The cholera toxin B subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.     

Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination

The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection

The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H. pylori urease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H. pylori gastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice against H. pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H. pylori infection.     

Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin

Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.     

Intranasal immunization of mice with group B streptococcal protein rib and cholera toxin B subunit confers protection against lethal infection

Intranasal immunization of mice with Rib, a cell surface protein of group B streptococcus (GBS), conjugated to or simply coadministered with the recombinant cholera toxin B subunit, induces systemic immunoglobulin G (IgG) and local IgA antibody responses and confers protection against lethal GBS infection. These findings have implications for the development of a human GBS vaccine.     

Evaluation of oral immunization with recombinant avian influenza virus HA1 displayed on the Lactococcus lactis surface and combined with the mucosal adjuvant cholera toxin subunit B

The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.     

Procholeragenoid: a safe and effective antigen for oral immunization against experimental cholera.

The immunogenicity and safety of procholeragenoid, a minimally toxic, heat-induced aggregate of cholera toxin (CT), were studied in enterically immunized rats and dogs. Although 99% less toxic than CT, procholeragenoid was only slightly less efficient in causing jejunal anti-CT responses in rats; in contrast, choleragenoid, the nontoxic B subunit pentamer of CT, was much less effective. The immunogenicity of procholeragenoid was due almost entirely to its large-molecular-weight components (MW = 10(6) to 10(7)) and was markedly reduced by preincubation with GM1 ganglioside or treatment with Formalin to eliminate residual toxicity. These findings suggest that molecular aggregation, binding to GM1 receptors on cell membranes, and stimulation of cellular adenylate cyclase each contributed to the effectiveness of procholeragenoid as a mucosal immunogen. In dogs, oral immunization with five 500-micrograms doses of procholeragenoid evoked vigorous anti-CT responses in jejunal mucosa without causing significant diarrhea. When subsequently challenged with virulent Vibrio cholerae, immunized dogs showed 83% protection against the development of severe or lethal diarrhea compared with non-immunized controls. These results confirm a protective role for mucosal antitoxin in experimental cholera and show that procholeragenoid is both safe and effective as an oral immunogen. Procholeragenoid, combined with other antigens of V. cholerae, may constitute a simple, safe, and effective oral vaccine for cholera.     

The adjuvant effect of CpG oligodeoxynucleotide linked to the non-toxic B subunit of cholera toxin for induction of immunity against H. pylori in mice

The present study was carried out to test the immunostimulatory and adjuvant effects of the non-toxic B subunit of cholera toxin (CTB), CpG oligodeoxynucleotide (ODN) and CpG ODN linked to CTB (CTB–CpG) for generation of immunity against H. pylori in mice. Herein, we showed that CTB–CpG induces more potent proinflammatory cytokine and chemokine responses in the cervical and the mesenteric lymph nodes (CLN and MLN, respectively) cells in vitro compared with those of CTB and CpG ODN. The adjuvant effects of these agents were examined following intranasal immunization of C57Bl/6 mice with H. pylori lysate in combination with CpG ODN, CTB or CTB–CpG. All three immunization regimes resulted in high H. pylori -specific IgG antibody responses; however, only the CTB–CpG and, to some extent, the CpG ODN immunized mice mounted a sustainable IgG2c antibody response. Importantly, mice immunized with H. pylori antigen and CTB–CpG or CpG ODN, but not CTB, developed strong H. pylori -specific proliferative and IFN-γ responses in their MLN CD4⁺ T cells upon recall antigen stimulation in vitro . These mice also had significantly lower bacterial load compared with the control-infected mice. Furthermore, the CTB–CpG and the CpG ODN immunized mice developed increased specific IgA antibody responses in their gastrointestinal tracts following H. pylori challenge. These results imply that CTB–CpG and CpG ODN, but not CTB, could serve as nasal adjuvants for induction of a H. pylori -specific Th1 type immunity in MLN and also a specific mucosal IgA antibody response in the gastrointestinal tract upon H. pylori challenge.     

Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice.

We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA. Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios. Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine. OVA-CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1. OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1. The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB. The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response. The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d. Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not. Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only. We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses.     

Adoptive transfer of gut mucosal antitoxin memory by isolated B cells 1 year after oral immunization with cholera toxin.

A protocol was elaborated for the adoptive transfer of lymphocytes from mice which were orally immunized with cholera toxin (CT) to enable the study of long-term gut mucosal immunological memory at the single-cell level. Mesenteric lymph node (MLN) cells were transferred 1 year after priming immunizations, and recipient animals were challenged perorally on days 1 and 2 with CT before sacrifice on day 6 to 7 following transfer of cells. Strong antitoxin ELISPOT spot-forming cell (SFC) responses were recorded in spleens, MLN, and laminae propriae (LP) of recipient mice. In contrast, no SFC were found in Peyer's patches. The magnitude of the response equaled that of the acute response seen after optimal oral CT immunization and was directly dependent on the number of transferred cells. The memory antitoxin response in MLN and LP required oral challenge with CT as opposed to the spleen SFC response, which could also be triggered by intravenous challenge with antigen. Spleen cells from mice immunized perorally with CT were as effective as MLN cells in transferring immunological memory detectable in the gut immune system. Irrespective of the tissue source of transferring immunological memory detectable in the gut immune system. Irrespective of the tissue source of the memory cells, the isotype distribution of the antitoxin SFC response in recipient mice was similar with predominantly immunoglobulin A (96%) in LP and immunoglobulin G (66%) in MLN and spleen. Transfer of antitoxic memory was completely abrogated by treatment of the cells with J11d monoclonal antibody and complement prior to their injection into recipient mice by was unaffected by treatment with anti-Thy-1.2 antibody and complement, suggesting that long-term gut mucosal memory is carried by B cells. Antitoxin B memory cells might help explain the long-term protection against recurrent disease seen in convalescents from cholera in cholera-endemic areas.     

Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis

Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response.     

The early cellular and humoral immune response to primary and booster oral immunization with cholera toxin B subunit

The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers. A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization. Using an immunospot assay, cells spontaneously secreting anti-toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization. There were 105 IgG- and 87 IgA-secreting cells per 2 x 10(6) mononuclear cells 7 days after the first immunization, and 282 IgG- and 413 IgA-secreting cells 5 days after the second immunization. A polyclonal increase in total IgM-secreting cells was observed. Few anti-toxin-secreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti-toxin-secreting cells after oral immunization.     

Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit.

Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).     

Reduction in oral immunogenicity of cholera toxin B subunit by N-terminal peptide addition.

The mucosal adjuvanticity of cholera toxin and the potential of the B subunit of cholera toxin (CtxB) to serve as an oral vaccine carrier have prompted interest in the coupling of immunogenic peptides to this protein. The purpose of this study was to determine how such fusions affect the function of CtxB. Oligonucleotides were genetically fused to the 5' terminus of the ctxB gene to encode additional amino acids of 8, 12, and 24 residues in length. None of these additions affected the ability of CtxB to oligomerize, as determined by nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Circular dichroism revealed no difference in conformation between the modified B subunits, regardless of the length of the addition. However, when compared with native CtxB, additions to the N terminus induced a consistent change in the net conformation of the protein. By using a competitive enzyme immunoassay, the affinity of the modified B subunits for GM1 ganglioside was shown to gradually decrease with increasing length of the N-terminal addition. A similar pattern was observed for the ability of the chimeras to inhibit proliferation of concanavalin A-stimulated spleen cells in vitro, which is a previously described functional property of CtxB that is dependent on its binding to cells. Lastly, the oral immunogenicity of these chimeras was found to be less than that of native CtxB. These results indicate that large fusions to the N terminus of CtxB can significantly affect its biological properties and could reduce its value as a mechanism for effective mucosal immunization.     

Direct interaction between cholera toxin and dendritic cells is required for oral adjuvant activity

Cholera toxin (CT) binds to GM1‐ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1‐ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1‐expressing hematopoietic cells in general, or specifically GM1‐expressing conventional DCs (cDCs), largely failed to elicit anti‐OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1‐ganglioside. These results could have important implications for the generation of novel oral adjuvants.     

Successful immunization against gastric infection with Helicobacter species: use of a cholera toxin B-subunit-whole-cell vaccine.

In previous studies we found that immunizing mice with a sonicate of Helicobacter felis and adjuvant cholera toxin (CT; 10 micrograms) protected the animals against challenge with viable H. felis. The aim of this study was to determine whether a low dose of CT or its nontoxic B subunit (CTB) was effective as an adjuvant in Helicobacter oral vaccines. Significant protection against viable H. felis challenge was achieved in the animals immunized with H. felis antigen plus the combination of 0.5 microgram of CT and 10 micrograms of CTB (96%), with H. felis antigen plus 0.5 microgram of CT (95%), and with H. felis antigen plus 10 micrograms of CTB (83%). No protective effect was found in the mice immunized with either H. felis antigen alone or adjuvant CTB and CT alone. Twenty-six percent of mice immunized with Helicobacter pylori antigen plus CT (10 micrograms) were protected against H. felis challenge, confirming the value of the model in predicting effects of immunization in humans. The observation that immunity can be induced with a nontoxic adjuvant CTB opens the way for human studies with H. pylori vaccines and is a further step along the road to effective strategies of prevention of gastroduodenal diseases of major world significance.     

Systemic and mucosal immune responses in mice after mucosal immunization with group B streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate vaccine

Group B streptococci (GBS) colonize the female genital and rectal tracts and can cause invasive infection in susceptible newborns. An optimally effective GBS vaccine should induce mucosal and systemic immunity. In this study, we investigate the local and systemic immune responses to GBS type III capsular polysaccharide (CPS) after mucosal vaccination of mice via intranasal, peroral, rectal, and vaginal routes, with GBS type III CPS conjugated with recombinant cholera toxin B subunit (GBS III CPS-rCTB). Cholera toxin (CT) was added as an adjuvant. Immunoglobulin G (IgG) and IgA antibodies to the CPS were tested in serum, lungs, and intestinal, rectal, and vaginal extracts by enzyme-linked immunosorbent assay. The conjugated CPS administered by intranasal, peroral, rectal, and vaginal routes was much more effective at inducing both mucosal and systemic antibody responses to GBS III CPS than was unconjugated CPS. The CPS-specific immune responses in various organs were dependent on the route of immunization. Generally, the highest levels of IgA and IgG were generated in the regions or sites of the conjugate exposure. Thus, intranasal vaccination elicited the highest anti-CPS IgA and IgG antibody levels in the lungs, whereas peroral administration in the intestinal site and vaginal vaccination elicited the highest antibody levels in the vagina. Rectal vaccination was superior to the other routes in inducing high antibody levels in the rectum. The four routes of mucosal vaccination also induced distant antibody responses to CPS. Rectal vaccination induced high specific IgA levels in the vagina and intestine, and oral administration induced high specific IgA levels in the lungs and rectum. All four routes of vaccination with the conjugate elicited similarly high levels of anti-CPS IgG in serum. Intranasal vaccination with different doses of the conjugate (10, 30, and 80 microg of CPS) did not have a significant influence on the anti-CPS specific antibody responses. Intranasal immunization induced better antibody responses when one dose of the conjugate was divided and given on three consecutive days compared to administration of the full dose on one occasion. In conclusion, rectal and vaginal vaccination may be the best way of stimulating anti-CPS immune responses in the rectal and vaginal tracts, while high levels of anti-CPS antibodies in the lungs can be achieved after intranasal administration. The vaccination regimen thus might influence the mucosal immune response to CPS. This conjugate may serve as an effective mucosal vaccine for preventing mucosal colonization and invasive infection caused by GBS.     

Comparison of serum humoral responses induced by oral immunization with the hepatitis B virus core antigen and the cholera toxin B subunit

The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.     

Comparative effectiveness of the cholera toxin B subunit and alkaline phosphatase as carriers for oral vaccines.

The purpose of this study was to determine whether the B subunit of cholera toxin (CtxB) has adjuvant activity over and above serving as a carrier protein for orally administered vaccines. An oligonucleotide that encodes an antigenic determinant (GtfB.1) from the glucosyltransferase B gene (gtfB) of Streptococcus mutans was genetically fused to the 5' terminus of either the CtxB gene (ctxB) or the Escherichia coli alkaline phosphatase gene (phoA). The resulting chimeric proteins were expressed in a phoA mutant strain of E. coli and then purified. The antigenicities of the proteins were confirmed by immunoblotting analysis using antisera specific for GtfB, CtxB, or PhoA. An equimolar amount of peptide on each carrier was administered by gastric intubation to mice three times at 10-day intervals. Antibody titers to the peptide, CtxB, and PhoA (in the serum, intestine, vagina, saliva, and bronchus) were determined by enzyme immunoassay. Antibody to the peptide was detected only in the sera of mice immunized with the peptide fused to CtxB. No antipeptide antibody was detected in mice immunized with the peptide fused to PhoA. The lack of detectable levels of antipeptide antibody in intestinal lavage fluid was attributed to dilution of the sample beyond the sensitivity of the assay. This was confirmed by cultivation of Peyer's patch and mesenteric lymph node tissue from mice orally immunized with the GtfB.1::CtxB chimera. Using this method, antipeptide antibody was detected in the culture fluid. We conclude that CtxB possesses unique properties that allow it to act as more than a simple carrier protein.     

Effect of oral immunization with recombinant urease on murine Helicobacter felis gastritis.

The ability of oral immunization to interfere with the establishment of infection with Helicobacter felis was examined. Groups of Swiss Webster mice were immunized orally with 250 micrograms of Helicobacter pylori recombinant urease (rUrease) and 10 micrograms of cholera toxin (CT) adjuvant, 1 mg of H. felis sonicate antigens and CT, or phosphate-buffered saline (PBS) and CT. Oral immunization with rUrease resulted in markedly elevated serum immunoglobulin G (IgG), serum IgA, and intestinal IgA antibody responses. Challenge with live H. felis further stimulated the urease-specific intestinal IgA and serum IgG and IgA antibody levels in mice previously immunized with rUrease but activated primarily the serum IgG compartment of PBS-treated and H. felis-immunized mice. Intestinal IgA and serum IgG and IgA anti-urease antibody responses were highest in rUrease-immunized mice at the termination of the experiment. Mice immunized with rUrease were significantly protected (P < or = 0.0476) against infection when challenged with H. felis 2 or 6 weeks post-oral immunization in comparison with PBS-treated mice. Whereas H. felis-infected mice displayed multifocal gastric mucosal lymphoid follicles consisting of CD45R+ B cells surrounded by clusters of Thy1.2+ T cells, gastric tissue from rUrease-immunized mice contained few CD45R+ B cells and infrequent mucosal follicles. These observations show that oral immunization with rUrease confers protection against H. felis infection and suggest that gastric tissue may function as an effector organ of the mucosal immune system which reflects the extent of local antigenic stimulation.