Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans

The induction of immune responses to rectally administered recombinant cholera toxin B subunit (CTB) in humans was studied. Three immunizations induced high levels of CTB-specific antibody-secreting cells, particular of the immunoglobulin A isotype, in both rectum and peripheral blood. Antitoxin antibody responses in rectal secretions and serum were also found.     

Expression and mutagenesis of recombinant cholera toxin A subunit

ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Escherichia coli (LT-I) share two short regions of sequence similarity with Bordetella pertussis toxin (PT). Previous studies have indicated that substitution of arginine for lysine 7 within the first region of CT drastically decreases ADP ribosyltransferase activity. We have more closely defined the role of other amino acids in this region by generating modified proteins in which arginine 7 was replaced with lysine (R7K), aspartate 9 was replaced with arginine (D9R), glycine was substituted for proline 12 (P12G), amino acids 6 to 13 were deleted (Δ613) or the C-terminal KDEL sequence was changed to NEDL. The modified proteins R7K, D9R and Δ613 exhibited undetectable ADP ribosyltransferase activity. Comparison of the tryptic digest of R7K with native CT suggested that changes in protein conformation may be responsible for the loss of ADP-ribosylation activity.     

Immune response induced by recombinant Mycobacterium bovis BCG producing the cholera toxin B subunit

The pentameric form of the cholera toxin B subunit (CTB) is known to be a strong mucosal adjuvant and stimulates antigen-specific secretory immunoglobulin A (IgA) and systemic antibody responses to antigens when given by mucosal routes. To deliver CTB for prolonged periods of time to the respiratory mucosa, we constructed a Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain that produces and secretes assembled pentameric CTB. Mice immunized intranasally (i.n.) with recombinant BCG (rBCG) developed a stronger anti-BCG IgA response in bronchoalveolar lavage fluids (BALF) than mice immunized with nonrecombinant BCG. The total IgA response in the BALF of mice immunized with rBCG was also stronger than that in BALF of mice immunized with the nonrecombinant strain. The induction of IgA was well correlated with an increased production of transforming growth factor beta1. Simultaneous administration of intraperitoneally delivered ovalbumin and of i.n. delivered CTB-producing BCG induced a long-lasting ovalbumin-specific mucosal IgA response as well as a systemic IgG response, both of which were significantly higher than those in mice immunized with nonrecombinant BCG together with ovalbumin. These results suggest that the CTB-producing BCG may be a powerful adjuvant to be considered for future mucosal vaccine development.     

Priming immune response to cholera toxin induced by synthetic peptides

A systematic study has been conducted of the priming effect in the immunization against cholera toxin (CT). We demonstrate that a priming phenomenon can be achieved by synthetic peptides of the CT B subunit, leading (after a subsequent booster with a subimmunizing dose of the intact toxin) to an efficient anti-CT neutralizing antibody response. This effect is obtained even upon a single administration of a peptide conjugate and even by peptides that as such are not able to induce CT cross-reactive antibodies whatsoever. This effect is specific and dose dependent. A macromolecular carrier as well as an adjuvant are essential for the induction of antitoxin response. In this respect, a totally synthetic priming agent, CTP3-poly (DL -alanyl)–poly(L -lysine), was adequate for an effective priming response. The specificity of the antibodies formed after the booster was mainly towards the whole CT molecule and only a small fraction of them were specific towards the peptide used for priming. The ability of synthetic peptides to prime the immune system towards a secondary stimulus with whole organism or native protein might be of general practical value, especially in endemic areas where the population is probably constantly exposed to a low level of a particular infectious agent. This exposure, which has no influence on the unprimed immune system, could serve as a booster in the case of individuals primed with an appropriate peptide, leading to a secondary immune response.     

Kinetics of local and systemic immune responses after vaginal immunization with recombinant cholera toxin B subunit in humans

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.     

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.     

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.     

Adjuvant effect of cholera toxin on the mucosal immune response to soluble proteins. Differences between mouse strains and protein antigens

We examined the effect of orally administered cholera toxin (CT) on the immune response to keyhole limpet haemocyanin (KLH) and ovalbumin (OVA) in high (C57Bl/6 H2b), medium (CBA H2k), and low (BALB/c H2d) responder I-A haplotypes to CT. Mice were fed OVA or KLH on three occasions at 10-day intervals and the effect of simultaneous feeding of CT determined. Isotype-specific antibody levels were assayed in serum samples collected 7 days after the last immunization. Antibody was also measured in the supernatant of gut explant cultures incubated at either 4 or 37 degrees C. Increased antibody levels in cultures kept at 37 degrees C indicated release of local intracellular antibody. Cholera toxin exerted an adjuvant effect on the mucosal response of all three strains to KLH and OVA. Overall, the responses to CT and the second protein were not correlated; we interpret these findings to indicate that while CT had an effect on the mucosal immune system which enhances the immune response to itself and other protein antigens, the final outcome of the response to the second antigen is dependent on differences in the ability of the strains to process, recognize, and respond to a particular antigen.     

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.     

Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon

Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFβ-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFβ-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.     

Induction and distribution of intestinal immune responses after administration of recombinant cholera toxin B subunit in the ileal pouches of colectomized patients

The induction and dissemination of mucosal immune responses to recombinant cholera toxin B subunit (rCTB) administered into the ileal pouches of patients, who had been colectomized because of ulcerative colitis, was analyzed. Biopsies from the duodenum and ileal pouch were collected, along with peripheral blood and ileostomy fluids. Two immunizations induced strong CTB-specific immunoglobulin A (IgA) antibody-secreting cell (ASC) responses in the duodenum in five of five patients, whereas weaker and less-frequent ASC responses were noted in the ileal pouch. Intestine-derived CTB-specific IgA ASCs were found in peripheral blood in three of the five patients. The vaccination also induced significant IgA antitoxin titer rises in ileostomy fluid in all of the patients. Increased production of gamma interferon in cell cultures from the ileal pouch was found in four of five patients after the vaccination. These results clearly indicate that rCTB administered into the distal ileum is capable of inducing B-cell responses in the "entire" small intestine and that homing of immunocompetent cells occurs preferentially to the duodenum.     

Suppression of the intestinal immune response to cholera toxin by specific serum antibody.

The possibility that preexisting specific serum antibody could suppress a defined mucosal immune response to a topically applied antigen was studied in rats. Hyperimmune serum antibody induced by parenteral immunization of rats with cholera toxoid markedly suppressed the mucosal immune response to enterically applied cholera toxin. Such antibody was far more suppressive than antibody induced by primary parenteral immunization, apparently due to its greater avidity. Transfusion of small amounts (25 to 100 microliter) of hyperimmune serum suppressed the primary mucosal antitoxin response, the development of specific memory in the mucosal immune system, and, somewhat less effectively, the secondary mucosal antitoxin response. Suppression was due largely to a direct effect of serum antibody upon the interaction of absorbed enteric antigen with lymphoid tissue in Peyer's patches and, possible, mesenteric lymph nodes; interference with antigen absorption played little or no role in the observed suppression. These results do not explain the previously reported suppressive effect of primary parenteral immunization on the mucosal immune response to cholera toxin. However, they support the notions that repeated parenteral immunization can evoke avid serum antibody without necessarily stimulating mucosa-associated lymphoid tissue and that such antibody can markedly suppress primary and secondary phases of the local immune response to mucosally applied antigen. Thus, a mechanism is demonstrated by which repeated parenteral immunization may adversely affect efforts to initiate or sustain protective mucosal immune responses.     

Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae

Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3). Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O₁ ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F’ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.     

Interaction of cholera toxin and toxin derivatives with lymphocytes. II. Modulating effects of cholera toxin on in vivo humoral and cellular immune responses.

The in vivo effects of cholera toxin on lymphoid organ structure and function in mice were investigated. It was found that within a day following intravenous injection of 1 mug of toxin, thymus as well as spleen weight decreased but the animals remained healthy. Histological studies suggested that the involution of lymphoid organs was due to cell death. Injection of cholera toxin into adrenalectomized mice was lethal within 36 h. In these animals no decrease in lymphoid organ weight was noted. Thymus cells from toxin-treated mice were found to be much inferior to thymocytes of untreated animals in their in vitro response to Concanavalin A, whereas the response of spleen cells from toxin-treated animals to mitogens was slightly increased. 1 mug of cholera toxin increased primary antibody formation when given to mice together with antigen (sheep erythrocytes) and decreased primary antibody formation when given before or after the antigen. The toxin also increased secondary antibody formation when injected simultaneously with or after the booster antigen dose, and decreased the antibody formation when given a few days before the booster injection. Treatment of mice with toxin was found to increase the capacity of spleen cells from these animals to induce the parental effect on antibody formation and to induce graft-versus-host reactions. The mechanisms behind the observed effects are discussed. It is suggested that cholera toxin affects different types of cells involved in immune responses primarily by a direct inhibitory action on cellular proliferation but also indirectly by causing release of adrenal gland hormones.     

Mucosal immune response to cholera toxin in ageing rats. I. Antibody and antibody-containing cell response.

Although ageing is accompanied by systemic immunodeficiencies, the status of the mucosal immune system in the elderly remains unresolved. The gastrointestinal mucosal immune response was evaluated in young, mature and old male rats subjected to intra-intestinal immunization with cholera toxin (CTx). Five days following secondary immunization, the alpha-CTx-IgA titre in the bile of immunized rats was markedly reduced, i.e. the values measured in young rats were approximately five-fold higher than those of old animals. alpha-CTx-IgA levels in non-immunized rats were negligible and age-related shifts in other antibody titres (alpha-CTx IgG and IgM) were not significant. The antibody response to CTx was not reflected in the total IgA content of the samples. The number of alpha-CTx antibody-containing cells (ACCs) in the small intestinal lamina propria was significantly reduced in old immunized rats in comparison with the young or mature animals. These data suggest that ageing compromises both non-immune cell (antibody transport by hepatocytes) and immune cell (number of ACCs in the gut wall) functions in response to cholera toxin immunization in this animal model.     

Role of Peyer''s patch in the intestinal immune response to cholera toxin in enterically immunized rats.

Cholera toxin has been widely used to obtain insight into the cellular dynamics of the antigen-specific mucosal immune response. The present study was undertaken to clarify the influence of the organized intestinal lymphoid tissue (Peyer's patches [PP]) on the distribution of anti-cholera-toxin-containing cells (ACC) after intraperitoneal immunization and intraduodenal challenge with purified cholera toxin. This was done in rats which were surgically deprived of all visible PP. In comparison with sham-operated animals, each PP-deprived rat had nearly the same amount of ACC in the spleen, the mesenteric lymph nodes, and, surprisingly, the thoracic duct lymph. In contrast, the ACC in the duodenum, the jejunum, and the ileum of each PP-deprived animal were drastically reduced. Therefore the PP are suggested as an important organizing structure for the buildup of a local antigen-specific immune response.     

重组霍乱毒素B亚基与恶性疟原虫多表位融合蛋白的免疫原性研究

目的　评价霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白的免疫原性。方法　在大肠杆菌中表达重组霍乱毒素与恶性疟原虫多价抗原的融合蛋白 ,通过免疫小鼠评价融合蛋白的免疫原性。结果　通过亲和层析纯化霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白 ,不加任何佐剂 ,以 5 0 μg/只的剂量免疫C5 7BL/ 6J小鼠 ,3次免疫后 ,CTB抗体滴度达 1∶6 40 0 ,抗疟原虫抗体滴度 1∶16 0 0 ,其CTL活性为 2 0 7%。结论　霍乱毒素具有较好的佐剂作用 ,经融合蛋白免疫的小鼠能产生良好的体液和细胞免疫 ,为评价该抗原的免疫保护作用打下了基础     

Oral immunization of mice with a live recombinant Yersinia enterocolitica O:9 strain that produces the cholera toxin B subunit.

The 70-kilobase pYV plasmid of Yersinia enterocolitica encodes a set of proteins called Yops that are produced during infection. To use Y. enterocolitica as a live carrier to present the cholera toxin B (CT-B) subunit to the immune system, we constructed an operon fusion between ctxB and the yop51 gene. This operon fusion was either cloned on an RSF1010-derived plasmid or integrated into the pYV plasmid itself. In Y. enterocolitica, both constructions directed the synthesis of free CT-B only under conditions of Yops production, i.e., at 37 degrees C in a medium deprived of Ca2+. Bacteria containing both types of recombinant plasmids were given orally to mice. A serum antibody response against CT-B was detected in both cases. A secretory immunoglobulin A activity specific to CT-B was also observed in the intestinal secretions. According to immunoblot analysis, the serum antibody response was only directed against the polymeric form of the B subunit. The ctxB gene was also inserted in frame within yop51, giving a chimeric Yop51-CT-B protein that was secreted into the surrounding medium. In this case, however, no antibody response was observed after oral inoculation of mice. This lack of response probably results from the inability of the hybrid protein to assemble into the polymeric form of the B subunit.     

Genetic control of immune response to staphylococcal exfoliative toxin A in mice.

Different inbred and congenic resistant strains of mice were immunized with staphylococcal exfoliative toxin A (ETA). In antibody responses measured in sera of mice by a passive hemagglutination technique, A/J, DBA/2, BALB/c, B10A, B10D2, B10S, and A.SW were high responders. C57BL/10 (B10), A.BY, and DBA/1 were low responders. The congenic C3H/HeJ and C3H.SW mice were, respectively, high and low responders. The observation that the immune responses of the mice to ETA were closely linked with the haplotypes of their H-2 complexes suggests the existence of an H-2-linked immune response (Ir) gene coding for the production of humoral antibodies to ETA. Four B10A recombinants were used to map this gene within the H-2 complex. The finding that B10A(2R) and B10A(4R) were high responders, whereas B10A(3R) and B10A(5R) were low responders, indicates that the gene controlling antibody response to ETA is located in the I-A subregion or the H-2K end within the H-2 complex. We wish to propose the name Ir-ETA for this gene. The function of Ir-ETA seems to be at least related to antigen recognition at the T-lymphocyte level. Neonatal mice are generally susceptible to ETA regardless of their H-2 haplotypes. However, the neonatal mice born to a high-responder mother immunized with ETA were resistant to the subcutaneous challenge of ETA, but those born to an immunized low-responder mother were susceptible to the challenge. This result suggests that if the mother is a high responder to ETA and is effectively immunized with ETA, the maternal immunity makes it possible to neutralize this toxin in neonatal mice.     

Intranasal administration of recombinant Neisseria gonorrhoeae transferrin binding proteins A and B conjugated to the cholera toxin B subunit induces systemic and vaginal antibodies in mice

The transferrin binding proteins (TbpA and TbpB) comprise the gonococcal transferrin receptor and are considered potential antigens for inclusion in a vaccine against Neisseria gonorrhoeae. Intranasal (IN) immunization has shown promise in development of immunity against sexually transmitted disease pathogens, in part due to the induction of antigen-specific genital tract immunoglobulin A (IgA) and IgG. Conjugation of antigens to the highly immunogenic cholera toxin B subunit (Ctb) enhances antibody responses in the serum and mucosal secretions following IN vaccination. In the current study, we characterized the anti-Tbp immune responses following immunization of mice IN with recombinant transferrin binding proteins (rTbpA and rTbpB) conjugated to rCtb. We found that both rTbpA-Ctb and rTbpB-Ctb conjugates administered IN induced antibody responses in the serum and genital tract. IN immunization resulted in both IgA and IgG in the genital tract; however, subcutaneous immunization mainly generated IgG. Surprisingly, rTbpA alone was immunogenic and induced serum and mucosal antibody responses similar to those elicited against the rTbpA-Ctb conjugate. Overall, rTbpB was much more immunogenic than rTbpA, generating serum IgG levels that were greater than those elicited against rTbpA. Bactericidal assays conducted with sera collected from mice immunized IN with TbpA and/or TbpB indicated that both antigens generated antibodies with bactericidal activity. Anti-TbpA antibodies were cross-bactericidal against heterologous gonococcal strains, whereas TbpB-specific antibodies were less cross-reactive. By contrast, antibodies elicited via subcutaneous immunization were not cross-bactericidal against heterologous strains, indicating that IN vaccination could be the preferred route for elicitation of biologically functional antibodies.