Immunostimulatory composition

Abstract

The present disclosure relates to: a composition for inducing germinal center migration of activated B-cells, the composition containing a substance that induces expression of CD11b in B-cells; a method for manufacturing the composition; and use of the composition. In one embodiment, the present disclosure pertains to immunostimulation and maintenance or improvement of mucosal flora using a substance that induces expression of CD11b in B-cells.

Description

[Technical field]

[0001] The present disclosure relates to a composition for inducing germinal center migration of activated B cells, a method for producing the composition, or a use thereof, comprising a substance that induces expression of CD11b in B cells. In one embodiment, the present disclosure relates to immune activation, maintenance or improvement of mucosal flora using a substance that induces expression of CD11b in B cells. [Background technology]

[0002] Research into the behavior of B cells and germinal centers (GCs) during immune responses is motivated by the possibility that they may be useful for vaccine development (Non-Patent Documents 1, 2). One of the important criteria for evaluating the efficacy of a vaccine is whether the vaccine can efficiently induce a germinal center (GC) response and generate high-affinity, long-lived plasma cells (PCs) and memory B cells to protect the host from invading pathogens (Non-Patent Documents 1, 2). During a GC response, B cells undergo three stages: pre-GC, GC, and post-GC (Non-Patent Documents 3-5). Upon antigen stimulation, newly activated naive B cells and activated memory B cells either directly differentiate into short-lived extrafollicular PCs or migrate to the interfollicular (IF) region to establish stable interactions with activated T cells (Non-Patent Documents 3-8). After interaction between TB cells, some B cells differentiate into low-affinity PCs, while others (pre-GC B cells) migrate to GCs (Non-Patent Documents 3, 4). These pre-GC B cells first migrate to the dark zone (DZ) of GCs and develop into GC B cells. B cells rapidly proliferate and undergo somatic hypermutation (SHM) to change the affinity of the B cell receptor (BCR) to bind to its specific antigen (Non-Patent Documents 3 and 4). Subsequently, B cells with mutations in their immunoglobulin (Ig) genes migrate from the DZ to the light zone (LZ), where they become T follicular helper (T FHAffinity selection is performed with the help of GC cells (Non-Patent Documents 3, 4, 8). B cells with high-affinity BCRs for antigens are selected as post-GC B cells, exit the GC, and eventually differentiate into long-lived PCs or memory B cells, whereas unselected B cells return to the DZ of GCs and can further accumulate SHM for higher-affinity BCRs (Non-Patent Document 7).

[0003] It has been reported that efficient activation of dendritic cells (DCs) is essential for GC responses (Non-Patent Document 3). Therefore, previous studies have focused on the selection of optimal adjuvants for stimulating DCs. However, Lycke et al. have shown that in DC-depleted mice, B cells collect antigens from microfold (M) cells and then migrate to GCs (Non-Patent Document 9), indicating that DC activation is not required for entry into GCs. Even if DCs are properly primed, only a small population of activated B cells can develop into pre-GC B cells and enter GCs (Non-Patent Documents 4, 6). Recent studies have reported that irf4, a transcription factor essential for GC formation, is highly expressed in pre-GC B cells (Non-Patent Documents 10, 11). However, the specific surface markers of pre-GC B cells are unknown. Therefore, the criteria for pre-GC B cells and what types of activated B cells enter GCs and produce high-affinity BCRs for specific antigens were not well understood. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Turner, JS et al. Human germinal centers engage memory and naive B cells after influenza vaccination. Nature. 586, 127-132 (2020). [Non-Patent Document 2] Andersen, TK et al. Enhanced germinal center reaction by targeting vaccine antigen to major histocompatibility complex class II molecules. npj Vaccines. 4, 9 (2019). [Non-Patent Document 3] Mesin, L., Ersching, J., & Victora, GD Germinal center B cell dynamics. Immunity. 45.3, 471-482 (2016). [Non-Patent Document 4] Kurosaki, T., Kometani, K., & Ise, W. Memory B cells.Nature Reviews Immunology. 15.3, 149-159 (2015). [Non-Patent Document 5] Chen, K., Magri, G., Grasset, EK & Cerutti, A. Rethinking mucosal antibody responses: IgM, IgG and IgD join IgA. Nature Reviews Immunology. 20, 427-441 (2020). [Non-Patent Document 6] Mesin, L. et al. Restricted clonality and limited germinal center reentry characterizing memory B cell reactivation by boosting. Cell. 180.1, 92-106 (2020). [Non-Patent Document 7] Chen, H. et al. BCR selection and affinity maturation in Peyer's patch germinal centers. Nature. 582, 421-425 (2020). [Non-Patent Document 8] Ise, W. et al. T follicular helper cell-germinal center B cell interaction strength regulates entry into plasma cell or recycling germinal center cell fate. Immunity. 48.4, 702-715 (2018). [Non-Patent Document 9] Komban, RJ et al. Activated Peyer′s patch B cells sample antigen directly from M cells in the subepithelial dome. Nat Commun. 10, 2423 (2019). [Non-Patent Document 10] Song, S. & Matthias, PD The Transcriptional Regulation of Germinal Center Formation. Front Immunol. 10.3389 (2018). [Non-Patent Document 11] Willis, SN. et al. Transcription factor IRF4 regulates germinal center cell formation through a B cell-intrinsic mechanism. J Immunol. 192(7), 3200-6 (2014). Summary of the Invention

[0005] Activated B cells can enter germinal centers (GCs) to undergo affinity maturation and produce high-affinity antibodies. However, it has remained unclear what type of activated B cells enter GCs. Here, we investigated the mechanism by which activated B cells enter GCs by targeting CD11b + IgA + We found a small population of B cells. + IgA +When B cells were injected into mice, they entered GCs 40 hours later, suggesting that CD11b is a pre-GC IgA + It was suggested that CD11b is a new surface marker for B cells. Furthermore, we found that the expression of CD11b on B cells was induced in vitro by bacterial antigens such as pam3CSK4 and heat-killed E. coli, independent of dendritic cell activation. Furthermore, oral administration of pam3CSK4 or heat-killed E. coli increased the number of GC B cells and enhanced mucosal antigen-specific IgA responses within 2 days. The results presented in this disclosure indicate that induction of CD11b on B cells is a promising marker for selecting effective mucosal vaccine adjuvants.

[0006] Based on the above findings, the inventors of the present disclosure have discovered that a substance that induces the expression of CD11b in B cells acts as an effective adjuvant for mucosal vaccines and can induce the production of high-affinity mucosal IgA antibodies, that this action activates the immunity of the mucosa and the like and can induce immunity against various antigens of external origin, and that this effect leads to the maintenance of an appropriate mucosal bacterial flora or improvement of the mucosal bacterial flora, and have completed the present disclosure.

[0007] In one aspect, the disclosure provides a composition for inducing germinal center migration of activated B cells, comprising an agent that induces expression of CD11b in B cells.

[0008] In one aspect, the disclosure provides a method for inducing expression of CD11b in B cells, inducing contact of the B cells with T cells and dendritic cells, and inducing germinal center migration of activated B cells, comprising preparing a composition comprising a substance that induces expression of CD11b in B cells and contacting the B cells with the substance.

[0009] More specifically, the present disclosure provides the following: [Item 1] A composition for inducing germinal center migration of activated B cells, comprising a substance that induces expression of CD11b in B cells. [Item 2] 2. The composition of claim 1, wherein the substance that induces expression of CD11b in B cells is a bacterium. [Item 3] 3. The composition according to item 2, wherein the substance that induces expression of CD11b in B cells is a bacterium of the class Gammaproteobacteria or the class Bacteroidetes. [Item 4] 4. The composition according to item 3, wherein the agent that induces expression of CD11b in B cells is Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Bacteroides vulgatus, or a combination thereof. [Item 5] 5. The composition according to any one of items 1 to 4, for treating a subject having a disease, disorder or condition that is treated by activation of germinal center migration of activated B cells. [Item 6] 5. The composition according to any one of items 1 to 4, for use in immunostimulation, maintenance or promotion of health, maintenance or improvement of mucosal bacterial flora, or treatment of an infectious disease. [Item 7] 5. The composition according to any one of items 1 to 4, which is a vaccine adjuvant. [Item 8] 8. The composition according to any one of items 1 to 7, which is administered mucosally. [Item 9] 7. The composition according to any one of items 1 to 6, which is in a form selected from the group consisting of medicines, foods, feeds, food additives, feed additives, and raw material compositions thereof. [Item 10] The composition according to item 1, for administration together with an antigen. [Item 11] 11. The composition according to item 10, which is administered simultaneously with an antigen. [Item 12] 11. The composition according to item 10, characterized in that it is administered separately from the administration of the antigen. [Item 13] 2. The composition according to item 1, which is a vaccine composition comprising an antigen. [Item 14] 14. The composition according to items 10 to 13, wherein the antigen is a viral antigen, a bacterial antigen, a parasitic antigen or a combination thereof. [Item 15] A method for inducing the expression of CD11b in B cells, inducing contact of the B cells with T cells and dendritic cells, and inducing migration of activated B cells to germinal centers, comprising preparing a composition containing a substance that induces expression of CD11b in B cells and contacting the B cells with the substance.

[0010] In another aspect, the present disclosure provides the following. [Item 16] A method for treating a subject having a disease, disorder, or condition that is treated by activating germinal center migration of activated B cells, the method comprising administering to the subject a composition comprising a substance that induces expression of CD11b in B cells. [Item 17] 17. The method of item 16, wherein the substance that induces expression of CD11b in B cells is a bacterium. [Item 18] 17. The method according to item 16, wherein the substance that induces expression of CD11b in B cells is a bacterium of the class Gammaproteobacteria or the class Bacteroidetes. [Item 19] 17. The method of item 16, wherein the agent that induces expression of CD11b in B cells is Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Bacteroides vulgatus, or a combination thereof. [Item 20] The method according to item 16, wherein the treatment of a disease, disorder or condition treated by activation of germinal center migration of activated B cells is immunostimulation, maintenance or promotion of health, maintenance or improvement of mucosal bacterial flora, or treatment of an infectious disease. [Item 21] 17. The method of claim 16, wherein the composition is formulated as a vaccine adjuvant. [Item 22] 17. The method of claim 16, wherein the composition is administered mucosally. [Item 23] Item 17. The method according to Item 16, wherein the composition is in a form selected from the group consisting of a medicine, a food, a feed, a food additive, a feed additive, and a raw material composition thereof. [Item 24] 17. The method according to item 16, wherein the composition is administered together with an antigen. [Item 25] 17. The method of claim 16, wherein the composition is administered simultaneously with the antigen. [Item 26] 17. The method of claim 16, wherein the composition is administered separately from the administration of the antigen. [Item 27] 17. The method of claim 16, wherein the composition is a vaccine composition comprising an antigen. [Item 28] 28. The method according to items 24 to 27, wherein the antigen is a viral, bacterial, fungal, or parasitic antigen. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing that only a portion of B cells enters the germinal center. [Diagram 2]Figure 2 shows that CD11b+IgA+ PP B cells are located outside GCs and migrate toward GCs. a, Representative flow cytometry data showing IgA and CD11b expression in PP B220+ B cells from WT mice. b,c, Flow cytometry of GC B cells in CD11b+IgA+ PP B cells stained with B220 and PNA (b) or B220 and Fas (c). a,c, Numbers indicate percentage of cells. d, Photomicrographs of PP cross sections stained with PNA, CD11b, IgA, and DAPI. Two CD11b+IgA+ double positive cells (yellow arrows) were found outside GCs (dashed circle). e, Images of SED, IF, and GC regions stained with ICAM-1, CD11c, CD4 (left photo), MAdCAM-1 (right photo), and DAPI. Each region is marked in the central schema. Scale bar, 500 μm. f, Images show that CD11b+IgA+ B cells (arrows, marked 1 to 7) are located in the indicated regions. GC and IF regions are indicated by dashed lines. g, CD11b-IgA+ B cells and CD11b+IgA+ B cells were directly injected into the PPs of IgA-Cre / YC3.60flox mice, respectively. Representative images showing the location of CD11b+IgA+ PP B cells (top) or CD11b-IgA+ PP B cells (bottom) engrafted into the PPs of IgA-Cre / YC3.60flox mice 1 h after injection. The GC border (dashed line) is the region enriched for IgA-YC3.60. Scale bar, 200 μm. h, 40 h after injection, PPs with injected CD11b+IgA+ B cells were observed under a microscope. Scale bar, 200 μm. Data are representative of three independent experiments. [Diagram 3]Figure 3 shows that CD11b+IgA+ B cells are pre-GC B cells that interact with CD4+ T cells. a, Heatmap showing log fold change (CD11b+IgA+ B cells / CD11b-IgA+ B cells) of the expression levels of the indicated genes by microarray analysis. b, Expression levels of the indicated GC-specific, non-GC-specific, and pre-GC genes were analyzed by qPCR and normalized to the expression level of the β-actin gene. The average relative expression level of CD11b-IgA+ B cells was set to 1. Each spot represents the average of technical triplicates. Bar graphs show the mean (±SD) of three to four independent measurements. c, Flow cytometry histograms showing the expression of CXCR4 and CD86 surface markers in CD11b+IgA+ and CD11b-IgA+ B cells. d, Flow cytometry data of CD4+ IgA+ cells conjugated with singlet IgA+ B cells. e, Flow cytometry data of CD4+ T cells conjugated with CD11b-IgA+ and CD11b+IgA+ PP B cells. f, Percentage of CD4+ T cells conjugated with CD11b+IgA+ and CD11b-IgA+ PP B cells. Bar graphs show the mean (±SD) of six independent measurements. g, Photograph of CD11b+IgA+ PP B cells conjugated with CD4+ T cells. Scale bar, 20 μm. [Figure 4-1]Figure 4-1 shows that bacterial antigens induce CD11b expression in B cells. a,f,Flow cytometric data of CD11b expression in splenic naive B cells stimulated for 3 days as indicated. b,g,Percentage of CD11b+ B cells after each stimulation. c,h,qPCR data of itgam of B cells isolated after the indicated stimulation. d,Flow cytometric data of CD11b+IgA+ and CD11b-IgA+ PP B cells isolated after 1 day of culture on 40LB cells. e,CD11b expression by Pam3CSK4 was lost by culture on 40LB cells. i,Percentage of CD11b+ B cells after each stimulation with the indicated inhibitors. b,c,g,h,i,Bar graphs show the mean (±SD) of at least three independent determinations. b, c, g, h, Data were analyzed by one-way ANOVA and Tukey's multiple comparisons. [Figure 4-2] FIG. 4-2 shows that heat-killed Proteobacteria bacteria Klebsiella pneumoniae and Bacteroides vulgatus induce CD11b expression in B cells. [Diagram 5]Figure 5 shows that CD11b-inducible bacterial antigens enhance pre-existing GC responses in PP. a, Flow cytometry data of CD11b expression in splenic naive B cells stimulated for 3 days with the indicated stimuli. b, Percentage of CD11b+ B cells at each stimulus. c, Balb / c mice were orally administered the indicated heat-killed bacteria, pam3CSK4, or PBS (control). Two days later, flow cytometry data (c) of PNAhiB220+ GC B cells, and the percentage (d) and absolute number (e) of PNAhighB220+ PP GC B cells were calculated for each mouse. f, Indicated iGB cells were isolated, labeled, and then injected intravenously into mice independently. Scale bar, 500 μm. g, Immunization schedule. h, OVA-specific IgA after immunization. b, d, e, h, Bar graphs show the mean (± SD) of at least three independent measurements. d,e,Data were analyzed by one-way ANOVA and Tukey's multiple comparison.h,Data were analyzed by two-way ANOVA and Tukey's multiple comparison. [Figure 6]Figure 6 shows the presence of CD11b+IgA+ B cells in mouse PPs. a, Flow cytometry analysis was performed using 8-12 week old WT Balb / c mice. Doublet cells were identified by FSC-H / FSC-W gating. Dead cells were excluded by propidium iodide (PI) staining. IgA and CD11b expression was analyzed in B220+ B cells. To confirm CD11b expression, fluorescence minus one (FMO) samples were prepared as negative controls. In live cells, GC B cells represent PNAhighB220+ cells, and non-GC B cells represent PNAlowB220+ cells. Representative data are from at least 20 experiments. b, Sorting purity was confirmed to be at least 90%. c, Representative mean fluorescence intensity (MFI) analysis of CD86 and CXCR4 expression in CD11b+IgA+ PP B cells and CD11b-IgA+ PP B cells. Bar graphs show the mean (±SD) of five independent measurements in WT mice. Data were compared by two-tailed Student's t test. [Figure 7] Figure 7 shows immunohistochemical confocal images confirming the expression of CD11b and IgA. Selected PP sections were stained with DAPI, CD11b, and IgA. The indicated area (dashed box) was amplified and analyzed for overlap of CD11b and IgA. Z-stack analysis was also performed to confirm the overlap of CD11b and IgA signals. Scale bar, 20 μm. [Figure 8] Figure 8 shows that ICAM-1 is expressed on HEVs. PP sections were stained with anti-ICAM-1, anti-CD4 (T cell marker), anti-CD11c (DC marker), and anti-MAdCAM-1 (HEV marker), respectively. SEDs were identified in areas rich in CD11c+ DCs. IF areas were identified in areas enriched in CD4+ T cells. HEVs expressing MAdCAM-1 were found in the IF areas. To analyze ICAM-1 expression, whole image and amplified display areas were shown. [Figure 9]Figure 9 shows the results of conjugation analysis of CD11b+IgA+ B cells and CD4+ T. Flow cytometry analysis was performed using 8-12 week old WT Balb / c mice. Dead cells were excluded by PI staining. Single cells and conjugated cells were selected using FSC-H / FSC-W gate. For single cells and conjugated cells, IgA+ cells were used to analyze conjugation with CD4+T cells. For single live cells and live conjugated cells, B cell gate was selected using B220+ cells. Expression of IgA and CD11b in B220+ cells was analyzed. Conjugation of CD4+T cells was analyzed in conjugated CD11b-IgA+ B cells and CD11b+IgA+ B cells. Single CD11b-IgA+ B cells and CD11b+IgA+ B cells were used as negative controls to analyze the conjugation of CD4+ T cells. Data are from six independent experiments. [Figure 10] Figure 10 shows that CD11b+IgA+ B cells with SHM are derived from newly born activated memory B cells. a, SHM frequency of PCR-amplified rearranged VDJ regions and downstream intronic sequences was analyzed. b, SHM frequency of rearranged VDJH3 and JH3 downstream intronic sequence (JH3-I) and rearranged VDJH4 and JH4 downstream intronic sequence (JH4-I) was analyzed in CD11b-IgA+ and CD11b+IgA+ B cells isolated from PPs of 26 WT mice. The segment size of the pie chart is proportional to the number of sequences with the number of mutations observed in each clone. The number in the center of the pie chart represents the number of clones analyzed. [Figure 11-1]Figure 11-1 shows the B cell proliferation and CD11b expression by in vitro stimulation. a, Purity and CD11b expression of sorted splenic naive B cells. b, Lymphocyte gate and number (right) of cultured splenic naive B cells. Representative data are obtained from at least three independent experiments. c, Lymphocyte gate of cultured splenic naive B cells after addition of the indicated heat-killed bacterial stimuli. d, Lymphocyte gate and CD11b expression of cultured splenic naive B cells. Representative data are obtained from three independent experiments. [Figure 11-2] Figure 11-2 shows the B cell proliferation and CD11b expression by in vitro stimulation. e, CD11b expression of splenic B cells stimulated with the indicated concentrations of each stimulus. The conditions selected for the experiments in Figures 4-1 and 5 are shown in light gray. [Figure 11-3] Figure 11-3 shows B cell proliferation and CD11b expression by in vitro stimulation. f, CD11b expression of purified splenic B cells stimulated with the indicated concentrations of each stimulus. g, CD11b expression of splenic B cells stimulated with pam3CSK4 (1 μg / ml) in the presence of the indicated concentrations of TLR1 / 2, TLR4, and NOD2 inhibitors. The conditions selected for the experiments in Figures 4-1 and 5 are shown in light gray. [Figure 12-1] Figure 12-1 shows that isolated CD11b+ B cells lost CD11b expression in the iGB culture system. a, CellTrace Violet-labeled CD11b+IgA+ and CD11b-IgA+ PP B cells were cultured in the iGB culture system with IL-21. CD11b expression was analyzed by flow cytometry on days 0 and 1. b, Approximately 50,000 naive splenic B cells and 5,000 sorted CD11b-IgA+ B cells labeled with CellTrace Violet were prepared as positive controls. 800 isolated CD11b+IgA+ PP B cells and CD11b-IgA+ PP B cells were prepared to monitor their proliferation. Representative data are from three independent experiments. [Figure 12-2]Figure 12-2 shows that isolated CD11b+ B cells lost CD11b expression in iGB culture system. c, Splenic naive B cells were cultured with pam3CSK4 for 3 days to induce CD11b+ B cells. Then, pam3CSK4-induced CD11b+ B cells and CD11b- B cells were isolated. Approximately 1.5×105 isolated CD11b+ B cells and CD11b- B cells were separately seeded on 40LB cells supplemented with 1ng / ml IL-4. Negatively sorted splenic naive B cells were used as a control. CD11b expression of B cells seeded on 40LB cells was analyzed at D0 and D4. Representative data are from three independent experiments. [Figure 13] Figure 13 shows that B cells stimulated with IgM-40LB or CpG-40LB do not enter PP GC after intravenous injection in mice. The indicated iGB cells, including CpG-40LB cells (top) and IgM-40LB cells (bottom), were isolated, labeled, and then independently injected intravenously into mice. Anti-MAdCAM-1 antibody was injected intravenously along with the labeled iGB cells to identify HEV. Anti-IgA antibody was injected directly into the PP to stain IgA+ cells and identify GC (dashed circle). Scale bar, 500 μm. [Figure 14] FIG. 14 shows that the adjuvant composition of the present disclosure did not increase serum TNFα levels at all. [Figure 15] FIG. 15 is a schematic diagram showing that B cells can sense bacterial antigens and determine their cell fate to pre-GC cells, independently of DCs or T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] To identify pre-GC B cells, we focused on mucosal immune-associated immune-inducing tissues, where GCs are constitutively active, allowing us to find pre-GC B cells residing in existing GCs at any time point. 7We hypothesized that integrin CD11b might be a potential marker for pre-GC IgA+ B cells in mucosal immune-associated inductive tissues. CD11b, a 165 kDa integrin αM, binds to CD18 to form a heterodimeric integrin known as macrophage-1 antigen (Mac-1). 12 CD11b is widely known as a marker for myeloid cells and is known to be involved in cell migration and adhesion. 12 Furthermore, previous studies have demonstrated complex functions of CD11b in B cells. 13,14 Ding et al. showed that CD11b negatively regulates BCR signaling and maintains tolerance of autoreactive B cells. 13 Kunisawa et al. reported that CD11b is involved in the IgA expression in the lamina propria (LP) of mouse intestinal mucosa. + CD11b is an early marker of PC + IgA + The PP structure is essential for PC production. 15 These studies motivated us to investigate the expression of CD11b on B cells in mucosal immune-related immune-inducing tissues.

[0013] In the present disclosure, IgA expressing integrin CD11b + We identified a small population of B cells as pre-GC B cells, which were located outside of GCs and highly expressed irf4. + IgA + B cells are transformed with IgA - Cre / YC3.60 flox When the cells were injected into the mucosal immune-related immune induction tissue of reporter mice, the injected cells did not enter the existing GCs directly, but were located around the GCs, and entered the GCs 40 hours later. This suggests that pre-GC IgA is involved in the mucosal immune-related immune induction tissue of mice. +We found that B cells were the primary B cells that expressed CD11b. Furthermore, we found that several bacterial antigens, including pam3CSK4, lipopolysaccharide (LPS), and heat-killed Escherichia coli (E. coli) and Salmonella enterica (S. enterica), but not Bifidobacterium, induced the expression of CD11b in naive B cells in vitro. Expression of CD11b on B cells prior to interaction with TB or DC-B allowed intravenously injected B cells to enter pre-existing GCs in mucosal immune-associated immune-inducing tissues. Thus, B cells control their cell fate to become pre-GC B cells, independent of DC activation. Furthermore, oral administration of pam3CSK4 or heat-killed E. coli to mice increased the number of B cells that entered GCs in mucosal immune-associated immune-inducing tissues within 2 days and enhanced mucosal antigen-specific IgA responses. Induction of CD11b on activated B cells may be a promising marker of pre-GC B cells as well as a useful criterion for selecting effective mucosal vaccine adjuvants.

[0014] (Composition of the present invention) The composition of the present disclosure contains a substance that induces expression of CD11b in B cells, and has the effect of inducing germinal center migration of activated B cells. The composition of the present disclosure is expected to exert the above-mentioned effects and can be suitably used in the fields of medicines, foods, feeds, etc. Therefore, the composition of the present disclosure can be a medicine, a food composition, a food ingredient composition, a feed composition, or a feed ingredient composition.

[0015] (Substance that induces CD11b expression in B cells) The substance that induces the expression of CD11b in B cells used in the composition of the present disclosure can be obtained by a person skilled in the art based on a known method by referring to the examples shown in the present specification. For example, after culturing naive splenic B cells isolated from an animal with a candidate substance, the expression of CD11b in B cells can be +A candidate compound that increases the proportion of B cells can be used as a substance that induces the expression of CD11b in B cells.

[0016] In one embodiment, a non-limiting example of a substance that induces expression of CD11b in the B cells is a bacterium. In one embodiment, it is preferable to use a gram-negative bacterium. In one embodiment, it is preferable to use a bacterium belonging to the phylum Proteobacteria or Bacteroidetes. In one embodiment, it is preferable to use a bacterium belonging to the class Gammaproteobacteria as the bacterium belonging to the phylum Proteobacteria. In one embodiment, it is preferable to use a bacterium belonging to the order Enterobacteriaceae. In one embodiment, it is more preferable to use Escherichia coli, Salmonella enterica, or Klebsiella pneumoniae. In another embodiment, it is preferable to use a bacterium belonging to the class Bacteroidetes as the bacterium belonging to the phylum Bacteroidetes. In one embodiment, it is preferable to use a bacterium belonging to the order ... As the bacterium belonging to the phylum Proteobacteria or Bacteroidetes, various serotypes of a particular species of bacteria can be used.

[0017] In one embodiment, the bacterium used as a substance that induces expression of CD11b in the B cells is a bacterium having a 16S rDNA sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 98% or more, 99% or more, or 99.5% or more nucleotide sequence identity with the 16S rDNA sequences of representative serotypes of Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Bacteroides vulgatus.

[0018] In one aspect, the bacterium used as a substance that induces expression of CD11b in the B cells is a bacterium having a 16S rDNA sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 98% or more, 99% or more, or 99.5% or more of base sequence identity to the 16S rDNA sequence below. Escherichia coli 16S rDNA sequence: DNA sequence encoding 16S ribosomal RNA of GenBank accession number CP003278.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP017100) (sequence consisting of bases 225107 to 226662). Salmonella enterica 16S rDNA sequence: DNA sequence encoding 16S ribosomal RNA of GenBank accession number CP003278.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP003278.1) (complementary strand of the sequence containing bases from 287486 to 289015, or complementary strand of the sequence containing bases from 3396200 to 3397729). Klebsiella pneumoniae 16S rDNA sequence: A DNA sequence encoding 16S ribosomal RNA of GenBank accession number CP018306.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP018306) (the complementary strand of the sequence including bases at positions 503553-505106, the complementary strand of the sequence including bases at positions 1312903-1314456, the sequence including bases at positions 1818027-1819580, the sequence including bases at positions 1926864-1928417, the sequence including bases at positions 2018746-2020299, the sequence including bases at positions 2444117-2445670, or the sequence including bases at positions 2799428-2800981). Bacteroides vulgatus 16S rDNA sequence: 16S ribosomal DNA of GenBank accession number AP025232.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / AP025232) A DNA sequence encoding an RNA (the complementary strand of the sequence containing the bases at positions 294690-296214, the complementary strand of the sequence containing the bases at positions 626688-628212, the complementary strand of the sequence containing the bases at positions 1871179-1872703, the complementary strand of the sequence containing the bases at positions 1957949-1959473, the complementary strand of the sequence containing the bases at positions 2375844-2377368, the complementary strand of the sequence containing the bases at positions 4187249-4188773, or the complementary strand of the sequence containing the bases at positions 4496890-4498414).

[0019] The composition of the present disclosure has an effect of inducing the migration of activated B cells to germinal centers. In one embodiment, the composition of the present disclosure has an effect of inducing the migration of activated B cells to germinal centers of mucosal immunity-related immune-inducing tissues. The mucosal immunity-related immune-inducing tissues are not particularly limited, and include mucosal immunity-related immune-inducing tissues that bring about affinity maturation of activated B cells. For example, mucosal immunity-related immune-inducing tissues include mucosal immunity-related immune-inducing tissues in the digestive tract (intestine, oral cavity, etc.) and nasal cavity. In one embodiment, the mucosal immunity-related immune-inducing tissue is Peyer's patch, and the composition of the present disclosure has an effect of inducing the migration of activated B cells to germinal centers of Peyer's patch.

[0020] When bacteria are used as a substance for inducing CD11b expression in B cells, heat-sterilized or live bacteria can be used.

[0021] In the composition of the present disclosure, the amount of the substance that induces the expression of CD11b in B cells is not particularly limited as long as it is an amount that can exert an effect of inducing germinal center migration of activated B cells. For example, the substance that induces the expression of CD11b in B cells is blended in the composition so as to be in the range of 0.001 to 99.99% by weight.

[0022] Pharmaceutical Composition In one embodiment, the composition comprising the substance that induces the expression of CD11b in B cells of the present disclosure is provided as a pharmaceutical composition. The pharmaceutical composition of the present disclosure is typically used to stimulate immunity. In one embodiment, the pharmaceutical composition of the present disclosure is used to induce germinal center migration activation and affinity maturation of IgA-producing B cells and high affinity antibody production, and to provide antigen-specific protection in the mucosal tissue or a distant mucosal tissue, etc. In one embodiment, the pharmaceutical composition of the present disclosure is used to activate germinal center migration of IgA-producing B cells in a mucosal immunity-related immune-inducing tissue. The mucosal immunity-related immune-inducing tissue is not particularly limited, and includes a mucosal immunity-related immune-inducing tissue that brings about affinity maturation of activated B cells. For example, the mucosal immunity-related immune-inducing tissue includes a mucosal immunity-related immune-inducing tissue in the digestive tract (in the intestine, in the oral cavity, etc.) and in the nasal cavity. In one embodiment, the mucosal immunity-related immune-inducing tissue is a Peyer's patch, and the composition of the present disclosure is used to induce migration of activated B cells to the germinal center of Peyer's patch.

[0023] The antigen targeted by the immunity stimulated by the pharmaceutical composition of the present disclosure may be one that is already present in the mucosal tissue in the living body, or may be one that is administered together with the pharmaceutical composition of the present disclosure. In one embodiment, the pharmaceutical composition of the present disclosure is used to stimulate immunity. In one embodiment, the pharmaceutical composition of the present disclosure is preferably used to stimulate mucosal immunity, and in one embodiment, it is used to stimulate immunity in the digestive tract (in the intestine, in the oral cavity, etc.) or in the nasal cavity.

[0024] In one embodiment, the pharmaceutical composition of the present disclosure is provided as an adjuvant composition that does not contain an antigen. In one embodiment, the pharmaceutical composition of the present disclosure is administered together with an antigen. In one embodiment, the pharmaceutical composition of the present disclosure is administered simultaneously with an antigen. In one embodiment, the pharmaceutical composition of the present disclosure is administered separately from an antigen. When the pharmaceutical composition of the present disclosure and the antigen are administered separately, the interval between each administration can be appropriately selected, for example, the interval between each administration can be 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc., but is not limited thereto. The frequency of administration of the pharmaceutical composition of the present disclosure and the antigen may be the same or different. The form of the pharmaceutical composition of the present disclosure and the composition containing the antigen may be the same or different, for example, the pharmaceutical composition of the present disclosure and a composition containing the antigen in a food form may be combined. In one embodiment, the pharmaceutical composition of the present disclosure is provided as a vaccine composition containing an antigen. The antigen is not particularly limited, but may be selected from, for example, viral, bacterial, fungal, and parasitic antigens.

[0025] In one embodiment, the pharmaceutical composition of the present disclosure is used to treat a subject having a disease, disorder or condition that is treated by activating germinal center migration of activated B cells. In one embodiment, the composition of the present disclosure is used for maintaining or promoting health through immunostimulation (e.g., mucosal immunostimulation, intestinal immunostimulation), maintaining or improving mucosal flora (e.g., intestinal flora), or suppressing infection (e.g., suppressing infection by viruses, bacteria, fungi, or parasites).

[0026] Thus, in one aspect, the present disclosure provides a pharmaceutical composition for inducing germinal center migration of activated B cells, comprising a substance that induces expression of CD11b in B cells.

[0027] In one aspect, the present disclosure provides a pharmaceutical composition for treating a subject having a disease, disorder, or condition that is treated by activating germinal center migration of activated B cells, comprising a substance that induces expression of CD11b in B cells.

[0028] In one embodiment, the present disclosure provides a pharmaceutical composition for immune activation, comprising a substance that induces expression of CD11b in B cells. The activation of mucosal immunity includes, but is not limited to, activation of gastrointestinal immunity (e.g., intestinal immunity and oral immunity) and nasal immunity.

[0029] In one aspect, the present disclosure provides a pharmaceutical composition for stimulating antigen-specific immunity in mucosa, comprising a substance that induces expression of CD11b in B cells. The mucosa includes, but is not limited to, the digestive tract (e.g., intestinal mucosa and oral mucosa) and nasal mucosa.

[0030] In one aspect, the immune activation includes, but is not limited to, a health maintaining or promoting effect involving immune activation (e.g., mucosal immune activation, intestinal immune activation), a health maintaining or promoting effect involving maintenance or improvement of mucosal flora (e.g., intestinal flora), or treatment of infectious diseases (e.g., treatment of viral infections, bacterial infections, fungal infections, or parasitic infections).

[0031] Also, in one aspect, the subject in need of activating antigen-specific immunity in the mucosa is a subject in need of immune activation (e.g., mucosal immune activation, intestinal immune activation), a subject in need of health maintenance or promotion involving immune activation, a subject in need of maintenance or improvement of mucosal flora (e.g., intestinal flora), a subject in need of health maintenance or promotion involving mucosal flora, or a subject in need of treatment of an infectious disease (e.g., treatment of a viral infection, bacterial infection, fungal infection, or parasitic infection).

[0032] Also, in one aspect, the present disclosure provides a composition for maintaining or promoting health through immune activation, comprising a substance that induces expression of CD11b in B cells.

[0033] Also, in one aspect, the present disclosure provides a composition for maintaining or improving mucosal flora (eg, intestinal flora) through immune stimulation, the composition comprising a substance that induces expression of CD11b in B cells.

[0034] In one aspect, the present disclosure provides a pharmaceutical composition for treating an infectious disease (e.g., treating a viral infection, bacterial infection, fungal infection or parasitic infection), comprising a substance that induces expression of CD11b in B cells.

[0035] As used herein, "treatment" has its general meaning in the art and refers to the administration of a composition of the present disclosure to a subject for the purposes of ameliorating, eradicating, or curing any disorder or disease state, preventing or delaying the onset of such disorder or disease state (prophylaxis), or preventing or reducing the likelihood of recurrence of such disorder or disease state (recurrence prevention).

[0036] In one embodiment, the pharmaceutical composition of the present disclosure is a pharmaceutical composition for stimulating mucosal immunity.In one embodiment, the pharmaceutical composition of the present disclosure is a composition used as a mucosally administered vaccine adjuvant.In one embodiment, the pharmaceutical composition of the present disclosure is a composition used to promote the expression of high affinity mucosal IgA antibody.

[0037] The pharmaceutical composition of the present disclosure may contain a substance that induces expression of CD11b in B cells. For example, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the pharmaceutical composition can be appropriately set to be in the range of 0.001 to 99.99% by weight, taking into consideration the type of disease to be treated, dosage form, administration method, subjects, the severity of symptoms in subjects, the degree of effect exerted by administration, and the like. In one aspect, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the pharmaceutical composition is 0.001 to 99.99% by weight, 0.01 to 99.99% by weight, 0.1 to 99.99% by weight, 0.2 to 99.99% by weight, 0.3 to 99.99% by weight, 0.4 to 99.99% by weight, 0.5 to 99.99% by weight, 0.6 to 99.99% by weight, 0.7 to 99.99% by weight, 0.8 to 99.99% by weight, 0.9 to 99.99% by weight, 1.0 to 99.99% by weight, 2.0 The weight percentage may be up to 99.99%, 3.0 to 99.99% by weight, 4.0 to 99.99% by weight, 5.0 to 99.99% by weight, 6.0 to 99.99% by weight, 7.0 to 99.99% by weight, 8.0 to 99.99% by weight, 9.0 to 99.99% by weight, 10 to 99.99% by weight, 20 to 99.99% by weight, 30 to 99.99% by weight, 40 to 99.99% by weight, 50 to 99.99% by weight, 60 to 99.99% by weight, 70 to 99.99% by weight, 80 to 99.99% by weight, or 90 to 99.99% by weight. In one aspect, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the pharmaceutical composition may be 0.001 to 90% by weight, 0.001 to 80% by weight, 0.001 to 70% by weight, 0.001 to 60% by weight, 0.001 to 50% by weight, 0.001 to 40% by weight, 0.001 to 30% by weight, 0.001 to 20% by weight, 0.001 to 10% by weight, 0.001 to 9.0% by weight, 0.001 to 8.0% by weight, 0.001 to 7.0% by weight, 0.001 to 6.0% by weight, 0.001 to 5.0% by weight, 0.001 to 4.0% by weight, 0.001 to 3.0% by weight, 0.001 to 2.0% by weight, or 0.001 to 1.0% by weight.In one aspect, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the pharmaceutical composition is preferably, but is not limited to, 1.0 to 80% by weight, 5.0 to 70% by weight, 10 to 60% by weight, 20 to 50% by weight, or 30 to 40% by weight.

[0038] In the present disclosure, the term "effective amount" refers to an amount of a substance that induces CD11b expression in B cells according to the present disclosure that can induce germinal center migration of activated B cells in a living body.

[0039] The pharmaceutical composition according to the present disclosure may contain a pharma- ceutically acceptable carrier or additive together with the substance that induces the expression of CD11b in B cells. The pharma- ceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known carrier or additive may be used.

[0040] The subjects of administration are not particularly limited, and non-limiting examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, horses, deer, wild boars, sheep, and goats; poultry birds such as chickens, ducks, turkeys, and quails; reptiles such as snakes and turtles; amphibians such as frogs; salmonids, yellowtail, cobia, freshwater trout, sweetfish, sea bass, sea bream, and meagre fish; flounder, pufferfish, yellowtail, and kanchi. Examples of fish include Pacific bluefin tuna, horse mackerel, striped jack, tiger pufferfish, yellowtail amberjack, stone bream, filefish, rockfish, scorpion fish, black porgy, black rockfish, sea bass, red sea bream, grunt, black sea bream, chub mackerel, spotted grouper, grouper, eel, catfish, loach, sturgeon, tropical fish, tilapia, pangasius, Japanese bluefin tuna, bighead carp, silver carp, grass carp, and carp, as well as bivalve shells (oysters, scallops, clams, mussels, and pearl oysters), abalone, shrimp, and seaweed.

[0041] The dosage and administration method of the pharmaceutical composition of the present disclosure are appropriately selected depending on the type of disease suffered by the individual to be administered, the sex, species, age, general condition, severity of the disease, the degree of the desired effect, etc. The dosage may be appropriately set in the range of 0.001 to 100 mg / kg / day in general. For example, when bacteria are used as a substance that induces the expression of CD11b in B cells, for example, 10 mg / kg / day for a mouse weighing 20 g is used based on the amount of bacteria. 6 CFU / time or more, 10 7 CFU / time or more, 10 8 CFU / time or more, 10 9 CFU / time or more, 10 10 CFU / time or more, for a human weighing 60 kg, it is 3x10 9 CFU / time or more, 3x10 10 CFU / time or more, 3x10 11 CFU / time or more, 3x10 12 CFU / time or more, 3x10 13 The dosage may be appropriately set within the range of, but is not limited to, 1 to 3 times a day. In one embodiment, when used for a human weighing 60 kg, the dosage is 3x10 10 CFU / day~3x10 13 CFU / day, 3x10 11 CFU / day~3x10 12 It is preferable to administer in the range of, but not limited to, CFU / day.

[0042] The pharmaceutical composition according to the present disclosure may be administered in the above amount once a day or in several divided doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a therapeutic effect against the above diseases.

[0043] The method of administration of the pharmaceutical composition of the present disclosure is not particularly limited, but it is preferably administered directly to mucosal tissues such as the digestive tract, and examples of such transmucosal administration include oral administration, nasal administration, vaginal administration, and enteral administration. The composition of the present disclosure administered through the mucosa of these various organs induces affinity maturation of IgA-secreting B cells and production of high-affinity IgA antibodies in mucosal immunity-related immune-inducing tissues, resulting in antigen-specific protection in tissues such as the administered mucosal tissue and distal mucosal tissues.

[0044] Here, enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but may include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc. In addition, for example, the composition of the present disclosure may be mixed with known components and used as an intestinal lavage solution.

[0045] In one embodiment, the pharmaceutical composition of the present disclosure can be used in combination with other therapeutic agents, such as vaccines containing antigens, to the extent that the function of the composition is not impaired.

[0046] When the pharmaceutical compositions of the present disclosure are used in combination, or when they are used in combination with other therapeutic agents, these agents may be included in a single formulation or may be included in separate formulations. When these are provided in separate formulations, they may be provided in the form of a kit containing the combination of formulations, or each may be provided as a single agent to be combined with other formulations. These formulations may be administered simultaneously or sequentially. In one embodiment, the pharmaceutical composition of the present disclosure is provided in the form of a raw material composition for the final pharmaceutical composition.

[0047] (Food composition) In one embodiment, the composition comprising the substance that induces CD11b expression in B cells of the present disclosure is provided as a food composition. The food composition is a composition that is suitable for exclusively using the immunostimulatory composition of the present disclosure in the field of food.

[0048] The form of the food composition of the present disclosure is not particularly limited, and can be provided as food and drink such as supplements, solid foods, liquid foods, and beverages. The food composition of the present disclosure can be provided as special purpose foods such as foods for specified health uses, functional foods, and nutritional functional foods. The food composition of the present disclosure can be provided as health functional foods, foods for the sick, dairy products, fermented milk, modified milk powder, lactic acid bacteria drinks, acidic drinks, yogurt, cheese, bread, biscuits, crackers, pizza crust, modified milk powder, liquid foods, foods for the sick, nutritional foods, frozen foods, food compositions, processed foods, and other commercially available foods.

[0049] The food compositions of the present disclosure include general foods, as well as foods for specified health uses including conditional foods for specified health uses, nutritional supplements, functional foods, foods for the sick, etc. In one aspect, the food compositions of the present disclosure can be provided as food compositions labeled, for example, for immunostimulation, mucosal immunity stimulation, intestinal immunity stimulation, for maintaining or promoting health, for maintaining or improving mucosal bacterial flora, for maintaining or improving intestinal bacterial flora, for combating viral infections, for combating bacterial infections, for combating fungal infections, or similar, or as food compositions for use for these purposes.

[0050] In one embodiment, the food composition of the present disclosure is administered together with the antigen. In one embodiment, the food composition of the present disclosure is administered simultaneously with the antigen. In one embodiment, the food composition of the present disclosure is administered separately from the antigen. When the food composition of the present disclosure and the antigen are administered separately, the administration intervals of each can be appropriately selected, for example, the intervals between each administration can be 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc., but are not limited thereto. The administration frequencies of the food composition of the present disclosure and the antigen may be the same or different. The forms of the food composition of the present disclosure and the composition containing the antigen may be the same or different, for example, the food composition of the present disclosure may be combined with a pharmaceutical composition containing an antigen. In one embodiment, the food composition of the present disclosure is provided as a composition containing an antigen. The above antigen is not particularly limited, but for example, a virus, bacteria, fungus, or parasite antigen is selected.

[0051] The specific form of the food composition of the present disclosure is not particularly limited, but examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; frozen desserts such as ice cream, ice sorbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-based processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; seasonings such as sauces and dressings; soups, stews, salads, side dishes, furikake, pickles, bread, cereals, and the like. In the case of foods for specified health uses, nutritional supplements, functional foods, etc., they can be provided in the form of, for example, powder, granules, capsules, troches, tablets, syrups, etc.

[0052] In one embodiment, the composition of the present disclosure is provided as a food material. The shape of the food material of the present disclosure is not particularly limited, and may be, for example, liquid, paste, powder, solid, etc., appropriately set according to the type of food to be used. The food material of the present disclosure may be further formed into a desired shape by crushing, grinding, granulation, etc., as necessary.

[0053] The food composition of the present disclosure may contain a substance that induces the expression of CD11b in B cells according to the present disclosure. For example, the content of the antibody according to the present invention in 100% by weight of the composition can be appropriately set to a range of 0.001 to 99.99% by weight, taking into consideration the degree of the effect to be exerted, etc. In one aspect, the content of the substance that induces CD11b expression in B cells according to the present invention in 100% by weight of the food composition is 0.001 to 99.99% by weight, 0.01 to 99.99% by weight, 0.1 to 99.99% by weight, 0.2 to 99.99% by weight, 0.3 to 99.99% by weight, 0.4 to 99.99% by weight, 0.5 to 99.99% by weight, 0.6 to 99.99% by weight, 0.7 to 99.99% by weight, 0.8 to 99.99% by weight, 0.9 to 99.99% by weight, 1.0 to 99.99% by weight, 2.0 The weight percentage may be up to 99.99%, 3.0 to 99.99% by weight, 4.0 to 99.99% by weight, 5.0 to 99.99% by weight, 6.0 to 99.99% by weight, 7.0 to 99.99% by weight, 8.0 to 99.99% by weight, 9.0 to 99.99% by weight, 10 to 99.99% by weight, 20 to 99.99% by weight, 30 to 99.99% by weight, 40 to 99.99% by weight, 50 to 99.99% by weight, 60 to 99.99% by weight, 70 to 99.99% by weight, 80 to 99.99% by weight, or 90 to 99.99% by weight. In one aspect, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the food composition may be 0.001 to 90% by weight, 0.001 to 80% by weight, 0.001 to 70% by weight, 0.001 to 60% by weight, 0.001 to 50% by weight, 0.001 to 40% by weight, 0.001 to 30% by weight, 0.001 to 20% by weight, 0.001 to 10% by weight, 0.001 to 9.0% by weight, 0.001 to 8.0% by weight, 0.001 to 7.0% by weight, 0.001 to 6.0% by weight, 0.001 to 5.0% by weight, 0.001 to 4.0% by weight, 0.001 to 3.0% by weight, 0.001 to 2.0% by weight, or 0.001 to 1.0% by weight. In one aspect, the content of the substance that induces expression of CD11b in B cells according to the present invention in 100% by weight of the food composition is preferably, but is not limited to, 1.0 to 80% by weight, 5.0 to 70% by weight, 10 to 60% by weight, 20 to 50% by weight, or 30 to 40% by weight.

[0054] In one embodiment, the composition of the present disclosure is provided as a food additive to be added when the above-mentioned food is ingested, or as a raw material composition for producing the above-mentioned food or food additive.

[0055] (Feed composition) In one embodiment, the composition comprising the substance that induces expression of CD11b in B cells of the present disclosure is provided as a feed composition. The feed composition is a composition that is used exclusively in the field of feed, such as the feed composition of the present disclosure.

[0056] The specific form of the above-mentioned feed composition is not particularly limited, and for example, as long as the effect of the feed composition according to the present disclosure described above is not impaired, the feed composition may be prepared by mixing it with normal feed or, if necessary, by mixing it with components that can be incorporated into normal feed, or the feed composition itself may be used as feed.

[0057] The feed composition of the present disclosure can be provided as a feed composition labeled, for example, for immunostimulation, mucosal immunity stimulation, intestinal immunity stimulation, for maintaining or improving mucosal bacterial flora, for maintaining or improving intestinal bacterial flora, for maintaining or promoting health, for maintaining or improving intestinal bacterial flora, for combating viral infections, for combating bacterial infections, for combating fungal infections, or similar purposes, or as a feed composition for use for these purposes.

[0058] In one embodiment, the feed composition of the present disclosure is administered together with the antigen. In one embodiment, the feed composition of the present disclosure is administered simultaneously with the antigen. In one embodiment, the feed composition of the present disclosure is administered separately from the antigen. When the feed composition of the present disclosure and the antigen are administered separately, the administration intervals of each can be appropriately selected, for example, the intervals between each administration can be 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc., but are not limited thereto. The administration frequencies of the feed composition of the present disclosure and the antigen may be the same or different. The feed composition of the present disclosure and the composition containing the antigen may be in the same or different forms, for example, the feed composition of the present disclosure and the pharmaceutical composition containing the antigen may be combined. In one embodiment, the feed composition of the present disclosure is provided as a composition containing an antigen. The above antigen is not particularly limited, but for example, a virus, bacteria, fungus, or parasite antigen is selected.

[0059] The feed composition of the present disclosure may contain a substance that induces the expression of CD11b in B cells according to the present disclosure. For example, the content of the antibody according to the present invention in 100% by weight of the composition can be appropriately set to a range of 0.001 to 99.99% by weight, taking into consideration the degree of the effect to be exerted, etc. In one aspect, the content of the substance that induces CD11b expression in B cells according to the present invention in 100% by weight of the feed composition is 0.001 to 99.99% by weight, 0.01 to 99.99% by weight, 0.1 to 99.99% by weight, 0.2 to 99.99% by weight, 0.3 to 99.99% by weight, 0.4 to 99.99% by weight, 0.5 to 99.99% by weight, 0.6 to 99.99% by weight, 0.7 to 99.99% by weight, 0.8 to 99.99% by weight, 0.9 to 99.99% by weight, 1.0 to 99.99% by weight, 2.0 to 99.99% by weight, 3.0 to 99.99% by weight, 4.0 to 99.99% by weight, 5.0 to 99.99% by weight, 6.0 to 99.99% by weight, 7.0 to 99.99% by weight, 8.0 to 99.99% by weight, 9.0 to 99.99% by weight, 10.0 to 99.99% by weight, 11.0 to 99.99% by weight, 12.0 to 12.0% by weight, 13.0 to 12.0% by weight, 14.0 to 12.0% by weight, 15.0 to 12.0% by weight, 16.0 to 12.0% by weight, 17.0 to 12.0% by weight, 18.0 to 12.0% by weight, 19.0 to 12.0% by weight, 20.0 to 22.0% by weight, 21 The weight percentage may be up to 99.99%, 3.0 to 99.99% by weight, 4.0 to 99.99% by weight, 5.0 to 99.99% by weight, 6.0 to 99.99% by weight, 7.0 to 99.99% by weight, 8.0 to 99.99% by weight, 9.0 to 99.99% by weight, 10 to 99.99% by weight, 20 to 99.99% by weight, 30 to 99.99% by weight, 40 to 99.99% by weight, 50 to 99.99% by weight, 60 to 99.99% by weight, 70 to 99.99% by weight, 80 to 99.99% by weight, or 90 to 99.99% by weight. In one aspect, the content of the substance that induces CD11b expression in B cells according to the present invention in 100 wt% of the feed composition may be 0.001 to 90 wt%, 0.001 to 80 wt%, 0.001 to 70 wt%, 0.001 to 60 wt%, 0.001 to 50 wt%, 0.001 to 40 wt%, 0.001 to 30 wt%, 0.001 to 20 wt%, 0.001 to 10 wt%, 0.001 to 9.0 wt%, 0.001 to 8.0 wt%, 0.001 to 7.0 wt%, 0.001 to 6.0 wt%, 0.001 to 5.0 wt%, 0.001 to 4.0 wt%, 0.001 to 3.0 wt%, 0.001 to 2.0 wt%, or 0.001 to 1.0 wt%. In one aspect, the content of the substance that induces CD11b expression in B cells according to the present invention in 100% by weight of the feed composition is preferably, but is not limited to, 1.0 to 80% by weight, 5.0 to 70% by weight, 10 to 60% by weight, 20 to 50% by weight, or 30 to 40% by weight.

[0060] In one aspect, the composition of the present disclosure is provided as a feed additive to be added when the feed is ingested, or as a raw material composition for producing the feed or feed additive.

[0061] The subjects to which the food composition or feed composition of the present disclosure may be administered are not particularly limited, and non-limiting examples include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, horses, deer, wild boars, sheep, and goats; birds such as poultry, chickens, ducks, turkeys, and quails; reptiles such as snakes and turtles; amphibians such as frogs; salmon, fish species, yellowtail and cobia, freshwater trout, sweetfish, sea bass, sea bream, and meagre fish; flounder, Examples of fish include pufferfish, yellowtail, amberjack, bluefin tuna, horse mackerel, striped jack, tiger pufferfish, yellowtail amberjack, stone bream, filefish, rockfish, scorpion fish, black porgy, black rockfish, sea bass, red sea bream, grunt, black sea bream, chub mackerel, spotted grouper, grouper, eel, catfish, loach, sturgeon, tropical fish, tilapia, pangasius, bluefin tuna, bighead carp, silver carp, grass carp, carp, and other fish, bivalve shells (oysters, scallops, clams, mussels, pearl oysters), abalone, shrimp, and seaweed.

[0062] (Treatment or health maintenance / improvement methods) In one aspect, the present disclosure includes a treatment method or a health maintenance or promotion method using a substance that induces the expression of CD11b in B cells, or a pharmaceutical composition, food composition, or feed composition containing the same. The treatment or health maintenance or promotion method in the present disclosure includes administering to a subject the substance that induces the expression of CD11b in B cells of the present disclosure, or the "composition", "pharmaceutical composition", "food composition", or "feed composition" of the present disclosure. Specific disorders or diseases to be prevented or treated, subjects to be administered, administration methods, dosages, etc. may be as described in the above "composition", "pharmaceutical composition", "food composition", or "feed composition". In addition, the health maintenance or promotion effect obtained by the method of the present disclosure is not particularly limited as long as it is caused by the germinal center migration of activated B cells.

[0063] In one aspect, the disclosure provides a method for activating germinal center transfer of activated B cells in a subject in need of such activation, the method comprising administering to the subject a composition comprising an agent that induces expression of CD11b on B cells.

[0064] Also, in one aspect, the disclosure provides a method for treating a subject having a disease, disorder, or condition that is treated by activation of germinal center migration of activated B cells, comprising administering to the subject a composition comprising an agent that induces expression of CD11b on B cells.

[0065] Also, in one aspect, the present disclosure provides a method for immunostimulation in a subject in need thereof, the method comprising administering to the subject a composition comprising a substance that induces expression of CD11b in B cells.

[0066] In addition, in one embodiment, the immune stimulation is preferably mucosal immunity stimulation.Therefore, the present disclosure provides a method for stimulating mucosal immunity in a subject, comprising administering to the subject a composition comprising a substance that induces the expression of CD11b in B cells.The mucosal immunity stimulation includes, but is not limited to, gastrointestinal immunity (e.g., intestinal immunity and oral immunity) and nasal immunity stimulation.

[0067] In one embodiment, the immune activation is mucosal antigen-specific immune activation. Thus, the present disclosure provides a method for mucosal antigen-specific immune activation in a subject in need of mucosal antigen-specific immune activation, comprising administering to the subject a composition comprising a substance that induces CD11b expression in B cells. The mucosa includes, but is not limited to, the digestive tract (e.g., intestinal mucosa and oral mucosa) and nasal mucosa.

[0068] In one aspect, the immune activation includes, but is not limited to, a health maintaining or promoting effect involving immune activation (e.g., mucosal immune activation, intestinal immune activation), a health maintaining or promoting effect involving maintenance or improvement of mucosal flora (e.g., intestinal flora), or treatment of infectious diseases (e.g., treatment of viral infections, bacterial infections, fungal infections, or parasitic infections).

[0069] Also, in one aspect, the subject in need of activating antigen-specific immunity in the mucosa is a subject in need of immune activation (e.g., mucosal immune activation, intestinal immune activation), a subject in need of health maintenance or promotion involving immune activation, a subject in need of maintenance or improvement of mucosal flora (e.g., intestinal flora), a subject in need of health maintenance or promotion involving mucosal flora, or a subject in need of treatment of an infectious disease (e.g., treatment of a viral infection, bacterial infection, fungal infection, or parasitic infection).

[0070] Thus, in one aspect, the present disclosure provides a method of immune activation in a subject in need of immune activation (e.g., mucosal immune activation, intestinal immune activation), comprising administering to the subject a composition comprising a substance that induces expression of CD11b in B cells.

[0071] Also, in one aspect, the present disclosure provides a method for maintaining or promoting health promotion through immune activation in a subject in need thereof, the method comprising administering to the subject a composition comprising a substance that induces expression of CD11b in B cells.

[0072] Also, in one aspect, the present disclosure provides a method for maintaining or improving a mucosal flora (e.g., intestinal flora) through immune activation in a subject in need thereof, the method comprising administering to the subject a composition comprising a substance that induces expression of CD11b on B cells.

[0073] Also, in one aspect, the disclosure provides a method of treating an infectious disease (e.g., treatment of a viral, bacterial, fungal or parasitic infection) in a subject in need thereof, comprising administering to the subject a composition comprising an agent that induces expression of CD11b in B cells.

[0074] In one aspect, the disclosed method of treatment or health maintenance or promotion includes a method of inducing germinal center migration of activated B cells, comprising preparing a composition comprising a substance that induces expression of CD11b in B cells, and administering the composition to a subject in need of or desiring treatment or health maintenance or promotion.

[0075] In one embodiment, the method for treatment or health maintenance or promotion disclosed herein includes a method for immune activation, comprising preparing a composition comprising a substance that induces expression of CD11b in B cells and administering the composition to a subject in need of or desiring treatment or health maintenance or promotion. In one embodiment, the immune activation is preferably mucosal immunity activation, including intestinal immunity activation.

[0076] In one embodiment, the method of treatment or health maintenance or improvement of the present disclosure includes a method of maintaining or improving a mucosal microbiota, comprising preparing a composition comprising a substance that induces expression of CD11b in B cells and administering the composition to a subject in need of or desiring treatment or health maintenance or improvement. In one embodiment, the method of maintaining or improving a mucosal microbiota is preferably a method of maintaining or improving an intestinal microbiota.

[0077] In one aspect, the disclosed method of treatment or health maintenance or promotion includes a method of promoting high affinity IgA antibody expression, comprising preparing a composition comprising a substance that induces expression of CD11b in B cells, and administering the composition to a subject in need of or desiring treatment or health maintenance or promotion.

[0078] In one aspect, the disclosed method of treatment or health maintenance or promotion includes a method of obtaining vaccine adjuvant activity comprising preparing a composition comprising a substance that induces expression of CD11b in B cells and administering the composition to a subject in need of or desiring treatment or health maintenance or promotion.

[0079] As a substance that induces the expression of CD11b in B cells to be used in the treatment or health maintenance or improvement method of the present disclosure, any of the substances described above (Substances that induce the expression of CD11b in B cells) can be used.

[0080] A composition containing a substance that induces expression of CD11b in B cells, which is used in the treatment or health maintenance or improvement method disclosed herein, can be prepared by a method known to those skilled in the art. The composition can be prepared by mixing the substance that induces expression of CD11b in B cells with an excipient, carrier, etc., or by obtaining a composition in which the substance that induces expression of CD11b in B cells is mixed with an excipient, carrier, etc. in advance. EXAMPLES

[0081] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and do not limit the present disclosure.

[0082] method Mouse animals Balb / c mice (8-12 weeks old) were obtained from Japan CLEA. Mice were kept under specific pathogen-free conditions in the animal experiment facility of the Institute of Quantitative Biology (IQB), The University of Tokyo. All experiments were performed in accordance with the guidelines of the Animal Experiment Committee of IQB, The University of Tokyo. For B cell transfer experiments, IgA-Cre / YC3.60 mice were provided by Dr. Takahiro Adachi of Tokyo Medical and Dental University. flox Mice (8–12 weeks old) were used. IgA-Cre mice were designed based on the reference by Cris Allen. YC3.60 flox mouse 39,40 After mating with IgA +The cells are YC3.60 + The cells are identified as

[0083] Flow cytometry analysis and cell sorting Peyer's patches (PPs) were gently excised from the small intestine of Balb / c mice. Single cell suspensions prepared from PPs were incubated with the following antibody combinations: Phyacoerythrin-Cy7 (PE-cy7) anti-mouse / human B220 (eBioscience mAb RA3-6B2), PE anti-mouse / human B220 (Biolegend mAb RA3-6B2), FITC anti-mouse / human B220 (Biolegend mAb RA3-6B2), PE anti-mouse IgA (α-chain specific) (Southern Biotech), Alexa Fluor (AF)647 anti-mouse IgA (Southern Biotech), FITC anti-mouse CD11b (Biolegend mAb M1 / 70), and PE anti-mouse CD11b (Biolegend mAb M1 / 70). PE-cy7 anti-mouse CD11b (eBioscience mAb M1 / 70), biotinylated PNA (Vector Laboratories), APC-R700 hamster anti-mouse CD95 (Fas) (BD Biosciences mAb Jo2), AF488 anti-mouse CD86 (Biolegend mAb GL-1), APC anti-mouse CD184 (CXCR4) (Biolegend mAb L236F12), PE anti-mouse IgM (eBiosciences mAb eB121-15F9), AF647 anti-mouse CD4 (Biolegend mAb GK1.5), streptavidin PE (Biolegend), streptavidin APC (Biolegend), streptavidin AF488 (Lifetechnologies). FSC-H / FSC-W gates were used for single cell selection. Propidium iodide (PI) (Nacalai) was used to exclude dead cells. Flow cytometry analysis was performed using a Spectral Cell Analyzer SA3800 (SONY). Cell sorting was performed using a Cell Sorter SH800 (SONY).

[0084] Immunohistochemical analysis The isolated Peyer's patches were flash frozen in Optimum Cutting Temperature (OCT) compound (Sakura Fine Techno) and stored at -80°C. PP sections with a thickness of 6 μm were prepared and dried overnight. The next day, the PP sections were fixed in acetone (Nacalai) at -20°C for 10 min. After washing five times with phosphate-buffered saline (PBS), the PP sections were incubated in blocking buffer (PBS / 5% FCS (NICHIREI BIOSCIENCES INC.)) for 30 min. The PP sections were then incubated in the following antibody combinations in a dark box at RT for more than 30 min. AF488 anti-mouse / human CD11b (Biolegend mAb M1 / 70), PE anti-mouse / human CD11b (Biolegend mAb M1 / 70), AF488 anti-mouse CD4 (Biolegend mAb GK1.5), AF647 anti-mouse CD4 (Biolegend mAb GK1.5), DAPI solution (BD Bioscience), AF488 anti-mouse CD54 (ICAM-1) (Biolegend mAb YN1 / 1.7.4), AF647 anti-mouse CD54 (ICAM-1 mAb YN1 / 1.7.4) (Biolegend), biotin anti-mouse CD11c (eBioscience mAb N418), AF488 anti-mouse MAdCAM-1 (Biolegend mAb MECA-367), PE anti-mouse MAdCAM-1 (Biolegend mAb MECA-367). Biotinylated PNA, AF647 anti-mouse IgA (α-chain specific) (Southern Biotech), PE anti-mouse IgA (α-chain specific) (Southern Biotech), streptavidin AF488 (Lifetechnologies). Observation was performed using a microscope LSM880 (Carl Zeiss). Images were analyzed using software ZEN2009 (Carl Zeiss).

[0085] Microarray analysis 25,000 CD11b + IgA + B cells and 100,000 CD11b - IgA +RNA was extracted from B cells using NucleoSpin RNA XS (Takara). Microarray analysis was performed using SurePrint G3 Mouse GE v2 8x60K Microarray Chip (Affymetrix) to detect gene expression.

[0086] RNA purification and real-time PCR Approximately 1×10 4 PP GC B cells (PNA hi B220 + cells) and approximately 1 × 10 4 Non-GC PP B cells (PNAS lo B220 + ) were sorted. RNA was extracted from the isolated cells using NucleoSpin RNA XS (Takara), and cDNA was synthesized using GoScript® Reverse Transcriptase (Promega). Real-time PCR was performed in triplicate on a 384-well optical PCR plate (Roche) using a LightCycler 480 (Roche) with the KAPA SYBR® FAST qPCR Master Mix (2X) Kit (KAPA Biosystems). Gene expression levels were normalized to the expression level of the housekeeping gene β-actin. The following primers were used for analysis. β-actin fw:5'-CCAACCGTGAAAAGATGACC-3', β-actin rv:5'-CCAGAGGCATACAGGGACAG-3', itgam (CD11b) fw:5'-CAGATCAACAATGTGACCGTATGGG-3', itgam (CD11b) rv:5'-CATCATCATGTCCTTGTACTGCCGCTTG-3', aicda (AID) fw:5'-CCTACGCTACATCTCAGACT-3', AICDA (AID) rv:5'-CTTTGAAGGTCATGATCCCG-3', BCL6 (BCL6) fw:5'-TCCTCACGGTGCCTTTTTACA-3', bcl6(BCL6) rv:5'-TAACGACAAGCATGACGCAG-3', irf4(IRF4) fw:5'-AGGTCTGCTGAAGCCTTGGC-3', irf4 (IRF4) rv:5'-CTTCAGGGCTCGTCGTGGTC-3', s1pr2 (S1PR2) fw:5'-GTGACGGGACGCAGAGGT-3', s1pr2(S1PR2) rv:5'-AAATGTCGGTGATGTAGGCATG-3', bcl2(BCL2) fw:5'-TGAGTACCTGAACCGGCATCT-3', bcl2(BCL2) rv:5'-GCATCCCAGCCTCGTTAT-3', gpr183(EBI2) fw:5'-GACATCCTGTTTACCACAGCT-3', gpr183 (EBI2) rv:5'-AGACCAGAATCCAGACGGACA-3'

[0087] In vivo imaging For migration imaging, IgA-Cre / YC3.60 flox Mice (8-12 weeks old) were anesthetized with a mixture of three anesthetic agents as described in ref. 41. The abdominal wall was carefully incised to expose the small intestine. PPs were identified with the naked eye. Approximately 8,000 isolated CD11b + IgA + PP B cells and approximately 30,000 CD11b - IgA + PP B cells were labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen) and then infused with IgA-Cre / YC3.60 using a 25 μL syringe (Trajan Scientific and Medical). floxThe cells were directly injected into the PPs of transgenic mice. The PPs with transplanted cells were observed under an LSM880 microscope (Carl Zeiss). Images were analyzed with ZEN2009 software (Carl Zeiss). After observing the PPs under a microscope for 1 hour, the abdominal incision was carefully closed with an ELP skin stapler (Akiyama Co., Ltd.). Forty hours after transplantation, under anesthesia, the PPs containing the transplanted cells were observed again under an LSM880 microscope (Carl Zeiss), and the transplanted CD11b + IgA + PP B cell localization was confirmed and analyzed with ZEN2009 software (Carl Zeiss).

[0088] Conjugation Analysis CD11b + IgA + PP B cells and CD4 + Conjugates between PP T cells were analyzed by flow cytometry using a Cell Sorter SH800 (SONY). Singlet cells were selected using the FSCH / FSC-W gate. Singlet cells were selected using the FSCH / FSC-W gate, and conjugated cells were negatively selected using the singlet cell gate. Imaging revealed that CD11b of PP conjugates was positive for CD11b. + IgA + CD4 + Conjugate cells were isolated from the spleen and observed under a microscope (LSM880, Carl Zeiss) and analysis software (ZEN2009, Carl Zeiss).

[0089] CD11b in iGB culture system + IgA + Isolation of PP B cells Approximately 800 sorted CD11b + IgA + PP B cells and 800 sorted CD11b - IgA + PP B cells were stained with CellTrace Violet Proliferation Kit (Invitrogen). + IgA +The number of PP B cells was very low, so 5,000 CD11b - IgA + PP B cells and 5 × 10 4 Naive splenic B cells (negatively isolated by B cell isolation kit (Miltenyi Biotec)) were used as a positive control. - IgA + PP B cells, CD11b + IgA + PP B cells and naive splenic B cells were seeded in 6-well tissue culture dishes in the presence of 40LB cells pretreated with mitomycin C to inhibit growth. The cells were grown in RPMI-1640 medium (Wako) (10% FCS, 5.5 × 10 -5 The cells were cultured for 3 days at 37°C and 5% CO2 using a medium containing 10 mM 2-mercaptoethanol (ME) (Nacalai) and 10 mM HEPES (Nacalai). + IgA + PP B cells and CD11b - IgA + PP B cells were cultured with rIL-21 (10 ng / ml; PeproTech), and naive splenic B cells were cultured with rIL-4 (1 ng / ml; Biolegend). Flow cytometry analysis was performed to detect CellTrace Violet from day 0 to day 3 using the iGB culture system. CD11b expression in cultured cells was analyzed by flow cytometry on days 0 and 1 using a Cell Sorter SH800 (SONY) and a Spectral Cell Analyzer SA3800 (SONY).

[0090] Pam3CSK4 stimulated splenic CD11b on iGB culture system + B cells Splenic CD11b in the iGB culture system + For B cell culture, splenic naive B cells were cultured with pam3CSK4 (Invivogen) for 3 days to induce CD11b expression. 5CD11b + Splenic B cells and 1.5 × 10 5 CD11b splenic B cells were isolated and stained with CellTrace Violet (Invitrogen), then cultured for 4 days in the 40LB system with IL-4 (1 ng / ml; Biolegend), and then analyzed for CD11b expression by flow cytometry using a Cell Sorter SH800 (SONY) and a Spectral Cell Analyzer SA3800 (SONY).

[0091] Preparation of bacteria for stimulating B cells E. coli (DH5α) and S. enterica were inoculated from glycerol stocks into 5 ml of LB medium and grown at 37°C overnight with shaking at 180 rpm. On the second day, 0.5 ml of the cell suspension was resuspended in 10 ml of LB medium. The culture was grown at 37°C for 3 h with shaking, and the growth curve of the culture was observed by measuring the optical density at 600 nm (OD600) every 30 min. 100 μl of serial dilutions of the cell suspension were spread on LB plates, and CFU were calculated. Based on the growth curves, E. coli and S. enterica were grown, harvested, and heat killed in an autoclave (121°C, 15 min).

[0092] Similarly, B. bifidum and B. breve were cultured anaerobically at 37°C in Difico Lactobacilli MRS broth (Becton, Dickinson and Company) and then heat-killed in an autoclave.

[0093] Stimulation of splenic B cells (in vitro) Splenic B cells were negatively sorted from the spleens of 8-week-old Balb / c mice using a B cell isolation kit (Miltenyi Biotec). Splenic B cells (concentration: 5 × 10 5 13 independent stimuli were added to the cultured cells (100 / ml) in RPMI 1640 medium. To select the appropriate concentration of each stimulus, the stimuli were titrated, and the results are shown in Figure 11e-g.

[0094] 1. Anti-mouse IgM (15 μg / ml; Jackson Immuno-Research Laboratories) 2. Anti-mouse CD40 (HM40-3,500 ng / ml; Biolegend) 3.BAFF (50 ng / ml; Biolegend) 4.Pam3CSK4 (1μg / ml, Invivogen) 5.Poly I:C (25 ng / ml, Invivogen) 6.LPS (50ng / ml, Sigma Aldrich) 7. Flagellin (20ng / ml, Sigma Aldrich) 8. Imiquimod (R837, 5ng / ml; Invivogen) 9.CpG (20μg / ml, GeneDsign Inc.) 10. Heat-killed E. coli (10 7 CFU / ml) 11. Heat-sterilized S. Enterica (10 8 CFU / ml) 12. Heat-killed B. bifidum (10 7 CFU / ml) 13. Heat-killed B. breve (10 7 CFU / ml) CD11b expression in stimulated B cells was analyzed on days 0 and 3 using a Cell Sorter SH800 (SONY) and a Spectral Cell Analyzer SA3800 (SONY).

[0095] For MDP stimulation, purified splenic B cells (5 × 10 5 / ml) were cultured in RPMI 1640 medium supplemented with N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (1 μg / ml; Sigma-Aldrich).

[0096] For analysis of TLR inhibitors and NOD2 inhibitors, purified splenic B cells (5 × 105 / ml), pam3CSK4 (1 μg / ml, Invivogen), LPS (50 ng / ml; Sigma-Aldrich), CpG (20 μg / ml, GeneDsign Inc), heat-killed E. coli (10 7 CFU / ml) in RPMI 1640 medium containing the TLR1 / 2 antagonist, CU-CPT (1 μM; Sigma-Aldrich), the TLR4 inhibitor, TAK-242 (1 μM; Sigma-Aldrich), or the NOD2 signal inhibitor II, GSK717 (30 μM; Sigma-Aldrich).

[0097] immunization On days 0 and 16, mice were injected with OVA (1 mg), OVA (1 mg) + pam3CSK4 (10 μg, Invivogen), or OVA (1 mg) + heat-killed E. coli (10 in 100 μl). 9 CFU) were orally administered to the mice. Feces were collected on the 21st day and OVA-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA).

[0098] Detection of OVA-specific IgA ELISA Feces were suspended in 10-fold weight / volume (w / v) PBS. After centrifugation (8,000 g, 15 min), the supernatant was collected as fecal extract. Plates were precoated with 1 mg / ml OVA overnight, followed by blocking with 1% (w / v) bovine serum albumin in PBS at room temperature for 1 h. Serially diluted fecal extracts were then added and incubated at room temperature for 1 h. The relative binding affinity of IgA was detected with alkaline phosphatase-conjugated goat anti-mouse IgA (Southern Biotech). After overnight incubation at 4°C, the optical density (OD) values ​​at 405 nm were measured using a TriStar Multimode Reader LB 942 (BERTHOLD).

[0099] Oral administration of heat-killed bacteria to WT mice Balb / c mice (8-12 weeks old) were inoculated with heat-killed E. coli (10 9CFU diluted in 100 μl PBS), S. enterica (10 9 CFU diluted in 100 μl PBS), B. bifidum (10 9 CFU diluted in 100 μl PBS), B. breve (10 9 CFU diluted in 100 μl PBS) or pam3CSK4 (10 μg diluted in 100 μl PBS) were orally administered to mice. As a control, mice were orally administered 100 μl PBS. Two days later, PPs of the mice were excised and GC B cells were analyzed using a Spectral Cell Analyzer SA3800 (SONY) as described above.

[0100] Oral adjuvant administration with OVA as an antigen OVA (1 mg) was used as an antigen, and each adjuvant (CpG-K3 (GeneDsign Inc.) 10 μg, CpG-ODN1826 (Invirogen tlrl-1826) 10 μg, or heat-killed E. coli 10 9 C57BL / 6 mice were orally immunized with a mixture of 1000 μg / mL of 10000 cfu each three times every two weeks, and blood samples were taken two days after the third immunization. Serum TNF-α levels were measured according to the protocol of the mouse TNF-α ELISA kit (Invitrogen, BMS607-3).

[0101] In vivo imaging of transplanted iGB cells induced by pre-stimulation As described above, splenic naive B cells were cultured in 100% PBS containing pam3CSK4 (1 μg / ml, Invivogen), heat-killed E. coli (10 7 CFU / ml), CpG (20 μg / ml, GeneDesign Inc.), or anti-IgM-Ab (15 μg / ml, Jackson Immuno Research Laboratories) were used to culture the cells in vitro for 3 days. 5 B220 +B cells were isolated and then seeded onto 40LB feeder cells supplemented with rIL-4 (1 ng / ml; Biolegend) to induce iGB cells. As a control, unstimulated naive splenic B cells were seeded onto 40LB feeder cells supplemented with rIL-4 (1 ng / ml; Biolegend). After 4 days of culture, 3 × 10 5 iGB cells (B220 + Naïve B-40LB cells, pam3CSK4-40LB cells, E. coli-40LB, IgM-40LB and CpG-40LB cells were isolated and labeled with CellTracker Orange CMTMR fluorescent dye (Invitrogen), and then separately injected intravenously into mice (Balb / c, 8-12 weeks old) with 10 μg of AF488 anti-mouse MAdCAM-1 (Biolegend mAb MECA-367) to identify HEV. Under anesthesia, IgA was injected into mice for GC identification. + To stain the cells, 1 μg of AF647 anti-mouse IgA (Southern Biotech) was injected directly into the PP of the same mice that had been injected with iGB cells. The localization of the injected labeled iGB cells was observed using a microscope LSM880 (Carl Zeiss) and analysis software ZEN2009 (Carl Zeiss).

[0102] DNA amplification of rearranged VDJ regions and downstream introns The rearranged VDJ sequence with downstream intron sequences was amplified by two rounds of nested PCR using several different upstream primers (where "S" is C or G, "R" is A or G, "N" is A, G, C or T, "M" is A or C, and "W" is A or T). MH1, 5′-SARGTNMAGCTGSAGSAGTC-3′; MH2, 5′-SARGTNMAGCTGSAGSAGTCWGG-3′; MH3, 5′-CAGGTTACTCTGAAAGWGTSTG-3′; MH4, 5′--GAGGTCCARCTGCAACARTC-3′; MH5, 5′-CAGGTCCAACTVCAGCARCC-3′; MH6, 5′-GAGGTGAASSTGGTGGAATC-3′). MH7, 5′-GATGTGAACTTGGAAGTGTC-3′, was replaced with the JH4R primer (5′-GACTAGTCCTCTCCAGTTTCGGCTGAATCC-3′), which is complementary to a sequence located at the 5′ end of the IgH intron enhancer. 16,42 The second PCR used the same upstream primer together with the JH4R-2 primer (5'-CAGGTGGTGTTTTGCTCA-3'), which is complementary to the upstream sequence of the JH4R primer. Amplification was performed using PrimeSTAR Max DNA Polymerase (TaKaRa) with 20 cycles of first-round PCR and 30 cycles of second-round PCR (95°C 15 sec, 58°C 10 sec, 72°C 30 sec). The PCR product was cloned into the pGEM T vector (Promega), and then colony PCR was performed with the JH4F primer (5'-TAT GCT ATG GAC TAC TGG-3') and the JH4R-2 primer using EmeraldAmp® PCR Master Mix (TAKARA) to select clones containing the intron sequence.

[0103] DNA sequencing and SHM analysis Plasmid DNA was isolated and then sequenced using T7 and SP6 primers. The consensus IgH intron DNA sequence was obtained from Accession No. AJ 851868.3. The rearranged VDJ was analyzed by IgBLAST.

[0104] statistical analysis Unless otherwise stated, statistical analyses were performed using GraphPad Prism version 8.4.2 for Mac (GraphPad Software, San Diego, CA, USA). Differences between two groups were compared using a two-tailed Student's t test. When there were more than two groups, one- or two-way analysis of variance (ANOVA) was performed followed by Tukey's multiple comparison test. Details regarding statistical tests and number of groups and replicates are given in the figure legends. A p value of <0.05 was considered significant.

[0105] Example 1: IgA expressing CD11b in mouse PPs + A small population of B cells Flow cytometry revealed that PP expresses CD11b + IgA + A small population of B cells (approximately 0.1% to 0.2% of PP B cells) was identified (Figures 2a and 6a). + IgA + and CD11b - IgA + Both PP B cells and Peanut Agglutinin (PNA) high and Fas +16 These results suggest that GC B cells exhibited a typical phenotype (Fig. 2b, c).

[0106] Second, pre-GC B cells reside in the IF region (outside the GC) before entering the GC, and therefore express CD11b + IgA + B cell localization was examined 3,4Immunohistologically, GC regions were identified by the expression of both PNA and IgA (Fig. 2d). As a result, in the area outside the GC of the section, two CD11b + IgA + We found that only B cells (yellow cells) were present (Figure 2d, Figure 7). Next, we analyzed the localization of CD11b ligand. Intercellular adhesion molecule 1 (ICAM-1) is widely known as a ligand of CD11b / CD18 that mediates cell adhesion and migration. 17 Already reported 8,18 As shown in Fig. 1, GC B cells in the IF region and high endothelial venules (HEVs) both express ICAM-1. HEVs also express mucosal addressable cell adhesion molecule-1 (MAdCAM-1). 19 CD11b is also expressed (Fig. 2e, Fig. 8). + IgA + Although we could not confirm that PP B cells directly bound to HEV, seven CD11b + IgA + Six of the PP B cells were found to be located in the vicinity of the HEV (Fig. 2f). + IgA + PP B cells (No. 7) were located near GCs (Fig. 2f) but not within GCs, suggesting that they may migrate from IFs to GCs.

[0107] Next, CD11b + IgA + The question then became whether PP B cells would subsequently migrate from outside the GC into the GC. + IgA + and CD11b - IgA + PP B cells were isolated, labeled, and differentiated into IgA-Cre / YC3.60 flox These were then separately injected into the PP of reporter mice. GC in the PP expressed IgA + Since B cells were identified in the enriched areas (Fig. 2d), IgA +These reporter mice, whose cells express YC3.60, can easily identify GCs in the PP. One hour after injection, the injected CD11b - IgA + B cells are located inside the GC (Fig. 2g) and express CD11b - IgA + These results suggest that the B cells are GC B cells. + IgA + B cells did not enter GCs but instead surrounded them (Fig. 2g). + IgA + B cells also entered the GC (Fig. 2h). Thus, CD11b + IgA + B cells migrate to the GC.

[0108] Example 2: CD11b + IgA + Gene expression in PP B cells To further confirm the phenotype of pre-GC B cells, CD11b + IgA + and CD11b - IgA + PP B cells were isolated and gene expression was compared by microarray analysis (Figures 3a and 6b). + IgA + B cells are CD11b - IgA + Compared to B cells, they express GC genes at relatively low levels and non-GC genes at relatively high levels, and + IgA + B cells are CD11b - IgA + To confirm the microarray results, quantitative PCR (qPCR) was performed (Figures 3b and 6b) and CD11b + IgA + B cell gene expression and GC(PNA high B220 + ) and non-GC (PNA low B220 +We compared gene expression in PP B cells from GCs. We found that expression of bcl6 (a major transcription factor restricted in GC B cells), aicda (encoding activation-induced cytidine deaminase (AID), an enzyme that induces somatic hypermutation (SHM) and class switch recombination), and s1pr2 (a receptor that controls the location of B cells in and out of GCs) was significantly increased in GCs. 10,20-22 Three genes highly expressed in GC B cells, including , and two non-GC genes, gpr183 (a receptor that controls B cell positioning outside the follicle) and bcl2 (a transcription factor highly expressed in non-GC B cells). 10,23,24 CD11b+IgA+ and CD11b - IgA + The expression levels of these genes in PP B cells were distinct from those in non-GC B cells (Fig. 3b), indicating that both populations are likely GC B cells. Pre-GC B cells have increased expression of the transcription factor irf4, which has been demonstrated to be required for the initiation of the GC response. 10,11,25 We further analyzed the expression of irf4. + IgA + PP B cells express CD11b - IgA + These B cells expressed higher levels of irf4 than PP B cells (Fig. 3b), indicating that they were pre-GC B cells. However, considering that some GC light zone (LZ) B cells also highly expressed irf4, 8,25 , followed by CD11b + IgA + The expression of CXCR4 and CD86 on PP B cells was analyzed by flow cytometry. GC LZ B cells expressed CD86 high CXCR4 low B cell 3,8 Since it has been identified as CD11b - IgA + Compared to B cells In comparison, CD86 expression was low and CXCR4 expression was high (Fig. 3c, Fig. 6c), suggesting that CD11b + IgA + The B cells were shown to be pre-GC B cells, not GC LZ B cells.

[0109] Example 3: CD11b + IgA + PP B cells interact with T cells before entering the GC To enter the GC, pre-GC B cells need to interact with T cells and DCs, as previously reported. 3,26 Therefore, CD11b + IgA + PP B cells express CD4 before entering GC + We investigated whether IgA interacts with T cells. + PP B cells were CD4 + However, the conjugated B cells were not positive for IgA. + CD4 + There was a clear population of double-positive PP cells (Fig. 3d, Fig. 9), and these IgA + PP B cells are CD4 + It has been shown that CD11b interacts with T cells. + IgA + More than 40% of B cells are CD4 + It showed binding to T cells (Fig. 3e, f, Fig. 9) and CD11b + IgA + PP B cells are CD4 + It was suggested that CD11b interacts with T cells. - IgA + PP B cells are CD4 + T cells interact with each other, and in the GC LZ, CD4 + T follicular helper (T FH ) may interact with cells (Fig. 3e, f). 4,8 Confocal microscopy of the conjugated cells isolated from PP revealed that CD11b + IgA + PP B cells and CD4 + The interaction of CD11b T cells was further confirmed (Fig. 3g). + IgA +This clearly demonstrates that PP B cells are pre-GC PP B cells.

[0110] Example 4: CD11b + IgA + PP B cells accumulate SHM in Ig genes CD11b + IgA + To confirm whether the B cells were derived from newly activated naive B cells, CD11b + IgA + and CD11b - IgA + We sequenced both the rearranged VDJ region and downstream intron sequences of IgH from PP B cells and found that SHM had been introduced during the GC reaction (Fig. ​ (Fig.10a,b). 16 CD11b + IgA + PP B cells (21 of 25 clones) and CD11b - IgA + More than 80% of the sequences in PP B cells (32 of 39 clones) were mutated (Fig. 10b). + IgA + Our results indicate that PP B cells are derived not only from newly activated naive B cells, but also from memory B cells that have re-entered GCs, as previously reported. 6,7 .

[0111] Example 5: Bacterial antigens induce expression of CD11b on naive splenic B cells CD11b + IgA + Our findings on PP B cells raise another question: what triggers CD11b expression on pre-GC B cells? B cells can transform into pre-GC B cells not only by stimulation with antigen but also by interactions with DCs and T cells. 3,4,26To investigate what signals induce CD11b expression, we isolated naive splenic B cells from unimmunized mice and cultured them in vitro with anti-IgM for crosslinking of the BCR, B cell-activating factor (BAFF) for interaction with DCs, and anti-CD40 for interaction with T cells. However, these stimuli, which mimic TB and DC-B interactions, did not induce CD11b in naive B cells. We then stimulated naive B cells with different Toll-like receptor (TLR) ligands, including pam3CSK4 for TLR2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, imiquimod for TLR7, and CpG for TLR9. After 3 days of culture, the number of B cells stimulated with pam3CSK4, LPS, and CpG was significantly increased compared to B cells stimulated with anti-IgM, anti-CD40, BAFF, poly I:C, flagellin, and imiquimod (Fig. 11a, b). + The percentages of B cells were 27.6% and 8%, respectively, whereas the percentages of CD11b in B cells stimulated with other stimuli were + The proportion of B cells was less than 5% (Figure 4-1a, b). It is noteworthy that CpG activated the proliferation of B cells but did not induce the expression of CD11b. Furthermore, we confirmed that the expression of itgam (CD11b) on B cells increased only when they were stimulated with pam3CSK4 or LPS, which are known as bacterial antigens (Figure 4-1c). Next, we investigated the relationship between TLR2 and (or) TLR4. 27,28 When heat-killed E. coli was prepared, which strongly stimulates both TLR2 and TLR4, the heat-killed E. coli significantly induced CD11b expression in splenic B cells (Figure 4-1a). These results demonstrated that bacterial antigens, which are ligands for TLR2 and TLR4, induce CD11b in B cells.

[0112] Since B cells in PP GCs do not express CD11b (Fig. 2d), we hypothesized that CD11b expression on B cells may be transient and may converge via TB or DC-B interactions. 26We used an in vitro induced GC B cell (iGB) culture system to induce GC-like B cells by mimicking the role of T cells and DC cells, using 40LB cells expressing CD11b as a feeder layer. When cells were cultured in the iGB system, the isolated CD11b + IgA + PP B cells were isolated and expressed CD11b - IgA + The CD11b B cells were able to proliferate at a level similar to that of PP B cells and naive splenic B cells (Fig. 12a,b). + IgA + PP B cells lost CD11b expression after 1 day of culture in the iGB system (Fig. 4-1d, Fig. 12a). Similarly, the pam3CSK4-induced CD11b + Splenic B cells lost CD11b expression after 4 days of culture in the iGB system (Fig. 4-1e, Fig. 12c). Splenic naive B cells treated with anti-CD40 Ab and anti-IgM Ab significantly suppressed CD11b expression even in the presence of pam3CSK4 (Fig. 4-1f-h). This indicates that TB cell-TB cell interactions and BCR cross-linking downregulate CD11b expression on B cells. Thus, only a small number of CD11b cells are expressed in vivo. + IgA + It is reasonable that only PP B cells were found (Fig. 2 ).

[0113] To further confirm whether TLR2 and TLR4 are involved in the induction of CD11b expression in splenic B cells, splenic B cells were stimulated with pam3CSK4, LPS, CpG, and heat-killed E. coli in the presence of TLR1 / 2 inhibitors, TLR4 inhibitors, and NOD2 inhibitors. NOD2 is known to be involved in downstream signaling of TLR2. 29 Interestingly, when TLR1 / 2 inhibitors were used, pam3CSK4 and heat-killed E. coli upregulated CD11b +The NOD2 inhibitor increased the proportion of B cells, whereas the TLR4 inhibitor did not (Figure 4-1i). On the other hand, the NOD2 ligand muramyl dipeptide (MDP) did not induce CD11b expression, but the NOD2 inhibitor strongly suppressed CD11b expression in B cells stimulated with pam3CSK4 and heat-killed E. coli (Figure 11d). This result strongly suggests that CD11b expression is not directly induced by TLR2 or TLR4 activation, but is partially induced by bacteria-derived stimulation via NOD2 (Figure 4-1i).

[0114] Heat-killed Klebsiella pneumoniae, which belongs to the Proteobacteria phylum, and Bacteroides vulgatus, which belongs to the Bacteroidetes phylum, were also tested in the same way as E. coli, and it was confirmed that these bacteria also induced the expression of CD11b. + The proportion of B cells is shown in Figure 4-2. The results show that bacteria belonging to the phylum Proteobacteria and Bacteroidetes induce the expression of CD11b in naive splenic B cells.

[0115] Example 6: Enhancement of GC responses by oral administration of heat-treated CD11b-inducing bacteria Next, we addressed the question of what bacteria could induce pre-GC B cells via CD11b expression. We selected E. coli and S. enterica as harmful bacteria, and Bifidobacterium bifidum (B. bifidum) and B. breve as representative beneficial bacteria. Upon in vitro stimulation, E. coli and S. enterica induced CD11b expression, but B. bifidum and B. breve did not (Fig. 5a, b). Oral administration of pam3CSK4 or heat-killed bacteria to mice increased the number and percentage of PP GC B cells within 2 days (Fig. 5c-e). This suggests that only harmful bacteria can enhance ongoing GC responses in vivo.

[0116] Example 7: Transient expression of CD11b allows entry of cultured B cells into GCs. Next, in vitro induced CD11b + We examined whether B cells could enter pre-existing GCs. To induce GC B cells in vitro, naive splenic B cells were stimulated in vitro with pam3CSK4 or heat-killed E. coli to induce CD11b and then cultured on 40LB cells for 4 days. iGB cells pre-stimulated with pam3CSK4 or heat-killed E. coli, which are stimuli that induce CD11b, mostly entered GCs when intravenously injected into mice, whereas iGB cells pre-stimulated with CpG or anti-IgM, which did not express CD11b, did not enter GCs (Fig. 5f, Fig. S13). These results indicate that expression of CD11b on B cells prior to interaction with TB or DC-B is essential for pre-GC B cells to enter GCs. This is consistent with previous reports that bacterial antigens, which are non-self antigens, are important for triggering GC responses. 30 .

[0117] Example 8: CD11b-inducible heat-killed bacteria function as effective mucosal adjuvants and induce antigen-specific mucosal IgA Adjuvants based on TLR2 or TLR9 ligands are already widely used in vaccination to efficiently stimulate DCs. 31,32However, our results indicate that B cells can commit to pre-GC B cells before receiving DC or T cell help. We therefore hypothesized that pam3CSK4 and heat-killed E. coli might function as adjuvants for mucosal vaccines. We orally administered ovalbumin (OVA) as an antigen to mice, and orally administered pam3CSK4 or heat-killed E. coli as an adjuvant (Fig. 5g). As expected, OVA-specific IgA in feces increased in mice co-administered with OVA and heat-killed E. coli or pam3CSK4, but not in mice administered OVA alone (Fig. 5h). Thus, this study revealed that CD11b, a pre-GC surface marker on B cells, is a promising marker for selecting effective mucosal vaccine adjuvants, particularly for enhancing GC-derived high-affinity mucosal IgA.

[0118] On the other hand, as a result of measuring the cytokine concentration after the third immunization, the adjuvant composition of the present disclosure did not increase the serum TNFα concentration at all. On the other hand, CpG-K3 and CpG-ODN1826, which are known vaccine adjuvants, caused an increase in the serum TNFα concentration, and were considered to induce an inflammatory response (FIG. 14). These results confirmed that the adjuvant composition of the present disclosure has extremely advantageous properties in that it has high antigen-specific binding IgA induction capacity without inducing an inflammatory response.

[0119] (Consideration) In this study, CD11b + IgA + By examining different populations of B cells, we found that CD11b mediates the expression of pre-GC IgA +We showed that CD11b is a novel surface marker for B cells. Induction of CD11b on B cells depends on stimulation with harmful bacterial antigens but is independent of activated DCs. Oral administration of these CD11b-inducing bacterial antigens to mice enhanced mucosal antigen-specific IgA responses in vivo. These results indicate that transient expression of CD11b on activated B cells before they enter GCs is a critical step for selecting activated B cells suitable for GC responses.

[0120] Regarding the function of CD11b in B cells, Yan's group revealed that CD11b is important for regulating BCR signaling through the Lyn-CD22-SHP-1 negative feedback pathway. 13 CD11b- / - mice exhibited enhanced antibody production and GC responses mediated by autoreactive B cells. 13,14 In addition, mutations in the ITGAM gene, which encodes CD11b, have been reported as a high-risk factor for developing autoimmune diseases such as systemic lupus erythematosus (SLE). 33 In this disclosure, we confirmed that CD11b expression is a marker of B cells before they enter GC. Presumably, transient expression of CD11b is sufficient, but not necessary, for them to enter GC. When B cells are activated by bacterial stimulation, they express CD11b and proliferate to prevent the production of autoreactive antibodies. B cells then initiate interactions with T cells at the IF region. After TB interaction, activated B cells lose CD11b expression, enter the GC DZ, rapidly proliferate, and undergo SHM to obtain high affinity. CD11b may play an important role in controlling activated B cells to mount beneficial GC responses, but also to avoid harmful self-reactions.

[0121] Do human B cells also express CD11b in response to microbial antigens? Dutra's group reported that B cells from Chagas' disease patients stimulated with a protein-enriched fraction derived from Trypanosoma cruzi expressed CD11b. + Demonstrated higher B cell frequencies34 .

[0122] We also clarified what kind of signal induces the expression of CD11b. First, the TLR2 ligand pam3CSK4 and heat-killed E. coli strongly induced CD11b on B cells in vitro, suggesting that CD11b is induced by TLR2 stimulation. E. coli contains peptidoglycan (PGN), a component of the cell wall, which acts as a ligand for TLR2. 35 Unexpectedly, however, the combined use of pam3CSK4 and TLR1 / 2 inhibitors promoted the expression of CD11b on B cells. On the other hand, the expression of CD11b induced by pam3CSK4 and heat-killed E. coli was suppressed by NOD2 inhibitors (Fig. 4-1i), suggesting that NOD2 regulates CD11b. This result is in part consistent with previous findings that NOD2 acts as a negative regulator of TLR2. 35 Furthermore, NOD2 contributes to immune tolerance and homeostasis by suppressing TLR2 / 4-mediated induction of inflammatory cytokine production, which is closely related to Crohn's disease (CD). 36 Thus, it was suggested that the TLR2-NOD2 pathway may regulate not only inflammatory responses but also GC responses via CD11b. However, other studies have shown that the TLR1 / 2 signaling pathway is not related to NOD2. 37 It has been shown that TLR2 inhibitors promote the induction of CD11b, and further studies are expected to elucidate the details.

[0123] Regarding CD11b-induced bacterial stimulation, examples of harmful bacteria include S. enterica and E. coli, which have been shown to induce CD11b expression in vitro and enhance GC responses in vivo, and Klebsiella pneumoniae and the Bacteroidetes bacterium Bacteroides vulgatus have been shown to induce CD11b expression in vitro, but not Bifidobacterium.

[0124] In summary, the experiments of the present disclosure reveal the following: 1. CD11b + IgA + B cells express the GC B cell surface markers PNA and FAS. + IgA + B cells highly express GC B-specific genes but do not express non-GC genes. 2. CD11b + IgA + B cells highly express the pre-GC gene irf4. 3. CD11b + IgA + B cells are located outside the GC and attempt to enter the GC. 4. CD11b + IgA + B cells express CD4 outside of GCs. + It interacts with T cells. 5. In vitro stimulation with bacterial antigens induces expression of CD11b on splenic B cells. 6. Orally administered bacterial antigens stimulate GC responses in vivo within 2 days. 7. Heat-treated E. coli can be used as an adjuvant in oral vaccines to enhance antigen-specific responses by inducing pre-GC B cells.

[0125] That is, the present disclosure reveals the following: (1) CD11b + IgA+ B cells are pre-GC B cells (2) Induction of CD11b on B cells is a promising marker for selecting effective mucosal immune vaccine adjuvants. It was also suggested that B cells could sense bacterial antigens and determine their own cell fate to pre-GC cells, independently of DCs and T cells (Figure 15).

[0126] Mucosal immunity-related immune-inducing tissues perform affinity maturation of IgA+ B cells that recognize antigen molecules taken in from the mucosal tissue epithelium, etc., and secrete high-affinity antibodies from the mucosal epithelium, thereby having an important function in providing antigen-specific defense in mucosal tissues. Mucosal immunity-related immune-inducing tissues are present in various mucosae, such as intestinal mucosa (Peyer's patches) and nasal mucosa, and immune cells activated in these tissues provide antigen-specific defense in the mucosa or tissues such as distal mucosa (e.g., J. Otol. 114:843-850, 2011, Vaccine Vol. 30, pp. 180-188, 2012, Frontiers in Immunology, 2021, Vol. 12, Article 635471). Each embodiment of the present disclosure uses Peyer's patch germinal center as a model for mucosal immunity-related immune-inducing tissues, and CD11b + IgA + We have shown that B cells enter germinal centers (GCs) and undergo affinity maturation to produce high-affinity antibodies. + We demonstrated that heat-killed bacteria that induce CD11b expression in B cells can be used as a highly effective mucosal immune vaccine adjuvant with reduced induction of inflammatory responses. The present disclosure relates to CD11b + IgA + These results show that B cell-induced adjuvants are extremely useful for affinity maturation of B cells in the germinal centers of various mucosal immune-related immune-inducing tissues and for inducing high-affinity IgA antibody production, and that heat-sterilized bacteria can be used as such adjuvants. Mucosal secretory IgA antibodies are known to exhibit polyreactive reactivity against bacteria, and can provide antigen-specific immune protection against a wide range of antigens compared to IgG antibodies, which are produced and induced by general vaccines (e.g., Journal of Enterobacteriaceae 31:151-157,2017, Science.2017 Oct 20; 358(6361): eaan6619., Cell Reports 36, 109655,2021). In one aspect, the adjuvant of the present disclosure is extremely useful in that it can provide polyreactive antigen-specific immune protection while suppressing inflammatory responses, and will make a significant contribution in the fields of medicine, food, feed, and the like.

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Claims

1. A composition which is a mucosally administered vaccine adjuvant for immune activation, maintenance or promotion of health, maintenance or improvement of mucosal bacterial flora, or treatment of infectious diseases by inducing migration of activated B cells to germinal centers, comprising a substance which induces expression of CD11b in B cells, wherein the substance which induces expression of CD11b in B cells is a bacterium.

2. The composition of claim 1 , wherein the substance that induces expression of CD11b in B cells is a bacterium of the class Gammaproteobacteria or the class Bacteroidetes.

3. 3. The composition of claim 2, wherein the agent that induces expression of CD11b in B cells is Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Bacteroides vulgatus, or a combination thereof.

4. A composition according to any one of claims 1 to 3 for treating a subject having a disease, disorder or condition that is treated by activation of germinal centre migration of activated B cells.

5. The composition according to any one of claims 1 to 4, which is in a form selected from the group consisting of medicines, foods, feeds, food additives, feed additives, and raw material compositions thereof.

6. The composition of claim 1 , which is administered together with an antigen.

7. The composition according to claim 6, which is administered simultaneously with an antigen.

8. The composition according to claim 6, characterized in that it is administered separately from the administration of the antigen.

9. The composition of claim 1 which is a vaccine composition comprising an antigen.

10. The composition according to any one of claims 6 to 9, wherein the antigen is a viral, bacterial, fungal or parasitic antigen.

11. A method for inducing expression of CD11b in B cells, inducing contact of the B cells with T cells and dendritic cells, and inducing germinal center migration of activated B cells, comprising preparing a composition containing a substance that induces expression of CD11b in B cells, and contacting the B cells with the substance, wherein the substance that induces expression of CD11b in B cells is a bacterium, and the contact does not occur within the human body.

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