Recombinant Protein Tat-TPI-induced CD8 ~ + T Cell Response To Protective Immunity Against Schistosoma Japonicum Infection

Schistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. Of the three major schistosome species that infect humans, Schistosoma japonicum is recognized as the most difficult to control. China is one of the countries most affected by Schistosoma japonicum. Although in the past six decades, a series of comprehensive measures have been adopted to control schistosomiasis japonica in China and we have achieved good control effect, there remain many challenges we need to face. According to the latest statistics, about 0.11 million people are infected and more than 251 million people living in 453 counties remain endemic for S. japonicum in China until 2014. Praziquantel is the effective drug for treatment, but it does not prevent post-treatment reinfection and long-term application might lead to increase the drug resistance. Therefore, development of safe and effective vaccines is an attractive goal.Generally, the studies of protective mechanisms against schistosomiasis are concentrated on CD4+T cells and antibody responses. First, CD4+T-cell-produced IFN-y is considered as the key factor. IFN-y activates host macrophages to produce NO, which help eliminate parasites efficiently. Secondly, schistosome specific IgG antibody levels were positively correlated with the protection level against S. mansoni infection. However, the role of CD8+T cells played in schistosome infection has been debated.Tat-fused protein could efficiently transduce into cells and was processed by proteasomes for MHC class I-dependent presentation to CD8+T lymphocytes. In this study, we evaluated the immunogenicity and efficacy of the fusion protein containing the protein transduction domain of HIV-1 TAT and the Schistosoma japonicum antigen Sj-TPI in a mouse model. The approach we used was to express this fused antigen to facilitate the entry of protein into cells and enhance CD8+and CD4+T-cell response.The main results are as follows:1. Expression and purification of fusion protein Tat-TPI and TPI The recombinant plasmids pET-32a(+)-Tat-TPI and pET-32a(+)-TPI were successfully constructed. Then the recombinant plasmids were transformed into E. coli strain BMRosetta (DE3) for protein expression. After induced expression by IPTG and subsequent purification with Ni-NTA Sepharose FF, the purified fusion protein Tat-TPI and TPI were obtained.2. The fusion proteins could be recognized by S. japonicum infected-mice serum Purified fusion proteins Tat-TPI and TPI could be recognized by S. japonicum infected-mice serum using western blot. It suggested that these proteins have good antigenicity.3. Immune response in the draining popliteal lymph nodes To investigate the immune effect induced by Tat-TPI and TPI, we injected these two proteins to mice footpad respectively, and observed T-cell response in the draining popliteal lymph nodes. On day 4 post injection, Tat-TPI and TPI vaccination can both evoke host’s Tel response in the draining popliteal lymph nodes compared to PBS controls (P< 0.01). More importantly, CD4+IFN-y+ T-cell response could be quickly evoked by Tat-TPI instead of TPI.4. CD8+T cells could help Thl response To ascertain if CD8+T cells could help Thl response, we used anti-mouse CD8a antibody to block CD8+T cells. Flow cytometry showed Tat-TPI stimulated splenocytes produced higher CD3+CD4+IFN-y+cells than TPI stimulation. When CD8+T cells were blocked, T-TPI stimulated spleen cells expressed lower levels of CD4+IFN-γ+ cells than other groups.5. Immune protection associated with Tat-TPI and TPI vaccination At six weeks after S. japonicum cercariae challenge, we calculated the numbers of adult worm recovered and eggs in the livers. Results showed that Tat-TPI-immunised group could induce high levels of Thl, Tc1 and IgG responses and significantly decrease hepatic egg granulomatous area though it did not achieve the ideal reduction rate of worm (11.9%) and egg (12.4%) burden.In conclusion, we successfully expressed fusion protein Tat-TPI. Tat-coupled TPI immunisation could activate CD8+T cells in mice, which assist CD4+T cells to produce more IFN-y. Although this vaccine strategy with Tat-TPI immunisation did not achieve the ideal worm and egg reduction, it did reduce the liver pathological damage in mice after Schistosoma japonicum infection.