Screening And Identification Of B-Cell Epitopes In Urease B Subunit Of Helicobacter Pylori

Helicobacter pylori(H.pylori), a spiral shaped, microaerophilic, Gram-negative bacterium. It has been classified as a group I carcinogen by the World Health Organization. Infection with H.pylori is highly associated with chronic active gastritis, gastric ulceration, and duodenal ulceration, lymphoma of mucosa-associated lymphoid tissue and gastric cancer. Currently, combinatorial chemotherapies is effective in the majority of patients. However, this eradication treatment has several disadvantages, such as adverse effects of antibiotics, increased emergence of drug-resistant strains of H.pylori due to antibiotic overuse, possible recurrence of infection, and, especially, the high cost of the treatment; all of which make the treatment impractical for global control. Therefore, developing an effective prophylactic vaccination has been suggested as a superior strategy over the use of antibiotics for the control of H.pylori infection. UreB is the main functional unit and shows protection superior to UreA. Animal experiments have demonstrated its safety and effectiveness as a vaccine antigen. Thus, the identification of protective B cell epitopes of UreB seems important to develop an effective H.pylori vaccine. The aim of this study was to identify the epitopes by MAbs A1H10, B3D9, A3C10 and characterize their role in the immunogenicity. This research include the following three parts:1. Identification of antigenic epitopes in UreB by MAbs A1H10, B3D9, A3C10A series of 19 partially-overlapping fragments of the UreB gene were expressed through multiple truncations. And on this basis, the progressive approach adopted by western blot to fine localization of the linear B-cell epitopes in the UreB which were recognized by monoclonal antibody (MAbs) A1H10, B3D9, A3C10. We have successfully constructed 19 expression vectors and expressions are achieved. Three linear B-cell epitopes were indentified by western blot, covering a stretch of 15 amino acid (AA) residues with the names for the UP32 (GGGTGPADGTNATTI), UP35 (WMLRAAEEYSMNLGF), and UP38 (TLHDMGIFSITSSDS) and localized in the AA regions 158-172,181-195,349-363 of UreB, respectively. The three identified epitopes to be further confirmed by ELISA, the purified fusion epitope protein occurrence of the specific binding reaction with the corresponding monoclonal antibody and positive serum from clinical H.pylori infected patients. For further description of the epitopes, the puried fusionprotein GST-UP32, GST-UP35 and GST-UP38 were subjected to indirect ELISA, which showed that MAbs A1H10, B3D9, A3C10 were recognized by GST-UP32, GST-UP35, GST-UP38, respectively, and the fusion peptides were recognized well by sera from H. pylori infected patients.In addition, to further identify the MAbs-defined epitopes, three peptides were synthesized commercially (Scilight Biotechnology, LLC) and comprised 15 aa which included aa 158-172 (GGGTGPADGTNATTI),181-195 (WMLRAAEEYSMNLGF), and 349-363 (TLHDMGIFSITSSDS) of UreB, named UP32, UP35, UP38, were assessed (by HPLC) to be in excess of 90% pure. These epitopes were further confirmed from MAbs binding to synthetic peptides by ELISA or Dot blot method, ELISA results showed that the reactivity observed was specific for the relevant MAbs with synthetic peptides and did not cross-react with non-specific peptide sequences, indicated the UP32, UP35, UP38 were the epitopes recoganized by MAbs A1H10, B3D9 or A3C10, respectively; the binding reaction of synthetic peptide with sera from H. pylori infected patients also occurred, stating that these epitopes had good reactivity. Dot blot assays showed that MAb B3D9 reacted with synthetic peptide UP35, which also confirmed this epitope。2. Immunological evaluation of antigenic epitopes in UreB8-week-old female BALB/c mice were immunized with purified fusion proteins for three times to raise polyclonal antibody. Serum samples were assayed by ELISA, western blot, and urease inhibition assay. ELISA assay titer of antiserum from vaccinated BALB/c mice reached 1:51200, indicating that the fusion epitopes proteins elicited effective humoral response. ELISA results showed that the antisera from fusion epitope-immune mice were also found to possess reactivity to synthetic peptides, native UreB of H. pylori strains NCTC 11637, H. pylori strains 26695, and rUreBM. Western blot performed to further characterize the specific response of antiserum, with rUreBM and BSA used as the positive and negative controls, respectively, showed that antisera from fusion epitope-immune mice responded to native UreB of H. pylori strains 26695 and the rUreBM. These results were in agreement with those determined by ELISA analysis. To confirm whether the MAbs and fusion peptide-specific antibody could inhibit H. pylori urease activity, the H. pylori urease inhibition test was performed by preincubation of purified MAbs with urease from strain NCTC 11637. When the amount of urease was fixed, inhibition by A1H10, B3D9, and A3C10 was dependent on the dose of each MAbs. The 3 MAbs effectively inhibited the activity at a high dose, with the maximum inhibition up to≈70%. The epitope-specific antiserum also could neutralize urease activity to a certain extent, a comparison with purified MAbs showed that the fusion epitope-specific IgG antibody showed<50% inhibition of urease activity, even at a high dose of 30μg of IgG/well.3. Surface expression and immunological evaluation of serial epitope geneThrough gene recombination approach, we knocked serial epitope gene fragment in c-term of the outer membrane protein A (OmpA) gene on S. ty2ΔrfaHΔssaV derivatives chromosome has been constructed previously in our laboratory, resulting in a recombinant strain S.ty2ΔrfaHΔssaV-U258, by western blot analysis confirmed that the target protein was expressed in the form of fusion protein. To evaluate the effectiveness of this Salmonella vector expression system, mice were inoculated with this recombinant strain via the orogastric route. After orogastrical inoculation, both high-and low-dose groups are effectively stimulated the immune response against foreign antigens UreB. Anti-UreB IgG titers as high as 1:3200, anti-vecter IgG titers were 1:800-1:3200. Secretory IgA against UreB could be detected, indicating that the two groups raised effective mucosal immune response.In conclusion, we used a combination of molecular biology methods and immunological techniques identified three neutralizing B-cell epitopes in UreB recognized by MAbs A1H10, B3D9, A3C10 successfully, animal experiments have demonstrated they with good immunogenicity. The newly identified epitopes provided an important theoretical basis and experimental evidence to construction of new multi-joint poly-epitope vaccine and.