The Study Of Antigenic Epitopes From Toxoplasma Gondii Excreted/Secreted Antigens And Surface Protein 1

Toxoplasma gondii is an obligate intracellular parasite capable of infecting a variety of animals and causing toxoplasmosis. Infections of T. gondii in livestock have economic importance due to abortions, stillbirth and neonatal losses, especially in sheep and goats, and even death in pigs. It is essential to establish a rapid, highly specific and accurate method for diagnosing T. gondii infection and to develop effective vaccine against toxoplasmosis.Several vaccination trials have confirmed that diagnostic method and vaccine using single antigen are not satisfactory because of the complexity of the parasite life cycle. The diagnostic method and vaccine based on multi-epitope peptides have become an attractive strategy. In this study, based on the immune protection study of excreted/secreted antigens (ESA) and multi-epitope peptide, the linear B cell antigenic epitopes derived from major protein were identified. The results of this study lay a foundation for further study of diagnostic method and vaccine for toxoplasmosis. The main results are summarized as following:1) The ESA is able to stimulate a better cell-mediated immune response as compared to soluble or cysts antigen. Therefore, this antigen is a good candidate for development of immunizing agents against T. gondii infection. In the present study, we have evaluated the immunogenicity of EAS antigens of the the Gansu Jingtai strain (GJS) of T. gondii after administration in the pigs. The important parameters (cellular and humoral immunity, the amount of parasites in blood, the clinical symptoms, pathological changes and pathological anatomy) in immunization experiments were evaluated. The response elicited by ESA was capable of protecting against acute T. gondii infection in pigs.2) For the first time, we investigated murine immune responses to one linear B-cell epitope (derived from conserved regions of SAG1) when conjugated to two other defined T-cell epitopes (from conserved regions of GRA1 and GRA4) situated in tandem through the GG spacer sequence, with the latter positioned adjacent to a polylysine core. Immunization of BALB/c and Kunming mice with the MAP construct in Freund's adjuvant induced not only a humoral immune response but also a cellular response. After challenge with lethal doses of T. gondii (1×103), vaccinated mice had increased survival time in comparison with unvaccinated controls. Our data demonstrated that a MAP construct could trigger strong humoral and cellular responses against T. gondii, and that the MAP is a vaccine candidate worth of further study.3) The B cell epitopes of SAG 1 and GRA1 were screened and identified by prediction of linear B cell epitopes and pepscan technique. The two new highly conserved linear B cell epitopes: 91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120 and 151PVTTQTFVVGCIKGDDAQSCMVTVTVQARA180 derived from SAG1 were identified successfully. The three new highly conserved linear B cell epitopes: 134YSEVGNVNMEEVIDTMKSMQ153,164NKGETVEEAIEDVAQAEGLN183 and 214LEKDKQQLKDDIGFLTGERE233 derived from GRA1 were identified successfully. A good reactivity was monitored with sera from pig infected with T. gondii. The results of this study may aid in the design of an epitope vaccine against T. gondii and the development of diagnostic reagents for toxoplasmosis.4) In this study, we developed a Pep- ELISA using peptides, 91PTLAYSPNRQICPAGTTSSCTSKAVTLSSL120 and 164NKGETVEEAIEDVAQAEGLN183, that can detect antibody against T. gondii. The sensitivity and specificity were 88.06% and 97.87% respectively. It is noteworthy that the diagnostic sensitivity of the Pep-ELISA may be improved by the inclusion of other peptides, originating from the same or other proteins. The Pep-ELISA described here may constitute an important tool for the diagnosis of toxoplasmosis.