Transcutaneous immunization with toxin-coregulated pilin A induces protective immunity against Vibrio cholerae O1 El Tor challenge in mice

Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.     

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.     

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.     

Distribution of Monoclonal Antibodies in Intestinal and Urogenital Secretions of Mice Bearing Hybridoma 'Backpack' Tumours

Mice bearing IgA hybridoma 'backpack' tumours have been used to demonstrate that secretion of a single monoclonal IgA can protect against mucosal infection, but the relevance of this model to normal IgA protection is not clear. The authors analysed the distribution of specific monoclonal and total antibodies in bile, local intestinal secretions, cervical-vaginal secretions, urine and serum of mice bearing anti-cholera toxin (CT) IgA and IgG backpack tumours, with and without bile duct ligation. Backpack tumours resulted in high levels of both anti-CT and total IgA or IgG in serum, and IgA (but not IgG) in bile. Secretions recovered by absorbent filter 'wicks' from mucosal surfaces throughout the intestines of backpack tumour mice contained significant concentrations of monoclonal anti-CT IgA, but total IgA levels were as in normal mice. Neither monoclonal nor total IgA levels on mucosal surfaces were altered by bile duct ligation. Furthermore, anti-CT monoclonal IgA levels in local intestinal secretions of backpack tumour mice were comparable to specific polyclonal IgA levels previously elicited by mucosal immunization with CT. Thus, IgA-mediated protection against enteric challenge in the backpack tumour model may be a valid predictor of protection provided by natural mucosal immunization in vivo . High monoclonal IgA levels in bile, urine and the female genital tract, however, may not reflect the situation in normal immunized mice.     

Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants

Transcutaneous immunization (TCI) is a new technique that uses the application of vaccine antigens in a solution on the skin to induce potent antibody responses without systemic or local toxicity. We have previously shown that cholera toxin (CT), a potent adjuvant for oral and nasal immunization, can induce both serum and mucosal immunoglobulin G (IgG) and IgA and protect against toxin-mediated mucosal disease when administered by the transcutaneous route. Additionally, CT acts as an adjuvant for coadministered antigens such as tetanus and diphtheria toxoids when applied to the skin. CT, a member of the bacterial ADP-ribosylating exotoxin (bARE) family, is most potent as an adjuvant when the A-B subunits are present and functional. We now show that TCI induces secondary antibody responses to coadministered antigens as well as to CT in response to boosting immunizations. IgG antibodies to coadministered antigens were also found in the stools and lung washes of immunized mice, suggesting that TCI may target mucosal pathogens. Mice immunized by the transcutaneous route with tetanus fragment C and CT developed anti-tetanus toxoid antibodies and were protected against systemic tetanus toxin challenge. We also show that bAREs, similarly organized as A-B subunits, as well as the B subunit of CT alone, induced antibody responses to themselves when given via TCI. Thus, TCI appears to induce potent, protective immune responses to both systemic and mucosal challenge and offers significant potential practical advantages for vaccine delivery.     

Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant

The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.     

Influence of the murine oestrous cycle on the induction of mucosal immunity

PROBLEM: To determine if the stage of oestrous cycle, at the time of immunization, affects the magnitude of mucosal and systemic immunity. METHOD OF STUDY: Female BALB/c mice were immunized with tetanus toxoid and cholera toxin by the oral, intranasal and transcutaneous routes. Groups of mice were immunized at proestrus, oestrus, postestrus and diestrus. Antibodies in serum and mucosal secretions were determined by ELISA and T cell responses by lymphocyte proliferation assay. RESULTS: Oral immunization at the oestradiol dominant stage of cycle (oestrus and proestrus) significantly enhanced TT-specific IgG and IgA levels in female reproductive tract (FRT) secretions and TT-specific IgA levels in faecal extracts. Transcutaneous immunization at diestrus enhanced TT-specific IgG in faecal extracts. TT-specific T cell proliferation is greatest following intranasal immunization at proestrus and transcutaneous immunization at diestrus, particularly in the caudal and lumbar lymph nodes draining the FRT and colon. CONCLUSIONS: Reproductive cycle-associated changes in the endogenous sex hormones oestradiol and progesterone influence the levels of vaccine-induced immunity in the FRT and distal colon following oral and transcutaneous immunization.     

Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A

BACKGROUND: Clostridium difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis. We studied whether antibody responses to C. difficile toxins affect the risks of colonization, diarrhea, and asymptomatic carriage. METHODS: We prospectively studied C. difficile infections in hospitalized patients who were receiving antibiotics. Serial stool samples were tested for C. difficile colonization by cytotoxin assay and culture. Serum antibody (IgA, IgG, and IgM) levels and fecal antibody (IgA and IgG) levels against C. difficile toxin A, toxin B, and nontoxin antigens were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 271 patients, 37 (14 percent) were colonized with C. difficile at the time of admission, 18 of whom were asymptomatic carriers. An additional 47 patients (17 percent) became infected in the hospital, 19 of whom remained asymptomatic. The baseline antibody levels were similar in the patients who later became colonized and those who did not. After colonization, those who became asymptomatic carriers had significantly greater increases in serum levels of IgG antibody against toxin A than did the patients in whom C. difficile diarrhea developed (P<0.001). The adjusted odds ratio for diarrhea was 48.0 (95 percent confidence interval, 3.4 to 678) among patients with colonization who had a serum level of IgG antibody against toxin A of 3.00 ELISA units or less, as compared with patients with colonization who had a level of more than 3.00 ELISA units. CONCLUSIONS: We find no evidence of immune protection against colonization by C. difficile. However, after colonization there is an association between a systemic anamnestic response to toxin A, as evidenced by increased serum levels of IgG antibody against toxin A, and asymptomatic carriage of C. difficile.     

Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge

In this study, the potential of the bare skin as a non-invasive route for vaccination was examined. Following application of heat-labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti-LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes. In addition, LT enhanced the serum and mucosal antibody and proliferative T-cell responses to the model protein antigen beta-galactosidase (beta-gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect. When a peptide representing a T-helper epitope (aa 307-319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide- and virus-specific T cells, as measured in vitro by the interleukin-2 (IL-2) secretion assay. LT was shown to be as immunogenic as CT. Binding activity to GM1 gangliosides was essential for effective induction of anti-CT serum and mucosal antibody responses. Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin. These findings give further support to a growing body of evidence on the potential of skin as a non-invasive route for vaccine delivery. This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical.     

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.     

Interferon-gamma receptor-deficient mice exhibit impaired gut mucosal immune responses but intact oral tolerance.

Interferon-gamma (IFN-gamma) receptor knock-out (IFN-gamma R -/-) mice were used to analyse the role of IFN-gamma in mucosal immune responses following oral immunization. We found that the IFN-gamma R -/- mice demonstrated 50% reduced spot-forming cell (SFC) responses in the gut lamina propria and spleen after oral immunization with keyhold limpet haemocyanin (KLH) plus cholera toxin (CT) adjuvant. The IFN-gamma R -/- mice exhibited 10-fold reduced total serum KLH-specific antibody levels compared with wild-type mice after oral immunization, while after intravenous immunization, no such difference was seen, suggesting a selective impairment of mucosal immune responses. Moreover, oral immunizations resulted in impaired interleukin-4 (IL-4), IL-10 and IFN-gamma production by spleen T cells from IFN-gamma R -/- mice, indicating that no reciprocal up-regulation of Th2-activities had occurred despite the lack of IFN-gamma R function. No reduction in Th1 or Th2 cytokines was observed following systemic immunizations. Despite potentially strong modulating effects of IFN-gamma on epithelial cell IgA transcytosis and electrolyte barrier functions, CT-immunized IFN-gamma R -/- mice demonstrated unaltered protection against CT in ligated intestinal loops together with normal anti-CT IgA and total IgA levels in gut lavage. Oral feeding with KLH followed by parenteral immunization resulted in strongly suppressed SFC numbers and reduced cell-mediated immunity in both wild-type and IFN-gamma R -/- mice. CT-adjuvant abrogated induction of oral tolerance in both IFN-gamma R -/- and wild-type mice. Collectively, our data argue that the two major response patterns induced by oral administration of protein antigen, i.e. active IgA immunity and oral tolerance, are differently regulated. Thus, IFN-gamma R -/- mice have impaired mucosal immune responses while induction of oral tolerance appears to be unaffected by the lack of IFN-gamma functions.     

Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes.

A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1). The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT. MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes. Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats. No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb. When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected. There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats. Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2. This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.     

Intranasal immunization with liposomes induces strong mucosal immune responses in mice

BALB/c mice were immunized intranasally with either soluble ovalbumin (OVA) or OVA entrapped in liposomes. The effect of adding Sigma cholera toxin B subunit (sCT-B), which contained low amounts of cholera holotoxin (CT), or recombinant CT-B (rCT-B) which was free from CT, as mucosal adjuvants was also investigated. The mucosal [lung enzyme-linked immunospot assay (ELISPOT), lung washing] and systemic (serum antibody and spleen ELISPOT) responses of immunized mice to OVA and CT-B were determined. Results showed that soluble OVA and liposome-entrapped OVA were poor inducers of mucosal or systemic responses unless CT-B was added as adjuvant. The types of responses augmented by sCT-B and rCT-B were different. CT-B containing low levels of CT (i.e. sCT-B) boosted both mucosal and systemic IgA and IgG responses, whereas rCT-B only increased IgG responses, unless antigen was entrapped in liposomes. Although rCT-B was unable to adjuvant IgA responses against soluble OVA, it was able to induce IgA responses against itself. These data show that mucosal responses can be increased by addition of CT-B containing low levels of CT to antigen preparations given intranasally, suggesting a direct role for CT-A in isotype switching. Furthermore, the ability of CT-B to adjuvant IgA responses against added antigens and its ability to induce responses against itself appear to be separate phenomena. The results from this study should assist the rational formulation of mucosal vaccines which induce potent mucosal and systemic immune responses.     

Intranasal coadministration of the Cry1Ac protoxin with amoebal lysates increases protection against Naegleria fowleri meningoencephalitis

Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 x 10(4) live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.     

Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c⁺ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC–B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP–TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal 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.     

Transcutaneous Immunization with a Vibrio cholerae O1 Ogawa Synthetic Hexasaccharide Conjugate following Oral Whole-Cell Cholera Vaccination Boosts Vibriocidal Responses and Induces Protective Immunity in Mice

A shortcoming of currently available oral cholera vaccines is their induction of relatively short-term protection against cholera compared to that afforded by wild-type disease. We were interested in whether transcutaneous or subcutaneous boosting using a neoglycoconjugate vaccine made from a synthetic terminal hexasaccharide of the O-specific polysaccharide of Vibrio cholerae O1 (Ogawa) coupled to bovine serum albumin as a carrier (CHO-BSA) could boost lipopolysaccharide (LPS)-specific and vibriocidal antibody responses and result in protective immunity following oral priming immunization with whole-cell cholera vaccine. We found that boosting with CHO-BSA with immunoadjuvantative cholera toxin (CT) or Escherichia coli heat-labile toxin (LT) following oral priming with attenuated V. cholerae O1 vaccine strain O395-NT resulted in significant increases in serum anti-V. cholerae LPS IgG, IgM, and IgA (P < 0.01) responses as well as in anti-Ogawa (P < 0.01) and anti-Inaba (P < 0.05) vibriocidal titers in mice. The LPS-specific IgA responses in stool were induced by transcutaneous (P < 0.01) but not subcutaneous immunization. Immune responses following use of CT or LT as an adjuvant were comparable. In a neonatal mouse challenge assay, immune serum from boosted mice was associated with 79% protective efficacy against death. Our results suggest that transcutaneous and subcutaneous boosting with a neoglycoconjugate following oral cholera vaccination may be an effective strategy to prolong protective immune responses against V. cholerae.     

The haemopoietic growth factor,Flt3L,alters the immune response induced by transcutaneous immunization

Topical application of antigen induces antigen-specific humoral and cellular immune responses. In this study we examined whether expansion of dendritic cells (DC) by Flt3 ligand (Flt3L) treatment influences the induction of immune responses following transcutaneous immunization. Mice were treated intraperitoneally with Flt3L or phosphate-buffered saline (PBS) and immunized transcutaneously with hen egg lysozyme (HEL). Flt3L-treated mice developed lower HEL-specific cellular and humoral immune responses than PBS-treated mice. However, in the presence of cholera toxin (CT), a potent adjuvant for mucosal and transcutaneous immunization, Flt3L-treated mice developed significantly higher cellular and humoral immune responses to HEL when compared to PBS-treated mice. We assessed whether the immunomodulatory effects of CT were a result of activation of epidermal dendritic cells (Langerhans' cells; LC). Our results indicate that within 8-12 hr of topical application of CT, epidermal LC cells lose their dendritic morphology and become rounder in appearance. In addition, we observed enhanced expression of major histocompatibility complex (MHC) class II, and of adhesion molecules CD11c and intracellular adhesion molecule-1 (ICAM-1). Our observations support the concept that the state of activation of DC in the skin is central to the regulation of immune responses. This information is relevant to the design of effective transcutaneous vaccination strategies.     

Specific-Antibody-Secreting Cells in the Rectums and Genital Tracts of Nonhuman Primates following Vaccination

To determine optimal strategies to induce specific-antibody-secreting cells (specific ASC) in the rectal and vaginal mucosae, we immunized monkeys with a prototype mucosal immunogen, cholera toxin (CT), given locally or via gastric or parenteral administration. Repeated rectal or vaginal CT immunizations induced strong mucosal and systemic ASC responses. The mucosal responses were, however, confined to the immunization sites and comprised high levels of both specific antitoxin immunoglobulin A (IgA) and IgG. Large numbers of specific IgA and IgG ASC were detected in cell suspensions from dissociated genital and rectal tissues, demonstrating local accumulation of effector B cells at these sites. Intragastric immunization with CT did not per se give rise to cervicovaginal or rectal ASC responses but did prime for a rectal IgA ASC response to local booster immunization. Both rectal and vaginal immunizations also induced circulating blood IgG ASC and IgA ASC. In conclusion, these results show that local administration of antigen to the rectal or vaginal mucosa results in higher ASC responses than systemic or distant mucosal delivery. Furthermore, both the vaginal and the rectal mucosae can serve as inductive sites for systemic ASC responses. These observations should be relevant to the development of vaccines against sexually transmitted diseases such as that caused by human immunodeficiency virus.     

Intestinal resistance to cholera toxin in mouse. Antitoxic antibodies and desensitization of adenylate cyclase

Mice were immunized perorally with cholera toxin (CT), cholera B-subunit (CB), or buffer as control. The response of anti-CT antibodies of the IgG, IgA and IgM class in bile, IgA being predominating, were similar in both immunized groups. The same number of anti-CT containing plasma cells (ACC) were determined in the intestinal lamina propria of CT - as well as of CB-immunized mice 20 days after the last immunization, while ACC at day 4 in the CB group were 50% higher than in the CT group. In contrast to the vigorous antibody response to CT in both groups of immunized mice, only animals immunized with CT displayed resistance to CT-induced intestinal hypersecretion and to CT stimulation of adenylate cyclase. The CB-treated group responded to CT with fluid accumulation and enzyme activation similar to controls. The results suggest that intestinal resistance to CT in mouse is due to desensitization of adenylate cyclase rather than to CT-neutralizing antibodies.