Application of Immunoproteomics and Bioinformatics to coccidioidomycosis Vaccinology

Coccidioidomycosis is a respiratory disease of humans caused by the desert soilborne fungal pathogen, Coccidioides spp. Recurrent epidemics of this mycosis in 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 2DPAGE gels, trypsin-digested and sequenced by tandem mass spectrometry. The fulllength 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 class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced interferon-gamma production by immune T lymphocytes. The T cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with C. posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive. 43 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 Southwestern United States and parts of Mexico, Central and South American (8). Inhalation of the airborne fungal spores (arthroconidia) can lead to onset of a disease (coccidioidomycosis, 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 endemic regions have been reported (33). The increased numbers of visitors, retirees, military recruits, etc. from non-endemic regions who have moved to the Southwest over the past 10 years has resulted in a large immunonaive population at risk of infection (8). Coccidioidomycosis in Arizona is currently the fourth most commonly reported infectious disease; only gonorrhea, chlamydial and hepatitis C infections are reported more frequently (33). Examinations of the incidence of coccidioidal infections in Arizona between 1998 and 2001 have indicated that persons 65 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 those 0-18 years old (33).