Heterologous Prime-Boost Vaccination with GRA35-Encoding DNA and mRNA Vaccines Enhances Protective Immunity Against Toxoplasma gondii Infection in Mouse Models

Toxoplasma gondii is an obligate intracellular protozoan Parasite that causes toxoplasmosis, posing a significant threat to human health and livestock production worldwide. Although monovalent DNA or mRNA vaccines often confer only partial protection, whether these platforms can be effectively integrated into a heterologous prime-boost regimen against T. gondii remains to be fully elucidated. Here, we constructed GRA35-encoding DNA and mRNA vaccines and evaluated their immunogenicity and protective efficacy, administered either alone or in heterologous prime-boost combinations, in C57BL/6 and BALB/c mice. Both vaccines induced strong antigen-specific immune responses, with the heterologous prime-boost regimen eliciting the strongest effects and conferring the most robust and consistent protection across both mouse strains. Immunization triggered a predominantly Th1-skewed response characterized by significantly elevated IFN-γ production, accompanied by balanced antigen-specific IgG responses. Moreover, vaccinated mice developed rapid and potent cytotoxic T lymphocyte (CTL) responses. Following challenge with the RH and PRU strains, vaccinated mice exhibited prolonged survival and significantly reduced brain cyst burdens following PRU challenge compared with control groups. Collectively, these findings indicate that GRA35-based nucleic acid vaccines, particularly when administered in a heterologous prime-boost regimen, elicit multifaceted protective immune responses and represent promising vaccine candidates against T. gondii Infection.