Louise Sjökvist Ottsjö

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.

CD4+ T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection. However, the effector mechanisms leading to reductions in the gastric bacterial loads of vaccinated mice remain unclear. We have investigated the function of IFN-γ and IL-17A for vaccine-induced protection and inflammation (gastritis) using IFN-γ-gene-knockout (IFN-γ-/-) mice, after sublingual or intragastric immunization with H. pylori lysate antigens and cholera toxin. Bacteria were enumerated in the stomachs of mice and related to the gastritis score and cellular immune responses. We report that sublingually and intragastrically immunized IFN-γ-/- mice had significantly reduced bacterial loads similar to immunized wild-type mice compared to respective unimmunized infection controls. The reduction in bacterial loads in sublingually and intragastrically immunized IFN-γ-/- mice was associated with significantly higher levels of IL-17A in stomach extracts and lower gastritis scores compared with immunized wild-type mice. To study the role of IL-17A for vaccine-induced protection in sublingually immunized IFN-γ-/- mice, IL-17A was neutralized in vivo at the time of infection. Remarkably, the neutralization of IL-17A in sublingually immunized IFN-γ-/- mice completely abolished protection against H. pylori infection and the mild gastritis. In summary, our results suggest that IFN-γ responses in the stomach of sublingually immunized mice promote vaccine-induced gastritis, after infection with H. pylori but that IL-17A primarily functions to reduce the bacterial load.

Summary There is a current lack of effective mucosal vaccines against major gastroenteric pathogens and particularly against Helicobacter pylori , which causes a chronic infection that can lead to peptic ulcers and gastric cancer in a subpopulation of infected individuals. Mucosal CD 4 + T‐cell responses have been shown to be essential for vaccine‐induced protection against H. pylori infection. The current study addresses the influence of the adjuvant and site of mucosal immunization on early CD 4 + T‐cell priming to H. pylori antigens. The vaccine formulation consisted of H. pylori lysate antigens and mucosal adjuvants, cholera toxin ( CT ) or a detoxified double‐mutant heat‐labile enterotoxin from Escherichia coli (dm LT ), which were administered by either the sublingual or intragastric route. We report that in vitro , adjuvants CT and dm LT induce up‐regulation of pro‐inflammatory gene expression in purified dendritic cells and enhance the H. pylori ‐specific CD 4 + T‐cell response including interleukin‐17A ( IL ‐17A), interferon‐ γ ( IFN ‐ γ ) and tumour necrosis factor‐ α ( TNF ‐ α ) secretion. In vivo , sublingual immunization led to an increased frequency of IL ‐17A + , IFN ‐ γ + and TNF ‐ α + secreting CD 4 + T cells in the cervical lymph nodes compared with in the mesenteric lymph nodes after intragastric immunization. Subsequently, IL ‐17A + cells were visualized in the stomach of sublingually immunized and challenged mice. In summary, our results suggest that addition of an adjuvant to the vaccine clearly activated dendritic cells, which in turn, enhanced CD 4 + T‐cell cytokines IL ‐17A, IFN ‐ γ and TNF ‐ α responses, particularly in the cervical lymph nodes after sublingual vaccination.

Immunological memory ensures life-long protection against previously encountered pathogens, and in mice and humans the spleen is an important reservoir for long-lived memory B cells (MBCs). It is well established that integrins play several crucial roles in lymphocyte survival and trafficking, but their involvement in the retention of MBCs in secondary lymphoid organs, and differences between B cell subsets in their adhesion capacity to ICAM-1 and/or VCAM-1 have not yet been confirmed. Here, we use an autoimmune mouse model, where MBCs are abundant, to show that the highest levels of LFA-1 and VLA-4 amongst B cells are found on MBCs. In vivo blockade of VLA-4 alone or in combination with LFA-1, but not LFA-1 alone, causes a release of MBCs from the spleen into the blood stream. In humans, we find that in peripheral blood, spleens and tonsils from healthy donors the highest expression levels of the integrins LFA-1 and VLA-4 are also found on MBCs. Consistent with this, we found MBCs to have a higher capacity to adhere to ICAM-1 and VCAM-1 than naïve B cells. In patients with the autoimmune disease rheumatoid arthritis, it is the MBCs that have the highest levels of LFA-1 and VLA-4; moreover, compared with healthy donors, naïve B and MBCs of patients receiving anti-TNF medication have enhanced levels of the active form of LFA-1. Commensurate levels of the active L subunit can be induced on B cells from healthy donors by exposure to the integrin ligands. Thus, our findings establish the selective use of the integrins LFA-1 and VLA-4 in the localization and adhesion of MBCs in both mice and humans.