Streptococcus oral cavity ZRSOR-5 as well as related products, method and application thereof

Abstract

The invention provides oral streptococcus ZRSOR-5 as well as a related product, a method and application thereof. The preservation number of the streptococcus oral cavity ZRSOR-5 provided by the invention in the Guangdong Microbial Culture Collection Center is GDMCC No: 65352, and the preservation number of the streptococcus oral cavity ZRSOR-5 provided by the invention is CGMCC No: 65352. Experiments prove that the SOR preparation is prepared by diluting the SOR preparation with normal saline and inhaling the SOR preparation into mice in advance, so that the survival rate of influenza virus infected mice can be remarkably increased, and lung inflammation and virus load can be reduced. When the ZRSOR-5 preparation provided by the invention is used in advance, pneumonia caused by influenza viruses can be resisted.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and specifically discloses oral streptococcus ZRSOR-5 and related products, methods and applications thereof. Background Art

[0002] Community-acquired pneumonia (CAP) is a lung infection that occurs outside of hospitals and has a high morbidity and mortality rate, especially in the elderly, infants, and people with pre-existing health problems. The traditional treatment strategy for CAP focuses on eliminating the pathogens that cause the disease. However, studies have shown that typical pathogens can be detected in less than 50% of CAP patients, indicating the important role of other respiratory microorganisms in the pathogenesis of CAP.

[0003] In recent years, more and more studies have found that the balance of respiratory microbiota is crucial to the health of the respiratory tract. Microbiome dysbiosis, that is, the reduction of microbial diversity and the overgrowth of potential pathogens, is closely related to the development of respiratory diseases such as CAP. Studies have shown that CAP patients usually show lower microbial diversity, increased bacterial load, and enrichment of Streptococcus and Veillonella.

[0004] In some patients with severe infections such as sepsis and acute respiratory distress syndrome (ARDS), studies have also found that gut-related bacteria are enriched in the lung microbiota and significantly correlated with pulmonary inflammatory mediator TNF-α. However, there is currently a lack of systematic research on the prevention of CAP and immune regulation by respiratory symbiotic bacteria, and no key symbiotic bacteria categories have been found. Summary of the invention

[0005] The technical problem to be solved by this application is how to improve the survival rate after influenza virus infection and reduce lung inflammation and viral load.

[0006] In order to solve the above technical problems, the present invention provides a streptococcus for the first time, wherein the streptococcus is oral streptococcus ( Streptococcus oralis ) ZRSOR-5, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCCNo: 65352.

[0007] The present invention also provides a composition, which contains the streptococcus.

[0008] The composition may be a culture, and the culture is a substance obtained by culturing the streptococcus in a microbial culture medium.

[0009] The active ingredient of the above composition may be the streptococcus and / or its metabolite and / or its culture.

[0010] The metabolite can be obtained from the shaking culture of the streptococcus. The metabolite can be a bacterial metabolite of the streptococcus.

[0011] The culture may be a substance obtained by culturing the streptococcus in a microbial culture medium. The substance may be a fermentation product, such as a fermentation liquid containing the streptococcus and a substance secreted into a liquid culture medium, or a solid fermentation product containing the streptococcus and a substance secreted into a solid culture medium.

[0012] The composition may also be a bacterial agent. The active ingredients of the bacterial agent may also contain other biological components or non-biological components, and those skilled in the art may determine the other active ingredients of the bacterial agent according to the effects of the bacterial agent.

[0013] A bacterial agent refers to a live bacterial preparation made by expanding the target microorganisms and using a carrier as an adsorbent to adsorb the fermentation liquid or solid fermentation products of the bacteria.

[0014] The above-mentioned bacterial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0015] The bacterial agent may further include a carrier as required, and the carrier may be a solid carrier or a liquid carrier.

[0016] The carrier may include a pharmaceutically acceptable carrier. The material of the carrier includes, but is not limited to, a water-soluble carrier material (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), a poorly soluble carrier material (such as ethyl cellulose, cholesterol stearate, etc.), an enteric carrier material (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, a water-soluble carrier material is preferred. Using these materials, a variety of dosage forms can be made, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; adhesives such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form as a suppository, various carriers known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In addition, colorants, preservatives, spices, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations as needed.

[0017] In one embodiment, the carrier may be 20% glycerol saline.

[0018] The above-mentioned bacterial preparations can be used as human medicine or animal medicine (veterinary medicine). The above-mentioned bacterial preparations include but are not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal preparations, buccal tablets, suppositories or freeze-dried powder injections.

[0019] The bacterial agent may also contain pharmaceutical excipients. The pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to active ingredients. In addition to excipients, carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetics, and fully synthetics. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. The same pharmaceutical excipients can be used in drug preparations with different administration routes and have different functions and uses.

[0020] The composition may be any of the following: A1) Compositions for preventing and treating viral pneumonia caused by viral infection; A2) a composition for reducing viral load after viral infection; A3) compositions that limit viral infection; A4) A composition for improving the survival rate after virus infection; A5) A composition for reducing the magnitude of body weight loss after viral infection; A6) A composition for increasing the expression of immune regulatory genes after viral infection; A7) Compositions for inhibiting viral transmission after viral infection; A8) A composition for reducing lung damage caused by viruses; A9) A composition for reducing damage to the alveolar capillary barrier after viral infection; A10) A composition for improving the permeability of alveolar capillary membranes after viral infection; A11) A composition for reducing the level of IL-6, an inflammatory marker after viral infection; A12) A composition for reducing the level of TNF-α, an inflammatory marker after viral infection; A13) A composition for reducing the infiltration of inflammatory cells caused by viruses; A14) A composition for promoting timely activation of innate immune response to viruses; A15) A composition for preventing immune-mediated pathological damage following viral infection.

[0021] The present invention also provides a method for preparing a composition, wherein the composition is the above composition, and the method comprises the step of using the streptococcus as a component of the composition.

[0022] The present invention also provides application of the streptococcus or the composition in preparing products.

[0023] The product should have at least one of the following properties: B1) Prevent and treat viral pneumonia caused by viral infection; B2) Reduction of viral load after viral infection; B3) Limit viral infection; B4) Improve survival rate after viral infection; B5) Reduce the weight loss after viral infection; B6) Increased expression of immune regulatory genes after viral infection; B7) inhibiting viral transmission after viral infection; B8) Reduce lung damage caused by viruses; B9) Reduce the damage of alveolar capillary barrier after viral infection; B10) Improves alveolar capillary membrane permeability after viral infection; B11) Reduce the level of IL-6, an inflammatory marker after viral infection; B12) Reduce the level of TNF-α, an inflammatory marker after viral infection; B13) Reduce the infiltration of inflammatory cells caused by viruses; B14) promotes timely activation of innate immune responses to viruses; B15) Prevent immune-mediated pathological damage after viral infection.

[0024] In the above applications, the product can be administered via intranasal and / or intra-airway inhalation inoculation.

[0025] The present invention also provides a method for preventing and / or treating viral infection, which comprises contacting the nasal cavity and / or airway with the streptococcus or the composition to prevent and treat viral pneumonia caused by the virus.

[0026] In the above, the virus may be an influenza virus.

[0027] The influenza virus may specifically be A / Puerto Rico / 8 / 1934 (H1N1) influenza virus.

[0028] The viral pneumonia may be community-acquired pneumonia (CAP).

[0029] The present invention longitudinally collects sputum from CAP patients and combines it with the host immune response data to find that oral streptococci are related to the patient's innate immune protection. A commensal bacterium was isolated using Columbia blood culture medium and found to have the ability to activate the host's innate immunity and resist pneumonia.

[0030] The present invention relates to the field of microbial technology, and specifically discloses the preparation of a respiratory symbiotic oral Streptococcus ZRSOR-1 probiotic preparation and its application in preventing pneumonia. The present invention first observed an increase in the abundance of Streptococcus in patients with community-acquired pneumonia (CAP), and combined with host immune response data, found that oral Streptococcus ( Streptococcus oralis , SOR) is related to the host's innate immune response pathway. In the second step, a strain of SOR was successfully isolated using Columbia blood culture medium, which was diluted with physiological saline and pre-inhaled into mice to prepare SOR preparations, which significantly improved the survival rate of mice infected with influenza virus and reduced lung inflammation and viral load. The ZRSOR-5 preparation of the present invention can resist pneumonia caused by influenza virus when used in advance.

[0031] Collection Instructions Bacteria name: Oral Streptococcus Latin name: Streptococcus oralis Strain ID: ZRSOR-5 Depository: Guangdong Microbial Culture Collection Center Depository Abbreviation: GDMCC Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou Deposit date: October 25, 2024 Registration number of the depository center: GDMCC No: 65352 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Dynamic changes of sputum microbiome at the species level in patients with CAP.

[0032] Figure 2 The differentially expressed genes between CAP patients and healthy controls are shown. The x-axis represents the log2 fold change (Log2FC), and the y-axis represents the -log10 value of the FDR-adjusted q value. The top 10 gene names with the largest fold changes between CAP patients and healthy controls are labeled.

[0033] Figure 3Figure 2 shows the gene-microbe interactions in CAP patients. a is the network visualization of the significant associations between S. oralis and S. mitis and the genes in the red module, and the remaining six bacteria in microbial cluster 5 formed an independent network; b is the gene enrichment analysis of the biological processes, cellular components, and molecular functions of the genes related to S. oralis and S. mitis in the red module.

[0034] Figure 4 Schematic diagram of the experimental design for the mouse experiments. Mice were pre-inoculated four times with S. oralis (SOR) via the intranasal route and subsequently infected with IAV. Lung and bronchoalveolar lavage fluid (BALF) samples were collected on days 2 and 6 post infection.

[0035] Figure 5 The mortality and weight loss caused by IAV in treated mice; SOR refers to ZRSOR-5 treatment; IAV refers to influenza virus; mock refers to saline control and PBS challenge control.

[0036] Figure 6 The performance of mice after infection; c is the lung virus load (PFU) of the four groups of mice 48 hours after infection; d is the level and total protein concentration of inflammatory markers IL-6 and TNF-α in the BALF of mice on the 6th day after infection; e is HE staining of lung sections of IAV-infected (IAV) and uninfected (Mock) mice pretreated with S.oralis (SOR) or saline (Saline) on the 6th day.

[0037] Figure 7 Differentially expressed genes and pathways in the lungs of mice pretreated with S.oralis compared with those of control mice, where f is the second day after infection and g is the sixth day.

[0038] Figure 8 This is the colony image of purified streptococci.

[0039] Fig. 9 This is the mass spectrometry identification diagram of oral Streptococcus ZRSOR-5. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0042] In the following embodiments, the influenza virus (IAV) is A / Puerto Rico / 8 / 1934 (H1N1) influenza virus (Jia J, Li H, Huang Z, Yu J, Zheng Y and Cao B (2023) Comprehensive immune landscape of lung-resident memory CD8+ T cells after influenza infection and reinfection in a mouse model. Front. Microbiol. 14:1184884.doi: 10.3389 / fmicb.2023.1184884). The public can obtain the biological material from the applicant in accordance with the relevant provisions of the national biosafety. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0043] The following examples used GraphPad Prism 10.2.3 statistical software to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0044] Example 1. Discovery and identification of oral Streptococcus ZRSOR-1 1. Discovery of oral Streptococcus ZRSOR-1 1.1 Sample collection In January 2023, hospitalized CAP patients aged 18 years and above were recruited in Beijing during the peak period of respiratory infections. Clinical samples included sputum and whole blood, which were collected within 24 hours of admission (T1), followed by a second sample every 2-3 days (T2), and the last sample was collected 1-2 days before discharge (T3). Patients were followed up 4 months after discharge, and samples were collected if they reported no respiratory symptoms and no antibiotics within one month. Induced sputum from 33 healthy individuals (no respiratory symptoms and no antibiotics in the past month) served as the control group, and the specific collection method was referred to previous studies. Sputum samples were liquefied with 0.1% dithiothreitol, aliquoted and stored at -80°C. Whole blood samples were collected into PAXgeneBlood RNA tubes (BD, USA) and stored at -20°C. All patients were enrolled in Zibo Municipal Hospital, and the study was approved by the ethics committees of Zibo Municipal Hospital and China-Japan Friendship Hospital.

[0045] 1.2 Metagenomic sample processing and sequencing DNA was extracted from sputum samples and paired-end sequencing was performed with a length of 150 bp using the Illumina HiSeq 4000 platform. Taxonomic analysis of metagenomic data included quality control of sequences, removal of low-quality sequences and sequences less than 100 bp in length and host sequences. Species-level classification was analyzed using Kraken2 v2.1.3 and the PlusPF database, which covers archaea, bacteria, viruses, plasmids, humans, protozoa, and fungi, released in December 2022. The association between microbiota and CAP was performed using generalized linear mixed models (GLMMs) in the lme4 package of R, adjusting for age and antibiotic use as covariates, and subject identity as a random effect.

[0046] 1.3 The proportion of streptococci in CAP patients is increased, and the pathogenic gene modules of patients are increased Among the 252 major bacterial species investigated in this cohort, Streptococcus was the most frequently detected enriched taxon (n = 16), followed by Veillonella (n = 4) and Prevotella (n = 4). The enrichment of Streptococcus in CAP patients persisted during the follow-up period, whereas the abundance of Veillonella, Campylobacter, and Roseburia returned to the level of healthy controls at 4 months after discharge ( Figure 1 ). Figure 1 Figure 2 Dynamic changes of sputum microbiome in CAP patients at the species level. Compared with healthy controls, Streptococcus was the most enriched species among the bacterial species significantly enriched in CAP patients.

[0047] A total of 3968 microbial genes were identified in the functional genes of the microorganisms, of which 287 were more abundant in CAP patients and 961 were more abundant in healthy controls ( Figure 2 ). Two genes related to microbial DNA repair and stress response, mcsA and mcsB, were most highly elevated in CAP patients. This suggests that bacteria in the respiratory tract of CAP patients are experiencing significant stress, possibly caused by host immune responses or antibiotic treatment. In addition, the rfbP gene was also one of the top ten genes with significantly increased abundance in CAP patients. This gene is involved in the biosynthesis of O antigen, which is an important component of the bacterial outer membrane and plays a key role in immune escape, allowing pathogens to persist and cause infection. In contrast, in healthy controls, the abundance of genes related to genetic mobility (insB) and nutrient metabolism (fucR, gltI, aatJ) was higher. This suggests that the respiratory microbiota is in a normal symbiotic state, maintaining a balanced microbial community without triggering a pathogenic response (see Figure 2 ). Figure 2 The volcano plot in the figure shows the differentially expressed genes between CAP patients and healthy controls, where the x-axis represents the log2 fold change (Log2FC) and the y-axis represents the -log10 value of the FDR-adjusted q value. The text labels the names of the top 10 genes with the largest fold changes between CAP patients and healthy controls.

[0048] 1.4 Integrative analysis of host response and respiratory microbiota To explore the interaction between the respiratory microbiota and host response in CAP patients, we performed weighted gene co-expression network analysis (WGCNA) to identify co-expressed gene modules and their associations with microorganisms. We found that two streptococcal species, oral Streptococcus (S. oralis, SOR) and Streptococcus mitis (S. mitis), were directly associated with 6 host genes in a gene module and indirectly associated with other genes. Gene enrichment analysis showed that the genes in this module were mainly involved in the activation of innate immune response, viral processes and cytokine response, suggesting that these two streptococci may play a role in regulating host immune response during viral infection ( Figure 3 ). Figure 3 The gene-microbe interactions in CAP patients are shown, where a is a network visualization of the significant associations between S.oralis and S.mitis and the genes in the red module, and the remaining six bacteria in microbial cluster 5 formed an independent network; b is a gene enrichment analysis of the biological processes, cellular components, and molecular functions of genes related to S.oralis and S.mitis in the red module.

[0049] 2. Isolation and identification of strain ZRSOR-5 Sputum samples are smeared on Columbia blood agar medium using a sterile inoculation loop and cultured at 37°C in an incubator containing 5% CO2 for 24-48 hours. Observe the colony morphology. Streptococci usually appear small, grayish white, translucent, round, with neat edges, accompanied by α- or β-hemolysis. Suspected streptococcal colonies are selected based on colony morphology and purified for culture (see Figure 8 ).

[0050] The colonies were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purified colonies were placed on the MALDI target plate, and the mass spectrometry analysis results were compared with the database. The identification results were Fig. 9 .

[0051] DNA from the selected colony ZRSOR-5 was extracted using a bacterial DNA extraction kit and then compared after next generation sequencing. The gene sequence to be identified was compared in the NCBI database using the BLAST tool. According to the molecular biology results, the Latin name of the strain was Streptococcus oralis , the strain was determined to be oral streptococcus. ZRSOR-5 was collected in Guangdong Microbial Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code: 510070) on October 25, 2024, with the collection number GDMCC No: 65352, hereinafter referred to as ZRSOR-5.

[0052] Example 2: Application of ZRSOR-5 preparation to resist viral pneumonia 1. Preparation of oral Streptococcus ZRSOR-5 preparation ZRSOR-5 was inoculated with Columbia blood agar medium (Thermofisher, PB0123A) and cultured at 37°C in an incubator containing 5% CO2 for 24 h. The cultured ZRSOR-5 was collected into 1 ml of saline using a cell scraper and centrifuged at 1000 g for 5 min. The supernatant was discarded. 1 ml of saline was added to suspend the precipitate, and the supernatant was removed by centrifugation. 1 ml of 20% glycerol saline was added to the precipitate to prepare a uniform suspension preparation with a concentration of 3.3×10 7 CFU / mL, stored at -80 degrees Celsius for future use.

[0053] 2. Divide into four groups for respiratory tract bacterial intervention.

[0054] All experimental mice were 8-9 weeks old female C57BL / 6 mice. Four groups of mice were selected, each with 15 mice. Two groups of mice were given 30ul ZRSOR-5 bacterial suspension containing 1×10 5 ZRSOR-5 bacteria were inoculated into mice by nasal drops once every two days for a total of 4 times. The other two groups of mice were only given normal saline as a control. The experimental and control mice were cultured at a temperature of 23±2℃, a relative humidity of 50-55%, indoor lighting simulating day and night, and a 12-hour light-dark cycle. Each mouse was uniformly fed 5-8g of feed per day. In the following text, S.oralis (SOR) pre-inoculation or pretreatment refers to giving mice 30ul ZRSOR-5 bacterial suspension, which contains 1×10 5 ZRSOR-5 bacteria.

[0055] After the last intranasal administration of bacteria (the control group received saline), one group of mice in the ZRSOR-5 group and the control group were given 10 3 CFU of influenza virus A / Puerto Rico / 8 / 1934 (H1N1), and another group was given PBS. Figure 4 Schematic diagram of the experimental design for mice, which were pre-inoculated four times with S. oralis (SOR) via the intranasal route and subsequently infected with IAV. Lung and bronchoalveolar lavage fluid (BALF) samples were collected on days 2 and 6 post infection.

[0056] The survival rate and weight changes of mice were observed for 10 days, and it was found that the weight of the two groups of mice that were not vaccinated with influenza virus did not decrease. The weight of the two groups of mice that were given influenza virus decreased, but the weight loss percentage of mice that were vaccinated with ZRSOR-5 intranasally in advance was significantly lower than that of mice that were vaccinated with saline intranasally. Among the 15 mice that were vaccinated with ZRSOR-5 intranasally in advance, no one died, while 5 of the 15 mice that were vaccinated with saline intranasally in advance died, indicating that ZRSOR-5 has a preventive effect on lethal influenza pneumonia in mice ( Figure 5 ). Figure 5 The results showed that pretreatment with ZRSOR-5 significantly reduced the weight loss and mortality caused by IAV in mice, where SOR was pretreated with ZRSOR-5; IAV was influenza virus inoculation; and mock was saline control and PBS challenge control.

[0057] also, Figure 6Figure c shows the lung viral load (PFU) of the four groups of mice 48 hours after infection with influenza virus. Compared with mice that were not pretreated with bacteria, the number of infectious virus particles in the lungs was also reduced within 48 hours after infection. The method for detecting lung viral load (PFU) is to infect MDCK cells with the sample after a series of gradient dilutions. After the virus is adsorbed, it is covered with a layer of melted semi-solid nutrient agar to allow the virus to spread limitedly in the monolayer cell culture. When white spots appear, they can be stained with neutral red. Add 1 mL of neutral red solution diluted 20 times with PBS to each well (the storage solution concentration is 3.3 mg / mL) and incubate at 37°C for 4 hours or overnight. Count the plaques after aspirating the dye. The titer was calculated by counting the plaques. The average viral load of mice treated with SOR was 15*10 3 pfu / ml, while the average viral load of mice in the control group was 39*10 3 pfu / ml, there was a significant difference between the two groups.

[0058] Figure 6 Figure d shows the levels of inflammatory markers IL-6 and TNF-α and total protein concentration in BALF of mice on the 6th day after influenza virus infection. Compared with mice not pretreated with bacteria, SOR pretreatment also led to a decrease in the levels of inflammatory markers IL-6 and TNF-α in BALF on the 6th day after infection, and at this time, the weight loss of mice in the SOR group was the largest after influenza virus attack; SOR also reduced the damage to the alveolar capillary barrier, as manifested by a lower total protein concentration in BAL. The total protein concentration was detected using a kit with catalog number A55860 from Thermofisher, reflecting an improvement in the permeability of the alveolar capillary membrane. IL-6 (Thermofisher, BMS603-2) and TNF-α (Thermofisher, BMS607-3) were detected using ELISA kits according to the instructions. First, the sample was added to the ELISA 96-well plate, which was embedded with specific antibodies to capture cytokines. The enzyme-labeled secondary antibody was added for color development, and the absorbance value was read by an enzyme reader. The concentration of cytokines in the sample was calculated based on the standard curve.

[0059] Figure 6 Figure e shows the stained sections of lung tissues of IAV-infected (IAV) and uninfected (Mock) mice pretreated with S.oralis (SOR) or saline on day 6. Mouse lung tissues were immediately fixed in 10% buffered formalin for at least 1 week after collection. The fixed tissues were routinely processed, embedded in paraffin, sliced, and stained with hematoxylin-eosin (H&E) for histopathological analysis. Pathological analysis of mouse lungs collected on day 6 after infection showed that SOR pretreatment reduced the infiltration of inflammatory cells and lung damage.

[0060] To explore the protective mechanism of SOR in preventing IAV infection, transcriptome analysis was performed on lung tissues of mice pretreated with SOR before IAV infection and untreated mice on days 2 and 6 after infection. Transcriptome sequencing data were quality filtered using Fastp v0.20.0 and aligned to the human genome GRCh38 using Hisat2 v2.1.0. Subsequently, the gene expression matrix was obtained using FeatureCounts v4.8.1. Differentially expressed genes were identified by EdgeR, and gene enrichment analysis was subsequently performed using the clusterProfiler v4.8.1 package. The enrichment score of each pathway for each sample was calculated using the GSVA v1.48.0 package. Figure 7 Middle f shows differentially expressed genes and pathways in the lungs of mice pretreated with S.oralis (SOR) compared with mice not treated with SOR at day 2 post infection. Pre-inoculation with SOR significantly increased the expression of multiple immune regulatory genes, including IL17F, LY6I, and C1RB, at day 2 post infection, which enhanced the activation of pathways related to bacterial infection, viral protein-cytokine interaction, cytokine and chemokine signaling, and complement response. These upregulated pathways play a key role in host resistance to respiratory infections, and their high activation in the early stages of IAV infection inhibited the spread of the virus and improved the prognosis of mice. Figure 7 Figure f shows differentially expressed genes and pathways in the lungs of mice pretreated with S.oralis (SOR) compared with mice not treated with SOR on day 6 after infection. When mice began to recover on day 6 after infection, these pro-inflammatory genes were no longer significantly different between the SOR-treated and untreated groups. Similarly, the pro-inflammatory pathways that were highly activated on day 2 were not enriched on day 6.

[0061] These findings suggest that pre-vaccination with the ZRSOR-5 formulation resulted in timely activation of innate immune responses that effectively limited IAV infection and that these pathways were rapidly resolved during the recovery phase to prevent immune-mediated pathological damage.

[0062] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Streptococcus, characterized in that The streptococcus is oral streptococcus ( Streptococcus oralis ) ZRSOR-5, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC No: 65352.

2. A composition, characterized in that The composition contains the streptococcus according to claim 1.

3. The composition according to claim 2, characterized in that: The composition is a culture, and the culture is a substance obtained by culturing the streptococcus in a microbial culture medium.

4. The composition according to claim 2, characterized in that: The composition is a bacterial agent.

5. A composition according to any one of claims 2 to 4, characterized in that The composition is any one of the following: A1) Compositions for preventing and treating viral pneumonia caused by viral infection; A2) a composition for reducing viral load after viral infection; A3) compositions that limit viral infection; A4) A composition for improving the survival rate after virus infection; A5) A composition for reducing the magnitude of body weight loss after viral infection; A6) A composition for increasing the expression of immune regulatory genes after viral infection; A7) Compositions for inhibiting viral transmission after viral infection; A8) A composition for reducing lung damage caused by viruses; A9) A composition for reducing damage to the alveolar capillary barrier after viral infection; A10) A composition for improving the permeability of alveolar capillary membranes after viral infection; A11) A composition for reducing lung inflammation following viral infection; A12) A composition for reducing the level of IL-6, an inflammatory marker after viral infection; A13) A composition for reducing the level of TNF-α, an inflammatory marker after viral infection; A14) A composition for reducing the infiltration of inflammatory cells caused by viruses; A15) A composition for promoting timely activation of innate immune response to viruses; A16) A composition for preventing immune-mediated pathological damage following viral infection.

6. A method for preparing a composition, characterized in that The composition is the composition according to any one of claims 2 to 5, and the method comprises the step of using the streptococcus according to claim 1 as a component of the composition.

7. Use of the streptococcus described in claim 1 or the composition described in claims 2-5 in preparing products.

8. The use according to claim 7, characterized in that: The product has at least one of the following properties: B1) Prevent and treat viral pneumonia caused by viral infection; B2) Reduction of viral load after viral infection; B3) Limit viral infection; B4) Improve survival rate after viral infection; B5) Reduce the weight loss after viral infection; B6) Increased expression of immune regulatory genes after viral infection; B7) inhibiting viral transmission after viral infection; B8) Reduce lung damage caused by viruses; B9) Reduce the damage of alveolar capillary barrier after viral infection; B10) Improves alveolar capillary membrane permeability after viral infection; B11) Reduce lung inflammation following viral infection; B12) Reduce the level of IL-6, an inflammatory marker after viral infection; B13) Reduce the level of TNF-α, an inflammatory marker after viral infection; B14) Reduce the infiltration of inflammatory cells caused by viruses; B15) promotes timely activation of innate immune responses to viruses; B16) Prevent immune-mediated pathological damage after viral infection.

9. A method for preventing and / or treating pneumonia caused by influenza virus, characterized in that: The method comprises contacting the nasal cavity and / or airway with the streptococcus described in claim 1 or the composition described in claims 2-5 to prevent and treat viral pneumonia caused by viruses.

10. The use according to claim 8, wherein the product is for intranasal and / or intratracheal inhalation vaccination.

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