Proteome-wide Humoral Immune Profiling in Central Nervous System Coccidioidomycosis Using Programmable Phage Display
Christine L. Boutros1, Ravi Dandekar2, Mark Voorhies3, Christina Homer4, George R. Thompson5, Satya Dandekar5, Anita Sil3, Michael R. Wilson2
1Neuroscience, 2Neurology, 3Microbiology & Immunology, 4Department of Medicine, University of California, San Francisco, 5Medical Microbiology and Immunology, University of California, Davis
Objective:
Comprehensively assess the humoral immune response to Coccidioides infection using a new high-resolution, proteome-wide antibody profiling assay in patients with and without coccidioidal meningitis.
Background:
Valley Fever is caused by inhalation of the soil-based fungus Coccidioides, which is endemic to the southwestern U.S. Most infections are asymptomatic, but 5-10% result in chronic complications, and sometimes death. CNS dissemination often necessitates surgical interventions and lifelong anti-fungal therapy. Because few Coccidioides antigens are known a) current diagnostic assays are variably sensitive and specific, b) vaccine design efforts are limited, and c) differences in the humoral immune response between patients with and without disseminated disease are unknown.
Design/Methods:
Known protein and predicted protein coding regions of the C. immitis and C. posadasii genomes were computationally tiled into overlapping 50 amino acid segments and collapsed on 95% sequence similarity. Oligonucleotides were synthesized, cloned, and packaged into T7 bacteriophage so the encoded peptides would be displayed on the phage surface. Multiple rounds of immunoprecipitation with patient-matched cerebrospinal fluid (CSF) and serum were performed (n=20 patients), and immunoprecipitated phage were sequenced. Peptides were counted by aligning sequences to the synthetic Coccidioides peptide library. Peptide enrichment was calculated relative to uninfected and no template controls.
Results:
To assess the quality of the new Coccidioides phage display library, the library was sequenced. On average, 94% of the 202,919 designed Coccidioides peptides were displayed. Preliminary immunoprecipitations with coccidioidal meningitis CSF enriched multiple proteins, including two known extracellular antigens: SOWgp and a CFEM domain containing extracellular membrane protein.
Conclusions:
We successfully designed and built a new assay that enables agnostic anti-Coccidioides antibody profiling. In preliminary experiments, we validated the immunogenicity of two Coccidioides cell surface proteins and anticipate future experiments will provide significant insights into the humoral response to coccidioidomycosis, especially in patients with CNS disseminated disease.