| Background and Objective:Helicobacter pylori (H. pylori) is present in the gastic mucosa of about half of the world's population and is associated with the development of peptic ulcer, gastrc adenocarcinoma, and B-cell lymphoma. The current standard of practice is to cure H. pylori infection in patients with peptic ulcer disease. The current recommended treatment for H. pylori infection is a triple therapy based on antibiotics. However, the rapid emergence of antibiotic resistance and the vast number of patients to be treated mandate the development of other forms of disease control, including vaccines. Most of H. pylori vaccines on researching are for crowd of H. pylori infection negative now. It was emergency to manufacture safe and effective therapeutic H. pylori vaccine to resolve such high infectious rate of H. pylori infection. Several vaccines based on H. pylori whole cell had demonstrated some therapeutic efficacy in the animal experiments. So , we plane to study inactivation H. pylori therapeutic vaccine. This study will be focus on prepare of antigen, and selection of adjuvants, dose, approach, periods, efficacy and the mechanisms of immune clearance.Study design: Firstly, prepare antigen: culture H. pylori by liquid culture and quantitative H. pylori by counting of the number of bacterial colonies per plate and production, inactivation H. pylori by formalin and examine it. Secondly, select adjuvants: Immunization H. pylori infected gerbils by orally administered antigen with CT, LT and CpG adjuvant. Then, administered inactivation H. pylori whole cell with freund's adjuvant, aluminium hydroxide and CpG by injection. And observe the immune therapeutic efficacy. Thirdly, Definition the immunizing dose, approach and periods: Administered inactivation H. pylori whole cell with aluminium hydroxide adjuvant(H. pylori therapeutic vaccine, HpTV) by Injection and find out the best way of injection immune. Fourthly, observe therapeutic efficacy and immune memory of HpTV: Immunization H. pylori infected gerbils with HpTV, then challenge after a month of the last immunization, and observe the immune therapeutic and resistance reinfcction. At last, investigate mechanisms of immune clearance: Detect the difference of INF-γ, IL-4 and IL-12p40 expression in gerbils's gastric mucosa by RT-PCR. Then, detect the level of H. pylori specific serum IgG, IgA and saliva sIgA in BalB/c mice by ELISA, which immuned in the same way.Results: Firstly, the best harvest by liquid culture was inoculation at 106 CFU/ml bacterium, and when inactivation 1×109 CFU/ml for 12 hours at 25℃ by 1:2000 formalin. H. pylori were inactivated completely and forms no change, and has good antigenicity. The inactivated H. pylori cells are very stable and can be stored more than 12 months at 4℃. Secondly, immunization by injection administered inactivated whole H. pylori cell combine with aluminium hydroxide adjuvant appears more effective. Thirdly, we found that immunization vaccin by muscle injection was better than abdominal injection, and immunization of 1×108CFU formalin inactivation H. pylori whole cell at 0,1,2,3 weeks appears more effective. Fourthly, clearance was 93.3% and protect rat from reinfection challenge was 90.0% by immunization of H. pylori infected gerbils with HpTV. At last: the expression of INF-γ, IL-4 and IL-12p40 in gerbils's gastric mucosa all arise after immunization, and the level of H. pylori specific serum IgG, IgA and saliva sIgA of BalB/c mice all arise significantly after three weeks of immne.Conclusion: Immunization H. pylori infected gerbils by injection immune HpTV developing immune response enough to clearance H. pylori infection. Furthermore, it induced immune protection against reinfection. This result suggest the mechanism of HpTV is induce Th1/Th2 balanced type immune response, and induce the level of H. pylori specific serum IgG, IgA and saliva sIgA arise. This study also provid some experiment data for H. pylori therapeutic vaccine investigation.
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