Development of an HK-fbp1-based vaccine platform against Cryptococcus neoformans

Cryptococcal meningitis remains a leading cause of death among HIV/AIDS patients, and no antifungal vaccine is available in clinical use. We previously showed that mice immunized with a heat-killed preparation of Cryptococcus neoformans ( Cn ) fbp1 Δ mutant (HK-fbp1) are protected against cryptococcosis and other invasive mycoses, suggesting that HK-fbp1 is an intriguing vaccine candidate for further study. Recent studies have reported that heat-killed wild type H99 (HK-H99) can also offer some protection in other vaccination models. Here we set out to decipher the differential protective capacity of HK-fbp1 versus HK-H99 across multiple conditions. We observed that HK-fbp1 confers superior protection in both prophylactic and therapeutic intranasal immunization settings than HK-H99, which was associated with greater activation of pulmonary CD4+ T cells and stronger airway IFN-γ production. In addition, HK-fbp1 preparations were highly stable, retaining full protective efficacy even after 5 years of frozen storage. Mice intranasally immunized with two-dose HK-fbp1 were protected against challenge by up to 10 6 H99 cells, a 100-fold increase over the commonly used lethal inoculum. Addition of a CpG adjuvant ODN to HK-fbp1 (HK-bp1/ODN) led to a 5-fold reduction of the intranasal vaccine inoculum required for protection, and its single-dose intramuscular vaccination induced protection persisted for at least 3 months post vaccination. Together, these findings highlight the robust protective capacity and formulation versatility of HK-fbp1, supporting its further development as a stable, dose-sparing antifungal vaccine candidate.