Flagellin expressed by live Salmonella vaccine strains induces distinct antibody responses following delivery via systemic or mucosal immunization routes

Salmonella flagellin, expressed as flagella in live attenuated vaccine strains, elicits distinct systemic (IgG) and secreted (IgA) antibody responses in mice following delivery via mucosal (nasal/oral) or parenteral (intraperitoneal (i.p.)) immunization routes. Reduced flagellin-specific antibodies were detected either systemically or locally following delivery of flagellated derivatives of aroA Salmonella enterica serovar Dublin SL1438 via the nasal route, the most effective mucosal site for activation of immune responses in mice. In contrast, flagellin represents the most potent Salmonella antigen for the generation of specific serum antibody (IgG) responses following i.p. inoculations. The distinct immunogenic properties of Salmonella flagellin could not be ascribed to deficient colonization, reduced invasive ability or loss of the flagellin expression by the flagellated vaccine strains.     

Immunoglobulin immobilized liposomal constructs for transmucosal vaccination through nasal route

The aim of the present investigation was to evaluate the prospective of surface-engineered vesicular carriers for mucosal immunization via the nasal route. IgG antibody was immobilized on the surface of hepatitis B surface antigen (HBsAg) antigen–loaded liposomes. The developed formulations were characterized on the basis of physicochemical parameters, such as morphology, particle size, polydispersity index, entrapment efficiency, and zeta potential. Liposomal formulations were then evaluated for in-process antigen stability and storage stability. In vivo studies were conducted to visualize targeting potential, localization pattern, and immunogenicity. In addition, immune response was compared with alum-HBsAg vaccine injected intramuscularly. The serum anti-HBsAg titer, obtained from the postnasal administration of IgG-coupled liposomes, was significantly higher than plain liposomes. Moreover, IgG-coupled liposomes generated both humoral (i.e., systemic and mucosal) and cellular immune responses upon nasal administration, while the alum-adsorbed antigen displayed neither cellular (cytokine level) nor mucosal (IgA) response. The formulation also displayed enhanced transmucosal transport, improved in vitro stability, and effective immunoadjuvant property. To conclude, IgG antibody-coupled liposomes may serve as novel carriers to augment the secretory immune response of antigen encapsulated in the liposomes, apparently by escalating liposome uptake via M cells, thereby rationalizing their use as a carrier adjuvant for nasal subunit vaccines.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza A virus

The antiviral neuraminidase inhibitor oseltamivir (OSV) is widely used to suppress viral replication in the treatment of influenza. Here, we report that OSV administration significantly suppressed respiratory mucosal secretory IgA responses with respect to antigen (Ag)-specific antibody (Ab) production and also the induction of Ag-specific IgA Ab-forming cells, but not systemic IgG responses, in weanling mice as a model of pediatric influenza. Neutralizing activities of the airway fluids in oral OSV-treated mice were significantly less than those of sham-treated mice. Our findings suggest the risk of re-infection in patients showing a low mucosal response following OSV treatment.     

Serum antibody response and nasal lymphoid tissue (NALT) structure in the absence of IL-4 or IFN-gamma

Immunization via the nasal route is effective for inducing not only mucosal immunity but also antibody (Ab) response in serum. Nasal lymphoid tissue (NALT) is important for induction of systemic immunity. It remains controversial which T effector cell response is important for serum Ab response after nasal immunization. We investigated serum Ab responses and NALT structures in interleukin (IL)-4 gene targeted (IL-4(-/-)) and interferon (IFN)-gamma gene targeted (IFN-gamma(-/-)) mice. Mice were immunized via nostrils with ovalbumin (OVA) and cholera toxin as adjuvant and serum Ab titers were measured 1 week after final antigen challenge. OVA-specific IgG titers in sera of IL-4(-/-) mice indicated a Th(1) type response, whereas titers in IFN-gamma(-/-) mice and wild-type mice indicated a Th(2) type response. Enhanced serum Ab responses were observed in IL-4(-/-) mice but not IFN-gamma(-/-) mice. OVA-specific Ab-forming cells were detected in the cervical draining lymph nodes but were rare or absent in and around the NALT of all strains of mice. Numbers of OVA-specific Ab-forming cells in cervical lymph nodes were significantly higher in IL-4(-/-) mice than in wild-type and IFN-gamma(-/-) mice. Germinal centers of lymphoid follicles were present in NALT of IL-4(-/-) and other mice. Immunohistochemistry for B and T cell markers revealed that NALT of all mice had approximately the same cellular compositions. Although the absence of IL-4 had no effect on NALT structure, IL-4 may suppress induction of serum Ab responses by nasal immunization.     

Antibodies in the intestinal secretions of rats. Primary and secondary responses to polymeric flagellin.

The antibody responses in serum and secretions obtained from the mucosal surfaces of the small intestine of rats immunized by a parenteral and intestinal route have been compared. Though no significant differences in the mean serum titres were found, the responses of animals immunized via the latter route to large doses of antigen were far less uniform. Apart from the first few days of the primary response, antibody activity was found in three major immunoglobulin classes (IgG2, IgA and IgM), irrespective of the route of immunization. Significant antibody activity appeared in the intestinal surface secretions only after two injections of antigen. In rats immunized parenterally the activity was found only in the IgG2 component. Whilst activity was found in both IgG2 and IgA fractions of the secretions obtained from intestinally immunized rats, it was predominantly of the IgG2 class. The possible significance of this observation is discussed.     

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Adjuvant synergy: the effects of nasal coadministration of adjuvants

Modern peptide and protein subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. However, none of the currently licensed adjuvants can elicit cell-mediated immunity or are suitable for mucosal immunization. In this study we explored the immunological effect of nasal co-administration of adjuvants with distinct functions: cholera toxin subunit B, a potent mucosal adjuvant that induces strong humoral responses, muramy di-peptide (MDP), an adjuvant known to elicit cell mediated immunity but rarely used nasally, and chitosan, an adjuvant that achieves specific physiological effects on mucosal membranes that improve antigen uptake. Groups of five female BALB/c mice received on days 1 and 56 nasal instillations of the recombinant Helicobacter pylori antigen urease admixed to single or multiple adjuvant combinations. Serum IgG kinetics were followed over 24 weeks. At the conclusion of the experiment, local antibody responses were determined and antigen-specific recall responses in splenocyte cultures were assayed for proliferation and cytokine production. The combination of adjuvants was shown to further contribute to the increased antigenicity of recombinant H. pylori urease. The data presented here outline and support facilitation of increased immunomodulation by an adjuvant previously defined as an effective mucosal adjuvant (chitosan) for another adjuvant (MDP) that is not normally effective via this route.     

Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice.

The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.     

DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic

Background

Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance.

Principal Findings

Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance.

Conclusion

Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.     

Impairment of mucosal immunity by total parenteral nutrition: requirement for IgA in murine nasotracheal anti-influenza immunity

Secretory IgA (SIgA) is the primary mucosal Ig and has been shown to mediate nasotracheal (NT) mucosal immunity in normal immune BALB/c mice. This finding has been challenged by a report of NT immunity without IgA in knockout mice, suggesting that IgA may not be necessary for the protection of mucosal surfaces. Although other protective mechanisms may become active in the congenital absence of SIgA, these mechanisms are not the primary means of protection in normal mice. In this paper we show that feeding chemically defined total parenteral nutrition (TPN) to genetically normal, immune ICR mice by the i.v. route results in loss of nasal anti-influenza immunity and a significant drop in influenza-specific SIgA in the upper respiratory tract compared with chow-fed mice (p < 0.005), while the serum influenza-specific IgG titer is unaffected. Loss of upper respiratory tract mucosal immunity is not related to serum Ab, because 10 of 13 TPN-fed mice shed virus into their nasal secretions despite adequate serum anti-influenza IgG titers. The number of IgG Ab-secreting cells in the nasal passages and spleens of TPN-fed mice was unaffected, while both the number and the percentage of splenic IgA-secreting cells were decreased relative to those in chow-fed animals. The loss of immunity is due to the route of nutrition, not the composition of the diet, because TPN solution fed orally via gastrostomy instead of i.v. maintains NT anti-influenza mucosal immunity. We hypothesize that delivery of nutrition via the gut triggers the release of gastrointestinal neuropeptides necessary for maintenance of the mucosal immune system.     

Differential induction of mucosal and systemic antibody responses in women after nasal,rectal, or vaginal immunization: influence of the menstrual cycle

A cholera vaccine containing killed vibrios and cholera toxin B subunit (CTB) was used to compare mucosal immunization routes for induction of systemic and mucosal Ab. Four groups of women were given three monthly immunizations by the rectal immunization (R(imm)) route, nasal immunization (N(imm)) route, or vaginal immunization route during either the follicular (V-FP(imm)) or luteal (V-LP(imm)) menstrual cycle phase. N(imm) was performed with 10-fold less vaccine to determine if administration of less Ag by this route can, as in rodents, produce mucosal Ab responses comparable to those induced by higher dose R(imm) or vaginal immunization. Concentrations of Ab induced in sera and secretions were measured by ELISA. None of these routes produced durable salivary Ab responses. N(imm) induced greatest levels of CTB-specific IgG in sera. R(imm) failed to generate CTB-specific IgA in genital tract secretions. N(imm), V-FP(imm), and V-LP(imm) all produced cervical CTB-specific IgA responses comparable in magnitude and frequency. However, only V-FP(imm) induced cervical IgA2-restricted Ab to the bacterial LPS vaccine component. V-FP(imm), but not V-LP(imm), also induced CTB-specific IgA in rectal secretions. N(imm) was superior to V-FP(imm) for producing rectal CTB-specific IgA, but the greatest amounts of CTB-specific IgA and LPS-specific IgA, IgG, and IgM Ab were found in rectal secretions of R(imm) women. These data suggest that in women, N(imm) alone could induce specific Ab in serum, the genital tract, and rectum. However, induction of genital tract and rectal Ab responses of the magnitude generated by local V-FP(imm) or R(imm) will likely require administration of comparably high nasal vaccine dosages.     

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.     

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.     

Induction of antigen-specific immune responses by oral vaccination with Saccharomyces cerevisiae expressing Actinobacillus pleuropneumoniae ApxIIA

An effective way of inducing both mucosal and systemic immune responses to protect against Actinobacillus pleuropneumoniae serotype 2 Korean isolate was examined in mice by oral immunization using Saccharomyces cerevisiae expressing the ApxIIA protein. The immunogenicity of the yeast-derived ApxIIA antigen was confirmed by the challenge test and ApxIIA-specific IgG antibody response assay. The group subcutaneously immunized with the protein extracted from the yeast expressing ApxIIA showed a higher survival rate after challenging with A. pleuropneumoniae serotype 2 isolate and IgG antibody level in serum than the group injected with that prepared from the yeast harboring vector only. Feeding the yeast expressing ApxIIA to mice induced both systemic and mucosal immune responses against the antigen. ApxIIA-specific IgA antibody titers and the number of IgA-secreting cells of mice vaccinated with S. cerevisiae expressing ApxIIA dose-dependently increased from the third immunization in both intestine and lung (P<0.01). A similar tendency of ApxIIA-specific IgG antibody responses was observed in the sera. The protective efficacy of the oral immunization was then evaluated by a challenge with a minimal lethal dose (MLD, 4.5 x 10(7) CFU/ml) of the A. pleuropneumoniae serotype 2 isolate. Fifty percent of the 30 mg administered group and 30% of the 15 mg administered group survived while none of the mice in the control groups survived after 36 h. These results suggest that feeding animals the yeast expressing the antigen can be an effective strategy to induce protective immune responses against A. pleuropneumoniae infection.     

Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100- Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Mucosal inoculation of Lactobacillus expressing hCGbeta induces an anti-hCGbeta antibody response in mice of different strains

To show that an anti-human chorionic gonadotrophin-beta (hCGbeta) antibody response can be induced by inoculating Lb. expressing hCGbeta through different mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 10(8), 10(9), and 10(10)Lb.hCGbeta (a recombinant Lactobacillus expressing hCGbeta). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGbeta IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGbeta antigen-specific antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 10(9) and 10(10)Lb.hCGbeta inoculations induced similar anti-hCGbeta antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 10(9) and 10(10)Lb. hCGbeta was able to neutralize more than 100 ng/ml hCG antigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P<0.05). The numbers of anti-hCGbeta IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGbeta antibody levels in the reproductive tract.     

The ovine nasal mucosa: an alternative tissue site for mucosal immunization

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.