Immunological Events Correlated With High Level Protection Induced By Attenuated Cercariae Against Schistosoma Japonicum In Pigs

Schistosomiasis japonica is still one of the public health problems, which seriously harms people's health and hinders the economic development of endemic areas in China. Current schistosomiasis control is based on limited methods such as community chemotherapy, snail control and environmental modifications. Therefore, development of vaccines to protect both human and the domestic animals is an attractive goal. Protective immunity to schistosomiasis induced by radiation-attenuated cercariae has been achieved in a variety of schistosome/host species. The results of vaccination experiments in a number of animal models (murine, artiodactyl and primates, etc) in laboratory and field trials have shown that radiation-attenuated vaccines can induce high level and stable protection against the challenge with Schistosoma mansoni (S. mansoni). Great progress has been made in the immunological mechanisms against S. mansoni, particularly in mouse model. Both antibody and CD4~+ T-cell-mediated, IFN-γ-dependent e?ector can be induced by vaccination with attenuated S. mansoni cercariae. In mice with depletion of IFN-γduring challenge using neutralizing monoclonal antibody or in IFN-γknockout (KO) or IFN-γReceptor KO mice, the protection induced by attenuated cercaria vaccine had a modest reduction. But the B-cell-deficient mice transferd the serum from singly vaccinated wild type mice could restore the high protection. In primates, repeated vaccination induced early IFN-γproduction, but type 2 cytokines were dominant by the time of challenge, and vaccination also induced parasite-specific IgG and reached high levels, which correlated with the protection. This indicated that antibody played a major role in irradiated vaccine immunity in primates. In studies on Schistosoma japonicum (S. japonicum), the majority papers reported low and unstable level protections in different mouse strains induced by attenuated S. japonicum cercariae. However, attenuated S. japonicum cercariae were always able to induce high level of protection in the domestic animals (artiodactyl), above 60% in pig and cattle. Till now, the protective immunity against S. japonicum had not been fully understood. Therefore, the purpose of this study is to investigate the humoral and celluar immune response levels in correlation to high level protection induced by UV-attenuated S. japonicum cercariae (UVAC) in pigs.Limited progress has been made into defining and validating the protective antigens involved in high level protection induced by radiation-attenuated vaccines. Another purpose of this study is to screen the suitable antigens as vaccine candidates using the radiation-attenuated cercariae model. It was considered that protective immunity generated by radiation-attenuated cercariae vaccines was mainly mediated by the antigen specific Th1 response. Our experiments showed that IgG2 antibody level in pigs immunized with UVAC increased significantly at the acute stage of infection , and the IgG2 subclass was considered to be associated with a Th1-cell-controlled immune response. Thus, some certain antigens derived from soluble worm antigen which was specifically recognized by IgG2 antibody in pig serum of vaccinated and challenged pigs could predominantly induce the Th1 response. In addition, the Th1, CTL and B cells epitopes in these selected antigens were further analysed using compute softwares, which would provide guidance on further appropriate vaccine research in the future.Using Landrace/Yorkshire/Duroc crossbred pigs modes, we investigated the protective effect induced by UV-attenuated cercariae and the relevant immunological events. To evaluate the protective effection, we counted the worms, eggs number in feces and observe the liver pathological reaction in the liver section at 8 weeks post-infection. To explore the humoral and cellular immune responses in the host, we detected S. japonicum antigen-specific IgM, IgG, IgG1 and IgG2 antibodies levels in sera of different pig groups by indirect ELISA, and tested Th1/Th2 cytokines in culture supernatant of peripheral blood mononuclear cells (PBMC). To observe the difference of early CD4+ T and CD8+ T functions in skin-draining lymph nodes (sdLNs) following percutaneous exposure to attenuated or normal cercariae, some gene transcription in sdLNs was tested by semi-quantitative RT-PCR on day 7 after immunization or infection. Then we further compared the gene expression profiles of the mesenteric lymph nodes from UVAC-vaccinated and cercarie-challenged group and Infection-control group at 8 weeks post-infection using microarray. Finally, we screened the antigens combined with the IgG2 antibodies in sera with high antibody titer using immunoprecipitation techniques. Then these antigens were predicted the subcellular localization, and the Th1-, CTL and B cells epitopes of protective antigens using bioinformatics softwares.The main results are as follows:1. The vaccination with UV-attenuated cercariae induced high protection against Schistosoma japonicum in pigs. UVAC vaccination (vaccination-challenge group, abbreviated to"Vac-Cha"group) led to a 63.84% reduction in worm number, 71.82% reduction in hepatic egg burden, decreased egg number in feces and alleviated liver pathology compared to the infection control (Inf-Con) group.2. The IgM, IgG, IgG1 and IgG2 antibodies levels increased gradually in Vac-Cha group after immunization, especially the significantly increased expression in IgG2 was observed in the acute stage of infection, compared with Inf-Con group.3. IFN-γconcentrations in response to SWAP and SEA began to increase after immunization and remained higher level at the early infection stage in Vac-Cha group. At the acute stage of infection, the cellular response exhibited a mixed Th1/Th2 phenotype by single vaccination. 4. UVAC vaccination induced the stronger gene transcription related to early CD4 and CD8 responses (ifnγ, il12p40, il4, il10, granzyme b, nk-lysin) in skin-draining lymph nodes (sdLNs) compared to normal cercariae infection.5. Some cytotoxicity related genes (granzyme b,nk-lysin) were markedly up-regulated in the mesenteric lymph nodes of UVAC-vaccinated pig at week 8 post-challenge. The mesenteric lymph nodes were used to analyze the gene expression profile with Affymetrix oligo probe arrays. In Vac-Cha group, there were 185 genes with a greater 1.5-fold increase in signal intensities compared to Inf-Con group, which were involved in cytotoxic effect and the activation of T cell and so on. On the other hand, the signal intensities of 207 genes in Inf-Con group were significantly higher than those in Vac-Cha group. Among of them were those genes of inflammatory factors, chemokine genes and their receptors, etc.6. Five antigens derived from SWAP specially combined with the IgG2 antibody in sera of Vac-Cha groups were identified. And the epitopes of five protective antigens were predicted using computer software. The results showed 11 Th1 cell-epitopes, 12 CTL-epitopes and 10 B cell-epitopes in them.The significances of this study are as follows: 1) we systematically investigated the dynamic characteristics of humoral and celluar immune responses in pigs vaccinated with UV-attenuated cercariae, which combined and supplied the individual resports on the immune responses induced by radiation attenuated cercariae in big animals. Especially, the pigs have close biologic similarities to human in anatomical, physiological and immunological properties, so we will understand the relevant immune responses in human according to the immune responses in pigs. 2) We proved that the effectors mediated by IFN-γ, IgG2 antibody were the key factors related with high protection. At the same time we provided the experimental evidence that CTL effector was associated with the high protection. These results suggested that the vaccine would induce higher protection if we aimed the target antigens and effectively combined the effectors of IFN-γ, IgG2 antibody and cytotoxicity. 3) We screened the antigens related to the protection in the pig model vaccinated with UVAC by immunoprcipitation and mass spectrum techniques. These results provided the new epitopes of the candidated vaccines and will be a basis to design and construct the epitope vaccine of S. japonicum.