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WO1995026362A1 - Peptides a usage diagnostique et therapeutique - Google Patents

Peptides a usage diagnostique et therapeutique Download PDF

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Publication number
WO1995026362A1
WO1995026362A1 PCT/AU1995/000176 AU9500176W WO9526362A1 WO 1995026362 A1 WO1995026362 A1 WO 1995026362A1 AU 9500176 W AU9500176 W AU 9500176W WO 9526362 A1 WO9526362 A1 WO 9526362A1
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Prior art keywords
sequence
protein
polypeptide
peptide
amino acid
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PCT/AU1995/000176
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English (en)
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John Rodney Warmington
Kathleen Mary Franklyn
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Curtin University Of Technology
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Priority to EP95913823A priority Critical patent/EP0759034A4/fr
Priority to AU21063/95A priority patent/AU684009B2/en
Publication of WO1995026362A1 publication Critical patent/WO1995026362A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • THIS INVENTION relates to peptides, polypeptides, or proteins or portions thereof, the amino acid sequences of which correspond to antigenic segments of an immunologically important protein of Candida albicans. These peptides, polypeptides or proteins are useful as diagnostic reagents for detecting the presence of antibodies reactive with Candida albicans and may also be useful as therapeutic agents as well as immunogens in compositions and methods to illicit antibodies against Candida albicans.
  • Yeasts of the genus Candida are commensals or saprophytes of warm blooded animals.
  • the Candida species are part of the normal endogenous microbial flora and are frequently present on the normal mucous membranes of the gastro- intestinal tract, upper respiratory tract, and genital tracts of humans.
  • the yeast Candida albicans is an opportunistic pathogen which can exploit any weakness or disability in the body's immune defences to cause disease. It has few equals in terms of the variety and severity of infections for which it is responsible.
  • Candida albicans The most common infection caused by Candida albicans is thrush, a benign rash of the skin and mucous areas especially the throat and vagina.
  • C. albicans infections are becoming increasingly common in association with HIV (Human Immunodeficiency Virus) infections.
  • C. albicans infection The most severe form of C. albicans infection is systemic candidiasis.
  • Systemic candidiasis has a mortality rate of over 70% amongst immunocompromised and debilitated patients (Gold, 1984).
  • C. albicans overwhelms the already weakened body's defences and invades the tissues and organs.
  • a severe infection results, which is frequently fatal.
  • Candida species have assumed increasing prominence as major pathogens.
  • the increase in Candida infections can be largely attributed to advances in modern medicine.
  • the improved survival of patients with underlying neoplasms, collagen vascular diseases, extensive trauma and other immunocompromising illness has increased the reservoir of patients at risk of Candida infections.
  • Candida species predominantly C. albicans, are responsible for approximately 10% of all nosocomial infections (Edwards, 1991).
  • vaginal candidiasis It is estimated that 75% of women will experience at least one clinically significant episode of vaginal candidiasis during their lifetime (Sobel, 1993). Based on the number of prescriptions written to treat C. albicans infections in the US from 1980-1990, the incidence of vaginal candidiasis has almost doubled (Kent, 1991). In 1990 over 13 000 000 US females received treatment for vaginal candidiasis.
  • Candidiasis is a world wide problem, with the most dramatic increase in incidence in "Westernised” countries. It is difficult to accurately estimate the number of individuals suffering from, or at risk from, candidiasis. However, based on the reported incidence of C. albicans infections, the estimated number of infected individuals is likely to be several hundred million worldwide.
  • the present invention seeks to provide proteins, polypeptides, peptides or fragments corresponding to immunologically reactive portions of the protein enolase and processes for production thereof which may be useful in the diagnosis of Candida albicans infection and for use as a therapeutic agent and/or a vaccine in the treatment of the infection. It is envisaged that such proteins, polypeptides, peptides or fragments thereof may be used in diagnostic assays or as therapeutic agents and/or vaccines which have advantages over the diagnostic assays, vaccines and therapeutic agents presently available .
  • the invention provides a protein, polypeptide or peptide which is immunologically reactive with Candida albicans induced antibodies.
  • the protein, polypeptide or peptide is a recombinant protein, polypeptide or peptide.
  • the protein of the invention may be of various forms provided it is immunologically reactive with Candida albicans induced antibodies.
  • the protein is the protein enolase and more preferably the protein is the protein encoded by the amino acid sequence presented as Sequence ID No. 1.
  • polypeptide of the invention may also be of various forms provided it is immunologically reactive with Candida albicans induced antibodies.
  • the polypeptide is provided by a portion of the protein enolase.
  • the polypeptide may comprise a portion of the amino acid sequence presented as Sequence ID No 1.
  • the polypeptide comprises the polypeptide encoded by amino acid numbers 93 to 249 of the amino acid sequence presented as Sequence ID No 1.
  • the peptide of the invention may be of various forms provided it is immunologically reactive with Candida albicans antibodies.
  • the peptide comprises any one or more of the peptides encoded by any one or more of the amino acid sequences presented as Sequence ID No's 3 to 18.
  • the present invention also provides a protein, polypeptide, or peptide of the general formulae X-Y-Z, wherein X and Z each represent independently of each other a hydrogen atom or a residue of an amino acid, of a protected amino acid, or a peptide, or of a polypeptide and Y represents one of the amino acid sequences presented as Sequence ID No 1 or a portion thereof or one of the amino acid sequences presented as Sequence ID No's 3 to 18 or portions thereof. Such sequences may be glycosylated or non glycosylated.
  • at least one of X and Z comprises at least one of the sequences presented as Sequence ID No's 3 to 18 which may be the same as or different from Y.
  • the present invention also encompasses functionally equivalent proteins, polypeptides and peptides of the above proteins, polypeptides and peptides, which have at least a immunological property of the aforementioned sequences.
  • proteins, polypeptides and peptides which have altered sequences which do not significantly affect the antigenic properties of the protein polypeptide or peptide are encompassed within the scope of the present invention.
  • the terms are to be interpreted to encompass fragments, functionally equivalent fragments, homologues and analogues of the protein, polypeptide or peptide.
  • the peptides encoded by the amino acid sequences presented as Sequence ID No. 1 and Sequence ID No's 3 to 18 are useful, alone or in combination, uncoupled or coupled to other molecules, in selective diagnostic methods for detecting fungal infections including those caused by Candida, as therapeutic agents for the treatment of Candida infections in immunisation against fungal infections including Candida infections, and in the production of polyclonal and monoclonal antibodies. Of course, their use is not to be limited to these methods.
  • the protein, polypeptide or peptide of the invention preferably comprises at least one epitope or antigenic determinant reactive with Candida albicans induced antibodies.
  • the epitope or antigenic determinant is preferably reactive with enolase and even more preferably is reactive with Candida albicans enolase.
  • the epitope may comprise at least one continuous (linear) epitope. Alternatively the epitope may comprise a conformational epitope.
  • the protein, polypeptide or peptide provided by the invention comprise oligo peptides having the amino acid sequences presented as Sequence ID No. 3-18 containing therein sequences which comprise continuous (linear) epitopes reactive with Candida specific antibodies.
  • the present invention also provides DNA sequences and/or polynucleotide sequences coding for the proteins, polypeptides or peptides of the present invention.
  • the invention resides in an isolated and purified antibody, either monoclonal or polyclonal, induced by one or more of the proteins, polypeptides or peptides of the present invention.
  • the present invention also provides a process for the production of a protein, polypeptide or peptide comprising the steps of; culturing a host organism transformed with a vector including a gene coding for a precursor of said protein, polypeptide or peptide; cleaving said precursor to produce said protein, polypeptide or peptide; and recovering said protein, polypeptide or peptide.
  • the invention resides in a polynucleotide sequence, preferably a DNA sequence, coding for a protein polypeptide or peptide of the present invention.
  • the polynucleotide preferably comprises the nucleotide sequence of Sequence ID No 2 or a portion thereof. DNA having this sequence may be obtained as described in the examples hereinafter and preferably the protein is Candida albicans enolase.
  • the present invention also provides for an expression vector including the above polynucleotide or a portion thereof.
  • the vector preferably includes the DNA sequence presented as Sequence ID No 2.
  • Sequence ID No 2 may also be used in the design of DNA probes for use in various hybridisation techniques to detect presence of fungal nucleic acid.
  • Other segments of the DNA sequence presented as Sequence ID No. 2 may be useful as primers for use in Polymerase Chain Reaction (PCR) procedures enabling the specific amplification of fungal DNA sequences to aid their detection.
  • PCR Polymerase Chain Reaction
  • the invention resides in a therapeutic agent, a vaccine and/or pharmaceutical composition comprising an effective dosage of the protein, polypeptide or peptide of the present invention, for inducing protection in a living mammal against a fungal infection, or treating disease caused by a fungus, preferably the fungus is Candida albicans and preferably the fungal infection is caused by Candida albicans.
  • a pharmaceutically acceptable carrier In the case of the vaccine there may further be provided a pharmaceutically acceptable carrier.
  • the therapeutic agent, vaccine and/or pharmaceutical compositions may be formulated with a suitable pharmaceutical carrier and preferably contains a dosage of the protein enolase or a fragment or homologue or analogue thereof.
  • the invention resides in a method for the in vivo production of antibodies against a protein, polypeptide or peptide comprising the step of contacting an antibody producing cell in vivo with said protein, polypeptide or peptide to raise said antibodies.
  • the protein, polypeptide or peptide may comprises at least one amino acid sequence selected from an amino acid sequence presented as Sequence ID No's 3-18.
  • the protein, polypeptide or peptide may comprise at least the sequence of Sequence ID No 1 or the protein enolase or a fragment or homologue'or analogue thereof.
  • the present invention also provides a diagnostic kit for use in diagnosing Candida albicans infections comprising; at least one protein, polypeptide or peptide which is immunologically reactive with Candida albicans induced antibodies; means for detecting the binding of said antibodies to the protein, polypeptide or peptide.
  • the protein, polypeptide or peptide is encoded by a portion of the amino acid sequence presented as Sequence ID No. 1.
  • the portion comprises amino acids 93 to 249 of the amino acid sequence presented as Sequence ID No. 1.
  • the protein, polypeptide or peptide utilised in the kit comprises the protein, polypeptide or peptide encoded by any one or more of the amino acid sequences presented as Sequence ID No's 3 to 18.
  • the present invention also provides a method of detecting Candida albicans induced antibodies in a sample comprising: reacting the sample with a protein , polypeptide or peptide of the invention and detecting the immunological complex.
  • Fig 1 The identification of antigenic components of C. albicans by enzyme labelled anti-IgG antibodies by immunoblot analysis. Lanes 1-3 were incubated with human sera from vaginitis, normal and AIDS patients, respectively. Lane 4 was incubated with hyperimmune rabbit serum. Lanes 5 and 6 were incubated with affinity-purified antibody prepared against the 48 kDa antigen and the recombinant 48 kDa antigenic determinant, respectively.
  • Fig 2 An immunoblot of affinity-purified anti-48 kDa antigen antibody against SDS-PAGE fractionated proteins from lanes 1-4, C. albicans clinical isolates; lane 5, S. cerevisiae: lane 6, C. krusei: lane 7, C. tropicalis.
  • Fig 3 The location of the epitopes using a matrix plot of the individual peptide reactivity with four different " sera samples from patients with vaginal candidiasis.
  • a differential screening strategy was used.
  • a complete set of eight-mer amino acid sequences were synthesised such that subsequent sequences overlapped by six amino acids, and together the sequences covered the entire region of the enolase protein identified as the major IgG reactive region.
  • Such extra amino acids are useful for coupling the sequences to each other, to another peptide, to a large carrier protein or to a solid support.
  • Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamine, aspartic acid, cysteine and derivatives thereof.
  • Additional protein modification techniques may be used, example NH ⁇ -Acetylation or COOH-terminal amidation, to provide additional means for coupling the peptides to another protein or peptide molecule or to a support.
  • Analogues are peptides which are functionally equivalent to the present peptides but which contain non naturally occurring amino acids.
  • homologues are peptides which have constitutively substituted amino acids which correspond to the peptides encoded by the genome of any other fungal isolates.
  • the polypeptides or peptides comprising an amino acid sequence (a) to (p) set out above can be used at least alone or in combination with other methods for detection of antibodies to Candida albicans.
  • the antibodies thereby generated may also be used to detect Candida albicans associated antigens.
  • Antibodies can be found in biological samples including but not limited to sera, other body fluids, tissue samples and other samples which may contain antibodies to Candida albicans.
  • the peptides may also be useful as vaccines to protect against future infection by Candida albicans and/or as therapeutic agents to treat a Candida albicans infection.
  • the methods which use the peptides to detect the presence of Candida albicans specific antibodies in the sample involve contacting the sample with a peptide comprising at least one of the amino acid sequences (a) to (p) under conditions which allow the formation of an immunological complex between the peptide and any antibodies to Candida albicans that may be present in the sample.
  • the formation of an immunological complex, if any, indicating the presence of antibodies to Candida albicans in the sample is then detected and measured by suitable means.
  • Such detection methods include but are not limited to homogeneous and heterogeneous binding immuno assays, such as enzyme linked immunosorbant assays (ELISAs), radioimmuno assays (RIA), and Western Blot Analysis. Further assay protocols using the peptides allow for competitive and non competitive binding studies to be performed. The screening methods are rapid, efficient and allow for simultaneous screening of numerous samples.
  • ELISAs enzyme linked immunosorbant assays
  • RIA radioimmuno assays
  • Western Blot Analysis Western Blot Analysis
  • the peptides may be labelled or unlabelled depending on the type of assay used.
  • Labels which may be coupled to the peptides are those known in the art and include but are not limited to enzymes, radio nuclear nuclides, fluorogenic and chromogenic substrates, co- factors, biotin-avidin, colloidal gold and magnetic particles.
  • the peptides can be coupled by any means known in the art to other peptides, solid supports and carrier proteins.
  • Such solid supports include but are not limited to polystyrene or polyvinyl microtitre plates, glass tubes or glass beads and chromatographic supports, such as paper, cellulose and cellulose derivatives, and silica.
  • Carrier proteins include but are not limited to bovine serum albumen (BSA) and keyhole hemosianine (KLH).
  • Preferred assay techniques especially for large scale clinical screening of patient sera and blood and blood derived products are Agglutination, Western Blot techniques and ELISAs.
  • ELISAs are particularly preferred for speed, the ability to test numerous samples simultaneously and ease of automation.
  • a signal generating means may be added to aid detection of complex formation.
  • a detectable signal is produced if Candida albicans specific antibodies are present in the sample.
  • Agglutination assays are commonly used and may utilise latex to which is bound to at least one of the peptides for use in a latex agglutination test.
  • the coated latex beads can be mixed with a small volume of patient serum and examined for agglutination. If Candida albicans enolase specific antibodies are present in the patients serum agglutination (clumping) of the latex particles will be observed. While not as sensitive as an ELISA, latex agglutination assays are quick and easy to perform and would be suitable for medical practitioners or naturopaths as an initial screen.
  • An enzyme linked immunosorbent assay for the detection of antibodies to C albicans is another use for the peptides of the current invention.
  • the ELISA is a far more sensitive and quantifiable assay when compared with the latex agglutination assay.
  • a standard titre of antibody reactive to the C. albicans enolase in normal healthy individuals would be determined, a titre of antibody above this level would be diagnostic of a C. albicans infection.
  • the ELISA test is based on techniques currently in use for detection of other antibodies.
  • the peptides of the invention are conveniently bonded to the inside surface of micro titre wells.
  • the peptides may be directly bonded by hydrophobic interactions to the microtitre well, or attached covalently by means known in the art to a carrier protein, such as BSA, with the resulting conjugate being used to coat the wells.
  • C. albicans infections in patients with systemic Candidiasis are difficult. These patients are often immunosuppressed and produce low levels of antibody which are unlikely to be detected in a latex agglutination test. In patients with systemic Candidiasis, a decrease in antibody titre is frequently associated with a poor prognosis.
  • An ELISA enables the level of enolase specific antibodies to be monitored in these patients. The monitoring of the levels of C. albicans enolase specific antibodies over the period of an infection will facilitate a more rapid treatment of patients with C. albicans infections.
  • the peptides of the invention may also be formulated into pharmaceutical compositions or vaccines for use as immunogens.
  • immunbgens can be used as or to elicit production of antibodies.
  • the antibodies produced can be either polyclonal or monoclonal.
  • an immunogenically effective amount of at least one of the peptides is admixed with a physiologically acceptable carrier suitable for administration to a subject.
  • the peptides may be covalently attached to each other, to other peptides, to a protein carrier or to other carriers incorporated into liposomes or other such vessicles, or complexed with an adjuvant or absorbent as is known in the vaccine art.
  • the peptides are not complexed with the above and merely admixed with a physiologically acceptable carrier such as normal saline or a buffering compound suitable for administration to the subject.
  • a physiologically acceptable carrier such as normal saline or a buffering compound suitable for administration to the subject.
  • Antibodies made in response to the peptides and which recognise the peptides may also be formulated. Such antibodies can be either polyclonal or monoclonal. Methods for making antibodies are well known in the art.
  • Example 1 describes the preparation of a protein according to the invention.
  • Example 2 describes the determination of the complete nucleotide sequence according to the invention.
  • Example 3 describes the epitope mapping of a protein according to the invention, the preparation of particular peptide epitopes and their use in detecting Candida infections.
  • C. albicans A clinical isolate of C. albicans (KEMH5) was obtained from the Department of Pathology, King Edward Memorial Hospital, Subiaco, Perth, WA. The yeast was grown overnight in YEPD (1% yeast extract, 2% peptone, 2% glucose) at 30°C with aeration).
  • Candida albicans blastospores (10 cells) were resuspended in 1 mL of lysis buffer
  • Candida albicans antigen preparation was solubilized and reduced by boiling for 5 min in 62 mmol/L Tris HC1, PH 6.8 containing 700 mmol/L ⁇ -mercaptoethanol, 10% (v/v) glycerol, 2% (w/v) SDS and 0.001% (w/v) bromophenol blue and then fractionated on a 7.5-12.5% PAGE gradient gel and electroblotted on to nitrocellulose membrane (Bio-Rad Laboratories Pty Ltd, USA).
  • Protein molecular mass determinations were determined by comparison with molecular weight standards (Bio-Rad, USA) consisting of lysozyme, 14400 kDa; soybean trypsin inhibitor, 21500 kDa; carbonic anhydrase, 31000 kDa; ovalbumin, 45000 kDa; bovine serum albumin, 66200 kDa; and phosphorylase b, 92500 kDa.
  • the immunodetection was essentially performed as described by Mierendorf et al 1987, with the following modifications: free protein binding sites were blocked with 1% BSA in PBS- Tw (PBS pH 7.4, 0.05% Tween 20) at 25°C for 60 min. The nitrocellulose strips were washed three times for 5 min in PBS-Tw, then incubated with sera (primary antibody, 1/100 finaT dilution in PBS-Tw) at 25°C for 90 min. After a further three 5 min washes in PBS-Tw the strips were incubated with the relevant IgG-alkaline phosphatase conjugated secondary antibody (Silenus Laboratories Pty Ltd, Vic, Australia) at 25°C for 60 min. After three 5 min washes in PBS, the antigen-antibody complexes were detected by monitoring the deposition of purple stain following reaction with the substrates NBT and BCIP.
  • PBS- Tw PBS pH 7.4, 0.05% Tween 20
  • Candida albicans proteins (200 ⁇ g) were injected subcutaneously into a New Zealand white rabbit. A further injection was given 14 days later and serum was collected after 28 days. The serum contained high levels of Candida reactive/or specific antibodies as determined by Western blot analysis.
  • the C. albicans antigen preparation was fractionated by SDS-PAGE and electroblotted on to nitrocellulose.
  • the horizontal band containing the immunodominant antigen was excised, and affinity-purified antibody prepared as described by Matthews and Burnie, 1988.
  • Protein was isolated from recombinant Escherichia coli clones as described by Huynh et al, 1985. A positive clone was selected and affinity-purified antibodies prepared as previously discussed.
  • ⁇ DNA was purified from the positive clones and the cDNA insert from one clone excised and subcloned into M13mpl8.
  • the cDNA was sequenced in both orientations using the di- deoxychain termination method using a Taq polymerase DNA sequencing kit (Biotech International Limited, Perth, WA).
  • the deduced amino acid sequence was used in a homology search against protein sequences in the PIR data base.
  • Immunoblot analysis of serum from patients with vaginal candidiasis demonstrated an antibody response to a protein of C. albicans with an apparent molecular mass of 48 kDa (Fig 1), as estimated from semi-logarithmic plots of the position of molecular weight markers on gradient gels.
  • This antibody appeared to be identical to that found in sera from patients with HIV infection or AIDS (Fig 1).
  • Antibody to the 48 kDa antigen was also identified in hyperimmune rabbit serum (Fig 1).
  • the latter serum was used to prepare an antibody probe against the 48 kDa antigen, the specificity of which was confirmed by immunoblotting against SDS-PAGE fractionated C. albicans protein (Fig 1).
  • the affinity purified anti-48 kDa antibody also exhibited weak reactivity against a similar sized antigen present in C. krusei. C. tropicalis and Saccharomyces cerevisiae (Fig 2).
  • the nucleotide sequence of the 470 bp cDNA insert from the clone ⁇ l.l contained a single large translational open reading frame, in frame with the ⁇ gtll encoded ⁇ - galactosidase gene.
  • the 157 amino acid sequence encoded by the cDNA insert was used in a homology search with the Protein Information Resource (PIR) data base.
  • PIR Protein Information Resource
  • a 74% amino acid identity was found with the corresponding region of enolase from the yeast S. cerevisiae and 56% identity with enolase from chicken skeletal muscle.
  • the region of homology extends from amino acid position 93 to position 249 of the S. cerevisiae enolase (the carboxyl terminus being amino acid 433).
  • the degree of amino acid identity between the enolase of C. albicans and that of S. cerevisiae is in agreement with the closer evolutionary relatedness of the two yeasts relative to chicken.
  • the S. cerevisiae and chicken skeletal muscle enolases exhibit homologies of 97 and 93% respectively.
  • C. albicans genomic DNA was extracted from a clinical isolate KEMH5 using the aqueous method of Cryer et al. 1975.
  • a C. albicans genomic library was constructed in the vector ⁇ GEM12 (Promega), using a modification of the partial end fill method of Zabarovsky and Allikmets, 1986.
  • C. albicans genomic DNA (2 ⁇ g) was partially digested with Sau3AI to generate 10-20 kb fragments.
  • the Sau3AI-digested genomic DNA was partially end filled using E. coli DNA polymerase (Klenow fragment, 1 U/2mg DNA) with 50 uM dATP and dGTP for 30 min at room temperature.
  • the ⁇ GEM12 DNA was digested with Xhol and the ends filled with dCTP and dTTP and ligated with the end-filled genomic DNA. Following packaging, the library was plated using standard methods.
  • the C. albicans genomic DNA library was screened with the radiolabelled cDNA probe ⁇ l.l, corresponding to codons 93-249 of the C. albicans enolase coding sequence.
  • the ⁇ gen 6.1 DNA was digested with various restriction enzymes. The restriction fragments were subcloned into M13mpl8 and M13mpl9 vectors. The recombinant plaques were hybridised with radiolabelled ⁇ l.l cDNA. Positive clones were selected and the DNA sequenced by the dideoxy-chain termination method. Clones containing overlapping restriction fragments were identified by hybridisation studies. Where necessary, oligonucleotide primers were synthesised and used as primers in a cycle-sequencing reaction (using a fmol sequencing kit, Promega) with ⁇ gen 6.1 DNA as the template.
  • a 470-bp cDNA was identified encoding the major IgG immunoreactive epitope(s).
  • the deduced amino acid sequence of the cDNA clone had 80% identity with amino acid positions 93-249 of enolase from the baker's yeast, S. cerevisiae.
  • the cDNA was purified from the ⁇ gtll DNA, radiolabelled, and used as a probe to screen a C. albicans genomic DNA library constructed in the vector ⁇ GEM12. Several positive plaques were identified.
  • the 157 amino acid region of the enolase as determined by the DNA sequence of the ⁇ gtll clone ⁇ l.l was synthesised on polyethylene pins by the method of Geysen et al (1987). A complete set of 8-mer peptides were synthesised such that subsequent peptides overlapped by six amino acids.
  • Peptides coupled to the surface of the pins were tested against human sera for enolase immunoreactivity by an ELISA.
  • the human serum samples were diluted 1/100 in 0.01 M PBS, pH 7.2 (2mM Na 2 HP0 4 ,9mM NaH 2 P0 4 , 150mM NaCl) containing 2%
  • Sheep anti-human alkaline phosphatase conjugated IgG antibody (Silenus) was diluted 1/1 500 in 0.01 M PBS, 2% (v/v) BSA, 0.1%(v/v) Tween 20 (pre-coat buffer) prior to use.
  • Immunodetection was performed by immersing the pins containing the immobilised peptides in the appropriate solutions in 96 well microtitre trays.
  • the pins were blocked in 200 ⁇ l of pre-coat buffer for 60 min at 25°C with gentle agitation.
  • the pins were then incubated in 175 ⁇ l of diluted (1/100) human serum overnight at 4°C.
  • the pins were washed four times in a bath of 0.01 M PBS for 10 min each at 25°C with gentle agitation.
  • the pins were then incubated in 175 ⁇ l of diluted (1/1500) sheep anti- human alkaline phosphatase conjugated IgG antibody for 60 min at 25°C with gentle agitation.
  • the pins Prior to immunodetection, the pins were re-washed four times in a bath of 0.01 M PBS for 10 min each at 25°C with gentle agitation. Detection of immunoreactive peptides was performed by incubating the pins in 150 ⁇ l of 1 mg/ml p- nitro-phenyl phosphate in 10 mM diethanolamine, pH 9.5 containing 0.5 mM MgC . The plates were incubated for 10- 30 min at RT. The reaction was halted by removing the pins from the detection fluid. The plates were then read at 405 nm in a Titertek Multiskan plate reader. For each serum sample, a matrix plot of the individual peptide reactivity relative to each other peptides was determined.
  • Peptide bound antibody was removed by heat, denaturation and sonication.
  • the pins were sonicated in 0.1 M PBS, containing 1% (v/v) SDS and 0.1% (v/v) ⁇ -mercaptoethanol for 10 min at 60°C.
  • the pins were rinsed twice in distilled water at 60°C for 30 seconds, with a final wash in water at 60°C for at least 30 min with gentle agitation.
  • the pins were then immersed in boiling methanol (60°C) for at least 15 seconds. The pin were then ready to be re-used for immunodetection studies.
  • Epitopes reactive to IgG contained within specific 8-mer peptides were identified by using a matrix plot of the individual peptide reactivity against human sera samples as depicted in Fig. 3.
  • GAC GGT ACT CCA AAC AAA TCC AAA TTG GGT GCC AAT GCT ATC 485
  • GGT TTA AGA TCT GGT CAA ATC AAG ACT GGT GCT CCA GCC AGA 1367
  • CACATTTTAT TAATCGATGG CTAACGTCTA TTTACATATA TTCTTTAGAG 1722

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Abstract

L'invention a pour object des peptides, des polypeptides et des protéines ou fragments dérivés de ces derniers, dont les séquences aminoacides correspondent aux segments antigéniques d'une protéine de Candida albicans, importante du point de vue immunologique. Ces peptides, polypeptides ou protéines sont utiles comme réactifs diagnostiques pour détecter la présence d'anticorps réagissant avec Candida albicans et peuvent également servir d'agents thérapeutiques et d'immunogènes dans des compositions et des techniques destinées à obtenir des anticorps dirigés contre Candida albicans.
PCT/AU1995/000176 1994-03-25 1995-03-27 Peptides a usage diagnostique et therapeutique WO1995026362A1 (fr)

Priority Applications (2)

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EP95913823A EP0759034A4 (fr) 1994-03-25 1995-03-27 Peptides a usage diagnostique et therapeutique
AU21063/95A AU684009B2 (en) 1994-03-25 1995-03-27 Peptides for diagnostics

Applications Claiming Priority (2)

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AUPM4732 1994-03-25
AUPM4732A AUPM473294A0 (en) 1994-03-25 1994-03-25 Peptides for diagnostics and therapeutics

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WO1995026362A1 true WO1995026362A1 (fr) 1995-10-05

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EP (1) EP0759034A4 (fr)
AU (2) AUPM473294A0 (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2304347A (en) * 1995-08-11 1997-03-19 Boeringer Ingelheim Vetmedica Antigenic preparations
WO1999021877A1 (fr) * 1997-10-24 1999-05-06 University Of Ottawa Peptides histoganine et utilisation de ces peptides
US20100119533A1 (en) * 2007-03-07 2010-05-13 Cornelius Joseph Clancy Polynucleotides and Polypeptides Identified by IVIAT Screening and Methods of Use

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT400722B (de) * 1993-08-27 1996-03-25 Biomay Prod & Handel Rekombinante cladosporium herbarum allergene

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FEMS MICROBIOL. LETT., (1993), 111(1), K.M. FRANKLYN and J.R. WARMINGTON, "Cloning and Nucleotide Sequence Analysis of Candida Albicans Enolase Gene", pages 101-7. *
J. BACTERIOL., (1992), 174(21), P. SUNDSTROM and G.R. ALIAGA, "Molecular Cloning of cDNA and Analysis of Protein Secondary Structure of Candida Albicans Enolase, an Abundant, Immunodominant Glycolytic Enzyme", pages 6789-99. *
J. BACTERIOL., (1993), 175(9), A.B. MASON, H.R. BUCKLEY and J.A. GORMAN, "Molecular Cloning and Characterisation of the Candida Albicans Enolase Gene", pages 2632-9. *
See also references of EP0759034A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2304347A (en) * 1995-08-11 1997-03-19 Boeringer Ingelheim Vetmedica Antigenic preparations
US6379678B1 (en) 1995-08-11 2002-04-30 Boehringer Indgelheim Vetmedica Gmbh Antigenic preparations
US7090857B2 (en) 1995-08-11 2006-08-15 Boehringer Ingelheim Vetmedica Gmbh Antigenic preparations
WO1999021877A1 (fr) * 1997-10-24 1999-05-06 University Of Ottawa Peptides histoganine et utilisation de ces peptides
US6566327B1 (en) 1997-10-24 2003-05-20 University Of Ottawa Histogranin peptides and their analgesic use
US6855692B2 (en) 1997-10-24 2005-02-15 University Of Ottawa Histogranin peptides and their analgesic use
US20100119533A1 (en) * 2007-03-07 2010-05-13 Cornelius Joseph Clancy Polynucleotides and Polypeptides Identified by IVIAT Screening and Methods of Use

Also Published As

Publication number Publication date
AUPM473294A0 (en) 1994-04-21
AU684009B2 (en) 1997-11-27
EP0759034A1 (fr) 1997-02-26
EP0759034A4 (fr) 1999-04-21
AU2106395A (en) 1995-10-17

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