Methods and kits for enhancing immune responses

Abstract

This provides a novel means of enhancing the immune response in mammals. 【means】 The present invention provides a method for enhancing a vaccine-induced immune response in a non-human mammal, a method for producing a non-human mammal with an enhanced vaccine-induced immune response, and a kit for enhancing a vaccine-induced immune response in a non-human mammal, comprising vaccinating the non-human mammal with a vaccine and orally administering butyrate-producing bacteria to the non-human mammal. According to the present invention, it is possible to enhance the vaccine-induced immune response and thereby increase the effectiveness of the administered vaccine.

Description

[Technical Field]

[0001] The present invention relates to a method for enhancing the immune response of non-human mammals, and a kit for enhancing the immune response of non-human mammals. [Background technology]

[0002] In dairy farming, pneumonia and enteritis caused by infectious diseases account for more than 60% of deaths in calves, leading to significant economic losses. Calves are given a variety of vaccines in sequence from about one month of age instead of antibiotics, but existing vaccines are not always sufficient to protect against infectious diseases in calves. Therefore, the development of methods to enhance the effectiveness of vaccines is desired.

[0003] In recent years, research on the use of probiotics as a disease control measure has been progressing in the livestock sector. For example, Non-Patent Document 1 reports that probiotics such as lactic acid bacteria are effective in preventing diarrhea in calves, and Non-Patent Document 2 reports that the activation of the innate immune response in cattle by lactic acid bacteria is involved in this diarrhea prevention effect.

[0004] Butyrate-producing bacteria are known as probiotics with immunomodulatory effects (Non-Patent Literature 3), and because the butyrate they produce suppresses intestinal mucosal immunity and immune responses mediated by the intestinal tract, they are being studied as "immunosuppressants" for allergies and autoimmune diseases (Non-Patent Literature 4 and 5). On the other hand, butyrate-producing bacteria are also attracting attention as "immunostimulants" that enhance the effects of cancer immunotherapy. It has been reported that administering butyrate-producing bacteria to patients with kidney cancer or lung cancer treated with immune checkpoint inhibitors extends the patients' overall survival and progression-free survival, and enhances the effects of cancer immunotherapy (Non-Patent Literature 6 and 7).

[0005] Regarding the use of butyrate-producing bacteria in the livestock field, for example, Non-Patent Document 8 reports that they promote the growth of cattle, and Non-Patent Document 9 reports that they increase the number of CD4-positive T cells in the blood of perinatal cattle. [Prior art documents]

Non-licensed literature

[0006]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides a new means capable of enhancing the immune response of mammals.

Means for Solving the Problems

[0008] The present inventors have found that oral administration of Clostridium butyricum enhances the vaccine-induced immune response.

[0009] The present disclosure provides the following. Item 1. A method for enhancing a vaccine-induced immune response in a non-human mammal, comprising inoculating the non-human mammal with a vaccine and orally administering Clostridium butyricum to the non-human mammal. Item 2. The method according to Item 1, wherein the non-human mammal is a cow or a pig. Item 3. The method according to Item 1 or 2, further comprising orally administering additional probiotics. Item 4. The method according to Item 3, wherein the additional probiotics are selected from the group consisting of lactic acid bacteria, Bifidobacterium, and saccharifying microorganisms. Item 5. The method according to any one of Items 1 to 4, wherein a beverage or feed containing Clostridium butyricum is orally administered. Item 6. The method according to Item 3 or 4, wherein a beverage or feed containing additional probiotics is orally administered. Item 7. A method for producing a non-human mammal with an enhanced vaccine-induced immune response, comprising inoculating the non-human mammal with a vaccine and orally administering Clostridium butyricum to the non-human mammal. Item 8. The method according to any one of Items 7, wherein the non-human mammal is a cow or a pig. Item 9. The method according to Item 7 or 8, further comprising orally administering additional probiotics. Item 10. The method according to Item 9, wherein the additional probiotics are selected from the group consisting of lactic acid bacteria, Bifidobacterium, and saccharifying microorganisms. Item 11. The method according to any one of Items 7 to 10, wherein a beverage or feed containing Clostridium butyricum is orally administered. Item 12. The method according to Item 9 or 10, wherein a beverage or feed containing additional probiotics is orally administered. Item 13. A kit for enhancing a vaccine-induced immune response in a non-human mammal, comprising a vaccine and Clostridium butyricum. Item 14. The kit according to Item 13, wherein the non-human mammal is a cow or a pig. Item 15. The kit according to Item 13 or 14, further comprising additional probiotics. Item 16. The kit according to Item 15, wherein the additional probiotics are selected from the group consisting of lactic acid bacteria, Bifidobacterium, and saccharifying microorganisms.

Advantages of the Invention

[0010] According to the present invention, it becomes possible to enhance a vaccine-induced immune response and improve the effect of the administered vaccine.

Brief Description of the Drawings

[0011] [Figure 1] A plot showing the percentage of CD25+CD69+ cells in CD4+ T cells and CD8+ T cells when peripheral blood mononuclear cells of calves in the negative control group (Group A), the Clostridium butyricum-administered group (Group B), and the live bacteria-containing mixed feed-administered group (Group C; also referred to as the zeosap KB-administered group in the following figures) 4 weeks after the vaccine booster immunization were stimulated with BVDV-1 or BRSV antigen. The vertical axis is the value obtained by subtracting the percentage of CD25+CD69+ cells under unstimulated conditions from the percentage of CD25+CD69+ cells under antigen stimulation. [Figure 2]This plot shows the percentage of CD25+ cells among CD4+ T cells and CD8+ T cells when peripheral blood mononuclear cells from calves stimulated with BVDV-1 or BRSV antigen in a negative control group (Group A), a group fed butyrate-producing bacteria (Group B), and a group fed a mixed feed containing live bacteria (Group C), four weeks after booster vaccination. The vertical axis represents the percentage of CD25+ cells under antigen stimulation minus the percentage of CD25+ cells under unstimulated conditions. [Figure 3] This plot shows the production levels of IFN-γ, TNF-α, and IL-6 when calf peripheral blood mononuclear cells were stimulated with BVDV-1 or BRSV antigen in a negative control group (Group A), a group fed butyrate-producing bacteria (Group B), and a group fed a mixed feed containing live bacteria (Group C) four weeks after booster vaccination. [Figure 4] This plot shows the changes in neutralizing antibody titers in calves from a negative control group (Group A), a group fed butyrate-producing bacteria (Group B), and a group fed a mixed feed containing live bacteria (Group C). Antibody titers <2 are shown as 2. [Figure 5] This plot shows the weight gain rates of calves in the negative control group (Group A), the butyrate-producing bacteria-fed group (Group B), and the live bacteria-containing mixed feed group (Group C). The weight gain rate is expressed as the percentage of weight gain during the study period relative to the weight at the start of the study. [Modes for carrying out the invention]

[0012] The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges represented using "~" or "-" mean ranges that include the numbers at both ends as the upper and lower limits, respectively, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be combined in any way.

[0013] The present invention provides a method for enhancing a vaccine-induced immune response, which involves vaccinating a non-human mammal with a vaccine. A vaccine that can be used in this invention is, for example, a vaccine against an infectious disease. A vaccine against an infectious disease contains an antigen that elicits an immune response against a pathogen (such as an infectious microorganism, virus, or protozoan). For example, vaccines for bovine infectious diseases include those that cause respiratory infections such as bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, bovine parainfluenza virus, bovine respiratory syncytial virus (RSV), bovine adenovirus, bovine epidemic fever virus, Mannheimia haemolytica, Pasteurella multocida, and Histoophilus somni; those that cause gastrointestinal infections such as coronavirus, rotavirus, Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, and Salmonella enterica subsp. enterica serovar Dublin; those that cause abnormal births such as Akabane virus, Chuzan virus, Aino virus, and Ibaraki virus; those that cause mastitis such as Escherichia coli and Staphylococcus aureus; and those that cause sudden death such as Clostridium chauvoei, Clostridium septicum, Clostridium novyi, Clostridium perfringens, Clostridium sordellii, and Clostridium It may contain an antigen that elicits an immune response to at least one of the tetani species.For example, a vaccine for infectious diseases in pigs may contain an antigen that elicits an immune response to at least one of the following: swine reproductive and respiratory syndrome virus, swine influenza virus, Mycoplasma hyopneumoniae, Bordetella bronchiseptica, Pasteurella multocida, and Actinobacillus pleuropneumoniae, which cause respiratory infections; swine infectious gastroenteritis virus, swine epidemic diarrhea virus, and Lawsonia intracellularis, which cause gastrointestinal infections; Japanese encephalitis virus, Aujeszky's disease virus, swine parvovirus, and swine getavirus, which cause abnormal births; swine circovirus, which causes stunted growth; Erysipelothrix rhusiopathiae, Clostridium perfringens, and Clostridium tetani, which cause sudden death, etc.; Streptococcus suis, which causes sepsis, etc.; and Haemophilus parasuis, which causes meningitis, etc.

[0014] Vaccines should be administered to non-human mammals according to the recommended or specified timing, method of use, dosage, and other conditions for each vaccine; no special conditions are required for administration.

[0015] In the present invention, non-human mammals may be any mammal other than humans that is susceptible to infectious diseases, such as primates other than humans; ruminants, such as cattle, yaks, buffalo, sheep, and goats (Bovidae); reindeer, red deer (Cervidae); dromedary camels, llamas, alpacas (Camelidae); and even domestic animals such as horses and pigs, and pet animals such as dogs and cats. The non-human mammal is preferably a cattle or a pig.

[0016] The present invention provides a method for enhancing a vaccine-induced immune response, which involves orally administering butyrate-producing bacteria to a non-human mammal. Butyrate-producing bacteria are bacteria that have the ability to produce butyrate and are found in the natural environment as well as in the digestive tract of mammals, including humans. The butyrate-producing bacteria available in the present invention are not limited as long as they do not have the ability to produce toxins or pathogenicity. Examples include Clostridium butyricum, Faecalibacterium prausnitzii, Anaerobutyricum soehngenii, Butyricicoccus pullicaecorum, Roseburia intestinalis, and Butyrivibrio fibrisolvens, with Clostridium butyricum being preferred.

[0017] Clostridium butyricum is an obligate anaerobic, spore-forming, Gram-positive, butyrate-producing rod-shaped bacterium. Clostridium butyricum available in this invention include, for example, Clostridium butyricum NT (FERM P-14285), Clostridium butyricum FERM P-11868, Clostridium butyricum FERM P-11869, Clostridium butyricum FERM P-11870, Clostridium butyricum ATCC859, Clostridium butyricum NBRC3315, Clostridium butyricum ATCC860, Clostridium butyricum ATCC19398, Clostridium butyricum NIP1020 (FERM BP-5794), Clostridium butyricum NIP1021 (FERM BP-5795), and Clostridium butyricum MIYAIRI 588 (FERM Examples include BP-2789), Clostridium butyricum MIYAIRI 585 (FERM BP-06815), Clostridium butyricum MIYAIRI 595 (FERM BP-06816), and Clostridium butyricum MIYAIRI 630 (FERM BP-06817). These strains can be obtained from strain preservation institutions or purchased from the manufacturers of each strain.

[0018] The butyrate-producing bacteria used in this invention may be live cells (vital bacteria) or spores. The butyrate-producing bacteria may be the culture itself, cultured under suitable culture conditions commonly used or known for butyrate-producing bacteria cultivation, or they may be isolated from the culture by means of centrifugation or other means. Butyrate-producing bacteria spores can be formed, for example, by placing the cultured cells of butyrate-producing bacteria under conditions suitable for spore formation, such as nutrient-poor conditions, high temperatures, and dryness. In addition, butyrate-producing bacteria that are commercially available as intestinal regulators or as additives to microbial preparations may be used as is.

[0019] The dosage of butyrate-producing bacteria may vary depending on factors such as the target mammal (species, age, sex, health status, etc.), the form of the butyrate-producing bacteria-containing preparation used, the method of administration, and other components other than butyrate-producing bacteria, but for mammals, for example, 10 per day 3 ~10 10 cfu / kg body weight, preferably 10 4 ~10 9 cfu / kg body weight, fu10 5 ~10 8 The dosage can be set to correspond to the cfu / kg body weight.

[0020] The present invention's method for enhancing the vaccine-induced immune response may include further oral administration of additional probiotics other than butyrate-producing bacteria. Probiotics are live microorganisms that, when ingested in appropriate amounts, have beneficial effects on the host. Examples of additional probiotics available in the present invention include lactic acid bacteria, bifidobacteria, and saccharifying microorganisms, from which one or more types of microorganisms can be selected and used.

[0021] Examples of lactic acid bacteria that can be used in the present invention include Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus delbrueckii subsp. bulgaricus, etc., Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, etc., and Lactiplantibacillus genus (Lactiplantibacillus rhamnosus, etc.). plantarum, etc.), Levilactobacillus genus (Levilactobacillus brevis, etc.), Lactococcus genus (Lactococcus lactis, etc.), Examples of fungi that can be found include those of the genera Ligilactobacillus (e.g., Ligilactobacillus salivarius), Streptococcus (e.g., Streptococcus thermophilus), Enterococcus (e.g., Enterococcus faecalis, Enterococcus faecium), and Heyndrickxia (e.g., Heyndrickxia coagulans), one example being Lactiplantibacillus plantarum HOKKAIDO (FERM P-19645).

[0022] Examples of Bifidobacterium usable in the present invention include Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum.

[0023] A saccharifying microorganism is a microorganism that produces carbohydrases (such as amylases and glucoamylases) that decompose complex carbohydrates such as starch into monosaccharides or oligosaccharides. By secreting carbohydrases, saccharifying microorganisms can assist in the digestion of nutrients in the intestine and promote the growth of other beneficial microorganisms. Examples of saccharifying microorganisms that can be used in the present invention include bacteria of the genus Lactobacillus (such as Bacillus subtilis and Bacillus subtilis subsp. natto), and yeasts of the genus Saccharomyces (such as Saccharomyces boulardii and Saccharomyces cerevisiae).

[0024] The additional probiotics used in the present invention may be live bacterial cells (live bacteria), or in the case of spore-forming bacteria, spores. The probiotics may be the culture itself cultured under appropriate culture conditions generally used or known for culturing the probiotics, or may be separated from the culture by means such as centrifugation. Spores of probiotics can be formed, for example, by placing the cultured bacterial cells of the probiotics under conditions suitable for spore formation such as oligotrophic conditions, high temperature, and dryness. Also, the probiotics may be used as they are, if they are commercially available as intestinal regulators or as additives such as microbial preparations. The dosage of the additional probiotics can be set to be, for example, 10 3 ~10 9 cfu / kg body weight, preferably 10 4 ~10 8 cfu / kg body weight, more preferably 10 5 ~10 7 cfu / kg body weight per administration per species.

[0025] The butyrate-producing bacteria used in this invention can enhance the immune response induced by vaccines administered to non-human mammals (vaccine-induced immune response). Furthermore, the additional probiotics used in this invention can further enhance the enhancement of the vaccine-induced immune response by the butyrate-producing bacteria. Here, a vaccine-induced immune response refers to an immune response to a pathogen induced by pathogen-derived antigens contained in a vaccine. Enhanced immune response refers to, for example, the promotion of proliferation or differentiation of immune cells involved in cellular immunity, such as natural killer (NK) cells, CD4-positive T cells, and CD8-positive T cells, or the enhancement of the expression of activation markers such as CD25 and CD69 in these cells; an increase in the proportion of said immune cells in immune cell populations such as peripheral blood mononuclear cells (PBMCs); an increase in the absolute number of said immune cells; the formation of pathogen-specific memory T cells or memory B cells; the promotion of Th1 cytokine production such as interferon-γ (IFN-γ) and interleukin-2 (IL-2); the promotion of Th2 cytokine production such as interleukin-4 (IL-4); or the enhancement of pathogen-specific immunoglobulin G (IgG) production or an increase in neutralizing antibody titers. If at least one of these phenomena is observed in a vaccinated non-human mammal, the vaccine-induced immune response in that animal is enhanced.

[0026] In the method of the present invention, butyrate-producing bacteria and optionally additional probiotics can be administered to non-human mammals in the form of an oral preparation. The oral preparation is not limited in form as long as it can be administered orally, and may be in the form of, for example, a powder, granules, tablets, suspension, paste, etc. Depending on its form, the oral preparation may also contain components other than butyrate-producing bacteria and additional probiotics, such as dispersants, emulsifiers, stabilizers, excipients, binders, disintegrants, lubricants, etc.

[0027] Furthermore, butyrate-producing bacteria and optionally additional probiotics may be administered in the form of compositions containing them, such as beverage compositions and feed compositions. These compositions can enhance vaccine-induced immune responses in non-human mammals. The feed composition may be a mixed feed containing butyrate-producing bacteria and optionally additional probiotics, along with adsorbents, nutrients (minerals, vitamins, oligosaccharides), etc., and can be used by mixing it with feeds such as concentrated feed, roughage, and compound feed.

[0028] Butyrate-producing bacteria, and optionally additional probiotics, oral preparations and compositions containing these (beverage compositions and feed compositions) can be orally administered to mammals before vaccination (e.g., 2 to 4 weeks before vaccination), after vaccination, or in parallel with vaccination. Oral administration may be carried out by orally ingesting the oral preparation or the compositions by mammals, or by forced oral administration using instruments such as syringes and tubes.

[0029] Oral formulations and the compositions described above can enhance the effects of vaccines, such as preventing the onset of infectious diseases (e.g., respiratory infections), suppressing exacerbations, promoting healing, and reducing the number of treatments, by enhancing the vaccine-induced immune response in mammals.

[0030] The present invention further provides a method for producing non-human mammals with an enhanced vaccine-induced immune response, comprising vaccinating non-human mammals with a vaccine and orally administering butyrate-producing bacteria and optionally additional probiotics to the non-human mammals. The present invention also further provides a kit for enhancing a vaccine-induced immune response in non-human mammals, comprising a vaccine, butyrate-producing bacteria, and optionally additional probiotics. The kit may further include articles such as instructions for use, equipment for vaccinating with the vaccine, and equipment for administering butyrate-producing bacteria and optionally additional probiotics.

[0031] The present invention will be described in more detail by the following examples, but the present invention is not limited thereto. [Examples]

[0032] [Materials and Methods] (1) Butyrate-producing bacteria administration and vaccination A study was conducted to evaluate the enhancement of vaccine-induced immune responses in calves using Clostridium butyricum NT as the butyrate-producing bacterium, and Enterococcus faecalis NT and Bacillus subtilis var. natto NT as additional probiotics. Calves (Holstein, male, 2 months old) were divided into three groups (Group A: 20 calves, Group B: 18 calves, Group C: 19 calves). Group A was given a negative control diet (composition: rice bran oil meal 71.5%, zeolite 28.5%), and Group B was given a diet supplemented with butyrate-producing bacterium (composition: rice bran oil meal 71.5%, zeolite 28.5%, butyrate-producing bacterium NT strain 10). 11 (pieces / kg), mixed feed containing live bacteria for group C (product name: Zeosapo KB, manufactured by Microbial Science Research Institute Co., Ltd. and Sasaeya Pharmaceutical Co., Ltd., composition: rice bran oil cake 71.5%, zeolite 28.5%, butyrate-producing bacteria NT strain 10 9 pieces / kg, lactic acid bacteria NT strain 10 9 pieces / kg, Bacillus subtilis NT strain 10 9 The bacteria (units / kg) were fed at a dose of 30g daily (equivalent to 1% of the total feed) for a continuous 16-week trial period. During the trial period, the amount of bacteria administered to calves in group B was approximately 3 × 10⁶ units per calf for the butyrate-producing bacterium NT strain. 9 pieces / day(6.3 x 10 7 In calves of group C, approximately 3 × 10¹⁶ cfu / kg body weight were present per calf for each strain of butyrate-producing bacteria NT, lactic acid bacteria NT, and Bacillus subtilis NT. 7 pieces / day(6.3 x 10 5 The value was cfu / kg body weight.

[0033] A mixed attenuated live virus vaccine (product name: "Kyoto Biken" Calfwin 6, manufactured by the Institute of Microbial Science Co., Ltd. and Sasaeya Pharmaceutical Co., Ltd.) was administered intramuscularly four weeks after the start of feed administration (primary immunization) and eight weeks after (booster immunization). This vaccine contains attenuated strains of bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus type 1 (BVDV-1), bovine viral diarrhea virus type 2 (BVDV-2), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine adenovirus type 7 (BAdV-7) as antigens.

[0034] (2) Isolation and culture of peripheral blood mononuclear cells Blood samples were collected five times: at the start of feeding, and at 4, 8, 12, and 16 weeks after the start of feeding. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. The PBMCs were suspended in RPMI-1640 medium (containing 10% FBS, antibiotics, and L-glutamine), seeded in 96-well plates, and cultured at 37°C and 5% CO2 for 5 days under conditions without antigen stimulation or stimulated with UV-inactivated antigens of BRSV or BVDV-1 contained in the vaccine. After culturing, the cells and culture supernatant were separated. The cells were used for flow cytometry analysis, and the culture supernatant was used for cytokine production measurement.

[0035] (3) Analysis of immune response The cells collected in (2) were incubated in FACS buffer (PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin) containing 10% goat serum (Thermo Fisher Scientific). The cells were then washed with FACS buffer and the following antibodies were added: Alexa Fluor 488-labeled anti-bovine CD25 monoclonal antibody (clone name CACT116A; VMRD), Alexa Fluor 647-labeled anti-bovine CD69 monoclonal antibody (clone name KTSN7A; Washington State University Monoclonal Antibody Center), PerCp / Cy5.5-labeled anti-bovine CD3 monoclonal antibody (clone name MM1A; Washington State University Monoclonal Antibody Center), PE / Cy7-labeled anti-bovine CD4 monoclonal antibody (clone name CC30; Bio-Rad), and PE-labeled anti-bovine CD8 monoclonal antibody (clone name CC63; Bio-Rad). The mixture was incubated at 4°C for 20 minutes. Antibodies MM1A and CC8 were conjugated with the corresponding fluorescent dyes using Lightning-Link Conjugation Kits (Abcam). Antibodies CACT116A and KTSN7A were labeled with Zenon Mouse IgG1 Labeling Kits (Thermo Fisher Scientific). After reaction with the antibodies, the cells were washed with 1% BSA-PBS and flow cytometry analysis was performed using FACSLyric (BD Biosciences) and FACSuite software (BD Biosciences). In addition, IFN-γ was measured using ELISA Flex: Bovine IFN-γ (HRP) (Mabtech), TNF-α using ELISA Flex: Bovine TNF-α (HRP) (Mabtech), and IL-6 using Bovine IL-6 Uncoated ELISA Kit (Thermo Fisher Scientific) in the culture supernatant collected in (2).

[0036] (4) Measurement of neutralizing antibody titer Blood was collected 16 weeks after the start of feeding (12 weeks after the first vaccination), and serum was obtained by centrifugation. The serum was inactivated by heating at 56°C for 30 minutes, and each serum was serially diluted 2-fold in E-MEM (containing 10% FBS, Tryptose Phosphate Broth) in a 96-well plate. 50 μl of the serum dilution prepared in the 96-well plate was used to test BRSV (NMK7 strain) 200 TCID diluted in E-MEM. 50 Mix with 100 μl / well and sensitize overnight at 22°C. Place 2 × 10⁶ Vero cells in each well of a 96-well plate containing the mixture. 4 Cells (100 μl) were added and cultured at 37°C and 5% CO2 for 7-8 days. After 7-8 days, the presence or absence of cytopathic effects was determined, and the highest dilution factor of serum in which no cytopathic effects were observed was defined as the neutralizing antibody titer.

[0037] (5) Statistical analysis All statistical tests were performed using GraphPad Prism 10 (GraphPad Software). A p-value less than 0.05 was considered statistically significant.

[0038] [result] CD4 count 4 weeks after booster immunization + T cells and CD8 + Figures 1 and 2 show the percentage of T cell activation marker (CD25, CD69) positive cells in T cells. In the butyrate-producing bacteria-fed group (Group B) and the live bacteria-containing mixed feed-fed group (Group C), an increase in activation marker positive cells was observed compared to the negative control group (Group A), particularly under BRSV stimulation. + Enhanced T cell response was observed.

[0039] Figure 3 shows the cytokine production levels of PBMCs collected 4 weeks after booster immunization. In the butyrate-producing group, increased IFN-γ and TNF-α production was observed under BVDV-1 stimulation and BRSV stimulation, and further increased IFN-γ and TNF-α production was observed even without antigen stimulation. It was confirmed that butyrate-producing bacteria enhance the Th1 cytokine response in an antigen-stimulation-dependent and independent manner. In addition, in the Zeosapo KB-producing group, the median IFN-γ level was higher than in the negative control group, showing a similar trend to the butyrate-producing group.

[0040] Figure 4 shows the neutralizing antibody titers against BRSV. At the end of the study (12 weeks after the first vaccination), the median neutralizing antibody titer tended to be higher in the Zeosapo KB-treated group than in the negative control group.

[0041] Figure 5 shows the weight gain rate of the calves at the end of the experiment. Significant weight gain was observed with both butyrate bacteria alone and with Zeosapo KB feeding. Furthermore, when the number of times the calves were treated for pneumonia exhibiting clinical symptoms was examined, the negative control group (Group A, 20 calves) received treatment 10 times, the butyrate bacteria-fed group (Group B, 18 calves) received treatment 8 times, and the group fed mixed feed containing live bacteria (Group C, 19 calves) received treatment 6 times. In particular, the group fed mixed feed containing live bacteria (Group C) tended to receive fewer treatments for pneumonia than the other groups.

[0042] This example demonstrates that the administration of butyrate-producing bacteria to calves, and the administration of butyrate-producing bacteria along with additional probiotics, reduces the CD4 levels against vaccines. + The study demonstrated enhanced T-cell and Th1 responses, and suggested a reduction in the incidence of clinically symptomatic pneumonia.

Claims

1. A method for enhancing a vaccine-induced immune response in a non-human mammal, comprising vaccinating the non-human mammal and orally administering butyrate-producing bacteria to the non-human mammal.

2. The method according to claim 1, wherein the non-human mammal is a cattle or a pig.

3. The method according to claim 1 or 2, further comprising orally administering an additional probiotic.

4. The method according to claim 3, wherein the additional probiotic is selected from the group consisting of lactic acid bacteria, bifidobacteria, and saccharifying microorganisms.

5. The method according to claim 1 or 2, wherein a beverage or feed containing butyrate-producing bacteria is administered orally.

6. The method according to claim 1 or 2, wherein a beverage or feed containing additional probiotics is administered orally.

7. A method for producing non-human mammals with an enhanced vaccine-induced immune response, comprising vaccinating non-human mammals with a vaccine and orally administering butyrate-producing bacteria to non-human mammals.

8. A kit containing a vaccine and butyrate-producing bacteria for enhancing vaccine-induced immune responses in non-human mammals.

9. The kit according to claim 8, wherein the non-human mammal is a cow or a pig.

10. The kit according to claim 8 or 9, further comprising additional probiotics.

11. The kit according to claim 10, wherein the additional probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, and saccharifying microorganisms.

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