The immune-stimulating particle adjuvant (ISPA) as a versatile adjuvant platform for recombinant vaccines against...

Toxoplasma gondii is a widespread intracellular parasite that establishes chronic infection through the persistence of tissue cysts, for which no effective treatment or licensed vaccines for humans are currently available. Preventive vaccination of animal hosts represents a promising strategy to reduce parasite transmission and disease burden. In this study, we evaluated the immunogenicity and protective efficacy of recombinant subunit vaccines based on T. gondii GRA7, ROP2, or profilin (TgPF), formulated with the Immune-Stimulating Particle Adjuvant (ISPA), a saponin-based particulate adjuvant structurally related to ISCOMATRIX. C57BL/6 mice were immunized intradermally and orally challenged with a sublethal dose of T. gondii ME49 cysts. Vaccines formulated with ISPA induced robust antigen-specific immune responses, characterized by increased IgG levels with a mixed IgG1/IgG2b profile, enhanced splenocyte proliferation, and the activation of IFN-γ-producing CD4⁺ and CD8⁺ T cells, accompanied by regulated IL-10 production. Importantly, all ISPAadjuvanted formulations significantly reduced brain cyst burden compared with control groups, whereas recombinant proteins or ISPA administered alone failed to confer protection. These results provide the first evidence supporting the use of ISPA as an adjuvant in T. gondii subunit vaccines and highlight its potential as a safe and effective platform for inducing balanced humoral and cellular immunity against intracellular pathogens.