Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen.

Detection of anti-Coccidioides complement-fixing (CF) antibody is a valuable diagnostic and prognostic aid in coccidioidomycosis. The CF antibody response is directed against a heat-labile antigen that has chitinase activity, hereafter referred to as the CF/chitinase protein. To identify and clone this immunoreactive enzyme, we constructed a Coccidioides immitis cDNA lambda ZAP expression library from spherule RNA and detected fusion peptides expressing CF epitopes by immunoscreening. A cDNA clone consisting of 1,623 bp was identified, sequenced, and found to contain a single open reading frame that encodes a protein of 47 kDa with 427 amino acids. Deduced amino acid sequence analyses showed that the cloned CF/chitinase cDNA contains a 35-amino-acid region, beginning at Ser-18 and ending at and ending at Arg-52 which has 92% homology with the reported N-terminal amino acid sequence of authentic CF/chitinase protein. The first 17 amino acids in the deduced sequence of the cloned cDNA are not present on the mature CF/chitinase protein, suggesting that it may be a signal peptide. Expression of the CF/chitinase cDNA insert by using the pGEX-4T-3 vector yields a fusion peptide that bears CF-specific epitopes and shows chitinase activity. The CF/chitinase clone will enable large-scale production of the recombinant CF antigen for use in immunoassays and facilitate studies on the role of chitinase in the morphogenesis of C. immitis.     

Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen.

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, greater than 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.     

Molecular and biochemical characterization of a Coccidioides immitis-specific antigen.

Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections.     

Suppression of T-lymphocyte response by Coccidioides immitis antigen.

Intravenous injection of BALB/c mice with coccidioidin or an alkali-soluble cell wall extract of Coccidioides immitis mycelia resulted in the induction of a splenic cell population(s) that suppressed delayed-type hypersensitivity response to coccidioidal antigen. To determine whether the levels of C. immitis antigen produced during the course of active coccidioidal disease might also cause suppression of T-lymphocyte response, BALB/c mice were infected by intranasal instillation of arthroconidia, and 2 weeks later, their sera were evaluated for suppression of T-lymphocyte response in syngeneic recipients. Intravenous transfer of sera, which were shown to contain high levels of coccidioidal antigen by an enzyme-linked immunoadsorbent assay, suppressed the delayed-type hypersensitivity response of recipients to immunization with coccidioidin. Solid-phase immunoadsorption of the sera with goat antibodies to C. immitis antigens removed the suppressive component(s). To determine whether the suppressive effect of circulating coccidioidal antigen(s) was associated with the activation of a splenic suppressor cell(s), as was observed in mice injected intravenously with coccidioidal antigen, spleen cell lysates were prepared from infected donors, and after filtration to remove viable fungi, the lysates were transferred to syngeneic mice. Recipients of lysates from infected but not noninfected donors were suppressed in their response to immunization with coccidioidin. Collectively, these results provide evidence that depressed T-cell responses observed in coccidioidomycosis are associated with, and may be attributable to, the activation of a suppressor cell or factor by circulating C. immitis antigens.     

Identification of a Coccidioides immitis antigen 2 domain that expresses B-cell-reactive epitopes.

Antigen 2 (Ag2), a major immunoreactive component of Coccidioides immitis mycelium- and spherule-phase cell walls, was recently cloned in our laboratory and was shown to elicit T-cell responses in Coccidioides-immune mice. In this investigation, we evaluated recombinant Ag2 (rAg2) and PCR-generated Ag2 truncations for expression of B-cell-reactive epitopes in enzyme-linked immunosorbent and immunoblot assays with sera from patients with active coccidioidomycosis, a hyperimmune goat anti-Ag2 serum, and a murine anti-Ag2 monoclonal antibody that recognizes a conformational epitope. The results established that rAg2 expresses both linear and conformational B-cell-reactive epitopes which are localized to a domain comprised of amino acids 19 through 96 (designated A19-96). Truncations designed to identify epitopes within the A19-96 domain yielded fragments that either were nonreactive (A62-194, A19-61, and A49-79) or showed reduced reactivity (A19-79). Hence, A19-96 was the shortest domain expressing epitopes recognized by the panel of antibodies. The prevalence of antibodies to the A19-96 domain was evaluated in enzyme-linked immunosorbent assays of sera from 28 coccidioidomycosis patients. Antibody reactivity was detected in 79% of the patients' sera, and the level of antibody reactivity was directly correlated with disease severity. Whereas patients with pulmonary disease showed a mean response (A405) of 0.16 +/- 0.04, patients with disseminated coccidioidomycosis showed a mean response of 0.69 +/- 0.17 (P < 0.05). No reactivity was detected with sera from histoplasmosis or blastomycosis patients. The production of a recombinant peptide that expresses C. immitis-specific Ag2 epitopes provides a useful reagent for examining the role of anti-Ag2 antibodies in coccidioidomycosis.     

Production and characterization of a monoclonal antibody to the complement fixation antigen of Coccidioides immitis.

Detection of complement-fixing antibody to coccidioidin by using the complement fixation test or an immunodiffusion assay for complement-fixing antibody (IDCF) is widely viewed as the most useful immunodiagnostic test for coccidioidomycosis. In this investigation, we report the production of an immunoglobulin G subclass 1 (IgG1) monoclonal antibody (MAb) to the IDCF antigen for use as a biospecific ligand for purifying the IDCF antigen on solid-phase immunosorbents and for use as a reagent for screening genomic or cDNA expression libraries from Coccidioides immitis. BALB/c mice were immunized by intramuscular injections of coccidioidin in adjuvant, followed by an intrasplenic booster injection of coccidioidin in saline. The spleen cells were fused with SP2/0 Ag14 myeloma cells, and the fusion products were screened for IgG antibody to coccidiodin by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned and evaluated for reactivity to the IDCF antigen by two-dimensional immunoelectrophoresis and by immunoblotting. An IgG1 Mab was produced that was specific for the IDCF antigen when evaluated by two-dimensional immunoelectrophoresis and immunoblotting. The epitope recognized by the MAb was heat labile (60 degrees C, 30 min) and susceptible to enzymatic digestion with pronase but was resistant to treatment with lipase, alpha-mannosidase, glucose oxidase, and endoglycosidase H. This heat-labile peptide epitope appears to be specific to C. immitis, as judged by the fact that the MAb was not reactive in immunoblots or enzyme-linked immunosorbent assays of histoplasmin or blastomycin.     

Development and characterization of a monoclonal antibody against the tube precipitin antigen of Coccidioides immitis.

Primary infection with Coccidioides immitis is commonly accompanied by the production of an immunoglobulin M precipitin antibody which is detected by the tube precipitin (TP) assay or by the immunodiffusion assay for TP antibody (IDTP assay). In the present investigation, spleen cells from spherulin-immunized BALB/c mice were fused with SP2/O Ag14 myeloma cells, and the resulting hybridomas were screened for antibody to the IDTP antigen by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned by limiting dilution and injected into pristane-primed mice for ascites production. Characterization of antibody reactivity was accomplished with the IDTP assay, two-dimensional immunoelectrophoresis, and immunoblotting. An immunoglobulin G1 monoclonal antibody which reacts with the IDTP antigen of C. immitis is described. The epitope that is recognized by the monoclonal antibody is also present, but to a lesser extent, on a second coccidioidal antigen which has been designated antigen 2. The monoclonal antibody was not reactive in immunoblots of histoplasmin or blastomycin, indicating that the epitope recognized by this antibody may be specific for C. immitis.     

Isolation and characterization of an extracellular proteinase of Coccidioides immitis.

A proteinase isolated from the respiratory pathogen, Coccidioides immitis, was shown to have collagenolytic and elastinolytic activity, as well as the ability to cleave human serum immunoglobulin G and secretory immunoglobulin A. Proteolytic activity was demonstrated with a bovine casein digestion assay in conidial culture exudates, mycelial and spherule culture filtrates, conidial and spherule wall material, and Sephacryl S-300 fractions of the isolated soluble conidial wall material described previously. One of the latter fractions (fraction 2) demonstrated high proteolytic activity. The proteinase was purified from this chromatographic fraction by cold acetone extraction followed by Sephadex G-50 gel filtration and was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By means of tandem two-dimensional immunoelectrophoresis, the proteinase was identified as antigen 11 on the basis of its reaction in the coccidioidin/anticoccidioidin reference system. The proteinase is characterized by a broad substrate specificity, optimal activity at 35 to 40 degrees C (pH 8.0) in the presence of human collagen, elastin, or hemoglobin, an isoelectric point of pH 4.5, and inhibition by organofluorides, N-tosyl-L-phenylalanine chloromethyl ketone, chymostatin, and alpha-1-antitrypsin. These features of the enzyme are comparable to those of chymotrypsinlike serine proteinases. Demonstration that the proteinase can cleave human immunoglobulins and digest ubiquitous tissue structural proteins (e.g., collagen and elastin) suggests that it may play a role in the virulence of the fungal pathogen.     

Molecular approaches to the study of Coccidioides immitis

The study of the molecular biology of Coccidioides sp. is only just beginning. As the importance of coccidioidomycosis grows as a public health problem, our need for understanding of pathogenesis, immune responses, and improved antifungal therapy also increases in proportion. Tools have now become available to study gene manipulation in this pathogen and this will allow molecular approaches to be used. Genetic experiments will also be accelerated by the availability of the whole coccidioidal genome, expected to be made public in the spring of 2003 (see http://www.tigr.org/tdb/tgi/cigi/GenInfo.html). Thus, there seems to be several reasons to expect considerable progress in the coming years.     

Collagenolytic activity of Coccidioides immitis.

Coccidioides immitis appears to be unable to digest particulate collagen when cultured on collagen-containing semisolid culture media. However, all C. immitis strains solubilized collagen when the fungus was grown in liquid suspension cultures. Moreover, sterile culture filtrates were collagenolytic in collagen-buffer-agar plate assays.     

Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum.

Detection of antibody to Coccidioides immitis F antigen is of proved value in the diagnosis of coccidioidomycosis. This antibody is demonstrable by use of an immunodiffusion assay with reference coccidioidin antigen and antiserum to C. immitis. Using a combination of lectin affinity and immunoaffinity chromatography, we isolated the F antigen from coccidioidin and prepared monospecific antibody to the purified antigen. The availability of these reagents will enable the development of a sensitive and specific assay for detecting serologic reactivity to this antigen.     

Immunoaffinity isolation and partial characterization of the Coccidioides immitis antigen detected by the tube precipitin and immunodiffusion-tube precipitin tests.

The antigen participating in the tube precipitin (TP) serologic test for coccidioidomycosis was isolated from mycelial-phase antigen (coccidioidin) by immunoaffinity and characterized by various analytical procedures. This was accomplished by first preparing the antigen-antibody precipitate by using antigen and human serum positive for TP (immunoglobulin M) antibody and then liberating the antigen by digestion with pronase. This protease destroyed the antibody and left the antigen intact as indicated by immunodiffusion-TP. The coccidioidal antigen was isolated from the proteolytic digest by using size exclusion chromatography. DEAE chromatography of this antigen yielded two fractions with immunodiffusion-TP reactivity which had average molecular sizes of 225 and 140 kilodaltons, respectively. The presence of carbohydrate and amino acids indicated that the antigen(s) is a glycopeptide. Compositional analysis showed that one fraction contained 3-O-methylmannose, mannose, and glucose in a ratio of 8:1.2:1, whereas the second fraction contained 3-O-methylmannose, mannose, glucose, and galactose in a ratio of 1:1:1:1. The amino acids glycine, alanine, serine, threonine, aspartic acid plus asparagine, and glutamic acid plus glutamine constituted 60 to 70% of the amino acids in both glycopeptides. Neither antigen could be detected entering the gel in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lectin affinity provided evidence of a high-mannose asparagine-linked glycopeptide in the first peak and an asparagine-linked glycopeptide with a biantennary complex-type structure in the second peak.     

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.     

Genetic Vaccination against Coccidioides immitis: Comparison of Vaccine Efficacy of Recombinant Antigen 2 and Antigen 2 cDNA

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.     

Production of a murine monoclonal antibody that recognizes an epitope specific to Coccidioides immitis antigen 2.

Antigen 2 (Ag2) has been implicated as a T-cell-reactive component of the pathogenic fungus Coccidioides immitis. We report the production of a murine monoclonal antibody (MAb) of the immunoglobulin G2a isotype that recognizes an epitope specific to C. immitis Ag2. This specificity was evidenced by the finding that the MAb did not recognize other antigens present in coccidioidin or spherulin and did not show reactivity with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis. The epitope was labile to enzymatic digestion with pronase but resistant to treatment with glycolytic enzymes and to periodate oxidation. This peptide epitope appears to require conformational structure on the basis that it was not recognized by the MAb in immunoblots of antigen that had been electrophoresed in polyacrylamide gels under denaturing, reducing conditions. Immunoaffinity chromatography of spherulin on columns containing the MAb established that the MAb was effective as a ligand for isolating Ag2 from heterogeneous extracts. The production of a MAb which recognizes an Ag2-specific epitope and its utility as a ligand for isolating Ag2 will provide a valuable reagent for studies of this immunologically important antigen.     

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis.

The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28- and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50- to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band.     

Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii.

The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.     

Elastase activity of Coccidioides immitis

Twenty-two strains of Coccidioides immitis were tested for the ability to hydrolyse elastin. Screening assays with Czapek's or Tryptic Soy Agar supplemented with 0.5% elastin demonstrated that 21 strains (95%) were elastolytic. In broth cultures, elastase activity was induced by incorporation of insoluble elastin into the medium and induction was suppressed by supplementation with yeast extract. C. immitis appears to be unique amongst dimorphic fungal pathogens in its digestion of elastin.     

Recombinant Coccidioides immitis complement-fixing antigen: detection of an epitope shared by C. immitis, Histoplasma capsulatum, and Blastomyces dermatitidis.

We undertook an investigation to assess the utility of a recombinant Coccidioides immitis complement-fixing (CF) antigen for detecting CF antibody in sera from patients with coccidioidomycosis. Enzyme-linked immunosorbent assays established that recombinant CF antigen and, for comparison, a commercially available coccidioidin were reactive with 19 of 19 sera from patients with active coccidioidomycosis. The recombinant antigen was significantly more sensitive than coccidioidin. The median titer obtained when patients' sera were assayed against recombinant CF antigen was 1:51,200 compared to 1:25,600 with coccidioidin (P < 0.027). The recombinant antigen was also more effective in distinguishing the antibody levels in sera from patients with pulmonary coccidioidomycosis than in sera from those with disseminated disease. Whereas patients with pulmonary disease showed a median antibody titer of 1:25,600, those with multifocal disease showed a median titer of 1:102,400 (P < 0.028). The recombinant CF antigen was found, however, to express an epitope(s) that reacted with sera from 6 of 12 patients with histoplasmosis and 2 of 12 patients with blastomycosis.     

An immunoreactive apoglycoprotein purified from Coccidioides immitis.

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and greater than 200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.