Protective Efficacy Of A Toxoplasma Gondii Vaccine Encoding On Multi-stage Antigens Conjugated To Ubiquitin

Toxoplasma gondii is a widely prevalent intracellular parasite that could infect almost all the warm-blooded animals and birds. The infections of parasites in healthy people usually presents asymptomatic latent infection. However, in pregnant women the parasites can cause miscarriage, premature birth and stillbirth, and survivors often left severe neonatal neurological and retinopathy. Moreover, these animals which serve as intermediate hosts for T. gondii, may be a potential source of infection for humans and cause zoonotic toxoplasmosis. Research into vaccines is promising as there are no effective drugs to treat toxoplasmosis.Many efforts have been made to develop vaccines against T. gondii including killed vaccines, live attenuated vaccines, and genetic engineering vaccines. However, this kind of vaccine pose a risk of infection to animals handling the vaccines for the reason of virulence restoring. The genetical engineered vaccine based on different antigens expressed in Toxoplasma gondii infection stages earns people more and more attention due to its safty and efficiency. However, the complex life cycle of T. gondi has three major infectious stages:tachyzoites, bradyzoites (in tissue cysts) and sporozoites (in oocysts). Vaccines based on antigens expressed in the single stage can’t induce complete protective immunity against T. gondii. Thus, vaccine contains antigens derived from different stages of the parasite life cycle is required.In order to prevent humans and animals from the infection of different T. gondii infective stages, we selected SAG3101-144, ROP18347.396, MIC6288-347, GRA7182-224, MAG158-125, BAG1156-211 and SPA142-200, and a specific CD4+ T cell epitope, AS15, derived from antigens in tachyzoite, bradyzoite and sporozoite stages of T. gondii, and linked them to construct the multistage antigen segments (MAS) of T. gondii. We further constructed a recombinant DNA vaccine and an adenovirus vaccine encoding multi-stage antigen of T. gondii linked to ubiquitin molecules and vaccinated B ALB/c mice with different strategy. Enzyme-Linked Immunosorbent Assays (ELISA), Flow Cytometry (FCM) were used to detect the cellular and humoral immune response. Protective efficacy was evaluated by challenging immunized mice with type Ⅰ and type Ⅱ parasite.Our results indicated that the DNA vaccine had the advantage of inducing a stronger humoral response, including much higher levels of IgG and IgG2a compared with recombinant adenovirus vaccine immunized group. Whereas the adenovirus-vectored vaccine resulted in higher levels of IFN-γ, IL-2, lymphocyte proliferation and the percentages of CD8+ T cells, these results indicated that the recombinant adenovirus vaccine could effectively improve the cellular immune response. Priming with the DNA vaccine and boosting with the adenovirus-vectored vaccine led to highest levels of cellular and humoral immune response, as well as the effective protection with increased survival rate (67%) and subsequent decreased brain cyst burden (296±92).The heterologous vaccination of the DNA vaccine followed by recombinant adenovirus vaccine encoding ubiquitin-conjugated multi-antigens derived from different infective stages was proved to be a potential vaccine protect mice against acute and chronic infection of T. gondii. In the future, diversity of antigens from different stages should be considered to vaccine construction to induce more comprehensive protection against parasites infection.