Sukanya Raghavan

Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3−CD25− CD4+ Regulatory T Cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (Treg). We found that total Treg, KJ1–26+ Treg and CTLA-4+ Treg were all increased in Peyer’s patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-β in serum. This could be abolished by coadministering cholera toxin or by treatment with anti-TGF-β mAb. CD25+ Treg, but also CD25−CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ Treg from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ Treg. Furthermore, in Rag1−/− mice that had adoptively received highly purified Foxp3−CD25−CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ Treg cells, which expressed Foxp3 more strongly than naturally developing Treg and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25− T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-β and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ Treg together with the generation of both Foxp3+ and Foxp3−CD25− CD4+ Treg.

Protection against Experimental Helicobacter pylori Infection after Immunization with Inactivated H. pylori Whole-Cell Vaccines

ABSTRACT The protective effect of therapeutic oral immunization with homologous and heterologous formalin-inactivated Helicobacter pylori cells given together with cholera toxin as an adjuvant was evaluated with C57BL/6 mice infected with H. pylori Sydney strain 1 (SS1). The bacteria used for immunization were strains that were either homologous or heterologous with regard to the O antigen (i.e., the Lewis antigen [Le antigen]) expressed by the lipopolysaccharide of the infecting H. pylori SS1 strain. We found that repeated oral immunization with inactivated H. pylori SS1 cells can significantly inhibit an existing infection (P < 0.001) and that the protection induced by such therapeutic immunization extends to protection against reinfection (P < 0.001). A similar level of protection was also achieved by immunization with another inactivated H. pylori strain having the same O antigen (Le antigen) as the infecting H. pylori SS1 strain. In contrast, immunization with inactivated strains expressing a heterologous O antigen, Lex, provided less protection or no protection. Immunization with H. pylori lysate preparations, on the other hand, resulted in significant comparable protection whether the lysates were prepared from an Lex strain or an Ley strain. Postimmunization gastritis was seen in mice that were protected after vaccination but not in unimmunized or unprotected mice. In conclusion, therapeutic immunization with inactivated H. pylori whole-cell vaccines may provide strong protection both against experimental H. pylori infection and against later reinfection.

Enhanced M1 Macrophage Polarization in Human Helicobacter pylori-Associated Atrophic Gastritis and in Vaccinated Mice

Background Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined. Methodology/Principal Findings By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion. Conclusions/Significance These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.

Orally Administered CpG Oligodeoxynucleotide Induces Production of CXC and CC Chemokines in the Gastric Mucosa and Suppresses Bacterial Colonization in a Mouse Model of Helicobacter pylori Infection

ABSTRACT Bacterial DNA and unmethylated CpG oligodeoxynucleotides (CpG ODN) are known to be potent stimulators of the innate immune system in vitro and in vivo. We therefore investigated if oral administration of CpG ODN could enhance innate immunity in the gastric mucosa and control the extent of Helicobacter pylori infection in mice. Intragastric administration of a single dose of CpG ODN significantly increased local production of the CC chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES and the CXC chemokine gamma interferon-inducible protein 10 in the stomach and/or the small intestine. Importantly, intragastric administration of CpG ODN to mice with an already established H. pylori infection, in the absence of any coadministered antigen, was found to reduce the bacterial load in the stomach compared to the load in H. pylori-infected control mice, while similar administration of non-CpG ODN had no effect on the bacterial load. The reduction in the bacterial numbers in the stomachs of mice treated with CpG ODN was associated with enhanced infiltration of immune cells and increased RANTES production in the gastric mucosa compared to the infiltration of immune cells and RANTES production in H. pylori-infected control animals. These findings suggest that intragastric administration of CpG ODN without antigen codelivery may represent a valuable strategy for induction of innate immunity against H. pylori infection.

Sublingual Immunization Protects against Helicobacter pylori Infection and Induces T and B Cell Responses in the Stomach

ABSTRACT Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4+ T cells and CD19+ B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.

Effects of oral vaccination and immunomodulation by cholera toxin on experimental Helicobacter pylori infection, reinfection, and gastritis

ABSTRACT Therapeutic vaccination is an attractive strategy to control infection and disease caused by Helicobacter pylori. In mice infected with H. pylori we have studied the protective effect of oral immunization with an H. pylori lysate preparation given together with the mucosal adjuvant cholera toxin (CT), both against the initial infection and against a later reinfection challenge. We have also examined the effects of treatment with the CT adjuvant alone on H. pylori infection and reinfection. Specific immunization with lysate was found to result in a sixfold reduction of the extent (bacterial load) of the primary infection and also to provide similar levels of protection against reinfection. However, these effects were associated with severe postimmunization gastritis. In contrast, oral treatment with CT alone at the time of initial infection, while unable to suppress the initial infection, gave rise to a 20-fold reduction in bacterial load upon reinfection without causing any associated gastric inflammation. Both the infected animals that were specifically immunized and those that were treated with CT only displayed increased in vitro proliferative responses of mononuclear cells to H. pylori antigens. Antibody levels in response to H. pylori were on the other hand only marginally increased after treatment with CT, whereas they were markedly elevated after immunization with lysate plus CT, with a rise in both (Th2-driven) immunoglobulin G1 (IgG1) and, especially, (Th1-driven) IgG2a antibodies. The results illustrate the complex balance between protection and harmful inflammation after postinfection vaccination against H. pylori as studied in a mouse model.

A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

ABSTRACT Helicobacter pylori infection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine against H. pylori infection is available to date. In mice, mucosal vaccination with H. pylori antigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses against H. pylori infection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutant Escherichia coli heat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimental H. pylori vaccine and compared it to CT in promoting immune responses and protection against H. pylori infection in mice. Immunization via the sublingual or intragastric route with H. pylori lysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhanced in vitro proliferative and cytokine responses from spleen and mesenteric lymph node cells to H. pylori antigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine against H. pylori infection.

Proinflammatory Cytokine Gene Expression in the Stomach Correlates with Vaccine-Induced Protection against Helicobacter pylori Infection in Mice: an Important Role for Interleukin-17 during the Effector Phase

ABSTRACT CD4+ T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection in mice. Less is known about the relative contributions of individual cell subpopulations, such as Th1 and Th17 cells, and their associated cytokines. The aim of the present study was to find immune correlates to vaccine-induced protection and further study their role in protection against H. pylori infection. Immunized and unimmunized mice were challenged with H. pylori, and immune responses were compared. Vaccine-induced protection was assessed by measuring H. pylori colonization in the stomach. Gastric gene expression of Th1- and/or Th17-associated cytokines was analyzed by quantitative PCR, and contributions of individual cytokines to protection were evaluated by antibody-mediated in vivo neutralization. By analyzing immunized and unimmunized mice, a significant inverse correlation between the levels of interleukin-12p40 (IL-12p40), tumor necrosis factor alpha (TNF), gamma interferon (IFN-γ), and IL-17 gene expression and the number of H. pylori bacteria in the stomachs of individual animals after challenge could be demonstrated. In a kinetic study, upregulation of TNF, IFN-γ, and IL-17 coincided with vaccine-induced protection at 7 days after H. pylori challenge and was sustained for at least 21 days. In vivo neutralization of these cytokines during the effector phase of the immune response revealed a significant role for IL-17, but not for IFN-γ or TNF, in vaccine-induced protection. In conclusion, although both Th1- and Th17-associated gene expression in the stomach correlate with vaccine-induced protection against H. pylori infection, our study indicates that mainly Th17 effector mechanisms are of critical importance to protection.

A truncated form of HpaA is a promising antigen for use in a vaccine against Helicobacter pylori.

HpaA is a Helicobacter pylori-specific lipoprotein that has been shown to be an effective protective antigen for mucosal vaccination against H. pylori infection in mice. However, detergents are needed for the purification of full-length HpaA (HpaA(full)), which might confer toxicity, thus making HpaA(full) unsuitable for use in a human vaccine. We here describe a recombinantly produced truncated version of HpaA (HpaA(trunc)), which is easily purified without the use of detergents. Evaluation in the murine H. pylori infection model showed that sublingual immunization with HpaA(trunc) was equally immunogenic and protective as immunization with HpaA(full). Immunization with a combination of HpaA(trunc) and recombinant UreB protein induced strong immune responses to both antigens and importantly had a strong synergistic effect on protection, associated with synergistically increased expression of IL-17 in the stomach. Notably, sublingual immunization with HpaA(trunc) and UreB was superior to corresponding intragastric immunization with regard to the level of protection induced. In conclusion, HpaA(trunc) is a promising, readily produced, non-toxic recombinant antigen for inclusion in a mucosal vaccine against H. pylori infection, which may preferably be given sublingually together with UreB.

Immune modulation by regulatory T cells in Helicobacter pylori-associated diseases.

Regulatory T cells (Treg) have the ability to suppress the activity of most other lymphoid cells as well as dendritic cells through cell-cell contact dependent mechanisms, which have not yet been fully defined. Treg are a key component of a functional immune system, and Treg deficiency is associated with severe autoimmunity and allergies. Antigen-specific Treg accumulate in gastric tissue during both Helicobacter pylori-induced gastritis and peptic ulcer disease (PUD). Several studies suggest that the local Treg response protects the gastric mucosa from exaggerated inflammation and tissue damage, and the risk of PUD is inversely related to Treg frequencies. At the same time the reduction of the inflammatory response achieved by Treg leads to increased bacterial density. Furthermore, the inability to mount a protective inflammatory response will lead to chronic infection and in some patients to the development of atrophic gastritis and gastric cancer progression. Treg actively infiltrate gastric adenocarcinomas and are predicted to promote tumor escape from cytotoxic immune responses. In addition, the presence of a potent Treg response will probably be an obstacle when constructing a future therapeutic vaccine against H. pylori. In this article, we will review the proposed mechanisms of action for Treg, their accumulation in the gastric mucosa in the different H. pylori-associated diseases, and how they may affect the immune response induced by H. pylori infection and the course of PUD and gastric adenocarcinomas.