verbatim) The applicants have used a novel in vivo microbial expression technology called In Vivo Induced Antigen Technology (IVIAT) to study the pathogenesis of oropharyngeal candidiasis (OPC) in HIV-infected patients. IVIAT uses anti-Candida albicans antibodies in the sera of HIV-infected patients to identify antigens that are expressed in vivo by C. albicans but are not expressed during routine in vitro growth. In preliminary studies, they have confirmed that IVIAT can identify virulence factors for C. albicans. In this proposal, they propose to modify IVIAT to identify antigens expressed by C. albicans during OPC but not expressed during either colonization of the oral mucosa by C. albicans or during in vitro growth. They hypothesize that some of the antigens expressed by C. albicans exclusively during OPC are important virulence factors, and identifying these antigens will provide insight into the mechanisms by which C. albicans is transformed from a harmless commensal organism into an invasive pathogen. This application has five specific aims. In the first specific aim, two separate pooled batches of sera, one from HIV-infected patients with OPC and the other from HIV-infected patients with colonization, will be exhaustively adsorbed with in vitro grown clinical C. albicans isolates to remove all antibodies that react with antigens expressed in vitro. The adsorbed sera will be used for differential screening of a C. albicans genomic expression library.
In specific aim 2, the plasmid DNA will be purified from clones that are reactive with the OPC sera but not with the colonization sera and the open reading frames (ORFs) responsible for the serum reactivity will be determined.
In specific aim 3, they will confirm that the IVIAT antigens are not expressed by C. albicans in vitro.
In specific aim 4, they will clone C. albicans genes encoding IVIAT antigens and purify the proteins expressed by these genes. Lastly, in specific aim 5, they will confirm that the IVIAT antigens are present within thrush samples recovered from HIV-infected patients by light microscopic immunohistochemistry using antibody raised against the purified proteins. They hope that this study will identify new C. albicans virulence factors, increase our understanding of the humoral response to OPC, and lead to potential applications for drug, vaccine or diagnostic test development. With the experience from this project, IVIAT should be readily adaptable to study other candidal infections and other fungal pathogens. Potential advantages of IVIAT over existing technologies include the use of the human immune response to identify in vivo expressed genes rather than animal models, its relative technical simplicity, and its ability to study differential gene expression in different types of C. albicans infections in humans.
