 6.

Figures 113 to 116: Results of affinity ED50 studies of SARS N-protein peptide - HLA-B*1501 binding. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figures 117 to 120: Results of off-rate half life [XM. or tl/2; hrs) studies of SARS N-protein peptide - HLA-B*1501 binding. For Figure 117, Tl/2 of positive control is 4.2 hrs. Peptide numbers indicated in X-axes and tables correspond to 9-mer peptide numbers listed in Figure 6.

Figure 121: Summary of results of SARS N-protein peptide - HLA-B*1501 binding. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 122: Rank comparison of iTopia vs. SYFPEITHI for SARS N-protein peptide - HLA-B*1501. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6.

Figure 123: Summary of results of SARS N-protein peptide - HLA-B*1501 studies. 27 epitopes (listed in table) were identified in respect of B*1501. Peptide numbers indicated correspond to 9-mer peptide numbers listed in Figure 6. DETAILED DESCRIPTION OF THE INVENTION

In the studies described herein, HLA class I alleles were studied for binding of SARS N peptides (i.e. peptides derived from the SARS-CoV nucleocapsid protein) and convalescent SARS patient peripheral blood mononuclear cells were used to demonstrate functional CD4 and CD8 reactivity to N protein epitopes- As a result, a number of immunodominant peptides were identified that elicit proliferative and functional responses from convalescent SARS patient peripheral blood leukocytes. The peptides identified also showed high affinity binding to MHC class I alleles and reactivity among various HLA groups inducing T cell proliferation and cytokine production. Additionally, these identified peptides correspond to peptides sharing overlapping amino acid sequences which exhibit high affinity for HLA class I alleles. Accordingly, in a first aspect, the invention relates to an epitope of the SARS-CoV N protein, i.e. an immunogenic polypeptide or fragment derived from the SARS-CoV N protein, as well as to variants or fragments of the polypeptide. In embodiments, the polypeptide or variants or fragments thereof have or are capable of effecting or eliciting an activity including but not limited to an induction of lymphocyte (e.g. T-cell [e.g. CD8+ or CD4+] ) activation or proliferation, induction of T-cell lytic activity, binding to an MHC class I molecule, induction of cytokine (e.g. IFNγ, IL-2,4, 5, 6, 10, 12, 13, TGF-β, and TNF- α) or chemokine expression or production, an alteration in the expression or an immunoregulatory surface molecule, or any combination thereof. Examples of methods to determine such activities are set forth in the Examples below. In embodiments, the polypeptide may be less than 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids in length. In embodiments the polypeptide is greater than or equal to 5, 8 or 9 amino acids in length. In embodiments the polypeptide is 9 or 15 amino acids in length. In embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43-46, 52-54, 61, 62, 64, 66, 67, 69-75, 78, 79, 85, 87, 90, 93, 111, 117, 119, 122, 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181- 186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245- 248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352, 366, ' 372, 374, 376, 377, 380-382, 384, 386-390, 392-397, 400, 405, 409, 410, 411, 412, 419, 426, 432-434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505; or a functional (e.g. immunogenic) variant or fragment thereof, with the proviso that the polypeptide is not the full length SARS CoV N-protein set forth in SEQ ID NO: 496. In embodiments, the polypeptide comprises 1-5, 1-6, 1-10, 1-11, 1-15, 1-16, 1-21, 1-30 or 1-36 amino acid additions to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43-46, 52-54, 61, 62, 64, 66, 67, 69-75, 78, 79, 85, 87, 90, 93, 111, 117, 119, 122, 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181-186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245-248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352, 366, 372, 374, 376, 377, 380-382, 384, 386-390, 392-397, 400, 405, 409, 410, 411, 412, 419, 426, 432- 434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505. In a further embodiment, the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 19, 21-25, 27, 29, 30, 41, 43-46, 52-54, 61, 62, 64 , 66, 67 , 69-75 , 78 , 79 , 85 , 87 , 90 , 93 , 111 , 117 , 119 , 122 , 125, 126, 129, 130, 132, 134, 145,147, 164-167, 174, 175, 181- 186, 191-193, 200, 203, 211, 212, 216, 219, 231, 240-242, 245- 248, 250, 262, 264, 268, 282, 290, 296, 300-304, 307, 319, 321, 322, 330, 335, 338, 341, 343, 347, 348, 350-352, 366, 372, 374, 376, 377, 380-382, 384, 386-390, 392-397, 400, 405, 409, 410, 411, 412, 419, 426, 432-434, 436, 442, 443, 460, 466, 484, 486-488 and 499-505. In a further embodiment, the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 44,66, 70, 186, 303, 412, 426 and 432. In embodiments, the polypeptide consists essentially of 5-10, 9, 5-15, 15, 5-20, 5-25, 5-30, 30, 5-35, 5-40, 5-45, 45 or 5-50 contiguous amino acids of SEQ ID NO: 496.' The invention further provides a pharmaceutical composition, such as a vaccine or immunogenic composition, comprising the polypeptide and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises an adjuvant. In a further embodiment, the composition further comprises an MHC molecule. The invention further provides a multimer (i.e. 2 or more) of MHC peptide complexes, whereby each MHC complex comprises a polypeptide of the invention and an MHC molecule. In an embodiment, the MHC complex comprises a polypeptide of the invention, an MHC class I heavy chain and β2 microglobulin. Such multimer systems are known in the art, and the complexes may for example be associated together via suitable interactions with a multivalent entity, e.g. biotin- (strept) avidin (which are tetravalent thus resulting in a tetramer) interactions (see US patent 5,635,363 [June 3, 1997]; Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer- Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen-specific T lymphocytes.Science. 1996 Oct 4;274 (5284) :94-6. Erratum in: Science 1998 Jun 19;280 (5371) :1821. ) . Such multimers can also be used to label, detect, isolate, and stimulate T cells, as well as to discover other epitopes. ■ In an embodiment, a composition (e.g. a vaccine or immunogenic composition) of the invention may comprise a plurality of the peptides of the invention. In an embodiment the composition may comprise a second polypeptide capable of eliciting a SAR-CoV immune response, such as an additional SARS-CoV polypeptide or fragment thereof. The invention further provides an antibody against, or which recognizes, or is capable of specifically binding to a polypeptide of the invention. The invention further provides an isolated nucleic acid or polynucleotide which encodes the polypeptide of the invention (such as a fragment of SEQ ID NO: 495 or a sequence which differs therefrom but still encodes the same polypeptide by virtue of the degeneracy of the genetic code) . The invention further provides a vector comprising the nucleic acid operably linked to a transcriptional regulatory or expression control sequence (e.g. a promoter) . The invention further provides a host cell comprising the nucleic acid or vector. The invention further provides prophylactic and therapeutic methods, for preventing or treating SARS or SARS- CoV infection, comprising administering a polypeptide, composition, or MHC complex (comprising a polypeptide of the invention and one or more MHC molecules [e.g. MHC class I heavy chain, β2 microglobulin] ) or vector of the invention to an animal (e.g., a mammal, e.g., a human) . In embodiments, such methods comprise using administering a polypeptide, composition or vector of the invention to vaccinate or immunize (i.e. generate an immune response) in an animal. The invention further provides diagnostic methods for the diagnosis and detection of SARS or SARS-CoV infection. Such methods may utilize as a reagent a polypeptide of the invention, a multimer comprising 2 or more MHC peptide complexes as noted above, or an antibody which binds specifically to a polypeptide of the invention. In embodiments, the method comprises contacting a tissue or body fluid (e.g. blood, lymphocytes) of an animal with the reagent. The invention further provides a peptide array or microarray comprising a polypeptide of the invention, and optionally other components such as an MHC molecule, which may be used in the just-noted diagnostic methods. The invention further provides a use of the polypeptide, MHC complex (comprising a polypeptide of the invention and one or more MHC molecules [e.g. MHC class I heavy chain, β2 microglobulin] ) or vector of the invention for the preparation of a medicament or vaccine, e.g. for the prevention or treatment of SARS or SARS-CoV infection. The invention further provides a use of the polypeptide, composition or vector of the invention for the prevention or treatment of SARS or SARS-CoV infection.

The invention further provides a commercial package comprising a polypeptide, composition or vector of the invention together with instructions for the prevention or treatment of SARS or SARS-CoV infection. The invention further provides a commercial package comprising a polypeptide, composition or antibody of the invention together with instructions for the diagnosis and detection of SARS or SARS-CoV infection. The invention further relates to a fusion polypeptide. As used herein, a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the 21 invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail) . A simple way to obtain such a fusion polypeptide is by translation of an in-frame fusion of the polynucleotide sequences, i.e., a hybrid sequence. The hybrid sequence encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell. Alternatively, the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present. Such vectors and instructions for their use are commercially available, e.g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention. An advantageous example of a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such1 as subunit B of either cholera toxin or E. coli heat-labile toxin. To effect fusion, the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity. Alternatively, a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity. Consistent with the above, the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention. By "heterologous signal peptide" is meant a signal peptide that is not found in naturally-occurring precursors of polypeptides of the invention. Polynucleotide molecules according to the invention, including RNA, DNA, or modifications or combinations thereof, have various applications. A DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating SARS or SARS-CoV infection, and (iii) as a vaccine agent (as well as an RNA molecule) , in a naked form or formulated with a delivery vehicle. Accordingly, a further aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture. Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term "recombinant" means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term "recombinant" when made in reference to genetic composition refers to a gamete or progeny or cell or genome with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as 'recombinant' therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events. In another aspect of the invention, an isolated nucleic acid, for example a nucleic acid sequence encoding a polypeptide of the invention, or homolog, fragment or variant thereof, may further be incorporated into a recombinant expression vector. In an embodiment, the vector will comprise transcriptional regulatory sequences or a promoter operably- linked to a nucleic acid comprising a sequence capable of encoding a peptide compound, polypeptide or domain of the invention. A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. A recombinant expression system is selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris) , mammalian cells (e.g., COSl, NIH3T3, or JEG3 cells) / arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells. A preferred expression system is a procaryotic host such as E. coll. Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland) . Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells. The choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form. One skilled in the art would readily understand that not all vectors and expression control sequences and hosts would be expected to express equally well the polynucleotides of this invention. With the guidelines described below, however, a selection of vectors, expression control sequences and hosts may be made without undue experimentation and without departing from the scope of this invention. In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance. In selecting an expression control sequence, a number of variables are considered. Among the important variables are the relative strength of the sequence (e.g. the ability to drive expression under various conditions) , the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription) . In selecting the host, unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product. The choice of the expression cassette depends on the host system selected as well as the features desired for the expressed polypeptide. Typically, an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3' terminal region (translation and/or transcription terminator) . The signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame. The signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression. The open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coll (as described in U.S. Patent No. 5,028,530 and in Cagnon et al., (Cagnon et al. , Protein Engineering (1991) 4 (7) : 843)); the promoter of the gene of bacteriophage T7 encoding RNA polymerase, that is functional in a number of E. coll strains expressing T7 polymerase (described in U.S. Patent No. 4,952,496); OspA lipidation signal peptide ; and RIpB lipidation signal peptide (Takase et al., J. Bact. (1987) 169:5692) . The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system. Expression vectors (e.g., plasmids or viral vectors) can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987) . Suitable expression vectors can be purchased from various commercial sources. Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al. , (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain. Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below. In an aspect, a polypeptide of the invention is substantially purified. A "substantially purified polypeptide" as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized. For example, a substantially purified polypeptide is free from cytoplasmic polypeptides. Those skilled in the art would readily understand that the polypeptides of the invention may be chemically synthesized, produced by recombinant means, or generated from a natural source. As used herein, the immunogenic or vaccine compositions of the invention are administered by conventional routes known the vaccine field, in such as to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g. via a patch) . The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred. The choice also depends upon the nature of the vaccine agent. For use in a composition of the invention, a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990) . Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below. A polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below. Accordingly, a further aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against SARS-CoV in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing SARS-CoV infection of non-human animals), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to SARS-CoV; and particularly, (v) a method for preventing and/or treating a SARS-CoV infection and SARS, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an infected individual. Additionally, the invention further provides a use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating SARS-CoV infection and SARS. As used herein, a vaccine vector expresses one or several polypeptides or derivatives of the invention. The vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) , that enhances the immune response (adjuvant effect) . It is understood that each of the components to be expressed is placed under the control of elements required for expression in a mammalian cell. The invention further provides a composition comprising several polypeptides or derivative thereof of the invention or vaccine vectors (each of them capable of expressing a polypeptide or derivative of the invention) . A composition may also comprise an additional SARS-CoV antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12 (or vaccine vector (s) capable of their expression) . "Vaccine" as used herein refers to a composition or formulation comprising one or more polypeptides of the invention, or a vaccine vector of the invention. Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine or vaccine vector of the invention to be administered by any conventional route. Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine or vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like) . Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette-Guerin (BCG), and Streptococcus. An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Patent No. 4,920,209. Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively. (Also see, e.g., 31 Tartaglia et al. , Virology (1992) 188:217) for a description of a vaccinia virus vector and Taylor et al, Vaccine (1995) 13:539 for a reference of a canary pox.) Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of vaccine viral vector, for therapeutic or prophylactic use, can be of from about IxIO4 to about IxIO11, advantageously from about IxIO7 to about IxIO10, preferably of from about IxIO7 to about IxIO9 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart- It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself. Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551 and U.S. Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced. WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations. WO 94/01533 describes a deletion mutant lacking functional ctxA and attRSl DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about IxIO5 to about IxIO9, preferably about IxIO6 to about IxIO8, viable bacteria in a volume appropriate for the selected route of administration. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally. Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally. Other bacterial strains used as vaccine vectors in the context of the present invention are described for Shigella flexneri in High et al. , EMBO (1992) 11:1991 and Sizemore et al. , Science (1995) 270:299; for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci. USA (1995) 92:6868; and forι Bacille Calmette Guerin in Flynn J.L., Cell. MoI. Biol. (1994) 40 (suppl. I) :31, WO 88/06626, WO 0 90/00594, WO 91/13157, WO 92/01796, and WO 92/21376. In bacterial vectors, the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid. The composition comprising a polypeptide or vaccine vector of the present invention may further contain an adjuvant. A number of adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below. Accordingly, a further aspect of the invention provides (i) a composition of matter comprising a polypeptide or polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polypeptide or polynucleotide of the invention; (iii) a method for inducing an immune response against SARS-CoV in a mammal by administration of an immunogenically effective amount of a polypeptide or polynucleotide of the invention to elicit a protective immune response to SARS-CoV; and particularly, (iv) a method for preventing and/or treating a SARS-CoV infection or SARS, by administering a prophylactic or therapeutic amount of a polypeptide or polynucleotide of the invention to an infected individual. Additionally, the invention further provides a use of a polypeptide or polynucleotide of the invention in the preparation of a medicament for preventing and/or treating SARS-CoV infection or SARS. Use of the polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes. Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, such a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al.r Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403) . Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al. , Human Gene Therapy (1996) 7:1205. Polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide. In the precursor form, the signal peptide is either homologous or heterologous. In the latter case, a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred. Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention. For use as a vaccine, a polynucleotide of the invention is formulated according to various methods outlined below. One method utililizes the polynucleotide in a naked form, free of any delivery vehicles. Such a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, 'with or without a carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20%, sucrose. An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake. Examples of such agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides. Anionic and neutral liposomes are well-known in the art (see, e.g. , Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides. Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin™ also known as DOTMA (N- [1- (2, 3-dioleyloxy)propyl]- NjN/N-trimethylammonium chloride), DOTAP (1, 2-bis (oleyloxy) -3- (trimethylammonio)propane) , DDAB {dimethyldioctadecylammonium bromide) , DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (3 beta- (N- (N' ,N'- dimethyl aminomethane) -carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine) , as described in WO 90/11092 as an example. Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds. A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane- permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768) . Gold or tungsten microparticles are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152. The microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263. The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal) , the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg and, more preferably, from about 25 μg to about 250 μg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals. Although not absolutely required, such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Patent No. 5,057,546. Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In an embodiment, a polypeptide of the invention, administered as a vaccine, is administered by a ' mucosal route in an amount from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 μg. When used as vaccine agents, polypeptides and polynucleotides of the invention may be used sequentially as part of a multistep immunization process. For example, a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT) . A polypeptide or variant or derivative thereof of the invention is also used in accordance with a further aspect of the invention as a diagnostic reagent for detecting the presence of anti-SARS-CoV antibodies, e.g. r in a blood sample. Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below. Adjuvants useful in any of the vaccine compositions described above are as follows. Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT) , are used in parenteral administration. Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT) , the Clostridium difficile toxin A and the pertussis toxin (PT) , or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB) . Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7- Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant) . Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-llO-Asp, and Glu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coll, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration. Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415) , DC-chol (3 b- (N- (Nf ,N'-dimethyl aminomethane) -carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336) . Any pharmaceutical composition -of the invention containing a polypeptide, a polypeptide derivative, a polynucleotide or an antibody of the invention, is manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time * necessary, to achieve the desired therapeutic result, such as a reduction of SARS disease symptoms and in turn a reduction in SARS-related disease progression and an improvement in SARS prognosis. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting SARS onset or progression. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration- The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, a polypeptide of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG) . Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. A further aspect of the invention provides an antibody that recognizes the polypeptide of the invention.

An antibody of the invention is either polyclonal or monoclonal. Antibodies may be recombinant, e.g., chimeric [e.g., constituted by a variable region of murine origin associated with a human constant region) , humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monoclonal antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab) !2, Fab or Fab' fragments'. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.

Antibodies against the polypeptide of the present invention are generated by immunization of a mammal with a partially purified fraction comprising the polypeptide. Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see Harlow and Lane (1988) and Yelton et al. (1981) , both of which are herein incorporated by reference. For monoclonal antibodies, see Kohler and Milstein (1975), herein incorporated by reference.

The antibodies of the invention, which are raised to a partially purified fraction comprising the polypeptide of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al. (1994), herein incorporated by reference) . The antibodies are used in diagnostic methods to detect the presence of a SARS-CoV N protein or fragment thereof or SARS-CoV in a sample, such as a tissue or body fluid. The antibodies are also used in affinity chromatography for obtaining a purified fraction comprising the polypeptide of the invention.

Accordingly, a further aspect of the invention provides (i) a reagent for detecting the presence of a SARS- CoV N protein polypeptide or fragment thereof and/or SARS-CoV in a tissue or body fluid; and (ii) a diagnostic method for detecting the presence of a SARS-CoV N protein polypeptide or fragment thereof and SARS-CoV in a tissue or body fluid, by contacting the tissue or body fluid with an antibody of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of a SARS-CoV N protein polypeptide or fragment thereof and/or SARS-CoV in the sample or the organism from which the sample is derived.

Those skilled in the art will readily understand that the immune complex is formed between a component of the sample and the antibody, and that any unbound material is removed prior to detecting the complex. It is understood that an antibody of the invention is used for screening a sample, such as, for example, blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia and fibroblasts, for the presence of a SARS-CoV N protein polypeptide or fragment thereof and/or SARS-CoV.

Similarly, a polypeptide of the invention may be used as a reagent to detect the presence of an antibody to a SARS-CoV N protein or fragment thereof and/or SARS-CoV in a tissue or body fluid, and therefore the invention further provides such a reagent, as well as a diagnostic method for detecting the presence of a an antibody to a SARS-CoV N protein or fragment thereof and/or SARS-CoV, by contacting the tissue or body fluid with a polypeptide of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of a SARS-CoV N protein or fragment thereof and/or SARS-CpV in the sample or the organism from which the sample is derived.

For diagnostic applications, the reagent (e.g, the polypeptide or antibody of the invention) is either in a free state or immobilized on a solid support, such as a tube, a bead, a plate or well thereof, or any other conventional support used in the field (such as a peptide microarray) . Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By "indirect means" is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system. Alternatively, a peptide tail is . added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail.

Such diagnostic agents may be included in a kit which also comprises instructions for use. The reagent is labeled with a detection means which allows for the detection of the reagent when it is bound to its target. The detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horseradish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as 125I or 51Cr. Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein. Throughout this application, various references are referred to describe more fully the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

EXAMPLES

Example 1: Materials and Methods

Generation of Peptides For the T cell proliferation assay (see below) , we utilized 15-mer peptides overlapping by 10 amino acids of the N protein (see Figure 5) synthesized by AnaSpec International (Kelowna, Taiwan, R.O.C.) . The peptides were generated using solid phase peptide synthesis using F-moc chemistry and was conducted on an automated peptide synthesizer symphony according to the manufacturer's standard protocols. The peptides were cleaved from the solid support by treatment with liquid trifluoroacetic acid (TFA) in the presence of phenol, thiocresole, anisole, and methyl sulfide. The crude products had been extracted with TFA and precipitated with diethyl ether. All peptides are analyzed by HPLC and Mass spectroscopy (MS) to verify their identity, and then stored in lyophilized form at -200C. Peptides were dissolved in DMSO and diluted to 5% DMSO in ddH20. For the Affinity Binding assays (see below) , we utilized 9-mer peptides overlapping by 8 amino acid of the N protein (see Figure 6) synthesized by SynPep Corporation (Dublin, CA) . The peptides were generated using Fmoc chemistry prepared by 9-fluoronylmethoxycarbonyl synthesis on Rainin/PTI Symphony™ automated machines. Peptide identity was verified by HPLC and MS analysis. For confirmation of binding results, fifteen of the 9-mer peptides were synthesized by New England Peptide Inc. (Gardner, MA) and tested for affinity to HLA class I complexes. For the studies corresponding to the results presented in Figures 9 to 123, overlapping 9-mer peptides of the N-protein (see Figure 6) were synthesized using standard methods by Jerini AG (Berlin , Germany , (http://www.jerini.de/)).

Cloning, expression and. purification of recombinant SARS-CoV Nυcleocapsid gene The single stranded RNA sequence of the SARS coronavirus nucleocapsid gene was obtained from genbank (accession number: NC_04718 region: 28120-28388) . Forward (GGA ATT CCA TAT GTC TGA TAA TGG ACC CA; SEQ ID NO: 497) and reverse (TAT CGC GGA TCC TTA TGC CTG AGT TGA ATC A; SEQ ID NO: 498) PCR primers were designed to amplify the open reading frame (1258 base pairs) of the nucleocapsid gene and incorporate restriction endonuclease sites Ndel and BamHl respectively. Primers were synthesized at McMaster University's central molecular biology facility. The nucleocapsid gene was amplified from reverse-transcribed SARS RNA and separated by agarose gel electrophoreses to confirm the presence of the amplified gene. PCR products were purified (QIAgen, Valencia, CA)", digested and ligated into the pET-28a (+) plasmid (Novagen, Madison, WI) . Competent JM109 cells (Promega, Madison, WI) were transformed with 10 ng of plasmid DNA by the Promega heat shock method. Plasmid containing bacteria were selected on kanamycin (34 μg /mL) containing LB agar plates. Colonies were verified to contain the nucleocapsid gene by PCR amplification using T7 forward and reverse primers. A single nucleocapsid-containing colony was grown in 9 mL of LB broth (34 μg/mL kanamycin) overnight at 370C, and plasmid DNA was isolated by the mini-prep procedure (QIAgen, Valencia, CA) . Rosetta DE3 (BL21) pLysS cells (Novagen) were transformed with 10 ng of plasmid DNA by the heat shock method, and selected on LB agar plates containing kanamycin (34 μg/mL) and choramphenicol (30 μg /mL) . A single colony was placed in 3 mL of starter LB broth (containing kanamycin and chloramphenicol) , grown overnight and used to seed 1 L of LB broth with antibiotics until AΘOO was between 0.6-0.8. IPTG (Sigma-Aldrich, St. Louis, MO.) was added to obtain a concentration of 1 mM, and the culture was incubated for 2 hours at 370C. Cells were pelleted by centrifugation at 6000 X g for 20 minutes at 4 0C, resuspended in binding buffer (0.02 M sodium phosphate buffer, 0.5 M NaCl, pH=7.4) before being lysed in a French press (Spectronic instruments, Madison, WI) . Lysates were centrifuged at 50 000 X g for 45 minutes to separate insoluble material, and applied to a 5 mL Ni charged Hi-trap Chelating HP column (Amersham-Pharmacia, Uppsala, Sweden) at 4 mL/min. Proteins were eluted in binding buffer with an imidazole gradient from 0-300 mM. Fractions were analyzed for the presence of Nucleocapsid protein by SDS- PAGE and coomassie blue staining against a size specific protein standard, followed by western blot analysis using an anti-His primary antibody at a dilution of 1:10 000 (Stressgen, Santa Cruz, CA) . Nucleocapsid protein was purified using an Akta Prime HPLC (Amersham-Pharmacia) equipped with a SP Sepharose Fast Flow cation exchange column (Amersham- Pharmacia) . Proteins were eluted in modified binding buffer (0.02 M sodium phosphate, pH=7.4, 0.5 mM DTT) with a salt gradient from 0-1 M. Presence of Nucleocapsid protein was confirmed in individual fractions using coomassie staining of SDS-PAGE gels and western blot using an anti-His (Stressgen, Santa Cruz, CA) primary antibody. Fractions containing Nucleocapsid protein were concentrated in pH=7.4 phosphate- buffered saline (PBS) using Amicon ultra centrifugal filter devices (Millipore, Bedford, MA) . Concentrated samples were stored at -800C in 15% glycerol with EDTA-free complete protease inhibitors (Roche, Laval, Quebec, Canada) prior to use.

PBMC isolation and storage Consenting convalescent Toronto SARS patients donated 60-80ml of blood drawn into heparin CPT tubes (Bd Biosciences, San Jose,CA). Peripheral blood mononuclear cells (PBMCs) were isolated according to manufacturer's protocol. Briefly, following blood collection, CPT tubes were centrifuged in an Allegra 6R centrifuge (Beckman Coulter, Inc., Fullerton, CA) at 1800 X g for 20 minutes at room temperature. Buffy coat layer was removed, washed once in 50 ml of Ix PBS, pH 7.4. PBMCs were frozen, in 2ml of 12.5% DMSO, 12.5% Human Serum Albumin (Sigma-Aldrich, St. Louis, MO) RPMI and cryopreserved in liquid nitrogen.

Proliferation Assay The proliferation assays were carried out according to the methods utilized by Younes et. al. , 2003 21. Briefly, convalescent PBMCs were labeled with 1.5 uM 5-6- Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) for 20 minutes at room temperature. The labeling reaction was quenched with one volume of 10% Human AB serum RPMI. The cells were washed in Ix phosphate-buffered saline (PBS) , resuspended at lxlθδ/ml in 10% Human AB serum' RPMI and 1 ml of cell suspension was distributed into 96 Deep well pates (BD Biosciences, San Jose, CA) . Cells were incubated at 37°C for 7 days in the presence of 10 μg of 15-mer peptides or 6 μg of whole protein. After 7 days of culture cells were stained for CD4 and CD8 expression. Cells were washed twice in IxPBS containing 2% fetal calf serum and stained with anti-human CD4-APC and CD8-PerCP, and analysed using a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA) .

Cytokine Analysis To analyze the concentration of cytokines we utilized the human Thl/Th2 and Chemokine Cytometric Bead Arrays™ (BD Biosciences, San Jose, CA) according to the manufacturer's specifications. Briefly, Specific capture beads for cytokines and multiple phycoerythrin-conjugated detection antibodies were incubated at room temperature for 3 hours with 50 μl of culture supernatants of the proliferation assay taken at day 4 or day 7 of incubation. Following acquisition of sample data using a FACSCaIibur™ flow cytometer and the results were analyzed using the BD CBA Analysis Software™.

HLA-Typing Peripheral blood collected into blood collection tubes containing EDTA were obtained from convalescent patients. Genomic DNA was isolated using the QIAamp 96 DNA Blood DNA isolation kit (Qiagen, Valencia, CA) . Typing was performed by the Canadian Network for Vaccine and Immunotherapeutics (CANVAC) HLA typing core. High resolution, four digit HLA class I (ABC) typing was determined by PCR-SSO, using the INNOLiPA™ reverse dot-blot hybridization method according to manufacturer's specifications (Innogenetics, Gent, Belgium) .

Affinity binding and Off rate assay nonameric Nucleocapsid peptides to HLA class I complexes Binding affinity and rate of dissociation of overlapping 9-mer peptides of N protein was assessed by using the iTopia™ Epitope Discovery System (Beckman Coulter, Inc., Fullerton, CA) to screen 8 major histocompatibility complex class I alleles, according to the manufacturer's instructions. Alleles tested were A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501. These eight alleles represent close to 90% of the human population (March, S. et al, HLA Facts Book, Academic Press, 2000) . The estimated gene frequencies of these HLA alleles is shown in Table 1.

Table 1: Estimated gene frequencies of HLA antigens included in this study* Caucasian African Asian Latino Al 15.18 5.72 4.48 7.40 A2- 28.65 18.88 24.63 28.11 A3 13.38 ' 8.44 2.64 8.07 All 6.17 1.58 17.31 4.83 A24 9.32 2.96 22.03 13.26 B7 12.17 10.59 4.26 6.44 B8 9.40 3.83 1.33 3.82 B15 6.49 3.52 12.21 5.29 *From: http://www.ashi-hla.org/publicationfiles/archives/prepr /motomi.htm ; HLA Gene and Haplotype Frequencies in the North American Population: The National Marrow Donor Program Donor Registry Briefly, this system entails the binding of a properly folded and biotinylated MHC class I monomer containing a bound peptide to an avidin-coated well of a microtiter plate. The buffer conditions are then changed to allow the dissociation of the folded complex, whereby the initial peptide and β2 microglobulin protein are washed away, however the MHC heavy chain remains bound to the well by virtue of the avidin-biotin interaction. The test peptide of interest is then added with additional β2 microglobulin and a labeled anti-HLA class I detector antibody. The sample wells are then incubated, washed and subjected to detection of the label. As the detector antibody binds to a properly folded complex, antibody binding indicates that the test peptide is capable of binding to the MHC molecule (see Figure 8) . Identified binding peptides were diluted 1:90 and 20ul added to allele specific plates. The plates were incubated at 21°C for 18 hours. After 18 hours plates were washed and placed at 37°C to begin the time course. The off rate of the identified peptides was determined by measurement of the decay of association at 0, 0.5, 1, 1.5, 2, 4, 6 and 8 hours. Identified peptides are also tested at decreasing peptide concentration in order to evaluate relative affinity. In respect of the results of Figures 9 to 123, peptide binding was studied as follows, testing the 414 N- protein 9-mers across the eight alleles noted above. Positive control peptides were provided in the iTopia™ Epitope Discovery system (Beckman Coulter, Inc., Fullerton, CA) . The protocol was followed according to manufacturer' s instructions:

Binding: 1:90 dilutions for each peptide were prepared. The allele coated plate was stripped and the peptide dilution was added with renaturation buffer provided by the manufacturer. Plates were then incubated for 18 hrs at 21"C with shaking. Subsequently, the plates were washed and read, and the % binding was calculated using iTopia™ software (iTopia™ Epitope Discovery System, Beckman Coulter, Inc., Fullerton, CA) .

Affinity assay: 8 serial dilutions were prepared for each peptide. The allele coated plate was stripped and the serial dilutions were added with renaturation buffer provided by the manufacturer. Plates were then incubated for 18 hrs at 21°C with shaking. Subsequently, the plates were washed and read, and the ED50 (concentration at which 50% of peptide is bound) was calculated using iTopia™ software.

Off rate assay: 1:90 dilutions for each peptide were prepared. The allele coated plate was stripped and the peptide dilution was added with renaturation buffer provided by manufacturer. Plates were then incubated for 18 hrs at 21°C with shaking. Subsequently, the plates were washed and a time decay study was performed: plates were read at T=O, 0.5hr, l.Ohr, 1.5hrs, 2.0hrs, 4.0hrs, β.Ohrs and 8.0hrs, and the % binding was calculated using iTopia™ software.

iScore: Using iTopia™ software, % binding, ED50 and off rate Tl/2 were utilized to generate an iScore for each peptide. iScore was in turn used to rank peptides.

Example 2: Results

To determine T cell immune responses mounted against the SARS coronavirus proteins in patients who had contracted SARS, we examined peripheral blood mononuclear cells from convalescent probable SARS patients for CD4 and CD8 positive T cell proliferative responses to the N protein and N peptides generated from the SARS Tor2 strain. We initially screened seven patients for reactivity against pools of N peptides or the entire N protein. The peptide pools consisted of 10 consecutive overlapping 15 amino acid peptides (Figure 5) encompassing the entire N protein. We found that all patients showed a two fold or greater proliferative response to one or more of the peptide pools, 3, 5, 7 and 8 (Figure IA and 2A) . The proliferative responses of CD4 and CD8 positive cells from two patients, 6 and 8, to N pools are shown in figure 1 and 2 respectively. Individual peptides from pools that stimulated proliferative responses were examined for their ability to stimulate CD4 and CD8 proliferative responses. We screened two 15-mer peptide pools, 3 and 7, for the ability to induce proliferative responses (Figure IB) . Of the constituting peptides of pools 3 and 7, a significant proliferative response from both CD8 and CD4 cells was elicited by peptide 70 (SEQ ID NO: 70; Figure IB, right panel) . CD4 and CD8 from patient 8 robustly proliferated when challenged with peptide 44 from pool 5 (Figure 2B left panel) . A modest CD8 proliferative response was observed when peptides 72 (SEQ ID NO: 72) and 73 (SEQ ID NO: 73) in pool 7 were used to stimulate PBMCs from patient 8 (Figure 2 B, left panel) . Many of the peptides that induced T cell proliferation in patients 6 and 8 could also induce IFN-γ expression (Fig. 1 C and Fig. 2 C) . The screening of the seven SARS patients identified eighteen peptides that were able to stimulate CD4 or CD8 proliferation in PBMCs from one or more patients (Table 2) . Of these 18 peptides ten could also induce expression of IFN-γ. We also screened PBMCs from four healthy volunteers. No peptides or pools of peptides were able to stimulate a two fold or greater proliferative response in PBMCs from healthy controls. We next screened an additional six patients with the ten peptides that induced proliferative responses of CD4 or CD8 cells in SARS PBMCs and cytokine stimulation. All of the additional patients showed functional responses to one or more of these peptides (Table 2, patients 12, 14, 15, 17, 19 and 20) . Interestingly patient 19 did not show a CD4 proliferative responses to any of these ten peptides. Table 2: Assessment of proliferative responses of PBMCs from

SARS patients to 15-mer peptides

To identify HLA Class I epitopes in the N protein we screened a library of N peptides composed of 9-mer peptides overlapping by 8 amino acids (Figure 6) for affinity and dissociation to six HLA class I alleles, A2, A3, All, A24, B7 and B15. Fifteen peptides were identified in the preliminary screen (Table 3) . Affinity and on off rates for A2, A3, All and A24 are shown in Figure 3. Interestingly, the sequence of all of the fifteen 9- mer peptides identified in the affinity and dissociation screen corresponded to 15. mer peptides that elicited CD8 proliferation (Table 2) . Two 9-mer peptides showing affinity for the A2 allele, 223 and 332 (sequences LLLDRLNQL [SEQ ID NO:303] and LTYHGAIKL [SEQ ID NO:412J, respectively) corresponded to the 15-mer peptides 44/45 and 66/67 (Table 3), three patients (#11, 15, and 20) with HLA A2 alleles showed reactivity to these peptides (Table 3) . Table 3: Assessment of affinity and dissociation of 9-mer

peptides to HLA class I alleles

The 15-mer peptides 66 (SEQ ID NO: 66) and 67 (SEQ

ID NO: 67) elicited CD8 proliferation in two patients (patient

14 and 20) with A3 alleles (Table 2 and 3) .

The All class I allele displayed similar affinity to N

peptides as A3 allele. The 9-mer peptides 120 (ASLPYGANK [SEQ ID NO: 200]) and 362 (KTFPPTEPK [SEQ ID NO: 442]) showed high affinity for the All allele and maintained stable association with the complex over the -8 hour dissociation assay (Figure 3c7 right panel) . Two All patients showed CD8+ reactivity towards 15-mer peptide 73 (SEQ ID NO: 73) which corresponds to the 9 mer 362 (SEQ ID NO: 442) . Furthermore, 3 patients had CD8+ and CD4+ reactivity towards 15-mer peptide 66 (SEQ ID NO: 66) or 67 (SEQ ID NO: 67) . One A24 patient displayed reactivity toward the 15- mer peptides corresponding to the A24 binding 9-mer peptides 307 (SEQ ID NO: 387) and 346 (SEQ ID NO: 426) (15 mer peptide 61 [SEQ ID NO: 61]- and 70 [SEQ ID NO: 70], respectively) . To verify the identity of the N protein peptides capable of eliciting strong CD8+ and CD4+ responses, we tested the response to N peptides of PBMCs of 9 additional SARS convalescent patients. Of the ten 15-mer peptides that we originally identified the peptides that induced significant responses from different patients were 15mer peptides 22, 43, 44, 66 and 70 (SEQ ID NOs: 22, 43, 44, 66 and 70, respectively) (Figure 4) . The demonstration of high affinity binding coupled with the functional proliferation and cytokine production from convalescent SARS patient PBMCs stimulated with the identified peptides indicates that several CD8 N epitopes are recognized across various HLA A alleles and elicit functional T cell responses. The data herein demonstrates that immunodominant epitopes of the SARS-CoV reside in the N protein. As noted above, the binding affinity and rate of dissociation of the 414 N-protein derived peptide 9-mers (see Figure 6) was assessed across the eight major histocompatibility complex class I alleles using the iTopia™ Epitope Discovery System (Beckman Coulter, Inc., Fullerton, CA) . A schematic representation of the assay is shown in Figure 8 and results are shown in Figures 9 to 123. The

correspondence of the epitopes identified with MHC class I

alleles is shown in Table 4.

Table 4:ι Correspondence of 9-mer epitopes identified with MHC

class I alleles tested

The N-protein peptides/epitopes identified in the

studies described herein are summarized in Table 5:

Table 5: SARS N-protein peptides/epitopes identified herein

61 *Start and end positions are in reference to N protein amino

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