[go: up one dir, main page]

WO1993011157A1 - VACCIN ET PEPTIDES CONTRE LA MALARIA COMPRENANT UN EPITOPE DE CELLULE T HUMAINE DE PROTEINE CIRCUMSPOROZOITE DE $i(P.VIVAX) - Google Patents

VACCIN ET PEPTIDES CONTRE LA MALARIA COMPRENANT UN EPITOPE DE CELLULE T HUMAINE DE PROTEINE CIRCUMSPOROZOITE DE $i(P.VIVAX) Download PDF

Info

Publication number
WO1993011157A1
WO1993011157A1 PCT/AU1992/000639 AU9200639W WO9311157A1 WO 1993011157 A1 WO1993011157 A1 WO 1993011157A1 AU 9200639 W AU9200639 W AU 9200639W WO 9311157 A1 WO9311157 A1 WO 9311157A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
peptide
acid sequence
protein
region encompassing
Prior art date
Application number
PCT/AU1992/000639
Other languages
English (en)
Inventor
Michael Good
Janine Bilsborough
Original Assignee
The Council Of The Queensland Institute Of Medical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Council Of The Queensland Institute Of Medical Research filed Critical The Council Of The Queensland Institute Of Medical Research
Publication of WO1993011157A1 publication Critical patent/WO1993011157A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • MALARIAL VACCINE AND PEPTIDES COMPRISING HUMAN T-CELL EPITOPE OF CIRCUMSPOROZOITE PROTEIN OF P . VIVAX
  • the present invention relates generally to a malarial vaccine and peptides useful for same. More particularly, the present invention is directed to a peptide comprising a human T-cell epitope of the circumsporozoite protein of Plasmodium vivax.
  • Plasmodium vivax the causative agent of benign tertian malaria, although producing much less mortality than Plasmodium falciparum. characteristically causes relapsing fevers generating an incapacitating disease.
  • the bite of an infected mosquito injects malaria sporozoites and initiates the parasite life cycle within the host that culminates in clinical disease.
  • Sporozoites of P. vivax differentiate, after invading die liver, either into early, primary schizonts or into hypnozoites, the latter being responsible for late relapses of the infection.
  • An ideal malaria vaccine for sporozoite stage infection should contain both B and T-cell epitopes to stimulate the production of neutralising antibodies, specific memory helper cells and effector T-cells which could operate via production of IFN- ⁇ , IL-6 or other lymphokines, or by direct killing of infected hepatocytes.
  • the major surface protein of sporozoites, the circumsporozoite (CS) protein, is one such candidate for vaccine development (Good, 1990).
  • one aspect of the present invention provides a peptide comprising a human T-cell epitope of the circumsporozoite (CS) protein of P. vivax or a part, fragment, derivative, analogue and/ or homologue thereof.
  • CS circumsporozoite
  • the present invention is particularly directed to P. vivax strains Belem and NK (Arnot et el, 1982). However, the present invention extends to other strains of P. vivax comprising a CS protein of analogous amino acid sequence and/or having analogous T-cell epitopes.
  • the full length amino acid sequence of strains of P. vivax can be found in Qari (1992).
  • peptide is used in a general sense to cover molecules ranging from a few amino acid residues to several hundred amino acid residues.
  • peptide is considered herein to cover a polypeptide.
  • the peptides of the present invention are henceforth referred to as "CS peptides" for circumsporozoite peptides and are those which carry a human T cell epitope or a portion thereof from CS protein.
  • the CS peptides of the present invention may be recombinant, synthetic or fragments parts, derivatives, analogues or homologues of the naturally occurring CS protein and, hence, reference to "CS peptides" in the specification and claims includes all such molecules.
  • a peptide comprising a T-cell epitope of a CS protein is defined as a peptide which is capable of inducing T cell proliferation.
  • the ordinary skilled artisan can determine the ability of the peptides of the present invention to induce T cell proliferation by methods known in the art. For exmaple, T cell proliferation can be assessed by comparing tritiated thymidine incorporation into newly synthesised DNA of control cells and peptide treated cells. Other parameters which are associated with T cell proliferative responses, such as production of interferon-gamma, can also be measured to determine the ability of the peptides of the present invention to induce T cell proliferation.
  • the T-cell epitopes or parts thereof are said herein to be “carried” on the CS peptides of the present invention.
  • the presence of the T-cell epitope of the peptide is readily determined as herein described.
  • the CS peptides of the present invention may comprise an amino acid sequence exactly corresponding to a sequence in a region of the naturally occurring CS protein or may contain single or multiple amino acid substitutions, deletions and/or additions to the naturally occurring sequence.
  • the CS peptides of the present invention are prepared by recombinant means and techniques for making mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis.
  • Other suitable techniques as described in Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual- Cold Spring Harbor Laboratories, Cold Spring Harbor, NY and include random mutagenesis.
  • nucleic acid isolate comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a CS peptide or precursor form thereof.
  • the sequence of the nucleic acid may correspond substantially to the naturally occurring DNA sequence of P. vivax or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto.
  • the nucleic acid may encode a single CS peptide or a repeated series of the same CS peptide for subsequent cleavage for into individual peptides.
  • the nucleic acid may also encode a series of different CS peptides.
  • precursor is meant any polypeptide or peptide from which the CS peptides can be derived and include fusion proteins.
  • the nucleic acid molecule is in a vector such as an expression vector capable of expression in a prokaryotic and/or eukaryotic host.
  • the nucleic acid isolate is in an attenuated viral vector such as an attenuated recombinant Salmonella viral vector. Such a vector is useful for delivery of a CS peptide in vivo.
  • the CS peptides are a biologically pure preparation meaning that they have undergone some purification away for other proteins and/or non-proteinacous material.
  • the purity of the preparation may be represented as at least 40% of the peptide, preferably at least 60%, more preferably at least 75%, even more preferably at least 85% and still more preferably at least 95% relative to non-CS peptide material as determined by weight, activity, amino acid homology or similarity, antibody reactivity or other convenient means.
  • a preparation of a CS peptide of the present invention which has undergone at least some purification is referred to herein as an "isolated peptide" or a "peptide isolate".
  • the CS peptides of the present invention are non-naturally occurring since they are non-full length molecules of the CS protein and/or have undergone some purification and/ or are recombinant or synthetic.
  • Amino acid insertional derivatives of the CS peptides of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in - 5 - its place. Typical substitutions are those made in accordance with the following Table 1:
  • CS peptide is derivatised by amino acid substitution
  • the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like.
  • Amino acid substitutions are typically of single residues.
  • Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues.
  • deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
  • the CS peptides contemplated herein may also be chemically synthesized such as by solid phase peptide synthesis or may be prepared by subjecting the CS protein to hydrolysis or other chemically disruptive processes to produce fragments of the molecule.
  • the peptides are made by in.xiiE ⁇ . or in. ⁇ Y ⁇ . recombinant DNA synthesis.
  • the peptides may need to be synthesised in combination with other proteins and then subsequently isolated by chemical cleavage or the peptides or polyvalent peptides may be synthesised in multiple repeat units.
  • multiple antigen peptides or polyvalent peptides could also be prepared according to Tarn (1988). 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.
  • analogues extends to any functional chemical equivalent of the CS peptides of the present invention characterised by its possession of one or more human T-cell epitopes to the CS protein of P. vivax.
  • analogue is also used herein to extend to any amino acid derivative of the peptides as described above.
  • Analogues of CS 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.
  • 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 N-1BH4; 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 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. - 8 -
  • 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 e 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.
  • peptides could be conformationally constrained by, for example, incorporation of C a and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p 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 therefore, extends to isolated, recombinant and /or synthetic peptides or polypeptides and amino acid and/or chemical analogues, homologues, derivatives, fragments and parts thereof corresponding to one or more regions of the CS protein acting as a human T-cell epitope. All such molecules are encompassed by the term "CS peptide".
  • the preferred CS peptides of the present invention cover amino acid residues 1 to 373 and preferably 90 to 370 of the CS protein of P. vivax strains Belem and NK (Arnot et al, 1988). Even more preferably, the peptides encompass amino acid residues 230 to 368 such as 232 to 251, 289 to 308, 339 to 358 and 349 to 368, including any derivatives or analogues as contemplated above as well as multiple repeats and/or combinations of these sequences. In a most preferred embodiment of the present invention, the peptides are defined by the following amino acid sequences which correspond to peptides 14, 16, 19, 24 and 25, respectively in Table 2:
  • the present invention also extends to peptides and polypeptides having at least 40%, preferably at least 55%, more preferably at least 65%, still more preferably at least 75% and even more preferably 80-85% or greater than 90- 95% similarity or homology to the naturally occurring "equivalent" sequence.
  • the present invention further contemplates a method for vaccinating a human - 10 - subject against P. vivax infection comprising administering to said subject a T- cell immunity developing effective amount of a peptide comprising a human T- cell epitope of CS protein of P. vivax or a part, fragment, derivative, homologue or analogue thereof for a time and under conditions sufficient for said immunity to develop or at least partially develop.
  • a peptide has the same meaning as set forth above.
  • this aspect of the present invention can be accomplished by a vaccine composition.
  • another aspect of the present invention contemplates a vaccine useful in the development of immunity to P. vivax and in particular for stimulating T-cell immunity to P. vivax CS protein, said vaccine comprising a peptide carrying a human T-cell epitope of CS protein of P. vivax or a part, fragment, derivative, homologue or analogue thereof.
  • the vaccine may also comprise a suitable adjuvant as discussed below.
  • Such a vaccine is particularly directed to the sporozoites phase of P. vivax infection.
  • the vaccine may be referred to as a "recombinant vaccine”.
  • the vaccine may be referred to as a "synthetic vaccine”.
  • the vaccine may contain a single peptide type or a range of peptides covering different or similar T-cell epitopes.
  • a single polypeptide may be provided with multiple T-cell epitopes.
  • the latter type of vaccine is referred to as a "polyvalent vaccine”.
  • the present invention contemplates a pharmaceutical composition or vaccine comprising a immunity developing effective amount of CS peptide or its parts, fragments, derivatives, homologues or analogues and/or combinations thereof including other active molecules and one or more - 11 - pharmaceutically acceptable carriers and/or diluents.
  • the vaccine may alternatively comprise an attenuated live vector encoding the CS peptide(s).
  • an attenuated recombinant Salmonella vector may be used.
  • the active ingredients of a pharmaceutical composition comprising the CS peptide or a vector encoding same are contemplated to exhibit excellent therapeutic activity, for example, in the development of T-cell immunity to ⁇ _____.
  • vivax when administered in amount which depends on the particular case. For example, from about 0.5 ug to about 20 mg per patient or per kilogram of body weight of the patient per day, week, or month may be administered.
  • Dosage periods 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. Depending on the patient or other conditions more preferred dosages comprise lO ⁇ g to lOmg, 20 ⁇ g to 5mg or lOO ⁇ g to lmg per patient or per kilogram of body weight of the patient per administration.
  • the active compound including attenuated live vector 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).
  • the active ingredients which comprise a CS 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.
  • a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
  • these may potentially be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis.
  • CS peptides may be coated by, or administered with, a material to prevent its inactivation.
  • CS 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 (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyediylene 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 an effective amount of CS peptide as hereinbefore described.
  • the active component is an attenuated live vector, a sufficient amount of CS peptide must be synthesised.
  • 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 flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • 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
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint
  • 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 flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • 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.
  • Dosage unit form 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 ⁇ g to about 2000 mg includes l.O ⁇ g to 200mg, lO ⁇ g to 20mg and lOO ⁇ g to lOmg.
  • 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 CS peptides.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the CS protein or may be specifically raised to the CS peptides. In the case of the latter, the peptides may need first to be associated with a carrier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments.
  • the present invention extends to recombinant and syndietic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.
  • the antibodies and/or CS peptides of the present invention are particularly useful for immunotherapy and vaccination and may also be used as a diagnostic tool for malaria infection or for monitoring the progress of vaccination or therapeutic mita.
  • the CS peptides can be used to screen for naturally occurring antibodies to CS protein.
  • specific antibodies can be used to screen for CS protein or CS peptides. The latter would be important, for example, as a means for screening or purifying CS peptides made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of CS protein but particularly those regions covered by a CS peptide.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of CS peptide, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981; Kohler and Milstein, Nature.256; 495-499, 1975; European Journal of Immunology & 511-519, 1976).
  • CS peptide or more commonly the presence of CS protein (indicative of infection) may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen- labelled antibody.
  • the sample is one which might contain antibodies and include serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • a first antibody having specificity for the CS peptide or CS protein, or antigenic parts thereof, is either covalentiy or passively bound to a solid surface.
  • the solid surface 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 of 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 covalentiy binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 °C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody- second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • 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 color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorecein and rhodamine
  • fluorecein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • kits for example, to conduct a lymphocyte proliferation assay.
  • the kit comprises a compartment adapted to contain one or more CS peptides and may further comprise in the same or different compartments the reagents for the lymphocyte proliferation assay.
  • Figure 1 is a schematic representation showing the structure of the recombinant vivax proteins used in this study.
  • Figure 2 is a graphical representation showing the percentage of exposed (top panel, 52 subjects) and non-exposed (lower panel, 24 subjects) individuals responding to each of the synthetic peptides and recombinant proteins by T- cell proliferation.
  • Response is defined as a stimulation inex of at least 3.
  • the single hatch represents the percent who responded maximally at a concentration of 30 ⁇ g/ml for the peptide of 15 ⁇ g/ml for the recombinant proteins (VI -V3), whereas the open box represents the percentage who responded maximally at the lower concentrations of 3 ⁇ g/ml for peptide and 5 ⁇ g/ml for recombinant proteins.
  • Figure 3 is a graphical representation showing the percentage of exposed (upper panel, 25 subjects) and non-exposed (lower panel, 9 subjects) responding to the synthetic peptides and recombinant proteins by lymphoproliferation (bars) or ⁇ -IFN production (diamonds).
  • Peptides were tested at 30 ⁇ g/ml and recombinant proteins at 15 ⁇ g/ml. Supematants were taken from the wells at day 4 and replaced with fresh medium. The wells were then pulsed with 3 H-methyl thymidine at day 6.
  • peptides 3, 6, 7, 12, 15 and 20 were tested with only 3, 20, 10, 14, 11 and 5 subjects respectively, and in the lower panel peptides 3, 6 and 20 were tested with only 3, 8 and 5 subjects respectively.
  • Figure 4 is a graphical representation of malaria-exposed individuals ranked in order of their time since most recent exposure to vivax sporozoites (defined as time since leaving an endemic area).
  • the number of vivax sporozoite-specific T cell epitopes recognised by both proliferating (•) and interferon- ⁇ -secreting cells ( ⁇ ) was enumerated.
  • the average number of epitopes recognised by a rolling average of 11 individuals (1-> 11, 2- > 12, 3- > 13 etc) was then calculated - 22 - and plotted.
  • 52 individuals were tested, only 48 were included in this analysis since the other 4 were not certain of the time of their most recent exposure to vivax sporozoites. Forty-eight individuals were included for the analysis of proliferating responses and 24 for the analysis of interferon- ⁇ - secreting responses.
  • peripheral blood mononuclear cells PBM
  • peripheral blood mononuclear cells EMEM
  • EMEM Eagles' Minimal Essential Medium
  • Twelve negative control wells containing no antigen were included for each person tested.
  • the cultures were incubated for 7 days in a 5% CO 2 humidified atmosphere at 37 °C.
  • IFN- ⁇ concentrations were maximal at this time. Quantitative analysis of IFN- ⁇ was performed using a solid phase sandwich enzyme immunoassay (Human ⁇ -Interferon Test Kit, Commonwealth Serum Laboratories, Melbourne, Australia). However, for this report, supematants are classified as either +ve or -ve for IFN- ⁇ production. A +ve result is defined as one where the absorbance of the well with antigen exceeded the absorbance of control wells (no antigen) by 0.1.
  • SI stimulation index
  • Lymphocyte subsets were purified using the magnetic cell sorting (MACS) technique as previously described (Miltenyi et al, 1990).
  • PBM magnetic cell sorting
  • PBS/BSA PBS supplemented with 1% bovine serum albumin and 5mM EDTA
  • MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD4 or CD8 antibodies were then washed and resuspended in 500 ⁇ L PBS/BSA and applied to the top of the separation columns (Miltenyi Biotec).
  • the non-magnetic fraction was collected and subjected to E-rosetting (Van Oers et al, 1977) to deplete remaining T cells. This non-T population was irradiated (25 Gy) and 10 4 cells were added to each well as APCs. The purified subsets (magnetic fraction) were washed and added to wells at 2x10 s cells per well (quadruplicate) in 0.2 ml of EMEM with 10% NHS. The cells were then cultured, pulsed, and results determined as described above.
  • FACScan FACScan.
  • the subsets were stained directly with biotinylated anti-human CD4 or CD8 monoclonal antibodies (Becton-Dickinson). Erythrocytes, dead cells and debris were excluded by gating on forward and side scatter. Purity of subsets was >95%.
  • IFN- ⁇ interferon- ⁇
  • supematants were collected at day four during the course of the lymphocyte proliferation assays, as preliminary experiments indicated IFN- ⁇ concentration were maximal at this time.
  • Quantitative analysis of IFN- ⁇ was performed using a solid phase sandwich enzyme immunoassay (Human Interferon- ⁇ Test Kit, CSL Limited, Melbourne, Australia).
  • IL-4 was measured in the supe atants of cultures taken at 24 hr using an ELISA kit from Genzyme (USA). Preliminary experiments showed tiiat in these primary stimulations levels were maximal at this time.
  • Fig. 2 Initial comparison of both the exposed and non-exposed populations' responses to the synthetic peptides tested indicated two types of responses: (i) peptide- specific responses which were evident in both groups; and (ii) those which were unique to the malaria-exposed individuals (Fig. 2).
  • 14 different peptides stimulated significant levels of T cell proliferation among the malaria-exposed group but not from the non-exposed group, with peptides 25, 19, 24, 16, and 14 each stimulating T cells from greater than 30% of the exposed population (Fig. 2).
  • Fig. 2 also illustrates the lymphocyte pro ⁇ ferative responses of both groups to the recombinant CS proteins, Vl-3. There was no significant difference in the responses of both populations to these recombinant proteins.
  • Immunodominant T cell epitopes were initially identified by measuring proliferation of PBM to a series of 26 HPLC-purified synthetic peptides (20-mers) which overlapped and spanned the Belem and NK strains of vivax CS protein (Table I). Peptides were tested at two concentrations, 3 ⁇ g/ml and 30 ⁇ g/ml, with the latter concentration being optimal in most cases.
  • Supe atants (day 4) were taken from all the peptide-specific cultures of a random 25 vivax-exposed and 9 control individuals and tested for the IFN- ⁇ . A similar pattern of response was observed as for the proliferative responses, and the percentage of individuals whose T cells secreted IFN- ⁇ is plotted in Figure 5 with the proliferative T cell data for that subset of indiviuals. Clearly, T cells from more exposed individuals secreted IFN- ⁇ , the range of the responses were 0.2-6.12 IU/ml for the exposed individuals, and 0.4-1.5 IU/ml for the non-exposed individuals. In most cases, the percentages of responders were similar whether measuring proliferation or IFN- ⁇ production.
  • Rolling averages are used rather than simply the number recognised by each individual since there is considerable individual to individual variation in the number of epitopes recognised per individual (perhaps as a result of MHC or other differences). Neither the age of the individuals nor the duration of an individuals exposure was correlated witii responsiveness to the defined epitopes.
  • a vaccine is prepared as hereinbefore described by admixing one or more CS peptides and one or more pharmaceutically acceptable carriers and/or diluents.
  • the vaccines may optionally further comprise an adjuvant.
  • the CS peptide selected as vaccine candidates are those encompassing residues 1 to 373, 90 to 370, 230 to 368, 232 to 251, 289 to 308, 339 to 358 and 349 to 368 of CS protein.
  • Individuals with no prior history of exposure to P. vivax are then selected and given the vaccine in convenient form and with a range of doses. For example, individuals may be given from about 0.5 ⁇ g to about 20 mg/kg of body weight. Administration of the vaccine occurs at least once for each individual but preferably the individual receives a "booster" dose approximately 3 weeks to 6 weeks after the first administration.
  • the antibody titres of the vaccinated individuals to the specific peptides and die T cell responses are monitored over time. Immune cells from vaccinated individuals can then be used in die human lymphocyte proliferation response assay hereinbefore described.
  • Vaccinated humans and animals are then subjected to P. vivax infection via infected mosquitos to show the protective effect of the subject CS peptides.
  • the peptides chosen for each subject were those to which these individuals responded to 3SI or above.
  • the purity of all T cell subsets was >95% as assessed by FACS analysis. - 33 -

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne globalement un vaccin contre la malaria et des peptides utiles pour ce dernier. Plus spécifiquement, il s'agit d'un peptide comprenant un épitope de cellule T humaine de la protéine circumsporozoïte de Plasmodium vivax ou bien des parties, des fragments, des dérivés, des homologues ou des analogues de ce dernier. Cette invention concerne également des anticorps contre ce type de peptide et leur utilisation dans une détermination de diagnostic servant à rechercher l'infection par P.vivax.
PCT/AU1992/000639 1991-11-27 1992-11-27 VACCIN ET PEPTIDES CONTRE LA MALARIA COMPRENANT UN EPITOPE DE CELLULE T HUMAINE DE PROTEINE CIRCUMSPOROZOITE DE $i(P.VIVAX) WO1993011157A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPK9720 1991-11-27
AUPK972091 1991-11-27

Publications (1)

Publication Number Publication Date
WO1993011157A1 true WO1993011157A1 (fr) 1993-06-10

Family

ID=3775845

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1992/000639 WO1993011157A1 (fr) 1991-11-27 1992-11-27 VACCIN ET PEPTIDES CONTRE LA MALARIA COMPRENANT UN EPITOPE DE CELLULE T HUMAINE DE PROTEINE CIRCUMSPOROZOITE DE $i(P.VIVAX)

Country Status (1)

Country Link
WO (1) WO1993011157A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010787A3 (fr) * 1996-09-11 1998-07-30 Patrick T Prendergast Therapie a but immunitaire
EP1487852A4 (fr) * 2002-03-01 2006-02-22 Queensland Inst Med Res Vaccin anti-protozoaires
EP1846023A4 (fr) * 2005-01-18 2009-09-30 Us Army Proteine circumsporozoite hybride de plasmodium vivax et son vaccin

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2570984A (en) * 1983-01-28 1984-08-15 New York University Protective peptide antigen
AU6129286A (en) * 1985-07-12 1987-02-10 New York University Immunogenic peptide antigen corresponding to plasmodium vivax circumsporozoite protein
AU1791488A (en) * 1987-03-30 1988-11-02 Chiron Corporation Immunogenic recombinant yeast expression product and method for purifying it
AU1593688A (en) * 1987-03-30 1988-11-02 New York University Immunogenic polypeptide and method for purifying it
AU3065489A (en) * 1988-02-01 1989-08-25 Wyeth Holdings Corporation T-cell epitope as carriers molecule for conjugate vaccines
EP0354611A1 (fr) * 1988-08-05 1990-02-14 ENIRICERCHE S.p.A. Antigènes synthétiques pour le diagnostic de malaria induite par plasmodium vivax

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2570984A (en) * 1983-01-28 1984-08-15 New York University Protective peptide antigen
AU6129286A (en) * 1985-07-12 1987-02-10 New York University Immunogenic peptide antigen corresponding to plasmodium vivax circumsporozoite protein
AU1791488A (en) * 1987-03-30 1988-11-02 Chiron Corporation Immunogenic recombinant yeast expression product and method for purifying it
AU1593688A (en) * 1987-03-30 1988-11-02 New York University Immunogenic polypeptide and method for purifying it
AU3065489A (en) * 1988-02-01 1989-08-25 Wyeth Holdings Corporation T-cell epitope as carriers molecule for conjugate vaccines
EP0354611A1 (fr) * 1988-08-05 1990-02-14 ENIRICERCHE S.p.A. Antigènes synthétiques pour le diagnostic de malaria induite par plasmodium vivax

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
E. NARDIN et al., J. IMMUNOLOGY, Volume 146, Number 5, issued March 1991, pages 1674-1678, "T Cell Epitopes of the Circumporozoite Protein of Plasmodium Vivax". *
E.H. NARDIN, IMMUNOLOGY LETTERS, Volume 25, 1990, pages 43-48, "T Cell Responses in a Sporozoite-Immunized Human Volunteer and a Chimpanzee". *
F. ZAVALA et al., J. EXP. MED., Volume 157, issued June 1983, pages 1947-1957, "Circumsporozoite Proteins of Malaria Parasites Contain a Single Immunodominant Region with Two or More Identical Epitopes". *
J. FERN et al., J. IMMUNOLOGY, Volume 148, Number 3, issued February 1992, pages 907-913, "Promiscuous Malaria Peptide Epitope Stimulates CD 45 Ra T Cells from Peripheral Blood of Nonexposed Donors". *
P.J. BARR et al., J. EXP. MED., Volume 165, issued April 1987, pages 1160-1171, "Expression in Yeast of a Plasmodium Vivax Antigen of Potential Use in a Human Malaria Vaccine". *
S. HERRERA et al., J. IMMUNOLOGY, Volume 148, Number 12, issued June 1992, pages 3986-3990, "Human Recognition of T Cell Epitopes on the Plasmodium Vivax Circumsporozoite Protein". *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010787A3 (fr) * 1996-09-11 1998-07-30 Patrick T Prendergast Therapie a but immunitaire
EP1487852A4 (fr) * 2002-03-01 2006-02-22 Queensland Inst Med Res Vaccin anti-protozoaires
EP1846023A4 (fr) * 2005-01-18 2009-09-30 Us Army Proteine circumsporozoite hybride de plasmodium vivax et son vaccin
US7790186B2 (en) 2005-01-18 2010-09-07 The United States Of America As Represented By The Secretary Of The Army Plasmodium vivax hybrid circumsporozoite protein and vaccine
US8258280B2 (en) 2005-01-18 2012-09-04 The United States Of America As Represented By The Secretary Of The Army Plasmodium vivax hybrid circumsporozoite protein and vaccine
US8697856B2 (en) 2005-01-18 2014-04-15 The United States Of America As Represented By The Secretary Of The Army Plasmodium vivax hybrid circumsporozoite protein and vaccine

Similar Documents

Publication Publication Date Title
KR100358271B1 (ko) 보호성면역반응및il-12생산의자극과증강을위한화합물및방법
US6288035B1 (en) Method of treating cancer with a 43 kD human cancer antigen
EP0787139B1 (fr) Peptides de synthese et vaccins les contenant
JP2004536885A (ja) 抗原性ポリペプチド
Smulian et al. Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization
JP2005535619A (ja) 免疫寛容を誘導するための物質および方法
US7455984B2 (en) Immunoreactive and immunotherapeutic molecules
US7820395B2 (en) Orientia tsutsugamushi truncated recombinant outer membrane protein (R47) and (R56) vaccines diagnostics and therapeutics for scrub typhus
US5958416A (en) Heat shock protein peptides and methods for modulating autoimmune central nervous system disease
US20070141076A1 (en) Antigen fragments for the diagnosis of Toxoplasma gondii
WO1990002206A1 (fr) Detection de l'hepatite non a, non b
WO1993021220A1 (fr) Peptides synthetiques utilises dans un vaccin contre l'infection par les streptocoques et pour le diagnostic de cette infection
JPH04500066A (ja) ポリペプチドの百日咳トキシンのワクチン
US5698204A (en) Recombinant allergenic proteins from ragweed pollen
Chen et al. Baculovirus expression of the major surface antigen of Toxoplasma gondii and the immune response of mice injected with the recombinant P30
WO1993011157A1 (fr) VACCIN ET PEPTIDES CONTRE LA MALARIA COMPRENANT UN EPITOPE DE CELLULE T HUMAINE DE PROTEINE CIRCUMSPOROZOITE DE $i(P.VIVAX)
US5578453A (en) Cloning and expression of toxoplasma antigens and use of recombinant antigens
JPH10511541A (ja) Ｔ細胞応答を調節するための方法および組成物
CA2313609C (fr) Antigene de trypanosoma cruzi, gene codant pour celui-ci, et methodes de detection et de traitement de la maladie de chagas
WO2002010340A1 (fr) Isolat de $i(neospora caninum)
KR0184715B1 (ko) 자궁암 면역치료제 조성물
AU2001277390B2 (en) Neospora caninum isolate.
CA2297955A1 (fr) Facteur de stimulation lymphocytaire tire de trypanosome
WO1990015621A1 (fr) Vaccin contre la malaria
CA2566828A1 (fr) Molecules immunoreactives et immunotherapeutiques interagissant chez des sujets atteints du diabete sucre insulinodependant (iddm)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase