CD4+ T cells recognize antigenic epitopes in the context of MHC II molecules on APCs. Many investigations of H.pylori infection have demonstrated that the protective immune response is mediated by CD4+ T cells but not by CD8+ T cells. Therapeutic immunization reduced H.pylori colonization in stomach in mice lacking B cells, suggesting that T cell is protective. Oral immunization with H. pylori whole-cell lysates reduced infection in wild-type and MHC I -/- mice, but it had no effect on MHC II -/- mice. Anti-H.pylori antibody levels in serum showed a dominant IgG in immunized wild-type and MHC I -/- mice but no detectable IgG in MHCII-/- mice. CD4+T cells from H.pylori antigen immunized mice were sufficient to transfer protective immunity to the immunodeficient recipients. Taken together, identification and characterization of the Th cell eptioptes of Lpp20 would contribute to a better understanding of protective immunity to H.pylori and facilitate the development of effective immunotherapeutic and immunoprophylactic strategies.
The identification of CD4+ T epitiopes has traditionally been done either by sequencing of eluted peptides bound to specific MHC II molecules from APCs or by screening panels of overlapping peptides. These two methods were successfully performed to identify many T epitopes; however, the method of sequencing eluted peptide is potentially cumbersome to identify epitopes from multiple processed antigens but not specifically a single antigen. The overlapping peptide method needs synthesis of a series of overlapping peptides, thus making it an expensive, laborious and time-consuming process. In addition, this method possibly misses junctional epitopes that might be present in the overlapping regions, though spanning the entire length of the antigens. These considerations lead us to use the combination of prediction and experiments to identify Th cell epitopes. Although it is possible to miss potential epitopes, this method represents a quick and effective approach to identify epitopes. Here we used algorithm program with T cell biological analysis to identify Th cell epitopes on Lpp20. Eight epitopes were selected to be synthesized and measured, of which two (L1 and L2) were identified to be Th cell epitopes, which were located at residues 83-97aa (L1) and 58-72aa (L2). Interestingly, L2, which was predicted to be both restricted by I-Ad and I-Ed, effectively stimulated more proliferation of splenic CD4+ T cells than L1, which was predicted to be I-Ad restriction. Our results indicate that the combination of prediction and experiments is an easy and effective way to identify Th cell epitopes. The present findings may be valuable for the development of epitope-based vaccines against H.pylori.
CD4+ T cells can polarize to Th1 or Th2 cells based on their profile of cytokine. Th1 cells produce IFN-γ, IL-2 and TNF-β that mediate cellular immunity. In contrast, Th2 cells produce IL-4, IL-5, IL-10 and IL-13, which are responsible for humoral immunity. It is generally believed that polarized Th1-type response is involved in the pathogenesis of H.pylori infection[14–17]. But Whether Th1 or Th2 type immune response is responsible for protective immunity is still unclear. Some studies support that Th2 response characterized by IL-4 secretion is important to clear H.pylori[18–21]. On the contrary, some studies reveal that protection against H.pylori is mediated by predominantly Th1-type immune responses independent of IL-4[22, 23]. Moreover, some researchers demonstrate that a mixed Th1-Th2 phenotype is correlated with the protective immunity against H.pylori infection. Therefore, identification of Th1 and Th2 type epitopes may help us investigate the role of Th1 or Th2 type responses on pathogenesis and immunity of H.pylori infection. Meanwhile, it is also a pivotal step for a rational modulation of immune response by developing effective multiple Th1 or/and Th2 epitope vaccine. In this study, we found that L1 and L2 were both Th1-type epitopes and induced CD4+ T cell to mainly secrete IFN-γ.
In conclusion, we have identified two Th1 cell epitopes on Lpp20. Peptide L1 is I-Ad restricted epitope and peptide L2 is both I-Ad and I-Ed restricted epitopes. These two peptides both evoked Th1-type response. In addition, the two pooled peptides stimulated significantly more elevated T cell responses, showing an additive effect. We have identified one B cell epitope of Lpp20 using phage displayed library previously. Next, we will combine these two Th epitopes and one B cell epitope to evaluate whether the combined epitopes can induce protective response and prevent H.pylori infection better than the whole Lpp20 protein in BALB/c mice. The data from our mouse model will provide significant information for further study of epitope-based vaccine.