Identification Of A Neutralizing B-cell Epitope Of Helicobacter Pylori Urease Subunit B

ObjectiveHelicobacter pylori (Hp) is a gram negative, spiral, microaerophylic bacterium that infects more than 50% of the world population and even 70% in developing country. There is a contradictory problem existing in anti-infecting therapy, because the single-drug therapy hardly eliminates Hp, while the costs of multi-drugs are expensive. The most important thing is that the current therapeusis gives low healing rate, and can not prevent reinfection. Besides, the increasing of drug resistance strain makes anti-Hp treatment more and more complex. Now vaccine provides a promising prophylactic way against Hp. When Helicobacter pylori (Hp) infection occurs,the immue tolerance exists in host body and the immune responses induced by Hp infection fail to clear Hp. To protect the human from chronic Hp infection by immunization, we must design vaccine antigen different from natrual antigen to elicit the effective immune responses. Epitope vaccine is a special type of vaccine to combat the infection disease, cancer and autoimmune disease. Epitope based vaccine design can promote the protective immune response and exclude the effect of non-protective and inhibitory immune response. So selecting the protective B cell epitopes of antigen and designing the epitope vaccine reasonably can avoid the side effect of the inhibitory epitope on natural antigen.H. pylori produces a large amount of urease, which is essential for the survival and pathogenesis of the bacterium. The urease enzyme consists of two major subunits, UreA(30kD) and UreB (63kD), at a stoichiometric ratio 1:1,and urease subunit B in particular has proven to be more protective. In our lab, some one has prepared mAb 6E6 against UreB of H. pylori and initially indentified the activity of mAb 6E6, the results confirmed that 6E6 has higher titer and can have responses with the antigen of UreB specially. In this work, we want to identify the neutral activity and epitope of mAb 6E6, then observe the immunogenicity of the epitope of mAb 6E6 on synthetic peptides and to provide experimental evidence of the possibility to develop epitope vaccine against H. pylori.Methods1. The neutralization of the monoclonal antibody (mAb) 6E6 which raised against H. pylori was detected by urease inhibition assay.2. The protein structure of urease B subunit (UreB) of H. pylori was analyzed by the DNASTAR software and DeepView/Swiss-PdbViewer 3.7 (SP5); Based on the above results, in order not to destroy the high antigenicity, hydrophilicity, accessibility and flexibility, five truncated antigens of UreB named as U12, U13, U47, U15 and U16 were cloned and constructed. In order to align the epitope of mAb 6E6 exactly, four epitope peptides were synthesized.3. The genes of U12, U13, U47, U15 and U16 were amplied from Helicobacter pylori genome, cloned into expression vector pET-11c(+) and the recombinant plasmids were transformed into Escherichia coli BL21(DE3), When OD600 reached 0.6, the productions were induced by the introduction of IPTG for 4h. Tris-tricine PAGE was used for the expression of proteins. The epitope domain recognized by mAb 6E6 were identified by Western blot analysis.4. The consecutive peptides overlapping the UreB sequence of H. pylori strain (each peptide contains 15 residues with five residues overlapping with the adjacent peptides) were designed to mimic and cover the entire region of epitopes mapped by the subtractive analyses of epitope mapping . By means of ELISA and Dot blot, the B-cell epitope of mAb 6E6 was located exactly.5. In order to increase the molecular weight of B-cell epitope for an antigen, the B-cell epitope was coupled with BSA. BALB/c mice were immunized by the conjugate, the epitope alone and PBS control with an subcutaneously injection of 100μg. The antigens were emulsified in Complete Freund's Adjuvant (CFA) at 1:1 ratio and injected subcutaneously at several sites at the back for the primary immunization. These were followed by two subcutaneous injections of the immunogens dissolved 1:1 in Incomplete Freund's Adjuvant (IFA) at two-week intervals. 35 days later, antiserum were collected and detected by ELISA and Western blot.6. Antergy assay was used to determine the ability of B-cell epitope to antergia the binding of mAb 6E6 to the urease of H. pylori; Serologic analysis from H. pylori infected patients was also done to identify the presence of epitope-specific antibodies against B-cell epitope of H. pylori in human serum.Results1. The mAb 6E6 can inhibit the activity of urease of H. pylori, and the inhibition percentage was changed by the dose of mAb. It was also demonstrated that the inhibited effect changed weakly by time.2. The analysis of molecular structure of UreB by bioinformatics software showed that the amino acids of 200, 230, 250, 260, 300 and 390 had low antigenicity, hydrophilicity, accessibility and flexibility, so we selected these sites as the truncated position and cloned five genes of U12(1～300aa),U13(1～200aa), U47(250～390aa),U15(1～230aa) and U16 (1～260aa).3. Five recombinant plasmids of pET11c-U12 , pET11c-U13 , pET11c-U47 ,pET11c-U15 and pET11c-U16 were constructed successfully; the electrophoresis of Tris- Tricine PAGE showed the recombinant proteins U12, U13, U47, U15 and U16 were successfully expressed in Escherichia coli BL21; the proteins molecular weight were 33.0kD, 22.0kD, 15.4kD, 25.3kD and 28.6kD, respectively. The recognition of expressed truncated proteins by mAb 6E6 was analyzed by Western blotting assay, it indicated that the epitope recognized by mAb 6E6 was located in UreB 200～230aa.4. Synthesize the peptides of U201-215, U206-220, U211-225 and U216-230, epitope mapping results showed that mAb 6E6 reacted with the peptide U211-225, suggesting that U211-225 ( IEAGAIGFKIHEDWG) was the B-cell epitope of H. pylori .5. When the conjugate of epitope-BSA immunized to BALB/c mice, it produced high titer antiserum which reached to 1:32000, but antiserum titer of epitope and PBS control were very low. The antiserum of conjugate of epitope-BSA could also response with the UreB of four different native Hp strains.6. Antergy assay showed that the epitope U211-225 could block the mAb to neutralize the activity of urease in dose manner; serologic analysis indicated that the epitope U211-225 could response with 37 anti-serum which from 57 H. pylori infected patients.Conclusion1. The neutral activity of the mAb 6E6 was identified, and confirmed that it is a neutral antibody of Hp.2. The B-cell epitope of mAb 6E6 was located at amino acids from 211 to 225 in UreB.3. The B-cell epitope U211-225 is a predominant epitope of Helicobacter pylori, it makes a basic foundation for the development of specific diagnostic agents and effective vaccine candidates to fight Helicobacter pylori.