Streptococcus oralis zrsor-5 and related products, methods and uses

Abstract

The application provides oral streptococcus ZRSOR-5 and related products, methods and applications. Streptococcus oralis The preservation number of the oral streptococcus (ZRSOR-5) provided by the application in the Guangdong Microbial Culture Collection Center is GDMCC No: 65352. Experiments prove that by diluting with physiological saline and pre-inhaling the SOR preparation to mice, the survival rate of the influenza virus infected mice can be significantly improved, and lung inflammation and virus load can be reduced. The ZRSOR-5 preparation provided by the application can be used in advance to resist pneumonia caused by the influenza virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, and specifically discloses oral streptococcus ZRSOR-5 and related products, methods and applications. BACKGROUND

[0002] Community-acquired pneumonia (CAP) is a lung infection disease that occurs outside the hospital, with a high morbidity and mortality, especially in the elderly, infants and people with previous health problems. The traditional treatment strategy of CAP mainly focuses on eliminating the pathogen that causes the disease. However, studies have shown that less than 50% of CAP patients can detect typical pathogenic bacteria, indicating the important role of other respiratory tract microorganisms in the pathogenesis of CAP.

[0003] In recent years, more and more studies have found that the balance of respiratory tract microbiota is crucial for respiratory health. Microbiota imbalance, i.e. reduction of microbial diversity and overgrowth of potential pathogenic bacteria, is closely related to the development of respiratory diseases such as CAP. Studies have shown that CAP patients usually exhibit lower microbial diversity, increased bacterial load and enrichment of Streptococcus and Veillonella.

[0004] In some severe infections such as sepsis and acute respiratory distress syndrome (ARDS) patients, studies have also found that lung microbiota is enriched with gut-related bacteria, which is significantly correlated with lung inflammatory mediator TNF-α. However, there is currently a lack of systematic research on the prevention of CAP and immune regulation by respiratory commensal bacteria, and the key commensal bacteria category has not been found. SUMMARY

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

[0006] To solve the above technical problems, the present application provides a streptococcus for the first time, which is oral streptococcus ZRSOR-5, with a preservation number of GDMCC No: 65352 in Guangdong Microbial Culture Collection Center. Streptococcus oralis )ZRSOR-5, with a preservation number of GDMCC No: 65352 in Guangdong Microbial Culture Collection Center.

[0007] The present application also provides a composition containing the above-mentioned streptococcus.

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

[0009] The active ingredient of the above-mentioned composition can be the streptococcus or / and metabolites of the streptococcus or / and culture of the streptococcus.

[0010] The metabolite can be obtained from the shake culture solution of the streptococcus. The metabolite can be a metabolite containing the streptococcus.

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

[0012] The composition can also be a microbial agent. The active ingredients of the microbial agent can also contain other biological or non-biological ingredients, and the other active ingredients of the microbial agent can be determined by those skilled in the art according to the effect of the microbial agent.

[0013] A microbial agent refers to a live microbial preparation obtained by processing the fermentation broth or solid fermentation product of the target microorganism after expansion using a carrier as an adsorbent.

[0014] In the above microbial agent, the dosage form of the microbial agent can be various, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder, or water dispersible granule, etc.

[0015] If necessary, the microbial agent can also include a carrier. The carrier can be a solid carrier or a liquid carrier.

[0016] The carrier can include a pharmaceutically acceptable carrier. The materials for the carrier include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (e.g., ethyl cellulose, cholesterolesterate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, the water-soluble carrier materials are preferred. Using these materials, various dosage forms can be prepared, including, but not limited to, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal preparations, buccal tablets, suppositories, lyophilized powder injections, etc. They can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle drug delivery systems. In order to prepare unitary drug delivery dosage forms into tablets, various carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol triestearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further prepared 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 prepare unitary drug delivery dosage forms into pills, various carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare unitary drug delivery dosage forms into suppositories, various carriers known in the art can be widely used. Examples of the carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In addition, if necessary, coloring agents, preservatives, flavors, flavoring agents, sweetening agents, or other materials can also be added to the pharmaceutical preparations.

[0017] In one embodiment, the carrier can be 20% glycerol in normal saline.

[0018] The above-mentioned microbial agents can be used as human or animal (veterinary) medications. These microbial agents include, but are not limited to, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, microspheres, transdermal preparations, lozenges, suppositories, or lyophilized powder injections.

[0019] The microbial agent may also contain pharmaceutical excipients. Pharmaceutical excipients refer to the excipients and additives used in the production of drugs and the preparation of formulations. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of drugs. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their functions and uses, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. The same pharmaceutical excipient can be used in drug formulations with different routes of administration and has different functions and uses.

[0020] The composition may be any one of the following:

[0021] A1) A composition for preventing and treating viral pneumonia caused by viral infection;

[0022] A2) Compositions that reduce viral load after viral infection;

[0023] A3) Compositions that limit viral infection;

[0024] A4) Compositions that improve survival rates after viral infection;

[0025] A5) A composition that reduces the rate of weight loss after viral infection;

[0026] A6) A composition that increases the expression of immune regulatory genes after viral infection;

[0027] A7) A composition that inhibits viral transmission after viral infection;

[0028] A8) A composition that reduces lung damage caused by viruses;

[0029] A9) A composition for reducing damage to the alveolar-capillary barrier after viral infection;

[0030] A10) A composition that improves alveolar capillary membrane permeability after viral infection;

[0031] A11) A composition that reduces the level of IL-6, an inflammatory marker after viral infection;

[0032] A12) A composition that reduces the level of TNF-α, an inflammatory marker, after viral infection;

[0033] A13) A composition that reduces the infiltration of inflammatory cells caused by viruses;

[0034] A14) A composition that promotes the timely activation of the innate immune response to the virus;

[0035] A15) A composition for preventing immune-mediated pathological damage following viral infection.

[0036] The present invention also provides a method for preparing a composition, wherein the composition is the above-described composition, and the method includes the step of using the streptococcus as a component of the composition.

[0037] The present invention also provides the use of the streptococcus or composition in the preparation of products.

[0038] The product should have at least one of the following properties:

[0039] B1) Prevention and treatment of viral pneumonia caused by viral infection;

[0040] B2) Reduce viral load after viral infection;

[0041] B3) Restricting viral infection;

[0042] B4) Improves survival rate after viral infection;

[0043] B5) Reduce the rate of weight loss after viral infection;

[0044] B6) Increases the expression of immune regulatory genes after viral infection;

[0045] B7) Inhibits viral transmission after infection;

[0046] B8) Reduces lung damage caused by the virus;

[0047] B9) Reduces damage to the alveolar-capillary barrier after viral infection;

[0048] B10) improves alveolar capillary membrane permeability after viral infection;

[0049] B11) Reduces the level of IL-6, an inflammatory marker following viral infection;

[0050] B12) Reduces the level of TNF-α, an inflammatory marker following viral infection;

[0051] B13) Reduces the infiltration of inflammatory cells caused by the virus;

[0052] B14) promotes the timely activation of the innate immune response to the virus;

[0053] B15) Prevents immune-mediated pathological damage following viral infection.

[0054] In the above applications, the product can be administered via intranasal and / or intratracheal inhalation.

[0055] The present invention also provides a method for preventing and / or treating viral infections, the method comprising contacting the streptococcus or composition with the nasal cavity and / or airway to prevent viral pneumonia caused by the virus.

[0056] The virus mentioned above could be the influenza virus.

[0057] The influenza virus in question may specifically be the A / Puerto Rico / 8 / 1934 (H1N1) influenza virus.

[0058] The viral pneumonia mentioned may be community-acquired pneumonia (CAP).

[0059] This invention, through longitudinal collection of sputum from CAP patients and combining it with host immune response data, discovered that oral streptococci are associated with the innate immune protection of patients. A symbiotic bacterium was isolated using Columbia blood culture medium and found to have the ability to activate the host's innate immunity and resist pneumonia.

[0060] This invention relates to the field of microbial technology, specifically disclosing the preparation and application of a respiratory tract symbiotic oral streptococcus ZRSOR-1 probiotic preparation for the prevention of pneumonia. This invention first observed increased abundance of Streptococcus spp. in patients with community-acquired pneumonia (CAP), and combined with host immune response data, discovered that oral streptococci (… Streptococcus oralis ZRSOR (Solar Oral Receptor) is associated with the host's innate immune response pathway. In the second step, a strain of ZRSOR was successfully isolated using Columbia blood culture medium. After dilution with physiological saline, the ZRSOR preparation was pre-inhaled into mice, significantly improving the survival rate of mice infected with influenza virus and reducing lung inflammation and viral load. Pre-administration of the ZRSOR-5 preparation of this invention can resist pneumonia caused by influenza virus.

[0061] Preservation Instructions

[0062] Bacterial species name: Oral streptococci

[0063] Latin name: Streptococcus oralis

[0064] Strain number: ZRSOR-5

[0065] Preservation Institution: Guangdong Provincial Center for Microbial Culture Collection

[0066] Abbreviation for depository institution: GDMCC

[0067] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

[0068] Deposit date: October 25, 2024

[0069] Registration number at the Depository Center: GDMCC No.: 65352 Attached Figure Description

[0070] Figure 1 To understand the dynamic changes of the sputum microbiome at the species level in CAP patients.

[0071] Figure 2 This section shows differentially expressed genes between CAP patients and healthy controls. The x-axis represents the log2 fold change (Log2FC), and the y-axis represents the -log10 value of the q-value adjusted for FDR. The names of the top 10 genes with the largest fold changes in CAP patients and healthy controls are labeled.

[0072] Figure 3 Gene-microbe interactions in CAP patients. a) Visualization of the network showing significant associations between *S. oralis* and *S. mitis* and genes in the red module; the remaining six bacteria in microbial cluster 5 form a separate network. b) Gene enrichment analysis of biological processes, cellular components, and molecular functions of genes associated with *S. oralis* and *S. mitis* in the red module.

[0073] Figure 4 This is a schematic diagram of the experimental design for mice. Mice were pre-inoculated with S. oralis (SOR) four times via intranasal route, followed by IAV infection. Lung and bronchoalveolar lavage fluid (BALF) samples were collected on days 2 and 6 post-infection.

[0074] Figure 5 The study included data on mortality and weight loss in mice after IAV treatment; SOR represented ZRSOR-5 treatment; IAV represented influenza virus; and mock represented saline control and PBS challenge control.

[0075] Figure 6 The results show the effects of infection in mice; c represents the viral load (PFU) in the lungs of the four groups of mice 48 hours after infection; d represents the levels of inflammatory markers IL-6 and TNF-α and the total protein concentration in the BALF of mice on day 6 after infection; e represents the HE staining of lung sections from infected (IAV) and uninfected (Mock) mice on day 6 after pretreatment with S. oralis (SOR) or saline.

[0076] Figure 7 Differentially expressed genes and pathways in the lungs of S. oralis-pretreated mice compared to control mice were identified, with f representing day 2 post-infection and g representing day 6.

[0077] Figure 8 This is a colony diagram of purified Streptococcus.

[0078] Figure 9 Mass spectrometry image of oral streptococci ZRSOR-5. Detailed Implementation

[0079] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] In the following examples, the influenza virus (IAV) is the 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 this biological material from the applicant in accordance with the relevant national biosafety regulations. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0082] The following examples used GraphPad Prism 10.2.3 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0083] Example 1: Discovery and Identification of Oral Streptococcus ZRSOR-1

[0084] 1. Discovery of oral streptococcus ZRSOR-1

[0085] 1.1 Sample Collection In January 2023, during the peak season for respiratory infections, hospitalized CAP patients aged 18 years and older were recruited in Beijing. Clinical samples, including sputum and whole blood, were collected within 24 hours of admission (T1), followed by a second sampling every 2-3 days (T2), with the final sampling performed 1-2 days before discharge (T3). Patients were followed up for 4 months after discharge. If they reported no respiratory symptoms and had not used antibiotics within one month, samples were collected again. Induced sputum from 33 healthy individuals (without respiratory symptoms and no antibiotic use in the past month) served as a control group; the specific collection method was the same as in previous studies. Sputum samples were liquefied using 0.1% dithiothreitol, aliquoted, and stored at -80°C. Whole blood samples were collected in PAXgeneBlood RNA tubes (BD, USA) and stored at -20°C. All patients were enrolled at Zibo Municipal Hospital, and the study was approved by the ethics committees of Zibo Municipal Hospital and China-Japan Friendship Hospital.

[0086] 1.2 Metagenomic Sample Processing and Sequencing

[0087] DNA was extracted from sputum samples and sequenced at 150 bp paired ends using the Illumina HiSeq 4000 platform. Taxonomic analysis of metagenomic data included sequence quality control, removing low-quality sequences, sequences shorter than 100 bp, and host sequences. Species-level classification was analyzed using the Kraken2 v2.1.3 and PlusPF databases, which cover archaea, bacteria, viruses, plasmids, humans, protozoa, and fungi, and were released in December 2022. The association between the microbiome and CAP was performed using a generalized linear mixture model (GLMM) from the lme4 package in R, adjusting for age and antibiotic use as covariates, and participant identity as a random effect.

[0088] 1.3 The proportion of streptococci was increased in CAP patients, and the pathogenic gene modules in these patients were also elevated.

[0089] Of the 252 major bacterial species investigated in this cohort study, *Streptococcus* was the most frequently detected enriched taxa (n=16), followed by *Veillonella* (n=4) and *Prevotella* (n=4). The enrichment of *Streptococcus* in CAP patients persisted into the follow-up period, while the abundance of *Veillonella*, *Campylobacter*, and *Rochetomyces* returned to the levels of healthy controls at a 4-month follow-up after discharge. Figure 1 ). Figure 1This study presents the dynamic changes in the sputum microbiome at the species level in patients with community-acquired pneumonia (CAP). Streptococci were the most predominantly enriched bacterial species in CAP patients compared to healthy controls.

[0090] 3968 microbial genes were identified in the functional genes of microorganisms, of which 287 were more abundant in CAP patients and 961 were more abundant in healthy controls. Figure 2 Two genes associated with microbial DNA repair and stress response, mcsA and mcsB, were elevated by the highest fold in CAP patients. This suggests that bacteria in the respiratory tract of CAP patients are experiencing significant stress, possibly induced by the host immune response or antibiotic treatment. Furthermore, the rfbP gene was also among the top ten genes with significantly increased abundance in CAP patients. This gene is involved in the biosynthesis of O antigen, a crucial component of the bacterial outer membrane, which plays a key role in immune escape, enabling pathogens to persist and cause infection. In contrast, in healthy controls, the abundance of genes associated with 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 shows differentially expressed genes between CAP patients and healthy controls, with the x-axis representing the log2 fold change (Log2FC) and the y-axis representing the -log10 value of the q-value adjusted for FDR. The names of the top 10 genes with the largest fold changes in both CAP patients and healthy controls are labeled.

[0091] 1.4 Integrated 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—*Streptococcus oralis* (SOR) and *Streptococcus mitis*—were directly associated with six host genes in a certain 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 responses, viral processes, and cytokine responses, suggesting that these two streptococci may play a role in regulating the host immune response during viral infection. Figure 3 ). Figure 3Gene-microbe interactions in CAP patients are shown, where a) is a network visualization of significant associations between S. oralis and S. mitis and genes in the red module, and the remaining six bacteria in microbial cluster 5 form an independent network; b) is a gene enrichment analysis of biological processes, cellular components and molecular functions of genes associated with S. oralis and S. mitis in the red module.

[0092] 2. Isolation and Identification of Strain ZRSOR-5

[0093] Sputum samples were spread onto Columbia blood agar using a sterile inoculation loop after liquefaction and incubated at 37°C for 24-48 hours in a 5% CO2 incubator. Colony morphology was observed; streptococci typically appeared as small, grayish-white, translucent, round colonies with regular edges, accompanied by α- or β-hemolysis. Suspected streptococcal colonies were selected based on colony morphology and purified (see [link to article]). Figure 8 ).

[0094] Bacterial colonies were analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purified colonies were placed on a MALDI target plate, and the mass spectrometry results were compared with database analyses. The identification results were as follows: Figure 9 .

[0095] DNA was extracted from selected ZRSOR-5 colonies using a bacterial DNA extraction kit, and then subjected to next-generation sequencing for comparison. The gene sequences to be identified were compared with the NCBI database using a BLAST tool. Based on the molecular biology results, the Latin name of the strain was determined. Streptococcus oralis The strain was identified as oral streptococci. ZRSOR-5 was deposited on October 25, 2024, at the Guangdong Microbial Culture Collection Center (GDMCC, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070), with accession number GDMCC No: 65352, hereinafter referred to as ZRSOR-5.

[0096] Example 2: Application of ZRSOR-5 formulation in combating viral pneumonia

[0097] 1. Preparation of oral streptococcal ZRSOR-5 formulation

[0098] ZRSOR-5 cells were inoculated onto Columbia blood agar medium (Thermofisher, PB0123A) and cultured at 37°C for 24 h in a 5% CO2 incubator. The cultured ZRSOR-5 cells were collected in 1 ml of physiological saline using a cell scraper, centrifuged at 1000g for 5 min, and the supernatant was discarded. The precipitate was resuspended in 1 ml of physiological saline, centrifuged again to remove the supernatant, and the precipitate was further dissolved in 1 ml of 20% glycerol saline to prepare a homogeneous suspension with a concentration of 3.3 × 10⁻⁶. 7 CFU / mL, store at -80 degrees Celsius for later use.

[0099] 2. Divide into four groups for respiratory tract bacterial intervention.

[0100] All experimental mice were 8-9 week old female C57BL / 6 mice. Four groups of mice were selected, with 15 mice in each group. Two groups were given 30 μL of ZRSOR-5 bacterial suspension containing 1 × 10⁻⁶ bacteria. 5 Mice were inoculated with ZRSOR-5 bacteria via intranasal drip every two days for a total of four times. Two other groups of mice served as controls, receiving only physiological saline. Both experimental and control mice were cultured under conditions of 23±2℃, 50-55% relative humidity, simulated day-night cycle with 12-hour light-dark cycles, and each mouse was fed 5-8g of feed daily. In the following text, S. oralis (SOR) pre-inoculation or pretreatment refers to administering 30ul of ZRSOR-5 bacterial suspension to mice, containing 1×10⁻⁶ bacteria. 5 One ZRSOR-5 bacterium.

[0101] The mice were given 10 doses of antibiotics via nasal administration the day after their last nasal administration (the control group received saline). One group from each of the ZRSOR-5 and control groups was then given 10 doses. 3 CFU was administered A / Puerto Rico / 8 / 1934 (H1N1) influenza virus, while another group received PBS. Figure 4 This diagram illustrates the design for mouse experiments, in which mice were pre-inoculated with S. oralis (SOR) four times via intranasal route, followed by IAV infection. Lung and bronchoalveolar lavage fluid (BALF) samples were collected on days 2 and 6 post-infection.

[0102] Survival rate and weight changes in mice were observed over 10 days. It was found that the weight of mice in the two groups not inoculated with influenza virus did not decrease. The weight of mice in both groups administered influenza virus decreased, but the percentage of weight loss in mice pre-inoculated intranasally with ZRSOR-5 was significantly lower than that in mice pre-inoculated intranasally with saline. Of the 15 mice pre-inoculated intranasally with ZRSOR-5, none died, while 5 of the 15 mice pre-inoculated intranasally with saline died, indicating that ZRSOR-5 has a preventive effect against lethal influenza pneumonia in mice. Figure 5 ).Figure 5 The results showed that ZRSOR-5 pretreatment significantly reduced IAV-induced weight loss and mortality in mice, where SOR was ZRSOR-5 pretreatment; IAV was influenza virus inoculation; and mock was saline control and PBS challenge control.

[0103] also, Figure 6 In Figure c, the viral load (PFU) in the lungs of four groups of mice 48 hours after infection with influenza virus was shown. Compared with mice that had not undergone bacterial pretreatment, the number of infectious viral particles in the lungs was also reduced within 48 hours after infection. The method for detecting lung viral load (PFU) involved serially diluting the sample and infecting MDCK cells. After virus adsorption, a layer of melted semi-solid nutrient agar was applied to allow limited viral diffusion in the monolayer cell culture. Once white spots appeared, neutral red staining was performed. 1 mL of 20-fold diluted neutral red solution (stock solution concentration 3.3 mg / mL) was added to each well and incubated at 37°C for 4 hours or overnight. After removing the stain, the empty spots were counted. The titer was calculated by statistically analyzing the empty spots. The average viral load in mice treated with SOR was 15*10⁻⁶. 3 pfu / ml, while the average viral load in the control group mice was 39*10 3 The pfu / ml level showed a significant difference between the two groups.

[0104] Figure 6 In the figure, d represents the levels of inflammatory markers IL-6 and TNF-α, and the total protein concentration in the alveolar pulmonary artery lavage fluid (BALF) of mice on day 6 post-infection with influenza virus. Compared with mice that had not undergone bacterial pretreatment, SOR pretreatment also led to a decrease in the levels of inflammatory markers IL-6 and TNF-α in BALF on day 6 post-infection, and at this time, the SOR group mice experienced the largest weight loss after influenza virus challenge. SOR also alleviated damage to the alveolar-capillary barrier, as evidenced by a lower total protein concentration in the BALF. The total protein concentration was detected using a Thermofisher kit (product number A55860), reflecting improved permeability of the alveolar-capillary membrane. IL-6 (Thermofisher, BMS603-2) and TNF-α (Thermofisher, BMS607-3) were detected using an ELISA kit according to the instructions. First, the samples were added to a 96-well ELISA plate containing specific antibodies to capture cytokines. After adding enzyme-labeled secondary antibody, color development was performed, and the absorbance values ​​were read using an ELISA reader. The concentration of cytokines in the samples was calculated using a standard curve.

[0105] Figure 6Image e shows stained lung tissue sections from infected (IAV) and uninfected (Mock) mice on day 6, pretreated with *S. oralis* (SOR) or saline. Mouse lung tissue was immediately fixed in 10% buffered formalin for at least one week after collection. The fixed tissue underwent routine processing, was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological analysis. Pathological analysis of mouse lungs collected on day 6 post-infection showed that SOR pretreatment reduced inflammatory cell infiltration and lung damage.

[0106] To investigate the protective mechanism of SOR in preventing IAV infection, transcriptome analysis was performed on lung tissues from mice pretreated with SOR before IAV infection and untreated mice on days 2 and 6 post-infection. Transcriptome sequencing data were quality filtered using Fastpv0.20.0 and aligned to the human genome GRCh38 using Hisat2 v2.1.0. Gene expression matrices were then obtained using FeatureCounts v4.8.1. Differentially expressed genes were identified using EdgeR, followed by gene enrichment analysis using clusterProfiler v4.8.1. Enrichment scores for each pathway were calculated for each sample using GSVA v1.48.0. Figure 7 The figures (f) show the differentially expressed genes and pathways in the lungs of S. oralis (SOR) pretreated mice compared to untreated mice on day 2 post-infection. SOR pre-vaccination significantly increased the expression of multiple immunomodulatory genes on day 2 post-infection, including IL17F, LY6I, and C1RB. These genes enhanced the activation of pathways related to bacterial infection, viral protein-cytokine interactions, cytokine and chemokine signaling, and complement response. These upregulated pathways play a crucial role in host resistance to respiratory infections; their high activation in the early stages of IAV infection inhibited viral spread and improved mouse prognosis. Figure 7 In the figure, f represents the differentially expressed genes and pathways in the lungs of S. oralis (SOR) pretreated mice compared to untreated mice on day 6 post-infection. By day 6, when mice began to recover, these pro-inflammatory genes showed no significant difference 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.

[0107] These findings suggest that pre-vaccination with ZRSOR-5 formulations promotes timely activation of the innate immune response, effectively limiting IAV infection, and that these pathways are rapidly resolved during the recovery phase to prevent immune-mediated pathological damage.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of streptococci or compositions containing said streptococci in the preparation of pharmaceutical products for the prevention of influenza viral pneumonia caused by A / Puerto Rico / 8 / 1934 (H1N1) influenza virus infection and / or for the restriction of A / Puerto Rico / 8 / 1934 (H1N1) influenza virus infection; The streptococcus in question is oral streptococcus ZRSOR-5, which has the accession number GDMCC No: 65352 at the Guangdong Provincial Center for Microbial Culture Collection.

2. The application according to claim 1, wherein the drug is administered via intranasal and / or intratracheal inhalation.

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