The Studies Of Schistosoma Japonicum RNA Vaccine And Host Immunological Regulatory Mechanism Of Sj23 Antigen

Schistosomiasis is one of the most serious parasitic diseases which is epidemic in 76 countries and infects approximately 250 million people. Schistosomiasis japonica which is most hard to prevent is still endemic in China today.Vaccines are the most efficient method for controlling infectious diseases. A schistosome vaccine is believed to be feasible due to the fact that immunization with attenuated parasites can elicit protective immunity against parasite infection. In recent years, effort in the development of a schistosome vaccine has been mainly focused on the subunit vaccines in forms of either recombinant proteins or DNA plasmids containing parasite genes. Several vaccine candidates have been tested in both laboratory and clinical settings. However, the vaccines tested so far have not been very successful in term of immunogenicity and protection efficiency and the key protective antigen still un-definited ,in addition the significance anti-infection immunorective schistosome antigen always are membrane antigen which is usually un-facility expressed,furthemore the expressed recombinant protein has low solubility and always difference from the arch-framework.In this study, we explored a novel vaccine development approach by cloning two key schistosome antiges (GST and Sj23) and chimeric gene(GST-Sj23) into the SemLiki Forest virus (SFV) vaccine vector. Prepared three kinds of antigen's mRNA in vitro transcription.we place the three mRNA antigen which is coding Schistosoma japonicum into sequential downstream of RNA replicase.and by this we will get high expression protein. Moreover we insert a signal peptide in the upstream of antigenic genic and a across membrane sequence at downstream for the antigen can expressed on the cell surface and reinforce cellular immunologic response. At the same time as we prepare RNA we also make RNA from pSFV-Help S and Help-C-DNA,and the electroporation of RNA into BHK21 cells,prepared pseudovirion contain Schistosoma japonicum key antigen GST,Sj23 and GST-Sj23 chimera antigen.The immunogenicity and immunoprotection efficiency of an RNA-based schistosome vaccine in mice was studied.Immunofluorescence assay was used to confirm the expression of the recombinant proteins after virus infection in BHK21 cells. In vitro tests indicated that parasite antigens were expressed and displayed on the infected cell surface after addition of recombinant virus particles into the culture of BHK21 cells. Determination of recombinant virus titre by indirect immunofluorescens.The principle is if the target protein expressed on the cellsurface,then it will combine together with corresponding Anti-GST tag or Anti-His tag,and next step will to integrate with Anti-mouse-G488 which has green fluorescent light jabel.By the fluorescence microscope we can find green fluorescence display on pseudovirion infected cell surface,while un-infected cells have no fluorescence.This things can explain that the pseudovirion SFV-GST,SFV-Sj23 and SFV-GSI-Sj23 all have transmissibility, they can effectually transmiss recombiante RNA to intra-cellular and make coding GST,Sj23 and GST-Sj23's protein expressed.Among the total,SFV-Sj23 pseudovirion is more active.The titre determin of SFV-GST,SFV-GST-Sj23 and SFV-Sj23 result is 2.17×108IU/mL, 2.29×108IU/mL and 3.06×108IU/mL。In order to get more humoral immunity and cellular immunologic response, we designed a prime-boost immunization strategy. We used SPF mice as experimental animals to evaluate protection against cercarium infection. Results from in vitro studies indicated that Sj23 was very immunogenic and could stimulate rapid IgG production immediately after immunization. Similar results were also observed in mice infected with the parasites. However, the protection efficiency represented by egg and worm reduction in the SFV-Sj23, SFV-GST and SFV-GST-Sj23 immunized groups were 3.67%, 23.97%, 3.45% and 2.28%, 15.9%, 2.93% respectively. Thus, specific antibody responses to the antigens studies did not correlate with protection.To further understand the immunogenicity of Sj23 antigen, we extended the study by exploring the structural characterization of the molecule and its interactions with host non-immune immunoglobulins. Sj23 antigen is a member of the tetraspanin family and expressed in all infective stages of the parasite. It has a relatively unique structure compared to other tetraspanin molecules.The molecules of TSP family are all composed of three big alfa-helixes and three low complex regions.TSP-1has a typical CD81-like and the structures of the two EC2 rigions complete onerlap.TSP-2 has big low complex region compared with CD81,but the size of the helixes are similar.The similarity between Sj23 and CD81 is the least .Our data indicated that the reason this antigen was highly immunogenic was likely due to its affinity to non-immune IgG molecule, which may be beneficial to the process of antigen presentation. However, only low cytophilic antibodies (IgG2 in human and IgG2a in mice) to this antigen were generated after parasite invasion or immunization. IgG2 antibodies performed weak protection against parasite invasion and development in the host. In conclusion, Sj23 antigen is likely a pro-Th2 antigen utilized by S. japonicum to modulate host immune system and facilitate parasite invasion and immune evasion.