Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant.

In the present study we investigated immunological memory at the cellular level following oral immunization using cholera toxin (CT) as the mucosal adjuvant. We found that memory cells, isolated from mice orally primed with keyhole limpet haemocyanin (KLH) admixed with CT adjuvant 8 months earlier, responded by increased proliferation to antigen-challenge in vitro. In contrast, unstimulated memory cells or KLH-stimulated cells from naive mice did not respond. Memory cells were isolated from different lymphoid tissues; spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) as well as the intestinal lamina propria (LP). Thus, oral immunization using CT adjuvant promoted the generation of memory cells that were present in both systemic and local intestinal lymphoid tissues. The demonstration of lymphokine production in the KLH-responsive cultures indicated the presence of antigen-specific memory T cells. Lymphokine production early in culture was dominated by interleukin-2 (IL-2), which peaked on day 2-3, followed by IL-5 and, in particular, interferon-gamma (IFN-gamma) which increased over time. Lamina propria memory cells were found to proliferate poorly to recall antigen in vitro compared to lymphocytes from SP or MLN. In contrast, very significant production of IL-5 and, in particular, IFN-gamma was demonstrable in LP cell cultures. The use of CT adjuvant also stimulated the generation of antigen-specific memory B cells following oral immunization. This was evidenced by KLH-specific antibody production in antigen-challenged memory lymphocyte cultures. The memory B cells produced IgM anti-KLH, while no detectable antigen-specific IgG or IgA was found. Unstimulated memory cells or naive cells failed to produce anti-KLH antibodies. These in vitro findings provide evidence that oral immunization using CT adjuvant stimulates both antigen-specific memory T and B cells. Furthermore, our data suggest the existence of memory B cells following oral CT adjuvant immunization which have retained the ability to produce IgM and which therefore probably have not undergone terminal isotype switch differentiation to other isotypes and thus have not deleted the mu constant heavy-chain gene. Finally, our data also suggest that memory T and B cells, either sessile in the various lymphoid tissues or recirculating, can be activated by antigen in situ in, for example, lymph nodes and spleen and, more importantly, in the intestinal LP itself.     

Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine.

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine.     

Mucosal priming of T-lymphocyte responses to fed protein antigens using cholera toxin as an adjuvant.

Cholera toxin is widely recognized as a potent stimulator of mucosal IgA responses after oral feeding. However, comparatively little is known of its ability to stimulate cellular responses. This is due in part to the direct inhibitory effects of cholera toxin on T lymphocytes, which can confound attempts to study primed T-cell responses in vitro. We have avoided this problem by using cholera toxin as an adjuvant to enhance the response to an unrelated protein fed simultaneously. Thus the simultaneous feeding of 10 micrograms of cholera toxin and 5 mg of keyhole limpet haemocyanin (KLH) results in the priming of T cells in the spleen, mesenteric lymph nodes, Peyer's patch and lamina propria that proliferate when restimulated with KLH in vitro. The feeding of KLH alone does not result in such responses. Both CD4- and CD8-positive antigen-specific T cells were involved in the response and the cells produced both interleukins 2 and 4 on antigen restimulation in vitro.     

Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens.

There is a great need for substances that can act as adjuvants on local mucosal immune responses to perorally (p.o.) administered immunogens and which could be included in future oral vaccines. In this study we show that in mice cholera toxin (CT) is a potent adjuvant on enteric mucosal immune responses to related (cholera B subunit) as well as unrelated (KLH) antigens presented by the p.o. route. The adjuvant action of CT was dose-dependent and was achieved only when CT was given p.o. and together with the antigen. Both priming (memory induction) and boosting of the gut mucosal immune system by the oral route were greatly potentiated by CT. High numbers of specific antibody-producing cells as well as substantial mucosal memory in the lamina propria were stimulated by p.o. priming immunizations if CT adjuvant was included. Anamnestic responses could be elicited by a single p.o. booster immunization for at least 10 weeks and probably much longer. The adjuvant action of CT is suggested to involve activation of adenylate cyclase and cyclic AMP-mediated signals with differential effects on B and regulatory T intestinal lymphocytes. The adjuvant-active dose of CT, 100-500 ng, was lower than the immunogenic dose (2 micrograms) and much below the p.o. dose needed for detectable net fluid secretion in mouse intestine (5-10 micrograms). Cholera B subunit (10 micrograms) administered p.o. together with 500 ng of CT was 50 times more effective in stimulating gut mucosal anti-toxin responses compared with B subunit vaccine alone. Our results suggest that CT or substances that use similar adjuvant mechanisms may substantially increase the mucosal immunogenicity and efficacy of non-replicating oral vaccines.     

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.     

The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant.

The in vitro production of a variety of cytokines by lymphocytes isolated from spleen mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria (LP) was measured, after oral immunization with keyhole limpet hemocyanin using cholera toxin as an adjuvant. LP responses were characterized by very high levels of interleukin (IL) 4, IL 5 and IL 6 with lower levels of IL 2 and interferon-gamma (IFN-gamma). The PP had lower levels of IL 4, IL 5 and IL 6 than LP but higher levels of IL 2, IFN-gamma was only present at very low levels in this organ. The MLN had a pattern of cytokine production similar to the PP but did produce IFN-gamma. The spleen produced all cytokines measured except IL 5. Antibody production was characterized by IgA in the LP and PP but IgG was the dominant isotype in the spleen, the MLN was a poor source of antibody-producing cells. We interpret the results to show that (a) the LP response to cholera toxin/keyhole limpet hemocyanin is dominated by Th2-type cytokines compared to a lower production of Th1 type and (b) that the PP has responses typical of an organ with a high proportion of resting lymphocytes which develop mainly into Th2-types cells. The spleen is less dominated by Th2-type cytokines than the mucosal sites and this difference is paralleled by IgA antibody production at the mucosal sites and IgG antibody dominance in the spleen.     

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin.

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.     

Intestinal and systemic immune responses to an oral cholera toxoid B subunit whole-cell vaccine administered during zinc supplementation

Zinc plays a critical role in the normal functioning of the immune system. We investigated whether zinc sulfate administered orally to adult zinc-replete volunteers modulates systemic and intestinal immune responses to an oral killed cholera toxoid B subunit (CTB) whole-cell cholera vaccine. The 30 participants were immunized twice, with a 17-day interval. The vaccinees in the intervention group ingested 45 mg of elemental zinc thrice daily for 9 days starting 2 days before each vaccine dose. The median serum anti-CTB immunoglobulin A (IgA) and IgG responses from day 0 to day 30, i.e. after two vaccine doses, were 13-fold lower (P value for identical distribution, <0.005) in the zinc-supplemented compared to the nonsupplemented vaccinees. The median serum vibriocidal responses from baseline to after one (day 0 to day 17) and two (day 0 to day 30) vaccine doses were at least sixfold (P = 0.033) and fourfold (P = 0.091) higher, while the median fecal anti-CTB IgA response after two doses was estimated to be fourfold higher (P = 0.084) in the zinc-supplemented vaccinees. These observations show that zinc reduces the antitoxin and may enhance the antibacterial responses in serum. Zinc may also improve the intestinal antitoxin immune response. Oral zinc administration has the potential to modify critical immune responses to antigens applied to mucosal surfaces.     

Influence of intravenous anesthesia on mucosal and systemic antibody responses to nasal vaccines

Inhalation of antigens may stimulate the immune system by way of the upper as well as the lower airways. We have shown that at least 1,000 times more live pneumococci were recovered from pulmonary tissue after being presented as drops of a liquid suspension onto the nares of anesthetized mice compared to the number of bacteria recovered from animals that were not anesthetized in the course of the challenge. Mice that were similarly immunized intranasally by inhalation of three different nonreplicating particulate vaccine formulations, i.e., a meningococcal outer membrane vesicle (OMV) vaccine, a formalin-inactivated whole-virus influenza (INV) vaccine, and the INV vaccine with OMVs as a mucosal adjuvant, during general intravenous anesthesia developed concentrations of vaccine-specific serum immunoglobulin G (IgG) antibodies that were four to nine times higher than in mice that were fully awake during immunizations. The concentrations of IgA antibodies in serum were also higher in anesthetized than in nonanesthetized mice and correlated positively with the corresponding levels of serum IgG antibodies in the anesthetized but not in the nonanesthetized mice. In saliva and feces, however, the concentrations of IgA antibodies were equally high whether or not the animals were dormant during immunizations. The results indicate that intrapulmonary antigen presentation, as a part of an intranasal immunization strategy, is of importance for systemic but not for mucosal antibody responses. A major portion of IgA antibodies in serum may thus be derived from nonmucosal sites.     

Recombinant cholera toxin B subunit is not an effective mucosal adjuvant for oral immunization of mice against Helicobacter felis.

Cholera toxin is a potent oral mucosal adjuvant for enteric immunization. Several studies suggest that commercial cholera toxin B subunit (cCTB; purified from holotoxin) may be an effective non-toxic alternative for oral immunization. The present study was performed, using an infectious disease model, to determine if the oral mucosal adjuvanticity of CTB is dependent on contaminating holotoxin. Mice were orally immunized with Helicobacter felis sonicate and either cholera holotoxin, cCTB or recombinant cholera toxin B subunit (rCTB). Serum immunoglobulin G (IgG) and intestinal immunoglobulin A (IgA) antibody responses were determined and the mice were challenged with live H. felis to determine the degree of protective immunity induced. All orally immunized mice responded with serum IgG antibody titres regardless of the adjuvant used. However, only mice immunized with either holotoxin or the cCTB responded with an intestinal mucosal IgA response. Consistent with the production of mucosal antibodies, mice immunized with either holotoxin or cCTB as adjuvants were protected from challenge while mice receiving H. felis sonicate and rCTB all became infected. cCTB induced the accumulation of cAMP in mouse thymocytes at a level equal to 0.1% of that induced by holotoxin, whereas rCTB was devoid of any activity. These results indicate that CTB possesses no intrinsic mucosal adjuvant activity when administered orally. Therefore, when used as an oral adjuvant, CTB should also include small, non-toxic doses of cholera toxin.     

Immunodulation of rat serum and mucosal antibody responses to Entamoeba histolytica trophozoites by beta-1,3-glucan and cholera toxin

Systemic and mucosal and immune responses can be manipulated with immunomodulators. Here we show the modulatory effects of cholera toxin (CT) and beta-1,3-glucan (GLU) on the rat antiamebic serum and fecal antibody responses to one or four intraperitoneal (IP) or intragastric (IG) doses of glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). One IP dose of GFT maximized serum IgM and IgG antiamebic antibodies on days 4 and 9, respectively; CT coadministration increased IgM antibodies, whereas IgG titers increased with CT or GLU; coproantibodies were undetectable after GFT alone or coadministered with GLU, whereas CT coadministration maximized fecal IgA antibodies on day 6. One IG dose of GFT alone increased serum IgM and IgG antibodies 2.5 times and no further increases were detected using GLU, whereas CT doubled serum IgG antibodies; GFT did not affect the coproantibody responses, whereas GLU coadministration maximized IgG coproantibody levels on day 6 and CT increased IgG and IgA coproantibody levels on the same day. On the other hand, four IG doses of GFT alone or with GLU induced tolerance, whereas GFT alone via the IP route increased serum antibodies slightly and GLU coadministration increased serum IgG antibody titers 300-fold. CT coadministration by both routes increased IgA coproantibodies, and simultaneous CT+GLU coadministration induced lower responses than either CT or GLU. Different antiamebic immune responses might therefore be attained through the use of different immunization routes and immunomodulators to induce protective immunity against intestinal or extraintestinal amebiasis.     

Enhanced systemic and mucosal antibody responses to a model protein antigen after intranasal and intratracheal immunisation using Bacillus firmus as an adjuvant

Bacillus firmus, a non-pathogenic Gram positive (G+) bacterium of the external environment was investigated for immunomodulatory properties. It stimulated an increase in anti-ovalbumin IgG in sera, bronchoalveolar lavages and intestinal washings after both intranasal (i.n.) and intratracheal (i.t.) immunisation, and enhanced anti-ovalbumin IgA in intestinal secretions and in bronchoalveolar lavage fluid after i.n. or i.t. immunisation, respectively. The immunomodulatory effect of B. firmus on antibody formation was antigen specific.     

H-2-unrestricted adjuvant effect of cholera toxin B subunit on murine antibody responses to influenza virus haemagglutinin.

Cholera toxin B subunit (CTB) has been shown to augment the antibody responses to influenza virus haemagglutinin (HA) in BALB/c mice immunized with HA vaccine together with CTB. In this study, mouse strain differences in the adjuvant effect of CTB on anti-HA antibody responses were investigated along with those in the antibody responses to CTB or HA, using various inbred and H-2 congenic strains. The antibody responsiveness to CTB depended on the H-2 haplotype of the strain: strains with the H-2b haplotype were high responders, those with H-2a, H-2k and H-2s were low responders, and those with H-2d were intermediate. The responsiveness to HA was also related to the H-2 haplotype: H-2a and H-2k strains were high responders, H-2b and H-2s strains were low responders, and H-2d strains were intermediate. However, the degree of the adjuvant effect of CTB on anti-HA antibody responses was almost constant, regardless of the H-2 haplotype or other genetic backgrounds of the strain. The lack of genetic restriction of the adjuvant effect would be favourable for application of CTB-combined HA vaccine to humans, who are genetically diverse. Moreover, these results suggest that the immunogenicity and adjuvanticity of CTB differ essentially in their mechanisms.     

Fluoride: an adjuvant for mucosal and systemic immunity.

Fluoride, the agent responsible for reduction of dental caries worldwide, and a recognized proliferative agent, is a potent adjuvant when given intragastrically to rats. Intragastric fluoride causes increases in the size and cellularity of the Peyer's patches and mesenteric lymph nodes as well as the number of plasma cells secreting IgG and IgA antibodies to ovalbumin given in their drinking water. Rats ingesting NaF and fed OA showed a significant increase in surface immunoglobulin expression on lymphocytes from the Peyer's patches and mesenteric lymph nodes. The frequency of CD4+ T cells in these lymphoid tissues was elevated while that of CD8+ T cells was significantly decreased. In separate experiments, rats parenterally immunized with myelin basic protein (MBP) and fed NaF twice weekly, had significantly elevated serum IgG antibody activity to MBP compared to similarly immunized rats not receiving NaF. The supplemental fluoride prescribed for infants and especially that which is inadvertently ingested by children and adults given fluoride gels, is within the concentration range of that which produced the effects we observed in rats. The adjuvant effect we describe thus has relevance for fluoride therapy worldwide.     

Zonula occludens toxin acts as an adjuvant through different mucosal routes and induces protective immune responses

Zonula occludens toxin (Zot) is produced by Vibrio cholerae and has the ability to increase mucosal permeability by reversibly affecting the structure of tight junctions. Because of this property, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. Here we show that Zot acts as a mucosal adjuvant to induce long-lasting and protective immune responses upon mucosal immunization of mice. Indeed, the intranasal delivery of ovalbumin with two different recombinant forms of Zot in BALB/c mice resulted in high Ag-specific serum immunoglobulin G titers that were maintained over the course of a year. Moreover, His-Zot induced humoral and cell-mediated responses to tetanus toxoid in C57BL/6 mice and protected the mice against a systemic challenge with tetanus toxin. In addition, we found that Zot also acts as an adjuvant through the intrarectal route and that it has very low immunogenicity compared to the adjuvant Escherichia coli heat-labile enterotoxin. Finally, by using an octapeptide representing the putative binding site of Zot and of its endogenous analogue zonulin, we provide evidence that Zot may bind a mucosal receptor on nasal mucosa and may mimic an endogenous regulator of tight junctions to deliver Ags in the submucosa. In conclusion, Zot is a novel and effective mucosal adjuvant that may be useful for the development of mucosal vaccines.     

Induction of systemic antifimbria and antitoxin antibody responses in Egyptian children and adults by an oral, killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine

We assessed serologic responses to an oral, killed whole-cell enterotoxigenic Escherichia coli plus cholera toxin B-subunit (ETEC-rCTB) vaccine in 73 Egyptian adults, 105 schoolchildren, and 93 preschool children. Each subject received two doses of vaccine or placebo 2 weeks apart, giving blood before immunization and 7 days after each dose. Plasma antibodies to rCTB and four vaccine-shared colonization factors (CFs) were measured by enzyme-linked immunosorbent assay. Immunoglobulin A (IgA) antibodies to rCTB and CFA/I were measured in all subjects, and those against CS1, CS2, and CS4 were measured in all children plus a subset of 33 adults. IgG antibodies to these five antigens were measured in a subset of 30 to 33 subjects in each cohort. Seroconversion was defined as a >2-fold increase in titer after vaccination. IgA and IgG seroconversion to rCTB was observed in 94 to 95% of adult vaccinees, with titer increases as robust as those previously reported for these two pediatric cohorts. The proportion showing IgA seroconversion to each CF antigen among vaccinated children (range, 70 to 96%) and adults (31 to 69%), as well as IgG seroconversion in children (44 to 75%) and adults (25 to 81%), was significantly higher than the corresponding proportion in placebo recipients, except for IgA responses to CS2 in adults. IgA anti-CF titers peaked after one dose in children, whereas in all age groups IgG antibodies rose incrementally after each dose. Independently, both preimmunization IgA titer and age were inversely related to the magnitude of IgA responses. In conclusion, serologic responses to the ETEC-rCTB vaccine may serve as practical immune outcome measures in future pediatric trials in areas where ETEC is endemic.     

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.     

Changes in intestinal fluid and mucosal immune responses to cholera toxin in Giardia muris infection and binding of cholera toxin to Giardia muris trophozoites.

The effect of Giardia muris infection on the diarrheal response and gut mucosal antibody response to cholera toxin was examined in mice. The results obtained showed that the fluid accumulation in intestinal loops exposed to cholera toxin was increased in mice infected with a low number (5 X 10(4) ) of G. muris cysts compared with the response in noninfected mice. This effect was associated with a marked reduction in absorption of oral rehydration fluid from the intestine. In contrast, mice infected with a high dose (2 X 10(5) ) of cysts showed a marked decrease in fluid accumulation in response to the toxin. This decrease might be related to the finding that both G. muris and Giardia lamblia trophozoites can bind significant amounts of cholera toxin. Evidence is presented which suggests that the gut mucosal antibody response, mainly immunoglobulin A but also immunoglobulin G, to an immunization course with perorally administered cholera toxin was depressed in mice infected with G. muris. The reduction in antibody levels was particularly evident when the primary immunization was made very early after infection. The serum antitoxin antibodies to the oral immunization with cholera toxin were, however, not affected. Likewise, the delayed-type hypersensitivity response against sheep erythrocytes in animals primed subcutaneously with sheep erythrocytes was not modified during the course of G. muris infection.     

Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates.

This study was designed to test local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. After preparing and characterizing dextran-cholera toxin B subunit (CTB) conjugates, we studied their immunogenicity in mice following systemic or mucosal immunizations. Dextran was chosen as a model polysaccharide antigen and conjugated via adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio)propionate to CTB. Mice were immunized either subcutaneously, intranasally, or perorally three times, and cholera toxin was used as an adjuvant for the mucosal immunizations. Three conjugates with different molecular weights for dextran (40,000 and 76,000) or varying dextran/CTB molar ratios were tested. Peroral immunizations with all conjugates evoked local immunoglobulin A (IgA) antibody responses against dextran in the small intestine, and intranasal immunizations did the same in the lung. Intranasal immunizations also elicited serum antibody titers that were significantly higher than or equal to those after subcutaneous immunizations. Intranasal immunizations evoked serum IgG antidextran titers which were dependent on the dextran/CTB molar ratio and inversely related to the local IgA response, which was not the case for subcutaneous immunizations. This is the first study of local and systemic immunity following mucosal immunization with a polysaccharide-protein conjugate. The results show that it is possible to evoke a local as well as a systemic antibody response against a polysaccharide by conjugating it to CTB and using an appropriate route of immunization.     

Safety and immune responses to attenuated Salmonella enterica serovar typhi oral live vector vaccines expressing tetanus toxin fragment C

Attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was used as a vector to deliver fragment C of tetanus toxin as a single-dose oral tetanus vaccine candidate to elicit protective levels of serum tetanus antitoxin. Twenty-one healthy adult volunteers received doses of 1.6 x 10(7) to 8.2 x 10(9) CFU of one of two strains, CVD 908-htrA(pTETnir15) or CVD 908-htrA(pTETlpp), which contained plasmid-encoded fragment C, with sodium bicarbonate, and the safety and immune responses to serovar Typhi antigens and tetanus toxin were assessed. No volunteer had fever or positive blood cultures after vaccination, although diarrhea occurred in 3 volunteers and vomiting in 2 volunteers within 3 weeks after vaccination. Most volunteers excreted the vaccine strain in the first 72 h after vaccination. Three of nine volunteers who received 10(8) CFU or higher doses of the CVD 908-htrA(pTETlpp) construct developed rises in serum antitoxin antibodies. The serum and cellular immune responses to serovar Typhi antigens were less frequent than those previously observed in volunteers who ingested the parent strain CVD 908-htrA. This study demonstrates that fragment C of tetanus toxin delivered orally to volunteers in an S. Typhi vector can elicit protective levels of serum antitoxin.