Vaccination With A DNA Vaccine Encoding T- And B-cell Epitopes Of SAG1,GRA2,GRA7 And ROP16 Elicits Protection Against Acute Toxoplasmosis In Mice

Background:Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects large variety of warm-blooded animals including humans, livestock, and marine mammals, and causes the disease toxoplasmosis. Although T. gondii infection rates differ significantly from country to country, the epidemic trend of toxoplasmosis is still on the rise. In these circumstances, developing an effective vaccine against T. gondii is urgently needed for preventing and treating toxoplasmosis.Objectives:In the present study, a multi-epitopes DNA vaccine and a recombinant plasmid pEGFP-RANTES encoding RANTES were constructed. Through evaluating the immune protective efficacy of the DNA vaccine with or without the genetic adjuvant RANTES against acute toxoplasmosis in mice, we supposed that the DNA vaccine and the genetic adjuvant might be new candidates for further vaccine development against T. gondii infection.Methods:Twelve T-and B-cell epitopes from SAG1, GRA2, GRA7 and ROP16 of T. gondii were predicted by bioinformatics analysis, and then a new gene fragment MEG was synthetized. MEG was cloned into the eukaryotic expression plasmid pEGFP-Cl to construct pEGFP-TgMEG RANTES gene fragment was amplified by PCR from cDN A of murine spleens using their respective primers, and then the RANTES PCR product was cloned into pEASY-Tl vector. After correct sequencing, the target gene RANTES was subcloned into the eukaryotic expression plasmid pEGFP-Cl to construct pEGFP-RANTES. After verified correctly by restriction enzymes digestion, PCR and sequencing, the two recombinant plasmids were transfected into HEK293T cells. The target protein expressions were detected by fluorescence microscopy; the target protein activity was detected by SDS-PAGE and Western blotting. The recombinant plasmids were prepared in large quantities for the preparation of DNA vaccine and genetic adjuvant. Through immunizing mice intramuscularly with DNA vaccine with or without the genetic adjuvant RANTES, the immune responses were evaluated. After challenged with high virulent T. gondii RH strain, the mice were observed with survival times.Results:Mice immunized with the multi-epitopes DNA vaccine gained higher levels of IgG titers and IgG2a subclass titers, significant production of gamma interferon (IFN-y), percentage of T lymphocyte subsets, and longer survival times against the acute infection of T. gondii compared with those of mice administered with empty plasmid and those in control groups. Furthermore, a genetic adjuvant pEGFP-RANTES (pRANTES) could enhance the efficacy of the multi-epitopes DNA vaccine associating with humoral and cellular (Thl, CD8+T cell) immune responses.Conclusions:The multi-epitopes DNA vaccine in this study was revealed that it could produce not only cellular but humoral immune response in mice, and could elicit partial protection against acute T. gondii infection. The genetic adjuvant pEGFP-RANTES (pRANTES) could enhance the efficacy of the multi-epitopes DNA vaccine. Thus, the DNA vaccine and the genetic adjuvant might be new candidates for further vaccine development against T. gondii infection.