清野 宏

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.

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.

Currently only limited information is available as to why dominant IgA isotype responses are supported by mucosal T cells in effector tissues. To address this issue directly, γδ and αβ T cells were isolated from the submandibular gland (SMG) of mice as an example of mucosal effector tissues. Freshly isolated CD3+ T cells from this tissue contained relatively high numbers of activated cells [approximately 10% interleukin‐2 receptor (IL‐2R)+ cells and 15% of cells in cycle stages S and G2 + M], of which 25% and 75% were γδ and αβ T cells, respectively. The cytokine‐specific quantitative reverse transcriptase‐polymerase chain reaction and enzyme‐linked immunospot analyses revealed that, although both γδ and αβ T cells were capable of producing an array of Th1 or Th2 cytokines following stimulation via the T cell receptor‐CD3 complex, these mucosal T cells were mainly committed to IL‐5 and IL‐6 expression in vivo (Th2 type). Both freshly isolated γδ and αβ T cells expressed mRNA and contained IL‐5 and IL‐6 spot‐forming cells (SFC) however, only the latter exhibited high mRNA levels and SFC for a Th1 cytokine (interferon‐γ). Taken together, the results show that freshly isolated CD3+ T cells from SMG contain activated γδ and αβ T cells which are programmed to produce IL‐5 and IL‐6. Thus, SMG, an example of an IgA effector tissue, can be characterized as a Th2‐dominant site. However, although both γδ and αβ T cells express cytokine profiles consistent with a Th2 phenotype, only the latter subset with a CD4+CD8− phenotype provided effective help for mucosal B cell responses in vitro. Copyright © 1995 WILEY‐VCH Verlag GmbH &amp Co. KGaA, Weinheim

The induction of effective mucosal immunity that also provides systemic immunity is a considerable challenge. Over the past two years, efforts to develop novel mucosal vaccine delivery systems to induce mucosal immunity against bacterial and viral diseases, including HIV, have dramatically increased. Here we cite novel vaccines and delivery systems being used to establish effective mucosal immunity. © 1994.

The induction of effective mucosal immunity that also provides systemic immunity is a considerable challenge. Over the past two years, efforts to develop novel mucosal vaccine delivery systems to induce mucosal immunity against bacterial and viral diseases, including HIV, have dramatically increased. Here we cite novel vaccines and delivery systems being used to establish effective mucosal immunity. © 1994.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2- and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2- and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

The immunofluorescence-linked immunospot (ILISPOT) assay associated with the immunofluorescence digital image processing (IDIP) system was originally developed in our laboratory to allow enumeration of immunoglobulin (Ig) producing, spot forming cells (SFC) in a more objective and quantitative manner. In this study, the ILISPOT-IDIP system was further advanced in order to analyze different sizes of SFC (e.g., IgA producing cells) including large (L), medium (M), and small (S) cells which correspond to high (&gt 2.4 pg), medium (1.2-2.4 pg) and low (&lt 1.2 pg) IgA secreting cells by the adaptation of real time image processor and intensified video camera system. When the ILISPOT-IDIP system was used to characterize the frequency of IgA secreting cells among mononuclear cells isolated from different parts of the murine gastrointestinal (GI) tract including the small (upper, middle and lower sections) and large (colon and rectum) intestine, the small intestine contained higher numbers of IgA SFC (∼ 8.4 × 105 SFC/106 cells) when compared with large intestine (∼ 1.3 × 105 SFC/106 cells). Among the 3 areas of small intestine, the upper (∼ 3.7 × 105 SFC/106 cells) and middle (∼ 2.4 × 105 SFC/106 cells) parts had higher numbers of IgA SFC when compared to the lower small (∼ 2.3 × 105 SFC/106 cells) intestine. When these IgA producing cells in different parts of the intestine were classified into three groups according to the size of individual spots, the upper and middle intestine contained higher frequencies of large (∼ 20%) and medium (∼ 20%) SFC which corresponded to high and medium IgA secretors in comparison to the lower small (∼ 9%) and large (∼ 6%) intestine. In contrast, the lower small and large intestine were dominated by small SFC since ∼ 85% of IgA producing cells were categorized as low secretors. Using the advanced ILISPOT-IDIP system, a unique distribution of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intestine. © 1993.

The immunofluorescence-linked immunospot (ILISPOT) assay associated with the immunofluorescence digital image processing (IDIP) system was originally developed in our laboratory to allow enumeration of immunoglobulin (Ig) producing, spot forming cells (SFC) in a more objective and quantitative manner. In this study, the ILISPOT-IDIP system was further advanced in order to analyze different sizes of SFC (e.g., IgA producing cells) including large (L), medium (M), and small (S) cells which correspond to high (&gt 2.4 pg), medium (1.2-2.4 pg) and low (&lt 1.2 pg) IgA secreting cells by the adaptation of real time image processor and intensified video camera system. When the ILISPOT-IDIP system was used to characterize the frequency of IgA secreting cells among mononuclear cells isolated from different parts of the murine gastrointestinal (GI) tract including the small (upper, middle and lower sections) and large (colon and rectum) intestine, the small intestine contained higher numbers of IgA SFC (∼ 8.4 × 105 SFC/106 cells) when compared with large intestine (∼ 1.3 × 105 SFC/106 cells). Among the 3 areas of small intestine, the upper (∼ 3.7 × 105 SFC/106 cells) and middle (∼ 2.4 × 105 SFC/106 cells) parts had higher numbers of IgA SFC when compared to the lower small (∼ 2.3 × 105 SFC/106 cells) intestine. When these IgA producing cells in different parts of the intestine were classified into three groups according to the size of individual spots, the upper and middle intestine contained higher frequencies of large (∼ 20%) and medium (∼ 20%) SFC which corresponded to high and medium IgA secretors in comparison to the lower small (∼ 9%) and large (∼ 6%) intestine. In contrast, the lower small and large intestine were dominated by small SFC since ∼ 85% of IgA producing cells were categorized as low secretors. Using the advanced ILISPOT-IDIP system, a unique distribution of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intestine. © 1993.