Human immune response towards recombinant Helicobacter pylori urease and cellular fractions.

Vaccination against Helicobacter pylori is of particular clinical interest. Recombinant urease, the major protein in H. pylori, has been used for mucosal vaccination trials in different animal models, but was found to be ineffective in humans. The current study therefore investigated the human immune response towards recombinant H. pylori urease A and B (rUreA/B) expressed in E. coli compared to different cellular fractions of H. pylori (cytosol, total, inner and outer membrane). Monocyte-derived dendritic cells (Mo-DC) were generated from monocytes isolated by magnetic antigen cell separation (MACS) from healthy volunteers and cultured in the presence of hrIL-4 and hrGM-CSF. Mo-DC were stimulated for 48h with the recombinant proteins (1 microg/ml) or cellular fractions (1-10 microg/ml) and cytokine release was determined in the culture supernatant by ELISA. rUreA and rUreB were effective in inducing IL-12 secretion (6-10 fold) and, to a much lesser extent (2 fold), IL-10 secretion from Mo-DC. Total and outer membrane preparations from H. pylori stimulated IL-12 secretion significantly, and were even more potent than intact bacteria. Mo-DCs pulsed with rUreA activated allogenic CD56+ NK-cells, as determined by TNF-alpha and IFN-gamma secretion, but not allogenic CD4+/CD45RA+ naïve T-cells. In contrast, Mo-DCs pulsed with H. pylori total membrane or outer membrane preparations activated allogenic naive T-cells in co-culture systems, as determined by increased TNF-alpha secretion. It appears that outer membrane preparations of H. pylori, but not recombinant urease are more effective in inducing a Th1 polarized response in humans in vitro.

Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori.

Outer membrane proteins (OMPs) are incorporated into the outer plasma membrane of Helicobacter pylori and are important for, e.g., ion transport, adherence, structural and osmotic stability, and bacterial virulence but may also be antigenic due to their surface exposure. Previous proteome-based approaches with H. pylori lysates determined a strong serological reaction towards two H. pylori OMPs, HpaA (TIGR HP0797) and Omp18 (TIGR HP1125). PCR was used to detect DNA encoding the two proteins, and a positive signal was found in all H. pylori strains tested. Proteins were cloned and expressed in the human kidney cell line HK293 with the QiaExpressionist system with a C-terminal His tag. Only sera from infected persons showed a positive reaction with the recombinant proteins. Recombinant HpaA (rHpaA) and rOmp18 were incubated with human peripheral blood mononuclear cells and induced secretion of interleukin-12 (IL-12) and IL-10 from these cells. To determine the effect on antigen-presenting cells, human blood monocytic and dendritic cells (DCs) were isolated by magnetic cell separation. rOmp18 and rHpaA strongly stimulated major histocompatibility class II and CD83 expression 7- to 10-fold on isolated DCs. rHpaA and rOmp18 failed to stimulate IL-8 secretion from monocytes but increased secretion of IL-12 and IL-10 from DCs significantly. In summary, HpaA and Omp18 are recognized by human dendritic cells and induce their maturation as well as antigen presentation. HpaA and Omp18 of H. pylori thereby appear to have a specific antigenic potential in humans.

Synergistic effect of Helicobacter pylori virulence factors and interleukin-1 polymorphisms for the development of severe histological changes in the gastric mucosa.

Polymorphisms of the IL-1B and IL-1RN genes (which encode interleukin [IL]-1beta and IL-1 receptor antagonist, respectively) have been associated with hypochlorhydria and gastric cancer. We investigated the influence of bacterial virulence factors and host IL-1 polymorphisms on the development of histologic abnormalities in 210 Helicobacter pylori-infected patients with chronic gastritis. cagA(+)/vacAs1(+) H. pylori strains were associated with intestinal metaplasia (IM), atrophic gastritis (AG), and severe inflammation. Carriers of the proinflammatory IL-1B -511T/-31C and IL-1RN*2 alleles had an increased risk for the development of AG, IM, and severe inflammation, with odds ratios (ORs) of 1.7 (95% confidence interval [CI], 0.8-3.4) to 4.4 (95% CI, 1.5-12.9). The highest prevalence of severe gastric abnormalities was found in patients with both host and bacterial high-risk genotypes (cagA(+)/vacAs1(+)/IL-1B -511T/IL-1RN*2), with ORs of 24.8 (95% CI, 5.2-117.3) for severe lymphocytic infiltration, 9.5 (95% CI, 2.8-32.1) for severe granulocytic infiltration, 6.0 (95% CI, 2.4-15.5) for IM, and 2.4 (95% CI, 0.93-6.2) for AG. Combined bacterial/host genotyping thus may provide a clinical tool to identify patients at high risk of developing cancer.