Experimental Study Of Helicobacter Pylori Therapeutic Vaccine By Attenuated Salmonella Typhimurium Carrier

Helicobacter pylori (H.pylori), as a spiral-shaped, Gram-negative bacteria, colonizes human gastric and duodenal mucosa and results in chronic gastritis, peptic ulcer and gastric cancer. Current treatment against H.pylori infection, consisting of combination therapy with two antibiotics and a proton-pump inhibitor, normally brings a good eradication of the bacteria and the healing of ulcers. However, there are some drawbacks to this treatment including antibiotic resistance, re-infection, patient compliance and high costs. Therapeutic vaccine could be an alternative or complementary method to antibiotic treatment against H.pylori infection. Several approaches have been adopted to investigate the development of vaccines against H.pylori infection.Whole-cell vaccines, include inactivated and lysates of H.pylori whole-cell, is very efficient. However, such an approach faces serious limitations for the entire antigenic repertoire of the bacteria, including the LPS and Lewis blood group. However, recombinant subunit antigens, even epitope vaccines had shown efficacy of against H.pylori. Several H.pylori protective antigens, such as UreB, CagA and VacA have been identified and used as candidate antigens to develop H.pylori vaccine. It is likely that effective immunity against the bacteria could be achieved by combining various antigens participating in different aspects of the pathogenesis of infection.The role of CD4+ T cells in protective immunity against H.pylori has been widely accepted. Most studies suggest that a strong Th1-biased environment, including increased gastritis, may be necessary for protection and clearance. Oral administration of Salmonella vector vaccines could elicits that classical Th1-type responses also induce significant mucosal sIgA responses. Besides, Salmonella could induce mucosal immune responses effectively through the transcytosis of microfold cell or directed antigen presentation of dendritic cells. It was reported that a large number of foreign antigens, expressed in live attenuated Salmonella, protect the animal models against a diversity of pathogens including virus, bacteria and parasites.Previous studies have demonstrated that orally administered recombinant Salmonella vaccines against H.pylori is safe and immunogenical. The intact proteins of H.pylori for vaccine antigens containing unneeded epitopes can result in a higher molecular weight, difficult to express and purify. Fusion protein is easy to express and purify. Fusion protein vaccines represent a new strategy for elevating a specific immune response against multi-antigenic components of selected antigenic regions.Objectives1. Try to evaluation the therapeutic effect of attenuated Salmonella vector vaccines, expressing different fusion protein, in H.pylori-infected mice model.2. Aim to elucidate Th1 or Th2 cells play more important role in immune clearance of H.pylori .Methods1. Attenuated Salmonella vector vaccines were constructed that expressed fusion proteins complexed with H.pylori CagA, VacA and UreB in different arrangements, and their therapeutic efficacy was evaluated in H.pylori-infected mice.2. The colonization of H.pylori was quantified by real-time quantitative PCR amplifying 16S rDNA of H.pylori. The antibody levels in the serum, gastric and small intestine samples were assessed by ELISA. IL-4 and IFN-γwere quantified by IL-4 and IFN-γ-specific sandwich ELISA kits.Results1. Oral therapeutic immunization with attenuated Salmonella, which expressed the fused protein CagA160-VacA62-UreB138 (CVU), significantly decreased H.pylori colonization in the stomach than immunization compared with vector control.2. Immunization with attenuated Salmonella live vector vaccine enhanced Th1 and H.pylori-specific antibody response significantly than immunization with vector control. And protection was related to specific CD4+ T cell Th1 type responses and serum IgG and mucosal sIgA antibody responses.ConclusionThese findings suggested that therapeutic efficacy was related to the arrangement of the fusion protein. The attenuated Salmonella vector vaccine, which expressed the fused protein arrangement CVU, is superior to others, and could be a candidate vaccine against H.pylori.