Antiviral agent

Abstract

According to the present invention, cultured cells are pretreated with a candidate strain and then infected with a virus, and a strain having an antiviral effect is selected by using, as an indicator, the expression of a viral gene or protein in the recovered cells. By the screening method, a Lactobacillus delbrueckii subsp. delbrueckii THU5 strain (accession number: NITE BP-04062) and a Limosilactobacillus reuteri THU6 strain (accession number: NITE BP-04063), which have a rotavirus infection inhibitory effect, were obtained. These strains, as immunobiotics, have an effect against viral infections, in particular, a rotavirus infectious disease.

Description

antiviral agents

[0001] The present invention relates to an antiviral agent containing, as an active ingredient, an antiviral probiotic, in particular, an immunobiotic having an immunomodulatory effect on mucosal immunity such as that of the intestinal mucosa.

[0002] Diarrhea caused by viral infections leads to growth retardation in infected livestock and, in the worst cases, death, resulting in significant economic losses. Rotavirus, in particular, is a common viral infection in both cattle and pigs, highly contagious, and poses a significant problem due to its severe watery diarrhea. Rotavirus is a major cause of diarrhea in young livestock, and livestock suffering from viral diarrhea are treated with drugs. However, drug treatment not only increases costs due to the use of drugs, but also poses problems such as the emergence of drug-resistant bacteria due to overuse. Therefore, there is a need to establish healthy rearing techniques that do not rely on drugs such as antibacterial agents to treat infectious diseases. However, the only preventive measure currently available is animal husbandry hygiene management. Rotavirus is also a major pathogen of infant diarrhea in humans, and since severe cases can be fatal, prevention is an important countermeasure.

[0003] The use of beneficial bacteria in food to promote health has been around for a long time. In 1989, Fuller defined probiotics as "live microorganisms that have beneficial effects by improving the balance of intestinal flora," and research into their use as functional ingredients has progressed. It is expected that the continuous intake of probiotics will increase resistance to viral infections and other diseases in livestock, thereby reducing damage.

[0004] Patent Documents 1 and 2 disclose the use of probiotics for the treatment and prevention of viral infections. Patent Document 1 describes that lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus paracasei, and Lactobacillus gasseri can be used for the treatment and prevention of viral infections. Patent Document 2 discloses the Lactobacillus plantarum CJLP475 strain, which has antiviral and immunopotentiating effects. As an example, it has been shown that the CJLP475 strain inhibits infection with porcine epidemic diarrhea virus (PEDV), which belongs to the coronavirus family.

[0005] Patent Document 1 discloses that some bacteria, including Lactobacillus plantarum, have antiviral effects via the cellular immune system. However, recent research has revealed that even within the same species, the effects vary greatly depending on the strain, and it is believed that some strains have weak antiviral effects. Patent Document 2 discloses that the strain CJLP475 is effective against porcine epidemic diarrhea virus, which belongs to the Coronaviridae family, but its effectiveness against rotavirus, the main cause of juvenile porcine diarrhea, has not been verified.

[0006] The present inventor has also disclosed an antiviral agent containing probiotics that effectively exerts an antiviral effect against viral infections (Patent Document 3). In Patent Document 3, the present inventor has disclosed Lactobacillus bacteria (three strains of Lactobacillus salivarius and nine strains of Lactobacillus plantarum) that have the effect of enhancing the expression of antiviral factors and reducing the expression of factors that downregulate antiviral factors, and has shown antiviral effects mediated by the immune system and a reduction in rotavirus-infected cells.

[0007] Patent Publication No. 2009-511470 Patent Publication No. 2020-529828 International Publication No. 2021 / 241728

[0008] Clancy, R. FEMS Immunol Med Microbiol, (2003). 38, 9-12. https: / / doi.org:10.1016 / S0928-8244(03)00147-0Lopez, T., etal., Journal of General Virology (2005), 86, 1609-1617. https: / / doi.org:10.1099 / vir.0.80827-0

[0009] As mentioned above, Patent Documents 1 and 2 do not disclose probiotics effective against rotavirus, a major cause of diarrhea in young livestock, and their effectiveness is unknown. The present inventor discloses multiple probiotics effective against rotavirus in Patent Document 3. However, the intestinal flora is composed of a large number of species of bacteria, and the bacterial species and balance vary greatly from individual to individual. Therefore, improvement of the intestinal flora cannot be achieved uniformly, and the effects obtained are thought to vary from individual to individual. Therefore, the probiotics that are effective may vary from individual to individual. Therefore, it is important to prepare multiple effective bacterial species for prevention and treatment. Furthermore, using multiple probiotics may be more effective.

[0010] The usefulness of lactic acid bacteria as probiotics has long been recognized. Widely used in fermented foods, "lactic acid bacteria" are bacteria that produce lactic acid through metabolism. They are a collective term for bacteria that are (1) Gram-positive, (2) bacillus or cocci in shape, (3) catalase-negative, (4) produce 50% or more of lactic acid from consumed glucose, (5) do not produce endospores, and (6) are generally non-motile. Among lactic acid bacteria, strains belonging to the former Lactobacillus genus are most commonly used as probiotics. With the recent advancement of 16S rRNA gene analysis technology, the classification was significantly revised in 2020. As a result of taxonomic verification based on genome analysis, 23 new genera were proposed, including the former Lactobacillus genus and other closely related genera and species, and the Lactobacillus genus was reorganized into 25 genera, including the existing genera Lactobacillus and Paralactobacillus. In the following, lactic acid bacteria refers in a broad sense to bacteria that have the six characteristics described above, and in a narrow sense to the former Lactobacillus genus, which was reclassified into 25 genera in 2020, and related genera and species.

[0011] An object of the present invention is to provide probiotics, particularly immunobiotics, that are effective in preventing and treating viral infections. Another object of the present invention is to discover antiviral immunobiotics that are effective against viral infections, particularly rotavirus infections, and to provide feed containing such immunobiotics as functional ingredients. Continuous administration of antiviral immunobiotics is expected to improve the disease resistance of livestock, leading to a reduction in damage caused by rotavirus infection. Furthermore, significant benefits are expected in terms of cost reduction and reduction in the burden of raising livestock.

[0012] As mentioned above, the present inventors have already isolated and disclosed strains with antiviral effects (Patent Document 3). As described in detail below, the present inventors conducted the following study to search for lactic acid bacteria other than Lactobacillus salivarius and Lactobacillus plantarum, which have already been isolated by the present inventors, that have antiviral effects against rotavirus. Furthermore, in order to evaluate the antiviral effect under conditions closer to those in vivo, an evaluation method using small intestinal organoid culture was developed.

[0013] The porcine-derived lactic acid bacteria library used in the following search is effective against rotavirus, but because it is effective against rotavirus, it is expected to be a useful immunobiotic for livestock other than pigs, and even for animals including humans. Furthermore, when the selected strain was applied to intestinal epithelial cells, enhanced expression of antiviral factors such as interferon was observed, suggesting that it is effective not only against rotavirus but also against other viral diseases. The active use of immunobiotics, which improve the host's antiviral capacity and prevent viral infections, as well as their metabolic products and post-immunobiotics, which are killed bacteria, will prevent viral and bacterial infections and reduce the use of drugs.

[0014] The present invention relates to the following antiviral agents and a method for screening bacterial strains: (1) An antiviral agent characterized by containing, as an active ingredient, Lactobacillus delbrueckii subsp. delbrueckii THU5 (accession number: NITE BP-04062) and / or Limosilactobacillus reuteri THU6 strain (accession number: NITE BP-04063). The inventors screened for bacteria that suppress rotavirus infection and obtained two strains. These strains suppress rotavirus infection, but because they also have the effect of enhancing the expression of antiviral factors such as IFN-β, they are thought to be effective against a wide range of infectious viruses. Their use as livestock feed, food, or beverages makes it possible to suppress viral infection.

[0015] (2) A method for screening bacterial strains with antiviral effects, in which cultured cells are pretreated with a candidate bacterial strain, infected with a virus, and bacterial strains with antiviral effects are selected using the expression of viral genes and / or proteins in the recovered cells as an indicator. This method makes it possible to screen for bacterial strains that suppress viral infection. Analysis by the present inventors has revealed that the effectiveness against viruses varies greatly depending on the strain, even within the same species. This screening method makes it possible to obtain bacterial strains that are effective against viruses.

[0016]

[0023] Figure 1 is a schematic diagram showing a screening method for antiviral probiotics. Graph showing the results of analyzing the changes in mRNA expression of viral structural protein VP6 and nonstructural proteins NSP1 and NSP5 over time after viral infection. Figure showing the results of analyzing the antiviral effect in screening 1 using 160 strains of a porcine-derived lactobacillus library, where samples were pretreated with a lactic acid bacteria strain, then infected with a virus, viral nonstructural protein NSP5 mRNA was amplified. Figure showing the correlation between the expression levels of NSP5 and VP6 or NSP1 genes after viral infection following pretreatment with each strain. Figure showing the results of evaluating the rotavirus infection-reducing ability of the selected strain THU5 by immunostaining. Figure showing the results of analyzing the antiviral effect in screening 2 using L. reuteri 107 strain, where samples were pretreated with a lactic acid bacteria strain, then infected with a virus, viral nonstructural protein NSP5 mRNA was amplified. 5 is a diagram showing the correlation between NSP5 and VP6 gene expression after pretreatment with each bacterial strain and viral infection. FIG. 6 is a diagram showing the results of evaluating the ability to reduce rotavirus infection by immunostaining of the selected bacterial strain THU6. FIG. 7 is a diagram showing the results of evaluating the ability to reduce rotavirus infection using killed bacterial cells. FIG. 8 is a diagram showing a schematic representation of a method for evaluating the effect of stimulating a lactic acid bacteria strain alone. FIG. 9 is a diagram showing the results of analyzing the changes in expression of antiviral factors IFN-β, IFN-λ3, OAS1, and Mx1 caused by the THU5 strain. FIG. 10 is a diagram showing the results of analyzing the changes in expression of viral receptors RIG-1 and MDA5 caused by the THU5 strain. FIG. 11 is a diagram showing a summary of the effects of stimulating the THU5 strain alone. FIG. 12 is a diagram showing a schematic representation of a method for evaluating the effect of stimulating a lactic acid bacteria strain against viral infection. FIG. 13 is a diagram showing the results of analyzing the changes in expression of antiviral factors IFN-β, IFN-λ3, OAS1, and Mx1 caused by the THU5 strain. A diagram showing the results of analyzing the expression changes of viral receptors RIG-1 and MDA5 caused by the THU5 strain. A diagram showing a summary of the effects of the THU5 strain on viral infection. A diagram showing the viral immune enhancing effect of the THU6 strain. A diagram showing the results of analyzing the acid and bile resistance of the THU6 strain. A diagram showing the results of analyzing the adhesion rate of the THU6 strain to porcine small intestinal epithelial cells (SIEC cells). Transmission electron microscope photographs of the THU6 strain and control strain No. 15. Microscopic images showing the results of analyzing the expression of cell markers in porcine small intestinal organoid cells.A schematic diagram showing the method for evaluating the effect of lactic acid bacteria on viral infection using porcine small intestinal organoid cells. A diagram showing the results of analyzing changes in the expression of small intestinal markers and viral markers due to stimulation with lactic acid bacteria and viral infection.

[0017] The antiviral agents described below are selected antiviral probiotic strains, used alone or in combination with multiple strains, as feed, food, beverage, or pharmaceutical preparations. Here, probiotics was redefined by the FAO / WHO Joint Expert Consultation in 2001 as "live microorganisms that, when ingested in sufficient amounts, confer a beneficial effect on the host." Postbiotics, as defined by the International Probiotics-Prebiotics Association, refers to "preparations of killed bacteria or their constituent components, including their metabolic products." As mentioned above, immunobiotics refer to bacterial species among probiotics that act on the intestinal immune system and have excellent immunostimulatory properties (Non-Patent Document 1). Here, killed bacteria were prepared and screened; however, when used as antiviral agents, either live or killed bacteria may be used. Killed bacteria can be more stably supplied as livestock feed, etc.

[0018] Furthermore, while the selection and testing of antiviral probiotics from porcine-derived lactic acid bacteria was conducted using pigs as the target here, the antiviral effects against rotavirus were verified and selected, and therefore the probiotics can be used not only in pigs but also in mammals in general, specifically as feed for livestock such as cattle, or as pharmaceuticals, or as human food and beverage products or pharmaceuticals. The selected bacteria may be mixed with additives and used in the form of a composition (a food and beverage composition or a pharmaceutical composition, or a feed composition for livestock). Examples of the above-mentioned food and beverage products include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally balanced foods, supplements, etc.) and health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.). When administered continuously as an antiviral agent, livestock feed and food and beverage products are preferred.

[0019] As used herein, "antiviral" means preventing and / or ameliorating (treating) viral infections by acting on the host to inhibit viral growth, inactivate viruses, reduce susceptibility to viruses, or the like. The lactic acid bacteria strains listed below are probiotics and can be used without causing side effects, unlike vaccines or antibiotics. Furthermore, there is no need to worry about the development of resistant bacteria, as there is with antibiotics.

[0020] The present invention will now be described with reference to data. 1. Screening 1 for lactic acid bacteria with antiviral effects: Selection of strains with antiviral effects was performed by treating cultured cells with various strains and then infecting them with rotavirus, using the ability to reduce rotavirus infection as an indicator. For screening, 160 strains (Table 1) of five species belonging to the former genus Lactobacillus were used to select strains with antiviral effects from the porcine lactobacillus library owned by the inventors. The strains were isolated from piglet feces, colostrum, breast milk, and vagina.

[0021]

[0022] Each bacterial cell was inoculated into MRS medium (Difco) and subcultured three times (37°C, 16 h). The prepared bacterial solution was washed three times with PBS (pH 7.2, Thermo Fisher Scientific) and then heated in a water bath at 75°C for 90 minutes to kill the bacterial cells. After that, the bacterial count was reduced to 2.5 × 10 ９ The cells were suspended in PBS to a concentration of 1000 cells / mL and stored at −20° C. until testing. Screening of the strains and evaluation of the selected strains were all carried out using killed cells.

[0023] Screening was performed as follows. Screening was performed using an immortalized porcine intestinal epithelial cell line, INV1-3 (transferred from the National Agriculture and Food Research Organization), cloned from the small intestine of a Duroc piglet. INV1-3 cells have enhanced NOD2 protein function, which recognizes muramyl dipeptide, a component of bacterial cell walls, and is responsible for the infection defense mechanism, and are known to be susceptible to viruses.

[0024] INV1-3 cells were cultured in collagen (Type I)-coated flasks or the like in a medium (hereinafter referred to as the medium) containing DMEM / F-12 (Thermo Fisher Scientific) supplemented with 10% FCS, 1% penicillin-streptomycin (Thermo Fisher Scientific), 1% insulin-transferrin-sodium selenite (ITS-Gx100) (Thermo Fisher Scientific), and 5 ng / mL human EGF (Sigma-Aldrich).

[0025] The screening method for strains with antiviral activity is shown in Figure 1A. INV1-3 cells were cultured in a collagen Type I-coated 24-well plate (Sumitomo Bakelite) at 3.0 × 10 ４ The cells were seeded at a concentration of 5.0 × 10 cells / mL and cultured for 4 days, after which pre-stimulation with bacterial cells was performed. ７ The bacterial solution and medium were added to give a total of 500 μL of cells / mL. After continuing stimulation with the bacterial cells for 48 hours, the culture medium was removed and the cells were infected with rotavirus.

[0026] The rotavirus used was the livestock-infectious group A rotavirus OSU strain (Ohio State University). ８．４ To 40 μL of rotavirus (MOI = 1), 10 μL of purified trypsin (100 μg / mL) (Trypsin Type I, SIGMA) was added, and the mixture was heated at 37°C for 30 minutes in a heat block to activate the virus. 10 mL of serum-free medium (DMEM-F12 supplemented with 1% penicillin-streptomycin) was then added to prepare a diluted rotavirus solution. The diluted rotavirus solution was added to a 24-well plate at 500 μL / well (MOI = 1), and incubated at 37°C in 5% CO. ２ The cells were cultured for 1 hour under the conditions described above. A control group (Mock) was used, in which no virus was inoculated. The cells were washed three times with serum-free medium heated to 37°C to wash away the virus, and then cultured again at 37°C and 5% CO ２ The cells were incubated in serum-free medium for 12 hours under these conditions until the time of harvest.

[0027] Twelve hours after virus infection, cells were lysed and collected using the following method. The 24-well plate was washed twice with PBS, and 500 μL / well of TRI Reagent™ (Molecular Research Center) was added. The cell lysate was collected and total RNA was extracted. Next, cDNA was synthesized from the total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc.). Rotavirus mRNA expression was analyzed by real-time PCR using the obtained cDNA as a template. The analytical instrument used was the CFX Connect™ Real-Time System (BIO-RAD). The premix reagent used was TB Green™ Premix Ex Taq II (Takara Bio Inc.). If the obtained total RNA or cDNA was not used immediately for analysis, it was stored at −80° C. until use.

[0028] The antiviral effect was evaluated based on the amount of viral gene mRNA in cells. Specifically, VP6, one of the six viral structural proteins, and NSP1 and NSP5, one of the six non-structural proteins, were amplified using the following primers to evaluate the degree of viral infection. Plasmids into which each viral factor had been cloned were serially diluted and used as standards to calculate the copy number.

[0029]

[0030] When using the above three viral mRNAs as screening indicators, we investigated the optimal time for detecting viral infection. INV1-3 cells were infected with rotavirus at the MOIs described above, and the mRNAs were amplified using the PCR primers listed in Table 2. The time-dependent changes in expression levels after viral infection were analyzed (Figure 1B). Because the expression levels of VP6, NSP1, and NSP5 mRNAs all peaked at 12 hours postinfection, we quantified their mRNA expression levels 12 hours postinfection using RT-qPCR and used them as screening indicators (see Figure 1A).

[0031] Using the above method, cells were pretreated with 160 strains of lactic acid bacteria, then infected with a virus, and NSP5 mRNA expression was analyzed. In Figure 2A, "Mock" indicates the level of NSP5 expression in cells not infected with the virus, and "Virus" indicates the level of NSP5 expression in cells infected with the virus alone without pretreatment with lactic acid bacteria (hereinafter referred to as the virus group). Of the 160 strains, 29 strains significantly reduced the level of NSP5 expression in rotavirus-infected INV1-3 cells, while 45 strains significantly enhanced the level of NSP5 expression, indicating that the number of strains with enhanced NSP5 expression was greater than the number of strains with reduced NSP5 expression. The strain that most suppressed the expression level of NSP5 was Lactobacillus delbrueckii THU5 strain (FeB206-6wR1-17), which suppressed the expression level by about 50% compared to the virus group. The strain THU5 was selected as an anti-rotavirus immunobiotic. Note that Lactobacillus delbrueckii subsp. delbrueckii THU5 strain is Lactobacillus delbrueckii subsp. The strain was deposited under the Budapest Treaty as L. delbrueckii THU5 on January 15, 2024 (original deposit date) at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan). The accession number is NITE BP-04062. Hereinafter, it will be simply referred to as strain THU5. The selected strain with antiviral activity will be referred to as the selected strain. Furthermore, 4FeB186, which significantly enhances NSP5 expression from the same species (L. delbrueckii), was selected as a control strain. The species and origin of the selected strains are shown in Table 3.

[0032]

[0033] After pretreatment with each strain, virus-infected INV1-3 cells were subjected to gene expression analysis for not only NSP5 but also VP6 and NSP1 (Figure 2B). The expression levels of viral factors were strongly correlated with each other, and THU5 significantly reduced the expression levels of both viral factors. It has been reported that rotavirus replication and assembly occur in a complex formed by NSP2 and NSP5, and that silencing NSP5 suppresses rotavirus replication (Non-Patent Document 2). Therefore, since strains that reduce NSP5 expression are considered to be strains that suppress rotavirus infection, the selection of antiviral agents based on NSP5 expression is considered highly relevant.

[0034] To confirm whether the selected strains suppressed rotavirus growth at the protein level, we evaluated virus infectivity by immunostaining (Figure 2C). INV1-3 cells were stimulated with THU5 or 4FeB186, infected with rotavirus for 16 hours, and fixed in 80% acetone at 4°C for 20 minutes. Staining was performed using standard methods with the primary antibody, porcine virus A Tohoku-2 strain gnotobiotic porcine infection-resistant serum (obtained from the Animal Health Research Institute, National Agriculture and Food Research Organization, diluted 1:100), and the secondary antibody, Alexa Fluor™ 488-cojugated AffiniPure Goat Anti-Swine IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.). Nuclear staining was performed using FLUOROSHIELD with DAPI (FLSD) (ImmunoBioScience Corp.) to stain rotavirus proteins and cell nuclei. Cells that emitted green fluorescence during fluorescence observation were counted as infected cells, and cells that emitted blue fluorescence due to DAPI were counted as the total number of cells. The number of infected cells was divided by the total number of cells, and the virus group was set at 100% to obtain a relative value to determine the virus infection rate (Figure 2C). The group pre-stimulated with THU5 showed a reduction in virus infection rate by approximately 70% compared to the virus group. On the other hand, the control strain 4FeB186 showed an infection rate almost equivalent to that of the virus group.

[0035] 2. Screening 2 for Lactic Acid Bacteria with Antiviral Effects Next, in order to select antiviral agents from different bacterial species, screening was performed using 107 strains of the porcine-derived lactic acid bacterium Limosilactobacillus reuteri. The selection method was the same as in Screening 1, using NSP5 expression as an indicator (FIG. 3A). Decreased NSP5 expression was observed in 40 of the 107 strains. Among them, Limosilactobacillus reuteri THU6 strain (205MR433), which showed the greatest decrease in expression, was selected as a strain with antiviral effects. The Limosilactobacillus reuteri THU6 strain was deposited under the Budapest Treaty on January 15, 2024 (original deposit date) at the National Institute of Technology and Evaluation (NITE Patent Microorganisms Depositary, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818). The deposit number is NITE BP-04063. Hereinafter, it will be referred to simply as the THU6 strain. Figure 3B shows the correlation between NSP5 and VP6 expression. Similar to the results obtained in Screening 1, there was a correlation between NSP5 and VP6 expression, and the THU6 strain also suppressed VP6 expression. Next, the infection rate was calculated by immunostaining in the same manner (Figure 3C). The results of protein expression confirmed that the infection rate was approximately 80% of that in the virus group after treatment with the THU6 strain. THU6 is a strain isolated from pig breast milk.

[0036] Using SIEC cells obtained from pig small intestinal epithelial cells, we selected strains that have the effect of inhibiting rotavirus proliferation. Heat-killed cells of lactic acid bacteria belonging to the genus Lactobacillus reuteri were added to the SIEC cells, and the expression level of NSP5, which is important for rotavirus proliferation, was quantified.

[0037] Each strain belonging to Lactobacillus reuteri was inoculated into MRS medium (Difco) and cultured at 37°C for 16 hours. After the culture, the medium was washed three times with sterilized 0.15 M sodium chloride-0.01 M phosphate buffer (PBS, pH 7.2), and the number of bacteria was 2.5 × 10 ９The cells were suspended in PBS to a concentration of 1000 cells / mL and heated at 70°C for 90 minutes to obtain heat-killed cells.

[0038] Next, porcine small intestinal epithelial cells (SIEC cells) were cultured in DMEM / F-12 medium (Wako Co., Ltd.) at a density of 3.0 × 10 ４ The resulting suspension was placed in a 48-well plate at 0.25 mL per well and cultured for 4 days. After medium replacement, 5.0 × 10 heat-killed bacteria were ７ The cells were added to a well at a concentration of 100 cells / mL and cultured for two days. As a control, wells containing PBS were prepared. After culture, rotavirus was added to a well at a concentration of 1 MOI and cultured at 37°C for one hour. The cells were then washed three times with DMEM / F-12 medium (Wako) and cultured for 11 hours.

[0039] After stimulation, the wells were washed twice with PBS. TRIzol reagent (Invitrogen) was added to each well to recover the cell lysate. RNA was extracted with phenol-chloroform and then precipitated with ethanol. cDNA was synthesized from the resulting RNA using the PrimeScript RT reagent Kit (Takara Bio). The expression level of NSP5 was measured as described above. The results showed that the THU6 strain significantly suppressed NSP5 expression. Furthermore, No. 15, which significantly enhanced NSP5 expression from the same species (L. dreuteri), was selected as a control strain (Figure 3D). The species and origins of the selected strains are shown in Table 4.

[0040] 3. Immunobiotic Function of the Selected Strains 3.1 Function of the THU5 Strain Next, to verify whether the selected strains function as immunobiotics, we examined the effects of the selected strains on the expression of immune-related factors using INV1-3 cells. First, cells were stimulated with the selected strains alone, and then harvested at 0, 6, 12, 24, and 48 hours. Gene expression analysis was performed by RT-qPCR (Figure 4A). Immune-related factors include antiviral factors IFN-α, IFN-β, IFN-λ3, OAS1 (2'-5'-oligoadenylate synthetase 1), RNase L, Mx1 (Myxovirus resistance 1), PKR (protein kinase R), inflammatory cytokines IL-6, MCP-1 (Monocyte chemotactic protein 1), TLR (Toll-like receptors) TLR2, TLR3, and TLR4, viral receptors RIG-I (retinoic acid-inducible gene-I), MDA5 (melanoma Gene expression analysis was performed for differentiation-associated protein 5, mitogen-activated protein kinase phosphatase 1 (MKP-1), a negative regulator of the TLR signaling pathway, A20, and Toll interacting protein (Tollip). Normalization was performed using β-actin expression. The PCR primers used are shown in Table 5.

[0041]

[0042] Figure 4B shows the results for IFN-β, IFN-λ3, OAS1, and Mx1, whose expression levels showed significant differences between the selected and control strains. The expression levels of IFN-β and IFN-λ3 increased starting 6 hours after THU5 stimulation. This suggests that the bacterial cells are recognized by some kind of receptor and induce interferon expression via a signal transduction system. No significant difference was observed in the expression levels of IFNα between the two strains. Furthermore, the expression of OAS1 and Mx1 was significantly enhanced by THU5 stimulation from 12 to 48 hours after bacterial cell stimulation. OAS1 and Mx1 are genes whose expression is induced by interferon. The enhanced expression of these genes by THU5 stimulation is thought to be due to the induction of expression by interferon enhanced by THU5 stimulation. PKR and RNase L tended to increase their expression after 12 hours, but no significant difference was observed compared to the control strain.

[0043] No significant differences were observed between the analyzed inflammatory cytokines and the control at any time point. Furthermore, although data are not shown here, the expression levels of TLR2 and TLR4, known receptors for lactic acid bacteria, tended to increase only in TLR2 after 24 hours of THU5 stimulation. Expression levels of the viral receptors RIG-I and MDA5 were also increased after 12 hours of THU5 stimulation. RIG-I and MDA5 are factors induced by interferon, and sustained expression enhancement was observed up to 48 hours after bacterial stimulation (Figure 4C). On the other hand, no significant differences were observed in the expression levels of TLR3, which has a similar function. Furthermore, no significant changes in the expression levels of the negative regulators MKP-1, A20, and Tollip were observed with THU5. Figure 4D summarizes the changes in each gene.

[0044] Next, we demonstrate the immunomodulatory effect of lactic acid bacteria on viral infection. INV1-3 cells were seeded, and lactic acid bacteria were added on the fourth day for stimulation for 48 hours. After the addition of virus, samples were collected 0, 3, 6, 12, and 24 hours later, and gene expression was analyzed (Figure 5A). Evaluation was performed using the same factors as in the analysis of stimulation with bacteria alone.

[0045] The expression of antiviral factors is shown in Figure 5B. THU5 stimulation enhanced the expression of IFN-β and IFN-λ3 beginning 6 hours after virus infection. It is known that rotavirus double-stranded RNA that enters cells is recognized by RIG-I and MDA5 and enhances interferon expression via signal transduction. The results of the aforementioned bacterial cell stimulation test showed that MDA-5 and RIG-I expression was enhanced after bacterial cell pre-stimulation, suggesting that bacterial cell stimulation renders cells more susceptible to virus recognition. The expression of interferon-induced antiviral factors OAS1 and Mx1 was enhanced by THU5 stimulation beginning 3 hours after virus infection, an early stage of infection, and continued to be enhanced for up to 12 hours. The enhancement at the early stage of infection (3 to 6 hours) is thought to be due to the enhancing effect of pre-stimulation with the selected bacterial cells, and is further enhanced by the antiviral factors induced after virus infection, leading to a sustained antiviral effect.

[0046] Similar to OAS1 and Mx1, the expression of the viral receptors RIG-I and MDA5 also tended to increase with THU5 stimulation from 3 to 12 hours after virus infection (Figure 5C). It is thought that RIG-I and MDA5, enhanced by THU5 pre-stimulation, recognize the virus and induce interferon from 6 hours after infection, and antiviral factors and viral receptors induced by interferon from approximately 12 hours after infection. An overview of the changes in each gene is summarized in Figure 5D. The THU5 strain significantly enhanced the expression of many antiviral factors and viral receptors even after virus infection. In contrast, the control strain showed almost no enhanced expression of these factors. While the results shown here are from the selected L. delbrueckii strain THU5, we have also confirmed that similar expression induction in response to interferon and other factors occurs with the selected L. reuteri strain THU6.

[0047] 3.2 Function of THU6 strain To verify the antiviral immune-enhancing effect of THU6 strain on cells, we analyzed the gene expression levels of IFN-β, Mx1, and Oas1 in SIEC cells supplemented with heat-treated THU6 strain and control strain No. 15.

[0048] Heat-killed cells of the THU6 strain and control strain No. 15, cultured according to the above culture method, were prepared and added to SIEC cells. After two days of incubation, the cells were infected with rotavirus. After stimulation, cDNA was synthesized and gene expression analysis of IFN-β, Mx1, and Oas1 was performed using qPCR (Figure 6A). The THU6 strain was able to induce the expression of IFN-β, Mx1, and Oas1 in SIEC cells using heat-treated cells. The letters on the graph indicate significant differences between the different letters.

[0049] The acid and bile tolerance of the THU6 strain was evaluated. The THU6 strain and control strain No. 15 were inoculated into MRS medium (Difco) and cultured at 37°C for 16 hours. After culture, the OD600nm was adjusted to 1, and the bacteria were collected by centrifugation (8000 x g, 5 min, 4°C). After centrifugation, MRS liquid medium adjusted to pH 2.0 or 0.3% bovine bile was added and cultured at 37°C for 2 hours. As a control, each strain was also tested with MRS liquid medium at pH 7.2 without bile. The bacterial solution after acid or bile treatment was serially diluted and plated on MRS agar medium, and the resulting colonies were counted. The survival rate after treatment was calculated by dividing the number of surviving colonies by the number of colonies in the untreated group. The THU6 strain was confirmed to have stronger acid tolerance than the control strain No. 15 (Figure 6B).

[0050] The adhesion rate to SIEC cells was analyzed. The THU6 strain and the control strain No. 15 were inoculated into MRS medium (Difco) and cultured at 37°C for 16 hours. After culture, the medium was adjusted to 1 mL at OD600nm = 1, and the bacteria were collected by centrifugation (8000 x g, 5 min, 4°C). After centrifugation, DMEM / F-12 medium was added to prepare a bacterial solution for cell culture. Next, porcine small intestinal epithelial cells (SIEC cells) were cultured in DMEM / F-12 medium (Wako) at a concentration of 3.0 x 10 ４The cells were suspended at a concentration of 1000 cells / mL, and 1 mL of this suspension was seeded into each well of a 12-well plate and cultured for 4 days. 0.5 mL of the prepared cell culture solution was added, and the cells were cultured at 37°C for 1 hour. After washing twice with PBS, 0.5 mL of PBS was added, and the cells were detached and collected. The cell collection solution was serially diluted and plated on MRS agar medium, and the number of colonies formed was counted. The cell adhesion rate was calculated by dividing the number of surviving bacteria by the number of bacteria before adding the cells. The cell adhesion rates of the strains were compared. The THU6 strain was confirmed to have stronger adhesion ability to SIEC cells than the control strain No. 15 (Figure 6C).

[0051] The THU6 strain was observed using a transmission electron microscope to determine whether it had morphological characteristics compared to the control strain. The THU6 strain and control strain No. 15 were inoculated into MRS medium (Difco) and cultured at 37°C for 16 hours. After culture, an equal volume of 5% glutaraldehyde (PBS) was added and the culture was left standing overnight at 4°C. The culture was then centrifuged (4000 x g, 5 min, 4°C), the supernatant was removed, washed once with PBS, and resuspended in 3 mL of PBS. Ultrathin sections of the lactic acid bacteria were prepared using standard methods, and the cell membrane and cell wall (surface structure) were observed (Figure 6D). The left image shows the THU6 strain, and the right image shows the control strain No. 15. It was confirmed that the THU6 strain had a thicker extracellular polysaccharide layer than the control strain No. 15.

[0052] 4. Evaluation of Probiotics Using Porcine Small Intestinal Organoids (PJO) Up to this point, evaluations have been performed using single cell systems. However, to evaluate post-immunobiotics in a model more closely resembling the small intestine, small intestinal organoids were established. Pig jejunal crypts were isolated and PJO (Porcine Jejunum Organoids) were prepared using standard methods. Crypts were cultured using Matrigel to maintain the crypt-like structure and villus tissue structure. The established PJO1-3 cells were cultured in a monolayer plate, either in a 3D intestinal-like structure or after disrupting the 3D structure using Matrigel.

[0053] Three-dimensionally cultured small intestinal organoids were confirmed by staining to form an intestinal-like structure and express small intestinal markers. Frozen sections were prepared from the three-dimensionally cultured small intestinal organoids, and HE staining and immunofluorescence staining were performed to confirm the expression of ZO-1, a protein that constitutes tight junctions, and Lgr5, which is expressed in epithelial stem cells (Figure 7A, upper panel). Immunostaining was performed in the same manner as described above, except that anti-ZO-1 antibody (Cosmo Bio Co., Ltd.) and anti-Lgr5 antibody (Invitrogen) were used as primary antibodies. It was confirmed that the small intestinal organoids that formed an intestinal-like structure formed tight junctions, expressed epithelial stem cell markers, and served as a model reflecting living tissue.

[0054] Furthermore, the cells were disorganized, passed through a cell strainer, and cultured in a monolayer. The expression of ZO-1 and Lgr5 was confirmed by immunofluorescence staining (Figure 7A, bottom panel). Even in monolayer plate culture, expression of both ZO-1 and Lgr5 markers was confirmed, suggesting that small intestinal organoid cells can be cultured in a monolayer plate and subjected to gene expression analysis in response to lactic acid bacteria or viral stimulation.

[0055] Porcine small intestinal organoid cells PJO1-3 were cultured in a monolayer plate and stimulated with lactic acid bacteria for 48 hours. After infection with rotavirus, two days later, the expression of small intestinal markers and viral markers was analyzed by RT-qPCR using the primers shown in Table 6 (Figure 7B).

[0056]

[0057] In addition to THU5 and THU6, which have been confirmed to have antiviral effects against rotavirus, we also stimulated the cells with other lactic acid bacteria strains, THU1 to THU4, and analyzed the expression of small intestinal cell markers and viral markers. The expression of the absorptive epithelial cell marker Villin, which constitutes the small intestine, the goblet cell marker Muc2, the stem cell marker Lgr5, and the Paneth cell marker Lyz was not significantly altered by lactic acid bacteria stimulation or viral infection. On the other hand, the expression of the rotavirus marker NSP5 was significantly reduced by stimulation with THU5 and THU6 (Figure 7C). Furthermore, some of the other four lactic acid bacteria strains showed enhanced NSP5 expression. Screening using a model more closely resembling the small intestine, rather than screening using a single cell, may enable the screening of highly effective strains.

[0058] Lactic acid bacteria have been said to have useful functions as probiotics, but their effectiveness varies greatly depending on the strain. L. delbrueckii subsp. delbrueckii THU5 and L. reuteri THU6, selected in this study based on their immunomodulatory activity against rotavirus, possess anti-rotavirus effects. Because rotavirus is the main cause of viral diarrhea, these two strains, which can suppress rotavirus infection, are extremely useful in the prevention and treatment of the disease.

Claims

1. An antiviral agent comprising, as an active ingredient, Lactobacillus delbrueckii subsp. delbrueckii THU5 (accession number: NITE BP-04062) and / or Limosilactobacillus reuteri THU6 strain (accession number: NITE BP-04063).

2. The antiviral agent according to claim 1, wherein the virus is rotavirus.

3. The antiviral agent according to claim 1 or 2, which is used in livestock feed or food and drink.

4. Lactobacillus delbrueckii subsp. delbrueckii THU5 strain, deposited under accession number NITE BP-04062.

5. Limosilactobacillus reuteri THU6 strain deposited under accession number NITE BP-04063.

6. A method for screening bacterial strains with antiviral effects, comprising pretreating cultured cells with a candidate bacterial strain, infecting them with a virus, and selecting bacterial strains with antiviral effects using the expression of viral genes and / or proteins in the recovered cells as an indicator.

7. A method for selecting a bacterial strain with antiviral effect as described in claim 6, further characterized in that the selected bacteria are evaluated using organoids.

8. The method for selecting a strain having antiviral activity according to claim 6, wherein the virus is rotavirus and the candidate strain is a lactic acid bacterium.

9. The screening method according to claim 6, characterized in that the cultured cells are organoid-constituting cells cultured in a monolayer plate.

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