ROP64-Deficient Toxoplasma gondii Strain Confers Protection Against Acute and Chronic Toxoplasmosis in Mice

Toxoplasmosis, caused by Toxoplasma gondii, remains an important zoonotic disease affecting both human and animal health, particularly in immunocompromised individuals and during pregnancy. Current treatments are unable to eliminate latent tissue cysts and are further limited by adverse effects and prolonged treatment regimens, highlighting the need for effective vaccines. In this study, we evaluated the virulence attenuation and protective efficacy of a CRISPR-Cas9-generated rhoptry protein 64 knockout strain (RHΔrop64) as a live attenuated vaccine candidate in Kunming mice. RHΔrop64 exhibited markedly reduced virulence, and mice tolerated doses of up to 1 × 10² tachyzoites without obvious clinical signs. Immunization provided substantial protection against acute challenge with the parental RHΔku80 and ToxoDB#9 (PYS) strains at 30 days post-vaccination and partial protection against type II (Pru) tachyzoites. In addition, all immunized mice survived following oral challenge with 10 or 40 Pru cysts and exhibited reduced brain cyst burdens among surviving mice. Vaccination also induced measurable humoral and cytokine responses, characterized by increased levels of T. gondii-specific IgG and cytokines that were maintained for at least 75 days post-vaccination. These results suggest that RHΔrop64 has the potential to serve as a live attenuated vaccine candidate, providing substantial, although incomplete, protection against acute challenge with the three T. gondii strains tested and being associated with reduced brain cyst burdens among surviving mice following chronic challenge. Further evaluation is required to determine its safety and applicability as a vaccine candidate for food-producing animals and cats.