Experimental Study On Therapeutic Epitope-vaccine Against Helicobacter Pylori

Helicobacter pylori (H.pylori) infection is a key aetiological factor in chronic gastritis, peptic ulcer disease, and is listed as a class I carcinogen due to its association with the development of gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT) lymphoma. The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces problems such as patient compliance, increasingly reported antibiotic resistance, side effects and possible recurrence of infection,which may limit the usefulness of the treatment. Most of the drawbacks of the pharmacological treatment could be overcome by the availability of efficacious vaccines and even more of vaccines able to work both prophylactically and therapeutically. An anti-H.pylori vaccine would be a promising method for the control of H.pylori infection.The fourth generation vaccine is epitope-based vaccine design (EBVD), and focus on the epitopes that induce immune response. The advantage of epitope-vaccine is its versatility and better specificity. Reasonable designation and combination of predominant epitopes will induce effective, safe and specific immune response. In recent years epitope- vaccine research has become a new trend in developing infective diseases, malign tumor and auto-immune diseases. Natural infection of H.pylori can escape the host's clearance by immune deviation,immune subvertion and immune deficiency, and then causes the host's immune tolerance to its natural proteins. Previous studies have proved that simply select the natural protein antigens of H.pylori can not break the host's immune tolerance and induce effective immune response. Therefore, natural antigens should be re-designed, modified and reformed. Protein antigens mainly display their specificity with their epitopes, and these epitopes include not only immune recognition relevant B cell epitopes, Th cell epitopes and CTL epitopes, but also toxic or suppressive eptiopes that are not favourable to immune protection. People must choose and design the vaccine at the level of epitope to promote the protection potency of protein antigens. By choosing H.pylori strong protection, specificity and immunogenicity epitopes in predominant antigens, and by the help of mucosal adjuvant to stimulate stronger, more specific and different natural infection mucosal immune response, it will be possible to break the immune tolerance and prevent or clear H.pylori infection.The protective mechanism of immune response induced by H.pylori infection is very complicated. Strong evidence has proved that CD4+ T cell is necessary but divergence appears when mentioning the relationships about helper T lymphocyte immune response (Th1 and Th2) with protective immune response. With the advancement in studying H.pylori vaccine treatment effect and mechanism it has been realized that the protection mechanism to against H.pylori infection may be a mode of balance between Th1/Th2, but the specific humoral immune response cannot be fully excluded. Based on this realization and previous study on H.pylori animal experiments in our lab, the basic strategy in therapeutic vaccine against H.pylori should keep to the balance between Th1/Th2 mediated by CD4+ T cell and including the specific humoral immunization.In all known protein antigens of H.pylori, UreB and HpaA are proved to be very immunogenic and have strong immune protective potency. In this studies, based on previous study in our lab in successfully screening and identification several Th epitopes of UreB, three predominant Th epitopes including two Th2 and one Th1 epitopes are chosen in addition to a intra-molecule adjuvant LTB. These epitopes are computed-aided designed, recombined, optimized and homology modeled, and finally the best theoretic combination is obtained. According to this combination mode, five epitopes of HUepi are combined with LTB to final epitope-vaccine fusing protein. These vaccines were analyzed about their antigenicity and epitope function, and then they were used to immunize BALB/c mice orally to cure H.pylori infection. The clearance of H.pylori and the relative immuneresponse were monitored. The methods, result and conclusion are as follows:1 Design of therapeutic epitope-vaccine Against Helicobacter pyloriThree predominant Th cell epitopes including two Th2 and one Th1 epitope and one documented B cell epitope of UreB, one B cell epitope of HpaA and an intra-molecule adjuvant LTB are chosen. Using RANKPEP and DNAstar software, theoretic optimal combination is obtained by analysis of various combinations of epitopes and the linker between epitopes and the intra-molecule adjuvant LTB. 3D structures are homologous modeled using Robetta, Modeller and SPDB Viewer software. 2 Construction, expression and purification of epitope-vaccine fusion proteins antigenAccording to the design in the first part, and based on successful construction of epitope fusing gene Uepi in our lab, genes encoding HUepi and ltB are cloned and these two genes are linked with a pre-designed linker by overlapping PCR to obtain a fusing gene HUepi-ltB encoding the epitopes and ltB. Using NcoⅠand XhoⅠrestriction digestion site, two plasmids pET22b-HUepi-ltB,pET22b-HUepi are constructed and transformed in BL21 to get prokaryotic expression bacteria pET22b-HUepi-ltB/BL21,pET22b-HUepi/BL21. IPTG induces efficient production of a 20.7kDa(HUepi-LTB)and 8.3 kDa(HUepi)recombinant proteins. Different purification methods are adopted to purify the recombinant fusing proteins HUepi-LTB and HUepi; and the purity of HUepi-LTB and HUepi are about 95.2% and 97.6% respectively.3 Epitope-vaccines fusion proteins antigenicity and epitopes′function analysisThe epitope-vaccine fusion proteins are used to immunize rabbits to prepare rabbit-anti HUepi-LTB and HUepi sera. Western blot and ELISA are used to analyze the reactivity of these specific antibodies to rUreB and rHpaA and LTB binding properties to GM1. The function of B cell epitopes is assayed by Urease inhibition test, H.pylori adhere to GES-1 cell and adherence inhibition assay and HA and HI test. These epitope-vaccine fusion proteins are also hypodermic injection immunize BALB/c mice, and Th cell epitopes′function were assayed by specifically lymphocyte proliferation assay. Results shows that the specific antibody induced by the epitope-vaccine fusion proteins and the antibody can react with rUreB, rHpaA and rLTB. The LTB component in the fusion protein retains its reactivity with GM1. Moreover, the antibody can inhibits urease activity, prevent H.pylori from adhering to GES-1 cell and inhibits H.pylori agglutination with akaryocyte. Specifically lymphocyte proliferation assay proves that the three Th epitopes,HUepi ,HUepi-LTB protein and rUreB can stimulate the lymphocytes from BALB/c mice immunized by HUepi ,HUepi-LTB protein to proliferate(SI>2).These experiments show that epitope-vaccine fusion proteins have good antigenicity, immuno-reactivity and retain epitopes′corresponding function.4 Evaluation of therapeutic epitope-vaccine in treating H.pylori infected BALB/c mouse and discussion of the therapeutic protection mechanism1) Evaluation the effects of therapeutic epitope-vaccine in treating H.pylori infected BALB/c mouseBALB/c mouse are infected by H.pylori strain SS1 to successfully establish a H.pylori infection animal model. Simultaneously, H.pylori quantitative culture method is established. Epitope-vaccine HUepi-LTB and HUepi+rLTB are orally administrated to H.pylori infected BALB/c mouse every 7~10 days and for four consecutive times. Thirty-eight days after the treatment, the effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori, Fast Uerase Test, and PCR test. The oral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice (P<0.01), and the clearance rate of HUepi-LTB and HUepi+rLTB are 64.2% and 61.5%, respectively.2) Evaluation the mechanism of epitope-vaccine therapeutic protection against H. pylori.Flow cytometry analyze the spleen lymphocyte (SPL) subgroups .Mucosal and systemic antibody responses were analyzed by ELISA in intestinal juice, saliva, feces and serum, respectively, and SPL and intestinal mucosal lymphocyte (GML) responses by an antigen specific proliferation assay.Cytokine production by the proliferating T lymphocyte were analyzed by ELISA. RT-PCR was used to detect mRNA level of IL-4 and IFN-γin SPL and GML.The results showed that CD4~+ T cell proliferated obviously in SPL and oral immunization epitope-vaccine resulted in markedly elevated levels of serum IgG, IgA and mucosal sIgA against H. pylori antigen. Spleen and intestinal mucosal lymphocytes from the vaccine immunized mice proliferated significantly and produce much more Th1 and Th2 cytokine comparing with that from the mice immunized with PBS,rLTB and HUepi (P<0.01). In addition, RT-PCR detected an obvious elevation of mRNA abundance of IL-4 and IFN-γin SPL and GML. These findings suggest that orally administration of H.pylori epitope-vaccine induce H. pylori specific local mucosal and systemic humoral immune response and combination Th1/Th2 cell immune response which may provide therapeutic protection against H. pylori.