| Toxoplasma gondii is an obligate intracellular protozoan parasite. As a significant human and animal pathogen, it was estimated to infect about one-third of the world's human population and all warm-blooded mammals which was frequently associated with congenital infection and abortion (Montoya & Liesenfeld,2004; Tenter et al.,2000). Therefore, T. gondii is of veterinary and medical importance. So far, drugs can not clean T. gondii from host. Thus, development of an effective and safe vaccine for controlling T. gondii infection in those animals is an important goal for scientists worldwide. However, due to T. gondii multiplicity immunity, the progress of new vaccines against T. gondii maintains is slowly.Therefore, in the present study, we explored the new vaccine design against T. gondii, and constructed "suicidal DNA vaccine" pSCA-MIC3 and the recombinant baculovirus vaccine Ac-V-MIC3, Ac-V-SAG1. Furthermore, the immune efficacy of the candidate vaccines was investigated in BALB/c mice. The research works were as following:1. Construction and immune effect of Toxoplasma gondii suicidal DNA vaccine pSCA-MIC3Among new kinds of vaccines, DNA vaccines become a focus as such vaccines have been shown to elicit potent, long-lasting humoral and cell-mediated immunity and low cost in manufacture,storage, and administration. But conventional DNA vaccines raise certain concerns such as potential integration into the host genome and cell transformation as well as their potency since they do not have the intrinsic ability to amplify in vivo as viral vaccines do. More recently, "suicidal DNA vaccine vector pSCA1", based on the replicon of alphaviruses, has been developed. Besides the advantages of conventional DNA vaccines, it has some prominent advantages. For example, it could break immunological tolerance by activating innate antiviral pathways, catalyzes cytoplasmic self-amplification of recombinant RNA,and high-level expression of the heterologous antigens.Furthermore,when a suicidal DNA vaccine is transfected into cells, it leads eventually to apoptosis of the transfected cells, which is particularly important in alleviating the concerns of potential integration and celltransformation generated by the use of conventional DNAvaccines. All these advantages indicate that suicidal DNA vaccine is a good delivery vehicle and could be used to replace conventional DNA vaccines.In this study, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferationresponse and IFN-y induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P< 0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P< 0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P> 0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.2. Construction and immune effect of recombinant baculovirus vaccine Ac-V-MIC3,Ac-V-SAG1Although AcMNPV are failing to replicate in vertebrate cells, it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene. Follwing these findings, baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells. Furthermore, it has been reported that a pseudotype baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope can extend the host range and increase the transduction efficiency in mammalian cells.so it was considered as an excellent expressing vector.In this study, the value of Bac-VSV-G in delivering T. gondii antigen was investigated. T. gondii MIC3,SAG1 gene was cloned into Bac-VSV-G, and recombinant baculovirus Ac-V-MIC3,Ac-V-SAG1 were obtained. Indirect immunofluorescence test showed Ac-V-MIC3,Ac-V-SAG1 were efficiently transduced and expressed in pig kidney cells. Then BALB/c mice were immunized with Ac-V-MIC3,Ac-V-SAG1 at different doses (1010pfu/mL,109pfu/mL,108pfu/mL/mouse) and challenged with T. gondii RH strain tachyzoites after immunization. The levels of specific T. gondii antibody, IFN-y, IL-4, IL-10 expression and release, and the survival rate of treated mice were evaluated. Compared with the mice immunized with DNA vaccine (pcDNA/SAG1,pcDNA/MIC3) encoding the same gene, Ac-V-MIC3,Ac-V-SAG1 induced higher levels of specific T. gondii antibody and IFN-y expression and the survival rate of mice with Ac-V-MIC3,Ac-V-SAG1 were significantly improved. These results indicated that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generation of vaccines against T. gondii infection. |
