Title:  Synthetic peptides and vaccines comprising same

United States Patent:  6,174,528

Assignee:  Counsel of the Queensland Institute of Medical Research (Herston, AU); Commonwealth Scientific and Industrial Research Organisation (Campbell, AU); The University of Melbourne (Victoria, AU); Walter and Eliza Hall Institute of Medical Research of Royal Melbourne (Victoria, AU); Biotech Australia PTY Limited (New South Wales, AU); CSL Limited (Victoria, AU)

Filed:  July 31, 1997

PCT NO:  PCT/AU95/00681

102(e) Date:  July 31, 1997

PCT PUB. Date:  April 25, 1996

Foreign Application Priority Data:  Oct 14, 1994[AU] (PM 8851)

The present invention relates generally to chimeric peptides comprising one or more protective epitopes in a conformation enabling inmunological interactivity and to vaccine compositions comprising same. The present invention is particularly directed to a chimeric peptide capable of inducing protecting antibodies against Group A streptococci.

Description of the Invention

The present invention relates generally to chimeric peptides comprising one or more protective epitopes in a conformation enabling immunological interactivity and to vaccine compositions comprising same. The present invention is particularly directed to a chimeric peptide capable of inducing protective antibodies against Group A streptococci.

Bibliographic details of the publications referred to in this specification by author are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Many proteins which may be useful vaccine candidates against several diseases have a coiled-coil structure, an important structural and biologically abundant motif found in a diverse group of proteins (Cohen and Parry, 1990, 1986). More than 200 proteins have now been predicted to contain coiled coil domains (Lupas et al., 1991). These include surface proteins of certain bacteria such as streptococcal protein A and M proteins; viruses such as influenza hemagglutinin and human immunodeficiency virus (HIV) glycoprotein gp45; and protozoa such as VSG of Trypanosomes. All coiled coil motifs share a characteristic seven amino acid residue repeat (a-b-c-d-e-f-g)_n. The x-ray structure of several coiled-coil domains have been solved and these include the leucine zipper portion of the yeast transcription factor GCN4 dimer (O'Shea et al. 1991), the repeat motif of .alpha.-spectrin (Yan, 1993), together with the GCN4 leucine zipper trimer (Harbury et al., 1994) and tetramer (Harbury et al., 1993) mutants.

In the development of a subunit vaccine based on these proteins, it is generally difficult to map epitopes within the coiled coil structure. Furthermore, protective epitopes may need to be presented in the correct conformation for immunological recognition, such as antibody binding. This is especially important in defining a stable minimal epitope and using it as a vaccine.

Group A streptococci (hereinafter referred to as "GAS") are the causative agent of several human diseases and can lead to acute rheumatic fever which causes serious heart disease. Rheumatic fever may represent an autoimmune illness initiated by cross-interactivity between the streptococcal M protein and cardiac antigens (Beachey et al., 1988). The M protein contains a seven-residue periodicity strongly suggesting that the central rod region of the molecule is in a coiled-coil conformation (Manula and Fischetti, 1980). Overlapping peptides have been made that span this region (see International Patent Application No. PCT/AU93/00131 [WO 93/21220]) and mouse antibodies raised against one synthetic 20mer peptide (designated "p145") from the highly conserved C-terminal region can opsonise and kill multiple isolates of GAS (Pruksakorn et al., 1994a). In addition, p145 can inhibit in vitro killing mediated by human sera. Of concern is that p145 may also stimulate heart cross-reactive T cells (Pruksakorn et al., 1992; 1994b). The B cell epitope within p145 is thought to be conformational because truncated peptides fail to elicit a protective antibody response (Pruksakorn, 1994). There is a need, therefore, to define the minimal region of p145 that is required to induce opsonic antibodies; this could then form the basis of a vaccine. Such a method would enable the identification of the minimal epitopic regions from a range of proteins from pathogens.

One method that has been used to map minimal epitopes from antigens is the PEPSCAN method (Geysen et al., 1987). However, the short peptides used only indicate sequential or continuous epitopes. Other methods to determine conformational epitopes, that is epitopes formed by the tertiary structure of the protein, rely upon mimotope strategies. A mimotope is a mimic of the epitope which induces the antibody. Peptides can be synthesised on polypropylene pins covering the total repertoire of octapeptides which can be made using the 20 common amino acids, i.e. 20⁸ peptides (Geysen et al., 1987). Alternatively, an epitope library consisting of a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface, can be surveyed for antibody recognition (Scott and Smith, 1990).

In accordance with the present invention, overlapping peptides derived from a conformational epitope are embedded within a peptide having a similar native conformation. This approach has the potential to be used in the mapping of a range of conformational epitopes and design of minimal epitopes as vaccine candidates against GAS and a variety of other pathogens.

Accordingly, one aspect of the present invention contemplates a chimeric peptide comprising a first amino acid sequence comprising a conformational epitope inserted within a second amino acid sequence wherein said first and second amino acid sequences are derived from peptides, polypeptides or proteins having similar native conformations.

In accordance with this aspect of the present invention, the second amino acid sequence constitutes a "framework peptide" and provides an appropriate conformation for the chimeric peptide. A framework peptide is selected or otherwise engineered to provide a similar conformation to the first amino acid sequence such as in its naturally occurring form. In its most preferred embodiment, the framework peptide assumes a .alpha.-helical coiled coil conformation and is, therefore, useful in presenting epitopes present in the first amino acid sequence in a similar conformation, i.e. an .alpha.-helical coiled coil conformation.

According to this preferred aspect of the present invention there is provided a chimeric peptide comprising a first amino acid sequence comprising a conformational epitope inserted within a second amino acid sequence wherein said second amino acid sequence folds to an .alpha.-helical coiled coil conformation.

The present invention is particularly exemplified herein by the first amino acid sequence being derived from the streptococcal M protein and in particular comprising a B-cell conformational epitope from within the following amino acid sequence (using the single letter abbreviation for amino acid residues):

L R R D L D A S R E A K K Q V E K A L E (SEQ ID NO:1),

or functional and/or chemical equivalents of one or more of these amino acid residues.

Accordingly, a particularly preferred embodiment of the present invention is directed to a chimeric peptide comprising a first amino acid sequence having at least three amino acids selected from within the following sequence:

L R R D L D A S R E A K K Q V E K A L E (SEQ ID NO:1),

wherein said at least three amino acids constitute a conformational B-cell epitope from streptococcal M protein and wherein said first amino acid sequence is inserted within a second amino acid sequence capable of folding to an .alpha.-helical coiled coil conformation. Preferably, the first amino acid sequence comprises at least five, more preferably at least ten and even more preferably at least fifteen contiguous amino acid residues.

Alternatively, non-contiguous amino acids may be selected such as those on the outside face of the helix and which are required or sufficient for activity.

The construction of a framework peptide is based on the seven amino acid residue repeat:

(a-b-c-d-e-f-g)_n

where a and d positions preferably have large apolar residues, positions b, c and f are generally polar and charged and positions e and g generally favour interchain ionic interactions. A particularly preferred framework peptide is based on the structure of a peptide corresponding to GCN4 leucine zipper (O'Shea et al., 1989; 1991) or its trimer (Harbury et al., 1994) or tetramer (Harbury et al., 1993) and the repeat motif of .alpha.spectrin (Yan, 1993). The GCN4 leucine zipper is particularly preferred and a model heptad repeat derived from the consensus features of the GCN4 leucine zipper peptide comprises the sequence:

V K Q L E D K (SEQ ID NO:2),

which gives a framework peptide of four heptad repeats denoted herein (GCN4)₄. Where required, the framework peptide could be or may need to be longer than the four repeats.

The first amino acid sequence is then embedded within the framework coiled coil peptide to give a chimeric peptide.

The chimeric peptides of the present invention may be produced by recombinant means or may be chemically synthesised by, for example, the stepwise addition of one or more amino acid residues in defined order using solid phase peptide synthetic techniques. Where the peptides may need to be synthesised in combination with other proteins and then subsequently isolated by chemical cleavage or alternatively the peptides or polyvalent peptides may be synthesised in multiple repeat units. The peptides may comprise naturally occurring amino acid residues or may also contain non-naturally occurring amino acid residues such as certain D-isomers or chemically modified naturally occurring residues. These latter residues may be required, for example, to facilitate or provide conformational constraints and/or limitations to the peptides. The selection of a method of producing the subject peptides will depend on factors such as the required type, quantity and purity of the peptides as well as ease of production and convenience.

The chimeric peptides of the present invention may first require their chemical modification for use in vivo since the peptides themselves may not have a sufficiently long serum and/or tissue half-life. Chemical modification of the subject peptides may also be important to improve their antigenicity including the ability for certain regions of the peptides to act as B and/or T cell epitopes. Such chemically modified chimeric peptides are referred to herein as "analogues". The term "analogues" extends to any functional chemical or recombinant equivalent of the chimeric peptides of the present invention characterised, in a most preferred embodiment, by their possession of at least one B cell epitope from the M protein of GAS and wherein an antibody reactive to the B cell epitope is only minimally reactive with human heart tissue. The term "analogue" is also used herein to extend to any amino acid derivative of the peptides as described above.

Analogues of the chimeric peptides contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues.

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 NaBH₄ ; 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 NaBH₄.

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 carbodiimide activation via O-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-chloromercuriphenylsulphonic 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-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide 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.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_.alpha. and N_.alpha. -methylamino acids, introduction of double bonds between C_.alpha. and C_.beta. atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention provides, therefore, a conformational epitope such as from streptococcal M protein in a hybrid molecule such that the epitope is provided in a functional conformational state such that it is capable of being immunologically interactive.

The present invention contemplates, therefore, a method for determining a minimal epitope on an antigenic peptide, polypeptide or protein, said method comprising determining native conformation of said peptide, polypeptide or protein or a portion thereof carrying a putative epitope; preparing peptide fragments of said peptide, polypeptide or protein; inserting or otherwise presenting said peptide fragments in a second peptide derived from or based on another peptide, polypeptide or protein having a similar native conformation to said first mentioned peptide, polypeptide or protein such that the putative epitope on the peptide fragment is presented in a conformation capable of immunological interactivity; and then screening said peptide fragments for immunological interactivity.

In a related aspect of the present invention there is provided a method for mapping regions of amphipathic helices on a peptide, polypeptide or protein which are recognised by antibodies, said method comprising determining native conformation of said peptide, polypeptide or protein or a portion thereof carrying a putative epitope; preparing peptide fragments of said peptide, polypeptide or protein; inserting or otherwise presenting said peptide fragments in a second peptide derived from or based on another peptide, polypeptide or protein having a similar native conformation to said first mentioned peptide, polypeptide or protein such that the putative epitope on the peptide fragment is presented in a conformation capable of immunological interactivity, then screening said peptide fragments for immunological interactivity.

Amphipathic helices which are recognised by antibodies may become valuable vaccine candidates. An amphipathic helix is a more common structural element in proteins and may be surface exposed (antigenic) or play a role in interactions with other proteins. A helical coiled coil is a more complex form of a helix which interacts to form homo-dimers, trimers and tetramers.

By "immunological interactivity" is meant any form of interaction with immune cells or immune effector cells and/or any form of immune response. Generally, immunological interactivity is measured by antibody binding or interactivity with the peptide fragment. However, the immunological interactivity also extends to measuring cellular immune responses.

It is important in therapeutic and diagnostic development to determine the minimal epitope capable of providing immunological interactivity and, for therapy, capable of inducing a protective immune response. Accordingly, the chimeric peptides of the present invention, including methods of their production, are particularly useful in vaccine development. Again, in its exemplified and preferred form, the present invention provides a chimeric peptide for use in a vaccine against GAS. This is done, however, with the understanding that the present invention extends to chimeric peptides useful in inducing a protective immune response against pathogenic microorganisms including bacteria, parasites, yeasts, fungi and protozoa or against viruses such as retroviruses, influenza viruses, hepatitis viruses and immunodeficiency viruses and in particular HIV.

Accordingly, a preferred aspect of the present invention provides a vaccine useful against Group A streptococci said vaccine comprising a chimeric peptide comprising a first amino acid sequence having at least three amino acids selected from within the following sequence:

L R R D L D A S R E A K K Q V E K A L E (SEQ ID NO:1),

wherein said at least three amino acids constitute a conformational B-cell epitope from streptococcal M protein and wherein said first amino acid sequence is inserted within a second amino acid sequence capable of folding to an .alpha.-helical coiled coil conformation, said vaccine further comprising one or more pharmaceutically acceptable carriers and/or diluents. The vaccine may further comprise an adjuvant and/or other immune stimulating molecules. Preferably, the second amino acid sequence forms a framework peptide derived from GCN4. Contiguous or non-contiguous amino acids from SEQ ID NO:1 may be selected as discussed above.

Another aspect of the present invention contemplates a vaccine useful in the development of humoral immunity to M protein but minimally cross reactive with heart tissue said vaccine comprising a chimeric peptide comprising a first amino acid sequence carrying at least one B cell epitope from the M protein wherein an antibody reactive with said B cell epitope is only minimally reactive with heart tissue, said first amino acid sequence inserted into a second amino acid sequence capable of folding into an .alpha.-helical coiled coil formation and said vaccine further comprising one or more pharmaceutically acceptable carriers and/or diluents.

The vaccine may contain a single peptide type or a range of peptides covering different or similar epitopes. In addition, or alternatively, a single polypeptide may be provided with multiple epitopes. The latter type of vaccine is referred to as a polyvalent vaccine. A multiple epitope includes two or more repeating epitopes.

The formation of vaccines is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA.

The present invention, therefore, contemplates a pharmaceutical composition or vaccine composition comprising a humoral immunity developing effective amount of a chimeric peptide (as hereinbefore defined) or its derivatives, analogues or homologues and/or combinations thereof including other active molecules and one or more pharmaceutically acceptable carriers and/or diluents. The active ingredients of a pharmaceutical composition comprising the chimeric peptide are contemplated herein to exhibit excellent therapeutic activity, for example, in the development of antibodies to M protein of streptococci but said antibodies being only minimally reactive with heart tissue when administered in amount which depends on the particular case. For example, from about 0.5 ug to about 20 mg per kilogram of body weight per day may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise a chimeric peptide may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of the chimeric peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer chimeric peptides by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, chimeric peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the chimeric peptides are suitably protected as described above, the active, compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

As used herein "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 .mu.g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 .mu.g to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Still another aspect of the present invention is directed to antibodies to the chimeric peptides. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the M protein or may be specifically raised to the chimeric peptides. In the case of the latter, the peptides may need first to be associated with a carrier molecule. The antibodies and/or chimeric peptides of the present invention are particularly useful for immunotherapy and vaccination and may also be used as a diagnostic tool for infection or for monitoring the progress of a vaccination or therapeutic regima.

For example, the chimeric peptides can be used to screen for naturally occurring antibodies to M protein. Alternatively, specific antibodies can be used to screen for M protein. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.

In accordance with this aspect of the present invention, the chimeric peptides are particularly useful in screening for antibodies to M protein and, hence, provide a diagnostic protocol for detecting streptococcal infection. Alternatively, biological samples, such as blood serum, sputum, tissue and tissue extracts can be directly screened for M protein using antibodies raised to the chimeric peptides.

Accordingly, there is provided a method for the diagnosis of streptococcal infection in a subject comprising contacting a biological sample from said subject with an antibody binding effective amount of a chimeric peptide for a time and under conditions sufficient for an antibody-chimeric peptide complex to form, and then detecting said complex.

The presence of M protein antibodies in a patient's blood serum, tissue, tissue extract or other bodily fluid, can be detected using a wide range of immunoassay techniques such as those described in U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. This includes both single-site and two-site, or "sandwich", assays of the non-competitive types, as well as in the traditional competitive binding assays. Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, a chimeric peptide is immobilised onto a solid substrate to form a first complex and the sample to be tested for M protein antibody brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an chimeric-peptide-antibody secondary complex. An anti-immunoglobulin antibody, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a tertiary complex of chimeric peptide-antibody-labelled antibody. Any unreacted material is washed away, and the presence of the first antibody is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations of the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. A similar approach is adopted to detect M protein. The antibodies used above may be monoclonal or polyclonal.

The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecule in this type of assay re either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, .beta.-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product.

Alternatively, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose. It will also be apparent that the foregoing can be used to label chimeric peptides and to use same directly in the detection of M protein antibodies.

Yet a further aspect of the present invention contemplates the use of the chimeric peptides as herein described in the manufacture of a medicament for use as a vaccine against GAS.

In a related embodiment, the present invention provides an agent comprising a chimeric peptide as herein described useful as a vaccine against GAS.

Claim 1 of 11 Claims

What is claimed is:

1. A chimeric peptide comprising:

(i) a first amino acid sequence which, in its native state, presents a conformational epitope, said conformational epitope not being present in the first amino acid sequence in an isolated state; and

(ii) a second amino acid sequence which has a conformation similar to the native conformation of the first amino acid sequence;

wherein the first amino acid sequence is inserted within the second amino acid sequence such that the first amino acid sequence presents the conformational epitope.

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