Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit

The level and distribution of isotype-specific antibodies in various secretions and of antibody-secreting cells in corresponding lymphoid organs and tissues were compared in mice immunized with Streptococcus mutans surface protein antigen I/II (AgI/II) conjugated to the cholera toxin B subunit (CTB), given intranasally (i.n.) or intragastrically (i.g.), with or without free cholera toxin (CT) as an adjuvant. Immunization i.n. induced stronger initial antibody responses to AgI/II in both serum and saliva than immunization i.g., but salivary immunoglobulin A (IgA)-specific antibody responses to immunization about 3 months later were not increased relative to total salivary IgA concentrations. Specific antibodies induced by i.n. immunization were as widely distributed in serum, saliva, tracheal wash, gut wash, and vaginal wash as those induced by i.g. immunization. Likewise, specific antibody-secreting cells were generated in the spleen, salivary glands, intestinal lamina propria, and mesenteric and cervical lymph nodes by either route of immunization. The strongest salivary IgA antibody response was induced by AgI/II-CTB conjugate given i.n., but the addition of CT did not further enhance it. However, free CTB could effectively replace CT as an adjuvant in i.n. immunization with unconjugated AgI/II. Booster i.n. immunization with AgI/II plus either free CT or CTB induced stronger recall serum antibody responses than conjugated AgI/II-CTB with or without CT as an adjuvant. Therefore, i.n. immunization with a protein antigen and free or coupled CTB is an effective means of generating IgA antibody responses expressed at several mucosal sites where protective immunity may be beneficial.     

Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin

We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA). Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages. Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes. These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.     

Systemic and mucosal immune responses of mice to aluminium-adsorbed or aluminium-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit

For the purpose of changing the immunization procedure of tetanus toxoid from intramuscular or subcutaneous injection, which has been in practice for a long time, to intranasal administration, we examined systemic and mucosal immune responses of mice to aluminium-adsorbed tetanus toxoid (aTT) and aluminium-non-adsorbed tetanus toxoid (nTT) inoculated intranasally with recombinant cholera toxin B subunit (rCTB). Intranasal immunization with aTT induced, at a concentration of 0.5 Lf, high levels of TT-specific serum IgG antibody titres and moderate levels of TT-specific serum IgA antibody titres in the presence and absence of rCTB. Induction of high or moderate levels of mucosal TT-specific IgA antibody responses was observed with and without rCTB in the lung, the nasal cavity, the small and large intestines and the vagina. Generally speaking, the co-administration of aTT and rCTB showed higher mucosal TT-specific IgA antibody titres when compared with the administration of aTT alone. In case of intranasal administration of nTT, the dose of 5 Lf was necessary and stimulated, only in the presence of rCTB (10 micrograms), high levels of tetanus toxoid (TT)-specific serum IgG antibody responses in all mice examined and moderate or slight levels of TT-specific IgA antibody responses in the nasal, pulmonary and small and large intestinal lavages of a few mice. All mice intranasally immunized with aTT alone or nTT and rCTB escaped onset of tetanus. This is the first report concerned with the mucosal adjuvant activity of an aluminium compound. Judging from these results, intranasal administration of aTT with and without rCTB or nTT with rCTB appears to be a very useful means for a vaccination against tetanus with respect to ease, safety, certainty, low cost and no need for an injection needle.     

Cholera toxin and cholera toxin B subunit induce IgA switching through the action of TGF-beta 1

Cholera toxin (CT) and its B subunit (CTB) are potent immunogens and adjuvants that, either alone or linked to protein Ags, can stimulate mucosal immune responses, modulate the induction of oral tolerance, and stimulate IgA isotype switching. The present studies addressed the mechanisms by which CT and CTB promote IgA switching. CT and rCTB, in the presence of IL-2, significantly increased IgA isotype switching at the clonal level in populations of purified and LPS-activated murine surface IgA- spleen B cells, as determined by ELISA, enzyme linked immunospot assays, and limiting dilution analysis. The IgA stimulatory effects of CT and CTB were independent of the A subunit of CT. CTB and CT did not increase the secretory rate of IgA-producing cells or the clonal burst size of IgA clones, and did inhibit B cell growth. Because TGF-beta 1 also inhibits B cell growth and promotes IgA switching, further studies tested whether the activity of CTB and CT on IgA isotype switching was mediated through TGF-beta 1. Anti-TGF-beta Ab and soluble TGF-beta 1 type IIR inhibited CTB- and CT-stimulated IgA isotype switching. Furthermore, increased TGF-beta 1 mRNA levels and bioactive TGF-beta 1, within a range shown to induce IgA isotype switching, were detected in cultures of surface IgA- B cells stimulated with CT or CTB and IL-2. These data indicate that CTB- and CT-stimulated IgA isotype switching are mediated through TGF-beta 1. The finding that CTB up-regulates TGF-beta 1 activity has important implications for understanding the mechanisms by which CTB promotes both IgA mucosal immunity and oral tolerance.     

Novel mucosal immunization with polysaccharide-protein conjugates entrapped in alginate microspheres

A novel mucosal immunization was examined using biocompatible and biodegradable alginate microspheres containing a conjugate of polysaccharide antigen and cholera toxin B subunit (CTB). In order to prepare the alginate microspheres with diameters of less than 5 microm, a new diffusion-controlled interfacial gelation technique was developed. Also, in order to improve the mucosal immune response, a pneumococcal capsular polysaccharide type 19 (PS19) was conjugated to the CTB (PS19-CTB). This conjugate was subsequently encapsulated into the alginate microspheres. The loading content of PS19-CTB to the alginate microspheres was 60%. An in vitro sustained release pattern was observed with the antigen-loaded microspheres, showing 80% antigen release within one day. Mucosal and systemic immunities following oral immunization with the alginate microspheres were studied. Balb/c mice were immunized perorally three times at intervals of two weeks. Peroral immunization with 25 microg of PS19-CTB entrapped in the alginate microspheres evoked both the mucosal IgA and systemic IgM responses to PS19 in small intestine and in sera, respectively. The results suggest that both the mucosal and systemic antibody responses could be induced by oral administration of the PS19-CTB antigen entrapped in alginate microspheres.     

Antibodies and antibody-secreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.     

A comparison of natural and recombinant cholera toxin B subunit as stimulatory factors in intranasal immunization

Cholera toxin B (CTB) is often envisaged and used as an immune stimulating agent in protocols for mucosal immunization. However, the nature of the CTB used (natural vs recombinant) is frequently not taken in consideration. This is important since the usage of natural CTB in mucosal immunization regimen and the mucosal response resulting from such an immunization can be effected by the presence of the CTA subunit in commercial CTB preparations. To clarify this, we have compared natural vs recombinant CTB in an intranasal (i.n.) mucosal immunization procedure using ovalbumin (OVA) as antigen. The results show that recombinant CTB induces similar immune responses like natural CTB. Furthermore, our experiments show that covalent coupling of OVA to CTB is not required for the induction of OVA specific mucosal and systemic immune responses upon i.n. immunization.     

Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.     

Antibody responses in serum and lung to intranasal immunization with Haemophilus influenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid

AIM: Cholera toxin B subunit (CTB) has previously been used as a mucosal carrier for various vaccine candidate antigens. The objective of this study was to see if coupling a bacterial polysaccharide, Haemophilus influenzae type b capsular polysaccharide (HibCPS), to CTB, either directly or through prior coupling to tetanus toxoid (TT), would improve the immunogenicity of HibCPS after nasal immunization. METHODS: HibCPS was conjugated to CTB, TT or via TT to CTB, using glutaraldehyde or 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The conjugates were characterized and used for intranasal (IN) and subcutaneous (SC) immunizations of mice. The anti-Hib, -TT and -CTB antibody titers in serum and lungs after the immunizations were measured with ELISA. RESULTS: The HibCTB was poorly immunogenic both given IN and SC compared with HibTT and HibTTCTB, probably because of inefficient coupling. In contrast, the conjugation of CTB to the HibTT conjugate resulted in a preparation which was superior both to the HibTT and the HibCTB conjugates in inducing local IgA and IgG anti-HibCPS antibodies in the lungs. The anti-HibCPS serum IgG titers after IN immunization with the HibTTCTB conjugate were similar to the titers after IN immunization with HibTT, or SC immunization with a commercial HibCRM conjugate vaccine. In contrast to the other conjugates, the HibTTCTB conjugate also gave rise to anti-Hib serum IgA titers. CONCLUSION: We conclude that appropriate conjugation to CTB increases the mucosal immunogenicity of HibCPS, and that intranasal immunization with such a conjugate can give rise to both local and systemic anti-HibCPS antibody responses.     

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

Receptor-dependent immune responses in pigs after oral immunization with F4 fimbriae

F4 receptor-positive (F4R+) and F4 receptor-negative (F4R-) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R+ animals, whereas immune responses were not detected in F4R- animals. Even after a subsequent oral infection with virulent F4(+) ETEC and a booster immunization with F4, the F4R- animals remained F4 seronegative whereas the unvaccinated F4R+ pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R+ pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R+ animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R+ pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Oral administration of an antigenic synthetic lipopeptide (MAP-P3C) evokes salivary antibodies and systemic humoral and cellular responses

Parenteral immunization with a tetravalent multiple antigen peptide (MAP) containing a gp120 sequence coupled to a synthetic lipophilic moiety (MAP-P3C) has been previously found to produce systemic antibody and cellular responses in mice. This study demonstrates that oral administration of MAP-P3C induced IgA antibodies in mucosal secretions and protein-specific IgG in the sera of the immunized mice. Moreover, intragastric delivery of MAP-P3C generated systemic T-lymphocyte stimulation and specific cytotoxic T-lymphocyte (CTL) activity. The CTL response was eliminated by treatment with CD8-specific antibody plus complement and was MHC class I-restricted. Therefore, presentation of lipid-linked synthetic peptides to the intestinal mucosal surface is effective in initiating humoral and cellular immunity both at mucosal sites and systemically.     

Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans

Numerous experiments performed in humans and animals have revealed that stimulation of mucosal lymphoid inductive sites such as intestinal Peyer's patches results in parallel immune responses manifested by the appearance of S-IgA antibodies in the external secretions of remote glands. However, recent experiments suggest that inductive sites associated with the upper respiratory tract, rectum, and perhaps genital tract may also function as sources of lymphoid cells that populate, with some selectivity, certain remote mucosal effector sites. Furthermore, antigen-specific IgA antibodies can be induced in certain secretions (e.g., female genital tract) not only by immunization in the vicinity of corresponding mucosal tissues (e.g., vagina and rectum) but also by oral and especially intranasal immunization. The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for effective delivery systems that would (a) increase the antigen absorption, (b) prevent its degradation, and (c) skew the outcome of immunization to a desired goal (protective response to infectious diseases vs. tolerance; B vs. T cell responses; mucosal vs. systemic). The induction of immune responses at a desired mucosal site can be accentuated with the use of a suitable antigen-delivery system including relevant bacterial or viral vectors, edible transgenic plants expressing microbial antigens, incorporation of antigens in biodegradable microspheres or liposomes, and linkage or coadministration of antigens with cholera toxin B subunit. However, only a few antigen-delivery systems extensively used in animal experimentation have been evaluated for their efficacy in humans. The combination of various immunization routes and the use of suitable antigen-delivery systems may accomplish an important task-the induction of mucosal immune responses at a location relevant to the site of entry of a given pathogen.     

Suppression of smooth muscle cell proliferation after experimental PTFE arterial grafting: a role for polyclonal anti-basic fibroblast growth factor (bFGF) antibody

The reciprocal regulation of T-helper cell (Th) subsets is widely documented in various animal models of infectious diseases. In this study IFN-gamma/IL-4 double knockout (DKO) mice were used to analyse the role of Th subsets in mucosal immune responses. We found that the DKO mice had normal IgA differentiation but impaired induction of specific gut mucosal antibody responses after oral immunization using cholera toxin adjuvant. Both Th1 and Th2 responses were reduced compared with wild-type mice. Despite the absence of both IFN-gamma and IL-4 in the DKO mice the overall results were similar to previous observations in IFN-gamma receptor-knockout (IFN-gammaR-/-) mice and did not suggest a strict cross-regulation of the two Th subsets in the gut mucosa. To further examine the role of IFN-gamma in mucosal immunity we compared two different mouse strains lacking IFN-gamma, i.e. IFN-gamma-/- (C57BL/6) and IFN-gammaR-/- mice (129/Sv). We found that IFN-gammaR-/- mice exhibited reduced mucosal antibody responses and decreased Th1 and Th2 activity after oral immunization, while IFN-gamma-/- mice had intact antibody responses and increased Th2 responses. Thus, genetic differences were found to critically affect the development of a specific gut mucosal immune response. An enhanced Th2 activity in the Peyer's patches following oral immunization was associated with an ability to mount strong intestinal IgA immunity.     

Tacrolimus therapy for persistent or recurrent acute rejection after lung transplantation

Purified native F1 antigen from Yersinia pestis was used to assess controlled-release vaccine delivery systems in poly(lactide-co-glycolide) (PLG) microparticles and liposomes. Antigen encapsulated in PLG microparticles induced high serum titres when injected i.p. in mice: mucosal IgA was also detected. Mice immunized with F1 in Alhydrogel or PLGs were protected against subcutaneous challenge with Y. pestis. F1 antigen surface-labelled onto liposome vesicles stimulated high serum titres in Balb/c mice and also induced a mucosal response: F1-labelled liposomes protected mice against challenge with up to 1 x 10(5) organisms. These findings indicate that a significant immune response is induced by immunizing with F1 formulated in PLGs and liposomes and that protection was achieved after only one dose.     

Intranasal immunization confers protection against murine Pneumocystis carinii lung infection

To evaluate the feasibility of mucosal immunization against Pneumocystis carinii (Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups (P < 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.     

Controlled lipidation and encapsulation of peptides as a useful approach to mucosal immunizations

To generate a useful strategy for mucosal immunization, we have developed an approach of lipidating a multiple Ag peptide (MAP) containing part of the V3 loop from HIV-1 gp120IIIB. In this work, we compare two delivery systems, lipidated MAP in PBS and encapsulation in poly(DL-lactide-co-glycolide) microparticles. Subcutaneous immunization, followed by intragastric administration of MAP peptide entrapped or not entrapped in microparticles, induced mucosal and systemic immune responses at local and distant sites, including mucosal IgA in saliva, vaginal secretions and feces, and IgG in blood. However, lipidated Ag delivered in microparticles induced higher levels of mucosal Abs, particularly of intestinal IgA, and generated CTL responses. In contrast, lipidated MAP delivered by nasal route microparticles was less effective in inducing CTL responses. These results demonstrate the feasibility of using a lipidated multimeric peptide for mucosal immunization to stimulate both systemic and mucosal immune systems, including the genital tract, irrespective of the route or method of delivery and without requiring the use of a carrier or an extraneous adjuvant.     

Analysis of the roles of antilipopolysaccharide and anti-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo

Secretory immunoglobulin A (IgA) antibodies (sIgA) directed against cholera toxin (CT) and surface components of Vibrio cholerae are associated with protection against cholera, but the relative importance of specific sIgAs in protection is unknown. A monoclonal IgA directed against the V. cholerae lipopolysaccharide (LPS), secreted into the intestines of neonatal mice bearing hybridoma tumors, was previously shown to provide protection against a lethal oral dose of 10(7) V. cholerae cells. We show here that a single oral dose of 5 to 50 micrograms of the monoclonal anti-LPS IgA, given within 2 h before V. cholerae challenge, protected neonatal mice against challenge. In contrast, an oral dose of 80 micrograms of monoclonal IgA directed against CT B subunit (CTB) failed to protect against V. cholerae challenge. A total of 80 micrograms of monoclonal anti-CTB IgA given orally protected neonatal mice from a lethal (5-micrograms) oral dose of CT. Secretion of the same anti-CTB IgA antibodies into the intestines of mice bearing IgA hybridoma backpack tumors, however, failed to protect against lethal oral doses of either CT (5 micrograms) or V. cholerae (10(7) cells). Furthermore, monoclonal anti-CTB IgA, either delivered orally or secreted onto mucosal surfaces in mice bearing hybridoma tumors, did not significantly enhance protection over that provided by oral anti-LPS IgA alone. These results demonstrate that anti-LPS sIgA is much more effective than anti-CT IgA in prevention of V. cholerae-induced diarrheal disease.     

Differential kinetics and distribution of antibodies in serum and nasal and vaginal secretions after nasal and oral vaccination of humans

Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well as the number of circulating antibody-secreting cells, before immunization and 1, 2, 3, 6, and 26 weeks thereafter. Nasal vaccination induced 9-fold CTB-specific immunoglobulin A (IgA) and 56-fold specific IgG antibody increases in nasal secretions, whereas no significant IgA increase was seen after oral vaccination. Both oral and nasal vaccination resulted in 5- to 6-fold CTB-specific IgA and 20- to 30-fold specific IgG increases in vaginal secretions. Strong serum responses to CTB were also induced by both routes of vaccination. A notable difference between nasal and oral vaccination was that the nasal route elicited a specific antibody response with a later onset but of much longer duration than did the oral route. We conclude from this study that the nasal route is superior to the oral route for administering at least nonliving vaccines against infections in the upper respiratory tract, whereas either oral or nasal vaccination might be used for eliciting antibody responses in the female genital tract.