Live-attenuated Toxoplasma gondii RHΔddx6 mutant establishes comprehensive immunity against acute, chronic, and congenital toxoplasmosis.

Toxoplasma gondii is an obligate intracellular parasite causing severe disease in immunocompromised individuals and congenitally infected infants. Despite decades of research, no licensed human vaccine exists. This study evaluates a novel live-attenuated vaccine candidate based on depletion of DDX6, a conserved DEAD-box RNA helicase involved in post-transcriptional gene regulation. A Δddx6 strain was generated in the type I virulent RH strain using CRISPR/Cas9, and its phenotype was comprehensively assessed. Although the mutant displayed only subtle defects in certain in vitro assays, it exhibited almost complete avirulence in mice even at high inoculums (106 tachyzoites), representing a dramatic attenuation phenotype not fully predicted by in vitro analyses. A single immunization with 1 × 106 Δddx6 tachyzoites induced robust Th1-biased immunity characterized by high T. gondii-specific IgG titers, CD4+/CD8+ T-cell activation, and IFN-γ production. Vaccinated mice achieved 100% survival against lethal RH challenge and showed a 99.5% reduction in brain cyst burden following PRU strain infection. Furthermore, the vaccine completely prevented vertical transmission in a pregnancy model. Transcriptomic analysis revealed widespread dysregulation in the Δddx6 strain, particularly downregulation of key virulence factors (ROPs, SAGs) and genes involved in intracellular transport. These findings demonstrate that targeting DDX6 generates a highly attenuated yet strongly immunogenic strain with exceptional cross-stage protective efficacy against acute, chronic, and congenital toxoplasmosis, representing a promising vaccine candidate worthy of further development.