Targeting sexual stage-specific flagellar proteins to develop vaccines for feline toxoplasmosis.

Toxoplasma gondii mainly relies on feline-shed oocysts for environmental transmission, making the blockade of oocyst shedding an effective strategy for toxoplasmosis control. Although cilia- and flagella-associated proteins (CFAPs) are essential for flagellar assembly, maturation, and motility in mammalian male gametes, their biological roles and transmission-blocking vaccine potential in apicomplexan parasites remain unexplored. Here, we identified CFAP221 as a sexual stage-exclusive antigen of T. gondii. CRISPR/Cas9-mediated knockout of CFAP221 did not affect tachyzoite growth or mouse virulence, but completely abolished oocyst shedding in cats, confirming its involvement in sexual development. To assess vaccine efficacy, cats were immunized with developmentally defective Δcfap221 tachyzoites or recombinant CFAP221 (rCFAP221) subunit vaccines. Comprehensive physical, hematological, and biochemical monitoring confirmed that all vaccine candidates were safe and well tolerated in cats. Upon oral challenge with PRU tissue cysts, daily fecal output was monitored from day 2 post-challenge for two weeks. Δcfap221 immunization reduced oocyst shedding by 70%, while unadjuvanted rCFAP221 decreased oocyst output by over 90%. Remarkably, adjuvanted rCFAP221 achieved complete blockage of oocyst shedding. These findings indicate CFAP221 as a highly promising transmission-blocking vaccine candidate against T. gondii.