Development Of Oral Schistosoma Japonicum Vaccine And Tumor Prodrug Therapy System Mediated By Attenuated Salmonella Typhimurium

Salmonella, one species of intracellular bacteria, could enter and survive in the antigen presenting cells. After attenuation of its virulence, it has been engineered to carry foreign antigen and delivered it to the antigen presenting cells. After oral administration of recombinant bacteria, promising antigen specific immune response could be induced. These bacterial-based vaccines provide a number of advantages over other antigen delivery strategies including low cost of production, the absence of animal products, genetic stability and safety. In addition, proteins delivered by this vector behave as endogenous antigens, are presented on the cell surface in the context of MHC class I molecules, and generate strong cell-mediated immune responses.On the other hand, Salmonella is facultative anaerobic bacteria which preferentially replicate in tumor necrotic region and exhibits high tumor targeting ablility (tumor:normal tissues> 1:1000-10000). It has been successfully tested for their potential to be applied in bacteria mediated tumor therapy. These bacteria have been extensively investigated for anticancer therapies as a single-agent therapy, tumor-targeted gene delivery vectors, or in combination with other antitumor strategies, such as chemotherapy, radiotherapy and angiogenesis inhibitor.This thesis is mainly about the following studies:delevelopment of an effective oral vaccine against Schistosoma japonicum infection based on attenuated Salmonella typhimurium; the development of a tumor targeting Salmonella typhimurium/prodrug therapy system for cancer treatment, in this system, use of attenuated Salmonella typhimurium as a natural tool for activation of prodrug 6-methylpurine 2’-deoxyriboside and test its anti-tumor effect in murine melanoma model.Schistosomiasis, a zoonotic disease caused by parasites such as Schistosoma japonicum, is one of the most important health problems in developing countries. An effective and cheap vaccine is needed to control this disease as a supplemental of chemotherapy. In the first part, we used an attenuated Salmonella typhimurium strain VNP20009, which has been demonstrated its safety in clinical cancer patients, to express and deliver SJ23LHDGST bivalent antigen from Schistosoma japonicum. For rational design of an effective vaccine strain, different type of promoters were used to express the antigen and the a-hemolysin secretion systemor or type Ⅲ secretion system was employed to secreted it into host cells. Among the recombinant constructs, An engineered salmonella strain which delivered antigen through nirB (an anaerobic condition induced promoter) driven type III secretion system elicited Thl-specific immune responses characterized by release of IFN-Y, IL-12 and IL-2 from stimulated splenocytes in vitro. Meanwhile, Up-regulation of CD44 and CD69 were also detected in splenocytes from mice orally immunized with this nirB strain. Besides, oral immunization of mice with this strain resulted in a considerable worm reduction rate of 41.69%and an egg reduction of 57.71%.In addition, to obtain stronger immune response to Sj23LHD-GST antigen delivered by recombinant nirB strain and enhance the protective efficacy, we immunized mice with recombinant S. typhimurium harboring nirB-sopE-sj23LHD-GST followed by booster immunization with the Sj23LHD-GST protein once and examined the protection. The results showed that the worm and egg reduction increased to 51.35% and 62.59% respectively when vaccination with a nirB prime-protein boosting strategy. Further more, we also tested the protection of recombinant salmonella delivering the Sj23LHD, GST, TPI antigen respectively or mixing these antigens expressing salmonella together as a cocktail oral vaccine. The results showed that the GST antigen delivered by this system is benefit to the protection. Vaccination of the cocktail which containing these three antigen together hold a 45.16% worm reduction and 55.13% egg reduction. In conclusion, oral delivery of antigen by inducible promoter (nirB) driven Salmonella typhimurium type Ⅲ secretion system is a novel, safe, cheap, efficient and convenient manner for schistosomes vaccine development.PNP/6-methylpurine 2’-deoxyriboside (6MePdR) system is an efficient gene directed enzyme prodrug therapy (GDEPT) system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates non-toxic 6MePdR into potent antitumor drug 6MeP. Salmonella typhimurium PNP (sPNP) gene has a 96% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In the second part, we used tumor-targeting S. typhimurium VNP20009 whose endogenous PNP gene is constitutively expressed, as a natural tool to activate prodrug 6MePdR and test the anti-tumor effect of combination treatment with bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8+T cells and CD4+T cells infiltration. In summary, our results demonstrate that combination therapy of S. typhimurium and prodrug 6MePdR is an effective strategy for cancer therapy.