WO2005012910A1

WO2005012910A1 - A peptide antigen

Info

Publication number: WO2005012910A1
Application number: PCT/GB2004/003266
Authority: WO
Inventors: Anthony Richard Fooks; Karen Louise Mansfield; Nicholas Johnson
Original assignee: The Secretary Of State For Environment, Food & Rural Affairs
Priority date: 2003-07-31
Filing date: 2004-07-29
Publication date: 2005-02-10

Abstract

A peptide antigen comprising SEQ ID NO 2, WXaXbXcDI where Xa, Xb and Xc may be any amino acid, has been found to be useful in the diagnosis of rabies vaccination or disease status of an animal, as well as being useful in vaccines, such as peptide marker vaccines.

Description

A Peptide Antigen

The present invention relates to a peptide antigen, derived from the rabies virus, and the use of this antigen in screening for rabies or rabies vaccination status, and for vaccinating against rabies infection. In particular, the peptide antigen may be used as, or as part of, a peptide marker vaccine in particular in rabies free areas .

Rabies virus (RAJ3V) is a member of the Rhabdoviridae family of the Lyssavirus genus, and comprises a single-stranded negative- sense RNA genome composed of five different proteins. Of these five proteins, the glycoprotein is the only surface exposed protein and has been postulated to form trimeric structures in the viral membrane (Gaudin et al . , Virology (1992), 187, 627-

632), although the crystal structure of the protein has not been elucidated. In immune responses following vaccination or infection, neutralising antibodies are directed primarily against the glycoprotein (Cox et al , (1911 ) Infection and Immunity, 16, 754-759) . This makes the glycoprotein the obvious target for vaccine development strategies. The glycoprotein is expressed as a 524 amino acid polypeptide, of which the first 19 residues form a signal peptide which is cleaved by host proteases as the protein enters the endoplasmic reticulum. The mature glycoprotein consists of a surface exposed 439-residue ectodomain, a 22-residue transmembrane domain and a 54-residue endodomain.

Previous studies of the antigenic structure of the rabies glycoprotein have identified both conformational and linear, non-conformational sites. All have relied upon the binding of neutralising monoclonal antibodies (MAbs) and subsequent sequencing of mutants that escape neutralisation. There are two major conformational sites, of which the immunodominant site is Antigenic Site II; formed from two regions, residues 34 to 42 and residues 198 to 200 (Prehaud et al , Journal of Virology, (1988) 62, 1-7) . The second site, Antigenic site III is between residues 330-338, and has arginine residue at position 333 (Seif et al, Journal of Virology (1985) , and has been shown to be important for neuroinvasion (Dietzschold et al . , Proc. Natl. Acad. Sci. (1983), 80, 70-74). It has been shown that 97% of monoclonal antibodies raised against the glycoprotein will bind to these immunodominant sites (Coulon et al , Onderstepoort Journal of Veterinary Research, (1993) 60, 371-275) . However, because these epitopes are conformational, many antibodies fail to recognise them in western blotting assays and the presence of antibodies against both sites have not been demonstrated in polyclonal sera raised against current vaccines. Further studies have identified nonconformational, neutralising epitopes, including work by Dietzschold et al . , (Journal of Virology (1990) 64, 3804-3809) who identified a linear epitope between amino acids 244-281. Additional work by Ni et al . , (Microbiology and Immunology (1995) 39, 693-702) confirmed a linear epitope in this region (between amino acids 249-268) and concluded that the glycoprotein has many linear epitopes most of which are not as strongly recognised as conformational epitopes. Luo et al . (Virus Research, (1997), 51, 35-41) have demonstrated a neutralising linear epitope in this region, located around the tryptophan at position 251. However, attempts to use a synthetic peptide to mimic this region failed to detect antibodies against it in the sera of vaccinated dogs. Therefore, the usefulness of any particular epitope in rabies diagnosis or vaccination is not clear.

Currently, screening for post vaccination responses to rabies is carried out by the OIE (Office International des Epizooties) approved Fluorescent Antibody Virus (FAVN) test. This requires the use of a fixed strain of rabies virus (RABV) and thus category III containment facilities. The test also requires two days to perform and requires dedicated staff, trained in working at cat III conditions. The development of an ELISA alternative to this would significantly reduce the cost and time required to measure anti- rabies titres. An ELISA test accepted by the OIE (May 2003) uses authentic antigen purified from RABV infected cells. This approach will not be acceptable for use in countries where the risks from animal contamination of products is an issue. The principal difficulty in developing such a test is the selection of a suitable antigen.

It would be expected that the detection of anti-rabies antibodies in an ELISA format might be carried out using whole inactivated virus and recombinant proteins (expressed in bacterial or eukaryotic cell culture systems) . However, whole inactivated virus incurs costs associated with growth and inactivation of a zoonotic agent. Recombinant proteins require detailed purification that incurs production costs.

Previous studies have suggested that the N-terminal region of the rabies glycoprotein contains antigenic sites detected by the sera of both rabbits (Dietzschold et al . , Journal of Virology, 1982, 44, 595-602) and dogs (Johnson et al . Journal of General Virology, 2002, 83, 2663-2669) that have received anti-rabies vaccine. Attempts at locating epitope sites using neutralisation escape mutants have identified a subset of antibodies which neutralise the rabies virus at acidic pH (Raux et al . , Virology (1995), 210, 400-408, Gaudin et al . , Journal of Virology, (1996) 70, 7371-7378) . These studies suggested that residues at the N- terminal of the glycoprotein (lysine 10, proline 13, serine 16) were critical to antibody binding.

The applicants have identified a different peptide antigen which appears to have a wide range of potential applications in the area of rabies diagnostics and vaccination. In particular, they have found that the sequence GPWSPIDIHHLS (SEQ ID NO 1) contains an epitope, and this epitope, and peptides including it form the subject of the invention. Thus in a first aspect, the invention provides the use of a peptide comprising SEQ ID NO 2, WX^aX^bX^cDI where X^a, X^b and X^c may be any amino acid, in the preparation of a reagent for the diagnosis of rabies vaccination or disease status of an animal.

Diagnosis in this context may mean the diagnosis of a disease (i.e. rabies) infected animal, or the detection of a positive response to a rabies vaccine, or the determination of the vaccine status of an animal (whether or not is has received a vaccine) .

In some instances, the epitope may also have beneficial protective effects.

Thus, in a second aspect, the invention provides the use of a peptide comprising SEQ ID NO 2 as defined above, in the preparation of a prophylactic or therapeutic vaccine against rabies infection.

The vaccine suitably comprises additional elements that produce a protective immune response in an animal to which it is administered. These may be peptides of proteins or even attenuated or killed organisms.

The term "peptide" as used herein generally refers to relatively short sequences of amino acids, for instance up to 20 amino acids, suitably up to 15 amino acids, and preferably up to 12 amino acids in length. However, it may also include larger moieties where these comprise multiple copies of the individual peptides. SEQ ID NO 2 was identified as being an epitopic sequence from a study as illustrated hereinafter, which identified SEQ ID NO 1 which is GPWSPIDIHHLS as being an antigenic region of the rabies glycoprotein. The epitope may comprise any number of amino acids within the sequence, but would be expected to comprise at least 3, for instance at least 6, or more likely at least 8 of the 12 amino acids found within the peptide of SEQ ID NO 1. These may be consecutive amino acids, or they may be spaced from each other. The amino acids constituting the epitope may be determined using routine methods. In particular, it is believed that the epitope requires the presence of at least three residues.

It is believed that at least one tryptophan, one aspartic acid and one isoleucine are important in terms of the epitope. The epitope can therefore be represented as SEQ ID NO 2 as shown above .

In particular however, X^a is selected from an amino acid having an uncharged polar side chain such as serine, asparagine, or an amino acid having an acidic side chain such as aspartic acid. It is preferably serine.

Examples of X^b included an amino acid with a non-polar side chain such as proline or leucine, or an amino acid having an uncharged polar side chain such as serine. Preferably X^b is proline.

Suitably X^c is an amino acid having a non-polar side chain such as isoleucine or valine or an amino acid having an uncharged polar side chains such as threonine. Preferably X^c is isoleucine.

As would be recognised in the art, amino acids with non-polar side chains are glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine. Amino acids with uncharged polar side chains are asparagine, glutamine, serine, threonine or tyrosine. Finally, amino acids with acidic side chains are aspartic acid and glutamic acid. Lysine, arginine and histine have basic side chains.

Epitopes of this general type have been previously described in connection with proteins from other organisms, for example in O03/050543 and US Patent No. 6,562,345, but they have not hitherto been associated with rabies.

Examples of peptides of SEQ ID NO 2 are peptides of SEQ ID NOs 2-12 as well as variants and fragments of any of these, as illustrated hereinafter. Preferred examples of peptides of SEQ ID NO 2 include SEQ ID NO 1 and variants thereof, and fragments of any of these, as set out above.

According to a further aspect of the invention, there is provided a peptide comprising SEQ ID NO 1, or variants thereof, of fragments of any of these.

As used herein, the expression "variants" includes peptides which differ from the base peptide in that one or more amino acids within the sequence are deleted or are substituted for other amino acids, but where biological activity and in particular the immunogenicity of the peptides is unchanged. Thus, antisera, which recognise the epitope described above, will also cross react with variants, and/or an immune response generated by a variant of the epitope will recognise rabies infections.

Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties, in particular, in terms of the acidity, basicity or polarity of the side chains. Non- conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably variants will be at least 75% identical, preferably at least 80% identical, and more preferably at least 90% identical to the base sequence.

Identity in this instance can be judged for example using the algorithm of Lipman-Pearson, with Ktuple:2, gap penalty: 4, Gap Length Penalty: 12, standard PAM scoring matrix (Lipman, D.J. and Pearson, W.R., Rapid and Sensitive Protein Similarity Searches, Science, 1985, vol. 227, 1435-1441).

Variants may also include structures which include more than one copy of the epitope, for example, lysine branched structures which may contain up to 12, suitably up to 8 copies of the epitope, linked for instance by their carboxy-terminal to a branched poly-lysine core.

The term "fragment" refers to any portion of the given amino acid sequence which has the same activity as the complete amino acid sequence. Fragments will suitably comprise at least 5 and preferably at least 10 consecutive amino acids from the basic sequence.

The peptides of SEQ ID NO 2, such as a peptide of SEQ ID NO 1 or variants thereof, can form the basis of an ELISA test for the detection of antibodies that may be indicative of the disease or vaccine status of an animal.

They provide a cost effective and convenient assay, as it is possible to work with reagents which can be prepared at low cost, with the benefit of not having to work with a human/animal pathogen in either the assay preparation or test application stages. Thus they are useful in screening for rabies or testing for the vaccination status of animals such as dogs. They may also have applications in the production of vaccines, which are protective against rabies. A particular potential application is as a "marker" vaccine. For instance, in rabies- free areas of the world, it would be desirable to vaccinate animals against the possibility of accidental exposure to rabies. However, if current vaccines, which are based upon inactivated rabies strains, are employed for this purpose, it then becomes impossible to distinguish between vaccinated animals and animals which have been infected naturally.

The peptides described above are immunogenic peptides which appears to mimic an epitope of a glycoprotein of rabies virus. However, animals who have been vaccinated using current vaccines do not all produce antibodies which recognise this peptide. Furthermore, as explained hereinafter, naturally infected animals may not respond to this antigenic site because it is believed to be hidden. The peptides can form the basis of a peptide marker vaccine in particular for use in rabies-free areas.

It may then be utilised in an ELISA test for the detection of antibodies to it, which allows vaccinated animals within the rabies-free area (assay positive) to be distinguished from any which were vaccinated but failed to respond, or animals infected naturally and/or introduced into the area (assay negative) .

It may therefore be useful in distinguishing between these animals, preferably as part of a "cocktail" or combination of a range of peptides, which will assist in confirming the results.

Furthermore, the fact that the peptide is a mimic of a natural epitope, means that it may produce or contribute towards a protective immune response in an animal to which it is administered in some cases. Such vaccines form a further aspect of the invention. Vaccines in accordance with the invention may be useful as prophylactic or therapeutic vaccines. Preferably however, the peptide is, or is a component of, a prophylactic vaccine. These will be discussed further hereinafter.

Most preferably the peptide is of SEQ ID NO 1 or a fragment thereof. SEQ ID NO 1 corresponds to the sequence on the mature RABV glycoprotein between residues 12 and 23. Suitable fragments comprise at least 6 and preferably at least

8consecutive amino acids, and comprise SEQ ID NO 2 defined above .

The peptide epitope/antigen of SEQ ID NO 1 is a short linear sequence of the RABV glycoprotein. It was identified by a phage display technique. SEQ ID NO 1 was derived from the Pasteur Virus (PV) strain of RABV.

SEQ ID NO 1, a peptide mimic of the epitope has been synthesized and a multimeric form used to inoculate rabbits and mice. The sera obtained could detect both the multimeric and monomeric forms of the peptide demonstrating the immunogenicity of the epitope. The monomer was also tested for reactivity with rabies- vaccinated dog sera, demonstrating antigenicity of this peptide mimic.

The site of SEQ ID NO 1 partially overlaps with the epitope site identified previously (Raux et al . , 1995, supra, Gaudin et al . , 1996 supra.). However it does appear to represent a novel linear site, not previously identified, because antibodies to the epitope of SEQ ID NO 2 showed different properties to antibodies to the previously identified epitope. In particular, those antibodies bound to conformational epitopes as evidenced by a failure to detect the glycoprotein by immunoblotting and could neutralise the virus; neither of which properties were shown by antibodies to the present epitope. The sequence appears to be highly conserved between rabies viruses and between the genotypes of phylogroup 1 of the lyssavirus family (Badrane et al 2001, J. Virol. 75, 3268-3276).

The peptide described above can be prepared by conventional methods. For example, it may be synthesised chemically, for example using a peptide synthesiser. Alternatively, a nucleic acid encoding the peptide may be incorporated into an expression vector, which may then be used to transform a host cell, which may be a prokaryotic or eukaryotic cell. Preferably the cell would be a conventional prokaryotic cell such as E. coli .

The applicants have found that the peptide binds to a range of ELISA plates and can be detected by sera using standard protocols.

Thus in a further embodiment, the invention provides a method for detecting the vaccination or disease status of an animal said method comprising contacting a sample taken from the animal with a peptide as described above, and detecting the presence of a complex formed between said peptide and an antibody present in the sample, and relating this to the vaccination or disease status of an animal.

The sample is suitably a serum sample, taken from an animal suspected of having been infected with rabies, or from an animal, which has been vaccinated against rabies, so that the efficacy of the vaccine can be tested.

Suitably the animal is a dog or cat, but samples from any other mammals including humans may be tested in this way.

Suitably the peptide or a binding agent therefore, is immobilised on a solid support, for example on an ELISA plate, but other forms of support, for example membranes such as those utilised in conventional "dip-stick" tests may also be employed. Detection of a complex between a peptide and an antibody can be detected using conventional methods, in particular immunological methods such as ELISA methods. Assay formats may take various forms including "sandwich" or "competitive" types.

In a typical sandwich assay, the peptide is immobilised on a support, such as an ELISA plate, where is it contacted with a sample suspected of containing anti-rabies antibodies, and in particular, an animal serum sample. Where present, these antibodies will bind the peptide and so become immobilised in their turn. The support is then separated from the sample, for example by washing. The presence of antibodies on the support can then be detected by application of secondary antibodies or binding fragments thereof, which bind to the target antibody, and are detectable, for example because they are labelled for instance with a visible label such as a fluorescent label, or a radiolabel, but preferably that they can be developed to produce a visible signal. A particular example of a secondary antibody is an antibody or binding fragment, that carries an enzymatic label, such as horseradish peroxidase, which can then be utilised to produce a signal by addition of the enzyme substrate, using conventional ELISA methodology.

In a particular competitive assay format, a primary antibody or a binding fragment thereof, which binds the peptide is immobilised on a support. In this instance, the peptide is added to the sample and incubated therewith, prior to contact with the support. Any antibody within the sample will bind the peptide of the invention, which will not then be available to bind to the immobilised antibody. Thus, the absence of peptide on the support is indicative of the presence of antibodies which recognise the peptide in the sample.

in this case, the peptide is suitably labelled so that it may be readily detected, for instance using a visible label such as a fluorescent or radiolabel. Alternatively, it may be detected using a secondary antibody or a binding fragment thereof, such as those discussed above in relation to sandwich assays, which binds the peptide in addition to the primary antibody.

Thus in a particular embodiment, the peptide described above carries a label, as discussed above.

Particular antibodies that may be used in these methods may be monoclonal or polyclonal antibodies, but in particular are monoclonal antibodies. They may be generated using conventional methods, and in particular are raised against the peptide as described above.

Polyclonal antibodies can be generated by immunisation of an animal such as a rabbit, rat, goat, horse, sheep etc, with the peptide described above. Monoclonal antibodies to the peptides described above may be obtained by conventional methods, for example using hybridoma cells, phage display libraries or other methods.

Suitable antibody fragments include F(ab)₂, Fab, FV, VH or VK fragments, as well as a single chain antibodies. Each of these types of antibody derivative and their acronyms are well known to the person skilled in the art.

Preferably the peptide of SEQ ID NO 2, such as the peptide or SEQ ID NO 1 or variants thereof are used as part of a "cocktail" of more than one peptide or protein which has diagnostic significance in the case of rabies. In this case, the different peptides and proteins are suitably differently labelled so that positive and negative responses to each can be readily distinguished.

A synthetic peptide of SEQ ID NO 1 was produced and used as the antigen component of an enzyme-linked immunosorbent assay (ELISA) to test the reactivity of dog sera vaccinated against rabies. Results suggest that the peptide can discriminate between vaccinated and non-vaccinated animals.

Subsequent immunisation studies in rabbits using a MAP-peptide variant of the epitope indicate that the peptide is immunogenic. The immune response may have some protective or therapeutic effects against rabies infection.

The invention also provides a peptide as described above for use as or in a prophylactic or therapeutic vaccine. Suitably, the peptide is for use as or in a prophylactic vaccine.

In particular, for vaccine applications, the peptide is administered in the form of a pharmaceutical composition, which further contains a pharmaceutically acceptable carrier. These compositions form a further aspect of the invention.

In addition to the pharmaceutically acceptable carriers, they may comprise other components, which produce or enhance an immune response in the animal that is protective against rabies.

Pharmaceutically acceptable carriers are well known in the art, and may be solid or liquid. The vaccine may be adapted for any suitable adminstration method, but is preferably adapted for parenteral or oral administration. Most preferably, the composition is suitable for parenteral administration for example for intravenous or intramuscular administration, in which case, the carrier is a liquid carrier such as water or saline.

The compositions of the invention will suitably comprise an appropriate dosage unit of the peptide. This may vary depending the nature of the patient, the condition being treated and other clinical factors. For example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used.

In general however, the composition of the invention will comprise approximately 2 to 10 wt% of peptide.

Vaccine compositions may further comprise an adjuvant in order to enhance the immune response to the biologically active material administered. Suitable adjuvants include pharmaceutically acceptable adjuvants such as Freund's incomplete adjuvant, alhydrogel, or aluminium compounds.

Live vaccines are also possible. In this case, a nucleic acid encoding a peptide as described above is incorporated into a live vaccine (pharmaceutically acceptable) vector, such as a viral or bacterial vector, which is then administered to the animal, in such a way that the peptide is expressed by the vector in vivo. Suitable viral vectors include vaccinia or adenovirus vectors. Suitable bacterial vectors include gut- colonising organisms such as Salmonella species, and in particular attenuated Salmonella species. Again suitable dosages will be determined using routine clinical practice.

As mentioned above, vaccines of this type may be particularly useful for vaccination in rabies-free areas. Sera from animals in this area can then be tested to detect antibodies to the peptide. Positive results would indicate that the animal had been vaccinated and had responded to the vaccine. Negative results would indicate that the animal had not responded to the vaccine, or may have entered the area from outside.

Protective results may be obtained by administering effective amounts of the pharmaceutical compositions or live vaccines as described above to the animal and such methods form yet a further aspect of the invention. Thus the vaccine status of an animal, as determined using the methods described above may be indicative of the place of origin of an animal .

As before, the sample is suitably a serum sample, taken from an animal suspected of having been infected with rabies, or from an animal, which has been vaccinated against rabies, so that the efficacy of the vaccine can be tested. Suitably the animal is a dog or cat, but samples from any other mammals including humans may be tested in this way. The peptide is preferably part of a diagnostic cocktail of more than one peptide, which together provide good discriminatory results.

Structural predication analysis suggests that the epitope of SEQ ID NO 2 is antigenic, whilst also indicating that it is not surface exposed, providing a possible explanation for the failure of antibodies raised against it to neutralise virus. However, the contribution of the N-terminal domain to the postulated properties of the rabies glycoprotein (receptor binding, membrane translocation etc.) are not known.

The epitope site is conserved between rabies virus isolates and lyssaviruses (phylogroup 1), but is probably hidden under native conditions. Roche and Gaudin (Virology, 2002, 297, 128-135) have suggested that an increase in acidity can cause conformational changes in the native glycoprotein conformation, resulting in exposure of previously hidden hydrophobic residues. Such conformational changes could occur during the inactivation process of vaccine production that might explain the presence of antibodies against a hidden site, detected in some vaccinated animals. However, it would suggest that it would not be recognized by the sera of naturally infected animals.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which: Figures la and lb show ELISA data of increasing dilution of peptide of SEQ ID No 1 (starting from a stock concentration of lOmg/ml in PBS) against increasing dilution of Mab;

Figure 2 is a graph illustrating the development of an antibody response to the peptide following a course of immunisation of rabbits with a peptide of the invention;

Figures 3a and 3b show the titration by ELISA of the two sera against increasing dilution of the monomeric form of the peptide (3a) and the MAP form (3b) used to inoculate the rabbits. In both cases the antisera generated binds to the peptide demonstrating that both animals have produced a specific antibody response against the epitope GPWSPIDIHHLS;

Figure 4 shows the results of 12 replicates of OIE naive sera (1), peptide immunised rabbit R356 (2) and peptide-immunised rabbit R357 (3) screened using the ELISA protocol;

Figure 5 shows the results of a peptide ELISA comparison between naive and immunised dog sera;

Figure 6 shows a comparison of FAVN values with peptide ELISA results from a panel of 33 post-vaccination dog serum samples; and

Figure 7 is a graph showing a summary of mouse antibody response to S2/4 peptides. (Groups as described in Table 5 below) .

Example 1

Identification of the Peptide

A phage display technique was used to identify peptide ligands for two monoclonal anti-rabies antibodies. Briefly, phage display is a selection technique employing random peptides expressed as fusions with the coat protein of a bacteriophage. The fused protein is displayed on the virion surface.

The PhD12 peptide library (New England Biolabs, Hitchin, UK,), which consists of random 12-mer peptides genetically fused to the coat-protein (pill) of filamentous bacteriophage M13, was used in this investigation.

A panel of antibodies was exposed to the library' of phage- bound peptides during a process called ^biopanning' . Unbound phage were washed away, and the bound phage eluted and amplified. The amplified phage were used as input phage for the next round of ^Spanning' .

Specifially, in order to perform four rounds of biopanning,

Maxisorp plates (Nunc, Life Technologies, Paisley, Scotland, UK) were coated with the target monoclonal antibody diluted in 0.1M NaHC0_3~ The coated plates were then blocked for 1 hour at 4°C, after which they were washed six times using Tris-buffered saline (pH 7.4) with 0.1% Tween 20 (TBST) . For the first round of biopanning, 3 X 1010 plaque forming units (PFU) of library diluted in TBST were added to the coated plate, and incubated for 1 hour at room temperature with agitation. The plate was then washed ten times, and a low-pH elution buffer added. This was incubated at room temperature for ten minutes with agitation, after which it was transferred to a tube and neutralised with Tris-HCL (pH 9.1).

E coli strain ER273 8 was used for amplification of eluted phage, which provided input phage for the next round of biopanning. Before and after amplification, the eluate was titred on LB-IPTG/XGAL plates. This blue/white screening allowed the counting of plaques, and an estimation of phage concentration in the eluate. Four rounds of biopanning were performed in total for each monoclonal antibody. This cycle of selection/amplification resulted in enrichment of the phage pool in favour of the tightest binding sequences.

After biopanning, the unamplified phage were titrated as before, and ten blue plaques were selected for sequencing. The selected clones were amplified, purified and converted to single-stranded DNA according to the manufacturer's protocols.

At this point, individual clones were characterised by DNA sequencing, and the target sequence deduced by alignment of the clone sequences using DNAstar/Megalign (Table 1) .

Sequencing was carried out using the ABI Prism® Big Dye- Terminator Cycle Sequencing Ready Reaction Kit (Applied

Biosystems, Foster City, California, USA), on a Perkin Elmer 9700, using the primer provided by New England Biolabs. The subsequent extension products were precipitated using 75% isopropanol, and the resultant DNA pellets were analysed by a commercial source (Lark Technologies Inc., Saffron Walden, Essex, UK) .

Sequences were annotated using the DNAstar program (DNAstar, Madison, USA) , complementary sequences to the ten clone sequences were determined and a consensus sequence for each antibody epitope was derived. Once aligned with the sequence of the rabies PV glycoprotein (GenBank Ml 3215), possible epitope sites for each monoclonal antibody could be determined.

Consensus sequences were achieved for each of the monoclonal antibodies panned against the peptide library. This is shown in Table 1, for one antibody. The amino acids Tryptophan (W) , Aspartic Acid (D) and Isoleucine (I) form a common consensus motif for this monoclonal antibody. Table 1

Illustration of how a consensus sequence for the epitope site is derived from ten phage clones (complementary sequences) , for monoclonal antibody MnAbl.

SEQ ID NO CLONE: 1 S N S V D I w H M S S 3 2 S N S V D I w H M S S 4 3 E L R w D L T D I Y N L 5 5 E L R w D L T D I Y N L 6 6 S w N S V D I w H M S S 7 7 S w N S V D I w H M S S 8 8 E L R w D L T D I Y N L 9 9 E L R D L T D I Y N L 10 10 E L R w D L T D I Y N L 11

CONSENSUS : E L R D L T D I Y N L 12

On alignment of the consensus sequences with the PV glycoprotein, it appeared that the binding sites of two of the antibodies analysed, designated MnAbl and MnAb2, were in a region near to the N-terminal (Table 2) , and contained a number of common motifs, notably, the amino acids tryptophan, aspartic acid and isoleucine. This region is a novel linear epitope. Table 2

Alignment of predicted epitope sites with the N-terminal of the glycoprotein, for monoclonal antibodies MnAbl and MnAb2.

K F P I Y T I P D K L G PW SP I D I H H L S C P N N L V V PVGlycoprotein (AA1-30) (SEQ ID NO 13) E L R W DL T D I Y N L S2 (SEQ ID NO 14) S W NS V D I W H M S S S4 (SEQ ID NO 15)

It was concluded that the epitope that the monoclonal antibodies targeted was a linear site between residues 12 and 22 of the mature rabies glycoprotein. This site is highly conserved between rabies viruses from throughout the world (Table 3) .

Table 3

Alignment of the first 40 residues of the rabies glycoprotein from viral isolated from throughout the world

PV Vaccine KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSG

Rv53 USA S C

Rv56 USA N

Rv63 Pol

Rv73 Belize Rvl03Mor

Rvl22 Zim Y S

Rvl28 Zim

Rv202 Tur

Rv245 Rus Rv277 Pak

Rv304 Rus

Rv313 Ger

Rv425 SA Rv440Rus

Rv464 Moz

Rv484 Bot

Rv629 Nig

Rv661 Fra Rv677 Fra

A synthetic 12-residues peptide was produced to mimic this site with the amino acid sequence GPWSPIDIHHLS.

A consensus sequence was obtained of 10 randomly selected phages isolated for each antibody with the residue sequence WxxxDIWHxS . This aligned closely with the native amino acid sequence of the rabies surface glycoprotein between residues 12 to 23 of the mature protein (Tordo et al, 1986 Proc. Natl. Acad. Sci. USA 83, 3914-3918).

Example 2

Use of Peptide in ELISA A synthetic 12-residues peptide was produced to mimic the site identified in Example 1, with the amino acid sequence GPWSPIDIHHLS (SEQ ID NO 1) .

The antigenic properties of the peptide mimic were confirmed by demonstrating binding of the original monoclonal antibodies to the peptide using a conventional ELISA technique.

A 96 well ELISA plate was coated with the peptide antigen of SEQ ID NO 1 at various concentrations in PBS for 2 hours at 37°C. The well was washed three times with phosphate buffered saline

(PBS) containing Tween 20 ^(RTM) (400μl/well) . The plate was then blocked with 5% skimmed milk (in PBS) for 1 hour at 37°C, followed by further washing. Dilute antibody samples (lOOμl/well) , were incubated at room temperature for 30 minutes, and added to the wells in triplicate. After 1 hour at 37°C, wells were washed six times. Protein A-hrp conjugate diluted 1 in 2000 in antibody diluent was added and the wells incubated for a further 1 hour at 37°C. After further washing (x3) , lOOμl TMB substrate (Sigma) was added and the reaction allowed to develop for up to 40 minutes. The reaction was then stopped with 50μl of weak acid solution and read at 450nm.

Figures la and lb demonstrates that the peptide is bound by both antibodies. The figures show ELISA data of increasing dilution of peptide (starting from a stock concentration of lOmg/ml in PBS) against increasing dilution of Mab. Both Mabs bind the peptide strongly down to a concentration of lμg/ml at low dilutions of antibody i.e. 1/1000.

Example 3

Use of Peptide to Elicit Immune Response

To demonstrate that the peptide was capable of inducing an antibody response a modified version of the peptide of SEQ ID NO 1 was prepared in the form of a Multiple Antigenic Peptide (MAP) . This consisted of eight identical peptides, each of SEQ ID NO 1, linked by their carboxy-terminal to a branched poly- lysine core. This preparation was dissolved in phosphate buffered saline (PBS) and mixed initially with Freunds complete adjuvant and used to inoculate two New Zealand White rabbits subcutaneously (code named R356 and R357) . Subsequent inoculations of peptide in incomplete Freunds adjuvant were given subcutaneously at 2, 4, 6 and 8 weeks. Test bleeds were carried out before and during the inoculations at 3 and weeks and the rabbits sacrificed at 10 weeks. These samples were subjected to an ELISA, substantially as described in Example 2 but using dilute serum samples (1 in 100 with PBS) instead of the antibody samples, and using the monomeric form of SEQ ID NO 1 as the antigen.

Both rabbits produced sera that could recognise the original epitope by ELISA. Figure 2 shows the development of the antibody response over the course of the immunisation.

Figures 3a and 3b show the titration by ELISA of the two sera against increasing dilution of the monomeric form of the peptide (3a) and the MAP form (3b) used to inoculate the rabbits. In both cases the antisera generated binds to the peptide demonstrating that both animals have produced a specific antibody response against the epitope GPWSPIDIHHLS.

This is indicative that this sequence contains a significant antigenic epitope.

Example 4

Immunogenicity Comparative study in rabbit The ELISA protocol described in Example 2 was tested using 12 replicates of OIE naive sera (1) , peptide immunised rabbit R356 (2) and peptide immunised rabbit R357 (3) , as described in Example 3.

The results show clearly that a recognisable immune response was generated in immunised rabbits as compared to the naive sera. Example 5

In order to evaluate the prototype peptide ELISA against both naive and hyperimmune dog sera, two panels were created. Hyperimmune dog sera was selected on the basis that the post- rabies vaccinated sera, when tested using the Fluorescent Antibody Virus Neutralisation (FAVN) assay, had a FAVN value of greater than 100 IU/ml. The FAVN assay is used to measure rabies virus neutralisation in animal sera submitted under the PETS travel scheme in the UK.

A naive panel (n = 16) with a mean FAVN value of 0.08 IU/ml (+/- 0.06) was compared a panel of vaccinated dog sera (N = 24) with a mean FAVN of 899.96 IU/ml (+/- 317).

For each sample tested, three wells of a Maxisorp plate were coated with peptide diluted to a final concentration of lOμg/ml in PBS (phosphate buffered saline) , with another three wells coated with PBS only; the coated plate was incubated at 37 °C for 2 hours. Plate contents were discarded and each well was filled with blocking buffer (PBS, 1% Tween 20, 1% non-fat milk), and the plate incubated at 37 °C for 1 hour. During the blocking incubation, samples were pre-diluted 1:250 in antibody diluent (PBS, 1% Tween 80, 2% foetal calf serum (FCS) , and incubated at room temperature for 30 minutes. Blocking was followed by 6 washes with 1% PBST (1 minute per wash, with agitation) . On addition to the coated plate (three peptide-coated wells and three uncoated wells per sample) , samples were incubated for 1 hour at 37°C, and then washed as before. Protein A-HRP (Sigma) diluted 1:6000 in antibody diluent was incubated with the plate for 1 hour at 37°, again followed by 6 washes with 1% PBST. 3', 3', 5 ' , 5' Tetramethylbenzidine (TMB) (Sigma) substrate was incubated with the plate for 25 minutes at room temperature in the dark. Reaction was stopped with IM sulphuric acid, and the plate was read at 450nm. All volumes were lOOμl per well, except the blocking step and wash steps where wells were completely fi l led .

Figure 5 displays the overall results of each sample following the ELISA described in Example 2. The naive group gave a mean OD450 of 0.24 units (+/- 0.1) whilst the vaccinated dog sera panel gave a mean OD450 of 0.74 IU/ml (+/- 0.5).

These results demonstrates that a proportion of vaccinated dogs produce a detectable response to peptide S2/4.

To further assess the response to peptide S2/4 within post- vaccination sera, the ELISA results of dog sera, grouped by FAVN titre were compared. Figure 6 shows that there appears to be a general trend for increasing FAVN values correlating with increased OD values.

However, this is believed to be due to the increased OD values given by the high titre sera only. This can be illustrated in Table 4 below. Only the high titre group (>100 IU/ml) show an increase in mean OD450. This suggests that , in this case, there is little correlation between response to the epitope mimicked by peptide S2/4 and virus neutralisation.

The presence of serum samples with a low FAVN titre, which on occasion produce high OD values, supports this conclusion. A more appropriate conclusion might be that not all vaccine recipients produce a response to this particular epitope. Where a strong response is observed, as evidenced by a high FAVN titre (>100 IU/ml) , it is more likely that a response to the epitope is seen (Figures 4& 5) . Table 4. Summary of mean FAVN and peptide ELISA values for four groups of Dog sera.

Example 6

Mouse immunisation studies Panels of mice were immunised with a range of peptide formulations, adjuvanted using monophosphoryl-lipid a + trehalose dicorynomycolate emulsion (MPL/TDM) (Sigma) . Serum samples were taken pre-immunisation and a final bleed taken after a course of 3 inoculations. The sera was assessed by peptide ELISA and FAVN. Table 5 shows the mean pre- and post- antibody response of each group of mice as tested by the monomer peptide ELISA and Figure 7 summarises this data.

Table 5. Detection of antibody responses in mice to peptide S2/4 (monomer) following inoculation with a panel of S2/4 peptides .

A clear response was detected for the two groups (C & D) who received peptide in the multiple antigenic conformation, reflecting its superior presentation of short peptides. A response was obtained with the peptide given in combination with the T cell epitope (Otvos, et al., 1995, Biochimica et Biophysica Acta 1267, 55-64) which adjoins the epitope encoded by peptide S2/4 within the rabies glycoprotein. It is possible that the T cell epitope has assisted the response to immunisation with this peptide. However, when the pre- and post-immunisation sera were screened using the FAVN assay, none of the serum samples were capable of neutralising the challenge virus standard (CVS) rabies virus (see Table 6 below) . This confirmed the inability of antibodies raised against the epitope/peptide mimic to neutralise the rabies virus. Table 6. Summary of peptide ELISA and FAVN tests on pre- and post mouse sera raised against S2/4 peptides.

Claims

1. The use of a peptide comprising SEQ ID NO 2, WX^aX^bX^cDI where X^a, X° and X° may be any amino acid, in the preparation of a reagent for the diagnosis of rabies vaccination or disease status of an animal.

2. The use according to claim 1 wherein the reagent is included in a kit further comprising other peptides or proteins which have diagnostic value for rabies antibodies.

3. The use of a peptide comprising SEQ ID NO 2 as defined in claim 1, in the preparation of a prophylactic or therapeutic vaccine against rabies infection.

4. The use according to any one of the preceding claims wherein the peptide is a peptide of SEQ ID No 1 GPWSPIDIHHLS or a variant thereof.

5. The use according to any one of the preceding claims wherein the peptide comprises more than one copy of SEQ ID NO 2 as defined in claim 1.

6. A peptide comprising SEQ ID NO 1, or variants thereof, or fragments of any of these.

7. A peptide according to claim 6 which is of SEQ ID NO 1.

8. A peptide according to claim 6 which comprises more than one copy of SEQ ID NO 1 or variants thereof.

9. A peptide according to claim 8 which comprises a lysine branched structure.

10. A nucleic acid which encodes a peptide according to any one of claims 6 to 9.

11. An expression vector which comprises a nucleic acid according to claim 10.

12. A host cell transformed with an expression vector according to claim 11.

13. A method for detecting the vaccination or disease status of an animal said method comprising contacting a sample taken from the animal with a peptide comprising SEQ ID NO 2 as defined in claim 1, and detecting the presence of a complex formed between said peptide and an antibody present in the sample, and relating this to the vaccination or disease status of an animal.

14. A method according to claim 13 wherein the peptide is a peptide according to any one of claims 6 to 9.

15. A method according to claim 13 or claim 14 wherein the sample is a serum sample.

16. A method according to any one of claims 13 to 15 wherein the sample is taken from a dog or cat.

17. A method according to any one of claims 13 to 16 wherein the peptide or a binding partner therefore is immobilised on a solid support.

18. A method according to claim 13 or claim 14 wherein the peptide is immobilised on a support, and comprising the steps of

(a) incubating a sample suspected of containing anti-rabies antibodies with said support, (b) separating the support from the sample, and

(c) detecting the presence of antibodies on the support.

19. A method according to claim 18 wherein in step (c) , antibodies are detected by application of secondary antibodies or binding fragments thereof, which bind to the target antibody, and are labelled with a detectable label.

20. A method according to claim 19 wherein detectable label is an enzymatic label, which can be developed by application of an enzyme substrate.

21. A method according to claim 13 or claim 14 wherein a primary antibody or a binding fragment thereof, which binds the said peptide is immobilised on a support, and which comprises the steps of (a) incubating a sample with the said peptide,

(b) contacting the product with a support, and

(c) detecting the presence of bound peptide on the support, wherein the absence of bound peptide is indicative of the presence of anti-rabies antibodies within the sample.

22. A method according to claim 21 wherein the peptide is labelled.

23. A peptide according to any one of claims 6 to 9 which carries a label.

24. A peptide according to claim 23 wherein the label is a visible label.

25. A peptide according to claim 24 wherein the visible label is a fluorescent label.

26. A peptide comprising SEQ ID NO 2 as defined in claim 1, for use as or in a prophylactic vaccine.

27. A pharmaceutical composition comprising a peptide comprising SEQ ID NO 2 as defined in claim l,in combination with a pharmaceutically acceptable carrier.

28. A pharmaceutical composition according to claim 27 which further comprises components which produce or enhance an immune response in the animal which is protective against rabies.

29. A pharmaceutical composition according to claim 27 or claim 28 which further comprises an adjuvant.

30. A live vaccine comprising a nucleic acid encoding a peptide of SEQ ID NO 2 as defined in claim 1 in combination with a pharmaceutically acceptable expression vector.

31. A live vaccine according to claim 30 which comprises a viral or bacterial vaccine vector.

32. A method of protecting an animal against rabies, or for treating a rabies infection in an animal, which comprises administering to said animal pharmaceutical composition according to any one of claims 27 to 29 or a live vaccine according to claim 30 or 31, so as to produce a protective or therapeutic immune response in said animal.