清野 宏

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCR delta(-/-)) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCR delta(-/-) mice. The TCR delta(-/-) mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toroid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toroid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCR delta(-/-) mice compared with dir orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta T cells in mucosal immunity.

We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which is most efficient for light emission in the 400-420 nm wavelength range. For this reason, we have chosen the bioluminescent molecule, aequorin, which upon the addition of Ca2+ undergoes a conformational change resulting in the emission of blue light at 469 nm, The high quantum yield is reflected by the fact that addition of Ca2+ to 1 ng of recombinant streptaequorin, a covalent conjugate of streptavidin and aequorin, resulted in the production of 7 x 10(8) relative light units, In this study, we show the superior sensitivity of biotin-streptaequorin when directly compared with biotin-streptavidin linked horseradish peroxidase commonly used for ELISA. For example, mice orally immunized once with cholera toxin (CT) did not exhibit detectable fecal IgA antibodies as determined by ELISA, whereas use of streptaequorin and the bioluminescent immunoassay revealed fecal IgA anti-CT-B subunit antibody titers of 1:24 500. In addition, no detectable anti-CT-B antibodies were noted in saliva samples by ELISA 7 days following oral immunization with CT, while IgA endpoint titers could be extrapolated to 1:393 000. The 21 day fecal IgA anti-CT-B titers were 1:512 by ELISA, whereas titers determined by luminometry reached 1:10(7) when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was > 10(4)-fold more sensitive when compared with ELISA for detection of mucosal and serum antigen- and isotype-specific antibody responses, Thus, the bioluminescent immunoassay is a more sensitive assay for detection of antibodies in dilute external secretions.

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The concept of the Collaborative Mucosal Immunization Research Group for AIDS (CMIG) was originally conceived by the AIDS Vaccine Branch, National Institute of Allergy and Infectious Diseases (NIAID) in order to provide support for a cooperative research environment for the development of mucosal immunity to AIDS. We have purposely organized five groups of investigators at five different locations to determine how effective mucosal immunity to AIDS can be optimally approached. CMIG recognizes that both rectal (homosexual) as well as vaginal (heterosexual) infections with HIV are two of the major ways that AIDS currently disseminates through the human population. Thus, we have chosen the SIV model of infection of rhesus macaques, but more importantly the CMIG have joined two of our five components in order to use the significant expertise developed for mucosal transmission of SIV and immunity. Thus, we have brought the extensive expertise with vaginal and rectal immunization and immunity to spread [Drs. Chris Miller and Marta Marthas, California Regional Primate Research Center (CRPRC), Davis and Drs. Thomas Lehner and Martin Cranage, United Medical and Dental School Guy's Hospital, London and the Centre for Applied Microbiology and Research (Guy's/CAMR)]. Two additional components were added in order to perform mucosal immune response studies required to develop and to optimize a mucosal vaccine. First, extensive CD4(+) T helper (Th) cell (e.g., Th1 and Th2) and CD8(+) T cell subset studies are a major effort of the coordinating group at the University of Alabama at Birmingham (Drs. Hiroshi Kiyono and Jerry R. McGhee). This component is closely interacting with both the CRPRC and Guy's/CAMR components in terms of SIV-specific CD4(+) and CD8(+) T cell subset responses. For example, SIV-specific CTL responses are jointly examined using different techniques by CRPRC, Guy's/CAMR and UAB investigators. Further, it is also important to examine a balance between SIV-specific and Th1 and Th2 cell responses following mucosal immunization since the Th cell-derived cytokines are essential for the induction of appropriate antigen-specific mucosal immune responses. This issue is currently being extensively examined by the CMIG effort and a summary of our findings is discussed in this review. A major question in mucosal immunity involves the functions of secretory IgA (S-IgA) antibodies and this area is of particular importance in rectal and reproductive tract immunity. A novel and exciting in vitro epithelial cell assay system is used to study how effectively S-IgA neutralizes SIV infection (Drs. John Huang, John Nedrud and Michael Lamm, Case Western Reserve, Cleveland). A clear advantage of this CMIG effort is the unique expertise in design of mucosal delivery systems for an AIDS vaccine. We are using state-of-the-art recombinant bacteria, i.e., rSalmonella and rVibrios for mucosal immunization [Drs. Yichen Lu and Bryan Roberts, Virus Research Institute (VRI), Boston]. In addition, we are also testing other mucosal delivery systems including DNA vaccine, microspheres, cholera toxin (CT) and CT-B, recombinant poliovirus, and immune complexes. These studies represent the first efforts to induce not only Th cell mediated S-IgA responses, but also CTL responses to SIV in primates immunized with different mucosal vector delivery systems. In order to focus our effort for the induction of SIV-specific immune responses following mucosal immunization, investigators from the CMIG are attempting to understand the induction and regulation of antigen-specific immune responses in rhesus macaques mucosally immunized with different preparations of SIV vaccines.

Despite pathophysiologic effects including diarrhea, cholera toxin (CT) is a potent mucosal immunogen and adjuvant. We investigated the influence of CT on T helper (Th)-type 1 (Th1) and Th2 cell-regulated Ag-specific B cell isotype and IgG subclass Ab responses elicited when the toxin was co-administered orally with different protein Ags. When mice were orally immunized with tetanus toroid (TT) and CT as adjuvant, this regimen induced TT-specific secretory IgA responses in the gastrointestinal tract as well as serum IgG, including IgG1 and IgG2b subclasses, and IgA responses. This oral regimen also induced TT- and CT-B-specific IgE responses. In addition, CT also elicited adjuvant effects for Ag-specific IgG1, IgE, and IgA responses when two other protein Ags, OVA and hen egg white lysozyme, were given by the oral route. Quantitative reverse transcriptase-PCR was performed to assess levels of mRNA for Th1 (IFN-gamma) and Th2 (IL-4) cytokine expression in TT-stimulated CD4(+) T cell cultures. Both Peyer's patches and splenic CD4(+) T cells expressed markedly increased levels of IL-4-specific message, but did not result in changes in IFN-gamma mRNA expression. To determine whether the route of immunization influenced IgE responses, mice were immunized s.c. with TT and CT as adjuvant. Significant increases in total and TT-specific IgE Abs were Induced when CT was co-administered. Taken together, these results show that CT acts as a mucosal adjuvant to enhance Th2-type responses and in particular, the IL-4 produced results in a characteristic Ab isotype pattern associated with this cytokine.

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The realization that induction of immune responses at mucosal surfaces may prevent colonization, invasion or dissemination of pathogenic microorganisms has spurred intensive efforts to develop vaccines which elicit effective mucosal immunity. In this paper, recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses. Oral administration of cholera toxin alone or with a co-administered protein vaccine tetanus toroid induces a strong T helper type 2 (TH2) cell response in both Peyer's patches and spleen. Both serum IgG and secretory IgA antibodies specific for cholera toxin or for the co-administered protein tetanus toroid were induced. When administered parentally, however, no mucosal antibody responses were evident and a mixed TH1- and TH2-type CD4(+) T cell response was noted in the spleen. Various vectors are being employed in an effort not only to induce mucosal immune responses but also to direct the response to a TH1-type response, thought to promote strong cell-mediated immune responses, or to a TH2-type response for maximum B cell antibody responses. The ability to manipulate the TH cell responses may provide a more rational approach for the design of vaccines. Although lymphoid tissues of the female reproductive tract differ from that of the gut, many of the strategies and evolving principles may be directly applicable to the development of vaccines designed to prevent sexually transmitted diseases.