清野 宏

We have directly compared enzyme-linked immunoassays (ELlSAs) 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 108 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 : 24500. 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: 393000. The 21 day fecal IgA anti-CT-B titers were 1: 512 by ELISA, whereas titers determined by luminometry reached 1 : 107 when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was &gt 104-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 extemal secretions.

We have directly compared enzyme-linked immunoassays (ELlSAs) 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 108 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 : 24500. 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: 393000. The 21 day fecal IgA anti-CT-B titers were 1: 512 by ELISA, whereas titers determined by luminometry reached 1 : 107 when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was &gt 104-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 extemal secretions.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of γδ T-cell receptor. positive T cells (γδ T cells). γδ T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into γδ(Dim) and γδ(Bright) fractions according to the intensity of γδ T-cell receptor expression. The γδ(Dim) T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas γδ(Bright) T cells did not express either receptor. Our study also revealed that recombinant murine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on γδ(Dim) T cells but not on γδ(Bright) IELs. Thus, treatment of γδ(Dim) T cells with rmIL-2 and rmIL- 7 resulted in high proliferative responses, whereas γδ(Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for γδ(Dim) T cells were neighboring epithelial cells (IL-7) and αβ IELs (IL- 2 and IL-7). Cytokine signaling by IL-2 and IL-7 from αβ T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of γδ T cells (e.g., γδ(Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of γδ T-cell receptor. positive T cells (γδ T cells). γδ T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into γδ(Dim) and γδ(Bright) fractions according to the intensity of γδ T-cell receptor expression. The γδ(Dim) T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas γδ(Bright) T cells did not express either receptor. Our study also revealed that recombinant murine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on γδ(Dim) T cells but not on γδ(Bright) IELs. Thus, treatment of γδ(Dim) T cells with rmIL-2 and rmIL- 7 resulted in high proliferative responses, whereas γδ(Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for γδ(Dim) T cells were neighboring epithelial cells (IL-7) and αβ IELs (IL- 2 and IL-7). Cytokine signaling by IL-2 and IL-7 from αβ T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of γδ T cells (e.g., γδ(Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.

Mucosal tissues of mice are enriched in T cells that express the γ/δ T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in γ/δ T cell receptor-deficient (TCRδ(- /-)) 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 TCRδ(-/-) mice. The TCRδ(-/-) mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid 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 toxoid 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(-/-) mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of γ/δ T cells suggests a specialized role for γ/δ T cells in mucosal immunity.

The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toxoid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4+ T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout IFN-γ(-/-) and IL-4(-/-)) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine L-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further, oral immunization of IFN-γ(-/-) and IL-4(-/-) mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-γ from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.

The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toxoid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4+ T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout IFN-γ(-/-) and IL-4(-/-)) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine L-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further, oral immunization of IFN-γ(-/-) and IL-4(-/-) mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-γ from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.

IgA and IgG binding factors (BF) can be found in the supernatant (T-h SUP) Of cultures containing macrophages and CD4(+) T cells stimulated with particulate antigens such as SRBC. Previous work indicated that these IgBF, when mixed with normal serum immunoglobulin, could block the activity of suppressor T cells (T-s) and allow IgA and IgG PFC responses in vibro. We present serologic and functional evidence that IgABF and IgGBF in T-h sup are soluble Fc alpha R and Fc gamma RII(or III), respectively. Th sup adsorbed on affinity columns containing anti-Fc gamma RII/III mAb or murine IgG failed to augment IgG PFC responses. Material eluted from either the IgG or anti-Fc gamma RII/ III columns could be added back, interchangeably, to the adsorbed T-h sup and restore IgG PFC. Recombinant murine Fc gamma RII (rFc gamma RII), added to the same adsorbed T-h sup at 0.01 to 0.5 ng/ml, resulted in a similar augmentation of IgG PFC. Interestingly, much higher concentrations of rFc gamma RII (10-100 ng/ ml) could not, augment IgG; PPC responses. Protein dot blots showed that T-h sup and the eluted material from murine IgG: columns contained structures reactive with the Fc gamma RII/III mAb. Similar studies using purified Fc alpha R revealed that IgABF eluted from IgA or anti-Fc alpha R columns was in fact Fc alpha R. Cross-adsorption studies indicated clearly that the IgGBF (Fc gamma RII/III) and the IgABF (FC alpha R) were separate molecules produced in the same T-h sup and that each regulated their respective Ig isotype independently. Thus, cultures of splenic macrophage and CD4(+) T cells, in the presence of particulate antigens such as SRBC, generate both Fc gamma RII/III and Fc alpha R. This soluble FcR in combination with serum Ig act to block isotype-specific T-s cells at low concentration in vitro. (C) 1996 Academic Press, Inc.

Liposome and cholera toxin (CT) are considered to be effective antigen delivery vehicles and adjuvants for mucosal vaccines. The effect of these antigen delivery systems on adjuvant responses to mucosally administered pneumococcal polysaccharide (Pnup) was investigated in this study. Both mucosal (e.g oral) and systemic (IP) immunization of mice with purified preparations of Pnup type 23F induced antigen-specific IgM responses in sera. Interestingly, oral immunization of as little as 10 μg of Pnup type 23F was sufficient to induce systemic IgM responses. Pnup-specific IgM antibodies peaked by day 7 and no booster responses were evident after a second dose on day 14. In order to examine whether IgG and IgA Pnup-specific immune responses are induced by mucosal immunization, the mucosal adjuvant CT was mixed with Pnup type 23 as an oral vaccine. Co-oral administration of CT and Pnup type 23F resulted in the induction of Pnup-specific faecal IgA antibodies. These results were confirmed by detecting antigen-specific IgA-spotforming cells in mononuclear cell suspensions prepared from the intestine of immunized mice. These findings suggest that oral immunization with Pnup in the presence of mucosal adjuvants, such as CT, could induce Pnup-specific IgA responses whereas Pnup alone did not. In an attempt to further enhance antigen-specific antibody responses, Pnup type 23F was encapsulated in liposomes and used as mucosal vaccine. However, immunogenicity of Pnup was not improved.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When n 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-Ab, and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.

IgA and IgG binding factors (BF) can be found in the supernatant (T-h SUP) Of cultures containing macrophages and CD4(+) T cells stimulated with particulate antigens such as SRBC. Previous work indicated that these IgBF, when mixed with normal serum immunoglobulin, could block the activity of suppressor T cells (T-s) and allow IgA and IgG PFC responses in vibro. We present serologic and functional evidence that IgABF and IgGBF in T-h sup are soluble Fc alpha R and Fc gamma RII(or III), respectively. Th sup adsorbed on affinity columns containing anti-Fc gamma RII/III mAb or murine IgG failed to augment IgG PFC responses. Material eluted from either the IgG or anti-Fc gamma RII/ III columns could be added back, interchangeably, to the adsorbed T-h sup and restore IgG PFC. Recombinant murine Fc gamma RII (rFc gamma RII), added to the same adsorbed T-h sup at 0.01 to 0.5 ng/ml, resulted in a similar augmentation of IgG PFC. Interestingly, much higher concentrations of rFc gamma RII (10-100 ng/ ml) could not, augment IgG; PPC responses. Protein dot blots showed that T-h sup and the eluted material from murine IgG: columns contained structures reactive with the Fc gamma RII/III mAb. Similar studies using purified Fc alpha R revealed that IgABF eluted from IgA or anti-Fc alpha R columns was in fact Fc alpha R. Cross-adsorption studies indicated clearly that the IgGBF (Fc gamma RII/III) and the IgABF (FC alpha R) were separate molecules produced in the same T-h sup and that each regulated their respective Ig isotype independently. Thus, cultures of splenic macrophage and CD4(+) T cells, in the presence of particulate antigens such as SRBC, generate both Fc gamma RII/III and Fc alpha R. This soluble FcR in combination with serum Ig act to block isotype-specific T-s cells at low concentration in vitro. (C) 1996 Academic Press, Inc.

Liposome and cholera toxin (CT) are considered to be effective antigen delivery vehicles and adjuvants for mucosal vaccines. The effect of these antigen delivery systems on adjuvant responses to mucosally administered pneumococcal polysaccharide (Pnup) was investigated in this study. Both mucosal (e.g oral) and systemic (IP) immunization of mice with purified preparations of Pnup type 23F induced antigen-specific IgM responses in sera. Interestingly, oral immunization of as little as 10 μg of Pnup type 23F was sufficient to induce systemic IgM responses. Pnup-specific IgM antibodies peaked by day 7 and no booster responses were evident after a second dose on day 14. In order to examine whether IgG and IgA Pnup-specific immune responses are induced by mucosal immunization, the mucosal adjuvant CT was mixed with Pnup type 23 as an oral vaccine. Co-oral administration of CT and Pnup type 23F resulted in the induction of Pnup-specific faecal IgA antibodies. These results were confirmed by detecting antigen-specific IgA-spotforming cells in mononuclear cell suspensions prepared from the intestine of immunized mice. These findings suggest that oral immunization with Pnup in the presence of mucosal adjuvants, such as CT, could induce Pnup-specific IgA responses whereas Pnup alone did not. In an attempt to further enhance antigen-specific antibody responses, Pnup type 23F was encapsulated in liposomes and used as mucosal vaccine. However, immunogenicity of Pnup was not improved.

One major concern about using adenoviral vectors for repetitive gene delivery to lung epithelial cells is the induction of an immune response to the vector, thus, impeding effective gene transduction. To assess the immune response to the adenoviral vector, repetitive intratracheal (i.t.) gene dosing was performed in CD-1 mice using the replication-deficient adenovirus 5 (Ade5) vector carrying the lacZ gene, and compared to the antibody responses induced by conventional intranasal (i.n.) and intraperitoneal (i.p.) routes of immunization. Kinetics of serum IgG, IgA, and IgM antibody responses to the adenoviral vector and to beta-galactosidase (beta-Gal) were evaluated. Two or three adenoviral vector doses given by i.t., i.n., or i.p. routes resulted in serum IgG titers in excess of 1:200,000, whereas serum IgM and IgA were moderately induced. Analysis of the predominant murine IgG subclass was determined to be IgG(2b) and IgG(2a). To determine the localization of this antibody response, the ELISPOT assay was employed. Lymphocytes were isolated from the lung, the lower respiratory lymph nodes (LRLN), the nasal passages (NP), and the spleen. For i.t- and i.n.-administered mice, the highest IgA spot-forming cell (SFC) response to Ade5 and beta-Gal was located in the NP and in the lung. Both the lung and the LRLN showed elevated numbers of IgG SFCs (4- to 12-fold greater than splenic IgG SFC response) for Ade5 and beta-Gal. This evidence suggests that the lung and associated lymphoid tissues were the source for serum antibodies. Further analysis of serum antibodies showed that the i.p.- and i.t.-administered groups yielded the greatest neutralization titers to Ade5, suggesting that the reduced effectiveness of repetitive gene transfer is in part due to circulating neutralizing antibodies. Thus, repetitive i.t. instillation will stimulate a localized and systemic antibody response to the vector.

Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is known to possess strong immunoregulatory potential in terms of inhibition of the induction of oral tolerance and adjuvanticity in oral immunization. We found that oral administration of an immunogenic peptide of LT [LT-B(26-45) spanning the residues 26-45 of LT-B] induced systemic unresponsiveness in BALB/c mice resulting in diminished serum IgG responses. It was also shown that the spleen (SP) CD4+ T cells of tolerized mice failed to proliferate, whereas the Peyer′s patches (PP) CD4+ T cells responded to the peptide. RT-PCR revealed that the SP CD4+ T cells did not generate IL-2 mRNA, while the PP CD4+ T cells expressed significant levels of IFN-γ, IL-2, IL-4, and TGF-β mRNA. Adoptive transfer of LT-B-specific intraepithelial lymphocytes to the tolerant mice abrogated the tolerance. In the reversed mice, LT-B(26-45)-stimulated SP CD4+ T cells expressed significant levels of IFN-γ, IL-2, IL-4, and IL-6 mRNA. These results indicate that PP CD4+ T cells induce oral tolerance due to systemic T cell anergy. © 1995 Academic Press, Inc.

Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is known to possess strong immunoregulatory potential in terms of inhibition of the induction of oral tolerance and adjuvanticity in oral immunization. We found that oral administration of an immunogenic peptide of LT [LT-B(26-45) spanning the residues 26-45 of LT-B] induced systemic unresponsiveness in BALB/c mice resulting in diminished serum IgG responses. It was also shown that the spleen (SP) CD4+ T cells of tolerized mice failed to proliferate, whereas the Peyer′s patches (PP) CD4+ T cells responded to the peptide. RT-PCR revealed that the SP CD4+ T cells did not generate IL-2 mRNA, while the PP CD4+ T cells expressed significant levels of IFN-γ, IL-2, IL-4, and TGF-β mRNA. Adoptive transfer of LT-B-specific intraepithelial lymphocytes to the tolerant mice abrogated the tolerance. In the reversed mice, LT-B(26-45)-stimulated SP CD4+ T cells expressed significant levels of IFN-γ, IL-2, IL-4, and IL-6 mRNA. These results indicate that PP CD4+ T cells induce oral tolerance due to systemic T cell anergy. © 1995 Academic Press, Inc.

Control of pandemic infection of human immunodeficiency virus type 1 (HIV-1) requires some means of developing mucosal immunity against HIV-1 because sexual transmission of the virus occurs mainiy through the mucosal tissues. However, there is no evidence as yet that the secretory immunoglobulin A (IgA) antibody induced by immunization with antigens in experimental animals can neutralize HIV-1. We demonstrate here that oral immunization with a new macromolecular peptide antigen and cholera toxin (CT) induces a high titre (1:211) of gut-associated and secretory igA antibody to HIV-1. Using three different neutralizing assays, we clearly demonstrate that this secretory igA antibody is able to neutralize HIV-1Hib, HIVSF2and HIV-1MN. Our new approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces against HIV-1. © 1995 Nature Publishing Group.