A lactobacillus salivarius and uses thereof

Abstract

The present application relates to the technical field of microorganism, and in particular to a saliva combined lactobacillus and its use. In order to develop a lactobacillus with the functions of anti-HPV infection, bacteriostasis and in-vivo immune activation, the present application provides a saliva combined lactobacillus KY-N2-E11. The strain has the effects of inhibiting the expression of E6 / E7 oncogenes of HPV16 and HPV18 positive cells, promoting immune activation and inhibiting pathogenic bacteria in a broad spectrum. The strain of the present application can be used for HPV auxiliary treatment and vaginal microecological regulation, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of Lactobacillus salivarius and its uses. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide. According to the World Health Organization (WHO) 2022 GLOBOCAN data, there are approximately 660,000 new cases and 350,000 deaths globally each year. In my country, there are approximately 150,000 new cases and nearly 60,000 deaths annually, indicating a still significant disease burden. Persistent infection with high-risk human papillomavirus (hr-HPV) is a necessary prerequisite for the development of cervical cancer, with HPV types 16 and 18 collectively responsible for approximately 70% of cervical cancer cases globally.

[0003] Currently, anti-HPV drugs mainly include immune enhancers (such as interferon) and antimetabolites (such as 5-fluorouracil). However, existing studies on adjuvant treatment of hr-HPV infection with interferon α2b are mostly small-sample, non-controlled trials, with reported HPV clearance rates varying considerably (approximately 30%-60%), and the confounding effect of spontaneous clearance is difficult to rule out. Currently, mainstream guidelines both domestically and internationally do not include it as a standard treatment, indicating a lack of sufficient evidence to support its efficacy. Furthermore, 5-fluorouracil has drawbacks such as severe local reactions, long treatment duration, and inability to prevent new infections or transmission. Therefore, there is an urgent need to develop safe and effective anti-HPV drugs.

[0004] The core molecular basis of high-risk HPV (hr-HPV) pathogenesis lies in the persistent expression of viral oncoproteins E6 and E7. E6 and E7 drive abnormal cell cycle proliferation by degrading tumor suppressor proteins such as p53 and pRb, which is the fundamental cause of carcinogenesis. The prerequisite for the long-term effect of E6 / E7 is viral escape from the host immune system and its clearance. Studies have shown that hr-HPV mainly achieves immune escape and persistent infection through the following mechanisms: on the one hand, the HPV life cycle does not cause cell lysis and lacks danger signals, leading to impaired maturation of dendritic cells (DCs); on the other hand, the E6 / E7 proteins themselves have immunomodulatory functions, directly inhibiting the type I interferon signaling pathway and reshaping the local cytokine microenvironment, resulting in a lack of virus-specific T-cell immunity.

[0005] Lactobacillus salivans ( Lactobacillus salivarius Lactobacillus salivarius is a common Gram-positive, facultative anaerobic probiotic widely found in the human gut and oral cavity. Chinese patent application CN121406499A discloses a strain called *Lactobacillus salivarius* ECON-1, which exhibits good antibacterial activity and the ability to stimulate dendritic cell (DC) maturation / activation. However, it does not inhibit HPV oncogenes, limiting its effectiveness in treating cervical cancer. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a *Lactobacillus saliva-associated* and its uses.

[0007] The novel *Lactobacillus salivarius* provided in this invention is *Lactobacillus salivarius* with accession number CCTCC NO: M 2026753, deposited by the China Center for Type Culture Collection. Ligilactobacillus salivarius KY-N2-E11.

[0008] Lactobacillus saliva-associated KY-N2-E11 ( Ligilactobacillus salivarius The strain KY-N2-E11 was deposited at the China Center for Type Culture Collection (CCTCC) on April 20, 2026, with accession number CCTCC NO: M2026753, located in Wuhan, China.

[0009] The present invention also provides a probiotic comprising the aforementioned live Lactobacillus salivarius KY-N2-E11. Preferably, the live count of the Lactobacillus salivarius is 1 × 10⁻⁶. 5 ~1×10 12 CFU / mL.

[0010] The present invention also provides a vaginal pharmaceutical composition, which is a preparation made of Lactobacillus salivarius KY-N2-E11 as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0011] This invention also provides the use of *Lactobacillus salivarius* KY-N2-E11 in the preparation of a medicament for treating cervical intraepithelial neoplasia or cervical cancer. The medicament is used to inhibit the expression of HPV viral oncogenic genes.

[0012] This invention also provides the use of *Lactobacillus salivarius* KY-N2-E11 in the preparation of immune enhancers. The immune enhancer is a substance that stimulates dendritic cell maturation / activation or systemically activates a Th1-type immune response.

[0013] This invention also provides the use of *Lactobacillus salivarius* KY-N2-E11 in the preparation of pharmaceuticals for regulating the vaginal microecological environment. Those skilled in the art will therefore recognize its potential for treating aerobic vaginitis (AV), bacterial vaginosis (BV), mixed vaginitis, etc.

[0014] This invention also provides the use of *Lactobacillus salivarius* KY-N2-E11 in the preparation of products that inhibit *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Shigella dysenteriae*, *Salmonella paratyphi B*, *Streptococcus agalactiae*, *Gardnerella vaginalis*, and / or *Atopobacterium vaginalis*. This product can be used as a pharmaceutical, food, medical device, or hygiene product, etc.

[0015] The present invention, *Lactobacillus saliva-associated* KY-N2-E11, has the following beneficial effects: (1) Inhibition of HPV oncogenes: The Lactobacillus salivarius KY-N2-E11 provided by this invention can significantly inhibit the expression of high-risk HPV E6 / E7 oncogenes. E6 / E7 is the core molecular basis of HPV pathogenesis. Its expression inhibition can block the degradation of p53 and pRb, thereby reversing abnormal cell cycle proliferation and curbing the carcinogenesis process from the source.

[0016] (2) In vivo immune activation: This invention demonstrates for the first time in a live model (zebrafish) that saliva-associated lactobacillus can systematically activate the Th1 immune response and significantly promote T cell proliferation and IL-12 and IFN-γ secretion. This discovery provides a breakthrough in the study of probiotic anti-HPV mechanisms, overcoming the limitation of traditional lactobacillus relying solely on in vitro data to infer immune function. This invention also demonstrates that saliva-associated lactobacillus KY-N2-E11 can effectively promote dendritic cell (DC) maturation / activation and reverse DC dysfunction caused by HPV infection; (3) Effective antibacterial activity: The KY-N2-E11 of Lactobacillus saliva provided by the present invention can effectively inhibit a variety of vaginal pathogens and help restore a healthy vaginal microecology with Lactobacillus as the dominant flora.

[0017] (4) Good biocompatibility: The Lactobacillus salivarius KY-N2-E11 of the present invention is derived from healthy human body, has good biocompatibility, no cytotoxicity, and is not prone to drug resistance.

[0018] The present invention, *Lactobacillus KY-N2-E11* in saliva, exhibits the following synergistic effects: First, it can inhibit the expression of high-risk HPV VE6 / E7 oncogenes, blocking their abnormal driving effect on the cell cycle; Second, it effectively promotes dendritic cell (DC) maturation / activation, reverses DC dysfunction caused by HPV infection, systematically activates the Th1 immune response, significantly promotes T cell proliferation and IL-12 and IFN-γ secretion, and enhances the killing efficacy of cytotoxic T lymphocytes (CTLs) against HPV-positive target cells; Third, it has a broad-spectrum inhibitory effect on pathogens, simultaneously inhibiting aerobic pathogens (such as *Staphylococcus aureus*, *Streptococcus agalactiae*, and *Escherichia coli*) and anaerobic pathogens (*Gardnerella vaginalis* and *Atopobacterium vaginalis*), effectively restoring vaginal flora balance. This strain provides a new option for developing safe and effective adjuvant HPV therapy and vaginal microecological regulation agents.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 Gram staining microscopic image of Lactobacillus salivarius KY-N2-E11. Detailed Implementation

[0022] Example 1: Isolation, identification, and safety assessment of *Lactobacillus saliva-associated* strains. Gram staining and microscopic examination of vaginal secretions from healthy female volunteers of childbearing age were performed, and Nugent scores were assessed. Healthy female volunteers with a Nugent score <3 were selected. Vaginal swabs from healthy volunteers were placed in MRS acidic liquid culture medium and incubated overnight at 37 °C for bacterial expansion. The enrichment broth was then serially diluted 10-fold to 10⁻⁶. -5 All dilution gradients were plated on 2% calcium carbonate-0.8% MRS agar and incubated at 37 °C for 36–48 h. Single colonies with distinct clear zones were picked from the incubated calcium carbonate-MRS agar to isolate single bacteria. The isolated single bacteria were cultured to the late logarithmic growth stage, and DNA was extracted and sequenced using a high-throughput sequencer. The predicted gene sequences were compared with the non-redundant protein database (NCBI-nr) using Blast software. Furthermore, the average nucleotide identity (ANI) value was obtained using FastANI (Version 1.33) software for species identification. The results of strain identification are shown in Table 1.

[0023] Table 1. Reference genomes of strains and *Lactobacillus salivarius* ( Ligilactobacillus salivarius FastANI analysis results of DSM20555 Table 1 shows that the average nucleotide identity (ANI) ratio reached 97.86%, confirming that the isolated strain was *Lactobacillus salivarius*, and named *Lactobacillus salivarius* KY-N2-E11. Ligilactobacillus salivarius Gram staining microscopic examination results of *Lactobacillus salivarius* KY-N2-E11 (KY-N2-E11). Figure 1 As shown. Lactobacillus salivarius KY-N2-E11 ( Ligilactobacillus salivarius The strain KY-N2-E11 was deposited at the China Center for Type Culture Collection on April 20, 2026, with accession number CCTCC NO: M 2026753, located in Wuhan, China.

[0024] To ensure the safety of medication use, a safety assessment of the isolated strains is necessary. This includes determining the presence of potential pathogenic factors and their relative risks; identifying antibiotic resistance genes to assess the potential impact of the strains in antibiotic-using environments, etc. The amino acid sequence of the predicted gene was compared with the Virulence Factors Database (VFDB) using Diamond (Version 2.1.6) software. A set of strict thresholds was used (sequence match ≥80%, sequence length coverage ≥60%, e-value ≤10). -5 The results showed that the Lactobacillus salivarius KY-N2-E11 strain did not contain any known pathogenic genes or key genes related to toxin synthesis, nor did it carry any drug resistance genes directly related to the drug resistance phenotype. Therefore, it can be determined that the strain used in this invention has no potential safety issues.

[0025] The beneficial effects of the present invention are illustrated below through experimental examples.

[0026] Experimental Example 1: Antibacterial activity of Lactobacillus KY-N2-E11 in salivary samples Using the isolation and purification method described in Example 1, Lactobacillus delbrueckii (hereinafter collectively referred to as DJS) isolated from Dingjunsheng, a commercially available microecological preparation of the same type, was used as the positive control strain of Example 1.

[0027] The classic bacterial cake test was used to evaluate the ability of the strain to inhibit eight common vaginal pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella paratyphi B, Streptococcus agalactiae, Gardnerella vaginalis, and Atobacillus vaginalis).

[0028] Experimental results: The results are shown in Table 2. The antibacterial ability of the present invention *Lactobacillus salivarius* KY-N2-E11 against 8 pathogenic bacteria was stronger than that of the control bacteria DJS, and it had a broad-spectrum inhibitory effect on common vaginal pathogens. Furthermore, the inhibition zone diameter of the present invention *Lactobacillus salivarius* KY-N2-E11 against *Pseudomonas aeruginosa* was significantly higher than the reported maximum value (the inhibition zone diameter of *Lactobacillus salivarius* S-5-6 against *Pseudomonas aeruginosa* was 20.20 mm; Li Jianzhou, Xiao Yaoting, Li Hongxiao, et al. Screening and mechanism study of lactic acid bacteria antagonizing *Pseudomonas aeruginosa* [J]. Food and Machinery, 2021, 37(10):6-12.). The inhibition zone diameter of *Lactobacillus salivarius* KY-N2-E11 against *Shigella dysenteriae* was significantly higher than the reported maximum (the inhibition zone diameter of *Lactobacillus salivarius* S54 against *Shigella dysenteriae* was 20.60 mm, Luo Xiyan, Ling Xin, Wang Qimeng, et al. Study on the probiotic characteristics of *Lactobacillus salivarius* S54 strain based on whole-genome analysis and phenotypic evidence [J / OL]. Food Industry Technology. https: / / doi.org / 10.13386 / j.issn1002-0306.2025070328.). The results of this experiment indicate that *Lactobacillus salivarius* KY-N2-E11 of this invention has a broad-spectrum inhibitory effect on common vaginal pathogens, and compared with other *Lactobacillus salivarius* strains, the inhibitory effect of the KY-N2-E11 strain of this invention on *Pseudomonas aeruginosa* and *Shigella dysenteriae* is significantly enhanced.

[0029] Table 2. Diameter of the inhibition zone of the present invention's *Lactobacillus saliva-associated* KY-N2-E11 against 8 pathogenic bacteria. Note: The size of the mushroom cake is 9mm. Only when the diameter of the inhibition zone is ≥9mm can it be considered to have an antibacterial effect.

[0030] Experimental Example 2: Inhibitory effect of saliva combined with Lactobacillus KY-N2-E11 on HPV oncogenes The E6 and E7 genes of HPV are key oncogenes leading to cervical cancer, playing a major role in the progression of vaginal / cervical intraepithelial neoplasia (CIN) and cervical cancer. Therefore, this invention uses qPCR to detect the effect of saliva combined with Lactobacillus KY-N2-E11 on the E6 and E7 genes.

[0031] Experimental methods: Caski (HPV16 positive) cells were cultured in 1640 medium containing 10% FBS and 1% penicillin antibiotics. Hela (HPV18 positive) cells were cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics. ECT1 / E6E7 (HPV16 positive) cells were cultured in Gibco Keratinocyte Serum Free Medium (K-SFM). SW756 (HPV18 positive) cells were cultured in L-15 medium containing 10% FBS and 1% penicillin antibiotics. 1 10 4 Cells were seeded per well in 96-well plates and allowed to adhere overnight.

[0032] The original culture of *Lactobacillus saliva-associated* strain KY-N2-E11 was cultured to the late logarithmic growth stage. After centrifugation and washing, the culture was resuspended in the appropriate cell culture medium and the concentration was adjusted to 1E+7 CFU / mL. 0.1 mL of the resuspended culture was added to the overnight adherent cells to make the MOI=100. After culturing for 24 h, Takara's CellAmp was used. TM The Direct RNA Prep Kit for RT-PCR extracts mRNA, then reverse transcribes it to obtain cDNA, which is then used for real-time quantitative PCR detection.

[0033] Grouping information: Blank control group, saliva-combined lactobacillus KY-N2-E11 group Using 2^ -△△Ct The relative changes in E6 / E7 gene mRNA expression compared to β-actin in each cell were calculated. The formula for calculating the relative expression rate of HPV oncogenes is: = 2^ -[（Ct目的基因，实验组-Ct β-actin，实验组）-（Ct目的基因，空白对照组-Ct β-actin，空白对照组）] .

[0034] Experimental results: The results showed that the Lactobacillus saliva-associated strain KY-N2-E11 reduced the mRNA expression levels of E6 and E7 genes in HeLa cells, Caski cells, ECT1 / E6E7 and SW756 cells compared with the blank control group (Table 3).

[0035] Table 3. Expression rates of HPV16 and HPV18 positive cell oncogenes relative to the blank control group by the salivary-associated Lactobacillus KY-N2-E11 of the present invention. Experimental Example 3: The ability of saliva combined with Lactobacillus KY-N2-E11 to stimulate dendritic cell (DC) maturation / activation Experimental methods: Thrp1 cells (ATCC) were induced into immature dendritic cells (iDCs) using cytokines GM-CSF and IL-4. iDCs were co-incubated with saliva and Lactobacillus KY-N2-E11 to stimulate iDC maturation. The expression level of the cell surface maturation marker CD86 was detected by flow cytometry, and the IL-12 level in the cell supernatant was detected by an IL-12 ELISA kit (Lianke Biotechnology).

[0036] Preparation of induction medium: Add 100 µg / mL GM-CSF and 100 µg / mL IL-4 to complete cell culture medium (RPMI 1640 + 10% inactivated complement FBS) (Gibco) and adjust to a final concentration of 100 ng / mL for both.

[0037] Thp1 cells were resuspended in induction medium (cell concentration 1.0 × 10⁶). 6 Cells (number per mL) were placed in a T25 flask for centralized induction, the medium was changed on day 3, and cell counting was performed on day 5. 2.1 × 10⁻⁶ cells / mL were then transferred to the flask. 5 Cells were seeded at 0.7 ml / well in 12-well plates and treated with either complete cell culture medium containing 20 ng / mL TNF-α (positive control group) or TNF-α induction medium plus 3.5 µL of saliva combined with live Lactobacillus KY-N2-E11 (MOI=1, final concentration 3E+5 CFU / mL) (experimental group) for 48 h. The negative control group consisted of 2.1 × 10⁻⁶ cells / well. 5 Cells were collected at 0.7 mL / well, with no other treatment. After 48 h of drug treatment, the cells and supernatant were collected together into a deep-well plate and centrifuged at 400 g for 5 min. The expression level of CD86 on the cell surface was detected by flow cytometry, and the IL-12 level in the cell supernatant was detected by the IL-12 ELISA kit according to the instructions.

[0038] Experimental Results: As shown in Tables 4 and 5, the saliva-based combination of Lactobacillus KY-N2-E11 increased the expression level of CD86, a maturation marker of dendritic cells (DCs), to 2.12 times that of the positive control group and 9.44 times that of the negative control group. Simultaneously, it also significantly increased the IL-12 level secreted by DCs, reaching 73.03 times that of the positive control group. The ability of *Lactobacillus salivarius* KY-N2-E11 to stimulate DC maturation / activation was significantly better than that of *Lactobacillus salivarius* W24 reported in the literature (the CD86 level in the *Lactobacillus salivarius* group was less than twice that of the negative control group (IDC group), and the IL-12 level in the *Lactobacillus salivarius* group was less than three times that of the positive control group (MF group, LPS group). Source: LEM Niers, MO Hoekstra, HM Timmerman, NO van Uden, PMA de Graaf, HH Smits, JLL Kimpen, GT Rijkers, Selection of probioticbacteria for prevention of allergic diseases: immunomodulation of neonataldendritic cells, Clinical and Experimental Immunology, Volume 149, Issue 2, August 2007, Pages 344–352, doi: 10.1111 / j.1365-2249.2007.03421.x). The salis-associated lactobacillus KY-N2-E11 of this invention exhibits excellent immunomodulatory capabilities and can enhance the host's resistance.

[0039] Table 4. Results of flow cytometry analysis of CD86 expression levels on cell surface Table 5. Results of IL-12 level detection in cell supernatant using the IL-12 ELISA kit. Experimental Example 4: In vivo model confirms that saliva combined with Lactobacillus KY-N2-E11 activates the immune system in zebrafish. A zebrafish immunodeficiency model was established by intravenous injection of vinorelbine tartrate. High-dose vinorelbine tartrate significantly suppressed bone marrow, leading to a decrease in platelet, erythrocyte, and leukocyte counts, ultimately resulting in immunodeficiency. Wild-type AB strain zebrafish with a 3dpf growth factor were selected and injected with vinorelbine tartrate according to the method reported in the literature to construct a zebrafish immunodeficiency model (Wang Tao, Dai Mingzhu, Li Yanchuan, et al. Study on the immune-enhancing effect and preliminary mechanism of compound traditional Chinese medicine extract [J]. Chinese Journal of Experimental Animals, 2022, 30(2): 198-207.). Then, bactericidal drugs (2×10) were administered in water. 8 The positive control group received Bailin capsules (15.0 μg / mL), while a model control group (injected only with vinorelbine tartrate) and a normal control group (not injected with vinorelbine tartrate) were also set up. Each well had a volume of 3 mL, and the treatment lasted for 48 h. The effects of the drug on the zebrafish immune system were determined by detecting the number of T cells (fluorescence intensity) and changes in immune and inflammatory factors (relative RNA expression levels of IL-12 and IFN-γ genes).

[0040] Experimental results: Table 6 shows that KY-N2-E11 can significantly improve T cell reduction and upregulate the relative expression levels of IL-12 and IFN-γ genes.

[0041] Table 6 Results of Zebrafish Drug Efficacy Model Detection In summary, this invention has achieved several key breakthroughs. First, it was discovered that the *Lactobacillus salivarius* KY-N2-E11 strain can inhibit the expression of the E6 / E7 oncogenes in high-risk HPV types (HPV16 and HPV18), blocking their abnormal driving effect on the cell cycle. Second, it effectively promotes dendritic cell (DC) maturation / activation, reversing DC dysfunction caused by HPV infection. Third, it exhibits broad-spectrum inhibitory effects against pathogens, simultaneously inhibiting both aerobic pathogens (such as *Staphylococcus aureus*, *Streptococcus agalactiae*, and *Escherichia coli*) and anaerobic pathogens (*Gardnerella vaginalis* and *Atopobacter vaginalis*), effectively restoring vaginal flora balance. This invention provides a new material basis for developing safe and effective adjuvant HPV treatments and vaginal microecological regulation agents.

Claims

1. A type of *Lactobacillus salivarius*, characterized in that: It is *Lactobacillus salivarius*, with accession number CCTCC NO: M 2026753, preserved by the China Center for Type Culture Collection. Ligilactobacillus salivarius KY-N2-E11.

2. A probiotic, characterized in that: It contains live Lactobacillus salivarius as described in claim 1.

3. The probiotic according to claim 2, characterized in that: The viable count of the *Lactobacillus saliva-associated* was 1 × 10⁻⁶. 5 ~1×10 12 CFU / mL.

4. A vaginal pharmaceutical composition, characterized in that: It is a formulation prepared using Lactobacillus salivarifolius as the active ingredient as described in claim 1, plus pharmaceutically acceptable excipients or auxiliary ingredients.

5. Use of the *Lactobacillus saliva-associated* as described in claim 1 in the preparation of a medicament for treating cervical intraepithelial neoplasia or cervical cancer.

6. The use according to claim 5, characterized in that: The drug is used to inhibit the expression of HPV virus carcinogenic genes.

7. The use of the *Lactobacillus saliva-associated* as described in claim 1 in the preparation of an immune enhancer.

8. The use according to claim 7, characterized in that: The immune enhancer is a substance that stimulates the maturation / activation of dendritic cells or systemically activates the Th1 immune response.

9. The use of the *Lactobacillus saliva-associated* as described in claim 1 in the preparation of a medicine for regulating the vaginal microecological environment.

10. The use of the *Lactobacillus saliva-associated* of claim 1 in the preparation of a product having the function of inhibiting *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Shigella dysenteriae*, *Salmonella paratyphi B*, *Streptococcus agalactiae*, *Gardnerella vaginalis* and / or *Atopobacter vaginalis*.

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