WO2005103259A1 - Epitopes de proteines nucleocapsidiques de sras-cov et utilisations - Google Patents

Epitopes de proteines nucleocapsidiques de sras-cov et utilisations Download PDF

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WO2005103259A1
WO2005103259A1 PCT/CA2005/000632 CA2005000632W WO2005103259A1 WO 2005103259 A1 WO2005103259 A1 WO 2005103259A1 CA 2005000632 W CA2005000632 W CA 2005000632W WO 2005103259 A1 WO2005103259 A1 WO 2005103259A1
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WIPO (PCT)
Prior art keywords
polypeptide
sars
seq
cov
peptide
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PCT/CA2005/000632
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English (en)
Inventor
David Kelvin
Desmond Persad
Cheryl Cameron
Kurtis R. Bray
Lori R. Lofaro
Camille Johnson
Rafick-Pierre Sekaly
Souheil-Antoine Younes
Pele Chong
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University Health Network
Beckman Coulter, Inc.
Universite De Montreal
National Health Research Institutes
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Publication of WO2005103259A1 publication Critical patent/WO2005103259A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • 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.
  • Severe acute respiratory syndrome 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) .
  • SARS-CoV SARS coronavirus
  • SARS-CoV Session in virus
  • 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 SI 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 SI 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 .
  • 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 xx .
  • 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 .
  • 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.
  • IBV infection bronchitis virus
  • T cells from animals inoculated with IBV to na ⁇ ve chicks provided CD8+ ⁇ specific protection 15 .
  • Rhesus macaques immunized with a cocktail of adenoviral vectors encoding SI fragment of Spike, M protein and N protein of the SARS-CoV led to the generation of antibody responses against the SI fragment of Spike protein i and T-cell responses against the N protein 16 .
  • the injection of a T helper type 1 (Thl) T cell line resistant to MHV into susceptible mice led to a marked increase in IFN ⁇ expression with a decrease in IL-4 production concomitant with complete protection against infection 17 .
  • 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.
  • 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.
  • the epitope is selected from the epitopes defined by the start and end positions within SEQ ID NO: 496 set forth in Table 5.
  • 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.
  • 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,
  • 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.
  • the isolated polypeptide is 50 amino acids or less in length.
  • the T-cell is selected from a CD8 + T-cell and a CD4 + T-cell.
  • the cytokine is selected from IFN ⁇ , IL-2,4, 5, 6, 10, 12, 13, TGF- ⁇ , and TNF- ⁇ .
  • the functional variant comprises 1-
  • 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,
  • 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.
  • the epitope is selected from the epitopes defined by the start and end positions within SEQ ID NO: 496 set forth in Table 5.
  • 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 .
  • 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.
  • 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.
  • the monomers are joined together into said multimer by virtue of a multivalent entity.
  • 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.
  • 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.
  • the nucleic acid comprises a fragment of a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 496.
  • 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 SARS, ⁇ 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.
  • 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 pr 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 Activa tion 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 lxlO 6 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:
  • the inflammatory cytokine, IFN ⁇ production was measured from culture media of patient (patient 6) cells stimulated with N peptides C) , left panel .
  • Figure 2 Activation of immunological responses of a convalescent SARS patient from stimula tion wi th peptides of N protein of SARS-CoV.
  • 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 lxlO 6 peripheral blood leukocytes for 7 days and analysed for expression of CD4+ and CD8+ cell surface receptors.
  • the inflammatory cytokine, IFN ⁇ 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) .
  • 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 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 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 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.
  • 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.
  • Figures 52 to 54 Results of off-rate half life (t*s 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 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.
  • 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 (t or tl/2; hrs) studies of SARS N-protein peptide - HLA-A*2402 binding.
  • 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 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 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 105 Results of off-rate half life (t ⁇ 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.
  • 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.
  • 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.
  • 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.
  • SARS N peptides i.e. peptides derived from the SARS-CoV nucleocapsid protein
  • convalescent SARS patient peripheral blood mononuclear cells were used to demonstrate functional CD4 and CD8 reactivity to N protein epitopes.
  • 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.
  • polypeptide is greater than or equal to 5, 8 or 9 amino acids in length. In embodiments the polypeptide is
  • 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, 3
  • 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,
  • 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.
  • the composition further comprises an adjuvant.
  • 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.
  • the MHC complex comprises a polypeptide of the invention, an MHC class I heavy chain and ⁇ 2 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,
  • a composition e.g. a vaccine or immunogenic composition
  • a composition may comprise a plurality of the peptides of the invention.
  • 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).
  • 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, ⁇ 2 microglobulin] ) or vector of the invention to an animal (e.g., a mammal, e.g., a human).
  • 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.
  • 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, ⁇ 2 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.
  • 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.
  • 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.
  • 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.
  • a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such 1 as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such 1 as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity.
  • a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity.
  • the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • 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., C0S1, 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. coli .
  • 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.
  • 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) .
  • 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.
  • 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. coli (as described in U.S. Patent No.
  • Expression vectors e.g., plasmids or viral vectors
  • 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
  • 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.
  • 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.
  • 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.
  • a substantially purified polypeptide is free from cytoplasmic polypeptides.
  • the polypeptides of the invention may be chemically synthesized, produced by recombinant means, or generated from a natural source.
  • 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) .
  • a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • 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 in
  • 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.
  • 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.
  • liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • VLPs virus-like-particles
  • 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 irfacrophages 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.
  • a mammal e.g.,
  • 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.
  • 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.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • 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 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 .
  • adenovirus vector 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)
  • 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.
  • the dose of vaccine viral vector, for therapeutic or prophylactic use can be of from about IxlO 4 to about lxlO 11 , advantageously from about IxlO 7 to about IxlO 10 , preferably of from about IxlO 7 to about IxlO 9 plaque-forming units per kilogram.
  • 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 IxlO 5 to about IxlO 9 , preferably about IxlO 6 to about IxlO 8 , 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 .
  • 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.
  • adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below.
  • 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.
  • 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.
  • 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.
  • 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).
  • CMV Cytomegalovirus
  • 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 . , 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.
  • polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide.
  • the signal peptide is either homologous or heterologous.
  • 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.
  • 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.
  • a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, 'with or without a carrier.
  • 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.
  • agents include (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 .
  • Liposomes A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes
  • Cationic lipids are also known in the art and are commonly used for gene delivery.
  • Such lipids include LipofectinTM also known as DOTMA (N- [1- (2, 3-dioleyloxy) propyl] - N,N,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- (W ,N' - dimethyl aminomethane) -carbamoyl) cholesterol).
  • DC-Choi 3 beta- (N- (W ,N' - dimethyl aminomethane) -carbamoyl) cholesterol.
  • 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.
  • spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane see, for example, WO 93/18759
  • 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
  • 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.
  • a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • 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.
  • 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.
  • a polypeptide of the invention, administered as a vaccine is administered by a ' mucosal route in an amount from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • the dose usually does not exceed about 1 mg, preferably about 100 ⁇ g.
  • polypeptides and polynucleotides of the invention may be used sequentially as part of a multistep immunization process.
  • 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.
  • 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., in a blood sample.
  • 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
  • Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli , Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
  • MPLA bacterial monophosphoryl lipid A
  • PLGA polylactide glycolide
  • Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b- (N- (N f ,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.
  • a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution such as phosphate buffer saline.
  • 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.
  • 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 .
  • 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.
  • 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, liposo e, 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.
  • 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.
  • 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.
  • Antibodies is either polyclonal or monoclonal.
  • Antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin i 4 1 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.
  • 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 are included in the sample or the organism from which the sample is derived.
  • 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.
  • 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.
  • the reagent e.g, the polypeptide or antibody of the invention
  • 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.
  • 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.
  • 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 125 I or 51 Cr.
  • T cell proliferation assay 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.
  • TFA liquid trifluoroacetic acid
  • 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
  • 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
  • iScore Using iTopiaTM 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.
  • LLLDRLNQL SEQ ID NO:303
  • LTYHGAIKL SEQ ID NO:412J, respectively
  • 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
  • CD8+ and CD4+ reactivity towards 15-mer peptide 66 (SEQ ID NO:
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454 (2003) . 7. Wong,S.K., Li,W., Moore, . J. , Choe,H. & Farzan,M. A 193- amino-acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J. Biol . Chem . (2003) .

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Abstract

La présente invention concerne des polypeptides, des acides nucléiques, des anticorps, des compositions, des vaccins, des microréseaux et des utilisations de ceux-ci dans la prévention et le traitement d'infections par SRAS et SRAR-CoV. La présente invention concerne également des utilisations desdits produits dans la détection et le diagnostic d'infections par SRAS et SRAR-CoV. La présente invention concerne par ailleurs des procédés et des ensembles commerciaux associés aux utilisations desdits produits.
PCT/CA2005/000632 2004-04-26 2005-04-26 Epitopes de proteines nucleocapsidiques de sras-cov et utilisations WO2005103259A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111440229A (zh) * 2020-04-13 2020-07-24 中国人民解放军军事科学院军事医学研究院 新型冠状病毒t细胞表位及其应用
CN111606980A (zh) * 2020-05-27 2020-09-01 中国医学科学院基础医学研究所 Sars-cov冠状病毒s2蛋白多肽及其应用
CN112341526A (zh) * 2020-09-24 2021-02-09 杭州医学院 新型冠状病毒核衣壳蛋白特异性抗原多肽及其应用
CN112961222A (zh) * 2020-02-04 2021-06-15 中国科学院微生物研究所 2019新型冠状病毒n蛋白线性表位肽和单克隆抗体及应用
WO2021174142A1 (fr) * 2020-02-26 2021-09-02 Tonix Pharmaceuticals Holding Corp. Vaccin à base de poxvirus recombinant contre le virus sars-cov-2
WO2021214248A1 (fr) * 2020-04-23 2021-10-28 F. Hoffmann-La Roche Ag Antigène corona nucléocapsidique destiné à être utilisé dans des dosages immunologiques d'anticorps
WO2021223647A1 (fr) * 2020-05-06 2021-11-11 The University Of Hong Kong Compositions et procédés de production et d'utilisation de vaccins contre la covid-19
WO2022086955A1 (fr) * 2020-10-23 2022-04-28 Siemens Healthcare Diagnostics Inc. Appareil de collecte d'échantillons et méthodes de test d'immunoessai
WO2022008973A3 (fr) * 2020-07-10 2022-05-12 Covid Diagnostics Ltd. Compositions, procédés et systèmes de détection de réponse immunitaire
WO2022221189A1 (fr) * 2021-04-12 2022-10-20 La Jolla Institute For Immunology Épitopes de lymphocytes t du coronavirus et utilisations associées
WO2022241760A1 (fr) * 2021-05-21 2022-11-24 Huiru Wang Vaccins plus sûrs
WO2022226534A3 (fr) * 2021-04-23 2023-01-26 Oakwood Laboratories, Llc Formulations de microsphères comprenant de multiples peptides non identiques et leurs procédés de fabrication

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN Z. ET AL: "Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein.", CLINICAL CHEMISTRY., vol. 50, no. 6, 30 March 2004 (2004-03-30), pages 988 - 995, XP002450358, DOI: doi:10.1373/clinchem.2004.031096 *
HE Q. ET AL: "Development of a Western Blot Assay for Detection of Antibodies Against Coronavirus Causing Severe Acute Respiratory Syndrome.", CLINICAL AND DIAGNOSIS LABORATORY IMMUNOLOGY., vol. 11, no. 2, 2004, pages 417 - 422 *
HE Y. ET AL: "Mapping of Antigenic Sites on the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome Coronavirus.", JOURNAL OF CLINICAL MICROBIOLOGY., vol. 42, no. 11, November 2004 (2004-11-01), pages 5309 - 5314 *
LI S.ET AL: "The Epitope Study on the SARS-CoV Nucleocapsid Protein.", GENO.,PROT & BIOINFO., vol. 1, no. 3, 2003, pages 198 - 206, XP002342345 *
LIN Y. ET AL: "Identification of an epitope of SARS-coronavirus nucleocapsid protein.", CELL RESEARCH., vol. 13, no. 3, 2003, pages 141 - 145, XP002339647, DOI: doi:10.1038/sj.cr.7290158 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961222A (zh) * 2020-02-04 2021-06-15 中国科学院微生物研究所 2019新型冠状病毒n蛋白线性表位肽和单克隆抗体及应用
WO2021174142A1 (fr) * 2020-02-26 2021-09-02 Tonix Pharmaceuticals Holding Corp. Vaccin à base de poxvirus recombinant contre le virus sars-cov-2
CN111440229A (zh) * 2020-04-13 2020-07-24 中国人民解放军军事科学院军事医学研究院 新型冠状病毒t细胞表位及其应用
CN111440229B (zh) * 2020-04-13 2021-08-03 中国人民解放军军事科学院军事医学研究院 新型冠状病毒t细胞表位及其应用
WO2021214248A1 (fr) * 2020-04-23 2021-10-28 F. Hoffmann-La Roche Ag Antigène corona nucléocapsidique destiné à être utilisé dans des dosages immunologiques d'anticorps
WO2021223647A1 (fr) * 2020-05-06 2021-11-11 The University Of Hong Kong Compositions et procédés de production et d'utilisation de vaccins contre la covid-19
CN111606980B (zh) * 2020-05-27 2021-10-26 中国医学科学院基础医学研究所 Sars-cov冠状病毒s2蛋白多肽及其应用
CN111606980A (zh) * 2020-05-27 2020-09-01 中国医学科学院基础医学研究所 Sars-cov冠状病毒s2蛋白多肽及其应用
WO2022008973A3 (fr) * 2020-07-10 2022-05-12 Covid Diagnostics Ltd. Compositions, procédés et systèmes de détection de réponse immunitaire
CN112341526A (zh) * 2020-09-24 2021-02-09 杭州医学院 新型冠状病毒核衣壳蛋白特异性抗原多肽及其应用
WO2022086955A1 (fr) * 2020-10-23 2022-04-28 Siemens Healthcare Diagnostics Inc. Appareil de collecte d'échantillons et méthodes de test d'immunoessai
WO2022221189A1 (fr) * 2021-04-12 2022-10-20 La Jolla Institute For Immunology Épitopes de lymphocytes t du coronavirus et utilisations associées
WO2022226534A3 (fr) * 2021-04-23 2023-01-26 Oakwood Laboratories, Llc Formulations de microsphères comprenant de multiples peptides non identiques et leurs procédés de fabrication
WO2022241760A1 (fr) * 2021-05-21 2022-11-24 Huiru Wang Vaccins plus sûrs

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