Nils Lycke

Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems.

Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects which alone or in combination might explain their strong adjuvant action in stimulating mucosal IgA and other immune responses to admixed unrelated antigens after oral immunization. These effects include, depending on animal species and experimental systems, enhanced antigen presentation by a variety of cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and complex stimulatory as well as inhibitory effects on T-cell proliferation and lymphokine production. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT and LT associated with enhanced cyclic AMP formation in the affected cells, and thus it may prove difficult to eliminate the enterotoxic activity without loss of adjuvanticity. However, through a separate mechanism, as an antigen-carrier system providing specific binding to epithelium including the M cells of intestinal Peyers patches, both CT and its non-toxic binding subunit moiety (CTB) have been shown to markedly enhance the mucosal immune response to various foreign antigens or epitopes covalently linked to these molecules. This gives promise for the future use of CTB or related non-toxic binding derivatives as vehicles to facilitate induction of mucosal immune responses to a broad range of antigens for human vaccination purposes.

Protection Against Helicobacter pylori Infection Following Immunization Is IL-12-Dependent and Mediated by Th1 Cells

The regulatory roles of Th1 and Th2 cells in immune protection against Helicobacter infection are not clearly understood. In this study, we report that a primary H. pylori infection can be established in the absence of IL-12 or IFN-γ. However, IFN-γ, but not IL-12, was involved in the development of gastritis because IFN-γ−/− (GKO) mice exhibited significantly less inflammation as compared with IL-12−/− or wild-type (WT) mice. Both IL-12−/− and GKO mice failed to develop protection following oral immunization with H. pylori lysate and cholera toxin adjuvant. By contrast, Th2-deficient, IL-4−/−, and WT mice were equally well protected. Mucosal immunization in the presence of coadministered rIL-12 in WT mice increased Ag-specific IFN-γ-producing T cells by 5-fold and gave an additional 4-fold reduction in colonizing bacteria, confirming a key role of Th1 cells in protection. Importantly, only protected IL-4−/− and WT mice demonstrated substantial influx of CD4+ T cells in the gastric mucosa. The extent of inflammation in challenged IL-12−/− and GKO mice was much reduced compared with that in WT mice, indicating that IFN-γ/Th1 cells also play a major role in postimmunization gastritis. Of note, postimmunization gastritis in IL-4−/− mice was significantly milder than WT mice, despite a similar level of protection, indicating that immune protection is not directly linked to the degree of gastric inflammation. Only protected mice had T cells that produced high levels of IFN-γ to recall Ag, whereas both protected and unprotected mice produced high levels of IL-13. We conclude that IL-12 and Th1 responses are crucial for H. pylori-specific protective immunity.

Gut IgA Class Switch Recombination in the Absence of CD40 Does Not Occur in the Lamina Propria and Is Independent of Germinal Centers

Conflicting findings have recently been presented as to the sites and sources of B cells that undergo class switch recombination (CSR) to IgA in the gut. In this study we provide compelling evidence in CD40−/− mice demonstrating that IgA CSR can be independent of CD40 signaling and germinal center formation and does not occur in the gut lamina propria (LP) itself. We found that CD40−/− mice had near normal levels of gut total IgA despite lacking germinal centers and completely failing to raise specific responses against the T cell-dependent Ags cholera toxin and keyhole limpet hemocyanin. The Peyer’s patches in CD40−/− mice expressed unexpectedly high levels of activation-induced cytidine deaminase mRNA and germline α transcripts, but few postswitch circular DNA transcripts, arguing against significant IgA CSR. Moreover and more surprisingly, wild-type mice exhibited no to low IgA CSR in mesenteric lymph nodes or isolated lymphoid follicles. Importantly, both strains failed to demonstrate any of the molecular markers for IgA CSR in the gut LP itself. Whereas all of the classical sites for IgA CSR in the GALT in CD40−/− mice appeared severely compromised for IgA CSR, B cells in the peritoneal cavity demonstrated the expression of activation-induced cytidine deaminase mRNA comparable to that of wild-type mice. However, peritoneal cavity B cells in both strains expressed intermediate levels of the germinal center marker GL7 and exhibited no germline α transcripts, and only three of 51 mice analyzed showed the presence of postswitch circular DNA transcripts. Taken together, these findings strongly argue for alternative inductive sites for gut IgA CSR against T cell-independent Ags outside of the GALT and the nonorganized LP.

The Cholera Toxin-Derived CTA1-DD Vaccine Adjuvant Administered Intranasally Does Not Cause Inflammation or Accumulate in the Nervous Tissues

Although highly effective, the use of GM1-receptor binding holotoxins as nasal mucosal adjuvants has recently been cautioned due to the risk for their accumulation in the brain and other nervous tissues. Therefore we have explored the efficacy of the CTA1-DD adjuvant for its ability to enhance nasal immune responses in mice. We found that despite the lack of a mucosal binding element, the B cell-targeted CTA1-DD molecule was an equally strong adjuvant as cholera toxin (CT). The potency of CTA1-DD was not a result of endotoxin contamination because more than a 50-fold higher dose of LPS was needed to achieve a similar enhancement. Moreover, the adjuvant effect was TLR4-independent and absent in mutant CTA1-E112K-DD, lacking enzymatic activity. The CTA1-DD adjuvant augmented germinal center formations and T cell priming in the draining lymph nodes, and contrary to CT, promoted a balanced Th1/Th2 response with little effect on IgE Ab production. CTA1-DD did not induce inflammatory changes in the nasal mucosa, and most importantly did not bind to or accumulate in the nervous tissues of the olfactory bulb, whereas CT bound avidly to the nervous tissues. We believe that the nontoxic CTA1-DD adjuvant is an attractive solution to the current dilemma between efficacy and toxicity encountered in CT-holotoxin adjuvant or Escherichia coli heat-labile toxin-holotoxin adjuvant strategies and provides a safe and promising candidate to be included in future vaccines for intranasal administration.

CTA1-M2e-DD: A novel mucosal adjuvant targeted influenza vaccine

At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.

The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens.

This study addresses the question of whether cholera toxin (CT) increases gut permeability for molecules > 3000 Da and whether such an effect is associated with an adjuvant function by CT on the gut immune response. We louiid that CT after oral administration gives rise to strikingly increased gut permeability for Dextran (Mw 3000) eoncomilantly with a strong enhancing effect on the anti‐keyhole limpet heniocyanin (KLII) specific immune response in the lamina propria after oral immunization with KLH plus Dextran and CT. In contrast, the B‐subunit of the hololoxin, which lacks the adenylate cyclase/cAMP‐activating property of CT. failed to increase gut permeability as well as local anti‐KLH immune responses. These results might suggest a causal linkage between the ability of CT to increase gut permeability and its adjuvant property on gut mucosai immune responses. In addition this finding supports the notion that the adenylate cyclase/cAMP system plays a regulatory role in gut permeability and is important in enhancing mucosal immune responses. Based on previous studies and the present data we propose that the mechanism for CTs adjuvant function on mucosal immune responses is by affecting antigen‐presenting cells, T and B cells in the gut to give a net enhancing effect on the stimulation of local immunity, and that the CT‐induced increase in gut permeability might be part of the adjuvant mechanism by facilitating luminal antigens to access the gut mucosal immune system.

Helicobacter pylori-specific antibodies impair the development of gastritis, facilitate bacterial colonization, and counteract resistance against infection.

In recent years, Abs have been considered a correlate rather than an effector of resistance against Helicobacter pylori infection. However, it is still poorly understood to what extent Ab production correlates with gastric immunopathology. Here we report that Abs not only are dispensable for protection, but they are detrimental to elimination of the bacteria and appear to impair gastric inflammatory responses. We found that the initial colonization with H. pylori bacteria was normal in the B cell-deficient (μMT) mice, whereas at later times (>8 wk) most of the bacteria were cleared, concomitant with the development of severe gastritis. In contrast, wild-type (WT) mice exhibited extensive bacterial colonization and only mild gastric inflammation, even at 16 wk after inoculation. Oral immunizations with H. pylori lysate and cholera toxin adjuvant stimulated comparable levels of protection in μMT and WT mice. The level of protection in both strains correlated well with the severity of the postimmunization gastritis. Thus, T cells were responsible for the gastritis, whereas Abs, including potentially host cell cross-reactive Abs, were not involved in causing the gastritis. The T cells in μMT and WT mice produced high and comparable levels of IFN-γ to recall Ag at 2 and after 8 wk, whereas IL-4 was detected after 8 wk only, indicating that Th1 activity dominated the early phase of protection, whereas later a mixed Th1 and Th2 activity was seen.

Cholera toxin enhances alloantigen presentation by cultured intestinal epithelial cells.

In the present study we show that cholera toxin (CT) strongly potentiates antigen presentation by intestinal epithelial cells, probably by enhancing co‐stimulation. This was demonstrated in an allogeneic system using cells from the IEC‐17 rat epithelial cell line as antigen presenting cells (APC). These cells were induced by optimal concentrations of IFN‐γ to express good amounts of Ia antigen and cultured for 24–48 h in the presence or absence of CT. Thereafter the cells were thoroughly washed and added to cultures containing MHC‐incompatible spleen cells as responder cells. Epithelial cells exposed to CT demonstrated greatly enhanced ability to trigger allogen‐specific T‐cell proliferation as compared with IEC‐17 cells treated with IFN‐γ alone. The mechanism for the enhanced APC function was investigated by analysing CT‐treated IEC‐17 cells for increased class II MHC antigen expression or enhanced production of cytokines with known co‐stimulatory function. We found no significant increase in class II MHC antigen expression. By contrast, CT strongly promoted, in a dose‐dependent fashion, the production of both IL‐1 and IL‐6 cytokines by IEC‐17 cells as compared with untreated epithelial cells. This effect of CT was specific and not due to contaminating endotoxin because excess amounts of soluble toxin receptor, ganglioside GM1, added to the IEC‐17 cultures completely abrogated the cytokine response to CT.

T Cell-Independent IgA Class Switch Recombination Is Restricted to the GALT and Occurs Prior to Manifest Germinal Center Formation

Recently, we reported that CD40−/− mice, exhibiting exclusively T cell-independent IgA class switch recombination (CSR), demonstrated near normal levels of IgA plasma cells in the gut lamina propria (LP), despite the complete lack of germinal centers (GCs). In this study, we have extended our analysis focusing on how to reconcile these findings using flow cytometry and molecular markers for IgA CSR. In agreement with our previous results with small intestinal LP, the colon LP was found to host IgA CSR only when lymphoid follicles were present. Thus, no IgA CSR was observed in the nonorganized colon LP. By contrast, the Peyer’s patch (PP) was the dominant IgA CSR site in both CD40−/− and wild type (WT) mice, and they both hosted similar levels of mRNA expression for B cell activating factor of the TNF family, a proliferation inducing ligand, and inducible NO synthase, potential switch-factors for IgA. Unexpectedly, we found that PP B cells undergoing IgA CSR were GL7-intermediate. These cells had not undergone somatic hypermutations (SHMs), whereas GL7-high cells in WT PP, which exhibited GCs, were heavily mutated. Moreover, IgA plasma cells in the LP of CD40−/− mice demonstrated few mutations in their Ig V regions, whereas WT LP B cells from different sites showed extensive SHMs, which were also clonally related. Therefore, IgA CSR can occur in PP at a stage preceding manifest GC (GL7-intermediate), whereas SHM require GC formations (GL7-high). These findings reconcile that IgA CSR can occur in PP in the absence of GC with the fact that CD40−/− mice host near normal levels of IgA plasma cells in the LP.

Local Intravaginal Vaccination of the Female Genital Tract

In a clinical trial the authors tested whether local intravaginal or oral vaccination would stimulate a mucosal immune response in the female genital tract. The whole cell/B subunit (CTB) oral cholera vaccine was used. Two groups of previously unimmunized volunteers were given three doses of vaccine at 2‐week intervals: a first group of seven women received oral immunizations and a second group of seven women were immunized locally in the genital tract by mixing the vaccine with a well defined gel, eldexomer, and applying it directly in the fornix of the vagina. The women were given the first vaccination on day 10 of the menstrual cycle. Sampling of peripheral blood and of cervical mucus (CM) using an Aspiglaire syringe was performed immediately prior to the first dose and at 8–10 days following the last immunization. The study showed that while only three of the seven orally immunized women responded with detectable IgA and IgG anti‐CTB antibodies in the genital tract, six out of the seven women in the locally vaccinated group responded with genital tract antibodies. The responses were also generally stronger and CM contained higher specific IgA and secretory component containing anti‐CTB titres in the locally vaccinated group. Of the orally vaccinated individuals all responded with increases in serum anti‐CTB IgG and 4/7 also exhibited specific IgA serum titres. By contrast, only 3/7 in the intravaginal group responded with increases in serum IgG and IgA anti‐CTB titers following immunization. The authors conclude that local intravaginal vaccination using a well‐defined gel appears to be the route of choice to stimulate immunity in the female genital tract.