WO1998046738A1 - Procedes d'obtention d'antigenes specifiques aux champignons, anticorps pour ces antigenes et diagnostic de maladie au moyen de ces antigenes et/ou anticorps - Google Patents

Procedes d'obtention d'antigenes specifiques aux champignons, anticorps pour ces antigenes et diagnostic de maladie au moyen de ces antigenes et/ou anticorps Download PDF

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Publication number
WO1998046738A1
WO1998046738A1 PCT/US1998/007728 US9807728W WO9846738A1 WO 1998046738 A1 WO1998046738 A1 WO 1998046738A1 US 9807728 W US9807728 W US 9807728W WO 9846738 A1 WO9846738 A1 WO 9846738A1
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Prior art keywords
fungus
antibody
target
antibodies
capsulatum
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PCT/US1998/007728
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English (en)
Inventor
Gary J. Weil
Ramaswamy Chandrashekar
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Barnes-Jewish Hospital
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Priority to AU71280/98A priority Critical patent/AU7128098A/en
Priority to CA002286335A priority patent/CA2286335C/fr
Publication of WO1998046738A1 publication Critical patent/WO1998046738A1/fr

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida

Definitions

  • the present invention generally relates to methods of detection of a fungus and fungal infections through the use of protein antigens specific for the fungus.
  • This invention also relates methods for identifying such antigens, to antibodies raised against these antigens, to 0 the use of such antigens and/or antibodies in assay methods for the determination of the presence of the fungus or diseases caused by the fungus in humans, other animals, plants, food, feed, and inorganic materials such as soil or air.
  • the invention also relates to assay kits 5 suitable for carrying out such diagnostic methods.
  • the invention particularly relates to the fungus Histoplasma capsulatum (hereinafter H.
  • H. capsulatum is a pathogenic dimorphic fungus that grows as multicellular mycelia in nature and as unicellular budding yeasts in humans and animals.
  • the most widely available tests are the immunodiffusion assay, which detects antibodies to heat- sensitive glycoproteins called H and M antigens, and the more sensitive complement fixation test, which is traditionally performed with yeast and mycelial antigens. More sensitive antibody assays such as radio-immunoassay and enzyme immunoassay have been used to detect IgM and IgG antibodies to crude fungal extracts. See, generally, references 39, 40, and 41. Attempts to develop antibody serology tests for diagnosis of histoplasmosis have been hampered by poor specificity caused by immunologic cross-reactivity between various fungal species. The problem of cross- reactivity to other fungi may be worsened in the diagnosis of histoplasmosis by the fact that H.
  • capsulatum is taxonomically closely related to two other pathogens, Coccidioides i mi tis, and Blastomyces dermati tidis . See Kwon-Chung, Science 177:368-369 (1972), and McGinnis et al . , Mvcotaxon 8:157-164 (1979). These fungi, which may be present along with H. capsulatum, cause coccidiomycosis and blastomycosis, diseases with etiologies similar to histoplasmosis. While H. capsulatum, C. immi tis, and B . dermati tidis are known as imperfect fungi due to their rare or nonexistent sexual stage, studies have shown that H.
  • capsulatum and B . dermati tidis are in the same telomorph genus, Ajellomyces, and those two fungi may be in the same taxonomic family as C. immi tis, the Gymnoascaceae, order Onygenales, of the Ascomycetes .
  • Another fungal pathogen, Candida sp. is also an ascomycete. See generally, Kwan- Chung et al . , Medical Mycology, Lea and Febiger,
  • antigens which are specific to a target fungus.
  • Such antigens are useful in diagnosis of diseases caused by the fungus and in determining the presence or absence of the fungus .
  • the present invention is directed to a method for identifying a protein antigen of a target fungus.
  • a cDNA gene expression library is obtained for the target fungus, and the library is expressed to form an array of target-fungus proteins.
  • Antisera to the target fungus and to a nontarget fungus are also obtained, each of which comprises antibodies to the target fungus and nontarget fungus, respectively.
  • the nontarget fungus has at least one antigenic determinant (e.g. a protein determinant or glycoprotein determinant) in common (ie, shared with) the target fungus.
  • a protein antigen specific to the target fungus is then identified by identifying a target-fungus protein which is bound by the antibodies to the target fungus, but which is not substantially bound by antibodies to the nontarget fungus. That is, while antibodies to the target fungus are immunoreactive with the identified protein antigen, antibodies to the nontarget fungus are not substantially immunoreactive with the identified protein antigen.
  • the invention is also directed to substantially purified or isolated antibodies or antibody fragments.
  • the antibody or antibody fragment is immunoreactive with a protein antigen identified according to the aforementioned method.
  • the antibody or antibody fragment is immunoreactive with an antigen of H. capsulatum, but which is not substantially immunoreactive with antigens of each of Coccidioides immi tis, Blastomyces dermati tidis or Candida sp .
  • antibody or antibody fragment is immunoreactive with a protein antigen having the amino acid sequence set forth in SEQ ID NO: 3 or with a portion thereof that is specific to H. capsulatum .
  • the invention is directed, moreover, to a method for determining the presence or absence of a target-fungus antibody in a vertebrate such as a mammal.
  • the method comprises obtaining an antibody- containing sample from the vertebrate, contacting the sample with a target-fungus protein antigen identified according to the method of claim 1, and determining whether an antibody in the sample immunoreacts with the target-fungus protein antigen.
  • the method comprises obtaining an antibody-containing sample from the mammal, contacting the sample with a protein antigen of H. capsulatum which is bound by antibodies to H.
  • the method comprises obtaining an antibody-containing sample from the mammal, contacting the sample with a protein antigen having an amino acid sequence as set forth in SEQ ID NO: 3, and determining whether an antibody in the sample immunoreacts with the protein antigen.
  • the invention is further directed to a method for determining the presence or absence of a target-fungus protein antigen in a sample.
  • the method generally comprises obtaining a sample to be tested for the presence or absence of the target-fungus protein antigen, contacting the sample with an antibody or antibody fragment which is immunoreactive with a target-fungus protein antigen identified according to the method of claim 1, and determining whether the antibody or antibody fragment immunoreacts with the target-fungus protein antigen.
  • the method comprises obtaining a sample to be tested for the presence or absence of the H. capsulatum protein antigen, contacting the sample with an antibody or antibody fragment which is immunoreactive with an antigen of H.
  • the method comprises obtaining a sample to be tested for the presence or absence of the H. capsulatum protein antigen, contacting the sample with an antibody or antibody fragment which is immunoreactive with a protein antigen having an amino acid sequence as set forth in SEQ ID NO: 3, and determining whether the antibody or antibody fragment immunoreacts with the protein antigen.
  • the invention is additionally directed to a kit that includes a reagent selected from, in one embodiment, one or more of the following: (i) a target-fungus protein antigen identified according to the aforementioned method, (ii) a fragment of a target-fungus protein antigen identified according to the aforementioned method wherein the fragment is bound by antibodies to the target fungus but is not substantially bound by antibodies to the nontarget fungus, and (iii) a target-fungus antibody or antibody fragment which immunoreacts with a target - fungus protein antigen identified according to the aforementiond method.
  • the reagent is selected from one or more of the following: (i) a protein antigen of H. capsulatum which is bound by H.
  • capsulatum antibodies but which is not substantially bound by antibodies to each of Coccidioides immi tis, Blastomyces dermati tidis or Candida sp. , (ii) a fragment of a H. capsulatum protein antigen wherein the fragment is bound by antibodies to H. capsulatum but is not substantially bound by antibodies to each of Coccidioides immi tis, Blastomyces dermati tidis or Candida sp . , and (iii) an antibody or antibody fragment which is immunoreactive with a H.
  • the reagent is selected from: (i) a protein antigen having an amino acid sequence as set forth in SEQ ID NO : 3 , (ii) a protein antigen that includes a portion of the amino acid sequence as set forth in SEQ ID NO: 3 wherein the included portion is bound by antibodies to H. capsulatum but is not substantially bound by antibodies to each of
  • Coccidioides immi tis, Blastomyces dermati tidis or Candida sp . (iii) an antibody or antibody fragment which is immunoreactive with a protein antigen having the amino acid sequence set forth in SEQ ID NO: 3, and (iv) an antibody or antibody fragment which is immunoreactive with a protein antigen that includes a portion of the amino acid sequence set forth in SEQ ID NO: 3 wherein the included portion is bound by antibodies to H. capsulatum but is not substantially bound by antibodies to each of Coccidioides immitis, Blastomyces dermatitidis or Candida sp ..
  • the kit also includes instructions for directing the use of the reagent for determining the presence or absence of the target fungus in a sample .
  • the instructions direct the use of the reagent for determining whether a mammal is presently infected or has been previously infected with the target fungus.
  • the instructions direct the use of the antibody reagent for determining the presence or absence of an antigen in a nonvertebrate sample or environment, such as a plant, food, feed, feed component, air, water, or other fluid sample.
  • FIG. 1 shows a hydropathy plot of the protein encoded by GH17. Hydropathy analysis was performed by the method of Hopp and Woods . Hydropathy values were averaged for a window of six amino acid residues.
  • FIG. 2 shows a comparison of the deduced amino acid sequence encoded by GH17 with threonine-rich regions of: (A) cellulase from the thermophilic bacterium Caldocellum saccharolyticum; (B) , Leishmania surface antigen; and (C) , integumentary mucin from Xenopus laevis using the NCBI BLAST analysis program. Identical residues are indicated with letters, and conserved residues are marked with a (+) .
  • FIG. 3 shows a Southern blot of genomic DNA of H. capsulatum probed with labeled cDNA insert from clone GH17.
  • Genomic DNA was digested with EcoRI (lane 1), PstI (lane 2) , and Sad (lane 3) , electrophoresed on a 1% agarose gel, and transferred to nylon membrane. The membrane was probed with peroxidase-labeled cDNA insert of GH17 and washed under high-stringency conditions.
  • FIG. 4 shows the results of an immunoblot analysis of the immunoreactivity and specificity of the ⁇ - galactosidase fusion protein of the recombinant clone GH17. The immunoreactive fusion protein band is indicated by an arrow.
  • FIG. 1 shows a Southern blot of genomic DNA of H. capsulatum probed with labeled cDNA insert from clone GH17.
  • Genomic DNA was digested with EcoRI (lane 1), PstI
  • FIG. 4A shows a representative immunoblot demonstrating the immunoreactivity of the fusion protein.
  • Bacterial cell-lysates from cells infected with GH17 were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose paper. The blot was developed with individual sera from patients with histoplasmosis (Lanes 1-18) . 16 of 18 sera had strong antibody reactivity with the fusion protein, and two sera were weakly reactive (Lanes 6 and 9) . Lane 19 was developed with a murine monoclonal antibody to ⁇ - galactosidase.
  • FIG. 4B demonstrates the antigenic specificity of recombinant H. capsulatum clone GH173- galactosidase fusion protein by immunoblot analysis .
  • FIG. 5 shows an SDS-PAGE and immunoblot analysis of expression of GH17-his in pPROEX-1TM protein expression vector.
  • 10% SDS-PAGE was loaded with E. coli extract without IPTG induction (lane 1) , and after IPTG induction for 3 hr (lane 2) .
  • the immunoblots were developed with human histoplasmosis serum pool (1:500), enzyme-labeled anti-human IgG secondary antibody, and substrate.
  • FIG. 5B shows immunoblot analysis of eluted fractions of GH17-his fusion protein separated by preparative SDS-PAGE in the model 491 BioRad Prep Cell.
  • FIG. 6 shows an immunoblot analysis of H. capsulatum yeast antigen extract developed with: lane 1, mouse antibody to GH17-his fusion protein (1:500); lane 2, normal mouse serum (1:500); and lane 3, mouse antibodies to H. capsulatum yeast extract (1:500) .
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed toward protein antigens which are specific to a target fungus, to methods of obtaining such antigens, to antibodies to such antigens, and to assays employing such antigens and antibodies .
  • An antigen specific to a particular target fungus is identified by a) obtaining a cDNA gene expression library for the target fungus, b) expressing target fungal proteins from the cDNA gene expression library, c) obtaining antisera to the target fungus which has antibodies to the target fungus, d) obtaining antisera to the nontarget fungus which has antibodies to the nontarget fungus and e) identifying a target-fungus protein which is bound by the antibodies to the target fungus, but which is not substantially bound by the antibodies to the nontarget fungus.
  • the steps of the above method need not be performed in any particular order.
  • the cDNA expression library is first screened with an antisera to the target fungus. Clones which are highly reactive to the target fungus are thus identified, and these selected clones are then screened with antisera to the nontarget fungus. The clones which produce a protein which is bound by antibodies in the antisera to the target fungus but not by antibodies in the antisera to other microorganisms (e.g. nontarget fungi) are then identified.
  • Various manipulations known in the art can utilize these clones to produce and substantially purify the protein antigens encoded by the clones. The protein antigens can then be used as discussed below.
  • target fungus denotes any fungus for which there is a particular interest.
  • target fungi are fungi which are pathogens of animals or plants, allergens, food spoiling agents, or food sources.
  • Preferred target fungi include Ascomycetes . More preferred target fungi are those fungi in the genus Gymnoascaceae. Even more preferred fungi are those in the genus Ajellomyces, and the most preferred target fungus is H. capsulatum .
  • a "nontarget fungus” may be any fungus that is desired to be distinguished from the target fungus, for identification or diagnostic purposes, by an immunoassay.
  • the nontarget fungus includes at least one antigenic determinant in common with the target fungus.
  • the nontarget fungus can cross react with an antibody that recognizes this shared antigenic determinant.
  • Exemplary nontarget fungi may be selected from fungi known to cause a disease with similar symptoms as the target fungus .
  • the cDNA gene expression library of the target fungus may be obtained from a commercial source or prepared by methods known in the art, using an appropriate morphological stage of the target fungus .
  • An easily culturable stage such as mycelium is preferred.
  • Common vectors used for this purpose are plasmid, bacteriophage, and mammalian cells.
  • a preferred expression library is one utilizing a phage such as bacteriophage ⁇ gtll, with poly (A) + mRNA.
  • the target fungus proteins are expressed by any method known in the art.
  • the proteins are generally expressed by induction with IPTG.
  • Antisera to the target fungus may be obtained in any manner known in the art.
  • Preferably such antisera are obtained by preparing an antigen which will, upon immunization of an animal with the fungus, yield an antisera which contains a population of antibodies which, in sum, will immunoreact to many antigens of the target fungus.
  • An unfractionated preparation of the target fungus is thus a preferred antigen preparation.
  • a fractionated preparation could also be employed.
  • the preferred screening antisera is serum from infected individuals.
  • a more preferred screening antisera is pooled serum from several infected individuals.
  • Antisera to the nontarget fungus can be obtained by methods similar to the methods used to prepare antisera to the target fungus.
  • Target-fungal proteins which are bound by the target-fungus antibodies but which are not substantially bound by the nontarget-fungus antibodies can be identified by screening each of the antisera by appropriate immunological assays known in the art .
  • immunological assays known in the art .
  • the terms "immunoreact”, “bind”, “are bound to” or grammatical derivations thereof are used interchangeably herein, and refer to the capability of an antibody or an antibody fragment to specifically attach to an antigen at the antibody's Fab binding site.
  • Exemplary screening assays include precipitin assays and label-based assays.
  • the antigen is identified from the expressed cDNA gene expression library by blotting the expressed proteins (e.g. from phage plaques) onto membranes then screening the membranes with the antisera made to each fungus .
  • the degree of immunological cross-reactivity for the identified target-fungus protein (generally referred to herein as a target-fungus specific antigen) is preferably sufficiently low so that tests that detect the antigen or antibodies to the antigen are useful for reliably distinguishing the fungus from nontarget fungi .
  • the degree of immunological cross-reactivity with nontarget fungi and/or other microorganisms of interest, when assessed by immunoassay (for example Western blot or ELISA) is preferably less than about 10%, more preferably less than about 5%, still more preferably less than about 2%, even more preferably less than about 1%, and most preferably less than about 0.1%.
  • the identified target-fungus specific protein antigen is more specifically characterized as follows, with the various aspects defining the protein to be considered both independently and in combination.
  • the target fungus-specific protein antigen is preferably substantially free from non-protein determinants such as carbohydrates, phosphorylcholine, and/or other moieties which, when attached to or otherwise associated with the protein, would reduce the immunological specificity of the protein.
  • telomere length As an example, an antigen of a target fungus which infects mammals would be specific if antibodies in serum produced by a target fungus-infected vertebrate bound to the antigen, but sera produced in vertebrates which are infected with a nontarget fungus do not substantially cross-react with the antigen.
  • the extent of cross-reactivity can be more specifically characterized with regard to a set (ie. group or population) of samples being evaluated.
  • a sample population known to comprise the target-fungus antigen or antibodies to the antigen the presence of the antigen or antibody is correctly determined in preferably at least about 90% of the samples, more preferably in at least about 95% of the samples and most preferably in at least about 99% of the samples.
  • the absence of the target fungus antigen or antibody thereto is correctly determined in preferably at least about 90% of the samples, more preferably in at least about 95% of the samples and most preferably in at least about 99% of the samples.
  • an antigen on a plant pathogenic fungus is specific if antisera made to that antigen does not substantially cross-react with antigens on other nontarget fungi which might be present in the same environment as the plant pathogenic fungus .
  • Target-fungus specific protein antigens with such cross-reactivity can be varied from the identified protein antigen, but will preferably have an amino acid sequence which has a sequence identity or, alternatively, a homology of at least about 75%, more preferably at least about 90%, even more preferably at least about 95% and most preferably at least about 98% relative to the amino acid sequence of the identified protein antigen or, alternatively, as encoded by the cDNA clone thereof.
  • the target fungus is H. capsulatum.
  • Preferred nontarget fungi are C. immi tis, B . dermati tidis, and Candida sp. H.
  • capsulatum proteins expressed from a cDNA expression library are screened, preferably by immunoblot, against antisera to H. capsulatum and at least one of the nontarget fungi.
  • the antisera are preferably provided by pooled sera from individuals infected with the target and nontarget fungi .
  • a preferred protein antigen has the amino acid sequence set forth as SEQ ID NO: 3. (See example) .
  • the protein is a H. capsulatum-specific protein antigen and has an amino acid sequence which includes at least a portion of the amino acid sequence set forth as SEQ ID NO: 3 which is specific for H. capsulatum, the included portion being, in a preferred protein, at least 5 amino acids in length.
  • the H. capsulatum-specific antigen identified in this manner is not substantially cross- reactive with antisera to the nontarget fungus. (See Example) .
  • the target-fungus specific antigen identified for example by the afore-described screening protocols, can be isolated and produced in substantially purified form according to methods known in the art. Briefly, the cDNA clone corresponding to the identified target-fungus specific antigen, or more generally any nucleic acid polymer encoding the target-fungus specific protein antigen, can be incorporated into an expression vector for recombinant production of the protein antigen, as discussed below.
  • the nucleic acid polymer can have the cDNA nucleotide sequence of the isolated cDNA clone.
  • the nucleic acid polymer can, alternatively, have a mRNA nucleotide sequence corresponding to the cDNA sequence.
  • the nucleic acid polymer preferably has the cDNA nucleotide sequence set forth as SEQ ID NO: 2 or a mRNA nucleotide sequence corresponding to the sequence set forth as SEQ ID NO: 2.
  • the nucleic acid polymer can encode a fungus-specific protein antigen and have an nucleotide sequence which includes at least a portion of the nucleotide sequence of the isolated cDNA, the included portion being at least 15 base pairs in length.
  • the nucleic acid polymer can be at least 15 base pairs in length and encode a fungus-specific protein antigen having an amino acid sequence which has a sequence identity or, alternatively, a homology of at least about 75%, more preferably at least about 90%, even more preferably at least about 95% and most preferably at least about 98% relative to the amino acid sequence encoded by the isolated cDNA.
  • the above-disclosed nucleic acid polymer which encodes a fungus-specific protein antigen is preferably used to create a vector which is used, for example, to replicate or translate the nucleic acid polymer.
  • Translation of the nucleic acid polymer is preferably accomplished by an expression vector by methods known in the art.
  • the expression vector can be, for example, a hybrid plasmid, a virus, or other nucleic-acid-polymer construct which is suitable for use in expressing the antigen in a eukaryotic or prokaryotic host-cell, in vitro, according to methods known in the art. In the case of H.
  • a host cell can be transformed with the above- disclosed vector for recombinant production of the target fungus-specific protein antigen.
  • the host cell can be, for example, a bacterial host cell such as E. coli , a yeast cell, a mammalian cell, or any other suitable host cell in which the antigen can be expressed and from which the antigen can be substantially isolated and purified.
  • the isolated fungus-specific protein antigen can be utilized to produce an antibody specific for the antigen.
  • the antigenic protein or fragment against which the antibody is raised and to which the antibody binds is preferably substantially purified, and is further characterized as set forth above, with the various aspects defining the protein antigen to be considered both independently and in combination.
  • the antibody may be a mono-specific antibody.
  • the monospecific antibody may be a monoclonal antibody produced, for example, by the method of Galfre et al . , Nature 266:550 (1977).
  • the monospecific antibody may be a recombinant antibody produced, for example, by the method of Lowman et al, Biochemistry 30:10832-10838 (1991) .
  • the antibody can also be a polyclonal antibody.
  • the polyclonal antibody can be prepared by immunizing a mammal such as a mouse or rabbit with the fungus-specific antigen and subsequently isolating the serum therefrom to obtain an antiserum that contains the polyclonal antibodies.
  • polyclonal antibodies reactive to the specific antigen are generally produced in the serum of an infected animal . That serum may be collected and utilized as a polyclonal antiserum to the fungus.
  • the target fungus-specific antigen, and antibodies made to that specific antigen can be utilized in assays to determine the presence or absence, in a sample, of antigens or antibodies which are indicative of or diagnostic for the target fungus.
  • any portion of the antigen which is specific for the fungus may be utilized for identifying the target fungus in a sample, and specific peptide sequences as small as five amino acids in length may be easily obtained by methods known in the art.
  • These specific fragments may, for example, be used alone or they may be engineered by methods known in the art to be part of a fusion protein, preferably comprising two domains, a first domain that includes at least a portion of the amino acid sequence encoded by the nucleic acid polymer, the included portion being at least 5 amino acids in length, and a second domain that includes the amino acid sequence of another protein or polypeptide.
  • the second domain includes the amino acid sequence of a protein from the expression vector, such as ⁇ - galactosidase or other protein incorporated in an expression system, which may facilitate expression and/or subsequent purification of the expressed antigen from the host-cell lysate.
  • a protein from the expression vector such as ⁇ - galactosidase or other protein incorporated in an expression system, which may facilitate expression and/or subsequent purification of the expressed antigen from the host-cell lysate.
  • the tests will preferably allow one to distinguish the fungal disease from other clinical conditions, especially from other fungal infections.
  • the target-fungus specific antigens result from the above-disclosed method because the expressed proteins from the cDNA expression library do not contain carbohydrate moieties which would be present in fungal antigen preparations prepared by prior art methods.
  • Prior art methods of immunizing vertebrates with components of the target fungus generally failed to identify specific antigens because the immune system of the immunized vertebrate would mount an immune response to the antigenic carbohydrate moieties of the immunogen target fungus.
  • the carbohydrate moieties of the immunogen target fungus are often also present in the nontarget fungi, thus leading to cross-reactivities with the nontarget fungi.
  • fungi which are closely related taxonomically such as H. capsulatum and B . derma ti tidis
  • fungi which are less closely related such as H. capsulatum and Agaricus bisporus , the common cultivated mushroom.
  • the present method is particularly suited for developing immunoassays for target fungi which might be confused for closely related nontarget fungi.
  • a test for histoplasmosis developed with an antigen specific for H.
  • H. capsulatum (as in the Example) allows a determination of the presence or absence of antibodies to H. capsulatum which do not immunoreact with nontarget fungi present in the sample -- particularly Coccidioides immi tis, Blastomyces dermatidi tis, and/or Candida species.
  • diagnostic methods i.e., assays
  • H. capsulatum provides diagnostic methods (i.e., assays) for determining whether a mammal has been infected with H. capsulatum.
  • an antibody assay comprises detecting the presence or absence of antibodies to a target fungus-specific antigen in a sample obtained from a vertebrate. The presence or absence of the target fungus-specific antibodies are detected by contacting the sample with an antigen specific to the target fungus and determining whether the sample contains antibodies that bind to the target fungus-specific antigen.
  • the preferred antigen is further characterized as set forth above, with the various aspects defining the antigen to be considered both independently and in combination.
  • Exemplary antibody assays include precipitin-based immunoassays, indirect label-based immunoassays, direct label-based immunoassays and inhibition/competitive-type label-based immunoassays.
  • the presence of target fungus-specific antibodies in a sample obtained from the mammal is evidence of current and/or past exposure or infection with the target fungus.
  • the method comprises detecting the presence or absence of antigens specific to the target fungus in a sample .
  • the presence or absence of target fungus-specific antigens is detected by contacting the sample with an antibody capable of binding to a target fungus-specific antigen and determining whether the antibody binds to the target fungus-specific antigen.
  • the preferred target fungus-specific antibody is as set forth above, with the various aspects defining the antibody to be considered both independently and in combination.
  • the target fungus-specific antigen being detected is further characterized as set forth above, with the various aspects defining the antigen to be considered both independently and in combination.
  • Exemplary antigen assays discussed in more detail below, include precipitin-based immunoassays, indirect label- based immunoassays, direct label-based immunoassays and inhibition/competitive-type label-based immunoassays.
  • the presence of antigens specific to the target fungus in a sample is evidence of the presence of the target fungus in the sample .
  • H. capsulatum As applied to H. capsulatum, the detection of antibodies which bind to the H. capsulatum-specific antigen in a sample from a mammal is evidence of past or current infection with H. capsulatum . Similarly, the detection of H. capsulatum- specif ic antigens in a sample from a mammal is strong evidence of current infection with H. capsulatum.
  • the following additional concerns are applicable to either of the aforementioned antibody assay or antigen assay as applied to a target fungus such as H. capsulatum.
  • the mammals from which a sample is obtained are preferably humans, domestic livestock and/or pets which are suspected of being or known to be susceptible to fungal infection by the target fungus (e.g. H. capsulatum) .
  • the sample can be a blood sample, a plasma sample, a serum sample, a urine sample, a sputum sample, a saliva sample or any other biological sample obtained from the mammal which is suspected of potentially having antibodies to the target fungus (e.g. H. capsulatum) or having target-fungus (e.g. H. capsulatum) antigens.
  • the sample can be pretreated prior to testing the sample in the assay.
  • exemplary pretreatment steps can include concentrating the sample and/or eliminating interfering substances (e.g. acid in urine or rheumatoid factors in serum) .
  • interfering substances e.g. acid in urine or rheumatoid factors in serum
  • Other pretreatment steps will be apparent to a person of skill in the art.
  • the extent of such binding can be quantitatively determined using methods known in the art .
  • the antibody assays of the present invention can be more specifically characterized according to a variety of formats set forth below and/or known in the art.
  • One approach includes the use of precipitin-based immunoassays.
  • the presence or absence of the antibodies can be detected by layering a first solution including the target fungus-specific antigen over the undiluted (i.e., neat) sample or over a second solution including the sample, the layered solution typically being formed in a container such as a test- tube, and observing the layered solution for the formation or the lack of formation of a precipitate comprising bound antigen and antibody.
  • the amount of target fungus-specific antigen in the first solution is preferably an amount which is necessary, on average, to form a precipitate with samples drawn from a vertebrate known to contain antibodies to the target fungus.
  • the steps of this approach can, alternatively, be repeated, in parallel or in series, using various amounts of the target fungus-specific antigen in the first solution, with the amount of antigen in the solution varying over a range which includes an amount which is about, on average, necessary to form a precipitate with samples drawn from mammals known to have histoplasmosis .
  • Precipitin-based immunoassays can also be carried out in gels such as agar or polyacrylamide gels or their equivalents known in the art, by methods typically referred to as immunodiffusion, immunoelectrophoresis, counterimmunoelectrophoresis and/or rocket electrophoresis, among others.
  • the presence or absence of the antibodies are detected by placing a first solution including the target fungus-specific antigen in a gel or in a well adjacent to a gel, placing the undiluted sample or a second solution including the sample, in a gel or in a well adjacent to a gel, allowing the target fungus-specific antigen and antibodies to diffuse in the gel, and observing the gel for the formation or the lack of formation of a precipitate comprising bound antigen and antibody.
  • Turbidometric or nephelometric-type assay formats can also be employed.
  • Precipitin-based immunoassays offer the advantage of not requiring a solid-phase matrix, and as such, may be suited for particular applications known in the art (e.g. automated systems) .
  • Another approach for the antibody assay of the present invention includes the use of label-based assay techniques, including direct, indirect and/or inhibition/competitive radioimmunoassays, enzyme- linked immunoabsorbant assays (ELISA) , immunofluorescent assays, immunochromatographic assays, and other techniques known in the art.
  • label-based assay techniques including direct, indirect and/or inhibition/competitive radioimmunoassays, enzyme- linked immunoabsorbant assays (ELISA) , immunofluorescent assays, immunochromatographic assays, and other techniques known in the art.
  • the presence or absence of the antibodies can be detected using an indirect label-based immunoassay by immobilizing the target fungus-specific antigen on a solid-phase, contacting the immobilized antigen with the undiluted sample or with a solution including the sample to allow any target fungus-specific antibody which may be present in the sample to specifically bind to the immobilized antigen, thereby forming a first immobilized complex which includes either solid-phase/target fungus-specific antigen or solid- phase/target fungus-specific antigen/antibody depending on whether the target fungus-specific antibody was present in the sample, washing the first immobilized complex to remove any unbound target fungus antibody and/or other serum components, contacting the first immobilized complex with a detectable secondary antibody capable of binding to the target fungus-specific antibody, thereby forming a second immobilized complex which includes either solid-phase/target fungus-specific antigen or solid-phase/target fungus-specific antigen/antibody/secondary-antibody depending on whether
  • the detectable secondary antibody can be labeled according to methods known in the art or later developed.
  • the secondary antibody can be a radiolabeled antibody (e.g. an antibody labeled with a gamma-emitting 125 I isotope) which is detected by radiographic methods or with instruments such as counters which measure the level of radioactivity present.
  • the secondary antibody can also be an enzyme-conjugated antibody (e.g. an antibody conjugated with alkaline phosphatase, horseradish peroxidase, or other enzyme) which is detected by contacting the enzyme-conjugated antibody with a color-producing enzyme substrate.
  • the secondary antibody can alternatively be tagged with biotin (or an equivalent) or with a fluorochrome (e.g. fluorescein and rhodamine) or a dye or other colored substance (e.g. colloidal gold) which can be detected visually or by known spectroscopic methods.
  • the presence or absence of target fungus-specific antibodies can be detected using a Western blot format.
  • This method is particularly advantageous in that it includes a step for separating the target fungus- specific antigen from other proteins in the sample in which it is present (e.g. for isolating recombinantly- produced target fungus-specific antigen present in a host-cell lysate) .
  • This method includes electrophoretically separating a target fungus-specific protein antigen electrophoretically, transferring the separated protein antigen to a solid-phase membrane (e.g.
  • a nitrocellulose or nylon membrane contacting the solid-phase/target fungus-specific antigen complex with an undiluted sample or with a solution including the sample to allow any target fungus-specific antibody which may be present in the sample to bind to the antigen, and to form a first complex which includes either solid- phase/target fungus-specific antigen or solid- phase/target fungus-specific antigen/antibody, depending on whether antibody was present in the sample, washing the first complex to remove any unbound antibody, contacting the first complex with a detectable secondary antibody capable of binding to the solid phase-bound antibody, thereby forming a second immobilized complex which includes either solid-phase/target fungus-specific antigen or solid-phase/target fungus-specific antigen/antibody/secondary-antibody depending on whether target fungus-specific antibody was present in the sample, washing the second immobilized complex to remove any unbound secondary antibody, and detecting the presence or absence of the secondary antibody on the second immobilized complex.
  • the detectable secondary antibody can, for example
  • the presence or absence of target fungus-specific antibodies can, in another exemplary method, be detected using a direct label-based immunoassay.
  • This method includes immobilizing a first anti-immunoglobulin antibody (e.g. IgG) capable of binding to the target fungus antibody being detected on a solid-phase (e.g.
  • contacting the immobilized anti-immunoglobulin antibody with an undiluted sample or with a solution including the sample to form a first complex which includes either solid- phase/anti-immunoglobulin-antibody or solid-phase/anti- immunoglobulin-antibody/target fungus-specific antibody, depending on whether target fungus-specific antibody was present in the sample, washing the first complex, contacting the first complex with a labeled target fungus-specific antigen to allow any target fungus- specific antibody present in the first complex to bind to the target fungus-specific antigen and to form a second complex comprising either solid-phase/anti- immunoglobulin-antibody or solid-phase/anti- immunoglobulin-antibody/target fungus-specific antibody/target fungus-specific antigen, depending on whether target fungus-specific antibody was present in the sample, washing the second complex to remove any unbound labeled target fungus-specific antigen, and detecting whether the labeled target fungus-specific antigen,
  • the target fungus-specific antigen can be labeled according to methods now known in the art or later developed, including, for example, being radiolabeled, enzyme- conjugated, tagged with a fluorochrome, dyed or otherwise associated with a colored material, as described above.
  • the presence or absence of target fungus-specific antibodies can, in a further exemplary method, be detected using a inhibition/competitive label-based immunoassays.
  • This method includes establishing a baseline reading for a control assay by immobilizing a target fungus-specific antigen on a solid-phase, contacting the immobilized target fungus-specific antigen with a detectable (e.g.
  • the method further includes, in a separate, independent test assay, immobilizing a target fungus-specific antigen on a solid-phase, contacting the immobilized target fungus-specific antigen with both (1) a detectable (e.g.
  • target fungus-specific antibody and (2) an undiluted sample or with a solution including the sample to allow any target fungus-specific antibody which may be present in the sample to bind to at least some of the immobilized target fungus-specific antigen and to thereby form a test complex in which at least some of the bound detectable target fungus-specific antibody may have been competitively inhibited from binding to the immobilized target fungus-specific antigen, depending on whether target fungus-specific antibody was present in the sample, washing the test complex to remove any unbound target fungus-specific antibody, detecting the level of detectable-target fungus-specific antibody bound to the test complex, and comparing the level of detectable- target fungus-specific antibody bound to the test complex to the baseline level of detectable target fungus- specific antibody bound to the control complex, a decrease in such level indicating the presence of target fungus-specific antibody in the sample.
  • the detectable target fungus-specific antibody can, for example, be radiolabeled, enzyme-conjugate
  • the assays can optionally include one or more blocking steps or proteins or detergents in the diluent to decrease the non-specific binding of antibodies, primary or secondary, to the solid-phase.
  • the assays of the invention can also be automated, with appropriate modifications to the described steps. All of the above antibody assays are effective in detecting target fungus- specific antibodies for any fungus, including H. capsulatum, which is capable of eliciting an antibody response in a vertebrate .
  • the antigen assays of the present invention are useful in any situation where a determination of the presence or absence of a target fungus is desired. Examples include the determination of the presence of a target fungus in an animal or plant suspected of being infected with the target fungus, in a food or feed suspected of being contaminated with the target fungus, in inorganic materials such as soil or air for the determination of the presence of an allergenic or pathogenic target fungus, and for the identification of a specific target fungus where the identity of the fungus is unknown.
  • the antigen assays of the present invention can be more specifically characterized according to a variety of formats set forth below and/or known in the art . One approach includes the use of precipitin-based immunoassays.
  • the presence or absence of the target fungus-specific antigens in the sample can be detected by layering a first solution including the target fungus-specific antibody over the undiluted sample or over a second solution including the sample, the layered solution typically being formed in a container such as a test-tube, and observing the layered solution for the formation or the lack of formation of a precipitate comprising bound antigen and antibody.
  • the amount of target fungus-specific antibody in the first solution is preferably an amount which is necessary, on average, to form a precipitate with samples previously known to comprise the target fungus.
  • the steps of this approach can, alternatively, be repeated, in parallel or in series, using various amounts of the target fungus-specific antibody in the first solution, with the amount of antibody in the solution varying over a range which includes an amount which is about, on average, necessary to form a precipitate with samples previously known to contain the target fungus-specific antigen.
  • Precipitin- based immunoassays can also be carried out in gels such as agar or polyacrylamide gels or their equivalents known in the art, by methods typically referred to as immunodiffusion, immunoelectrophoresis, counterimmunoelectrophoresis and/or rocket electrophoresis, among others.
  • the presence or absence of the antigens are detected by placing a first solution including the target fungus-specific antibody in a gel or in a well adjacent to a gel, placing the undiluted sample or a second solution including the sample, in a gel or in a well adjacent to a gel, allowing the target fungus-specific antibody and antigens to diffuse within the gel and observing the gel for the formation or the lack of formation of a precipitate comprising bound antigen and antibody.
  • Another approach for the antigen assay of the present invention includes the use of label-based assay techniques, including direct, indirect and/or inhibition/competitive radioimmunoassays, enzyme-linked immunoabsorbant assays (ELISA) , immunofluorescent assays, immunochromatographic assays, and other techniques known in the art.
  • label-based assay techniques including direct, indirect and/or inhibition/competitive radioimmunoassays, enzyme-linked immunoabsorbant assays (ELISA) , immunofluorescent assays, immunochromatographic assays, and other techniques known in the art.
  • the presence or absence of the antigens can be detected in a direct sandwich-type format by immobilizing target fungus-specific antibody on a solid-phase, contacting the immobilized antibody with an undiluted sample or with a solution including the sample to allow any target fungus-specific antigen which may be present in the sample to bind to the immobilized antibody, thereby forming a first immobilized complex which includes either solid-phase/target fungus-specific antibody or solid-phase/target fungus-specific antibody/target fungus-specific antigen, depending on whether the target fungus-specific antigen was present in the sample, washing the first immobilized complex to remove any unbound target fungus-specific antigen, contacting the complex with a detectable secondary antibody capable of binding to a different epitope on the target fungus-specific antigen, thereby forming a second immobilized complex which includes either solid- phase/target fungus-specific antibody or solid- phase/target fungus-specific antibody/target fungus-specific antigen/second
  • the presence or absence of target fungus-specific antigen can be detected using a Western blot format.
  • This method includes electrophoretically separating proteins contained in the sample in a gel (e.g. such as a polyacrylamide gel), electrophoretically transferring the separated proteins to a solid-phase -membrane (e.g.
  • a nitrocellulose membrane contacting the separated proteins with an unlabeled target fungus- specific antibody to allow any target fungus-specific antigen which may have been present in the sample to bind to the antibody and form a first complex with includes either solid-phase/target fungus-specific antigen or solid-phase/target fungus-specific antigen/target fungus- specific antibody, depending on whether target fungus- specific antigen was present in the sample, washing the first complex to remove unbound target fungus-specific antibody, contacting the first complex with a detectable secondary antibody to form second complex which includes either solid-phase/target fungus-specific antigen or solid-phase/target fungus-specific antigen/target fungus- specific antibody/secondary-antibody, depending on whether target fungus-specific antigen was present in the sample, and detecting the presence or absence of the secondary antibody bound to the antigen in the second complex.
  • the secondary antibody can, for example, be radiolabeled, enzyme-conjugated, tagged with biotin or an equivalent
  • the presence or absence of the antigens in the sample can be detected using a method which includes electrophoretically separating proteins contained in the sample in a gel (e.g. such as a polyacrylamide gel), transferring the separated proteins to a solid-phase membrane (e.g.
  • a gel e.g. such as a polyacrylamide gel
  • a solid-phase membrane e.g.
  • target fungus-specific antibody can, for example, be radiolabeled, enzyme-conjugated, tagged with a fluorochrome or dyed, as described above.
  • the presence or absence of the antigens in a sample can be detected in an inhibition/competitive-type format by mixing a solution including a detectable (e.g. labeled) target fungus- specific antibody with the undiluted sample or with a solution including the sample to allow any target fungus- specific antigen which may be present in the sample to bind with the detectable target fungus-specific antibody and to form a test solution which includes either unbound detectable target fungus-specific antibody or a detectable target fungus-specific antibody/target fungus- specific antigen complex depending on whether target fungus-specific antigen was present in the sample.
  • a detectable target fungus-specific antibody e.g. labeled
  • the method further includes immobilizing a target fungus- specific antigen on a solid-phase, contacting the immobilized target fungus-specific antigen with the test solution to allow any unbound detectable target fungus- specific antibody present in the test solution to bind with the immobilized target fungus-specific antigen and form an immobilized complex including solid-phase/target fungus-specific antigen or solid-phase/target fungus- specific antigen/detectable target fungus-specific antibody depending on whether target fungus-specific antigen was present in the sample, washing the solid- phase to remove any unbound target fungus-specific antibody, and measuring the presence or absence of the detectable target fungus-specific antibody on the immobilized complex.
  • an antigen assay can include immobilizing a target fungus-specific antibody on a solid-phase, contacting the immobilized antibody with both (1) a detectable (e.g.
  • target fungus-specific antigen labeled
  • target fungus-specific antigen an undiluted sample or a solution including the sample to allow any target fungus- specific antigen which may be present in the sample to bind to at least some of the immobilized target fungus- specific antibody and to thereby form a test complex in which at least some of the detectable target fungus- specific antigen may have been competitively inhibited from binding to the immobilized target fungus-specific antibody, depending on whether target fungus-specific antigen was present in the sample, washing the test complex to remove any unbound target fungus-specific antigen, and detecting the level of detectable target fungus-specific antigen bound to the test complex.
  • the level of detectable target fungus-specific antigen bound to the test complex can be compared to a baseline level of detectable target fungus-specific antigen bound to a control complex, with a decrease in such level indicating the presence of target fungus- specific antigen in the sample.
  • the detectable target fungus-specific antigen can, for example, be radiolabeled, enzyme-conjugated, tagged with biotin or an equivalent thereto, a fluorochrome, dyed or otherwise associated with a colored material as described above.
  • the assays can optionally include one or more blocking steps to decrease the non-specific binding of antibodies, primary or secondary, to the solid-phase.
  • the assays of the invention can also be automated, with appropriate modifications to the described steps. All of the above antigen assays are effective in detecting target fungus- specific antigens for any fungus, including H. capsulatum.
  • Kits are provided which are suitable for use in performing the aforementioned assay methods to facilitate diagnosis of histoplasmosis in humans and other mammals.
  • an assay kit of the present invention can include labeled and/or unlabeled target fungus-specific antigen, as described above, in quantities sufficient to carry out the assays of the present invention.
  • an assay kit can include labeled and/or unlabeled antibodies to an target fungus-specific antigen, as described above, in quantities sufficient to carry out the assays of the present invention.
  • an assay kit of the present invention can include both labeled and/or unlabeled target fungus-specific antigen and labeled and/or unlabeled antibody thereto, each as described above, in quantities sufficient to carry out the assays of the present invention.
  • an assay kit can also further comprise known positive and/or negative control samples, other reagents useful in carrying out the assays of the present invention (e.g. radiolabeled secondary antibodies and/or enzyme-conjugated secondary antibodies along with the corresponding color-producing enzyme substrate therefore) , and instructions for carrying out the assay methods.
  • Kits as provided above may be utilized for any target fungus, including H. capsulatum.
  • H. capsulatum G217B a North American isolate, was obtained from the American Type Culture Collection (ATCC #26032, Rockville, MD) .
  • Mycelial-phase organisms were cultured in a shaking water bath at 25°C in broth containing 2% glucose and 1% yeast extract.
  • Yeast-phase organisms were grown at 37°C in HMM broth (Gibco-BRL, Gaithersburg, MD) supplemented with 18.2 g of glucose, 1.0 g of glutamic acid (per liter), adjusted to pH 7.5 (17) . Human and animal sera.
  • Control human sera were obtained from healthy residents of St. Louis, MO.
  • Genomic DNA from yeast cells was isolated essentially as previously described (17,34). Briefly, yeast cells were pelleted and resuspended in TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA) . SDS was added to 1% final concentration. DNA was extracted using phenol-chloroform, ethanol precipitated, and washed with 70% ethanol. Mouse sera.
  • Antibodies to yeast antigen or to a histidine fusion protein of the recombinant clone GH17 were produced in 6 -week-old female BALB/c mice by foot-pad injection of 10 ⁇ g of yeast antigen or purified GH17-his in FCA followed by a second injection of antigen in IFA 4 weeks later. Sera were collected 1 week after the booster immunization. Yeast antigen.
  • Yeast cells were suspended in 0.01M Tris buffer (pH 8.3) containing protease inhibitors (1 mM phenyl methyl sulfonyl fluoride, 1 mM EDTA, 25 ⁇ g/ml N- tosyl-L-phenylalanine chloromethyl ketone, and 25 ⁇ g/ml N-a-p-tosyl-L-lysine chloromethyl ketone (all from Sigma Chemical Company, St. Louis, MO) .
  • the yeast homogenate was rocked at 4°C overnight and centrifuged at 15,000 x g for 10 min. The protein concentration in the supernatant was measured with a commercial kit (BCA; Pierce Chemical Co., Rockford, IL) .
  • a ⁇ gtll cDNA library was custom- synthesized (Clontech Lab. Inc., Palo Alto, CA) using Poly (A) + mRNA derived from the mycelial stage of the G217B strain of H. capsulatum. This library has a recombinant frequency of over 90% after amplification.
  • the DNA insert size range is 0.6-4.5 kb with an average size of 1.6 kb.
  • the library was immunoscreened to identify H. capsulatum-specific clones essentially as previously described (5,6) .
  • Clones that were reactive with antibodies in the histoplasmosis serum pool but not reactive with a normal human serum pool were selected and purified by repeated cycles of immunoselection.
  • the reactivity of serum pools to fusion proteins expressed by purified recombinant phage was studied by plaque-dot immunoblot analysis as previously described (5) .
  • PCR was employed to amplify the cDNA inserts of selected recombinant ⁇ gtll clones with the GenAmp DNA amplification kit (Perkin Elmer-Cetus, Norwalk, CT) as previously described (31) .
  • DNA dot hybridization was performed using peroxidase-labeled DNA fragments (14) to assess homology between the selected clones.
  • H. capsulatum genomic DNA (5 ⁇ g) was cut with selected restriction endonuclease enzymes. Digestion products were electrophoresed in a 1% agarose gel and transferred to Hybond-N+ nylon transfer membrane (Amersham, Arlington Heights, IL) by standard techniques, and blots were probed with labeled cDNA insert of GH17 (26) .
  • ⁇ gtll DNA purified from GH17 was digested with EcoRI and ligated into pBluescript II SK- (Strategene Cloning Systems, La Jolla, CA) by standard methods (26) , and plasmid DNA was prepared for DNA sequencing.
  • the dideoxynucleotide chain termination method (32) was used for double stranded DNA sequencing using the TaqTrack Sequencing System (Promega Corporation, Madison, WI) with T3 and T7 pBluescript primers and with synthetic oligonucleotides .
  • the PC/GENE DNA Sequence Analysis Software (Intelligenetics, Mountain View, CA) and the BLAST Program (NCBI, NLM, NIH, Bethesda, MD) were used to analyze nucleotide and deduced amino acid sequences and to determine sequence homologies with previously reported sequences in the GenBankTM data base .
  • GH17 fusion protein (GH17-his) was purified from bacterial lysates by continuous-elution electrophoresis using a BioRad Prep Cell (BioRad Laboratories, Hercules, CA) .
  • EDTA 10 mM EGTA, pH 7.0
  • the cells were frozen at -20°C overnight. Cells were thawed in cold water and lysed by mild sonication. Cellular debris was removed by centrifugation at 10,000 x g for 15 min. A 12% polyacrylamide gel was poured per the manufacturer's protocol for the BioRad Model 491 Prep Cell. Ten ml of sample in 1:1 loading buffer (0.0625M Tris-HCl, pH 6.8, 10% glycerol, 0.025% bromphenol blue) was loaded and the gel was run for 8 hrs at 12W constant power. Three ml fractions were collected and run on 12% SDS-PAGE minigels (21) .
  • the presumed initiation codon 36 bp downstream from the 5' end is the first ATG in the ORF.
  • the sequence also has a purine (adenine) in the -3 position (Kozak's rule), a prerequisite for an initiation codon (19) .
  • the initiation codon is followed by a hydrophobic sequence (predicted by hydropathy analysis, Fig. 2) which is consistent with a signal peptide sequence.
  • Two potential signal peptidase cleavage sites were identified by the method of von Heijne (37) which predicts cleavage after residues 20 and 24.
  • the sequence also contains a predicted transmembrane helix from amino acid 2 to 28
  • the 3' non-coding region has a poly (A) tail of 14 bp.
  • the ORF codes for a protein of 211 amino acids with a predicted size of 23.5 kDa and a calculated pi of 4.15. There are three potential N-glycosylation sites in the predicted amino acid sequence; these are located in the hydrophilic domains of the protein (boxed areas, Fig. 1) .
  • the GH17 sequence does not exhibit significant similarity to any proteins present in GenBank/EMBL sequence databases except for the similarity of the threonine-rich region to other threonine rich sequences such as cellulase of Caldocellum saccharolyticum (25) , Xenopus laevis integumentary mucin (12) , and a Leishmania surface antigen (27) (Fig. 2) .
  • GH17 has an internal SacI site. These results suggest a single location in the H. capsulatum genome for GH17. Sensitivity and specificity of IgG antibodies to recombinant H. capsulatum proteins. Immunoreactivity of recombinant H. capsulatum proteins produced by clones GH2 , 17, 22, 23 was assessed by Western blot with sera from patients with a variety of fungal infections. Most sera from histoplasmosis patients had easily visible antibody reactivity with all 4 recombinant proteins (Table 1, Fig. 4A) . The sensitivity of Western blot with these clones for histoplasmosis sera ranged from 89-100% (Table 1, Fig. 4A) . None of these clones was recognized by sera from humans and animals infected with other fungi (Fig. 4B) .
  • the cDNA insert of GH17 was expressed as histidine fusion in the pPROEXTM -1 protein expression system.
  • Plasmid pPROEX-1 consists of a Trc promotor for high level expression in E. coli , a prokaryotic ribosome binding site, and a 6X His affinity tag for ease of purification.
  • a fusion protein with an apparent size of 32 kDa was evident by SDS-PAGE and immunoblot (Fig. 5A) .
  • GH17-his failed to bind to a metal affinity column. Therefore, the fusion protein was purified from bacterial lysates by continuous-elution electrophoresis using a BioRad PrepCell.
  • Western blots were performed to select fractions of interest by immunoblotting with a human histoplasmosis serum pool (Fig. 5B) . Three consecutive fractions containing the band of interest were selected, pooled and dialyzed. This yielded a total of 500 ⁇ g of purified protein from approximately 700 ml of bacterial culture .
  • H. capsulatum-specific antigens can be identified, cloned, characterized, and produced using recombinant DNA methodologies and other methods, as described.
  • a recombinant H. capsulatum antigen was shown to have highly specific and sensitive immunodiagnostic potential.
  • Recombinant clones that expressed H. capsula turn- specific antigens were identified by several cycles of differential immunoscreening, and the most immunoreactive and specific clone (GH17) was selected for more detailed studies .
  • GH17 codes for the most promising recombinant diagnostic antigen for histoplasmosis that has been identified to date.
  • GH17 codes for a protein that corresponds to a 60 kDa native H.
  • capsulatum antigen There are three potential N-glycosylation sites [Asn- Asn/Lys-Thr] in the predicted amino acid sequence of GH17 (boxed areas, Fig. 1) . Glycosylation at these sites could account for the difference between the predicted polypeptide mass of 23.5 kDa and the observed size of the native yeast protein recognized by the mouse antibodies to GH17 histidine fusion protein (60 kDa) .
  • the protein encoded by GH17 is highly antigenic in humans with histoplasmosis. Chou-Fasman predictions based on the deduced amino acid sequence of GH17 (7) indicate that the protein is rich in potential B cell epitopes. These predictions are based principally on the hydrophilic character and accessibility of highly charged and exposed polar residues that comprise the turns and alpha helices within the predicted GH17 protein (13,20) . Our results with human sera are consistent with these predictions. GH17 produced a 140 kDa fusion protein that was recognized in Western blots by 18 of 18 sera from patients with histoplasmosis.
  • the present invention provides a method to isolate specific fungal antigens.
  • the present invention offers significant advantages over prior art fungal antigens, antibodies and diagnostic methods employing the same.
  • the specificity of the present target fungus antigens and antibodies make them particularly advantageous for reliable determination of the presence of the target fungus or antibodies which are specific to the target fungus.
  • target fungi which are pathogens of vertebrates, such as H. capsulatum
  • use of antigens and antibodies of the present invention are useful for providing reliable evidence of present or past infection with H. capsulatum.
  • Other features, objects and advantages of the present invention will be apparent to those skilled in the art.
  • Radioimmunoassay a sensitive screening test for histoplasmosis and blastomycosis . Am. Rev. Resp.
  • Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes . Cell 44:283- 292.
  • ORGANISM Histoplasma Capsulatum

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Abstract

La présente invention concerne un procédé d'identification d'un antigène de protéine dirigé contre un champignon cible. Le procédé comprend également une sélection de protéines exprimées provenant d'une bibliothèque d'expression de gènes d'ADNc à l'aide d'antisérums dirigés contre le champignon cible et une sélection croisée à l'aide d'un antisérum dirigé contre un champignon non cible. La présente invention concerne également des anticorps dirigés contre l'antigène de protéine. La présente invention concerne, en outre, des procédés de détection de la présence ou de l'absence des anticorps ou de l'antigène de protéine ainsi que des équipements permettant d'exécuter ces analyses. Selon un mode de réalisation préféré, le champignon cible est H. capsulatum.
PCT/US1998/007728 1997-04-15 1998-04-15 Procedes d'obtention d'antigenes specifiques aux champignons, anticorps pour ces antigenes et diagnostic de maladie au moyen de ces antigenes et/ou anticorps WO1998046738A1 (fr)

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CA002286335A CA2286335C (fr) 1997-04-15 1998-04-15 Procedes d'obtention d'antigenes specifiques aux champignons, anticorps pour ces antigenes et diagnostic de maladie au moyen de ces antigenes et/ou anticorps

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Cited By (5)

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WO2001011081A2 (fr) * 1999-08-06 2001-02-15 Ivigene Corporation Polynucleotides microbiens exprimes durant l'infection d'un hote
WO2001011366A1 (fr) * 1999-08-05 2001-02-15 Oy Medix Biochemica Ab Composants antigeniques de champignons indicateurs choisis et leur utilisation dans des procedes et trousses d'essais
US7033748B2 (en) 1999-08-06 2006-04-25 Ivigene Corporation Identification of microbial polynucleotides expressed during infection of a host
US7052837B2 (en) 2001-03-13 2006-05-30 The Board Of Trustees Of The University Of Arkansas Histoplasma capsulatum catalase sequences and their use in the detection of Histoplamsa capsulatum and histoplasmosis
US7217519B1 (en) 2002-11-21 2007-05-15 The Board Of Trustees Of The University Of Arkansas Histoplasma capsulatum chitin synthase sequences and their use for detection of Histoplasma capsulatum and histoplasmosis

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"ANTIBODIES TO MAJOR HISTOCOMPATIBILITY ANTIGENS PRODUCED BY HYBRID CELL LINES.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 266., 7 April 1977 (1977-04-07), United Kingdom, pages 550 - 552., XP002914004, ISSN: 0028-0836, DOI: 10.1038/266550a0 *
CHANDRASHEKAR R., ET AL.: "MOLECULAR CLONING AND CHARACTERIZATION OF RRECOMBINATION PARASITO ANTIGENS FOR IMMUNODIAGONOSIS OF ORCHOCERCIASIS.", JOURNAL OF CLINICAL INVESTIGATION, AMERICAN SOCIETY FOR CLINICAL INVESTIGATION, US, vol. 88., 1 November 1991 (1991-11-01), US, pages 1460 - 1466., XP002913999, ISSN: 0021-9738, DOI: 10.1172/JCI115455 *
CHANDRASHEKAR R., ET AL.: "MOLECULAR CLONING OF BRUGIA MALAYI ANTIGENS FOR DIAGNOSIS OF LYMPHATIC FILARIASIS.", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 64., 1 January 1994 (1994-01-01), NL, pages 261 - 271., XP002914003, ISSN: 0166-6851, DOI: 10.1016/0166-6851(94)00035-2 *
CHUNG K.: "EMMONSIELLA CAPSULATA: PERFECT STATE OF HISTOPLASMA CAPSULATUM.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 177., 28 July 1972 (1972-07-28), US, pages 368/369., XP002914008, ISSN: 0036-8075, DOI: 10.1126/science.177.4046.368 *
GEORGE R. B., ET AL.: "RADIOIMMUNOASSAY: A SENSITIVE SCREENING TEST FOR HISTOPLASMOSIS AND BLASTOMYCOSIS.", THE AMERICAN REVIEW OF RESPIRATORY DISEASE, AMERICAN THORACIC SOCIETY, US, vol. 124., no. 04., 1 October 1981 (1981-10-01), US, pages 407 - 410., XP002914007, ISSN: 0003-0805 *
KAUFMAN L.: "LABORATORY METHODS FOR THE DIAGNOSIS AND CONFIRMATION OF SYSTEMIC MYCOSES.", CLINICAL INFECTIOUS DISEASES, THE UNIVERSITY OF CHICAGO PRESS, CHICAGO, IL., US, vol. 14., no. SUPPL., 1 January 1992 (1992-01-01), US, pages 523 - 529., XP002914001, ISSN: 1058-4838 *
KUMAR B. V., ET AL.: "CROSS-REACTING HUMAN AND RABBIT ANTIBODIES TO ANTIGENS OF HISTOPLASMA CAPSULATUM, CANDIDA ALBICANS, AND SACCHAROMYCES CEREVISIAE.", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 48., no. 03., 1 June 1985 (1985-06-01), US, pages 806 - 812., XP002914002, ISSN: 0019-9567 *
LOWMAN H. B., ET AL.: "SELECTING HIGH-AFFINITY BINDING PROTEINS BY MONOVALENT PHAGE DISPLAY.", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 30., 1 January 1991 (1991-01-01), US, pages 10832 - 10838., XP002914005, ISSN: 0006-2960, DOI: 10.1021/bi00109a004 *
WHEAT L. J., ET AL.: "DIAGNOSIS OF DISSEMINATED HISTOPLASMOSIS BY DETECTION OF HISTOPLASMA CAPUSLATUM ANTIGEN IN SERUM AND URINE SPECIMENS.", NEW ENGLAND JOURNAL OF MEDICINE, MASSACHUSETTS MEDICAL SOCIETY, US, vol. 314., no. 02., 9 January 1986 (1986-01-09), US, pages 83 - 88., XP002914006, ISSN: 0028-4793 *
ZIMMERMANN C. R., ET AL.: "CLONING AND EXPRESSION OF THE COMPLEMENT FIXATION: ANTIGEN- CHITINASE OF COCCIDIOIDES IMMITIS.", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 64., no. 12., 1 December 1996 (1996-12-01), US, pages 4967 - 4975., XP002914000, ISSN: 0019-9567 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011366A1 (fr) * 1999-08-05 2001-02-15 Oy Medix Biochemica Ab Composants antigeniques de champignons indicateurs choisis et leur utilisation dans des procedes et trousses d'essais
WO2001011081A2 (fr) * 1999-08-06 2001-02-15 Ivigene Corporation Polynucleotides microbiens exprimes durant l'infection d'un hote
WO2001011081A3 (fr) * 1999-08-06 2002-07-11 Ivigene Corp Polynucleotides microbiens exprimes durant l'infection d'un hote
US7033748B2 (en) 1999-08-06 2006-04-25 Ivigene Corporation Identification of microbial polynucleotides expressed during infection of a host
US8034571B1 (en) 1999-08-06 2011-10-11 Oragenics, Inc. Microbial polynucleotides expressed during infection of a host
US7052837B2 (en) 2001-03-13 2006-05-30 The Board Of Trustees Of The University Of Arkansas Histoplasma capsulatum catalase sequences and their use in the detection of Histoplamsa capsulatum and histoplasmosis
US7217519B1 (en) 2002-11-21 2007-05-15 The Board Of Trustees Of The University Of Arkansas Histoplasma capsulatum chitin synthase sequences and their use for detection of Histoplasma capsulatum and histoplasmosis

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