Lactobacillus reuteri and uses thereof

Abstract

This invention relates to the field of microbial technology, specifically to a type of *Lactobacillus reuteri* and its uses. The *Lactobacillus reuteri* KY-N5-E2 of this invention inhibits the expression of E6 and E7 oncogenes in HPV16 and HPV18 positive cells, and also promotes dendritic cell (DC) maturation / activation. It works synergistically through a dual mechanism of "awakening the host immune system" and "inhibiting oncogenes at their source," providing a novel strategy for the adjuvant treatment of HPV infection and related lesions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of *Lactobacillus reuteri* 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. 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 accounting for approximately 70% of cervical cancer cases globally.

[0003] The core molecular basis of high-risk HPV (hr-HPV) pathogenesis lies in the persistent expression of viral oncoproteins E6 and E7, which drive abnormal cell cycle proliferation by degrading tumor suppressor proteins such as p53 and pRb. More importantly, hr-HPV achieves immune evasion and establishes persistent infection by inhibiting the functional maturation of dendritic cells (DCs) and remodeling the local cytokine microenvironment. Therefore, reversing HPV-mediated local immunosuppression is key to developing novel therapeutic strategies. Existing treatments such as surgery or interferon are insufficient to effectively address this immunodeficiency problem.

[0004] Recent studies have found that oral or topical application of specific probiotic strains can reduce HPV titers in patients and prevent virus-related cancers, making them promising new anti-HPV agents. (CHA MK, et al., Antiviral activity of) Bifidobacterium adolescentis SPM1005-A on human papillomavirus type 16[J]. BMC Medicine, 2012, 10: 72. et al. found that Bifidobacterium adolescentis SPM1005-A can inhibit the expression of E6 and E7 oncogenes of HPV16 in SiHa cells; ABDOLALIPOUR E, et al., Evaluation of the antitumor immune responses of probiotic Bifidobacterium bifidumIn a human papillomavirus-induced tumor model[J]. Microbial Pathogenesis, 2020, 145: 104-207. This article discloses that intravenous or oral administration of Bifidobacterium bifidum can effectively induce anti-tumor immune responses in mice and inhibit the growth of tumors expressing HPV16; intravenous injection of the probiotic Bifidobacterium bifidum into tumor-bearing mice can activate the production of tumor-specific IL-12 and IFN-γ, promote lymphocyte proliferation, and enhance CD8+. + The cell-dissolving reaction controls and eradicates tumor growth. These observations suggest that probiotics can achieve anti-cancer effects by activating and regulating the immune system.

[0005] Lactobacillus reuteri ( Limosilactobacillus reuteri ), Lactobacillus genus ( Limosilactobacillus Lactobacillus reuteri is a type of lactic acid bacteria naturally found in the gastrointestinal tract of humans and animals. It has a strong ability to adhere to the intestinal mucosa, can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and can also improve human function and enhance immunity. No reports have been found regarding the use of *Lactobacillus reuteri* for HPV treatment. Summary of the Invention

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

[0007] This invention provides a *Lactobacillus reuteri* strain, which is a *Lactobacillus reuteri* strain preserved by the China Center for Type Culture Collection (CCTCC NO: M 2026751). Limosilactobacillus reuteri KY-N5-E2.

[0008] This invention relates to *Lactobacillus reuteri* KY-N5-E2 ( Limosilactobacillus reuteri KY-N5-E2 was deposited at the China Center for Type Culture Collection (CCTCC) on April 20, 2026, with accession number CCTCC NO: M2026751, located in Wuhan, China.

[0009] The present invention also provides a probiotic comprising live bacteria of the aforementioned *Lactobacillus reuteri* KY-N5-E2. Preferably, the live count of the *Lactobacillus reuteri* is 1 × 10⁻⁶. 5 ~1×10 12 CFU / mL.

[0010] This invention also provides the use of *Lactobacillus reuteri* KY-N5-E2 in the preparation of medicaments for the prevention and / or adjunctive treatment of high-risk HPV infection. The HPV may be HPV16 and / or HPV18.

[0011] The present invention also provides the use of *Lactobacillus reuteri* KY-N5-E2 in the preparation of medicaments for the prevention and / or adjuvant treatment of cervical intraepithelial neoplasia or cervical cancer.

[0012] Preferably, the drug is a drug that inhibits the expression of HPV viral oncogenes and / or stimulates the maturation / activation of dendritic cells.

[0013] This invention also provides the use of *Lactobacillus reuteri* KY-N5-E2 in the preparation of immunomodulators.

[0014] The present invention also provides a pharmaceutical composition for the prevention and / or adjunctive treatment of high-risk HPV infection, which is a formulation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to Lactobacillus reuteri KY-N5-E2, which is also provided by the present invention, as the active ingredient.

[0015] Preferably, the preparation is a topical preparation.

[0016] The Lactobacillus reuteri KY-N5-E2 of this invention has the following beneficial effects: (1) Targeted inhibition of HPV oncogenes: The strain of the present invention can significantly inhibit the expression of high-risk HPV E6 / E7 oncogenes, block their degradation of p53 and pRb at the molecular level, thereby reversing abnormal cell cycle proliferation and inhibiting the carcinogenesis process.

[0017] (2) Unique immunomodulatory function: The *Lactobacillus reuteri* KY-N5-E2 provided by this invention can significantly promote the maturation and activation of dendritic cells (DCs), effectively reversing the immune recognition barrier caused by HPV virus through the "lack of danger signal" mechanism. This function lays the foundation for rebuilding Th1 antiviral immunity by precisely regulating the host immune response, and fundamentally solves the immunodeficiency problem of persistent HPV infection.

[0018] (3) Innovative dual mechanism of “gene silencing + immune awakening”: The strain of this invention does not rely on directly killing infected cells, but works synergistically through the dual mechanism of “awakening the host immune system” and “shutting down oncogenes from the source”. This unique mode of action provides a new strategy for adjuvant treatment of HPV infection and related lesions.

[0019] (4) Good biocompatibility: The Lactobacillus reuteri KY-N5-E2 of the present invention is derived from healthy human body, has good biocompatibility, no cytotoxicity, is not prone to drug resistance, can colonize for a long time, and reduces the risk of recurrence.

[0020] The *Lactobacillus reuteri* KY-N5-E2 strain of this invention exhibits the following synergistic effects: First, *Lactobacillus reuteri* KY-N5-E2 can inhibit the expression of E6 and E7 oncogenes in HPV16 and HPV18 positive cells, blocking their abnormal driving effect on the cell cycle; Second, this strain promotes dendritic cell (DC) maturation / activation, reversing the dendritic cell functional defects caused by HPV infection. The *Lactobacillus reuteri* KY-N5-E2 strain of this invention provides a new option for developing safe and effective adjuvant HPV therapy.

[0021] 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.

[0022] 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

[0023] Figure 1 Gram staining microscopic image of *Lactobacillus reuteri* KY-N5-E2. Detailed Implementation

[0024] Example 1: Isolation, identification, and safety assessment of *Lactobacillus reuteri* 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.

[0025] Table 1. Strain and reference genome of *Lactobacillus reuteri* ( Limosilactobacillus reuteri FastANI analysis results of ATCC23272) Table 1 shows that the average nucleotide identity (ANI) ratio reached 97.50%, confirming that the isolated strain was *Lactobacillus reuteri*, and named *Lactobacillus reuteri* KY-N5-E2. Limosilactobacillus reuteri Gram staining microscopic image of *Lactobacillus reuteri* KY-N5-E2. Figure 1 As shown. *Lactobacillus reuteri* KY-N5-E2 ( Limosilactobacillus reuteri The strain KY-N5-E2 was deposited at the China Center for Type Culture Collection on April 20, 2026, with accession number CCTCC NO: M 2026751, located in Wuhan, China.

[0026] 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 reuteri* KY-N5-E2 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.

[0027] The beneficial effects of the present invention are illustrated below through experimental examples.

[0028] Experimental Example 1: Inhibitory effect of *Lactobacillus reuteri* KY-N5-E2 on HPV oncogenes The E6 and E7 genes of HPV are key oncogenes leading to cervical cancer, playing a major role in vaginal / cervical intraepithelial neoplasia (CIN) and the progression of cervical cancer. Therefore, this invention uses qPCR to detect the effect of *Lactobacillus reuteri* KY-N5-E2 on the E6 and E7 genes.

[0029] 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 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.

[0030] The original culture of *Lactobacillus reuteri* strain KY-N5-E2 was cultured to the late logarithmic growth stage. After centrifugation and washing, the culture was resuspended in an equal volume of cell culture medium and adjusted to a concentration of 1E+7 CFU / mL. 0.1 mL of the resuspended culture was added to overnight adherent cells to achieve an MOI of 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.

[0031] Grouping information: Blank control group, Lactobacillus reuteri KY-N5-E2 group.

[0032] Using 2^ -△△Ct Calculate the changes in mRNA expression of E6 / E7 genes relative to β-actin in each cell type. The relative expression rate of HPV oncogenes = 2^ -[（Ct目的基因，实验组-Ctβ-actin，实验组）-（Ct目的基因，空白对照组-Ctβ-actin，空白对照组）] .

[0033] Experimental results: The results showed that the Lactobacillus reuteri KY-N5-E2 strain 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 2).

[0034] Table 2. Expression rates of oncogenes in HPV16 and HPV18 positive cells relative to the blank control group by *Lactobacillus reuteri* KY-N5-E2 of this invention. Experimental Example 2: The ability of *Lactobacillus reuteri* KY-N5-E2 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 Lactobacillus reuteri KY-N5-E2 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).

[0035] 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.

[0036] Thp1 cells were resuspended in induction medium (cell concentration 1.2 × 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 live Lactobacillus reuteri KY-N5-E2 (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.

[0037] Experimental Results: As shown in Tables 3 and 4, *Lactobacillus reuteri* KY-N5-E2 increased the expression level of CD86, a maturation marker of dendritic cells (DCs), to 3.16 times that of the positive control group and 14.02 times that of the negative control group. Simultaneously, it also significantly increased the level of IL-12 secreted by DCs, reaching 49.43 times that of the positive control group. The ability of *Lactobacillus reuteri* KY-N5-E2 to stimulate DC maturation / activation was significantly better than that of *Lactobacillus reuteri* ATCC PTA 6475 reported in the literature (CD86 levels in the ATCC PTA 6475 group were lower than those in the positive control group, while IL-12 levels in the ATCC PTA 6475 group were higher than those in the positive control group, visually estimated to be less than 20 times higher than those in the positive control group; Source: Bene KP, Kavanaugh DW, Leclaire C, Gunning AP, MacKenzie DA, Wittmann A, Young ID, Kawasaki N, Rajnavolgyi E and Juge N (2017). *Lactobacillus reuteri* Surface Mucus Adhesins Upregulate Inflammatory Responses Through Interactions With Innate C-Type Lectin Receptors. Front. Microbiol. 8:321. doi: According to 10.3389 / fmicb.2017.00321), the *Lactobacillus reuteri* KY-N5-E2 strain of this invention exhibits excellent immunomodulatory capabilities and can enhance the host's resistance.

[0038] Table 3. Results of flow cytometry analysis of CD86 expression levels on cell surface Table 4. Results of IL-12 level detection in cell supernatant using the IL-12 ELISA kit. In summary, the *Lactobacillus reuteri* KY-N5-E2 strain of this invention can significantly inhibit the expression of the E6 / E7 oncogenes of high-risk HPV types (HPV16 and HPV18), blocking their degradation of p53 and pRb at the molecular level, thereby reversing abnormal cell cycle proliferation and inhibiting the carcinogenesis process. Furthermore, this strain exhibits excellent immunomodulatory capabilities, significantly promoting the maturation and activation of dendritic cells and effectively reversing the immune recognition impairment caused by the "lack of danger signal" mechanism of HPV. This function, through precise regulation of the host immune response, lays the foundation for rebuilding Th1-type antiviral immunity, fundamentally addressing the immunodeficiency problem of persistent HPV infection. This strain demonstrates good biocompatibility, working synergistically through a dual mechanism of "awakening the host immune system" and "inhibiting oncogenes at their source," providing a novel strategy and material basis for adjuvant treatment of HPV infection and related lesions.

Claims

1. A type of *Lactobacillus reuteri*, characterized in that: It is *Lactobacillus reuteri*, with accession number CCTCC NO: M 2026751, preserved by the China Center for Type Culture Collection. Limosilactobacillus reuteri KY-N5-E2.

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

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

4. Use of the *Lactobacillus reuteri* of claim 1 in the preparation of a medicament for the prevention and / or treatment and / or adjunctive treatment of high-risk HPV infection.

5. The use according to claim 4, characterized in that: The HPV is HPV16 and / or HPV18.

6. Use of the *Lactobacillus reuteri* as described in claim 1 in the preparation of a medicament for the prevention and / or adjuvant treatment of cervical intraepithelial neoplasia or cervical cancer.

7. The use according to claim 4 or 5, characterized in that: The drug is a drug that inhibits the expression of HPV viral oncogenes and / or stimulates the maturation / activation of dendritic cells.

8. Use of the *Lactobacillus reuteri* as described in claim 1 in the preparation of immunomodulators.

9. A pharmaceutical composition for the prevention and / or adjunctive treatment of high-risk HPV infection, characterized in that: It is a formulation prepared using *Lactobacillus reuteri* as the active ingredient as described in claim 1, plus pharmaceutically acceptable excipients or auxiliary ingredients.

10. The composition according to claim 9, characterized in that: The preparation is for external use.

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