WO2005019246A1 - Coronavirus vaccines, therapeutics and diagnostics - Google Patents

Abstract

The present invention provides peptides useful in the prevention and diagnosis of coronavirus infection. The peptides may be used as a component of a vaccine as it is contemplated they will elicit a protective immune response. It is further contemplated that the peptides are useful for diagnosis of a coronavirus infection, and especially in respect of SARS infection.

Description

CORONAVIRUS VACCINES, THERAPEUTICS AND DIAGNOSTICS

FIELD OF THE INVENTION The present invention relates to the field of the treatment, prevention and diagnosis of viral infections. More specifically, the invention provides novel peptides relevant to coronavirus infection, and the use of such peptides in vaccines, therapeutics and diagnostics. BACKGROUND TO THE INVENTION

The first coronavirus (CoV) was isolated from chickens in 1937, and there are now about 15 species in this Family. Coronaviruses (CoVs) have a broad host range including man, cattle, pigs, rodents, cats, dogs and birds

CoV particles are irregularly-shaped (approximately 60-220nm in diameter), with an outer envelope bearing distinctive, 'club-shaped' peplomers (approximately 20nm long x 10nm at the wide distal end). This 'crown-like' appearance gives the family its name. The centre of particle appears amorphous in negatively stained electron micrographs, the nucleocapsid being in a loosely wound and disordered state.

The envelope carries three glycoproteins: The S (spike) protein functions in receptor binding, cell fusion, and as a major antigen. The E (envelope) protein is a small envelope-associated protein. The M (membrane) protein is a transmembrane protein involved in virus budding and envelope formation. In some CoVs there is a third glycoprotein, known as HE (haemaglutinin esterase).

It is now known that severe acute respiratory syndrome (SARS) is a type of pneumonia caused by CoV infection. Symptoms include fever, a dry cough, dyspnea (shortness of breath), headache, and hypoxaemia (low blood oxygen concentration). Typical laboratory findings include lymphopaenia (reduced lymphocyte numbers) and mildly elevated aminotransferase levels (indicating liver damage). Death may result from progressive respiratory failure due to alveolar damage. The typical clinical course of SARS involves an improvement in symptoms during the first week of infection, followed by a worsening during the second week. Studies indicate that this worsening may be related to patient's immune responses rather than uncontrolled viral replication.

Since the World Health Organisation (WHO) raised a global alert on March 12^th, 2003, research into the pathogenesis of SARS has seen unprecedented world wide collaboration. After collaborators had proved Koch's principles on a coronavirus isolate (Fouchier et al., 2003), the WHO declared the newly described CoV strain, TOR2 to be the pathogen.

Phylogenetic analysis (Drosten et al., 2003, Ksiazek et al., 2003) places the newly discovered CoV well apart from the other known CoVs, which cluster in three distinct groups. One of these, group 1 , includes both the human CoV strain, HcoV-229E (Genbank accession code: NC_002645), which is the cause of common cold in about 15-30% of the infected (Chilvers et al., 2001 ), and two porcine coronaviridae (Genbank accession codes: NC_002306, AAA46905).

Droplet infection is at present considered to be the major route of transmission for the SARS CoV (Seto et al., 2003). Given the clinical manifestation of SARS with respiratory disease and diarrhoea being the most common symptoms, and a correlation of the severity of disease with the viral load (Peiris et al., 2003), a strategy aiming at minimizing the viral load early on in the course of the disease seems attractive. To that end, intranasal inoculation of interferon alpha for high-risk persons has been suggested (Peiris et al., 2003), as this procedure has been shown to be effective in other human CoV infections (Turner et al., 1986). To date, there has been no evidence of any efficacy using this approach.

Neutralising IgA type antibodies have an early protective effect in viral respiratory disease, and indeed stimulation of production of specific IgA type antibodies has been shown to be a valid strategy for vaccination against another virus with respiratory pathogenicity, influenza (Watanabe et al., 2002). While elevated serum antibodies were correlated with pathogenesis of coronavirus-mediated feline infectious peritonitis, stimulation of mucosal IgA production was correlated with recovery (Gerber et al., 1990). This suggests a vaccination strategy aiming at early mucosal IgA production despite the observation that the histopathological examination of biopsy specimens of SARS victims hints at severe systemic inflammatory damage (Peiris et al., 2003).

It is an aspect of the present invention to alleviate or overcome a problem of the prior art by Identifying neutralising epitope regions in CoV S protein useful in vaccination strategies and diagnostic tests for CoV infection.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. SUMMARY OF THE INVENTION

In a first aspect the present invention provides a peptide comprising the amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (II) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

In another aspect the present invention provides a peptide consisting of the amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (11) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

Applicants propose that these peptides are immunologically relevant to CoV infection, having efficacy in eliciting neutralising antibodies in a host. The peptides are proposed to be especially relevant to SARS infection.

In another aspect the present invention provides a nuoleotide sequence capable of encoding a peptide as described herein. In another aspect the present invention provides a vector comprising a nuoleotide sequence as described herein.

In a further aspect the present invention provides a recombinant virus, bacterium that has been genetically altered to comprise a nuoleotide sequence as described herein.

In another aspect the present invention provides a composition comprising a peptide or nucleic acid as described herein in combination with pharmaceutically acceptable carrier and/or diluent.

In a further aspect the invention provides a composition comprising a peptide or nucleic acid as described herein in combination with an adjuvant.

Another aspect of the present invention provides a peptide or nucleic acid described herein in the preparation of a medicament for the treatment or prevention of a viral infection. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection

A further aspect of the present invention provides use of a peptide or nucleic acid described herein in the diagnosis of a virus infection. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection

A further aspect of the present invention provides a kit for diagnosing a virus infection comprising a peptide as described herein.

In another aspect the present invention provides a method of treating or preventing a virus infection comprising administering to a mammal in need thereof an effective amount of a composition described herein. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. The following standard single letter code for amino acids is used throughout this specification: Isoleucine 1 Leucine L Valine V Phenylalanine F Methionine M Cysteine C Alanine A Glycine G Proline P Threonine T Serine s Tyrosine Y Tryptophan w Glutamine Q Asparagine N Histidine H Glutamic acid E Aspartic acid D Lysine K Arginine R

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

(A) Comparative genomic analysis between SARS CoV isolates, and between SARS and non-SARS CoVs. The blue solid line represents (i) the average percent identity between the various SARS CoVs with respect to the Canada TOR2 isolate. This was computed by counting the average number of identical nucleotides between the multiple sequence alignment of the entire genome for various SARS CoV isolates: Urbani (AY278741 ), HKU (AY278491 ), CUHK (AY278554) with respect to the TOR2 sequence (NC_004718) within a rectangular sliding window of 50 nucleotides. The red dashed line shows (ii) the average percent identity between SARS CoV and the non-SARS CoVs. This was computed by counting the average number of identical nucleotides between the multiple sequence alignment of the entire genome for human CoV 229E (NC_002645), MHV (NC_001846), bovine CoV (NC_003045), avian infectious bronchitis virus (NC_001451 ), TGEV (NC_002306) with respect to SARS CoV (TOR2) within a rectangular sliding window of 500 nucleotides. Broad arrows indicate the positions of ORFs 1ab, encoding the non-structural polyproteins, and the ORFs encoding the S, M and N structural proteins.

(B) Comparative analysis of predicted S protein sequence between SARS CoV isolates, and between SARS and non-SARS CoVs. The blue solid line represents (i) the average percent identity between the S proteins of the various SARS CoVs with respect to the Canada TOR2 isolate. This was computed by counting the average number of identical nucleotides between the multiple sequence alignment of the entire genome for various SAR CoV isolates: Urbani, HKU, CUHK, Taiwan (AY29145), Beijing (AY278488), SIN2774 (AY28798), SIN2748 (AY283797), SIN (AY283796), SIN2677 (AY283795), and SIN2500 (AY283794) with respect to the TOR2 sequence within a rectangular sliding window of 50 nucleotides. The black (dotted) and red (dashed) lines represent the average percent (ii) similarity and (iii) identity, respectively, between the S proteins of the SARS and non-SARS CoVs. The average similarity score was computed using the JOHM930101 amino acid scoring matrix (Johnson and Overington, 1993) between the multiple sequence alignment of the S proteins from various CoVs: human CoV 229E, MHV, TGEV, avian infectious bronchitis virus, Bovine CoV, Porcine Respiratory CoV (AAA46905), Canine CoV (X77047), Feline enteric CoV (Z80799) with respect to that of SARS TOR2 CoV within a rectangular sliding window of 500 amino acids. All plots were based on multiple sequence alignment of the sequences generated using ClustalW (Higgins et al., 1994).

Figure 2: (A) PSIPRED secondary structure prediction. MHV S protein epitope (SPLLGCIGSTCAE, position 846-858, boxed red) and its flanking region is structurally similar to the corresponding putative epitope (SQILPDPLKPTKR, position 785-797, boxed red) in the SARS CoV S protein.

(B) Structural similarity between TGEV S protein epitope (SSFFSYGEI, position 379-388, boxed red) and the corresponding putative epitope (SGNFKHLRE, position 176-184, boxed red) in the SARS CoV S protein. The predicted secondary structures are annotated as follows: β-strands (yellow arrows, E), coils (black solid line, C), and helices (green cylinders, H). Figure 3

(A) 3D-PSSM structure-based sequence alignment. Predicted secondary structures (helices) for TGEV S protein epitope (boxed) and the putative SARS CoV S protein epitope (boxed) are indicated using solid black bars. The epitopes and flanking regions from these viral S proteins matched a common structural fold from a template protein (IFXK) in the fold library among the top hits. CORE values give a measure of how deeply buried a residue is. A score of zero indicates a surface orientation, and nine indicates a buried residue.

(B) Tertiary structure prediction using Threader3 v3.4 matched the epitope region (QLAKDKVNE, colored red) in SARS CoV S protein to a common structural fold on the template protein (1 EUC). The structural models of the TGEV and SARS epitope together with their 'framework' regions (colored green) were built using 1 EUC as the template structure. Visualisation was achieved using PyMOL (DeLane, 2002).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a peptide comprising of the amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (II) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

In another aspect the present invention provides a peptide consisting of the amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (II) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

Applicants propose that these peptides are immunologically relevant to CoV infection, having efficacy in eliciting neutralising antibodies in a host. The peptides are proposed to be especially relevant to SARS infection. It is contemplated that these peptides will have relevance to a broad range of SARS isolates. Genome-wide analysis of four SARS CoV isolates (Canada TOR2, Urbani, HKU and CUHK) shows remarkable sequence identity (Figure 1A, I). At present, only the Canada TOR2 sequence in Genbank is listed as an entry in NCBI's Reference Sequence (RefSeq) collection. Hence the TOR2 sequence was used as the reference SARS CoV sequence herein.

In contrast, comparison of the SARS CoVs (Canada TOR2) with various non-SARS coronaviruses (Figure 1A, ii) reveals both highly conserved regions within open reading frame (ORF) 1 b, as well as highly variable regions, such as within ORFs encoding the viral structural proteins - Nucleocapsid (N), Membrane (M) and Spike (S) proteins. Both the M and S proteins are exposed on the surface of various CoVs and CoV-infected cells, and therefore have a higher probability of eliciting a protective host immune response against CoV infection. Whilst the S protein in various CoVs, including transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV), has been found to elicit protective immunity, the ability of the M protein to do likewise is less clear (Wesseling et al., 1993).

The peptides of the present invention are derived from the S protein. The S protein forms peplomers of the virus and contains structural determinants that are important for viral entry, viral tropism, immune recognition, and viral pathogenesis. An analysis that includes the complete and confirmed sequences from eleven SARS CoV isolates

(Canada TOR2, Urbani, Taiwan, HKU, CUHK, Beijing and five Singapore isolates), showed that the S protein is very highly conserved (Figure 1B, i). There are three amino acid substitutions: G to D (position 77 in CUHK and Beijing), I to T (position

244 in CUHK and Beijing) (Ruan et al., 2003) and S to A (position 577 in Canada

TOR2). In contrast, the average similarity of the S protein between SARS CoVs and other non-SARS CoVs is low (of about 20-30%) along its entire predicted primary amino acid sequence. However there are also regions of higher similarity (around 40- 50%) (Figure 1 B, ii) within the C-terminal half of the S protein.

Some of the features of the S protein that are highly conserved between SARS CoVs and other non-SARS CoVs include: 12 conserved cysteine residues (implicated in intramolecular disulphide bond formation), a cystein-rich region straddling the membrane-spanning region and the cytoplasmic tail, and a highly conserved motif

KWPWY(W)VWL adjacent to a stretch of hydrophobic residues (trans-membrane spanning region) near the C-terminus which has been shown to be critical for membrane fusion and infectivity of MHV (Chang et al., 2000).

The skilled person will understand that it is not necessary to use the exact S protein amino acid sequences (I) (II) or (III) described above. For example, only a portion of the peptide may be necessary for utility. Accordingly, the present invention includes portions of the amino acid sequences described herein. In a preferred form of the invention the portion comprises 3 contiguous amino acids. For example, for peptide (I) the sequence may be SQI, QIL, LPD, or PDP and so on. In a more highly preferred form, the portion comprises 4 contiguous amino acids such as SQIL, QILP, ILPD, LPDP, and so on. In an especially preferred form, the portion comprises 5 or more contiguous amino acids.

Preferably the equivalent is equivalent in primary structure. The skilled person will also understand that it will not be absolutely necessary to use peptides having an identical amino sequence to peptides (I) (II) and (III). For example, it is known that conservative substitutions may be made to a peptide without materially affecting its biological function. For example, substituting a hydrophobic amino acid residue for another hydrophobic amino acid residue will have little effect on the properties of the resultant protein.

Preferably the equivalent is equivalent in terms of secondary structure. There are three common secondary structures in proteins, namely alpha helices, beta sheets, and turns. That which cannot be classified as one of the standard three classes is usually grouped into a category called "other" or "random coil". It is understood that it is possible to identify peptides having a similar secondary structure but with different amino acid sequences.

Preferably the equivalent is equivalent in tertiary structure. Tertiary structure describes the folding of the polypeptide chain to assemble the different secondary structure elements in a particular arrangement. While the peptide sequences (I) (II) and (III) may be too short to provide any significant tertiary structure, it must be considered that the peptides may be a part of a larger peptide sequence which does have the ability to form tertiary structures.

The peptides defined by sequences (I) (II) or (III) and equivalents may or may not be glycosylated. In nature, CoV S protein is glycosylated, however it would be a matter of routine experimentation for the skilled person to decide any effect of glycosylation on immunogenicity.

The equivalent to sequences (I) (II) or (III) may incorporate non-natural amino acids, modified amino acids, amino acid analogues and the like. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodimmide activation via 0-acylisourea formation followed by subsequent derivitisation for example to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide, performic acid oxidation to cysteic acid, formation of a mixed disulphides with other thiol compounds reaction with maleimide, maleic anhydride or other substituted maleimide, formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chioromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials, carbamoylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

Preferably the peptide provides an epitope relevant to a virus infection. In the context of a virus vaccine a relevant epitope would be one that could elicit a neutralising (or protective) antibody. Neutralising epitopes are a subset of all antigenic regions present on a virus, but are special in that they have the ability to elicit antibodies that can inhibit the ability of the virus to infect a host.

The peptide may define a predominantly B-cell epitope or predominantly a T-cell epitope. Preferably the peptide is a B-cell epitope relevant to a virus infection. Since there is increasing evidence to suggest that the respiratory failure following SARS- associated CoV infection is due to immunopathological damage in response to a high virus load (Nicholls et al., 2003, Peiris et al., 2003), vaccination strategies employing B-cell-epitope bearing peptides (or in combination with a suitable T-helper cell determinant) are attractive approaches for stimulating the appropriate antibody response to neutralise mucosal infection by SARS-associated CoVs. This strategy may effectively minimise the extent of immunopathological damage resulting from an aggressive cell-mediated immune response mounted against a high coronavirus load. Presented herein are three epitopes in the SARS CoV S protein that are highly conserved across the eleven SARS CoV isolates examined. In another aspect the present invention provides a nuoleotide sequence capable of encoding a peptide as described herein. The degeneracy of the genetic code provides flexibility in the sequences that will be useful in the context of the present invention. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same protein.

In another aspect the present invention provides a vector comprising a nuoleotide sequence as described herein. Those skilled in the art will be familiar with materials and methods for cloning a given nucleic acid sequence into a vector. Such methods are described in detail in the following citation: Maniatis, T., Fritsch, E., F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y. (1989). For example, a sequence encoding a peptides of the present invention may be cloned into an expression vector for large scale recombinant production of the peptide.

In a further aspect the present invention provides a recombinant virus or bacterium that has been genetically altered to comprise a nuoleotide sequence as described herein. As well as for the large-scale production of recombinant peptide, it is possible to genetically alter a bacterium or virus to express an epitope on the surface. Such genetically altered organisms could be used as a vaccine against virus infection.

In another aspect the present invention provides a composition comprising a peptide or nucleic acid as described herein in combination with pharmaceutically acceptable carrier and/or diluent. The formulation of such compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

In a further aspect the invention provides a composition comprising a peptide or nucleic acid as described herein in combination with an adjuvant. As a matter of routine the skilled person will be enabled to select an appropriate adjuvant from the many known in the art. Examples of adjuvants includes bacterial toxins such as cholera toxin. Alternatively, the peptides may be mixed with the B subunit of cholera toxin (CTB) or an E. coli heat-labile toxin (LT), or B subunit of E. coli heat-labile toxin (LTB) to form a vaccine composition. Other adjuvants such as cholera toxin, labile toxin, tetanus toxin or toxoid, poly[di(carboxyIatophenoxy)phosphazene] (PCPP), saponins Quil A, QS-7, and QS-21 , RIBI (HAMILTON, Mont.), monophosphoryl lipid

A, immunostimulating complexes (ISCOM), Syntax, Titer Max, M59, CpG, dsRNA, and CTA1-DD (the cholera toxin A1 subunit (CTA1) fused to a dimer of the Ig-binding D-region of Staphylococcus aureus protein A (DD)), are also contemplated.

The adjuvants discussed above may be used in a vaccine composition at a concentration effective to assist in the eliciting of an immune response against the peptides of the present invention from an immunized subject. The concentration of adjuvant included in the vaccine compositions of the present invention may range from about 0.01 .μg/ml to 1 mg/ml. In another embodiment, the concentration of adjuvant used in a vaccine composition may range from about 0.1 .μg/ml to 100 .μg/ml. In yet another embodiment, the concentration of adjuvant used in a vaccine composition may range from about 1.0 .μg/ml to 100 .μg/ml. In still another embodiment, the concentration of adjuvant used in a vaccine composition may be about 10.0 .μg/ml. These ranges are provided for the sake of guidance in practicing the present invention. It should be noted that other effective concentrations of adjuvants may be determined by one of ordinary skill in the art using experimental techniques well known in that art.

Preferably, the adjuvant is a mucosal adjuvant. The mucosal adjuvant which is optionally, and preferably, administered with the peptides or nucleic acids described herein to the host or potential host is preferably cholera toxin. Mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivates of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the peptides as described herein. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as E Coli heat labile toxin. Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran dextran and polyomithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.

In a further preferred embodiment the composition comprises a combination of peptides or nucleic acids as described herein.

Another aspect of the present invention provides a peptide or nucleic acid described herein in the preparation of a medicament for the treatment or prevention of a viral infection. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection

A further aspect of the present invention provides use of a peptide or nucleic acid described herein in the diagnosis of a virus infection. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection. Methods for detecting the presence of a protein or nucleic acid in a biological sample are well known to the skilled person an include Enzyme-Linked Immunosorbant Assay (ELISA), immunodiffusion assay, immunofluroescence assay, Polymerase Chain Reaction (PCR), Northern analysis, Southern analysis, and the like.

The viral infection may be also be diagnosed by detecting antibodies specific for CoV infection. As an example of this approach, an ELISA plate could be coated with a peptide of the present invention, and binding of antibody to the immobilised peptide detected . A further aspect of the present invention provides a kit for diagnosing a virus infection comprising a peptide as described herein.

In another aspect the present invention provides a method of treating or preventing a virus infection comprising administering to a mammal in need thereof an effective amount of a composition described herein. Preferably the viral infection is a coronavirus infection. More preferably the viral infection is a SARS infection.

The dosage regimen involved in a method for vaccination, including the timing, number and amounts of booster vaccines, will be determined considering various hosts and environmental factors, e.g., the age of the patient, time of administration and the geographical location and environment.

Also included in the present invention are methods of vaccinating mammals against coronavirus infection and disease with the novel peptides and vaccine compositions described above. The vaccine compositions may be administered by a variety of routes contemplated by the present invention. Such routes include intranasal, oral, rectal, vaginal, intramuscular, intradermal and subcutaneous administration. Preferably the infection is SARS.

Vaccine compositions for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions or emulsions, the protein vaccine, and an adjuvant as described herein. The composition may be in the form of a liquid, a slurry, or a sterile solid which can be dissolved in a sterile injectable medium before use. The parenteral administration is preferably intramuscular. Intramuscular inoculation involves injection via a syringe into the muscle. This injection can be via a syringe or comparable means. The vaccine composition may contain a pharmaceutically acceptable carrier. Alternatively, the present vaccine compositions may be administered via a mucosal route, in a suitable dose, and in a liquid form. For oral administration, the vaccine composition can be administered in liquid, or solid form with a suitable carrier.

Doses of the vaccine compositions may be administered based on the relationship between the concentration of the peptide contained in the vaccine composition and that concentration of fusion protein required to elicit an immune response from an immunized host. The calculation of appropriate doses to elicit a protective immune response using the peptide vaccine compositions of the present invention are well known to those of skill in the art.

The present invention also extends to the field of DNA vaccines. With DNA vaccines, the subject is not injected with the antigen but with DNA encoding the (protein) antigen. The DNA is incorporated in a plasmid which is then injected into a muscle just as conventional vaccines are. The plasmid is taken up by cells, and the gene encoding the antigen is transcribed and translated. The proteins (or fragments thereof) are exposed at the cell surface with Class I MHC where they serve as a powerful stimulant for the development of cell-mediated immunity.

As well as preventing infection, it is also contemplated that the present invention will be useful in treating existing infections by passive immunization. It is well known in the art that infection with viruses can be limited by the administration of neutralising antibodies raised against the virus. For example, the peptides of the present invention could be injected into a large animal (such as a horse) that would stimulate the production of antibodies. The animal is then bled, and the serum purified for the appropriate antibodies and formulated for administration.

It will be appreciated that the following examples are illustrative and are not intended to limit the scope of the present invention. One of skill in the relevant art would be able to use the teachings described in the following examples to practice to full scope of the present invention. EXAMPLES

EXAMPLE 1 : IDENTIFICATION OF EPITOPES

Various epitopes in the S protein of MHV and TGEV that have been shown to elicit neutralising antibodies were examined. Of particular interest is a peptide containing a B-cell epitope at residue position 846-858 (SPLLGCIGSTCAE) in the MHV S protein. When used in combination with a T-helper cell determinant, this was demonstrated to elicit an effective antibody response which protected mice against an acute fatal MHV infection (Koolen et al., 1990). A region of the SARS CoV S protein was identified that shares a similar secondary structural signature with the epitope region on the

MHV S protein, using the protein secondary structure prediction method, PSIPRED

(McGuffin et al., 2000) (Figure 2A). The corresponding putative epitope in the SARS

S protein falls within a very hydrophilic region SQILPDPLKPTKR (785-797). Using the fold-recognition method Threader3 v3.4 (Jones et al., 1992), the 3-D structure concurs partially with the secondary structure predicted by PSIPRED.

Two further epitopes on the TGEV S protein were also examined. These epitopes have been mapped using different groups (l-IV) of neutralising monoclonal antibodies (Correa et al., 1990). Group IV neutralising antibodies recognised 2 epitope regions at position 379-387 (SFFSYGEI) and position 1176-1184 (QLAKDKVNE) in the TGEV S protein (Posthumus et al., 1990, Posthumus et al., 1991). Antibodies raised against the first epitope region of the TGEV S protein are effective at binding and neutralising whole TGEV. The second epitope region was found to contribute significantly towards enhancing the antigenicity of the combination peptide containing amino acids of both the first and the second epitope regions versus each of the single epitope peptides alone, indicating composite neutralising determinants (Posthumus et al., 1991 ). ^l

Using PSIPRED, it was found that the first epitope and its immediate flanking regions in TGEV S protein share a structural signature or motif (rather than amino acid sequence similarity) with a highly hydrophilic region in the SARS CoV S protein (Figure 2B). The corresponding putative epitope in the SARS S protein is mapped to position 176-184 (SGNFKHLRE).

For the second epitope in the TGEV S protein, mapping of the corresponding putative epitope in the SARS CoV S protein was facilitated by comparative analysis of the predicted amino acid sequence across seven CoVs. This putative epitope was identified at position 1005-1013 (NLAATKMSE) within a highly conserved 'framework' region (residue position 948 to 1046) in the C-terminal half of the SARS CoV protein. In the absence of structural data, this framework region (residue position 1119-1217) containing the second epitope of TGEV S protein was compared with the corresponding region in the SARS coronavirus S protein, using fold-recognition methods to locate structural and contextual similarity. Two fold-recognition methods to locate structural and contextual similarity. Two fold-recognition methods were used: 3D-PSSM (Position-Specific Scoring Matrix) (Kelley et al., 2000) (Figure 3A) and Threader3 v3.4 (Jones et al., 1992) (Figure 3B and 3C). Both methods predicted that the TGEV epitope and its immediate flanking regions share a similar helical-rich structural fold with the corresponding putative epitope region in the SARS CoV S protein, suggesting that the latter might also be a potential epitope.

EXAMPLE 2: Human Vaccine Trial

A statistically significant number of volunteers are enrolled in a study to test the safety and efficacy of the peptide vaccine compositions of the present invention. A geographical location or locations for the test is selected on the basis that the area is known to have been the site of past SARS outbreaks. The ratio of vaccine to placebo groups is randomized to result in a range from at least 1 :1 to no more than 2:1 ratio within the group. This randomization is designed to provide appropriately large groups for statistical analysis of the efficacy of the vaccine.

The vaccine composition will consist of a sufficiently high concentration of peptide so as to be effective to induce a protective immune response when the composition is administered parenterally or mucosally. Parenteral administration will preferably be via intramuscular injection. In both cases any of the adjuvants which are disclosed in the specification can be used.

The placebo will consist of an equal volume of buffered saline and is also to be given mucosally or parenterally. Vaccine and placebo are supplied as individual doses that are stored at -20°C. and thawed immediately prior to use. To determine the amount of vaccine necessary, different concentrations will be administered experimentally to a mouse. An effective concentration will be extrapolated and a comparable amount used in human subjects.

Blood samples are collected from all of the subjects for use in various laboratory assays. For example, enzyme immunoassay may be performed to evaluate the extent of the immune response elicited in each of the vaccinated individuals in response to the vaccine or placebo administered. Such techniques are well known in the art. Individuals participating in this study are chosen who are healthy at the time of vaccination with either the test vaccine or the placebo. Test subjects are assigned to receive vaccine or placebo in a double-blind fashion using a block randomization scheme. An appropriate number of doses are administered over a given period of time, e.g., two months, to elicit an immune response.

Study participants are monitored throughout the following year to determine the incidence of coronavirus infection and the subsequent development of disease conditions. Participating subjects are contacted on a periodic basis during this period to inquire about symptoms of SARS disease, both in the test subject and in the subject's community. Local epidemiological surveillance records may also be accessed.

The results of the above described study are assessed using standard statistical methods. The vaccine is well tolerated at the effective dose. The epidemic curves of outbreaks of SARS in the geographic areas tested will be assessed and the distribution of episodes of SARS disease will be established. The incidence of SARS caused disease in immunized individuals will be reduced to a statistically significant extent as compared to those individuals receiving the placebo.

REFERENCES

Chang, K. W„ Sheng, Y. & Gombold, J. L. (2000). Coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein. Virology 269, 212-24.

Chilvers, M. A., McKean, M., Rutman, A., Myint, B. S., Silverman, M. & O'Callaghan, C. (2001 ). The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur. Respir. J. 18, 965-70.

Correa, I., Gebauer, F., Bullido, M. J., Sune, C, Baay, M. F., Zwaagstra, K. A., Posthumus, W. P., Lenstra, J. A. & Enjuanes, L. (1990). Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus. J. Gen. Virol. 71 , 271-9.

DeLano, W. L. (2002). The PyMOL Molecular Graphics System. In DeLano Scientific. San Carlos, CA, USA.

Drosten, C, Gunther, S., Preiser, W., Van DerWerf, S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kotesnikova, L., Fouchier, R. A., Berger, A.,Burguiere, A. M., Cinati, J., Eickmann, M., Escriou, N., Grywna, K.,Kramme, S., Manuguerra, J. C, Muller, S., Rickerts, V., Sturmer, M.,Vieth, S., Klenk, H. 0., Osterhaus, A. D., Schmitz, H. & Doerr, H. W. (2003). Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome. N. EngL J. Med.

Fouchier, R. A., Kuiken, T., Schutten, M., Van Amerongen, 0., Van Doomum, G. J., Van Den Hoogen, B. G., Peiris, M., Lim, W., Stohr, K. & Osterhaus, AD. (2003). Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423, 240.

Gerber, J. D., Ingersoll, J. D., Gast, A. M., Christianson, K. K., Seizer, N. L., Landon, R. M., Pfeiffer, N. E., Sharpee, R. L. & Beckenhauer, W. H. (1990). Protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive FIPV vaccine. Vaccine 8, 536-42. Higgins, 0., Thompson, J., Gibson, 1., Thompson, J. 0., Higgins, D. 0. & T.J., 0. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.

Jones, 0.1., Taylor, W. R. & Thornton, J. M. (1992). A new approach to protein fold recognition. Nature 358, 86-9.

Kelley, L.A., MacCallum, R. M. & Stemberg, M. J. (2000). Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Med. Biol. 299, 499- 520.

Koolen, M. J., Borst, M. A., Horzinek, M. C. & Spaan, W. J. (1990). Immunogenic peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection. J. Virol. 64, 6270-3.

Ksiazek, T. G., Erdman, 0., Goldsmith, C. S., Zaki, S. R., Peret, 1., Emery, S., Tong, S., Urbani, C, Corner, J. A., Lim, W., Rollin, P. E., Dowell, S. F., Ling, A. E., Humphrey, C. 0., Shieh, W. J., Guarner, J., Paddock, C. D., Rota, P., Fields, B., DeRisi, J., Yang, J. Y., Cox, N., Hughes, J. M., LeDuc J. W., Bellini, W. J. & Anderson, L. J. (2003). A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348,1953-66.

McGuffin, L. J., Bryson, K. & Jones, 0. T. (2000). The PSIPRED protein structure prediction server. Bioinformatics 16,404-5.

Nicholls, J. M., Poon, L L. M., Lee, K. C, Mg, W. F., Lai, S. T., Leung, C. Y., Chu, C. M., Hui, P. K., Mak, K. L, Lim, W., Yan, K. W„ Chan, K. H., Tsang, N. C, Guan, Y., Yuen, K. Y. & J.S.M., P. (2003). Lung pathology of fatal severe acute respiratory syndrome. Lancet 361 , 1773- 1778.

Peiris, J. S. M., Chu, C. M., Cheng, V. C. C, Chan, K. S., Hung, I. F. N., Poon, L L. M., Law, K. I., Tang, B. S. F., Hon, T. Y. W., Chan, C. S., Chan, K. H., Mg, J. S. C, Zheng, B. J., Mg, W. L, Lai, R. W. M., Guan, Y., Yuen, K. Y. & group (2003). Clinical progression and viral load in a community outbreak of coronavirus- associated SARS pneumonia: a prospective study. Lancet 361, 1767- 1772.

Posthumus, W. P., Lenstra, J. A., Schaaper, W. M., van Nieuwstadt, A. P.,Enjuanes, L. & Meloen, R. H. (1990). Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus. J. Virol. 64, 3304-9.

Posthumus, W. P., Lenstra, J. A., van Nieuwstadt, A. P., Schaaper, W. M., van der Zeijst, B. A. & Meloen, R. H. (1991 ). Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus. Virology 182, 371-5.

Ruan, Y. J., Chia, L. W., Ling, A. E., Vega, V. B., Thoreau, H., Se Thoe, S. Y., Jer-

Ming Chia, J.-M., Mg, P., Chiu, K. P., Lim, L., Zhang, T., Chan, K. P., Oon, L. L E.,

Mg, M. L, Leo, Y. S., Mg, L. F. P., Ren, E. C, Stanton, L. W., Long, P. M. & Liu, E. T. (2003). Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection. Lancet

361 , 1779-85.

Seto, W. H., Tsang, 0., Yung, R. W._f Ching, T. Y., Mg, T. K., Ho, M., Ho, L M. & Peiris, J. S. (2003). Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS). Lencet 361 , 151 9-20.

Turner, R. B., Felton, A., Kosak, K., Kelsey, 0. K. & Meschievift, C. K. (1986). Prevention of experimental coronavirus colds with intranasal alpha-2b interferon. J. Infect. Dis. 154, 443-7.

Watanabe, I., Hagiwara, Y., Kadowaki, S. E., Yoshikawa, T., Komase, K., Aizawa, C, Kiyono, H., Takeda, Y., McGhee, J. R., Chiba, J., Sata, T., Kurata, T. & Tamura, S. (2002). Characterization of protective immune responses induced by nasal influenza vaccine containing mutant cholera toxin as a safe adjuvant (CTI 12K). Vaccine 20, 3443-55.

Wesseling, J. G., Godeke, G. J., Schijns, V. E., Prevec, L., Graham, F. L., Horzinek, M. C. & Rather, P. J. (1993). Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirus vectors protect mice against a lethal infection. J. Gen. Virol 74, 2061-9.

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (II) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

2. As peptide consisting of in another aspect the present invention provides a peptide consisting of the amino acid sequence selected from the group consisting of (I) SQILPDPLKPTKR or portion or equivalent thereof, (II) SGNFKHLRE or portion or equivalent thereof, and (III) NLAATKMSE or portion or equivalent thereof.

3. A peptide according to claim 1 or claim 2 wherein the equivalent is equivalent in primary structure.

4. A peptide according to claim 1 or claim 2 wherein the equivalent is equivalent in terms of secondary structure.

5. A peptide according to claim 1 or claim 2 wherein the equivalent is equivalent in tertiary structure.

6. A peptide according to claim 1 or claim 2 wherein the peptide provides an epitope relevant to a virus infection.

7. A peptide according to claim 6 wherein the virus infection is a coronavirus infection.

8. A peptide according to claim 6 wherein the virus infection is a SARS infection.

9. A nucleic acid capable of encoding a peptide according to claim 1 or claim 2.

10. A vector comprising a nucleic acid according to claim 9.

11. A recombinant virus or bacterium genetically altered to comprise a nucleic acid according to claim 9.

12. A composition comprising a peptide according to claim 1 or claim 2, or a nucleic acid according to claim 9 in combination with pharmaceutically acceptable carrier and/or diluent.

13. A composition comprising a peptide according to claim 12 comprising an adjuvant.

14. A composition according to claim 13 wherein the adjuvant is a mucosal adjuvant.

15. A composition according to claim 14 wherein the mucosal adjuvant is a cholera toxin.

16. Use of a peptide according to claim 1 or claims 2, or a nucleic acid according to claim 9 in the preparation of a medicament for the treatment or prevention of a viral infection.

17. Use according to claim 16 wherein the viral infection is a coronavirus infection.

18. Use according to claim 16 wherein the viral infection is a SARS infection.

19. Use of a peptide according to claim 1 or claim 2, or a nucleic acid according to claim 9 in the diagnosis of a virus infection.

20. Use according to claim 19 wherein the viral infection is a coronavirus infection.

21. Use according to claim 19 wherein the viral infection is a SARS infection.

22. A kit for diagnosing a virus infection comprising a peptide according to claim 1 or claim 2, or a nucleic acid according to claim 9.

23. A method of treating or preventing a virus infection comprising administering to a mammal in need thereof an effective amount of a composition according to any one of claims 12 to 15.

24. A method according to claim 23 wherein the viral infection is a coronavirus infection.

25. A method according to claim 23 wherein the viral infection is a SARS infection.