Recombinant TgDDX3X DEAD-box protein confers partial protection in murine models of acute and chronic toxoplasmosis

Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, represents a global health concern for both humans and animals. This study evaluates the immunogenic potential and protective efficacy of a recombinant T. gondii DDX3X protein (rTgDDX3X; gene accession: TGGT1_226250) as a vaccine candidate. A gene fragment encoding residues 25–232 of TgDDX3X was amplified and inserted into the pET30α vector for expression in Escherichia coli BL21 (DE3) cells. The purified recombinant protein (∼30.1 kDa) was validated using SDS-PAGE and immunoblotting, which revealed specific reactivity with sera from T. gondii-infected mice. Antiserum produced in rTgDDX3X-immunized rats selectively recognized native TgDDX3X in tachyzoite lysates, and immunofluorescence analysis localized the protein primarily to the parasite cytoplasm. Vaccination elicited robust T cell activation, with progressive increases in CD4⁺ and CD8⁺ populations over 6 weeks. Elevated titers of anti-rTgDDX3X IgG antibodies were observed, predominantly of the IgG1 isotype (IgG1/IgG2a >1), indicating a Th2-skewed immune response. In challenge models, mice immunized with rTgDDX3X exhibited prolonged survival following infection with the virulent RH strain (mean: 12 days vs. 10 days in controls) and a reduced brain cyst burden after PRU infection (410 vs. 616 cysts/brain). Neutralization assays demonstrated that polyclonal antibodies against rTgDDX3X suppressed T. gondii proliferation In vitro and enhanced survival In vivo. Collectively, these findings indicate that rTgDDX3X induces measurable immune responses and confers partial protection, supporting its potential as a foundational antigen for toxoplasmosis vaccine development.