Vaccinating against Helicobacter pylori in the developing world.

Helicobacter pylori infects more than half the world's population and in developing nations the incidence can be over 90%. The morbidity and mortality associated with H. pylori-associated diseases including ulcers and gastric cancer therefore, disproportionately impact the developing world. Mice have been used extensively to demonstrate the feasibility of developing a vaccine for H. pylori infection, and for testing antigens, routes of immunization, dose, and adjuvants. These successes however, have not translated well in clinical trials. Although there are examples where immune responses have been activated, there are few instances of achieving a reduced bacterial load. In vivo and in vitro analyses in both mice and humans demonstrates that the host responds to H. pylori infection through the activation of immunoregulatory mechanisms designed to suppress the anti-H. pylori response. Improved vaccine efficacy therefore, will require the inclusion of factors that over-ride or re-program these immunoregulatory rersponse mechanisms.

Th1-mediated immunity against Helicobacter pylori can compensate for lack of Th17 cells and can protect mice in the absence of immunization.

Helicobacter pylori (H. pylori) infection can be significantly reduced by immunization in mice. Th17 cells play an essential role in the protective immune response. Th1 immunity has also been demonstrated to play a role in the protective immune response and can compensate in the absence of IL-17. To further address the potential of Th1 immunity, we investigated the efficacy of immunization in mice deficient in IL-23p19, a cytokine that promotes Th17 cell development. We also examined the course of Helicobacter infection in unimmunized mice treated with Th1 promoting cytokine IL-12. C57BL/6, IL-12 p35 KO, and IL-23 p19 KO mice were immunized and challenged with H. pylori. Protective immunity was evaluated by CFU determination and QPCR on gastric biopsies. Gastric and splenic IL-17 and IFNγ levels were determined by PCR or by ELISA. Balb/c mice were infected with H. felis and treated with IL-12 therapy and the resulting gastric bacterial load and inflammatory response were assessed by histologic evaluation. Vaccine induced reductions in bacterial load that were comparable to wild type mice were observed in both IL-12 p35 and IL-23 p19 KO mice. In the absence of IL-23 p19, IL-17 levels remained low but IFNγ levels increased significantly in both immunized challenged and unimmunized/challenged mice. Additionally, treatment of H. felis-infected Balb/c mice with IL-12 resulted in increased gastric inflammation and the eradication of bacteria in most mice. These data suggest that Th1 immunity can compensate for the lack of IL-23 mediated Th17 responses, and that protective Th1 immunity can be induced in the absence of immunization through cytokine therapy of the infected host.

Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective Helicobacter pylori immunity.

A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform.

Lack of genetic influence on the innate inflammatory response to helicobacter infection of the gastric mucosa.

Helicobacter pylori (H. pylori) is a bacterial pathogen that resides at the gastric mucosa and has a world-wide prevalence of over 50%. Infection usually lasts for the life of the host, and although all infected individuals will develop histologic gastritis only a subset will develop symptomatic gastritis, peptic ulcer disease, gastric MALT lymphoma, or gastric adenocarcinoma. The bacterial and host factors that determine clinical outcome and influence the development of widely varying diseases have not been elucidated. We compared disease in Helicobacter-infected severe combined immunodeficient (SCID) mice on different genetic backgrounds with their corresponding immunocompetent partners to determine if the genetics of the host significantly impacts the innate inflammatory outcome, independent of variations in bacterial virulence factors. BALB/c SCID and C57BL/6 SCID mice developed equivalent histologic gastritis by 8 weeks of infection. Immunocompetent BALB/c mice and C57BL/6 mice developed significantly lower or higher degrees of inflammation respectively. Innate inflammation in immunodeficient mice on the C57BL/6 background remained low even in the absence of the regulatory cytokine IL-10. These results demonstrate that adaptive immunity is not required for the generation of low level inflammation in response to Helicobacter infection and that the degree of inflammation is consistent among different genetic backgrounds. Additionally, this inflammation is limited even in the absence of regulatory T cells.

Laboratory maintenance of Helicobacter species.

Helicobacter species are Gram-negative bacteria that colonize the gastric or intestinal mucosa of many mammalian and avian hosts and induce histologic inflammation. The association of H. pylori with gastritis, peptic ulcer disease, and gastric cancers makes it a significant human pathogen. Animal models for these diseases are being used to explore the pathogenesis of H. pylori infection and in vaccine development. Both bacterial and host factors contribute to Helicobacter pathogenesis; therefore, the microbiology is very important. This unit describes how to culture the most commonly used gastric Helicobacter species, H. pylori, H. mustelae, and H. felis.

Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A.

BACKGROUND: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL-17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori-infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL-17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL-17A function is absent during the entire immune response using IL-17A and IL-17A receptor knockout (KO) mice immunized against H. pylori. MATERIALS AND METHODS: Gastric biopsies were harvested from naïve, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL-17A KO mice were also treated with anti-IFNγ or control antibodies. RESULTS: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL-17A KO and WT mice, whereas IL-17RA KO mice had on average a four-fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. CONCLUSION: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL-17A-CXC chemokine pathway.

Partial protection against Helicobacter pylori in the absence of mast cells in mice.

The goal of this study is to evaluate the contribution of mast cells to Helicobacter pylori immunity in a model of vaccine-induced protection. Mast cell-deficient Kitl(Sl)/Kitl(Sl-d) and control mice were immunized with H. pylori sonicate plus cholera toxin and challenged with H. pylori, and the bacterial loads, inflammatory infiltrates, and cytokine responses were evaluated and compared at 1, 2, and 4 weeks postchallenge. In vitro stimulation assays were performed using bone marrow-derived mast cells, and recall assays were performed with spleen cells of immunized mast cell-deficient and wild-type mice. Bacterial clearance was observed by 2 weeks postchallenge in mast cell-deficient mice. The bacterial load was reduced by 4.0 log CFU in wild-type mice and by 1.5 log CFU in mast cell-deficient mice. Neutrophil numbers in the gastric mucosa of immune Kitl(Sl)/Kitl(Sl-d) mice were lower than those for immune wild-type mice (P < 0.05). Levels of gastric interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-alpha) were also significantly lower in immune Kitl(Sl)/Kitl(Sl-d) mice than in wild-type mice (P < 0.001). Immunized mast cell-deficient and wild-type mouse spleen cells produced IFN-gamma and IL-17 in response to H. pylori antigen stimulation. TNF-alpha and CXC chemokines were detected in mast cell supernatants after 24 h of stimulation with H. pylori antigen. The results indicate that mast cells are not essential for but do contribute to vaccine-induced immunity and that mast cells contribute to neutrophil recruitment and inflammation in response to H. pylori.

Analysis of innate immune responses in a model of IgA nephropathy induced by Sendai virus.

In a model of IgA nephropathy (IgAN) induced by Sendai virus (SeV) without Th1/Th2 polarizing immunization, Th2-prone BALB/c mice develop more severe nephritis with acute renal insufficiency than Th1-prone C3H mice. To determine whether Th1 or Th2 predominance influences the severity of experimental IgAN in mice, we employed polarizing immunizations in a SeV-induced IgAN model in Th1-prone C57Bl/6 mice and Th2-prone BALB/c mice. C57Bl/6 mice, immunized with SeV +CFA or +IFA, showed: (1) clear cytokine polarity by splenocytes in recall assays. (2) Total serum IgA and especially SeV-specific IgA from the IFA group showed a selective defect in galactosylation, not seen in the CFA group, and (3) serum creatinine in the IFA group was higher than in the CFA group or nonimmune controls. However, BALB/c mice did not show clear cytokine polarity with CFA/IFA adjuvant. Moreover, spleen cells from naive BALB/c mice produce IFN-gamma (but not IL-2, -4, -5, or -13) upon stimulation with inactivated SeV in vitro. By flow cytometry, IFN-gamma producing cells are CD3(-), CD19(-), CD49b(+) natural killer cells. IFN-gamma production by naive splenocytes is blocked partially by anti-IL12 blocking Abs, and completely by anti-IL18R blocking Abs. In conclusion, C57Bl/6 mice with polarizing priming with SeV showed clear cytokine polarity and distinct kidney injuries. However, BALB/c mice did not show clear cytokine polarity in the same immunizing system, presumably due to the effects of innate responses to SeV upon antigen-specific lymphocytes. Natural IFN-gamma production may influence the risk of renal failure in IgAN.

Laboratory maintenance of helicobacter species.

Helicobacter species are Gram-negative bacteria that colonize the gastric or intestinal mucosa of many mammalian and avian hosts and induce histologic inflammation. The association of H. pylori with gastritis, peptic ulcer disease, and gastric cancers makes it a significant human pathogen. Animal models for these diseases are being used to explore the pathogenesis of H. pylori infection and in vaccine development (UNIT ). Both bacterial and host factors contribute to Helicobacter pathogenesis, and therefore the microbiology is very important. This unit describes how to culture the most commonly used gastric Helicobacter species, H. pylori, H. mustelae, and H. felis.

Differential effects of Sendai virus infection on mediator synthesis by mesangial cells from two mouse strains.

BACKGROUND: Recently, we observed that the severity of glomerulonephritis in an experimental model of immunoglobulin A nephropathy (IgAN) induced by Sendai virus differs between C57BL/6 and BALB/c mouse strains. The determinants of differing renal insufficiency are not understood. In the present study, we examine the capacity for mesangial cells to support Sendai viral replication and assess the direct effects of Sendai virus on the production of selected cytokines, chemokines, and eicosanoids by mesangial cells, comparing C57BL/6 to BALB/c mouse strains. METHODS: Sendai virus replication was measured by viral plaque assay using LLCMK2 cells. Production of cytokines [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)], chemokines (JE and KC), and eicosanoids [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)] in culture medium was evaluated by sandwich enzyme-linked immunosorbent assay (ELISA) or competitive enzyme immunoassay (EIA) after 48 hours' incubation with infectious or inactivated Sendai virus. RESULTS: Sendai virus replicates equally well in mesangial cells from both strains, and infection evokes increased IL-6, JE, KC, and PGE2 production in relation to viral dose. BALB/c mesangial cells produce significantly more IL-6 and JE than those from C57BL/6, and the dose response for KC is steeper in BALB/c mesangial cells than those from C57BL/6. Synthesis of PGE2 in BALB/c mesangial cells is higher than that of C57BL/6 mesangial cells, both under basal conditions and in response to infectious Sendai virus, again in a dose-dependent manner. There is no TNF-alpha or thromboxane response to viral stimulation. CONCLUSION: We conclude that different mesangial cell responses to this common mucosal viral pathogen might influence the severity of IgAN in our model system.

Vaccine-induced protection against Helicobacter pylori in mice lacking both antibodies and interleukin-4.

To test the hypothesis that a Th2 response to Helicobacter pylori is necessary for protection and to address the possibility that humoral and Th2 cellular responses may compensate for each other, we generated mice deficient in both interleukin-4 (IL-4) and antibodies. The immunized double-knockout mice were protected from H. pylori challenge, as were the parental strains and wild-type C57BL/6 mice. Neutralization of IL-4 in B-cell-deficient mice did not prevent protection. Immunized IL-5-deficient mice were also protected. Thus, IL-4 and IL-5 are not essential for protection.

Protective efficacy of anti-Helicobacter pylori immunity following systemic immunization of neonatal mice.

Helicobacter pylori infection of the gastric mucosa is a significant cause of morbidity and mortality because of its etiologic role in symptomatic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Infection occurs in young children; therefore, a prophylactic vaccine would have to be administered within the first year of life, a period thought to be immunologically privileged. We investigated vaccine formulations administered by different routes to confer protective anti-H. pylori immunity in neonatal mice. Neonatal mice immunized with a single dose of vaccine in complete Freund's adjuvant (CFA) generated antigen-specific gamma interferon-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in numbers similar to those in immunized adult mice, while vaccine administered to neonates in incomplete Freund's adjuvant (IFA) induced such cells in reduced numbers compared to those in adult mice. Both IFA and CFA, however, provided partial protection from a challenge with infectious H. pylori when the vaccine was administered subcutaneously. Neonatal immunized mice also had reduced bacterial loads when immunized intraperitoneally with CFA. In all cases, protection was equivalent to that achieved when adult counterparts were immunized. These studies suggest that an efficacious vaccine might be successfully administered to very young children to prevent perinatal infection of H. pylori.

Vaccine-induced reduction of Helicobacter pylori colonization in mice is interleukin-12 dependent but gamma interferon and inducible nitric oxide synthase independent.

Previous studies with mice have shown that major histocompatibility complex class II (MHC-II) is required for protection from Helicobacter pylori, while MHC-I and antibodies are not. Thus, CD4(+) T cells are presumed to play an essential role in protective immunity via secretion of cytokines. To determine which cytokines are associated with a reduction of bacterial load in immunized mice, gastric cytokine expression was examined by semiquantitative reverse transcription-PCR in protected (defined as > or =2-log-unit decrease in bacterial load) and unprotected mice 4 weeks after challenge. Elevated levels of mRNA for interleukin-12p40 (IL-12p40), gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) were associated with protection in immunized-challenged (I/C) mice, but Th2 cytokine (IL-4, IL-5, IL-10, and IL-13) and chemokine (KC, MIP-2, and MCP-1) expression was not associated with protection. Despite the association of IFN-gamma and iNOS message with protection, I/C mice genetically lacking either of these products were able to reduce the bacterial load as well as the wild-type I/C controls. The I/C mice lacking IL-12p40 were not protected compared to unimmunized-challenged mice. All I/C groups developed gastritis. We conclude that neither IFN-gamma nor iNOS is essential for vaccine-induced protection from H. pylori infection. The p40 subunit of IL-12, which is a component of both IL-12 and IL-23, is necessary for protection in immunized mice. These findings suggest a novel IFN-gamma-independent function of IL-12p40 in effective mucosal immunization against H. pylori.

Helicobacter pylori inflammation and immunity.

Gastric inflammation is a significant contributor to the disease process associated with Helicobacter pylori infection. It appears that both bacterial genes and differential host responses make interrelated contributions to gastritis and disease outcome after H. pylori infection. While the cag pathogenicity island (PAI) continues to be a focus for much of this investigation on the bacterial side, other bacterial genes/proteins are certainly important as well. On the host cell side, significant progress is being made defining the eucaryotic signaling cascades induced after host cells interact with H. pylori. The role of host cell cytokines, gastric acid, and mast cells is also being actively studied. Prospects for control of H. pylori associated disease continue to include vaccination. The mechanism(s) for vaccine-mediated control of H. pylori infection and disease remain ill-defined but recent evidence from animal models suggests that the inflammatory response may be involved. Manipulating the host response to H. pylori infection in humans to take advantage of the possible beneficial effects of inflammation, while minimizing its detrimental effects is a significant challenge for the future.

Clearance of Helicobacter pylori Infection and Resolution of Postimmunization Gastritis in a Kinetic Study of Prophylactically Immunized Mice.

Patients infected with Helicobacter pylori mount an immune response which fails to clear the infection and may contribute to disease. Mice can be protected by immunization. To further characterize the H. pylori-mouse model, stomachs of unimmunized or intranasally immunized C57BL/6 mice were quantitatively cultured 3 days and 1, 2, 4, 8, 16, 32, and 52 weeks after challenge with H. pylori. At 3 days and 1 week after challenge, colonization was the same in the immunized and unimmunized mice. By 2 weeks after challenge, the immunized mice had a >2-log decrease in bacterial load, and at all later time points, they either were culture negative or had at least a 2-log decrease in bacterial load. Gastritis in the immunized mice peaked at 1 to 2 weeks after challenge and was characterized by a mixed inflammatory infiltrate and epithelial proliferation centered at the transition between corpus and antrum. By 52 weeks postchallenge, the gastric histology in the immunized mice was not different from that in control unchallenged mice. The unimmunized group began to show a reduction in bacterial load as early as 16 weeks after challenge, and by 52 weeks seven of eight unimmunized mice had developed gastritis and reduced bacterial loads. These results indicate that prophylactic immunization does not prevent colonization by H. pylori but enables mice to clear the infection or significantly reduce the number of colonizing bacteria. The reduction in bacterial load is associated with gastric inflammation that subsides over time.