Increasers of IgA antibody content in breast milk

Abstract

The present invention aims to provide an intestinal commensal bacterium that increases IgA levels in breast milk. The agent for increasing IgA antibody levels in breast milk comprises one or more strains selected from the following: a Bacteroides strain having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 1 and increases IgA antibody levels in breast milk; and a Prevotella strain having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 2 and increases IgA antibody levels in breast milk.

Description

Technical Field

[0001] The present invention relates to an agent for increasing the IgA antibody (hereinafter sometimes referred to as "IgA") content in milk, comprising, as an active ingredient, intestinal commensal bacteria having an effect of increasing the IgA antibody content in milk. Background Art

[0002] Microorganisms are foreign to the host organism and are therefore likely to be eliminated by the immune system. However, intestinal commensal bacteria have the characteristic of not being actively eliminated by the intestinal immune system and remaining symbiotic within the intestine. Mammals, such as humans, are born in a sterile state and, through contact with the outside world, acquire a variety of bacteria, forming the intestinal flora. The human intestine contains over 1,000 species and approximately one trillion intestinal commensal bacteria, and it is expected that they have a significant impact on the intestinal environment.

[0003] Bacteroides bacteria was considered to be the less neutral bacteria (conditional pathogens) of usefulness in the past. In addition, because it is an anaerobic bacterium, it is difficult to cultivate, and there are more unclear points headed by the proliferation mechanism. By the research about Bacteroides bacteria in recent years, for example, it is reported that if the Peyer's patches cells as intestinal lymphoid tissue are cultivated in the presence of the heat-killed bacteria of Bacteroides bacteria, then compared with the situation of cultivating in the presence of the heat-killed bacteria of Lactobacillus (Lactobacillus) bacteria as lactic acid bacteria, the IgA amount secreted into the culture supernatant is more (non-patent literature 1,2). In addition, it is reported that Bacteroides acidifaciens (acidogenic Bacteroides) has therapeutic effect (patent literature 1) for the metabolic diseases with abnormal glucose and lipid metabolism as the cause. But, up to now, the correlation between intestinal symbiotic bacteria (that is, the bacterium constituting intestinal flora) and the IgA antibody content in milk is not known.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application No. 2017-538702

[0007] Non-patent literature

[0008] Non-patent literature 1: Journal of Enterobacteriaceae 27: 203-209, 2013

[0009] Non-patent literature 2: Biosci Biotechnol Biochem. 2009 Feb; 73(2): 372-7. Summary of the Invention

[0010] Problems to be solved by the invention

[0011] An object of the present invention is to provide an intestinal symbiotic bacterium having an effect of increasing the IgA content in breast milk.

[0012] Means used to solve problems

[0013] The inventors of the present invention have continued to conduct in-depth research to address the above-mentioned issues. During this process, they first discovered that in lactating mammalian mothers, the immune function of Peyer's patches (hereinafter sometimes referred to as "PP") is activated, causing IgA-producing plasma cells to migrate from the PP to the mammary glands, resulting in an increase in the IgA content in milk. They also confirmed that if the proportion of Bacteroides acidifaciens and Prevotella buccalis in the overall intestinal flora of lactating mammalian mothers decreases, the number of IgA-producing plasma cells in the mammary glands and the IgA content in the milk decrease. They further discovered that if lactating mammalian mothers in whom the proportion of these bacterial species in the overall intestinal flora decreases are orally inoculated with these bacterial species, the number of IgA-producing plasma cells present in the mammary glands and the IgA content in the milk increase. The present invention is based on these findings.

[0014] That is, the present invention is as follows.

[0015] [1] An agent for increasing the IgA antibody content in milk, characterized in that it contains one or more strains selected from the following Bacteroides strains and Prevotella strains, wherein the Bacteroides strain has a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 1 and has an effect of increasing the IgA antibody content in milk, and the Prevotella strain has a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 2 and has an effect of increasing the IgA antibody content in milk.

[0016] [2] The increasing agent according to [1] above, characterized in that the strain has the effect of activating the immune function of Peyer's patches and increasing the number of IgA antibody-producing plasma cells.

[0017] [3] The increasing agent according to [1] or [2] above, characterized in that it is inoculated into a mammalian mother in which the proportion of the intestinal flora of a Bacteroides strain having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 1 and a Prevotella strain having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 2 is reduced.

[0018] [4] The booster according to any one of [1] to [3] above, characterized in that it is administered orally.

[0019] In addition, other embodiments of the present invention include:

[0020] A method for increasing the level of IgA antibodies in breast milk, comprising the steps of inoculating one or more strains selected from the group consisting of the following Bacteroides strains (hereinafter sometimes referred to as the "present strains"), which have a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 1 and has an effect of increasing the level of IgA antibodies in breast milk, and the Prevotella strain (hereinafter sometimes referred to as the "present Prevotella strain"), which has a 16S rRNA gene that is at least 90% identical to the nucleotide sequence represented by SEQ ID NO: 2 and has an effect of increasing the level of IgA antibodies in breast milk, into a mammalian mother in need of increased IgA antibody levels in breast milk.

[0021] One or more strains of this invention for increasing the IgA antibody content in breast milk; or

[0022] Use of one or more strains of the present invention in the manufacture of an agent for increasing the IgA antibody content in milk.

[0023] Effects of the Invention

[0024] This strain has the effect of increasing IgA levels in breast milk. In particular, if this strain is administered to lactating mammalian mothers whose intestinal flora is disturbed due to factors such as stress or antibiotic administration, resulting in a decrease in the proportion of this strain in the overall intestinal flora and a consequent decrease in IgA levels in their milk, the effects of this strain increase the IgA levels in their milk. This can effectively prevent pathogenic bacterial or viral infections in pups (specifically, those who lack or have a low ability to produce IgA) who ingest this milk, contributing to improved productivity in the livestock industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 middle, Figure 1 A and 1B are graphs showing the results of analyzing the level of IgA antibody production and the expression level of CD93, a plasma cell marker, in mammary gland cells. Figure 1 The numerical value in A represents the ratio (%) of cells contained in the area surrounded by the square box relative to the total number of cells. Figure 1 B's "IgA high" means Figure 1The cells included in the "high" region of the region surrounded by the square frame in A (i.e., plasma cells producing IgA antibodies at high levels). Figure 1 B's "IgAlow" indicates Figure 1 The cells included in the "low" region of the region surrounded by a square frame in A (ie, plasma cells producing IgA antibodies at a low level). Figure 1 C is a graph showing the results of analyzing the level of IgA antibody production and the expression levels of four cell surface markers (B220, Ly6C, I-Ad, and CD11b) in mammary gland cells. Figure 1 The area surrounded by square boxes in C represents plasma cells that produce IgA antibodies at high levels. Figure 1 The numerical values ​​in C represent the ratio (%) of plasma cells producing IgA antibodies at high levels relative to the total number of cells.

[0026] Figure 2 middle, Figure 2 A is a graph showing the results of analysis using IgA and B220 as markers for the presence ratio of plasma cells in the mammary glands of three types of inbred (BALB / c) female mice [BALB / c mice ("non-deficient" in the figure), inguinal lymph node (hereinafter sometimes referred to as "ILN")-deficient mice ("ILN-deficient" in the figure), and PP-deficient mice ("PP-deficient" in the figure)]. Figure 2 B is based on Figure 2 A, measuring the presence of IgA-producing plasma cells in the mammary gland ( Figure 2 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 2 C is a graph showing the results (mean + standard deviation) of IgA antibody concentrations measured in the gastric contents of pups that ingested milk from the three inbred (BALB / c) dams described above. "**" and "****" in the graph indicate statistically significant differences (p < 0.01 and p < 0.0001, respectively) (the same applies hereinafter). In addition, white circles (O) in the graph represent the respective samples (the same applies hereinafter).

[0027] Figure 3 middle, Figure 3 A represents the three immunodeficient (CB-17 / Icr-scid / scidJcl) female mice [transplanted with wild-type CB-17 / Icr-+ / +Jcl mice (hereinafter sometimes referred to as "wild-type (Icr + / +Figure 2 shows the results of analysis of plasma cells in the mammary gland using IgA and B220 as markers in immunodeficient mice transplanted with monocytes from the ILN of wild-type mice ("ILN-derived monocytes transplanted"); immunodeficient mice transplanted with monocytes from the PP of wild-type mice ("PP-derived monocytes transplanted"); and immunodeficient mice not transplanted with these cells ("Not transplanted") [Figure 2]. Figure 3 B means based on Figure 3 A result, measuring IgA-producing plasma cells ( Figure 3 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 3 C is a graph showing the results (mean + standard deviation) of measuring IgA antibody concentrations in the gastric contents of pups that ingested milk from the three immunodeficient (CB-17 / Icr-scid / scidJcl) dams. "***" in the graph indicates a statistically significant difference (p < 0.001) (the same applies hereinafter). In this example, statistical processing was performed using a multiple comparison test using Prism 7 software.

[0028] Figure 4 middle, Figure 4 A is for two inbred strains (C57BL / 6) of female mice [Spibf lox / flox Mouse ("Spibf" in the figure) lox / flox ”) and based on Spibf lox / flox A diagram showing the results of analysis of plasma cells in the mammary gland of mice with intestinal epithelium-specific SpiB gene deficiency (hereinafter sometimes referred to as "SpiBcKO mice") ("SpiBcKO" in the figure) using IgA and B220 as markers. Figure 4 B means based on Figure 4 A result, measuring IgA-producing plasma cells ( Figure 4 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 4 C is a graph showing the results (mean + standard deviation) of measuring the IgA antibody concentration in the gastric contents of pups that ingested milk from the two inbred strains (C57BL / 6) of dam mice.

[0029] Figure 5The total number of intestinal bacteria (tuf gene) was analyzed using the bacterial tuf gene as an indicator after inbred (BALB / c) female mice were allowed to drink water containing three antibiotics [ampicillin ("Amp" in the figure), neomycin ("Neo" in the figure), and vancomycin ("Van" in the figure)] or their mixture ("Mix" in the figure) or water without antibiotics ("DW" in the figure). Figure 5 A) and the ratio of various bacteria that make up the intestinal flora ( Figure 5 B and Figure 5 C) is a graph of the results. Figure 5 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 5 B and Figure 5 The numerical values ​​(1 to 7) in C represent individual mice.

[0030] Figure 6 The results show that after inbred (BALB / c) female mice were allowed to freely drink water containing three antibiotics [ampicillin ("Amp"), neomycin ("Neo"), and vancomycin ("Van")] or their mixture ("Mix"), or water without antibiotics ("DW"), the proportion of four bacterial strains [Parabacteroides goldsteinii ( Figure 6 A), Bacteroides acidifaciens ( Figure 6 B), oral Prevotella buccalis ( Figure 6 C) and Escherichia albertii ( Figure 6 D)] is a graph showing the results of the ratio (mean + standard deviation). "*" in the graph indicates a statistically significant difference (p < 0.05) (the same applies hereinafter).

[0031] Figure 7 middle, Figure 7 A is a graph showing the results of analyzing plasma cells in the mammary gland using IgA and B220 as markers after inbred (BALB / c) female mice were allowed to freely drink five types of water [water containing ampicillin ("Amp" in the figure); water containing neomycin ("Neo" in the figure); water containing vancomycin ("Van" in the figure); water containing a mixture of these three antibiotics ("Mix" in the figure); or water containing no antibiotics ("DW" in the figure)]. Figure 7 B means based on Figure 7 A result, measuring IgA-producing plasma cells (in Figure 7A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 7 C is a graph showing the results (mean + standard deviation) of measuring the IgA antibody concentration in the gastric contents of pups that ingested milk from the above-mentioned inbred strain (BALB / c) mother mice that drank various types of water ad libitum.

[0032] Figure 8 The results show that after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of three antibiotics (ampicillin, neomycin, and vancomycin) (hereinafter sometimes referred to as "antibiotic mixture"), fecal microbiota transplantation (FMT; fecal microbiota transplantation, fecal microbiota transplantation) using feces of healthy inbred (BALB / c) female mice in lactation ("FMT+") or without FMT ("FMT-"), the total number of intestinal bacteria (tuf gene derived from bacteria) was analyzed using the tuf gene as an indicator. Figure 8 A) and the proportion of various bacteria that make up the intestinal flora ( Figure 8 B and Figure 8 C) is a graph of the results. Figure 8 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 8 B and Figure 8 The numerical values ​​(1 to 7) in C represent individual mice.

[0033] Figure 9 The results show that after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of three antibiotics (ampicillin, neomycin, and vancomycin) (hereinafter sometimes referred to as "antibiotic mixture"), FMT ("FMT+" in the figure) was performed using feces of healthy inbred (BALB / c) female mice during lactation, or FMT was not performed ("FMT-" in the figure), and the intestinal flora of four strains [Parabacteroides gordonii ( Figure 9 A), Bacteroides acidophilus ( Figure 9 B), oral Prevotella ( Figure 9 C) and Escherichia albertii ( Figure 9 D) Graph of the results (mean + standard deviation) of the proportion of]).

[0034] Figure 10 middle, Figure 10A is a graph showing the results of analyzing plasma cells in the mammary gland using IgA and B220 as markers, after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of the above three antibiotics, and then performing FMT ("FMT+" in the figure) using feces of healthy inbred (BALB / c) lactating female mice, or not performing FMT ("FMT-" in the figure). Figure 10 B means based on Figure 10 A result, measuring IgA-producing plasma cells (in Figure 10 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 10 C is a graph showing the results (mean + standard deviation) of measuring the IgA antibody concentration in the gastric contents of pups that ingested milk from the above-mentioned inbred (BALB / c) mother mice that underwent FMT ("FMT+" in the figure) or did not undergo FMT ("FMT-" in the figure).

[0035] Figure 11 middle Figure 11 A is a graph showing the results (mean + standard deviation) of measuring the IgA antibody concentration in the gastric contents of pups that ingested milk from three types of mother mice [SPF mice ("SPF" in the figure), GF mice ("GF" in the figure), and immunodeficient mice ("SCID" in the figure)]. Figure 11 B is a graph showing the results (mean + standard deviation) of analyzing the reactivity of IgA antibodies in the gastric contents of pups that ingested milk from SPF mice to microorganisms contained in the feces of three types of mother mice [SPF mice ("SPF" in the figure), GF mice ("GF" in the figure), and immunodeficient mice ("SCID" in the figure)].

[0036] Figure 12 middle, Figure 12 Figure A is a graph showing the results of hematoxylin-eosin staining of small intestinal tissue from three types of mother mice: wild-type mice ("wild type" in the figure); immunodeficient mice transplanted with mononuclear cells from the bone marrow of wild-type mice ("immunodeficient BMT+" in the figure); and immunodeficient mice that did not undergo BMT ("immunodeficient BMT-" in the figure). The figures (1 to 5) in the middle and lower sections are enlarged views of the areas (1 to 5) surrounded by square boxes in the figure in the upper section. The scale bar in the figure represents 500 μm. Figure 12B is a graph showing the results of immunohistochemical staining of small intestinal tissues of the three types of mother mice described above using anti-CD3 antibodies for detecting T cells and anti-B220 antibodies for detecting B cells. In the figure, areas surrounded by white lines indicated by arrows indicate the presence of B cells (B220-positive cells), and areas surrounded by white lines indicated by arrowheads indicate the presence of T cells (CD3-positive cells).

[0037] Figure 13 The total number of intestinal bacteria was analyzed using the bacterial tuf gene as an indicator for three types of mother mice [wild-type mice ("wild type" in the figure); immunodeficient mice that underwent BMT (transplantation) of mononuclear cells from the bone marrow of wild-type mice ("immunodeficient BMT+" in the figure); and immunodeficient mice that did not undergo BMT ("immunodeficient BMT-" in the figure)]. Figure 13 A) and the proportion of various bacteria that make up the intestinal flora ( Figure 13 B and Figure 13 C) is a graph of the results. Figure 13 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 13 B and Figure 13 The numerical values ​​(1 to 6) in C represent individual mice.

[0038] Figure 14 It means that the intake of two kinds of female mice [wild-type mice ( Figure 14 A) and immunodeficient mice ( Figure 14 Figure 2 shows IgA antibodies in the stomach contents of pups fed milk from wild-type mice (B) and the results (mean + standard deviation) of reactivity to microorganisms contained in the feces of three types of mother mice: wild-type mice ("Wild Type" in the figure); immunodeficient mice that underwent BMT of mononuclear cells derived from the bone marrow of wild-type mice ("Immunodeficient BMT+" in the figure); and immunodeficient mice that did not undergo BMT ("Immunodeficient BMT-" in the figure). "#" and "##" in the figure indicate statistically significant differences (p < 0.05 and p < 0.01, respectively) as determined by the t-test.

[0039] Figure 15 The figure shows the expression of four strains (Parabacteroides gordonii ( Figure 15 A), Bacteroides acidophilus ( Figure 15 B), oral Prevotella ( Figure 15 C) and Escherichia albertii ( Figure 15 D) is a graph of the results (mean + standard deviation) of the proportion of [D].

[0040] Figure 16 This figure shows that after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of three antibiotics (ampicillin, neomycin, and vancomycin), FMT ("wild-type FMT" and "immunodeficient FMT" in the figure) from feces of two types of mice [wild-type CB-17 / Icr-+ / +Jcl mice and immunodeficient (CB-17 / Icr-scid / scidJcl) mice] was performed. The total number of intestinal bacteria was analyzed using the tuf gene derived from bacteria as an indicator ( Figure 16 A) and the proportion of various bacteria that make up the intestinal flora ( Figure 16 B and Figure 16 C) is a graph of the results. Figure 16 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 16 B and Figure 16 The numerical values ​​(1 to 5) in C represent individual mice.

[0041] Figure 17 The figure shows that after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of the above three antibiotics, FMT was performed using feces from two types of mice (wild-type mice and immunodeficient mice) ("wild-type FMT" and "immunodeficient FMT" in the figure) to measure the intestinal flora of four strains [Parabacteroides gordonii ( Figure 17 A), Bacteroides acidophilus ( Figure 17 B), oral Prevotella ( Figure 17 C) and Escherichia albertii ( Figure 17 D) is a graph of the results (mean + standard deviation) of the proportion of [D].

[0042] Figure 18 A is a graph showing the results of analyzing the IgA antibody production levels and B220 expression levels of plasma cells in the mammary gland by FMT ("wild-type FMT" and "immunodeficient FMT" in the figure) using feces from two types of mice (wild-type mice and immunodeficient mice) after allowing inbred (BALB / c) female mice to freely drink water containing a mixture of the above three antibiotics. Figure 18 B means based on Figure 18 A result, measuring IgA-producing plasma cells ( Figure 18 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 18C is a graph showing the results (mean + standard deviation) of IgA antibody concentrations in the gastric contents of pups that ingested milk from the two inbred strains (BALB / c) of female mice that underwent FMT.

[0043] Figure 19 is for two strains [Bacteroides acidogenicus ( Figure 19 A) and oral Prevotella ( Figure 19 B)], using the tuf gene derived from the bacteria as an indicator, the strains that bind to IgA antibodies from the milk of BALB / c mother mice ("IgA" in the figure) were analyzed. + ”) and the non-binding strain (“IgA - The vertical axis represents the copy number of tuf genes derived from various bacteria per 1 mg of feces.

[0044] Figure 20 This indicates that inbred (BALB / c) female mice were allowed to freely drink water containing an antibiotic mixture and inoculated with three bacterial strains (Parabacteroides goldsteinii, Bacteroides acidophilus, and Prevotella oralis) ("P. Goldsteinii inoculation," "B. Acidifaciens inoculation," and "P. Buccalis inoculation" in the figure), or not inoculated with these strains ("Not inoculated" in the figure), and the total number of intestinal bacteria was analyzed using the bacterial tuf gene as an indicator ( Figure 20 A) and the proportion of various bacteria that make up the intestinal flora ( Figure 20 B and Figure 20 C) is a graph of the results. Figure 20 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 20 B and Figure 20 The numerical values ​​(1 to 8) in C represent individual mice.

[0045] Figure 21 A is a graph showing the results of analyzing plasma cells in the mammary gland using IgA and B220 as markers after inbred (BALB / c) female mice were allowed free access to water containing an antibiotic mixture and inoculated with three strains (Parabacteroides goldsteinii, Bacteroides acidophilus, and Prevotella oralis) ("P. Goldsteinii inoculation," "B. Acidifaciens inoculation," and "P. Buccalis inoculation" in the figure), or not inoculated with these strains ("Not inoculated" in the figure). Figure 21 B means based on Figure 21 A result, measuring IgA-producing plasma cells (in Figure 21 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 21C is a graph showing the results (mean + standard deviation) of measurement of IgA antibody concentrations in the gastric contents of pups that ingested milk from inbred (BALB / c) mother mice inoculated or not inoculated with the three strains described above.

[0046] Figure 22 The figure shows that BALB / c female mice were allowed to drink water containing an antibiotic mixture freely and were inoculated with three strains (P.goldsteinii, B.acifaciens, and Prevotella oralis) ("P.goldsteinii inoculation", "B.acidifaciens inoculation", and "P.buccalis inoculation" in the figure), or were not inoculated with these strains ("not inoculated" in the figure), and the titer of IgA antibodies against P.goldsteinii was measured ( Figure 22 A) IgA antibody titer against Bacteroides acidophilus ( Figure 22 B) and the titer of IgA antibodies against oral Prevotella ( Figure 22 C) Graph of the results.

[0047] Figure 23 The results show that two inbred (BALB / c) female mice [BALB / c mice ("non-defective") and PP-defective mice ("PP-defective")] were allowed to freely drink water containing an antibiotic mixture, and were inoculated with three bacterial strains (Parabacteroides goldsteinii, Bacteroides acidophilus, and Prevotella oralis) ("P. Goldsteinii inoculation", "B. Acidifaciens inoculation", and "P. Buccalis inoculation" in the figure), or not inoculated with these strains ("uninoculated" in the figure), and the total number of intestinal bacteria was analyzed using the bacterial tuf gene as an indicator ( Figure 23 A) and the proportion of various bacteria that make up the intestinal flora ( Figure 23 B and Figure 23 C) is a graph of the results. Figure 23 In A, the vertical axis represents the copy number of the tuf gene derived from various bacteria per 1 mg of feces. Figure 23 B and Figure 23 The numerical values ​​(1 to 8) in C represent individual mice.

[0048] Figure 24 The figure shows that two inbred (BALB / c) female mice [BALB / c mice ("non-defective") and PP-defective mice ("PP-defective")] were allowed to drink water containing an antibiotic mixture freely, and inoculated with three strains (Parabacteroides gordonii, Bacteroides acidophilus, and Prevotella oralis) ("P. Goldsteinii inoculation", "B. Acidifaciens inoculation", and "P. Buccalis inoculation"), or not inoculated with these strains ("uninoculated"), and the intestinal flora of four strains [Parabacteroides gordonii ( Figure 24 A), Bacteroides acidophilus ( Figure 24 B), oral Prevotella ( Figure 24 C) and Escherichia albertii ( Figure 24 D)] The results (mean + standard deviation) of the proportion of [D] are shown in the graph. "*", "***" and "****" in the graph indicate statistically significant differences (p < 0.05, p < 0.001 and p < 0.0001, respectively).

[0049] Figure 25 middle, Figure 25 A is a graph showing the results of analyzing plasma cells in the mammary gland using IgA and B220 as markers after allowing two inbred (BALB / c) female mice [BALB / c mice ("non-defective") in the figure and PP-defective mice ("PP-defective") to freely drink water containing an antibiotic mixture and inoculating them with three strains (Parabacteroides goldsteinii, Bacteroides acidophilus, and Prevotella oralis) ("P. Goldsteinii inoculation," "B. Acidifaciens inoculation," and "P. Buccalis inoculation" in the figure), or not inoculating them with these strains ("uninoculated" in the figure). Figure 25 B means based on Figure 25 A result, measuring IgA-producing plasma cells (in Figure 25 A graph showing the results (mean + standard deviation) of the number of cells contained in the lower right area of ​​each graph in A. Figure 25 Graph C shows the results (mean + standard deviation) of IgA antibody concentrations measured in the gastric contents of pups that ingested milk from the two inbred (BALB / c) dams inoculated or not with the three strains. "**," "***," and "****" in the figure indicate statistically significant differences (p<0.01, p<0.001, and p<0.0001, respectively).

[0050] Figure 26 The figure shows that two inbred (BALB / c) female mice [BALB / c mice ("non-defective") and PP-defective mice ("PP-defective")] were allowed to drink water containing an antibiotic mixture freely, and were inoculated with three strains (P.goldsteinii, B.acifaciens, and Prevotella oralis) ("P.goldsteinii inoculation", "B.acidifaciens inoculation", and "P.buccalis inoculation") or not inoculated with these strains ("uninoculated"), and the titer of IgA antibodies against P.goldsteinii was measured ( Figure 26 A) IgA antibody titer against Bacteroides acidophilus ( Figure 26 B) and the titer of IgA antibodies against oral Prevotella ( Figure 26C) is a graph showing the results. "***" and "****" in the graph indicate statistically significant differences (p < 0.001 and p < 0.0001, respectively). DETAILED DESCRIPTION

[0051] The agent for increasing the IgA antibody content in milk of the present invention is a preparation (hereinafter sometimes referred to as the "present agent"), designated for use as "for increasing the IgA antibody content in milk," containing one or more strains of the present invention (i.e., strains selected from the following genus: Bacteroides strains having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1 and has an effect of increasing the IgA antibody content in milk; and Prevotella strains having a 16S rRNA gene that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 2 and has an effect of increasing the IgA antibody content in milk). Here, the 16S rRNA gene is generally contained in the genomic DNA of the present strain.

[0052] This increasing agent can be used alone as a livestock feed, food or medicine (preparation) as a probiotic that increases the IgA antibody content in milk, or it can be mixed with additives and used as a composition (livestock feed composition, food and beverage composition or pharmaceutical composition). As the above-mentioned food and beverage, for example, health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally adjusted foods, supplements, etc.), health functional foods (specific health foods, nutritional functional foods, functional foods, etc.) can be cited. As this increasing agent, livestock feed and food and beverage are preferred.

[0053] The present Bacteroides strain may be either a living strain or an inactivated strain, as long as it has a 16S rRNA gene that is at least 90% identical to the entire nucleotide sequence of SEQ ID NO: 1 and has the effect of increasing the level of IgA antibodies in breast milk. The present Bacteroides strain also includes a Bacteroides strain that, while different from the acidogenic Bacteroides species whose effects were demonstrated in the present Examples described below, has a 16S rRNA gene that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1 and has the effect of increasing the level of IgA antibodies in breast milk. Examples of the Bacteroides strains of the present invention include Bacteroides acidogenes (e.g., JCM10556 strain [GenBank Accession No.: EU136694], NM70_E10 strain [GenBank Accession No.: MK929079], A40 strain [GenBank Accession No.: NR_028607], JJM0207-2 strain [GenBank Accession No.: KR364740]), Bacteroides caecimuris (e.g., I48 strain [GenBank Accession No.: CP015401], NM63_1-25 strain [GenBank Accession No.: MK929076]), Bacteroides thetaiotaomicron (e.g., BCRC strain 10624 [GenBank Accession No.: EU136679], strain 7330 [GenBank Accession No.: CP012937]), Bacteroides ovatus (e.g., BCRC strain 10624 [GenBank Accession No.: EU136679], strain 7330 [GenBank Accession No.: CP012937]), and Bacteroides ovatus. ovatus (e.g., ATCC strain 8483 [GenBank Accession No.: CP012938], strain V975 [GenBank Accession No.: LT622246]), etc.

[0054] The present Prevotella strain may be either a live strain or an inactivated strain, as long as it has a 16S rRNA gene that is at least 90% identical to the entire nucleotide sequence set forth in SEQ ID NO: 2 and has the effect of increasing the level of IgA antibodies in breast milk. The present Prevotella strain also includes a Prevotella strain that, while different from the oral Prevotella species whose effect was demonstrated in this Example described below, has a 16S rRNA gene that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 2 and has the effect of increasing the level of IgA antibodies in breast milk. Examples of the Prevotella strains herein include oral Prevotella (e.g., JCM12246 strain [GenBank Accession No.: NR_113098], DCW_SL_32A strain [GenBank Accession No.: MK424033], ATCC 35310 strain [GenBank Accession No.: NR_044630], SEQ186 strain [GenBank Accession No.: JN867261]), BV3C7 strain (GenBank Accession No.: JN809774), and BV3P1 strain (GenBank Accession No.: JN809764).

[0055] More specifically, the present Bacteroides strain and the present Prevotella strain have the effect of activating the immune function of Peyer's patches, which are intestinal lymphoid tissue, and increasing the number of IgA antibody-producing plasma cells.

[0056] The strains of the present invention may be naturally occurring Bacteroides strains and Prevotella strains of the present invention, or strains obtained by modifying naturally occurring Bacteroides strains and Prevotella strains of the present invention using genetic recombination techniques. Examples of such modified strains include modified strains expressing antigens in order to induce specific IgA antibodies against antigens of bacteria or viruses that cause infections (e.g., diarrhea) in breast milk, and modified strains expressing antigens in order to induce the production of IgA antibodies against antigens that cause infections (e.g., mastitis / mastitis) in the mammary glands of mammalian mothers. The additives of the present invention comprising these modified strains are also useful as maternal vaccination-type mother-to-child transfer vaccines or vaccines targeting the mammary glands (breasts) of the mother.

[0057] In this specification, "increase in the level of IgA antibodies in milk" means that the level of IgA antibodies in the milk of a lactating mammalian mother is increased compared to the level of IgA antibodies in the milk of a lactating mammalian mother serving as a comparison control (hereinafter sometimes referred to as a "comparison control mother"). The comparison control mother is not particularly limited and may be a lactating mammalian mother without any cause of intestinal flora disturbance such as stress or antibiotic administration. However, since the effect is demonstrated in the present Examples described later, a lactating mammalian mother in which the proportion of the present Bacteroides strain and / or the present Prevotella strain in the overall intestinal flora is preferably exemplified by a decrease in the proportion of the present Bacteroides strain and / or the present Prevotella strain in the overall intestinal flora compared to a lactating mammalian mother without any cause of intestinal flora disturbance such as stress or antibiotic administration (in other words, a mammalian mother with a normal intestinal flora). Regarding whether the IgA antibody content in breast milk is increased, the threshold (cutoff value) can be set to an arbitrary value. Examples of such thresholds include: the mean value of the IgA antibody content in the milk of control mothers; the mean + standard deviation (SD); the mean + 2SD; the mean + 3SD; the median; the interquartile range, etc. Furthermore, the threshold can be calculated using a ROC (Receiver Operating Characteristic) curve using statistical analysis software based on data on the IgA antibody content in the milk of lactating mammalian mothers inoculated with the present strain and data on the IgA antibody content in the milk of lactating mammalian mothers not inoculated with the present strain, so as to increase the sensitivity (the proportion of lactating mammalian mothers inoculated with the present strain that can accurately be determined as positive) and the specificity (the proportion of lactating mammalian mothers not inoculated with the present strain that can accurately be determined as negative).

[0058] In the present specification, mammals include humans, non-human mammals [e.g., monkeys, mice, rats, dogs, cats, livestock (e.g., rabbits, pigs, horses, cattle, sheep, goats, deer)], etc., and preferably humans and livestock are exemplified.

[0059] As the subject to be vaccinated with the present enhancing agent (also referred to as "administration" or "application" depending on the form of the present enhancing agent), any mammalian mother (preferably a lactating mammalian mother) who needs to increase the IgA antibody content in breast milk can be used. Since its effect is confirmed in the present embodiment described later, a mammalian mother in which the proportion of the present Bacteroides strain and the present Prevotella strain in the overall intestinal flora is reduced can be preferably exemplified.

[0060] In the present invention, "at least 90% identity to the nucleotide sequence represented by SEQ ID NO: 1 (or 2)" means that one or more nucleotides in the nucleotide sequence of SEQ ID NO: 1 (or 2) are substituted, deleted, inserted, added, or inverted, and that 90% or more of the entire nucleotide sequence of SEQ ID NO: 1 (or 2) is identical. Here, "a nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted, added, or inverted" refers to a nucleotide sequence in which, for example, 1 to 149 nucleotides are substituted, deleted, inserted, added, or inverted, preferably 1 to 100 nucleotides, more preferably 1 to 75 nucleotides, even more preferably 1 to 50 nucleotides, even more preferably 1 to 40 nucleotides, even more preferably 1 to 30 nucleotides, and even more preferably 1 to 15 nucleotides are substituted, deleted, inserted, added, or inverted.

[0061] In the present invention, “at least 90% identity” is preferably 91% or higher, more preferably 92% or higher, even more preferably 93% or higher, further more preferably 94% or higher, particularly preferably 95% or higher, particularly more preferably 96% or higher, particularly more preferably 97% or higher, particularly still more preferably 98% or higher, and most preferably 99% or higher (for example, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, 99.9% or higher, 100%). Nucleotide sequence identity can be determined using a program called BLASTX or BLASTP based on the BLAST algorithm of Kallin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl Acad Sci USA 90: 5873, 1993), or a program called BLASTN based on (Altschul SF, et al: J Mol Biol 215: 403, 1990). When using BLASTN to analyze a nucleotide sequence, the parameters are, for example, score = 100 and word length = 12.

[0062] The present supplement is broadly categorized into liquid and non-liquid forms. Liquid supplements can be produced by purifying the strain from its culture medium, adding appropriate physiological saline, supplements, or pharmaceutical additives as needed, and then filling the solution into ampoules or vials. Non-liquid supplements can be produced by adding an appropriate cryoprotectant (e.g., glycerol, dimethyl sulfoxide [DMSO], trehalose, dextran) to the liquid supplement, filling the solution into ampoules or vials, and then freezing or freeze-drying the solution.

[0063] The method of administering the enhancing agent of this invention may be oral administration or non-oral administration (e.g., intravenous administration, topical administration), but since its effect is confirmed in the present example described later, oral administration is preferably exemplified.

[0064] In this specification, examples of additives include pharmaceutically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives, coating agents, extenders, lubricants, glidants, solubilizers, smoothing agents, flavoring agents, sweeteners, solvents, gelling agents, and nutrients. Specific examples of such additives include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, rubber, talc, magnesium stearate, hydroxypropylcellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0065] The inoculation amount of the present strain contained in the present supplement varies depending on the species, age, weight, physical condition, etc. of the inoculated object (mammalian mother), so it cannot be generalized, but for example, it can be 10 per day per kg of body weight. 4 ~10 12 cfu (Colony Forming Unit), preferably 10 6 ~10 10 cfu. In addition, such an amount can be inoculated once or divided into multiple inoculations. In addition, when the present enhancer is a livestock feed composition, the amount of the present strain contained in the livestock feed composition is, for example, 10 per 1g of the livestock feed composition. 4 ~10 12 cfu / g, preferably 10 6 ~10 10 cfu.

[0066] Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.

[0067] Example

[0068] 1. Materials and Methods

[0069] [Strain]

[0070] Three bacterial strains (Parabacteroides gordonii [JCM13446], Bacteroides acidogenes [JCM10556], and Prevotella oralis [JCM12246]) were obtained from the Microbial Material Development Laboratory of the RIKEN BioResource Research Center. These bacteria were cultured overnight in GAM liquid medium under anaerobic conditions using a bacterial culture gas concentration regulator (AnaeroPack [registered trademark]) (manufactured by SUGIYAMA-GEN), and the bacterial count was counted.

[0071] [Model Animals]

[0072] Six types of mice (BALB / c [BALB / cCrSlc] mice; C57BL / 6 [C57BL / 6NCrSlc] mice; ICR [CB-17 / Icr-+ / +Jcl] mice; ICR mice with severe combined immunodeficiency [CB-17 / Icr-scid / scidJcl] (i.e., immunodeficient mice); and B6N.Cg-Tg [Vil-cre] 997Gum / J mice) were purchased from Japan SLC Co., Ltd., Japan CLEA Co., Ltd., The Jackson Laboratory, or Sankyo LABO Service Co., Ltd. and bred at the animal facilities of the Graduate School of Agriculture, Tohoku University or the Institute of Medical Science, University of Tokyo. In addition, B6-Tg (CAG-FLPe) 36 mice were distributed from RIKEN Bioresource Research. Figure 2 and Figures 23 to 26 The PP-deficient mice used in the experiment were prepared by intrauterine administration of 1 mg of anti-IL-7Rα antibody (A7R34) to BALB / c mice on day 14 of birth (refer to the literature "Yoshida et al., International Immunology, 11, 643-655.1999"). Figure 2 The ILN-deficient mice used in the experiments were created by surgically removing the inguinal lymph nodes. Figure 3 The mice used in the experiment were obtained by converting the PP-derived monocytes and ILN-derived monocytes obtained by the method described in the following [Isolation of Cells] into wild-type (Icr) at the beginning of mating of immunodeficient mice. + / + ) mice were administered intravenously to prepare the Figures 5 to 7The mice used in the experiment were prepared by allowing BALB / c female mice to freely drink water containing antibiotics (1 g / L ampicillin, 1 g / L neomycin, 500 mg / L vancomycin or a mixture thereof) from before mating to 14 days after birth. Figures 8 to 10 and Figures 16 to 18 The mice used in the experiment were given free access to water containing an antibiotic mixture from before mating to 7 days after birth by BALB / c female mice. The feces ( Figures 8 to 10 and Figures 16 to 18 ) or feces collected from immunodeficient mice ( Figures 16 to 18 ) is prepared by FMT. Figures 12 to 15 The mice used in the experiments were derived from wild type (Icr + / + ) mice were transplanted with 106 bone marrow mononuclear cells for bone marrow transplantation (BMT). Figures 20 to 22 The mice used in the experiment were given free access to water containing an antibiotic mixture from before mating to 7 days after birth by BALB / c female mice. 10 CFU of three strains (Parabacteroides gordonii, Bacteroides acidogenes, and Prevotella oralis) were prepared by oral administration of 500 μL of PBS. All animal experiments were conducted according to protocols approved by the in-house animal experiment committees of both Tohoku University and the University of Tokyo.

[0073] [Creation of SpiB cKO mice]

[0074] The targeting vector was designed by flanking exons 2 to 5 of the Spib gene at loxP sites and inserting a Frt-flanked neo resistance cassette in the intron between exons 5 and 6. After linearizing 30 μg of the targeting vector, it was transformed into JM8A1.N3 embryonic stem cells (obtained from the KOMP Repository). After selecting colonies of neomycin-resistant and ganciclovir-resistant embryonic stem cells, homologous recombinants targeting the Spib gene were screened by PCR using the primer set shown in Table 1 below or Southern blot analysis using the probe shown in Table 1 below. The screened homologous recombinants were microinjected into blastocysts of C57BL / 6 mice to produce chimeric mice. In order to delete the Neo resistance cassette and obtain floxedSpib mice, heterozygous F1 mice were mated with B6-Tg (CAG-FLPe) 36 mice. Then, the floxedSpib mice were mated with B6N.Cg-Tg(Vil-cre)997Gum / J mice to produce Spibflox / flox Mice. By making Spibf lox / flox The mice were mated with mice expressing Cre recombinase specifically in intestinal epithelial cells (Villin-Cre transgenic mice) to generate SpiB conditional knockout mice (ie, SpiB cKO mice).

[0075] [Table 1]

[0076]

[0077] [Cell separation]

[0078] Monocytes were isolated from the mammary gland, PP, and ILN of various mice. Specifically, the mammary gland and PP were digested with 0.5 mg / mL collagenase for 60 minutes at 37°C to isolate mammary gland-derived monocytes and PP-derived monocytes. In addition, ILN-derived monocytes were prepared by physically treating the ILN. A portion of the resulting monocytes was stained with Turk's solution, the cell number was counted, and then used for flow cytometry or intravenous administration.

[0079] [Flow cytometry]

[0080] Monocytes isolated from various tissues were blocked with 10 μg / mL anti-mouse CD16 / 32 (2.4G2) at 4°C for 15 minutes and incubated in the presence of five antibodies corresponding to five cell surface markers (B220, Ly6C, CD93, I-Ad, and CD11b) labeled with fluorescent substances (2 μg / mL BV421-labeled anti-CD45R [B220] antibody [RA3-6B2, BD Bioscience], 2 μg / mL PE-labeled anti-Ly6C antibody [HK1.4, Biolegend], 2 μg / mL PE-Cy7-labeled anti-CD93 antibody [AA4.1, Biolegend], 5 μg / mL Alexa647-labeled anti-I-Ad antibody [39-10-8, Biolegend], and 2 μg / mL BV510-labeled anti-CD11b antibody [M1 / 70, BD Bioscience]). Bioscience company]) in the presence of 5 μg / mL, antigen-antibody reaction treatment was performed at 4 ° C for 30 minutes. In addition, as isotype control antibodies, 4 antibodies (5 μg / mL of Alexa647 labeled mouse IgG3 antibody [MG3-35, Biolegend company], 2 μg / mL of BV421 labeled rat IgG2a [R35-95, BD Bioscience company], 2 μg / mL of PE-Cy7 labeled rat IgG2b [RTK4530, Biolegend company] and 2 μg / mL of PE labeled rat IgG2c [RTK4174, Biolegend company]) were used. In addition, in order to remove dead cells in flow cytometry analysis, 10 μg / times of cell viability solution (Cell Viability Solution) (BD Bioscience company) was added. In addition, to detect intracellular IgA antibodies, mononuclear cells isolated from various tissues were fixed in a 4% (w / v) paraformaldehyde solution for 20 minutes at room temperature, treated with a surfactant (0.1% [w / v] saponin) for 15 minutes at room temperature, and then subjected to antigen-antibody reaction in the presence of 5 μg / mL FITC-labeled anti-IgA antibody (C10-3, manufactured by BD Bioscience) for 30 minutes at room temperature. In addition, two antibodies (5 μg / mL FITC rat IgG1 [R3-34, manufactured by BD Bioscience] and 2 μg / mL BV421 rat IgG1 [R3-34, manufactured by BD Bioscience]) were used as isotype control antibodies.Flow cytometry was performed using Attune NxT AcousticFocusing Cytometer (manufactured by Thermo Fisher Scientific) or Accuri C6 flow cytometer (manufactured by BD Bioscience).

[0081] [ELISA method]

[0082] In order to measure the concentration of IgA antibodies in the milk of mother mice, the concentration of IgA antibodies (hereinafter sometimes referred to as "IgA antibodies derived from milk") in the gastric contents collected from pups that had ingested milk was measured using the ELISA method. Specifically, 1 mg of the gastric contents of the pups that had ingested milk was suspended in 10 μL of PBS, and the supernatant (hereinafter referred to as "the sample derived from milk") was recovered by centrifugation. Next, 100 μg / mL of anti-IgA antibody (manufactured by Bethyl Laboratries) was added to each well of a 96-well ELISA plate, and solid-phase treatment was performed by incubating overnight at 4°C. After blocking treatment was performed at room temperature for 1 hour in the presence of 1% (w / v) BSA, the sample derived from milk after two-stage dilution was added to each well and incubated at room temperature for 2 hours. After washing with PBS, 100 ng / mL of HRP-labeled IgA antibody (an anti-IgA antibody with a different epitope from the above-mentioned immobilized anti-IgA antibody) (manufactured by Bethyl Laboratries) was added to each well and incubated at room temperature for 1 hour. A signal derived from HRP was generated using a TMB (Tetramethylbenzidine) microwell horseradish peroxidase substrate system (manufactured by SeraCare Life Sciences). A standard curve for IgA antibody was prepared using mouse serum containing known concentrations of IgA antibody. Based on this standard curve, the IgA antibody concentration in the gastric contents of pups that had ingested milk was measured.

[0083] For milk-derived IgA antibodies, the ELISA method was used to analyze their reactivity to feces (including intestinal microorganisms) and three strains (Parabacteroides gordonii, Bacteroides acidogenes, and Prevotella oralis). Specifically, PBS containing 100 μg / mL of feces and PBS containing 10 μg / mL of the above three strains were filtered through a 100 μm pore size cell strainer, added to each well of a 96-well ELISA plate, and incubated at 4°C overnight for solid phase treatment. For the plate after the feces were solid phased, the plate was blocked in the presence of 1% (w / v) BSA and 1 μg / mL of anti-IgA antibodies for 1 hour at room temperature; for the plate after the above three strains were solid phased, the plate was blocked in the presence of 1% (w / v) BSA for 1 hour at room temperature. Then, a milk-derived sample diluted to 1:64 and an undiluted milk-derived sample were added to each well and incubated at room temperature for 2 hours. After washing with PBS, 100 ng / mL of HRP-labeled IgA antibody (Bethyl Laboratories) was added and incubated at room temperature for 1 hour. A signal derived from HRP was generated using the TMB microwell horseradish peroxidase substrate system (SeraCare Life Sciences). The titer of milk IgA against feces (including intestinal microorganisms) and the above three strains was calculated as OD 450 value.

[0084] [IgA-Seq]

[0085] To identify the intestinal bacterial species bound by IgA antibodies in milk, a partially modified IgA-Seq method described in the document "Palmet al., Cell 158, 1000-1010.2014" was used. Specifically, PBS (100 μg / μL) containing feces collected from healthy BALB / c mice was filtered through a 40 μm pore cell filter to remove debris, thereby preparing a fecal suspension containing intestinal microorganisms. After blocking treatment for 30 minutes at 4°C in the presence of 500 μg / mL anti-IgA antibody, 20% (v / v) normal rat serum, and 1% (w / v) BSA, the suspension was mixed with a milk-derived sample at a ratio of 1:1 and incubated at 4°C for 30 minutes. After washing, the suspension was incubated at 4 ° C for 30 minutes in the presence of 2 μg / mL of PE-labeled anti-IgA antibody (mA-6E1, Invitrogen) and 500nM of a nuclear staining reagent (SYTO 9), and then incubated at 4 ° C for 30 minutes in the presence of 30 μL / test Anti-PE MicroBeads UltraPure (Miltenyi Biotec). The bacteria bound to the IgA antibody were recovered using a magnetic cell sorter AutoMACS (Miltenyi Biotec), and genomic DNA from the bacteria was extracted using a stool DNA isolation kit (Stool DNA Isolation Kit) (Chiyoda Scientific Co., Ltd.). Using this genomic DNA as a template, the intestinal bacterial species that bind to the IgA antibody in milk were identified according to the method described in the following [Metagenomic Analysis] project.

[0086] [Metagenome Analysis]

[0087] Genomic DNA from bacteria was extracted from the feces of various mice using the QIAamp DNA Stool Mini Kit (Qiagen) and subjected to metagenomic analysis. Specifically, PCR was performed using PrimeSTAR HS DNA polymerase (Takara Bio) and the primers listed in Table 2 below, amplifying the V3 and V4 regions of the 16S rRNA gene of various bacteria using the extracted bacterial genomic DNA as a template.

[0088] [Table 2]

[0089]

[0090] The single underlined part in the table represents the linker tag sequence, and the double underlined part represents the spacer sequence.

[0091] For the PCR amplification products obtained from the first PCR, a second PCR was performed using a forward primer (5'-CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3') containing a 6-base index represented by "xxxxxx" and a reverse primer (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3') to identify individual samples, as described in the literature "Palmet al., Cell 158, 1000-1010.2014." The resulting PCR amplification products were sequenced using the MiSeq platform and MiSeq Reagent Kit v.2 (manufactured by Illumina). The resulting data were analyzed using BaseSpace (manufactured by Illumina) to identify the bacterial species.

[0092] [Quantitative PCR]

[0093] Quantitative PCR was performed using TB Green Premix Ex Taq II (Takara Bio) using bacterial genomic DNA extracted using the methods described in the [IgA-Seq] or [Metagenomic Analysis] sections as templates to quantify the copy number of the bacterial-specific tuf gene. All primers were designed using the Perfect Real-time Support System (Takara Bio).

[0094] [Tissue staining]

[0095] Small intestinal tissue was isolated from mice that had undergone BMT according to a common method, fixed in 4% (w / v) paraformaldehyde solution, and then embedded in paraffin. Tissue sections (5 μm) were blocked in TNB solution at room temperature for 30 minutes, and then subjected to antigen-antibody reaction at 4 ° C overnight in the presence of PE-labeled anti-CD45R (B220) antibody (RA3-6B2) and anti-human CD3 (SP2) antibody that cross-reacts with mouse CD3. After washing, the tissue sections were incubated at room temperature for 1 hour in the presence of HRP-labeled IgG antibody, and the signal from CD3 was amplified using TSA Plus fluorescein System (manufactured by PerkinElmer) at room temperature for 10 minutes. In addition, cell nuclei were stained with 1 μg / mL of DAPI. In addition, for histopathological analysis, tissue sections were stained with hematoxylin-eosin, and images were obtained using either BZ-9000 (manufactured by Keyence) or BX63 (manufactured by Olympus).

[0096] 2. Results

[0097] [Analysis of cell surface marker expression in IgA antibody-producing plasma cells]

[0098] It has been reported that in order to produce IgA in the mammary gland, the migration of IgA antibody-producing plasma cells to the mammary gland is indispensable (refer to the literature "Halsey et al., Ann NY Acad Sci 409, 452-460.1983" and "Niimi et al., Mucosal Immunol 11, 643-653.2018"). Therefore, in order to identify IgA-producing plasma cells in the mammary gland of female mice, flow cytometry using 6 plasma cell-associated markers (IgA antibodies and 5 cell surface markers [B220, Ly6C, I-Ad, CD11b and CD93]) was performed. As a result, plasma cells that produce IgA antibodies at high levels showed that 3 cell surface markers (Ly6C, I-Ad and CD93) were positive and two cell surface markers (B220 and CD11b) were negative (refer to Figure 1 Therefore, in subsequent experiments, B220-negative and IgA-positive plasma cells were used as indicators of IgA-producing plasma cells.

[0099] [Peyer's patches play a major role in the production of maternal IgA antibodies in the mammary gland]

[0100] The inguinal lymph nodes (ILN) are known to function as draining lymph nodes for the mammary gland (see "Leonhardt, Gene 94, 121-124. 1990"). Furthermore, Peyer's patches (PP) are known to play a crucial role in the mucosal immune system, particularly in the gastrointestinal tract, where IgA antibodies are most abundantly produced (see "Lindner et al., Nat Immunol 16, 880-888. 2015" and "Moro-Sibilot et al., Gastroenterology 151, 311-323. 2016"). Therefore, using two maternal mouse models (ILN-deficient mice and PP-deficient mice), the effects of maternal IgA antibody production in the mammary gland were analyzed. The results showed that the ratio and number of IgA-producing plasma cells in the mammary glands of ILN-deficient mice were almost unchanged compared to those in the mammary glands of non-deficient mice. In contrast, the ratio and number of IgA-producing plasma cells in the mammary glands of PP-deficient mice were significantly lower than those in the mammary glands of non-deficient mice (refer to Figure 2 A and B). In addition, it was shown that the concentration of IgA antibodies in the milk of ILN-deficient mice was almost unchanged compared to the concentration of IgA antibodies in the milk of non-deficient mice, whereas the concentration of IgA antibodies in the milk of PP-deficient mice was significantly lower than that of non-deficient mice (refer to Figure 2 C).

[0101] Furthermore, it was shown that even when immunodeficient (CB-17 / Icr-scid / scidJcl) female mice with T or B cell deficiency were transplanted with wild-type (Icr + / + ) mice, the ratio and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies in milk were almost unchanged compared to those of immunodeficient mothers without transplantation. In contrast, when mononuclear cells from the PP of wild-type mice were transplanted into immunodeficient mothers, the ratio and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies in milk were significantly higher than those of immunodeficient mothers without transplantation (refer to Figure 3 ).

[0102] These results indicate that PP, but not ILN, is required for the migration of IgA-producing plasma cells into the mammary gland.

[0103] [Antigen uptake by M cells in PP is required for the production of maternal IgA antibodies in the mammary gland]

[0104] Spib is known to be a transcription factor that participates in the differentiation of M cells that take up antigens present in follicle-associated epithelium (FAE) into mature B cells that produce IgA antibodies in the intestinal epithelium including follicle-associated epithelium (FAE) (refer to the literature "Kanaya et al., Nat Immunol 13, 729-736.2012" and "Sato et al., Mucosal Immunol 6, 838-846.2013"). On the other hand, the conditional knockout model mouse of the Spib gene (i.e., SpiB cKO mouse) is a mouse that lacks M cells in Peyer's patches (PP). Therefore, the correlation between Spib and IgA production in the mammary gland was analyzed using SpiB cKO mice. The results showed that the proportion and value of IgA-producing plasma cells in the mammary gland of SpiB cKO mice were significantly higher than those of Spibf. lox / flox The proportion and number of IgA-producing plasma cells in the mammary glands of mice (i.e., mice without SpiB gene defect) were significantly lower (refer to Figure 4 A and B) In addition, the concentration of IgA antibodies in the milk of SpiB cKO mice was compared with that in the milk of SpiB cKO mice. flox / flox The concentration of IgA antibodies in the milk of mice was significantly lower than that in Figure 4 C).

[0105] This result shows that Spib is involved in the production of IgA in the mammary gland. Figure 2 and Figure 3 The results showed that the immune function of PP was activated by antigens in the intestine taken up by M cells, the number of IgA-producing plasma cells increased, and the increased IgA-producing plasma cells migrated to the mammary gland, resulting in an increase in the IgA antibody content in milk.

[0106] [Gut microbes promote IgA antibody production in breast milk]

[0107] Considering that the intestinal flora establishes a balance in the body together with the host's immune cells in the gastrointestinal tract including PP, the intestinal flora was disturbed by administering various types of antibiotics, specifically ampicillin, neomycin, vancomycin or a mixture thereof, to BALB / c female mice during pregnancy to lactation, and the effect on maternal IgA antibody production was investigated. First, in order to investigate the effects of various antibiotic treatments on the intestinal flora, metagenomic analysis was performed. As a result, although the total number of intestinal bacteria was almost unchanged between the various antibiotic treatments and the untreated group (refer to Figure 5 A), but the composition of the intestinal flora varies greatly between the two (refer to Figure 5 B and Figure 5C), in particular, the proportions of four strains (Parabacteroides gordonii, Bacteroides acidogenicus, Prevotella oralis, and Escherichia albertii) relative to the intestinal flora changed significantly (refer to Figure 6 Specifically, when vancomycin or a mixture of three antibiotics (ampicillin, neomycin, and vancomycin) was administered to female mice through free drinking water, the three strains (Parabacteroides gordonii [ Figure 6 A], Bacteroides acidophilus[ Figure 6 B] and oral Prevotella [ Figure 6 C]) in the intestinal flora decreased significantly, compared with one strain (Escherichia albertii [ Figure 6 Furthermore, it was shown that when vancomycin or a mixture of the above three antibiotics was administered to mother mice via free drinking water, the ratio and number of IgA-producing plasma cells in the mammary glands of the mother mice were significantly lower than those of mother mice not administered antibiotics (refer to Figure 7 A and B), and the concentration of IgA antibodies from breast milk was also significantly lower (refer to Figure 7 C).

[0108] These results suggest that one of three strains (Parabacteroides gordonii, Bacteroides acidogenes, and Prevotella oralis) contained in the mammalian maternal gut microbiota is involved in IgA antibody production in the mammary gland.

[0109] In order to verify the correlation between the three strains of intestinal flora and the production of IgA antibodies in the mammary gland in more detail, a mixture of the three antibiotics was administered to female mice to disrupt the intestinal flora. Then, FMT was performed using feces collected from healthy BALB / c mice (in which the three strains coexisted). The results showed that the total number of intestinal bacteria in female mice that underwent FMT was slightly increased compared to female mice that did not undergo FMT (refer to Figure 8 A), but the proportion of bacteria that make up the intestinal flora varies greatly between the two (refer to Figure 8 B and Figure 8 C), the proportion of four strains (Parabacteroides gordonii, Bacteroides acidogens, Prevotella oralis, and Escherichia albertii), especially two strains (Bacteroides acidogens and Prevotella oralis), in the intestinal flora was significantly higher in mice that underwent FMT compared with those that did not (refer to Figure 9 ). In addition, it was shown that the ratio and number of IgA-producing plasma cells in the mammary gland of mother mice that underwent FMT were significantly higher than those of mother mice that did not undergo FMT (refer to Figure 10 A and B), and the concentration of IgA antibodies from breast milk was also significantly higher (refer to Figure 10 C).

[0110] To analyze the correlation between IgA antibody production in breast milk and intestinal flora, we analyzed the concentration of IgA antibodies derived from breast milk of GF (germ-free) mice, which have no intestinal flora, and SPF (special pathogen-free) mice, which have physiological intestinal flora.

[0111] Milk samples were collected from lactating mice maintained in either an SPF facility or a sterile facility. It was shown that the IgA antibody concentration in milk from GF mice was significantly lower than that in milk from SPF mice, and was close to that in milk from immunodeficient mice lacking all antibody subclasses (refer to Figure 11 A). In addition, although IgA antibodies derived from the milk of SPF mice showed reactivity to microorganisms contained in the feces of SPF mice, they showed almost no reactivity to microorganisms contained in the feces of immunodeficient mice (refer to Figure 11 B) Furthermore, no reaction was observed between IgA antibodies derived from milk of SPF mice and feces of GF mice, which were used as a control and did not contain microorganisms. Based on these results, considering that host immune cells, particularly lymphocytes, may contribute to the intestinal microbial environment involved in the production of IgA antibodies in milk, analysis was conducted using immunodeficient mice.

[0112] Peyer's patches are rarely observed in immunodeficient mice. However, when mononuclear cells from the bone marrow of wild-type mice are transplanted into immunodeficient mice (BMT), PP-like lymphoid structures containing sufficient B cells and T cells are confirmed (refer to Figure 12 ).

[0113] The results showed that although PP organogenesis has been reported to begin before birth (see the document "Honda et al., J Exp Med 193, 621-630.2001"), the immune system was reconstructed by transplanting mononuclear cells from the bone marrow of wild-type mice into immunodeficient mice after birth, resulting in the formation of PP-like intestinal lymphoid tissue.

[0114] Furthermore, the total number of intestinal bacteria in immunodeficient mice was almost unchanged between those transplanted with mononuclear cells derived from wild-type mouse bone marrow and those without transplantation (refer to Figure 13 A), but the composition of the intestinal flora was significantly altered by transplanting mononuclear cells from the bone marrow of wild-type mice (refer to Figure 13 B and Figure 13C) In addition, IgA antibodies derived from the milk of wild-type mice showed reactivity not only to microorganisms contained in the feces of wild-type mice, but also to microorganisms contained in the feces of immunodeficient mice transplanted with mononuclear cells derived from the bone marrow of wild-type mice (see Figure 14 A). On the other hand, the reactivity of milk from immunodeficient mice that do not contain IgA antibodies to microorganisms contained in the feces of wild-type mice and to microorganisms contained in the feces of immunodeficient mice transplanted with mononuclear cells derived from wild-type mouse bone marrow is at background level (refer to Figure 14 B) Furthermore, the proportion of two strains (Bacteroides acidophilus and Prevotella oralis) in the intestinal flora of immunodeficient mice was significantly increased by transplanting mononuclear cells from the bone marrow of wild-type mice (refer to Figure 15 ).

[0115] These results suggest that the immune system of lactating mice is required for both strains (Bacteroides acidogenicus and Prevotella oralis) to colonize the intestines of lactating mice and for the IgA antibody levels in milk to increase.

[0116] Furthermore, a mixture of the three antibiotics was administered to female mice, and FMT was performed using feces collected from healthy mice that were coexisting with the three strains and feces collected from immunodeficient mice that were not coexisting. The results showed that although the total number of intestinal bacteria was almost unchanged between the FMT using feces collected from healthy mice and the FMT using feces collected from immunodeficient mice (see Figure 16 A), but the structure of the intestinal flora changes between the two (refer to Figure 16 B and Figure 16 C), in particular, the proportion of two strains (Bacteroides acidogenicus and Prevotella oralis) in the intestinal flora was significantly higher when FMT was performed using feces of wild-type mice than when FMT was performed using feces of immunodeficient mice (refer to Figure 17 In addition, the proportion and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies derived from milk were significantly higher when FMT was performed using feces of wild-type mice than when FMT was performed using feces of immunodeficient mice (refer to Figure 18 ).

[0117] These results show that the mammalian mother's immune system regulates the intestinal environment created by two strains (Bacteroides acidophilus and Prevotella oralis) to produce IgA antibodies in the mammary gland.

[0118] Bacteroides acidogenus is a strain of the genus Bacteroides having a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 1. Furthermore, Prevotella oralis is a strain of the genus Prevotella having a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 2.

[0119] [The specificity of IgA antibodies in breast milk depends on the intestinal microbial environment]

[0120] Next, to analyze the specificity of IgA antibodies in milk for two strains (Bacteroides acidophilus and Prevotella oralis), IgA-Seq was performed using feces collected from healthy BALB / c mice and stomach contents of pups that had ingested milk from BALB / c mothers. The results showed that the proportion of strains that bound to IgA antibodies in milk for the two strains was higher than the proportion of strains that did not (refer to Figure 19 ).

[0121] These results indicate that the two strains (Bacteroides acidogenicus and Prevotella oralis) not only promote the production of maternal IgA antibodies overall in the mammary gland but also stimulate immune cells in the PP to promote the production of IgA antibodies against these strains.

[0122] [Oral inoculation of B. acidifaciens and P. buccalis increases IgA antibody production in breast milk]

[0123] The two strains (Bacteroides acidophilus and Prevotella oralis) were inoculated into BALB / c female mice administered with the antibiotic mixture to verify whether the production of IgA antibodies in milk increased. In addition, Parabacteroides gordonii was used as a control. As a result, the total number of intestinal bacteria was almost unchanged when these strains were inoculated compared to when the strains were not inoculated (refer to Figure 20 A), although changes can be seen in the structure of intestinal flora (refer to Figure 20 B and Figure 20 C), but when two strains (Bacteroides acidophilus and Prevotella oralis) were inoculated, the proportion and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies derived from milk were significantly increased compared with the case without inoculation (refer to Figure 21 On the other hand, when the control strain Parabacterium gordonii was inoculated, the ratio and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies derived from milk were almost unchanged compared to the case where the strain was not inoculated (refer to Figure 21 ). In addition, it was shown that when two strains (Bacteroides acidophilus and Prevotella oralis) were inoculated, the amount of milk-derived IgA antibodies specific to these strains increased significantly (refer to Figure 22 ).

[0124] These results indicate that inoculation of two bacterial strains (Bacteroides acidophilus and Prevotella oralis) into mammalian mothers with disturbed intestinal flora and decreased mammary IgA antibody production activates PP's immune function and increases mammary IgA antibody production.

[0125] [PP is essential for the increase in IgA antibody production in breast milk following oral inoculation with B. acidifaciens and P. buccalis]

[0126] Using PP-deficient mice, we verified whether PP is essential for the increase in IgA antibody production in the mammary gland that was confirmed when two strains (acidogenic Bacteroides and oral Prevotella) were inoculated into mammalian mothers with disturbed intestinal flora and decreased IgA antibody production in the mammary gland. In addition, Parabacteroides gordonii was used as a control. As a result, if the two strains (acidogenic Bacteroides and oral Prevotella) were inoculated into PP-non-deficient mice (BALB / c female mice) and PP-deficient mice, respectively, the total number of intestinal bacteria in both mice remained almost unchanged (refer to Figure 23 A), but in the case of B. acidifaciens inoculation, the proportion of B. acidifaciens in the intestinal flora of both mice increased significantly (refer to Figure 23 B. Figure 23 C and Figure 24 ). In addition, when PP-non-deficient mice were inoculated with the above-mentioned two strains, the proportion and number of IgA-producing plasma cells in the mammary gland and the concentration of IgA antibodies derived from milk significantly increased compared to the case where the strains were not inoculated; in contrast, even when PP-deficient mice were inoculated with the above-mentioned two strains, no increase in the proportion and number of IgA-producing plasma cells in the mammary gland and the increase in the concentration of IgA antibodies derived from milk were observed, and these were at the same level as when PP-non-deficient mice were inoculated with the control Parabacteroides gordonii (refer to Figure 25 ). In addition, when PP non-deficient mice were inoculated with the above two strains, the amount of IgA antibodies specific to these strains derived from milk increased significantly; in contrast, even when PP deficient mice were inoculated with the above two strains, no increase in the amount of IgA antibodies specific to these strains derived from milk was observed (refer to Figure 26 ).

[0127] These results indicate that PP (specifically, activation of the immune function of PP) is essential for the increase in IgA antibody production in the mammary gland observed when two strains (Bacteroides acidophilus and Prevotella oralis) are inoculated into mammalian mothers with disturbed intestinal flora and decreased IgA antibody production in the mammary gland.

[0128] Industrial Applicability

[0129] The present invention contributes to improving the productivity of the livestock industry and preventing pathogenic bacterial infection and viral infection of young animals.

Claims

1. Use of one or two strains selected from Bacteroides acidifaciens and Prevotella buccalis in the manufacture of an agent for increasing the IgA antibody content in milk, The Bacteroides acidifaciens has a 16S rRNA gene having a nucleotide sequence represented by SEQ ID NO: 1, The oral Prevotella buccalis has a 16S rRNA gene having a nucleotide sequence represented by SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The strain has the effect of activating the immune function of Peyer's patches and increasing the number of IgA antibody-producing plasma cells.

3. The use according to claim 1 or 2, characterized in that The increasing agent is used for inoculating mammalian mothers in which the proportions of Bacteroides acidifaciens and oral Prevotella buccalis in intestinal flora are reduced.

4. The use according to claim 1 or 2, characterized in that The booster is used for oral vaccination.

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