Safety and Immune protection of a RHΔompdcΔuprt mutant live-attenuated vaccine against Toxoplasmosis

Abstract

Abstract Background: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study explored the immunoprotective effects of a live attenuated vaccine in mice and cats. Methods: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated for the verification of its potential use in vaccination. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and the subsets of T lymphocytes. Finally, the immunoproctive effects were evaluated by challenging with tachyzoites of different strains in mice or cysts of ME49 strain in cats via monitoring the survival time of mice or determining the number and shedding period of oocysts in feline feces. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out in mice, as serum, CD19+ B cells, CD4+ T cells, CD8+ T cells, and total splenocytes were involved. Results: The ompdc and uprt genes were deleted by using CRISPR-Cas9 system to develop a uracil auxotrophic T. gondii strain RHΔompdcΔuprt. The intracellular replication, virulence and immune response of double knockout mutant were evaluated. Compared with the wild-type RH strain, the RHΔompdcΔuprt mutant notably reduced proliferation with limited intracellular escape. In addition, RHΔompdcΔuprt mutant strain exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. It’s worth noting that limited pathological change or tachyzoites were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, significantly high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) of mice were elicited by RHΔompdcΔuprt mutant, which were proven protective to reinfection with the T. gondii type I (RH), type II (ME49), and Chinese isolated strains (WH6). Remarkably, all mice vaccinated with RHΔompdcΔuprt survived a lethal challenge with RH and ME49, and WH6 strains. The immunized serum and splenocytes, especially CD8+ T cells, could significantly extend the survival time of mice challenged with RH strain compared with naïve mice. In addition, cats immunized with the mutant strain also produced high levels of IgG antibodies and notably decreased the shedding numbers of oocysts in feces (95.3%) than non-immunized cats. Conclusions: The avirulent RHΔompdcΔuprt mutant strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing safe and effective live attenuated vaccine.