Fungal Antigen Resources for Development of Vaccines and Diagnostics - Project Summary/Abstract Invasive fungal infections cause millions of deaths annually. Despite the obvious need, there are no licensed human fungal vaccines. Delays in diagnosing fungal infections are common due in part to suboptimal diagnostic tests. A major impediment to the development of vaccines and diagnostics is the paucity of publicly available, defined, and validated immunogenic fungal antigens. To address this resource deficiency, teams from three institutions (UMass Chan, UTSA, and UWM) will produce, validate, and provide relevant fungal antigens to the scientific community. Crude antigenic preparations from Cryptococcus neoformans, C. gattii, Candida albicans, C. auris, Aspergillus fumigatus, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, and C. posadasii will be manufactured. These include heat-killed whole fungi, Coccidioides formalin-fixed spherules, fractions from bead-beaten fungi, and fungal extracts. The biochemical composition and immunogenicity of the crude antigens will be determined. Purified fungal antigens will also be made. The UMass Chan, UTSA, and UWM teams selected 36 vaccine-candidate proteins (12 each from Cryptococcus, Coccidioides, and Blastomyces) for study. These 36 fungal proteins will be recombinantly produced in E. coli, formulated into vaccines, and tested for their ability to protect DR4 mice (which express a human MHC Class II allele) from a lethal fungal challenge. Based on the data obtained and aided by immunoinformatic analyses, four proteins/team will be down-selected for in-depth studies. Vaccine-mediated protection will be extended to include humanized DR3 mice (which express a different MHC Class II allele from DR4 mice). Overlapping peptide libraries will be synthesized and used to map the human MHC Class II epitopes that stimulate CD4+ T cell responses in vaccinated DR4 and DR3 mice. Proteins will be recombinantly expressed in yeast strains identical to or closely related to their strain of origin. A comparison of immune responses following vaccination with proteins recombinantly expressed in E. coli and yeast will enable determination of how epitope recognition is affected by yeast post-translational modifications. Finally, the ability of the recombinant proteins to stimulate CD4+ T cell responses in PBMCs from human subjects exposed to or infected with Cryptococcus, Coccidioides, Blastomyces, or Histoplasma will be determined. The fungal antigens manufactured and validated will be provided as resources to the scientific community. 1) Validated crude antigen preps will be deposited in the Biodefense and Emerging Infections Research Resources Repository (BEI) repository. 2) The E. coli and yeast recombinant antigens, along with the strains expressing these recombinant proteins, will be deposited to BEI. 3) The peptide epitopes and the corresponding MHC Class II alleles stimulated will be uploaded to the Immune Epitope Database (IEDB). These resources will be valuable to investigators studying fungal immunology, vaccinology, and diagnostics, particularly newcomers to the field and those who do not have the biosafety facilities or approval needed to study pathogenic fungi.
