Immune Protection Of The Cocktail DNA Vaccine Against Toxoplasma Gondii

Research of vaccines against Toxoplasma gondii experiences four stages: including whole protozoan body vaccine, parasite-specific composition vaccine, genetic engineering vaccine and DNA vaccine. However, an attenuated T. gondii strain S48 of tachyzoite stage is the only vaccine which has been commercialized for sheep toxoplasmosis and until now there is no vaccine against T. gondii available for human. More and more evidences had showed vaccination with stage-specific antigens can only lead to stage-limited protection and it was also reported multi-antigenic vaccine was more effective than single-antigenic vaccine. Therefore, it has been a trend to develop multi-antigenic vaccines against T. gondii. Moreover, the addition of adjuvant may play an important part in immune protection of the combined multi-antigenic vaccines. At present, the main vaccine candidates are as follows: T. gondii main surface antigens, SAGs; Dense Granule Antigens, GRAs; rhoptry proteins, ROPs; micronemes and so on. In the study, we constructed a combined multi-antigenic DNA vaccine containing GRA1, SAG1, ROP2 and AMA1 which use IL-12 plasmid as an adjuvant to enhance the immune protection.1. Construction and identification of plasmids GRA1, SAG1, ROP2 and AMA1.According to our previous study, DNA vaccines GRA1 and SAG1 provide partial immune protection against T. gondii infection. And it is also reported the candidate vaccine genes ROP2 and AMA1 have the same effects. In this study, we constructed recombinant plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2 and pVAX1-AMA1 used for cocktail DNA vaccine, and recombinant expression plasmids pVAX1-ROP2His and pVAX1-AMA1His.After transiently transfected into macrophages cells, the expressions of pVAX1-ROP2His and pVAX1-AMA1His proteins were assayed by Western blot. Our members have previously proved the proteins expressions of pVAX1-GRA1 and pVAX1-SAG1 by immunohistochemistry method. And the sequences of new recombinant plasmids pVAX1-GRA1 and pVAX1-SAG1 we constructed were in consistent with the past ones. After transiently transfected into macrophages cells, the protein expression of pNGVL3-mIL12 was confirmed by ELISA method. pNGVL3-mIL12 recombinant plasmid is the gift of Ph.D Alexander Rakhmilevich.2. Evaluation of immune protection provided by plasmids pVAX1-GRA1, pVAX1-SAG1, pVAX1-ROP2, pVAX1-AMA1 and pNGVL3-mIL12.Different strains of inbred mice have different levels of susceptibility to T. gondii caused mortality. The C3H, BALB/c, C57BL/6 etc. were the most frequently used mice for evaluation the effect of DNA vaccines against T. gondii. Because C3H/He (H-2k) are medium susceptible to T. gondii, it is expected to able to evaluate a long-term protection against T. gondii.In early experiments, mice were often challenged with high dose of T. gondii to determine protective effect of DNA vaccination, such as 104 or more of tachyzoites stage of T. gondii RH strain. And 104 tachyzoites has been considered a too high dose. These researches were always difficult to get ideal data, for instance survival rate. Challenge dose has been decreased from 105 to 104, then 500, finally 50 tachyzoites of T. gondii RH strain as recently reported. And prolongation of the survival time also has been replaced by the index of survival rate.The C3H mice were challenged in our research with 500 tachyzoites of T. gondii RH strain while BALB/c mice in Quan Liu et al.'s research was challenged 50 trophozoites. And that BALB/c mouse is more resistant to T. gondii than C3H mouse. Compared to Quan Liu et al.'s reports, lower survival rate was obtained in our research. It was maybe an influencing factor that the challenge dose of DNA vaccinated mice in our research was still too high. In the study, the mice of the group immunized with pVAX1 - GRA1 + SAG1 + ROP2 + AMA1 lived longer than the group immunized with pVAX1 - ROP2 + AMA1, which demonstrated that the first group produced higher immune protection compared to the other. Moreover, the co-immunization with IL-12 adjuvant enhanced the immune protection and prolonged survival time significantly. Even in the experimental group, one mouse among ten survived more than 30 days.The sera anti-TLA (Toxoplasma lysate antigen) IgG levels of the groups of mice immunized by the cocktail DNA vaccines were higher than the control one's. The IFN-γlevel of the group pVAX1-ROP2+AMA1 was higher than the pVAX1 control group, more genes group again elevate the IFN-γlevel, and IL-12 adjuvant still more elevate the IFN-γlevel. IL-4 levels of the experimental groups were no significant difference than the control group. The lymphocyte proliferation reactions of all experimental groups were higher than the pVAX1 control group. The ratio of CD4+/CD8+ T lymphocyte of pVAX1-GRA1+SAG1+ROP2+AMA1 + pNGVL3- mIL12 immunized group decreased. These demonstrated that the cocktail DNA vaccine induced specific immune response in C3H mice.The results confirmed the cocktail DNA vaccine has nice immune protection, combined immunity enhanced immune effect, and the effect was still elevated by the application of immune adjuvant.