Proteome-Wide Antigen Discovery Reveals Compartment-Specific Humoral Responses in Coccidioidomycosis

Coccidioidomycosis is a fungal infection of rising public health concern, with coccidioidal meningitis (CM) representing its most devastating manifestation. Diagnosis of CM remains challenging due to the limited sensitivity and technical demands of conventional cerebrospinal fluid (CSF) serology testing. To comprehensively characterize the humoral immune response across the full spectrum of coccidioidal disease, we designed and deployed a proteome-wide Coccidioides phage immunoprecipitation sequencing (PhIP-Seq) library tiling both C. immitis and C. posadasii proteomes. We profiled antibody reactivity in sera from 323 participants spanning six clinically defined disease severity categories, as well as CSF and matched serum from participants with confirmed CM (n=108) and Coccidioides -negative other neurologic disease (OND) controls (n=163). Serum profiling revealed a potential narrowing of the antigenic repertoire as disease severity increased, with subclinical participants mounting the broadest response (55 peptides from 55 proteins) compared to 3-7 peptides in symptomatic categories. Two proteins, spherule outer wall glycoprotein (SOWgp) and a previously uncharacterized Proline-rich Immunodominant Antigen (PIA1), emerged as immunodominant across disease categories. Enriched antigens were disproportionately proline-rich and repetitive, a structural feature associated with immunodominance in other pathogens. CSF profiling revealed a compartment-specific antibody signature in CM, with 94% of CSF-enriched seroreactive peptides absent from matched sera. To orthogonally validate these findings, we developed a five-antigen Luminex assay using SOWgp- and PIA1-derived peptides, achieving 100% sensitivity and 100% specificity in both a discovery cohort (CM n=20, OND n=20) and an independent, blinded validation cohort (34 CM and 36 OND CSF samples. These findings expand the repertoire of Coccidioides serological responses associated with disease severity and demonstrate that proteome-wide antibody discovery can be translated into a targeted, high-performance diagnostic platform.