CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11c(high) conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8(+) T cells in vivo, but the role of cDCs in CD4(+) T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4(+) T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11c(high) cDCs. Our results show that cDCs are absolutely required for activation of CD4(+) T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4(+) T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.

Sublingual immunization protects against Helicobacter pylori infection and induces T and B cell responses in the stomach.

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.

Preclinical immunogenicity and protective efficacy of an oral Helicobacter pylori inactivated whole cell vaccine and multiple mutant cholera toxin: A novel and non-toxic mucosal adjuvant.

Mucosal vaccines against Helicobacter pylori consisting of either whole cell bacteria or recombinant antigens can induce immune protection against challenge in mice only when co-administrated with a strong mucosal adjuvant such as cholera toxin (CT) or Escherichia coli heat labile enterotoxin (LT). The strong enterotoxicity of these adjuvants however preclude their use in human vaccines. The recently developed multiple mutant CT (mmCT) is a strong, yet practically non-toxic novel mucosal adjuvant which here was admixed with a formalin-inactivated H. pylori whole cell vaccine (WCV) as a potential vaccine candidate against H. pylori infection. We report that intragastric immunizations with H. pylori WCV together with mmCT, similar to immunization with WCV together with CT, resulted in 50-125-fold reduction in colonization of H. pylori in the stomach of mice associated with rises in both serum IgG and intestinal-mucosal IgA anti-H. pylori antibody responses and strong T cell and IFNγ and IL-17A cytokine responses. Data presented in this study also supports that the proposed vaccine can be grown in a bioreactor and would be effective against infection caused by a multitude of pathogenic H. pylori strains isolated from patients from various continents. The results warrant immunization studies in humans to evaluate the safety, immunogenicity and efficacy of the proposed H. pylori WCV and mmCT.

Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

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.

Construction of novel vaccine strains of Vibrio cholerae co-expressing the Inaba and Ogawa serotype antigens.

The approach of inducing protective immunity against cholera by oral vaccination with killed whole Vibrio cholerae cells is effective, but the complexity of current cholera vaccines makes them difficult and relatively expensive to manufacture, especially if recombinant cholera toxin B subunit is included in the formulation. In an effort to simplify the composition of a new generation of oral cholera vaccines we have generated a novel non-toxigenic candidate vaccine strain of V. cholerae O1 that stably expresses both the Ogawa and Inaba serotype antigens on its surface. This was done by introducing a functional wbeT gene without a functional promoter into the chromosome of an O1 Inaba strain. The resulting low levels of expression of the wbeT gene product allowed for the desired partial serotype switching. This strain (MS1342) can potentially replace the three virulent strains used in currently manufactured cholera vaccines. Oral immunization of mice with formalin-killed MS1342 bacteria gave rise to Ogawa-specific, Inaba-specific and cross-reactive serum antibodies that were detectable both by lipopolysaccharide (LPS)-specific ELISAs and as vibriocidal antibodies that are considered to predict protective efficacy. These responses as well as intestinal mucosal IgA anti-LPS antibody responses were fully comparable with those obtained by immunization with the internationally licensed oral cholera vaccine Dukoral(®). We propose that such a strain may form the basis of a single strain killed whole cell cholera vaccine protecting against cholera caused by either the Inaba or Ogawa serotype of V. cholerae O1.