藤橋 浩太郎

Cellular and molecular mechanisms of the immune system influencing oral bone metabolism remain to be elucidated. In this study, we characterize the mucosal cytokine production by CD4⁺ T cells in the inflamed gingiva of mice infected with Porphyromonas gingivalis (P. gingivalis). A murine periodontal disease model with alveolar bone loss showed significant levels of FimA-specific salivary secretory IgA and plasma IgG Ab responses. Further, IL-6 and TNF-α production was elevated when compared with sham-infected mice. The frequencies of Th1 (IFN-γ), Th2 (IL-4) and Th17 (IL-17) producing CD4⁺ T cells were also increased in P. gingivalis infected mice. Among these cytokines, a significantly higher frequency of IFN-γ producing CD4⁺ T cells was noted. The kinetics of intracellular cytokine analyses revealed a significantly increased frequency of IFN-γ-, IL-4- and IL-17-producing CD4⁺ T cells one day after infection. Further, the frequency of IFN-γ producing CD4⁺ T cells was maintained throughout the experimental period. Although IL-4 and IL-17 production was intact until 15 days after the infection, the numbers of CD4⁺ T cells producing these cytokines were reduced thereafter. These results indicate that Th1-type CD4⁺ T cells play distinct roles in the induction and regulation of periodontal disease when compared with Th2- and Th17-type cytokine-producing CD4⁺ T cells.

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. (C) 2010 Elsevier Masson SAS. All rights reserved.

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. © 2010 Elsevier Masson SAS.

This study was designed to investigate whether secretory-IgA (S-IgA) Abs induced by a pneumococcal surface protein A(PspA)-based nasal vaccine are necessary for prevention of streptococcal colonization. Mice nasally immunized with PspA plus a plasmid expressing Flt3 ligand (pFL) cDNA as a mucosal adjuvant showed significantly higher levels of PspA-specific S-IgA and IgG Ab responses in both plasma and nasal washes when compared with naive mice. Although IgA(-/-) mice given nasal PspA plus pFL had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in nasal washes and nasal passages. In contrast, vaccinated wild-type mice showed essentially no bacteria in the nasal cavity. Further, a nasal vaccine consisting of PspA plus pFL effectively reduced pre-existing Streptococcus pneumoniae in the nasal cavity. These results show that PspA-based vaccine-induced specific S-IgA Abs play a necessary role in the regulation of S. pneumoniae colonization in the nasal cavity. The Journal of Immunology, 2010, 185: 1755-1762.

P>The aim of this study was to evaluate the efficacy of an oral vaccine containing the 40-kDa outer membrane protein of Porphyromonas gingivalis (40K-OMP) and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) to control oral infection by P. gingivalis. Oral immunization with 40K-OMP plus CpG ODN induced significant 40K-OMP-specific serum immunoglobulin G (IgG), IgA, and saliva IgA antibody responses. The 40K-OMP-specific CD4+ T cells induced by oral 40K-OMP plus CpG ODN produced both T helper type 1 (Th1; interferon-gamma) and Th2 (interleukin-4) cytokines. Furthermore, increased frequencies of CD11c+ B220+ dendritic cells (DCs) and CD11c+ CD11b+ DCs with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II molecules were noted in spleen, Peyer's patches, and cervical lymph nodes. Immunized mice were then infected orally with P. gingivalis to determine whether the immune responses induced by oral 40K-OMP plus CpG ODN were capable of suppressing the bone resorption caused by P. gingivalis infection. Mice given 40K-OMP plus CpG ODN showed significantly reduced bone loss associated with oral infection by P. gingivalis. Oral administration of 40K-OMP together with CpG ODN induces Th1-type and Th2-type cells, which provide help for protective immunity against P. gingivalis infection. This may be an important tool for the prevention of chronic periodontitis.

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyer's patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naive, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c(+) dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-beta, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c(+) DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

We have previously shown that one of the minimal active regions of statherin, a human salivary protein, for binding to Fusobacterium nucleatum is a YQPVPE amino acid sequence. In this study, we identified the FomA protein of F. nucleatum, which is responsible for binding to the statherin-derived YQPVPE peptide. Overlay analysis showed that a 40-kDa protein of the F. nucleatum cell envelope (40-kDa CE) specifically bound to the YQPVPE peptide. The equilibrium association constant between the affinity-purified 40-kDa CE and the YQPVPE peptide was 4.30 x 10(6). Further, the purity and amino acid sequence analyses of the purified 40-kDa CE revealed approximately 98.7% (wt/wt) purity and a high degree of homology with FomA, a major porin protein of F. nucleatum. Thus, a FomA-deficient mutant failed to bind to the YQPVPE peptide. In addition, increased levels of a FomA-specific mucosal IgA antibody (Ab) and plasma IgG and IgA Abs were seen only in mice immunized nasally with cholera toxin (CT) and the purified 40-kDa FomA protein. Interestingly, saliva from mice that received FomA plus CT as a mucosal adjuvant nasally prevented in vitro binding of F. nucleatum to statherin-coated polyvinyl chloride plates. Taken together, these results suggest that induction of specific immunity to the 40-kDa FomA protein of F. nucleatum, which specifically binds to the statherin-derived peptide, may be an effective tool for preventing the formation of F. nucleatum biofilms in the oral cavity.

A T cell receptor transgenic mouse line reactive to a microbiota flagellin, CBir1, was used to define mechanisms of host microbiota homeostasis. Intestinal IgA, but not serum IgA, was found to block mucosal flagellin uptake and systemic T cell activation in mice. Depletion of CD4(+)CD25(+) Tregs decreased IgA(+) B cells, total IgA, and CBir1-specific IgA in gut within days. Repletion of T cell-deficient mice with either CD4(+)CD25(+) or CD4(+)foxp3(+) Tregs restored intestinal IgA to a much greater extent than their reciprocal CD4(+) subsets, indicating that Tregs are the major helper cells for IgA responses to microbiota antigens such as flagellin. We propose that the major role of this coordinated Treg-IgA response is to maintain commensalism with the microbiota.

Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system. (C) 2009 Elsevier Ltd. All rights reserved.

The eye is protected by the ocular immunosurveillance system. We show that tear duct associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/ differentiation 2, retinoic acid-related orphan receptor gamma t, lymphotoxin (LT) alpha 1 beta 2-LT beta R, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

In order to establish effective mucosal immunity against various mucosal pathogens, vaccines must be delivered via the mucosal route and contain effective adjuvant(s). Since mucosal adjuvants can simply mix with the antigen, it is relatively easy to adapt them for different types of vaccine development. Even in simple admixture vaccines, the adjuvant itself must be prepared without any complications. Thus, CpG oligodeoxynucleotides or plasmids encoding certain cDNA(s) would be potent mucosal adjuvant candidates when compared with other substances that can be used as mucosal adjuvants. The strategy of a DNA-based mucosal adjuvant facilitates the targeting of mucosal dendritic cells, and thus is an effective and safe approach. It would also provide great flexibility for the development of effective vaccines for various mucosal pathogens.

Although native cholera toxin (CT) is an extremely effective adjuvant, its toxicity prevents its use in humans. We report here that apple polyphenol extract (APE), obtained from unripe apples, reduces CT-induced morphological changes and cAMP accumulation. Based upon this finding, we have attempted to design a novel, effective and safe mucosal vaccine by using CT with several dosages of APE as nasal adjuvants. Mice nasally immunized with OVA plus CT and an optimal dosage of APE showed significantly reduced levels of inflammatory responses as well as total and OVA-specific IgE antibodies when compared with mice given without APE. However, levels of both mucosal and systemic OVA-specific antibody responses were maintained. Further, APE significantly down-regulated accumulation of CT in the olfactory nerves and epithelium. In summary, an optimal dosage of APE would take full advantage of mucosal adjuvanticity of native CT without any toxicity for application in humans. (C) 2009 Elsevier Ltd. All rights reserved.

All jawed vertebrates limit use of D(H) reading frames (RFs) that are enriched for hydrophobic amino acids. In BALB/c mice, DFL16.1 RF2 encodes valine and isoleucine. To test whether increased use of RF2 affects B cell function, we examined B cell development and Ab production in mice with an IgH allele (Delta D-D mu FS) limited to use of a single, frameshifted DFL61.1 gene segment. We compared the results of these studies to wild-type mice, as well as those previously obtained in mice limited to use of either a single normal D(H) or a single inverted D(H) that forces use of arginine in CDR-H3. All three of the mouse strains limited to a single D(H) produced fewer immature B cells than wild type. However, whereas mice limited to a single normal D(H) achieved normal B cell numbers in the periphery, mice forced to preferentially use RF2 had reduced numbers of mature B cells in the spleen and bone marrow, mirroring the pattern previously observed in mice enriched for charged CDR-H3s. There were two exceptions. B cells in the mice using RF2 normally populated the marginal zone and peritoneal cavity, whereas mice using inverted RF1 had increased numbers of marginal zone B cells and decreased numbers of B1a cells. When challenged with several T-dependent or T-independent Ags, Ag-specific Ab titers in the mice forced to use RF2 were altered. These findings indicate that B cell development and Ag-specific Ab production can be heavily influenced by the global amino acid content of the CDR-H3 repertoire. The Journal of Immunology, 2008, 181: 8409-8415.

Background & Aims: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. Methods: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-p sigma 1), p sigma 1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined. Results: A low dose of OVA-p sigma 1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-p sigma 1 than those fed p sigma 1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor beta 1 (TGF-beta 1) and interleukin-10 (IL-10) production compared with nontolerized mice. Conclusions: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta 1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.

We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than 25 weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8(+) DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly. (C) 2008 Elsevier Ltd. All rights reserved.

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma 1 (p sigma 1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via p sigma 1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus p sigma 1, termed OVA-p sigma 1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-p sigma 1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-p sigma 1 could be adoptively transferred using cervical lymph, node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta 1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-p sigma 1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta 1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using p sigma 1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Immunological memory induced by nasal immunization with adjuvant-combined influenza vaccine was analyzed in different ages and strains of mice. The memory activities were assessed by secondary nasal-wash IgA and serum IgG antibody (Ab) responses and protection against challenge infection with a lethal dose of influenza virus. Mice were primed with 0.1 mu g of vaccine and boosted with 0. 1 or 1.0 mu g vaccine I (short-term memory)- or 17 (long-term memory)-months later. Influenza-specific short-term memory responses in young adult BALB/c mice (2-month-old) were significantly higher than those of long-term memory activities in mice boosted at 19 months of age. However, those influenza-specific long-term memory responses provided protective immunity against influenza virus challenge and were higher than short-term memory in aged mice primed at 18-month-old and boosted I month later. These results show that the age at which initial nasal immunization is given is critically important in order to induce protective immunity in aged mice. Similar findings were noted in the C3H mouse strain; however, C57/BL/6 mice failed to induce influenza-specific immune responses in both young adult and aged mice. These results indicate that low doses of cholera toxin B subunit (supplemented with 0.2% of hole toxin) combined nasal vaccine may required further improvement in order to provide protective immunity in human use. (C) 2007Elsevier Ltd. All rights reserved.

alpha-Galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of alpha-Ga1Cer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and alpha-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given alpha-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, alpha-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and alpha-GalCer is a safe and effective means of preventing influenza infection. (c) 2007 Elsevier Ltd. All rights reserved.

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. ffigh numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA(+), CD5(+) B cells (B-la B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R a-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4(+) T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ra(+) B-la B cells and IL-5-producing CD4(+) T cells in the mucosal effector lymphoid tissues. The Journal of Immunology, 2007, 178: 6058-6065.

Background One of the pathological hallmarks of Alzheimer's disease (AD) is deposits of amyloid beta-peptide (A beta) in neuritic plaques and cerebral vessels. Immunization of AD mouse models with A beta reduces A beta deposits and improves memory and learning deficits. Because recent clinical trials of immunization with A beta were halted due to brain inflammation that was presumably induced by a T-cell-mediated autoimmune response, vaccination modalities that elicit predominantly humoral immune responses are currently being developed. Methods We have nasally immunized a young AD mouse model with an adenovirus vector encoding 11 tandem repeats of A beta 1-6 fused to the receptor-binding domain (la) of Pseudomonas exotoxin A (PEDI), AdPEDI-(A beta 1-6)(11), in order to evaluate the efficacy of the vector in preventing A beta deposits in the brain. We also have investigated immune responses of mice to AdPEDI-(A beta 1-6)(11). Results Nasal immunization of an AD mouse model with AdPEDI-(A 16)11 elicited a predominant IgG1 response and reduced A load in the brain. The plasma IL-10 level in the AD mouse model was upregulated after immunization and, upon the stimulation with PEDI-(A beta 1-6)(11), marked IL-10 responses were found in splenic CD4(+) T cells from C57BL/6 mice that had been immunized with AdPEDI-(A beta 1-6)(11). Conclusions These results suggest that the induction of Th2-biased responses with AdPEDI-(A beta 1-6)(11) in mice is mediated in part through the upregulation of IL-10, which inhibits activation of dendritic cells that dictate the induction of Th1 cells. Copyright (c) 2007 John Wiley & Sons, Ltd.

Botulism has classically been considered to be a food- and water-borne disease. However, it was recently classified by the US National Institute of Allergy and Infectious Diseases (National Institute of Health) and the US Centers for Disease Control and Prevention as a Category A agent. Thus, the botulinum exotoxin, a neurotoxin, could be easily disseminated by bioterrorists through the air-borne route with a high morbidity and mortality rate. In this regard, a high priority should be given to the development of a safe and effective mucosal vaccine to protect against botulinum neurotoxins (BoNTs) since it is well known that the mucosal immune system is the first line of defense against major pathogens. Further, mucosal immunization has been shown to induce both mucosai and systemic immunity to pathogens. By contrast, the current injection-type vaccine only provides protective immunity in the systemic compartment. Clearly, the development of a safe and effective mucosal vaccine against this toxin should be a high priority. In this regard, it has been shown that both nasal and oral immunization approaches have been taken in order to protect from BoNT intoxication. In this article, we will discuss the importance of the development of a mucosal vaccine against botulinum and introduce current aspects of BoNT mucosal vaccines, which show that they effectively prevent mucosal BoNT intoxication.

Purpose of review Abrogation of mucosal T cell homeostasis by exaggerated not only T helper 1, but also T helper 2 cells is a major problem that leads to intestinal inflammation. In this regard, it is important to understand these different aspects of mucosal inflammation. Recent findings Both T helper 1 and 2 cells play central roles in the induction of mucosal immune responses including secretory IgA antibody production, which would be the most beneficial aspect for the host defense mechanism. T helper 1- and 2-type responses, however, exhibit other roles in the abrogation of intestinal homeostasis. Although it has been shown that T helper 1-type immune responses are key players in the induction of intestinal inflammation in mice colitis models and also in inflammatory bowel diseases in humans, studies in murine colitis models clearly show that T helper 2-type responses are also involved in the pathophysiology of the intestinal inflammation. Both regulatory type T cells and T helper 17 cells are involved to down- or upregulate aberrant T helper 1 and 2 cell responses. Summary Understanding the cellular and molecular mechanisms of crosstalk among T helper 1, 2, 17 and T regulatory 1 cells is central for the prevention or treatment of inflammatory bowel diseases.

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hc beta tre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hc beta tre or Hc beta tre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hc beta tre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hc beta tre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hc beta tre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hc beta tre-dosed mice. Mice immunized with adjuvanted Hc beta tre-Ad2F or Hc beta tre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hc beta tre-Ad2F or Hc beta tre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hc beta tre Ab titers even in the absence of adjuvant. This study shows that the Hc beta tre structure can confer protective immunity and that use of Hc beta tre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study: we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Thl-type cytokine responses by OVA-specific CD4(+) T cells, dmCT E112K/KDEV exhibited significantly lower Thl-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain ( CDR-H3), the center of the classic antigen-binding site. To assess the role of D-H RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single D-H encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in D-H RF1 alters CDR-H3 content and impairs B cell development and antibody production.

Anthrax edema toxin (EdTx) is an AB-type toxin that binds to anthrax toxin receptors on target cells via the binding subunit, protective Ag (PA). Edema factor, the enzymatic A subunit of EdTx, is an adenylate cyclase. We found that nasal delivery of EdTx enhanced systemic immunity to nasally coadministered OVA and resulted in high OVA-specific plasma IgA and IgG (mainly IgG1 and IgG2b). The edema factor also enhanced immunity to the binding PA subunit itself and promoted high levels of plasma IgG and IgA responses as well as neutralizing PA Abs. Mice given OVA and EdTx also exhibited both PA- and OVA-specific IgA and IgG Ab responses in saliva as well as IgA Ab responses in vaginal washes. EdTx as adjuvant triggered OVA- and PA-specific CD4(+) T cells which secreted IFN-gamma and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines.

The immunodeficiency and increased risk of infection seen in elderly patients may be attributable to an alteration in the immune system. Since dendritic cells (DCs) play a key role for the induction of antigen (Ag)-specific immune responses, we hypothesize that upregulation of aged DCs function by flt3 ligand and CpG ODN abrogate for the induction of Ag-specific adjuvant immune responses. Mononuclear cells were isolated from spleen of BALB/c mice (>24 months of age) and were stained with an array of fluorescence-conjugated mAbs in order to determine the frequencies of DCs by fluorescence activated cell sorter (FACS). The spleen from aged mice contained reduced frequencies CD8⁺ DCs and pDCs. Further, aged splenic DCs showed impaired APC function when compared with DCs from young adult mice. To compensate this age-associated alterations, flt3 ligand (FL) and CpG ODN were employed. When aged splenic DCs were cultured with recombinant (r) FL and CpG ODN, significant proliferative responses were induced, which is essentially identical to that of splenic DCs from young mice. Further, aged mice given nasal OVA plus FL plasmid (pFL) and CpG ODN showed high levels of OVA-specific IgG and IgA Abs in plasma which are comparable to those of young adult mice. Thus, the frequencies of CD8⁺ DCs and plasmacytoid DCs in spleen of aged mice were significantly increased after the immunization with pFL and CpG OND. More importantly, aged splenic DCs expressed increased frequencies of the costimulatory molecules. These results showed that a combination of pFL and CpG ODN treatment was an effective strategy to enhance impaired immunity that is seen in aged mice.

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (013). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1 beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer(+) CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer(+) CD8 T cells expressed high levels of granzyme B and IFN gamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, postinfection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer(+) CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.

OVA-induced allergic diarrhea occurs as a consequence of over-expression of Th1 inhibitory IL-12p40 monomers and homodimers in the large intestine, establishing a dominant Th2-type environment. In this study, we demonstrate that intranasally administered murine IL-12p70 naked DNA expression plasmids resulted in the synthesis of corresponding cytokine in the large intestinal CD11c(+) dendritic cells, leading to the inhibition of Ag-specific Th2-type response for the prevention of allergic diarrhea and the suppression of clinical symptoms including OVA-specific IgE Ab synthesis. The nasal IL-12p70 DNA treatment proved effective even after the establishment of allergic diarrhea. These results suggest that the mucosal administration of naked IL-12p70 DNA plasmid should be considered as a possible preventive and therapeutic treatment for Th2 cell-mediated food allergic diseases in the intestinal tract.

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 10(3) LD(50) of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD(50) was determined. When mice were given an oral challenge of 5 mug (2 x oral LD(50)) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.

Studies using the enterotoxin cholera toxin and the related heat-labile toxin I of Escherichia coli as adjuvants have established that effective immunity can be provided in mucosal tissues and the general blood stream by vaccines administered by the oral or nasal route. Despite this knowledge and other recognized advantages, needleless mucosal vaccines rarely appear in the health care market and classic injected vaccines remain the norm. Safety issues related to the adjuvants needed for oral and nasal vaccines are the chief factor that hampers their generalized use in humans. Initial strategies to overcome the toxicity of enterotoxin adjuvants consisted of removing the enzymatic activity of these molecules. The binding of these toxins to their ganglioside receptors was later found to be a major source of safety concern regarding nasal vaccines. Non-ganglioside-binding bacterial toxins and their derivatives are being evaluated as alternatives for the development of effective and safe mucosal vaccines. The CTA1-DD adjuvant, which acts by B-cell targeting of the enzymatically active cholera toxin A subunit, represents one of the strategies to induce immunity by nasal immunization without targeting central nervous system tissues. More recently, we found that the edema toxin of Bacillus anthracis, which binds to target cells via the anthrax toxin receptors and delivers its adenylate cyclase subunit, is an effective adjuvant for nasal vaccines. Interestingly, the unique receptors of anthrax toxin derivatives appear to prevent their accumulation in the central nervous system.

Nasal antigen plus mucosal adjuvant delivery has emerged as perhaps the most effective route for induction of both mucosal and peripheral immune response. It has been shown that dendritic cells (DCs) played essential roles for the induction of both immunity and tolerance. Thus, nasal application of CpG ODN targeting plasmacytoid DCs effectively induced antigen-specific immunity. Our recent studies showed that nasal or oral immunization with protein antigen, ovalbumin plus plasmid encoding Flt3 ligand (pFL) cDNA as adjuvant effectively elicited OVA-specific immune responses in both mucosal and systemic lymphoid tissues. The numbers of (DCs) in mice given pFL as mucosal adjuvant were significantly increased. Further, these expanded DCs were activated and expressed MHC II and co-stimulatory molecules (CD40, CD80 and CD86). These results show that mucosal delivery of pFL is DCs targeting adjuvant system that effectively induce both mucosal and systemic antigen-specific immune responses.

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4(+)) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.

Age-associated dysregulation of the immune system of the gastrointestinal (GI) tract has been well documented for both secretory (S)-IgA immunity and oral tolerance. Thus, impaired antigen-specific Ab responses in aged animals and the elderly have been reported. Further, it has been shown that gut-associated lymphoreticular tissue (GALT) mediated immune responses are more susceptible to aging than are lymphoid tissues involved in peripheral immunity. Aging also impairs oral tolerance, which may be of central importance for maintaining GI homeostasis. Thus, as early as 6-8-month-old mice failed to establish systemic unresponsiveness to orally introduced antigens. Despite these studies, the precise mechanisms for impaired GI tract immune system responses remain unclear. The evidence of reduced sizes of Peyer's patches through aging suggests that age-associated mucosal dysregulation may be the result of mucosal inductive tissue dysfunction. Indeed, the frequencies of naive CD4(+) T cells and dendritic cells (DCs) in Peyer's patches of aged mice were reduced and this led to a lack of essential cytokine synthesis for the induction of either S-IgA immunity or oral tolerance. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

We have analyzed the gastrointestinal inflammation which develops following adoptive transfer of IL-4 gene knockout (IL-4(-/-)) CD4(+)CD45RB(Hi) (RBHi) T cells to severe combined immunodeficient (SCID) or to T cell-deficient, T cell receptor beta and delta double knockout (TCR-/-) mice. Transfer of IL-4(-/-) RBHi T cells induced a similar type of colitis to that seen in SCID or TCR-/- recipients of wild-type (wt) RBHi T cells as reported previously. Interestingly, transfer of both wt and IL-4(-/-) RBHi T cells to TCR-/- but not to SCID mice induced inflammation in the gastric mucosa. Notably, TCR-/- recipients of IL-4(-/-) RBHi T cells developed a more severe gastritis with erosion, apoptosis of the antral epithelium, and massive infiltration of macrophages. This gastritis was partially dependent on the indigenous microflora. Recipients of both wt and IL-4(-/-) RBHi T cells developed duodenitis with multinuclear giant cells, expansion of mucosal macrophages, and dendritic cells. Full B cell responses were reconstituted in TCR-/- recipients of RBHi T cells; however, anti-gastric autoantibodies were not detected. We have now developed and characterized a novel model of chronic gastroduodenitis in mice, which will help in our understanding of the mechanisms involved in chronic inflammation in the upper gastrointestinal tract of humans.

This study assessed the roles of the postnatal lymphotoxin-beta receptor (LTbetaR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTbetaR-dependent events by postnatal administration of the fusion protein of LTbetaR and IgG Fc (LTbetaR-Ig) reduced both the size and numbers of Peyer's patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTbetaR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTbetaR-Ig eliminated the formation of ILF. LTbetaR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTbetaR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.

IL-12 consists of two disulfide-linked subunits, p40 and P35, that form functionally active heterodimers for the induction of Th1 cells. In contrast to IL-12 heterodimers, p40 monomers and homodimers possess inhibitory effects on Th1 cells leading to the creation of a Th2 environment. Although it has been shown that IL-12p40 acts as antagonist of IL-12p70 in vitro, no evidence is currently available whether IL-12p40 is functional in vivo. We now report that IL-12p40 plays an important pathological role in an intestinal allergic disease. A high expression of IL-12p40 protein wits demonstrated in epithelial cells, dendritic cells, and macrophages in large but not small intestine of allergic diarrhea-induced mice. Interestingly, neutralization with anti-IL-12p40 mAbs reduced the incidence and delayed the onset of disease development. Lower levels of ovalbumin (OVA)specific IgE Abs in serum were detected in anti-IL-12p40 mAb-treated mice than in control Ab-treated mice. The secretion of Th2 cytokines and eotaxin by the mononuclear cells isolated from the large intestine of anti-IL-12p40 mAb-treated mice was significantly decreased. Finally, the removal of the IL-12p40 gene resulted in complete inhibition of disease development. These results show that over-expression of IL-12p40 is an important contributing factor for the generation of the dominant Th2-type environment in the large intestine of mice with allergic diarrhea.

Nasal immunization is an effective way to induce both mucosal and systemic immune responses. In this study, we assessed a cDNA vector for Flt3 ligand (FL) for its potential to enhance mucosal immunity or tolerance. Interestingly, tolerance was avoided and elevated levels of OVA-specific Ab responses were induced in nasal washes, fecal extracts, and saliva as well as in plasma when compared with mice given nasal OVA plus DNA plasmid without the FL gene. In addition, significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IL-4 and IL-2 production were noted in spleen and cervical lymph nodes. Further, marked increases in FL protein occurred in the nasal lamina propria and submandibular glands and the frequencies of CD11c(+)CD8(+) dendritic cells (DCs) significantly increased in the mucosal tissues. Moreover, these DCs expressed high levels of CD40, CD80, CD86, and MHC class II molecules. Nasal delivery of plasmid FL with OVA resulted in FL expression in both mucosal inductive and effector sites and resulted in expanded activated lymphoid DCs. Thus, nasal plasmid FL prevents mucosal tolerance and enhances active immunity when given by a mucosal route.

Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4(+) T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by upregulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.

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.

Background & Aims: T helper (Th):1 and Th2 cell subsets significantly influence the pathological features of inflammation in the gastrointestinal tract in a distinct manner. It is now established that the transfer of CD4(+)CD45RB(Hi) (RBHi) T cells to either severe combined immunodeficient (SCID) or recombinase activation gene 2-deficient (RAG(-/-)) mice results in a severe granulomatous hypertrophic colitis mediated by Th1 cells. We have modified this approach to address the role of Th2 cells. Methods: RBHi T cells from wild-type (Wt) mice or mice genetically predisposed to Th2 responses (interferon-gamma-defective [IFN-gamma(-/-)]) with or without B cells were transferred to T cell receptor (TCR)-beta and delta-chain-defective (TCR-/-) or SCID mice. Results: Transfer of Wt RBHi T cells induced wasting disease with severe colitis in the TCR-/- mice. In contrast, IFN-gamma(-/-) RBHi T cells induced severe weight loss and hypoalbuminemia without significant inflammation in the colon. The small intestine of these mice exhibited villus atrophy, a decrease in brush-border enzymes, reduced enterocyte proliferation, and an increased number of goblet cells. The presence of B cells was necessary for these changes, because SCID recipients required cotransfer of B cells, together with IFN-gamma(-/-) RBHi T cells for ileal lesions to develop. Treatment of TCR-/- recipients of IFN-gamma(-/-) RBHi T cells with anti-IL-4 mAb abrogated both the wasting disease and the villus atrophy. Conclusions: Dysregulated Th2 cells cause atrophic changes and goblet cell transformation in the small intestinal epithelium and wasting disease mediated by excess interleukin-4 and B cells.

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1-and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly. The Journal of Immunology, 2003.

Our past studies showed that Peyer's patches were required for the induction of oral tolerance to the protein antigen ovalbumin (OVA), but not to the hapten 2,4,6-trinitrobenzene sulfonic acid (TNBS). In the present study, the effects of immunosenescence on oral tolerance induction were assessed with these two toleragens. Significant reductions in OVA-specific serum IgG antibody and CD4(+) T cell responses to subsequent challenge were observed in OVA-fed, young adult mice. Importantly, these reduced anti-OVA antibody responses were associated with delayed-type hypersensitivity, and antigen-induced CD4(+) T(h)1- and T(h)2-type cytokine responses. On the other hand, aged mice fed OVA failed to develop oral tolerance. Thus, CD4+ T cells from Peyer's patches produced selected T(h)2- but no T(h)1-type cytokines. The TNP-specific serum IgG antibody and T cell responses were significantly diminished by prior TNBS feeding in young adult, 6- to 8-month-old and 12- to 14-month-old, but not in senescent, 2-year-old mice. Finally, we have directly assessed dendritic cell subsets and T cell responses in Peyer's patches, and their function in tolerance induction was impaired at an earlier stage of life. These results suggest that lack of oral tolerance to the protein OVA during aging is the result of dysfunctional Peyer's patches.

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB(5)-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4(+)) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4(+) Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4(+) T cells secreting IFN-gamma and IL-4-independent Tb2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4(+) Th2 cells secreting high levels of IL-4, but not IFN-gamma. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.

The mucosal immune system has evolved alongside, but separate, from the general systemic immune System. As a major consequence of this dichotomy, only immune responses initiated in mucosal inductive sites can result in effective immunity in mucosal tissues themselves. Oral tolerance, as usually assessed as orally-induced systemic unresponsiveness, contributes to mucosal homocostasis by preventing unwanted immune reactions to food or environmental antigens. It is now established that tolerance can also be induced by the nasal route and mucosally-induced tolerance is being actively investigated for immune therapy against a number of diseases. Nontoxic derivatives of cholera toxin and the heat labile toxin of Escherichi coli as well as chimeric enterotoxins have been developed. These molecules retain the mucosal adjuvant activity of native enterotoxins and are effective at inducing targeted Th1 or Th2-type immune responses. Mucosal delivery of cytokines as adjuvants represents a safer alternative to parenteral cytokine injection. Nasally administered cytokines such as IL-1 and IL-12 or chemokines including RANTES, lymphotactin, MIP-1beta, all act as mucosal adjuvants for co-administered antigens. Each of these cytokines promote specific pattern of CD4(+) T helper cell cytokine responses that could be exploited for targeted immune therapy. Although GALT and NALT are both parts of the Common Mucosal Immune System, there arc major differences between orally and nasally induced immune responses. Nasal vaccines more effectively promote protective immunity in the genitourinary tract than do oral vaccines. In addition, aging affects mucosal tolerance or immunity in GALT more than is seen in NALT. Therapeutic manipulation of mucosal immunity involves regulation of CD4(+) T cell cytokine responses and thus, should require a careful examination of the host status, including the occurrence of inflammatory bowel diseases.