Recombinant Toxoplasma gondii expressing OVA confers antigen-specific protection against Eimeria falciformis

Coccidiosis, caused by protozoan parasites of the genus Eimeria, remains a major enteric disease that requires effective antigen-specific cellular immunity for protection. In this study, we evaluated genetically engineered Toxoplasma gondii, an apicomplexan parasite closely related to Eimeria, as a live-vector platform for antigen delivery and induction of protective immunity against Eimeria infection. Recombinant RHΔku80 strains expressing ovalbumin (OVA) alone or fused with four tandem copies of complement fragment C3d (OVA-C3d₄) were generated using a CRISPR/Cas9-mediated strategy. Stable transgene integration and expression were confirmed by PCR, immunofluorescence assay, western blotting, and whole-genome resequencing. Phenotypic characterization demonstrated that expression of OVA or OVA-C3d₄ did not significantly affect parasite invasion, intracellular replication, plaque formation, or acute virulence in mice. Immunization with recombinant parasites induced robust antigen-specific cellular immune responses, as evidenced by increased frequencies of IFN-γ-secreting splenocytes following stimulation with OVA-derived CD4⁺ and CD8⁺ T-cell epitopes. Fusion with C3d₄ modestly influenced immune responses by altering antibody isotype profiles in an inversely dose-dependent manner, although total antibody titers were not substantially increased. Protective efficacy was evaluated using challenge infection with OVA-expressing Eimeria falciformis. Mice immunized with RHΔku80-OVA or RHΔku80-OVA-C3d₄ exhibited significantly reduced oocyst shedding and alleviated intestinal pathology compared with unimmunized controls. However, no significant additional protective benefit was observed for the C3d₄-containing recombinant strain compared with the OVA-expressing strain under the experimental conditions tested. Collectively, these findings demonstrate that recombinant T. gondii can serve as an effective live-vector platform for inducing antigen-specific cellular immunity and partial protection against OVA-expressing E. falciformis challenge. This study highlights the potential of protozoan-based vaccine platforms and supports a potential contribution of antigen-specific cellular immunity to protection against coccidiosis.