This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tarcha, E. J.
Right arrow Articles by Cole, G. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tarcha, E. J.
Right arrow Articles by Cole, G. T.

 Previous Article  |  Next Article 

Infection and Immunity, January 2006, p. 516-527, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.516-527.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Recombinant Aspartyl Protease of Coccidioides posadasii Induces Protection against Pulmonary Coccidioidomycosis in Mice

Eric J. Tarcha,1 Venkatesha Basrur,1 Chiung-Yu Hung,1 Malcolm J. Gardner,2 and Garry T. Cole1*

Department of Medical Microbiology and Immunology, Medical University of Ohio, Toledo, Ohio 43614,1 The Institute for Genomic Research, Rockville, Maryland 208502

Received 2 September 2005/ Returned for modification 5 October 2005/ Accepted 24 October 2005


arrow
ABSTRACT
 
Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogens Coccidioides spp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with Coccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.


arrow
INTRODUCTION
 
Coccidioides is an environmental pathogen and causative agent of a respiratory infection of mammals. The filamentous, saprobic form of the fungus occurs in desert soils of the southwestern United States and parts of Mexico and Central and South America (8). Inhalation of the airborne fungal spores (arthroconidia) can lead to onset of a disease (coccidioidomycosis or San Joaquin Valley fever) which is rarely life-threatening but causes significant morbidity in more than 40% of infected individuals. The disseminated form of this mycosis is difficult to treat; it usually necessitates long courses of antifungal drug administration, and relapse frequently occurs following the therapeutic regimen (2). The incidence of coccidioidomycosis in California and Arizona has been correlated with seasonal climatic changes, and recurrent outbreaks of the respiratory disease within populations which reside in the regions where it is endemic have been reported (33). The increased numbers of visitors, retirees, military recruits, etc., from regions where the disease is not endemic who have moved to the Southwest over the past 10 years have resulted in a large immunonäive population at risk of infection (8). Coccidioidomycosis in Arizona is currently the fourth most commonly reported infectious disease; only gonorrhea, chlamydia, and hepatitis C virus infections are reported more frequently (33). Examinations of the incidence of coccidioidal infections in Arizona between 1998 and 2001 have indicated that persons 65 years or older showed the highest rate of disease and hospitalization. However, it was also determined that the incidence of coccidioidomycosis in younger populations had increased, particularly in those 0 to 18 years old (33).

Recovery from infection with either of the two recognized species of Coccidioides, C. immitis or C. posadasii (15), usuallyconfers lifelong immunity to reinfection (41). Immunization of mice with an attenuated strain of the pathogen has been shown to protect the animals against infection following a lethal, intranasal (i.n.) challenge with Coccidioides (32). On the basis of these observations, it has been argued that generation of a vaccine against coccidioidomycosis is feasible and would be cost-effective (4, 8). Both clinical and experimental evidence have demonstrated that T-cell immunity is pivotal for defense against this respiratory disease (9). The ability of the host to elicit a strong delayed-type hypersensitivity response to the pathogen is essential. On the other hand, rising titers of antibody to Coccidioides antigen typically signal a poor prognosis. Our search for candidate vaccines against coccidioidomycosis using a murine model of the pulmonary disease has focused on T-cell-reactive proteins. Criteria used in this study for evaluation of protection include evidence that the vaccine candidate stimulates a T-helper 1 (Th1) pathway of immune response as measured by T-lymphocyte secretion of appropriate cytokines (28, 48), significant increase in survival of vaccinated mice compared to nonvaccinated controls after intranasal infection with a potentially lethal inoculum of Coccidioides, and demonstration that the majority of vaccinated survivors have cleared the organism from their lungs. In this paper we report a new bacterium-expressed recombinant vaccine candidate derived from a cell wall extract of C. posadasii and identified as an aspartyl protease homolog.


arrow
MATERIALS AND METHODS
 
Fungal growth conditions. The saprobic and parasitic phases of C. posadasii (strain C735) were grown in vitro as previously described (17). Arthroconidia were isolated from saprobic-phase cultures after 4 weeks of incubation as reported previously (28) and used to inoculate flasks of defined glucose-salts medium for growth of the parasitic (spherule-endospore) phase (19).

Isolation and protein extraction of the parasitic cell wall fraction. Spherules were isolated from pooled parasitic-phase cultures after incubation for 96 h (preendosporulation) or 132 h (postendosporulation) and mixed (1:1; vol/vol). The same number of arthroconidia (1 x 107) were used to inoculate each culture, which resulted in production of approximately equal numbers of first-generation, segmented, and endosporulating spherules (19). The cell pellet collected by centrifugation (3,000 x g) was washed four times with ice-cold disruption buffer (20 mM Tris-HCl, pH 7.4) which contained 2x protease inhibitor cocktail, set IV (Calbiochem, San Diego, Calif.). The parasitic cells suspended in buffer were disrupted in a BeadBeater (BioSpec Products, Inc., Bartlesville, Okla.) using precooled 0.5-mm-diameter zirconia-silica beads (Biospec). The homogenate was centrifuged (5,000 x g), and the pellet was washed five times with cold disruption buffer as described above and then frozen and stored at –80°C until ready for use. The cell wall isolate was incubated with 2% Triton X-114 (TX114; Sigma Chemical Co., St. Louis, Mo.) in extraction buffer (50 mM Tris-HCl [pH 6.8], 100 mM NaCl, and protease inhibitor cocktail) for 1 h at 4°C with vigorous shaking as previously described (17). The supernatant obtained after centrifugation (27,000 x g) was allowed to separate into the aqueous and detergent phases upon incubation at 30°C for 30 min without agitation. The detergent-phase fraction (TX114-DF) was collected, and the protein components were precipitated with ice-cold, absolute acetone as reported previously (17). The acetone-precipitated proteins were washed once with 80% acetone, resuspended in ultrapure MilliQ water (Millipore Corp., Bedford, Mass.), and then reprecipitated in absolute ethanol to remove any residual detergent. The protein fraction was resuspended in MilliQ water and used for vaccination studies described below.

Vaccination, animal challenge, and evaluation of protection. Immunoprotection experiments were conducted with C57BL/6 mice (females, 8 weeks old) supplied by the National Cancer Institute (Bethesda, Md.). Mice were immunized subcutaneously with either the total protein fraction extracted from the parasitic cell wall described above (TX114-DF; 14 µg [dry weight] per dose) or a purified, bacterium-expressed recombinant aspartyl protease (rPep1; 1 µg or 5 µg per dose) which is described below. The vaccination protocol was the same as previously reported (28). Mice were also immunized with a synthetic oligodeoxynucleotide (ODN) preparation containing unmethylated CpG dinucleotides that was used as an adjuvant (CpG ODN; Integrated DNA Technologies, Inc., Coralville, Iowa) (25). The CpG ODN sequence employed in this study was the same as we have previously reported (13). The oligonucleotides were dissolved in phosphate-buffered saline (PBS) (1 mg/ml) and used as a stock solution for the vaccination experiments. Mice were immunized two times (2 weeks apart) with either adjuvant alone (10 µg of CpG prepared in 50 µl of PBS plus 50 µl of incomplete Freund's adjuvant) as described previously (28) or selected concentrations of the test reagent (TX114-DF or rPep1) plus adjuvant. Mice were challenged by the i.n. route at 4 weeks after the second immunization with 80 to 90 viable arthroconidia obtained from 30-day-old saprobic-phase cultures as reported previously (13). Mice were scored for survival over a 50- to 90-day period postchallenge or evaluated for fungal burden in homogenates of their lungs obtained at 15, 30, 60, and 90 days after challenge as previously described (28). Survival differences between groups of i.n. infected mice (20 animals per group) were analyzed for statistical significance by the Kaplan-Meier method as reported previously (28). The fungal burden (CFU) in the lungs of vaccinated and nonvaccinated, infected mice was expressed on a log scale for individual animals, and the Mann-Whitney U test was used to compare the numbers of CFU in each group of mice (15 animals per group) as described previously (13). The limit of detection of the pathogen in organ homogenates is 102 CFU.

Internal amino acid sequence analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein components of the TX114-DF extract of the isolated parasitic cell wall fraction were initially separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblot analysis of sister gel separations of the TX114-DF was conducted by incubation with either pooled sera from surviving mice which had been vaccinated with the wall extract and challenged as described above or pooled human sera from patients with confirmed coccidioidal infection. The latter was conducted using goat anti-human immunoglobulin G (IgG)-specific secondary antibody (Southern Biotechnology Associates, Inc., Birmingham, Ala.) as reported previously (17). A 43-kDa, Coomassie blue-stained gel band was selected for further examination on the basis of its seroreactivity, prominence, and apparent electrophoretic separation from other protein components in the SDS-PAGE gel. The selected band was excised, destained, and subjected to in-gel digestion with sequencing-grade trypsin (Promega, Madison, Wis.) at 37°C overnight as reported previously (18). Peptides were extracted from the gel with 60% acetonitrile:0.1% trifluoroacetic acid and concentrated using an SPD1010 SpeedVac system (ThermoSavant, Holbrook, N.Y.). The peptides were applied to a reverse-phase high-pressure liquid chromatography column (Aquasil C18 Picofit column, 75-µm inside diameter by 5 cm, tapered to 15-µm inside diameter; New Objective, Woburn, Mass.), eluted using a binary gradient of 1% acetic acid-acetonitrile (5 to 95% acetonitrile in 35 min), and then introduced into an ion-trap mass spectrometer equipped with a nanospray source (LCQ Deca XP plus; Finnigan Corp., San Jose, Calif.). The tandem mass spectrometer was operated in the double play mode in which the instrument was set to acquire a full MS scan (400 to 2,000 m/z) and an MS/MS spectrum of the most intense ion. Collision-induced dissociation (CID) spectra were obtained that yielded amino acid sequences of the peptides. A search for matching sequences in the translated C. posadasii (strain C735) genome database (18) (www.tigr.org) was conducted using the TurboSEQUEST software package, version 3.0 (Finnigan). Details of the computational method used to match the nascent CID mass spectra of peptides to database sequences have been given previously (49). Sequence matches were also manually verified. On this basis of peptide sequence matches we identified an open reading frame (ORF) of a 1.4-kb gene in the C. posadasii genome database which revealed 99.8% nucleotide sequence identity to the cDNA of a previously reported gene that encodes an aspartyl protease of Coccidioides (23). The genomic and cDNA sequences of the gene identified in the C. posadasii database, which were confirmed by cloning and nucleotide sequence analysis as described below, are designated in this paper as PEP1.

The basic local alignment search tool (BLAST) (1) was used to search the Swiss-Prot/TrEMBL database (www.us.expasy.org/tools/blast) and the National Center for Biotechnology Information (NCBI) nonredundant protein database (www.ncbi.nih.nlm.gov) for proteins with sequence similarities to the translated, full-length PEP1 gene. Analysis of the predicted hydropathicity profile of Pep1 was performed as reported previously (26). The GPI-SOM algorithm (14) was used to examine the translated sequence of PEP1 for a putative glycosylphosphatidylinositol (GPI) anchor site, while the WoLF PSORT II algorithm was employed for prediction of a signal peptide and cellular localization (http://wolfpsort.seq.cbrc.jp/). The PROSITE algorithm was used to identify conserved motifs in the translated polypeptide with homology to reported proteins (16).

2D SDS-PAGE and immunoblot analysis. Approximately 150-µg aliquots of either the concentrated protein fraction of the TX114-DF extract described above or the protein fraction of the combined 96-h and 132-h parasitic-phase culture supernatants (CS) were subjected to two-dimensional (2D) PAGE separation. The CS fraction was obtained after centrifugation (27,000 x g, 10 min, 4°C), dialysis against distilled water, and lyophilization as previously reported (51). The TX114-DF or CS preparations were solubilized in 2D PAGE sample buffer which contained 8 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; a zwitterionic detergent; AmershamBiosciences, Piscataway, N.J.}, 40 mM Tris, 65 mM dithiothreitol (Sigma), and 0.5% carrier ampholytes (Amersham). Each sample was subjected to vortexing (30 s, three times), and insoluble material was removed by centrifugation (15,000 x g, 30 min, at 4°C). The supernatant was applied to an 18-cm Immobiline Drystrip gel (Amersham) overnight at room temperature. The gel was then subjected to isoelectric focusing (IEF) using an Ettan IPGphorII IEF system (Amersham). The IEF gel strips were first electrophoresed at 500 V for 2,500 V · h to remove interfering low-molecular-weight substances. IEF was performed at a maximum of 200 µA per strip in a two-step process: 3,500 V for 15,000 V · h followed by 5,500 V for 52,500 V · h. The gel strips were subsequently exposed to a reducing buffer at room temperature (50 mM Tris-HCI [pH 6.8], 6 M urea, 30% glycerol, 2% SDS, 0.5% dithiothreitol, and a trace of bromophenol blue) for 10 min, followed by alkylation with 2.5% (wt/vol) iodoacetamide for an additional 10 min. Electrophoresis in the second dimension was conducted in a 10% SDS-polyacrylamide gel at 50 mA using an Ettan Daltsix system (Amersham). Protein spots were visualized by Coomassie blue staining, and gel images were digitally recorded using a GS-700 imaging densitometer (Bio-Rad, Hercules, Calif.). Coomassie blue-stained protein spots in the 2D PAGE gels of the TX114-DF and CS fractions were selected for excision and LC-MS/MS analysis as described above on the basis of their seroreactivity in immunoblots of the respective 2D PAGE sister gels. The latter were incubated with pooled sera from 10 human patients with confirmed coccidioidal infection as described above.

ProPred prediction of promiscuous MHC class II-restricted epitopes. The web-accessible ProPred algorithm (www.imtech.res.in/raghava/propred/) (40), which was developed on the basis of the TEPITOPE program (43), was used to predict the presence of promiscuous, human major histocompatibility complex (MHC) class II-restricted epitopes in the proteins identified by immunoblot assays of the 2D PAGE gels. This algorithm has been previously employed to predict MHC class II epitopes in microbial and tumor antigens (5, 20). The ProPred algorithm contains matrix-based motifs of 51 human leukocyte antigen (HLA)-subregion DR alleles derived from an MHC class II pocket profile database (40). We used the algorithm to identify epitopes of the deduced proteins which were predicted to bind to each of the 51 HLA-DR molecules. The threshold for the ProPred analyses was set at a relatively high stringency of 5%. Under these conditions, promiscuous epitopes were defined as peptides that were predicted to bind to at least 80% of the MHC class II molecules expressed by the 51 HLA-DR alleles.

Real-time PCR. Levels of expression of PEP1 during different stages of in vitro development of first-generation parasitic cells were determined by quantitative real-time PCR as reported previously (18). Parasitic cells were isolated from cultures after 36 h, 96 h, and 132 h of incubation, and the majority of the fungal cells showed near-synchronous development as presegmented, segmented, or endosporulating spherules, respectively (19). PEP1-specific primers were designed using the LightCycler Probe Design software (version 1.0; Roche Diagnostics, Indianapolis, Ind.). The sequences of the sense and antisense primers were 5'-AAATCCTGGAACGGTCAATAC-3' and 5'-GAAAGCGTCTCCAAGAATGG-3', respectively. This primer pair amplified a 221-bp PCR product using single-stranded template cDNA generated by reverse transcription of total RNA as reported previously (13). RNA was isolated separately from the three developmental stages of C. posadasii as described previously (18). A 191-bp amplicon used for normalization of the assay was derived from the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of C. posadasii (GenBank accession no. AF288134) as previously reported (13). Confirmation that the GAPDH gene of this pathogen is constitutively expressed has been reported (50a). Data analysis was performed as described previously (18), and the results are presented as the ratio of PEP1 to GAPDH transcripts in each sample.

Expression of PEP1 by Escherichia coli. Oligonucleotide primers were designed to amplify the cDNA of PEP1 (1.2 kb), which encodes amino acids (aa) 1 to 399 (predicted full-length protein). The nucleotide sequences of the sense and antisense primers were 5'-GGCAGCCATATGGCTAGCATGAGGAACTCCATCCTGCTCGCAG-3', and 5'-GAATTCGGATCCTTACTCGAGTTAGTTCCCGGCTTTGGCAAGGCC-3', which contained engineered NdeI and BamHI restriction sites, respectively (underlined nucleotide sequences). The amplification parameters were as follows: an initial denaturation step at 94°C for 2 min, followed by 30 cycles which consisted of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2 min. The 1.2-kb amplicon was subcloned into the pGEM-TE cloning vector (Promega Corp., Madison, Wis.), and the nucleotide sequence of the insert was determined as previously reported (18). The pGEM-TE-PEP1 plasmid was digested with NdeI and BamHI to release the 1.2-kb insert, which was then subcloned into the pET28b expression vector (Novagen) and used to transform E. coli strain BL21(DE3) as described previously (19). Purification of the recombinant protein (rPep1) was conducted as reported previously (28). Confirmation of the identity of the protein was performed by LC-MS/MS sequence analysis of peptides generated by trypsin digestion of the purified rPep1 as described above. The endotoxin content of the stock solution that contained the purified recombinant protein (0.22 mg/ml in PBS [0.1 M, pH 7.4]) was determined by use of a Limulus amebocyte lyase kit (QCL-1000; BioWhittaker, Walkersville, Md.) as previously reported (28). The stock solution contained 3.0 to 3.3 endotoxin units per µg of protein.

In vitro assays of murine immune T-cell proliferation and cytokine production. Purified rPep1 (5 µg) plus the CpG ODN adjuvant was used to immunize five 8-week-old, female C57BL/6 mice as described above. Two weeks after the second immunization, the spleens were harvested, pooled, and macerated as reported previously (28). Separation of CD90+ T cells from the cell suspension was conducted using mouse CD90 (Thy 1.2) MicroBeads (Miltenyi Biotec Inc., Auburn, Calif.) as previously described (48). Antigen-presenting cells (APCs) were isolated from pooled splenocytes obtained from five naïve (untreated), age- and gender-matched C57BL/6 mice. This splenocyte suspension was subjected to antibody depletion of the CD90+ T cells followed by irradiation as previously described (6). All cells were incubated in RPMI 1640 medium containing 10% (vol/vol) fetal calf serum, ß-mercaptoethanol, penicillin, and streptomycin as previously reported (48). Isolated CD90+ T cells plus APCs were transferred to 96-well flat-bottomed plates (Costar, Cambridge, Mass.) for proliferation assays (2.5 x 105 T cells plus 5 x 105 APCs per well). Alternatively, the isolated T cells (1 x 106 cells per well) plus APCs (2.5 x 106 cells per well) were transferred to 48-well flat-bottomed plates (Costar) for assays of cytokine production. For proliferation assays, the cells were cultured in medium alone, medium plus 2 µg/ml of mitogen (concanavalin A [ConA]; Sigma), or medium plus a range of concentrations of purified rPep1 (0.1 to 10 µg/ml) and then pulsed with [3H]thymidine after 54 h of incubation and harvested 18 h later for determination of levels of radioisotope uptake as reported previously (28). For determination of levels of cytokine production, CD90+ T cells plus APCs were incubated for 48 h or 120 h in medium alone or medium to which ConA (2 µg/ml) or rPep1 (1 µg/ml or 5 µg/ml) had been added. The cytokine assays were conducted in the presence or absence of polymyxin B (10 µg/ml; Sigma) to assess the influence of endotoxin contamination on production of the selected cytokines. Concentrations of secreted gamma interferon (IFN-{gamma}), interleukin-4 (IL-4), IL-5, and IL-10 in the culture supernatants were determined using the OptEIA mouse cytokine assay kits (Pharmingen, San Diego, Calif.) as reported previously (48).

IFN-{gamma} ELISPOT assays. Purified rPep1 (5 µg) plus the CpG ODN adjuvant was used to immunize four 8-week-old female C57BL/6 mice or four 12-week-old HLA-DR4 (DRB1*0404) transgenic mice (a gift from Thomas Forsthuber, University of Texas at San Antonio). The immunization protocol was the same as described above. Two weeks after the second immunization, the spleens of the two groups of mice were separately harvested, pooled, and macerated as described above. Isolation of CD90+ T cells from the total splenocytes, collection of APCs, and cell culturing were performed as described above. IFN-{gamma} enzyme-linked immunospot (ELISPOT) assays were performed according to instructions of the kit manufacturer (MABTECH, Inc., Mariemont, Ohio). In brief, 96-well filtration plates (ELIIP 10SSP; Millipore) were coated with monoclonal anti-murine IFN-{gamma} antibody, washed with PBS, and blocked with RPMI containing 2% fetal calf serum. CD90+ immune T cells (4 x 105) plus APCs (2.5 x 105) were then added to each well and incubated with or without stimulatory reagents (ConA, rPep1, synthetic peptides, or medium alone) as described previously (48). Synthetic peptides spanning each of the five ProPred-predicted, promiscuous MHC class II-restricted epitopes of Pep1 were constructed using 9-fluorenylmethyloxycarbonyl chemistry and supplied by Mimitopes, Ltd. (Morris Plains, N.J.). A control peptide (18-mer) which corresponds to a region of Pep1 not predicted to contain a T-cell epitope was synthesized and included as a control. The identity and purity of the peptides were confirmed by the manufacturer using mass spectrometry and high-pressure liquid chromatography, respectively. The peptides were solubilized in 80% tissue culture-grade dimethyl sulfoxide (American Type Culture Collection, Manassas, Va.) at stock concentrations of 10 mg/ml. The peptide solutions were stored at –80°C in 0.1-ml aliquots until used for the assays.

After incubation with stimulating reagents, the cells were removed from the filtration membrane of each well, the plates were washed, and biotin-labeled anti-IFN-{gamma} monoclonal detection antibody (R4-6A2-biotin) was added to the wells followed by streptavidin-alkaline phosphatase according to the manufacturer's instructions. After incubation, the plates were washed and the substrate, 1-Step nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Pierce, Rockford, Ill.), was added for color development. Each membrane was analyzed using an automated ELISPOT reader system by Zellnet Consulting Inc. (Fort Lee, N.J.). The frequency of IFN-{gamma}-secreting antigen-specific CD90+ T cells was calculated as the number of spots per 4 x 105 CD90+ T cells seeded in the presence of antigen minus the number of spots per equal number of CD90+ T cells in medium alone.

Nucleotide sequence accession number. The genomic and cDNA sequences of the gene identified in the C. posadasii database, which were confirmed by cloning and nucleotide sequence analysis as described above, have been deposited in GenBank (accession no. DQ164306).


arrow
RESULTS
 
Immunization with a detergent extract of the parasitic cell wall (TX114-DF) confers protection against pulmonary coccidioidomycosis. C57BL/6 mice were vaccinated subcutaneously with a complex mixture of proteins extracted from isolated walls of two separate cultures of parasitic cells which had developed to near-synchronous stages of segmentation and endosporulation (13, 19). Our rationale for combining these cell types was to account for the recognized quantitative and qualitative differences in antigen production during the parasitic cycle (12), which could be important in our search for candidate vaccines. Mice immunized with two doses of the detergent-extracted cell wall protein fraction (TX114-DF; 14 µg each) plus CpG ODN were well protected (90% survival) against a potentially lethal inoculum of C. posadasii (80 viable arthroconidia) delivered via the i.n. route, compared to no survivors among the control mice immunized with adjuvant alone (Fig. 1). The latter group of animals began to die at 11 days postchallenge, followed by a sharp decrease in the number of survivors during the subsequent 5 days, and then a short lag period before the rest of the mice died. In the case of the vaccinated mice, two animals died at 13 days postchallenge, and the remaining 18 mice appeared to be healthy throughout the subsequent 37 days of the experiment. The same vaccination protocol was repeated twice using separate detergent extracts of cell wall proteins prepared in the same manner. The percentage of vaccinated mice which survived was not significantly different between the three experiments, suggesting that the TX114-DF extract of the cell wall contained immunogenic components which confer protective immunity against pulmonary challenge with C. posadasii. Compositional analyses of the detergent-soluble fraction were conducted by SDS-PAGE as described below.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. Representative comparison of the protective efficacy of a Triton X-114 extract of the isolated parasitic cell wall of C. posadasii (TX114-DF plus immunoadjuvant [Adj.], CpG ODN) versus adjuvant alone, both delivered subcutaneously to C57BL/6 mice. The animals were subsequently challenged with a potentially lethal inoculum (80 viable arthroconidia) of C. posadasii via the i.n. route.

A 43-kDa polypeptide identified as an aspartyl protease homolog is a major protein component of TX114-DF. The Coomassie blue-stained SDS-PAGE separation of the TX114-DF preparation revealed a prominent band at 43 kDa, as well as multiple lightly stained bands in the range of 34 kDa to >200 kDa (Fig. 2A). Immunoblot analysis of sister gel separations of this extract incubated either with pooled mouse sera from survivors reported in Fig. 1 or pooled sera from patients with confirmed coccidioidal infection both showed strong reactivity with the 43-kDa component (not shown). The seroreactive 43-kDa gel band appeared to be electrophoretically separated from other protein components in the SDS-PAGE gel and was selected for excision and trypsin digestion. The acquired tryptic peptides were subjected to amino acid sequence analysis by tandem mass spectrometry. The sequences were deduced by the mass differences between y- or b-ion "ladder" series resulting from the CID fragmentation spectra of the selected tryptic peptides as reported elsewhere (34). A representative CID spectrum for a 1,711.88-Da peptide is shown in Fig. 2B. Five good CID spectra were obtained, each of which yielded amino acid sequences of 16 to 26 residues. TurboSEQUEST searches of the translated C. posadasii genome database revealed that all five peptides matched a 1.2-kb translated ORF of a 1.4-kb gene (Fig. 2C). A BLAST search of the Swiss-Prot/TrEMBL and nonredundant NCBI protein databases using the translated ORF identified an aspartyl protease of C. posadasii which had previously been described (GenBank accession no. AF162132) (23). The earlier reported gene (PEP) was isolated from the Silveira strain of C. posadasii. The authors estimated that the molecular size of the mature protein in SDS-PAGE gels was 45 kDa and determined on the basis of its N-terminal sequence that two processing events occurred which resulted in cleavage of an 18-aa signal peptide and a 52-aa propeptide. Two N-glycosylation sites were predicted. The cDNA sequence of the PEP gene of the Silveira strain showed three nucleotide substitutions when aligned with the genomic sequence of the homolog in the database of strain C735. The 1.2-kb cDNA (ORF) of the C735 homolog was PCR amplified using primers described above in Materials and Methods and subjected to nucleotide sequence analysis. Its sequence matched that of the C. posadasii database. The nucleotide substitutions identified in the C735 cDNA homolog did not influence its translated amino acid sequence, which was identical to that of the Silveira strain. The isolated gene of C. posadasii strain C735 has been designated PEP1.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 2. Application of LC-MS/MS to the identification of a major protein component of the parasitic cell wall extract, TX114-DF. (A) Coomassie blue-stained SDS-PAGE separation of TX114-DF. (B) CID mass spectrum of 1,711.88-Da peptide [(M + 2H)2+ = 856. 94 Da] derived from the trypsin digest of the excised 43-kDa band in panel A. (C) Complete sequence of the putative C. posadasii aspartyl protease (Pep1). Five underlined peptide sequences are those identified by LC-MS/MS analysis and matched with the translated C. posadasii genome database using the TurboSEQUEST software package.

The WoLF PSORT algorithm predicted Pep1 to be cell wall associated, secreted into the culture medium, or both. The protein sequence contains two conserved aspartyl protease active sites identified by the PROSITE algorithm at D103XG105X S107 XXW110V111 and D287T288G289 (D is the active site residue) in accordance with the sequence analysis performed by Johnson and coworkers (23). The hydrophobicity profile of Pep1 predicted three hydrophobic domains (aa 180 to 222, 275 to 305, and 345 to 380). On the basis of the GPI-SOM algorithm (14), the C-terminal hydrophobic region is predicted to contain a GPI anchor signal sequence with a putative anchor site at G374.

2D PAGE/immunoblot analyses reveal that Pep1 is one of several seroreactive proteins in the parasitic cell wall extract and culture supernatant. 2D PAGE separations of the TX114-DF wall extract and CS were blotted and incubated with pooled patient sera. Prominent, seroreactive protein spots with an estimated molecular size of 43 kDa (Fig. 3A and B) were identified as Pep1 by LC-MS/MS in both preparations. These data support the prediction that Pep1 is both wall bound and released into the culture medium (37). Portions of the two immunoblots of the 2D PAGE gels in the pI range of 3 to 6 revealed additional seroreactive protein components of the TX114-DF and CS preparations. Each protein in the corresponding Coomassie blue-stained 2D PAGE gel was excised and sequenced by LC-MS/MS. Seven deduced proteins in the combined wall extract plus culture supernatant were identified in addition to Pep1, and their sequences have been deposited in the GenBank database (Table 1). One of the excised gel components (gel spot 6) failed to yield interpretable CID spectra, and two of the excised proteins in the CS preparation were components of the same polypeptide (gel spots 8 and 9). The latter was identified as a spherule outer wall glycoprotein (SOWgp), which has been previously reported in C. posadasii strain C735 and is characterized by prominent, seroreactive 82-kDa and 60-kDa bands (17). Recombinant SOWgp has proved not to be a candidate vaccine against coccidioidomycosis (18). SOWgp as well as another deduced protein listed in Table 1 (gel spot 5) is rich in proline, which may account for their contradictory molecular size estimates in the 2D PAGE gels compared to the predicted sizes based on the respective amino acid sequences (36). All but one of the deduced proteins were predicted to be cell wall associated or extracellular, and three of these were suggested to be GPI anchored. All of the deduced proteins except SOWgp were characterized by hydropathicity profiles with well-defined hydrophobic domains (not shown), as revealed by Pep1 and described above. Each of the deduced proteins in Table 1 was subjected to sequence analysis using the ProPred algorithm for prediction of promiscuous epitopes which bind to human MHC class II molecules. Four of the deduced proteins contained five to seven predicted promiscuous T-cell epitopes, and one of these was Pep1.



View larger version (103K):
[in this window]
[in a new window]
  		FIG. 3. Immunoblots of 2D PAGE-separated TX114-DF (A) and CS (B) proteins. Numbers correspond to protein spots identified by LC-MS/MS (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Summary of deduced seroreactive proteins of C. posadasii identified in 2D PAGE separations of the TX114-DF extract and culture supernatant

PEP1 is constitutively expressed during the parasitic cycle. We have previously demonstrated that spherule development during the first generation of the parasitic cycle of C. posadasii strain C735 is fairly well synchronized (18). Total RNA was isolated either from spherules prior to endospore release at incubation times of 36 h and 96 h or after endospore differentiation and the initiation of endospore release at 132 h. Results of quantitative real-time PCR analysis of PEP1 gene expression, compared to temporal expression of the constitutive GAPDH gene, revealed approximately equal amounts of transcript for each developmental stage examined (data not shown). However, the peak ratios of transcript levels of PEP1/GAPDH are about 10-fold lower than that of another C. posadasii gene (MEP1) which was recently described (18). However, in spite of the comparatively low amount of PEP1 transcript we have observed that the 43-kDa antigen is an abundant protein in SDS-PAGE separations of both the wall extracts and culture supernatants of parasitic cells. This suggests that the PEP1 transcript is stable and persistent during the parasitic cycle, and/or Pep1 accumulates both in the wall and in the culture supernatant during spherule development.

Bacterium-expressed recombinant Pep1 is seroreactive and stimulates immune T-cell response. E. coli transformed with the pET28b-PEP1 plasmid produced high levels of recombinant protein either in the presence or in the absence of IPTG (isopropyl-ß-D-thiogalactopyranoside; Fig. 4), albeit the amounts of total protein loaded onto the gel were not equal. The predicted and observed molecular size of the recombinant protein in the SDS-PAGE gel is 47 kDa, which includes the vector-encoded fusion peptide that contained the His tag at its N terminus. The 47-kDa rPep1 was isolated by nickel-affinity chromatography, subjected to electrophoresis, excised from the SDS-PAGE gel, trypsin digested, and processed for LC-MS/MS sequence analysis as described above. Five peptide sequences were obtained by this procedure, and each matched the translated sequence of the PEP1 gene. The recombinant protein was reactive with randomly selected sera from patients with confirmed coccidioidal infection, as demonstrated by the representative immunoblot in Fig. 4. On the other hand, pooled sera from healthy individuals (i.e., hospital admissions with no indication of fungal infection) did not recognize the purified rPep1.



View larger version (117K):
[in this window]
[in a new window]
  		FIG. 4. SDS-PAGE separation and immunoblot (Iblt.) of E. coli-expressed rPep1. Shown are standards (Std.), lysates of bacteria transformed with the pET28b-PEP1 plasmid vector in the presence (+) or absence (–) of IPTG, and the nickel-affinity-isolated rPep1 which was subsequently purified by electroelution from an SDS-PAGE gel as previously reported (13). The immunoblot of the rPep1 isolated by nickel-affinity chromatography was incubated with either pooled sera from patients with confirmed coccidioidal infection or pooled sera from control patients.

CD90+ T cells isolated from mice immunized with rPep1 plus the CpG ODN adjuvant were stimulated to proliferate in vitro in response to the presence of the purified homologous recombinant protein at concentrations of 1 to 10 µg/ml (Fig. 5A). Immune T cells incubated with rPep1 at a concentration of 0.1 µg/ml showed markedly lower response but significantly greater proliferation than immune T lymphocytes incubated in culture medium alone (P < 0.05). Immune T cells incubated with the mitogen ConA responded in an expected manner.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 5. (A) In vitro proliferative response of isolated CD90+ T cells derived from rPep1-plus-CpG ODN-immunized C57BL/6 mice stimulated with different concentrations of the homologous antigen. (B to D) ELISAs of secreted cytokines (IFN-{alpha}, IL-5, and IL-10) produced by immune T cells stimulated with ConA (positive control) or rPep1 (1 µg or 5 µg) in the presence (+) or absence (–) of polymyxin B (PMB). Assays were conducted with culture supernatants obtained after incubation for 48 h or 120 h. Immune T cells grown in medium alone served as a negative control. Mean values plus standard deviations for three separate determinations of both the proliferative response and ELISAs of cytokine concentrations (pg/ml) are reported.

In vitro T-cell immunoassays were also performed to evaluate the amount and type of cytokines secreted by the immune cells during their proliferative response to rPep1 (Fig. 5B to D). The two concentrations of recombinant protein tested (5 µg/ml and 1 µg/ml) were chosen on the basis that they stimulated comparable levels of immune T-cell proliferation (Fig. 5A). Polymyxin B was added to replicate assays to neutralize potential effects of endotoxin contamination on T-lymphocyte response. Immune T cells incubated with 5 µg/ml of rPep1 for 48 h and 120 h (± polymyxin B) showed levels of IFN-{gamma} production (Th1-type cytokine) which were equal to or higher than the concentrations of the same cytokine detected in the supernatant of immune T-cell cultures that had been incubated with the mitogen ConA (Fig. 5B). Incubation with 1 µg/ml of rPep1 under identical conditions resulted in significantly lower levels of IFN-{gamma} production compared to 5 µg/ml (P < 0.05) but still at concentrations which were above background. In contrast, T-cell stimulation with either 5 µg/ml or 1 µg/ml of the recombinant protein resulted in secretion of significantly lower concentrations of both IL-5 and IL-10 (Th2-type cytokines) compared to ConA stimulation under identical incubation conditions (Fig. 5C and D). Even lower concentrations of IL-4 were detected in the T-cell culture supernatants after 48 h and 120 h of incubation in the presence of rPep1 (not shown). On the basis of the results presented in Fig. 5 it appears that rPep1 is a T-cell-reactive antigen, and immune T lymphocytes respond to the presence of the recombinant protein by secretion of high levels of a Th1-type cytokine. The addition of polymyxin B to the reaction mixtures containing rPep1 had no significant effect on levels of cytokine production.

Identification of T-cell epitopes of Pep1. Pep1 sequence analysis using the ProPred algorithm identified five regions of the full-length polypeptide (range of 21 to 32 amino acids) which were predicted to contain ligands that can bind to human MHC class II molecules. Each of these five regions, which may include more than one T-cell epitope, was synthesized (P1 to P5; Table 2) and used separately to test the in vitro response of immune T cells obtained from rPep1-immunized C57BL/6 and HLA-DR4 transgenic mice (Fig. 5A and B, respectively). A synthetic peptide (18-mer; P6 in Table 2) not predicted to bind to any of the 51 HLA-DR molecules examined in the ProPred algorithm was included as a negative control. T-cell response to each of the synthetic peptides (5 µg/ml) was compared to that of the recombinant Pep1 (2.5 µg/ml) by IFN-{gamma} ELISPOT assays of the relative numbers of cytokine-producing CD90+ immune T lymphocytes. In both C57BL/6 and HLA-DR4 transgenic mice, significant responses were observed in the presence of synthetic peptides P1 and P2 compared to cells incubated in medium alone (P < 0.05 and < 0.01, respectively). On the other hand, the number of IFN-{gamma}-producing cells was much greater in the presence of the recombinant protein (Fig. 6A and B). Immune T cells from the C57BL/6 and transgenic mice showed no significant response to the other synthetic peptides (P3 to P5) representing putative epitopes or to the control peptide, P6. It is possible that induction of significant response would have been observed in the presence of P3 to P5 at higher concentrations of the synthetic peptides. These assays are planned for future studies. Nevertheless, results of the IFN-{gamma} ELISPOT assays confirm the ProPred prediction that P1 and P2 are promiscuous epitopes which can activate both murine and HLA-DR4 immune T cells.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Amino acid sequences of synthetic peptides selected from ProPred prediction of Pep1 ligands which bind to human MHC class II molecules



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 6. Assessment of IFN-{gamma} production by immune CD90+ T cells derived from C57BL/6 or HLA-DR4 (DRB1*0404) transgenic mice conducted by IFN-{gamma} ELISPOT assays. Asterisks indicate statistically significant differences between responses of T cells in the presence of peptides and those in the presence of medium alone. Results representative of three separate experiments are shown here.

Vaccination with rPep1 enhances survival and results in significant reduction of fungal burden in Coccidioides-challenged mice. Control C57BL/6 mice immunized with adjuvant alone and then challenged via the intranasal route with a lethal inoculum (90 viable arthroconidia) began to die at about 13 days postchallenge (Fig. 7A). Approximately 7 days later all the control animals had died. Groups of mice (20 per group) vaccinated with rPep1 (1 µg or 5 µg per dose) plus adjuvant and challenged with the same intranasal inoculum began to die at about 14 to 16 days but then showed significantly greater survival than the control mice (P values indicated in Fig. 7A). Comparison of the survival plots of TX114-DF-vaccinated mice in Fig. 1 and rPep1-vaccinated mice in Fig. 7A (5-µg dose) by Kaplan-Meier analysis revealed no statistically significant difference (P = 0.79). This survival plot is representative of three separate experiments using the same vaccination and challenge protocols. A trend was evident that the 5-µg dose provides better protection than the 1-µg dose, although the difference in percent survival between these two groups of mice was not statistically significant. However, on the basis of this apparent trend we decided to examine the fungal burden in the lungs and spleen of C57BL/6 mice vaccinated with the 5-µg dose of rPep1; inoculated by the i.n. route (90 viable arthroconidia); and sacrificed at 15, 30, 60, and 90 days postchallenge (10 animals randomly selected from survivors of 15 vaccinated/challenged mice per group). Control mice sacrificed at 15 days postchallenge showed high numbers of CFU in their lungs (105 to 107.8, Fig. 7B) and spleen (103; not shown). None of the vaccinated and challenged mice showed detectable organisms in their spleen between 15 and 90 days after inoculation. The vaccinated mice also revealed progressive pulmonary clearance of C. posadasii, resulting in significant reduction of the fungal burden between 15 and 90 days postchallenge as indicated in Fig. 7B.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 7. (A) Representative evidence of the protective efficacy of rPep1 (1 µg or 5 µg) plus CpG ODN adjuvant used to immunize C57BL/6 mice, which were subsequently challenged by the i.n. route with a lethal inoculum of C. posadasii. Control mice were immunized with adjuvant alone. Statistical significance (P values) of the difference in survival plots of the vaccinated versus nonvaccinated mice is shown. The results are representative of three separate vaccination/survival experiments using the same immunization and challenge protocols. (B) Plot of CFU of C. posadasii detected in dilution plate cultures of lung homogenates obtained from nonvaccinated (PBS + Adj.) or vaccinated (5 µg rPep1 + Adj.) C57BL/6 mice. The animals were inoculated intranasally with C. posadasii as described above and sacrificed at different times postchallenge as indicated. The P value indicates a significant difference between the fungal burden of vaccinated mice at 15 days postchallenge and that at 90 days postchallenge.


arrow
DISCUSSION
 
The majority of Coccidioides antigens which have so far been shown to induce a protective immune response in mice against coccidioidomycosis are derived from the parasitic cell wall (13, 27, 31, 39). This same observation has been reported for several other fungal pathogens (35, 47). The rationale for extraction of the isolated spherule wall with Triton X-114 was to release noncovalently linked proteins. Our reasons not to focus on proteins that are covalently bound to the glycan network of the parasitic cell wall (50) are that the majority of these polypeptides are highly glycosylated and, because of their tight association with structural wall components, may not be readily accessible to antigen-presenting cells for processing and subsequent T-cell activation. TX114 extraction was also selected because the detergent has been shown to at least partially solubilize the lipid-rich spherule outer wall (SOW) layer which coats the parasitic cells of C. posadasii (17). Finally, we chose to isolate, fractionate, and test the protective properties of the TX114-extracted proteins because we wanted to focus our analyses on polypeptides with hydrophobic domains. Temperature-dependent phase partitioning with Triton X-114 has proved to be a useful method for enrichment of hydrophobic proteins (52). Since hydrophobicity is considered to play an important role in binding of antigenic peptides to class II major histocompatibility complexes of CD4+ T lymphocytes (42), we argue that this extraction procedure enhanced our ability to identify candidate T-cell-reactive proteins.

The TX114 detergent extract of the isolated spherule wall material, together with the CpG ODN adjuvant preparation, induced a robust protective immune response in C57BL/6 mice against a lethal pulmonary challenge with C. posadasii arthroconidia. We base this conclusion on survival of 90% of the animals at 50 days postchallenge. In addition, we were able to reproduce this level of murine protection using the same vaccination protocol with separate preparations of the detergent extract of the isolated parasitic cell wall, suggesting that the TX114 fraction is a good source of vaccine candidates. A large body of evidence supports the concept that synthetic ODNs with CpG motifs provide enhanced immune response to codelivered antigens (25). CpG is suggested to directly activate dendritic cells and macrophages to enhance production of cytokines that create a Th1-like milieu in lymphoid tissue. The choice of adjuvant is a pivotal element in the evaluation of candidate vaccines (29).

The total protein subfraction of the solubilized parasitic cell wall material which was separated by SDS-PAGE and subjected to immunoblot analysis using pooled sera from patients with confirmed Coccidioides infection revealed multiple seroreactive bands. Although seroreactivity does not necessarily indicate T-cell reactivity of an antigen, the fact that the secondary antibody used for our immunoblot assays was anti-IgG specific argues that the seroreactive proteins contain T-cell epitopes (21). We initially selected the 43-kDa protein component of the parasitic cell wall extract for sequence analysis by LC-MS/MS based on its patient seroreactivity and abundance in Coomassie blue-stained SDS-PAGE separations of the TX114 fraction. Mass spectrometry is a valuable tool for studies of protein structure. Interfacing reverse-phase high-performance liquid chromatography with electrospray ionization has permitted efficient and accurate determination of the amino acid sequences of complex starting mixtures of tryptic peptides (49). With near-completion of the genome sequence and annotation of C. posadasii strain C735, we were able to unambiguously match peptide sequences obtained by LC-MS/MS with a specific gene in the Coccidioides database. Through bioinformatic analyses of the translated full-length gene sequence, conserved motifs were identified and a putative function was assigned to the deduced protein. The amino acid sequence of the 43-kDa polypeptide identified in immunoblots of both one-dimensional and 2D PAGE separations of the TX114-DF extract showed high homology to previously reported aspartyl proteases of other filamentous fungi. Sequence analysis of the C. posadasii protein (Pep1) revealed three defined hydrophobic domains, including a C-terminal GPI anchor signal sequence. The native Pep1 was also detected in the parasitic culture supernatant, demonstrating that the protein is both cell wall associated and extracellular. This contrasts with an Aspergillus fumigatus homolog (Pep2) which has been suggested to be bound to the fungal cell wall and not released into the culture supernatant (38). The reason for this apparent difference in localization of these structurally related proteins (93% sequence similarity) is unknown.

Two-dimensional electrophoresis, combined with immunoblot analysis and bioinformatics (10), has been applied for the first time in this report to the characterization of C. posadasii antigens. Incubation of 2D PAGE separations of the detergent extract and culture supernatant with patient sera revealed multiple seroreactive proteins in the respective immunoblots. For comparative purposes in this study, we examined a narrow pH range in the 2D gels that included the estimated pI of the native Pep1 (approximately 4.4 to 4.5). All seroreactive proteins were excised from the Coomassie blue-stained sister gel and trypsin digested, and the fractionated peptides were subjected to LC-MS/MS sequence analysis. Each of the deduced proteins examined in the TX114-DF extract and culture supernatant, except the SOWgp, revealed multiple hydrophobic domains, which may account for their partitioning into the detergent phase during the isolation procedure. Although SOWgp was not expected to be isolated in the detergent fraction (17), its association with the lipid layer at the surface of parasitic cells (7, 18) may account for its presence in the TX114-DF extract. The WoLF PSORT algorithm predicted that all but one of the deduced proteins were cell wall associated and/or extracellular. Patient seroreactivity, hydrophobicity, and parasitic cell wall association are features which suggested that these proteins were worthy of further examination as candidate T-cell-reactive antigens. An additional criterion for selection of antigens as vaccine candidates is the presence of epitopes which bind to MHC class II molecules (8). The ProPred algorithm has proved to be a valuable bioinformatics tool for identification of putative T-cell epitopes in microbial antigens and has permitted researchers to successfully progress from genome sequences to epitope-derived vaccine design (11). On the basis of ProPred analyses of the deduced proteins identified in the 2D PAGE gels, four cell wall-associated/extracellular antigens and one cytoplasmic antigen were shown to contain multiple promiscuous epitopes predicted to bind to at least 80% of the representative HLA-DR molecules in the algorithm. HLA-DR molecules account for more than 90% of the HLA class II isotypes expressed on APCs (43). The full-length sequence of Pep1 was predicted to contain five promiscuous epitopes.

The aspartyl protease (Pep1) of C. posadasii was originally isolated by Johnson and coworkers (23) from disrupted, formaldehyde-killed spherules. A formaldehyde-killed spherule vaccine has been shown to induce a protective response in mice and primates against coccidioidal infection but also revealed serious irritant properties which prevented its successful application as a human vaccine (30). Our interest in Pep1 was piqued by its abundance in SDS-PAGE separations of both parasitic cell wall extracts and culture supernatants of C. posadasii. In support of this observation, we found that expression of the PEP1 gene is constitutive during the parasitic cycle. Aspartyl proteases produced by other microbial pathogens have been proposed to be vaccine candidates. Immunization of mice with a secreted aspartyl protease of Candida albicans (Sap2) has been shown to significantly decrease severity of systemic candidiasis (46). Alum was used as the adjuvant. The authors proposed that the protective response was antibody mediated. An aspartyl protease isolated from Schistosoma japonicum, causative agent of schistosomiasis, has also been evaluated as a vaccine in a murine model of this disease (44). Immunization with the recombinant protein resulted in reduced worm burden in challenged mice but little to no effect in reducing the fecundity of the pathogen. The authors showed that the schistosome protease induced a mixed Th1/Th2 cytokine response (45). Immunization of C57BL/6 mice with recombinant Pep1 of C. posadasii induced a moderate in vitro proliferative response of isolated immune T cells in a recall experiment. Of particular interest, however, was that IFN-{gamma} was the most abundant secreted cytokine in the supernatants of the activated T lymphocytes. Only after incubation of the CD90+ T cells with antigen for 120 h was it possible to detect significant amounts of secreted IL-5 and IL-10. Results of clinical and animal model studies of coccidioidomycosis have supported the argument that Coccidioides antigens which stimulate a Th1 pathway of host response are essential components of a vaccine against this respiratory disease (9). Our data obtained from IFN-{gamma} ELISPOT assays of immune CD90+ T cells of both C57BL/6 and HLA-DR4 transgenic mice in the presence of the recombinant Pep1 provide additional evidence that this antigen stimulates a potent Th1 pathway of immune response. Two synthetic peptides (P1 and P2), which represent N-terminal regions of Pep1 that contain predicted MHC class II-binding epitopes, were shown to induce IFN-{gamma} production by immune T cells isolated from both strains of mice. These data suggest that promiscuous epitopes predicted to bind to human HLA-DR molecules also bind to murine MHC class II molecules and vice versa. P1 includes the signal peptide of the aspartyl protease. Protective epitopes of another vaccine candidate of C. posadasii, referred to as Ag2/Pra, have been demonstrated to be present within the N-terminal region of the protein, including the 18-residue signal peptide (22).

Results of rPep1 vaccination of C57BL/6 mice against a potentially lethal pulmonary infection of C. posadasii suggested that the animals had mounted a potent and durable cellular immune response against the pathogen. The majority of survivors cleared the fungus from their lungs over a 90-day period postchallenge. It is possible that immunization with rPep1activates both antibody- and cell-mediated immune defenses during the protective response against Coccidioides. Anti-rPep1antibody (both IgG2a and IgG1 isotypes) was detected by enzyme-linked immunosorbent assay (ELISA) in vaccinated mice just prior to infection and at 7 and 12 days postchallenge (data not shown). Humoral immunity may play a significant role in defense against coccidioidomycosis, particularly by opsonization of endospores which are small enough to be engulfed by host phagocytes (18). Since Pep1 is cell wall associated and produced throughout the parasitic cycle, this cell surface antigen may be involved in opsonization. Opsonins have been shown to contribute to the activation and binding of dendritic cells to Cryptococcus neoformans yeast, which results in enhanced antifungal activity (24). Evidence has been presented that dendritic cell activation is pivotal to defense against coccidioidal infection (3). Although the mechanisms by which vaccination with rPep1 influences innate and acquired immune response to C. posadasii infection are essentially unknown, the level of protection afforded by this single antigenic protein plus CpG ODN adjuvant engenders confidence that development of a recombinant vaccine against human coccidioidomycosis is feasible (8). This study has also demonstrated that combined applications of immunoproteomics and bioinformatics to compositional analyses of crude, protective cell wall extracts of Coccidioides represent an efficient method to screen for T-cell-reactive antigens and vaccine candidates.


arrow
ACKNOWLEDGMENTS
 
Support for this study was provided by Public Health Service grants AI19149 and UO1 AI50910 (Coccidioides genome sequencing project) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.


arrow
FOOTNOTES
 
* Corresponding author. Present address: Department of Biology University of Texas at San Antonio, Margaret Batts Tobin Building, Rm. 1.308E, 6900 North Loop 1604 West, San Antonio, TX 78249. Phone: (210) 458-7017. Fax: (210) 458-5658. E-mail: garry.cole{at}utsa.edu. Back

Editor: A. Casadevall


arrow
REFERENCES
 
    1
  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
    2. 2
  2. Anstead, G. M., G. Corcoran, J. Lewis, D. Berg, and J. R. Graybill. 2005. Refractory coccidioidomycosis treated with posaconazole. Clin. Infect. Dis. 40:1770-1776.[CrossRef][Medline]
    3. 3
  3. Awasthi, S., and D. M. Magee. 2004. Differences in expression of cell surface co-stimulatory molecules, Toll-like receptor genes and secretion of IL-12 by bone marrow-derived dendritic cells from susceptible and resistant mouse strains in response to Coccidioides posadasii. Cell. Immunol. 231:49-55.[CrossRef][Medline]
    4. 4
  4. Barnato, A. E., G. D. Sanders, and D. K. Owens. 2001. Cost-effectiveness of a potential vaccine for Coccidioides immitis. Emerg. Infect. Dis. 7:797-806.[Medline]
    5. 5
  5. Cochlovius, B., M. Stasser, O. Christ, L. Raddrizzani, J. Hammer, I. Mytilineos,and M. Zoller. 2000. In vitro and in vivo induction of a Th cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program. J. Immunol. 165:4731-4741.[Abstract/Free Full Text]
    6. 6
  6. Cole, G. T., T. N. Kirkland, M. Franco, S. Zhu, L. Yuan, S. H. Sun, and V. M. Hearn. 1988. Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis. Infect. Immun. 56:2695-2701.[Abstract/Free Full Text]
    7. 7
  7. Cole, G. T., K. R. Seshan, M. Franco, E. Bukownik, S. H. Sun, and V. M. Hearn. 1988. Isolation and morphology of an immunoreactive outer wall fraction produced by spherules of Coccidioides immitis. Infect. Immun. 56:2686-2694.[Abstract/Free Full Text]
    8. 8
  8. Cole, G. T., J. M. Xue, C. N. Okeke, E. J. Tarcha, V. Basrur, R. A. Schaller, R. A. Herr, J.-J. Yu, and C.-Y. Hung. 2004. A vaccine against coccidioidomycosis is justified and attainable. Med. Mycol. 42:189-216.[CrossRef][Medline]
    9. 9
  9. Cox, R. A., and D. M. Magee. 1998. Protective immunity in coccidioidomycosis. Res. Immunol. 149:417-428.[CrossRef][Medline]
    10. 10
  10. da Fonseca, C. A., R. S. A. Jesuino, M. S. S. Felipe, D. A. Cunha, W. A. Brito, and C. M. A. Soares. 2001. Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis. Microbes Infect. 3:535-542.[CrossRef][Medline]
    11. 11
  11. DeGroot, A. S., J. McMurry, L. Marcon, J. Franco, D. Rivera, M. Kutzler, D. Weiner, and B. Martin. 2005. Developing an epitope-driven tuberculosis (TB) vaccine. Vaccine 23:2121-2131.[CrossRef][Medline]
    12. 12
  12. Delgado, N., C.-Y. Hung, E. Tarcha, M. J. Gardner, and G. T. Cole. 2004. Profiling gene expression in Coccidioides posadasii. Med. Mycol. 42:59-71.[Medline]
    13. 13
  13. Delgado, N., J. Xue, J.-J. Yu, C.-Y. Hung, and G. T. Cole. 2003. A recombinant beta-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis. Infect. Immun. 71:3010-3019.[Abstract/Free Full Text]
    14. 14
  14. Fankhauser, N., and P. Maser. 2005. Identification of GPI anchor attachment signals by a Kohonen self-organizing map. Bioinformatics 21:1846-1852.[Abstract/Free Full Text]
    15. 15
  15. Fisher, M. C., G. L. Koenig, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73-84.[Abstract/Free Full Text]
    16. 16
  16. Hulo, N., C. J. Sigrist, V. Le Saux, P. S. Langendijk-Genevaux, L. Bordoli, A. Gattiker, E. De Castro, P. Bucher, and A. Bairoch. 2004. Recent improvements to the PROSITE database. Nucleic Acids Res. 32:134-137.
    17. 17
  17. Hung, C.-Y., N. M. Ampel, L. Christian, K. R. Seshan, and G. T. Cole. 2000. A major cell surface antigen of Coccidioides immitis which elicits both humoral and cellular immune responses. Infect. Immun. 68:584-593.[Abstract/Free Full Text]
    18. 18
  18. Hung, C.-Y., S. R. Kalpathi, J.-J. Yu, R. A. Schaller, J. Xue, V. Basrur, M. J. Gardner, and G. T. Cole. 2005. A metalloproteinase of Coccidioides posadasii contributes to evasion of host detection. Infect. Immun. 73:6689-6703.[Abstract/Free Full Text]
    19. 19
  19. Hung, C.-Y., J.-J. Yu, P. F. Lehmann, and G. T. Cole. 2001. Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated beta-glucosidase of Coccidioides immitis. Infect. Immun. 69:2211-2222.[Abstract/Free Full Text]
    20. 20
  20. Iwai, L. K., M. Yoshida, J. Sidney, M. A. Shikanai-Yasuda, A. C. Goldberg, M. A. Juliano, J. Hammer, L. Juliano, A. Sette, J. Kalil, L. R. Travassos, and E. Cunha-Neto. 2003. In silico prediction of peptides binding to multiple HLA-DR molecules accurately identifies immunodominant epitopes from gp43 of Paracoccidioides brasiliensis frequently recognized in primary peripheral blood mononuclear cell responses from sensitized individuals. Mol. Med. 9:209-219.[Medline]
    21. 21
  21. Janeway, C. A., P. Travers, M. Walport, and J. D. Capra. 1999. Immunobiology. The immune system in health and disease, 4th ed. Garland Publishing, New York, N.Y.
    22. 22
  22. Jiang, C., D. M. Magee, F. D. Ivey, and R. A. Cox. 2002. Role of signal peptide sequence in vaccine-induced protection against experimental coccidioidomycosis. Infect. Immun. 70:3539-3545.[Abstract/Free Full Text]
    23. 23
  23. Johnson, S. M., K. M. Kerekes, C. R. Zimmermann, R. H. Williams, and D. Pappagianis. 2000. Identification and cloning of an aspartyl proteinase from Coccidioides immitis. Gene 241:213-222.[CrossRef][Medline]
    24. 24
  24. Kelly, R. M., J. Chen, L. E. Yauch, and S. M. Levitz. 2005. Opsonic requirements for dendritic cell-mediated responses to Cryptococcus neoformans. Infect. Immun. 73:592-598.[Abstract/Free Full Text]
    25. 25
  25. Klinman, D. M. 2004. Immunotherapeutic uses of CpG oligodeoxynucleotides.Nat. Rev. Immunol. 4:249-258.[CrossRef][Medline]
    26. 26
  26. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132.[CrossRef][Medline]
    27. 27
  27. Lecara, G., R. A. Cox, and R. B. Simpson. 1983. Coccidioides immitis vaccine: potential of an alkali-soluble, water-soluble cell wall antigen. Infect. Immun. 39:473-475.[Abstract/Free Full Text]
    28. 28
  28. Li, K., J.-J. Yu, C.-Y. Hung, P. F. Lehmann, and G. T. Cole. 2001. Recombinant urease and urease DNA of Coccidioides immitis elicit an immunoprotective response against coccidioidomycosis in mice. Infect. Immun. 69:2878-2887.[Abstract/Free Full Text]
    29. 29
  29. Lima, K. M., S. A. dos Santos, J. M. Rodrigues, and C. L. Silva. 2004. Vaccine adjuvant: it makes the difference. Vaccine 22:2374-2379.[CrossRef][Medline]
    30. 30
  30. Pappagianis, D., et al. 1993. Evaluation of the protective efficacy of the killed Coccidioides immitis spherule vaccine in humans. Am. Rev. Respir. Dis. 148:656-660.[Medline]
    31. 31
  31. Pappagianis, D., R. Hector, H. B. Levin, and M. S. Collins. 1979. Immunization of mice against coccidioidomycosis with a subcellular vaccine. Infect. Immun. 25:440-445.[Abstract/Free Full Text]
    32. 32
  32. Pappagianis, D., H. B. Levin, C. E. Smith, R. J. Berman, and G. S. Kobayashi. 1961. Immunization of mice with viable C. immitis. J. Immunol. 86:28-34.[Abstract/Free Full Text]
    33. 33
  33. Park, B. J., K. Sige, V. Vaz, L. Komatsu, C. McRill, M. Phelan, T. Colman, A. C. Comrie, D. W. Warnock, J. N. Galgiani, and R. A. Hajjeh. 2005. An epidemic of coccidioidomycosis in Arizona associated with climatic changes, 1998-2001. J. Infect. Dis. 191:1981-1987.[CrossRef][Medline]
    34. 34
  34. Pitarch, A., J. Abian, M. Carrascal, M. Sanchez, C. Nombela, and C. Gil. 2004. Proteomics-based identification of novel Candida albicans antigens for diagnosis of systemic candidiasis in patients with underlying hematological malignancies. Proteomics 4:3084-3106.[CrossRef][Medline]
    35. 35
  35. Ponton, J., M. J. Omaetxebarria, N. Elguezabal, M. Alvarez, and M. D. Moragues. 2001. Immunoreactivity of the fungal cell wall. Med. Mycol. 39:101-110.[Medline]
    36. 36
  36. Proft, T., H. Hilbert, G. Layh-Schmitt, and R. Herrmann. 1995. The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH. J. Bacteriol. 177:3370-3378.[Abstract/Free Full Text]
    37. 37
  37. Rawlings, N. D., D. P. Tolle, and A. J. Barrett. 2004. MEROPS: the peptidase database. Nucleic Acids Res. 32:160-164.
    38. 38
  38. Reichard, U., G. T. Cole, R. Ruchel, and M. Monod. 2000. Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus. Int. J. Med. Microbiol. 290:85-96.[Medline]
    39. 39
  39. Shubitz, L., T. Peng, R. Perrill, J. Simons, K. Orsborn, and J. N. Galgiani. 2002. Protection of mice against Coccidioides immitis intranasal infection by vaccination with recombinant antigen 2/PRA. Infect. Immun. 70:3287-3289.[Abstract/Free Full Text]
    40. 40
  40. Singh, H., and G. P. Raghava. 2001. ProPred: prediction of HLA-DR binding sites. Bioinformatics 17:1236-1237.[Abstract/Free Full Text]
    41. 41
  41. Smith, C. E. 1942. Parallelism of coccidioidal and tuberculous infections. Radiology 38:643.
    42. 42
  42. Southwood, S., J. Sidney, A. Kondo, M.-F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chestnut, H. M. Grey, and A. Sette. 1998. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160:3363-3373.[Abstract/Free Full Text]
    43. 43
  43. Sturniolo, T., E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, and J. Hammer. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17:555-561.[CrossRef][Medline]
    44. 44
  44. Verity, C. K., D. P. McManus, and P. J. Brindley. 2001. Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum. Parasite Immunol. 23:153-162.[CrossRef][Medline]
    45. 45
  45. Verity, C. K., D. P. McManus, and P. J. Brindley. 2002. Cellular responses to Schistosoma japonicum cathepsin D aspartic protease. Parasite Immunol. 24:363-367.[CrossRef][Medline]
    46. 46
  46. Vilanova, M., L. Teixeira, I. Caramalho, E. Torrado, A. Marques, P. Madureira, A. Ribeiro, P. Ferreira, M. Gama, and J. Demengeot. 2004. Protection against systemic candidiasis in mice immunized with secreted aspartic proteinase 2. Immunology 111:334-342.[CrossRef][Medline]
    47. 47
  47. Wong, L.-P., P. C. Woo, A. Y. Wu, and K.-Y. Yuen. 2002. DNA immunization using a secreted cell wall antigen Mp1p is protective against Penicillium marneffei infection. Vaccine 20:2878-2886.[CrossRef][Medline]
    48. 48
  48. Xue, J., C.-Y. Hung, J.-J. Yu, and G. T. Cole. 2005. Immune response of vaccinated and non-vaccinated mice to Coccidioides posadasii infection. Vaccine 23:3535-3544.[CrossRef][Medline]
    49. 49
  49. Yates, J. R., J. K. Eng, A. L. McCormack, and D. Schieltz. 1995. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67:1426-1436.[Medline]
    50. 50
  50. Yin, Q. Y., P. W. J. de Groot, H. L. Dekker, L. de Jong, F. M. Klis, and C. G. de Koster. 2005. Comprehensive proteomic analysis of Saccharomyces cerevisiaecell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages. J. Biol. Chem. 280:20894-20901.[Abstract/Free Full Text]
    51. 51
  51. Yuan, L., and G. T. Cole. 1987. Isolation and characterization of an extracellular proteinase of Coccidioides immitis. Infect. Immun. 55:1970-1978.[Abstract/Free Full Text]
    52. 52
  52. Zuobi-Hasona, K., P. J. Crowley, A. Hasona, A. S. Bleiweis, and L. J. Brady. 2005. Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis. Electrophoresis 26:1200-1205.[CrossRef][Medline]

Infection and Immunity, January 2006, p. 516-527, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.516-527.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. (2009). An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection. FASEB J. 23: 3007-3019 [Abstract] [Full Text]  
  • Herr, R. A., Hung, C.-Y., Cole, G. T. (2007). Evaluation of Two Homologous Proline-Rich Proteins of Coccidioides posadasii as Candidate Vaccines against Coccidioidomycosis. Infect. Immun. 75: 5777-5787 [Abstract] [Full Text]  
  • Tarcha, E. J., Basrur, V., Hung, C.-Y., Gardner, M. J., Cole, G. T. (2006). Multivalent Recombinant Protein Vaccine against Coccidioidomycosis.. Infect. Immun. 74: 5802-5813 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tarcha, E. J.
Right arrow Articles by Cole, G. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tarcha, E. J.
Right arrow Articles by Cole, G. T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»