Experimental Study On The Vaccine Of Toxoplasma Gondii Encoding GRA10 Epitopes Combined With Chitosan Microsphere

T. gondii vaccine have undergone the development of killed parasites vaccines, attenuated parasites vaccines and the genetic vaccines. Killed parasites have a good security, but no ideal to induce long time protection. Attenuated vaccine have been proved to have a good immunity and induce protective response effectively. However, it may cause a reversion to virulence and a form to cysts. Genetic vaccines could induce a protective immune response in animals but are not considered enough sufficiently safe for use in humans. Vaccine based on epitopes, which is a developed vaccine trend, is considered to be the fourth generation vaccine. Epitope vaccine have the advantage of designing flexible, direct synthesis which can enhance antigen presentation efficiency and induce the host to produce efficient humoral and cellular responses.In this research, bioinformatics were used to predict B cell epitope, CD4+ and CD8+ T cell epitope of GRA10. Dense granules protein (GRAs) secreted by the toxoplasma dense granules participate in the composition of parasitophorous vacuole and cyst. GRA10 is proved to play an important role for the intracellular growth and proliferation of T. gondii. We constructed a multi-epitope peptide with dominant epitopes which selected by immunological method. A GRA10 multi-epitope peptide loaded chitosan microsphere was further constructed. Chitosan microsphere have drown much attention because of their biological safety and ease to synthesize and modification in vaccine researches.We observed the change in ability of antigen presenting in dendritic cell, and then discussed the immune mechanism of this vaccine. After vaccinated BALB/c mice, cellular and humoral immune response were detected and protective efficacy was evaluated by challenging immunized mice with the PRU strain of T. gondii. Our results indicated that the GRA10 multi-epitope peptide showed stronger immunogenicity than GRA10 single epitope peptide. GRA10 multi-epitope peptide loaded chitosan microsphere induced much higher levels of IgG and IgG2a, and thus indicated that the microsphere vaccine could effectively improve the humoral response. Furthermore, microsphere vaccine can significantly augment Th1-mediated cellular immune responses and higher level of IL-2, IFN-γ during the cellular response against toxoplasmosis. In addition, a significant reduction in the cyst burden was detected in the mice immunized microsphere.In summary, GRA10 multi-epitope peptide loaded chitosan microsphere is a potential candidate vaccine. It elicits both robust humoral and cellular immune responses, accompanied by a significant reduction in brain cyst burdens in vaccinated mice with the PRU strain of T. gondii. Therefore, the results from this research should contribute to development of an effective epitope-microsphere vaccine for preventing T. gondii.