Vaginal active lactobacillus and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to combined lactobacillus salivarius and application thereof in female genital tract health. The preservation number of the strain is CCTCC NO: M20231719, and the strain has good bacteriostatic ability and dendritic cell (DC) maturation / activation stimulation ability, has no potential safety problem, can regulate vaginal micro-ecological balance, prevent infection caused by exogenous bacteria, effectively improve tumor immunosuppression microenvironment, and has good application prospects. And the method has very important significance on prevention / treatment of vaginal dysbacteriosis patients, gynecological tumors and precancerous lesions thereof.

Description

Technical Field

[0001] This invention relates to a live lactobacillus for vaginal use. It is intended for use in the preparation of specific pharmaceutical products, medical devices, cosmetics, or hygiene products. Suitable for application to the vagina and / or vulva, it can be used for regulating vaginal flora and vaginal mucosal immunity, enhancing local resistance. It is applicable to bacterial vaginal infections and / or vaginal / cervical intraepithelial neoplasia, cervical cancer, endometrial cancer, and other gynecological tumors and precancerous lesions. Background Technology

[0002] The vaginal flora plays an important role in maintaining the vaginal microenvironment and controlling pathogenic microbial infections. It also helps coordinate the recruitment of the host's immune defense system and prevent the development of vaginal diseases in women. [2-3] .

[0003] The dynamic interaction between vaginal flora and HPV is crucial for the acquisition and persistence of HPV infection. Dysbiosis of various vaginal flora, including Atopobium vaginae, Gardnerella vaginalis, and Fusobacterium, contributes to the development of cervical intraepithelial neoplasia (CIN) and cervical cancer (CC). Increasing evidence suggests that persistent HPV infection depends on the regulation of the immune response and related factors such as hormones, human genetics, and vaginal flora interactions. Therefore, vaginal flora may be a clinical target for the prevention and treatment of persistent HPV and subsequent progression of CIN and CC. [2-3] .

[0004] In addition to its role in cervical cancer, the vaginal flora also influences cancer risk through mechanisms such as preventing tumor-related chronic infections. Vaginal flora is associated with bacterial vaginosis and pelvic inflammatory disease, potentially serving as an indirect risk factor for endometrial cancer. Ovarian cancer development may also be indirectly regulated by the vaginal flora through local inflammatory responses or immune surveillance. Studies show that the ovaries and fallopian tubes possess unique flora, and the composition of the upper genital tract flora differs between patients with epithelial ovarian cancer and healthy women. Furthermore, the composition of the uterine flora is associated with the development of endometrial cancer. *Atopobacterium vaginalis* and *Porphyromonas* also participate in the development of gynecological malignancies. [3] .

[0005] Vaginal probiotic preparations are effective in preventing and treating vaginal flora imbalance. Vaginal lactobacillus probiotic preparations have been successfully used in the prevention and treatment of bacterial vaginosis and can reduce recurrence rates. Similar to the prevention of gastrointestinal tumors by intestinal probiotics, vaginal probiotics can reduce the risk of gynecological tumors through potential mechanisms such as enhancing immune surveillance and stimulating Treg cells. In addition, probiotics can be directly injected into tumors to regulate the tumor microenvironment. In vitro studies have confirmed that probiotics can promote tumor cell apoptosis or induce dendritic cells and Treg cells to produce anti-inflammatory cytokines.[3] .

[0006] Currently, among commercially available vaginal lactobacillus probiotic preparations, only Dingjunsheng is widely used, but its efficacy is insufficient due to significant individual differences, indicating a large unmet clinical need. There are currently no vaginal probiotics used in immunotherapy, especially via vaginal administration. Probiotics have the advantages of good safety and no toxic side effects, perfectly compensating for the long-term toxic side effects of commercially available chemical drugs. Probiotics can provide numerous pathogen-associated molecular patterns (PAMPs), which, when combined with immunotherapy, complement each other and have a synergistic effect. This invention is of great significance for the prevention / treatment of patients with vaginal flora imbalance and gynecological tumors and their precancerous lesions. Summary of the Invention

[0007] The present invention aims to provide a probiotic, Ligilact obacillus salivarius ECON-1, the strain of which was identified by whole genome sequencing.

[0008] This invention provides a composition comprising 1 × 10⁶ live Lactobacillus salivae ECON-1. 5 CFU ~ 1×10 12 CFU can be administered to patients orally, via intratumoral injection, and / or topically via vagina.

[0009] In some specific embodiments, the aforementioned composition contains 1 × 10⁶ live Lactobacillus saliva-associated ECON-1 bacteria. 5 CFU ~ 1×10 9 CFU.

[0010] In some specific embodiments, the aforementioned composition further includes pharmaceutically acceptable excipients or carriers.

[0011] In some specific embodiments, the lactobacilli in the aforementioned composition exist in the form of live cells and / or inactivated, lysed, cultured metabolites, etc.

[0012] This invention also provides the application of Lactobacillus in the preparation of products that regulate the structure of the vaginal flora.

[0013] This invention also provides the application of lactobacillus in the preparation of products for the prevention or treatment of vaginal flora imbalance and bacterial infections in women, specifically including bacterial vaginosis, aerobic vaginitis, and mixed vaginitis.

[0014] This invention also provides the application of lactobacillus in the preparation of products that regulate vaginal mucosal immunity and enhance local resistance.

[0015] This invention also provides the application of lactobacillus in the preparation of products for the prevention or treatment of gynecological tumors and precancerous lesions, specifically including vaginal / cervical intraepithelial neoplasia, cervical cancer, endometrial cancer, etc.

[0016] This invention also provides an application of Lactobacillus in the preparation of products that improve the tumor immunosuppressive microenvironment.

[0017] The present invention also provides an application of Lactobacillus in the preparation of products that stimulate DC maturation / activation, regulate immune function, and enhance host resistance.

[0018] The beneficial effects of this invention are:

[0019] Compared with first-line antibiotics for treating vaginal infections, the lactobacillus of this invention has better safety, no side effects, no drug resistance, and does not affect the normal flora, thereby reducing recurrence. Compared with existing products, it can better inhibit pathogenic bacteria causing vaginal infections, such as Gardnerella vaginalis, Atobacillus vaginalis, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, and Shigella dysenteriae.

[0020] The *Lactobacillus* of this invention, as a novel immunomodulatory substance, has advantages over commercially available chemical drugs, including better safety, no side effects, and no drug resistance. It can be administered long-term via multiple routes, addressing the long-term treatment of complex tumor-related diseases. As a novel immunomodulatory substance, the *Lactobacillus* of this invention provides numerous pathogen-associated molecular patterns (PAMPs) that can be recognized by the host's innate immune system. Combined with existing immunotherapies, it has a synergistic therapeutic effect.

[0021] The strain preservation information provided by this invention is as follows:

[0022] The strain *Ligilactobacillus salivarius* ECON-1 was deposited at the China Center for Type Culture Collection (CCTCC) on September 18, 2023, with accession number CCTCC NO: M 20231719, located in Wuhan, China. Attached Figure Description

[0023] Figure 1 This image shows a Gram-stained microscopic image of Lactobacillus salivae ECON-1.

[0024] Figure 2 The hydrogen peroxide colorimetric reaction of Lactobacillus salivarii ECON-1 is shown. Detailed Implementation

[0025] Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be noted that, unless otherwise specified, all reagents or instruments used in this invention are commercially available. The culture medium preparation methods used in the following embodiments are all well-known methods.

[0026] Example 1: Isolation, Identification and Gene Safety Analysis of Lactobacillus

[0027] 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 cultured 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.

[0028] Table 1. FastANI analysis results of strain and the reference genome of *Ligilactobacillus salivarius* LPM01.

[0029] index numerical values ANI value (%) 97.94 Reference genome length (bp) 2,033,361 Query genome length (bp) 2,030,435 Number of all sequence fragments in the reference genome 676 Query the number of homologous fragments aligned in the genome 586

[0030] Based on molecular identification results, the isolated strain was identified as *Ligilactobacillus salivarius*, and named *Ligilactobacillus salivarius* ECON-1. Figure 1 Gram staining microscopic image of Lactobacillus salivae ECON-1.

[0031] 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 ) [4-5] The results showed that the Lactobacillus salivarius ECON-1 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.

[0032] Example 2: Antibacterial activity of Lactobacillus saliva-associated with ECON-1

[0033] Lactobacillus delbrueckii (hereinafter referred to as DJS), isolated from the commercially available microecological preparation Dingjunsheng using the same method, was used as the positive control strain in Example 1.

[0034] The classic bacterial cake test was used to evaluate the ability of the strain to inhibit different pathogenic bacteria. The results are shown in Table 2. The *Lactobacillus saliva-associated* ECON-1 strain of this invention showed stronger inhibitory effects against all eight pathogenic bacteria than the control strain DJS. In the field of vaginal bacterial infections, the strain of this invention shows broader application potential and potentially better clinical efficacy than *Dingjun Sheng*.

[0035] Table 2. Antibacterial activity of the present invention's saliva-associated Lactobacillus ECON-1 against 8 pathogenic bacteria.

[0036]

[0037] Note: The mushroom cake size is 9mm.

[0038] Example 3: Hydrogen peroxide production by saliva in conjunction with Lactobacillus ECON-1

[0039] Lactobacillus salivae strain ECON-1 was cultured to the late logarithmic growth stage. 2 μl of fermentation broth was inoculated onto 2% MRS agar plates. Each plate contained 5 mg of filtered and sterilized tetramethylbenzidine (TMB) and 0.2 mg of horseradish peroxidase (HRP). The plates were anaerobically cultured at 37°C for 36-48 h, followed by aeration for 20-30 min. HRP catalyzed the production of O2 from H2O2, and TMB stained the colonies blue in the presence of O2.

[0040] The results are as follows Figure 2The results showed that both *Lactobacillus saliva-associated* ECON-1 and the control strain DJS colonies exhibited a blue color reaction, indicating that they both produce H2O2. The production of H2O2 by *Lactobacillus* can regulate the vaginal microecological balance and prevent infections caused by the invasion of foreign bacteria.

[0041] Example 4: The ability of saliva-associated Lactobacillus ECON-1 to stimulate dendritic cell (DC) maturation / activation

[0042] Thrp1 cells (ATCC) were induced into immature dendritic cells (iDCs) using cytokines GM-CSF and IL-4. The iDCs were then co-incubated with saliva and Lactobacillus ECON-1 to stimulate their 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 using an IL-12 ELISA kit (Lianke Biotechnology).

[0043] Preparation of induction medium: Add 100ug / ml GM-CSF and 100ug / ml IL-4 to complete cell culture medium (RPMI 1640 + 10% inactivated complement FBS) (Gibco) and adjust to a final concentration of 100ng / ml for both.

[0044] Resuspend Thp1 cells in induction medium (cell concentration not exceeding 1.2 × 10⁻⁶). 6 Cells (number per ml) were transferred to T25 flasks for centralized induction, with the medium changed on day 3 and cell counting performed on day 5. 2.1 × 10⁻⁶ cells / ml were then added. 5 Cells were seeded at 0.7 ml / well in 12-well plates and treated with either TNF-α induction medium (positive control group) at 20 ng / ml or TNF-α induction medium plus 3.5 μL of saliva combined with live Lactobacillus ECON-1 (MOI=1) for 48 h. After 48 h of treatment, the cells and supernatant were collected together in deep-well plates, centrifuged at 400 g for 5 min, and the expression level of CD86 on the cell surface was detected by flow cytometry. The IL-12 level in the cell supernatant was detected by the IL-12 ELISA kit according to the manufacturer's instructions.

[0045] The results, as shown in Tables 3 and 4, indicate that *Lactobacillus salivarius* ECON-1 significantly increased the expression level of CD86, a maturation marker of dendritic cells (DCs), to 1.83 times that of the positive control group and 3.23 times that of the negative control group. Simultaneously, it also significantly increased the level of IL-12 secreted by DCs, reaching 68.5 times that of the positive control group and 155.67 times that of the negative control group. *Lactobacillus salivarius* ECON-1 demonstrates the ability to stimulate DC maturation / activation, exhibiting excellent immunomodulatory capabilities and enhancing host resistance.

[0046] Table 3. Results of flow cytometry analysis of CD86 expression levels on cell surface

[0047]

[0048] Table 4. Results of IL-12 level detection in cell supernatant using the IL-12 ELISA kit.

[0049]

[0050] Example 5: Depolarizing ability of Lactobacillus saliva-associated with M2 macrophages by ECON-1

[0051] M2 macrophages are associated with an immunosuppressive tumor microenvironment, and high recruitment of M2 cells facilitates tumor escape. Mouse RAW264.7 macrophages (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were cultured in DMEM high-glucose medium (Gipco) containing 10% FBS. When they were about to reach confluence, 2 × 10⁶ cells were added... 5 Cells were seeded in 12-well plates. After overnight cell adhesion, the cells were divided into three groups: C group, M2 group, and M2+ECON-1 group.

[0052] Group C: Treated with DMEM antibiotic-free medium for 72 hours;

[0053] M2 group: M2 macrophages were subjected to polarization treatment, that is, 1 μl of 20 μg / ml IL-4 and 20 μg / ml IL-13 were administered per well (1 ml), with a final concentration of 20 ng / ml IL-4 + 20 ng / ml IL-13, and the treatment lasted for 72 h;

[0054] M2+ECON-1 group: Cytokine treatment was performed in the same manner as the M2 group for 48 hours. In the drug administration group, 10 μL of saliva combined with Lactobacillus ECON-1 lysate was administered to each well (1 ml) to a final concentration of 1%, followed by treatment for another 24 hours.

[0055] After 72 hours of treatment, samples were collected, washed once with PBS, and 400 μl of Accutase enzyme was added to each well for digestion. After 5 minutes, the samples were centrifuged at 400g for 5 minutes to remove the supernatant. The expression level of CD206, an M2 cell marker molecule on the cell surface, was detected by flow cytometry.

[0056] The results, as shown in Table 5, indicate that Lactobacillus saliva-associated ECON-1 significantly reduced the expression level of the M2 cell marker molecule CD206, suggesting that the strain of this invention can improve the tumor immunosuppressive microenvironment and promote anti-tumor effects.

[0057] Table 5. Results of flow cytometry analysis of CD206 expression levels on cell surface

[0058]

[0059]

[0060] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.

[0061] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes, and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is defined only by the claims. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0062] References:

[0063] [1] Infectious Diseases Collaboration Group of Obstetrics and Gynecology Branch of Chinese Medical Association. Chinese Expert Consensus on Clinical Application of Vaginal Lactobacillus Live Bacteria Capsules (2023 Edition) [J]. Chinese Journal of Practical Gynecology and Obstetrics, 2023, 39(5): 537-546.

[0064] [2]Wang Y, Thakur R, Shen Q, et al. Influences of vaginal microbiota on human papillomavirus infection and host immune regulation: What we have learned? [J].Decoding Infection and Transmission,2023,1:null-null.DOI:10.1016 / j.dcit.2023.07.001.

[0065] [3]Champer M, Wong AM, Champer J, et al. The role of the vaginalmicrobiome in gynecological cancer: a review. [J]. Bjog An International Journal of Obstetrics&Gynaecology, 2017.

[0066] [4] Oh, YJet al. Integrated genome-based assessment of safety and probiotic characteristics of Lactiplantibacillus plantarum PMO 08 isolated from kimchi. Plos One 17, doi:10.1371 / journal.pone.0273986(2022).

[0067] [5] Chokesajjawatee, N. et al. Safety Assessment of a Nham StarterCulture Lactobacillus plantarum BCC9546 via Whole-genome Analysis. Scientific Reports 10, doi:10.1038 / s41598-020-66857-2(2020).

[0068] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein in their entirety for all purposes. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an admission or in any way an implication that they constitute valid prior art or are part of common general knowledge in any country of the world.

Claims

1. A type of *Ligilactobacillus salivarius* ECON-1, characterized in that, The accession number of the *Lactobacillus salivarius* mentioned is CCTCC NO: M 20231719 at the China Center for Type Culture Collection.

2. A composition, characterized in that, It includes the *Ligilactobacillus salivarius* ECON-1 as described in claim 1.

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

4. The composition according to claim 2 or 3, characterized in that, The composition further includes pharmaceutically acceptable excipients or carriers.

5. The use of the salivary lactobacillus ECON-1 as described in claim 1 in the preparation of products that regulate vaginal flora structure.

6. The use of the saliva-associated lactobacillus ECON-1 as described in claim 1 in the preparation of products for the prevention or treatment of vaginal bacterial infections in women.

7. The use of the saliva-associated lactobacillus ECON-1 as described in claim 1 in the preparation of products that regulate vaginal mucosal immunity and enhance host resistance.

8. The use of the saliva-associated lactobacillus ECON-1 as described in claim 1 in the preparation of products for the prevention or treatment of gynecological tumors and precancerous lesions.

9. The application as described in claim 8, characterized in that, Specific conditions include vaginal and cervical intraepithelial neoplasia, cervical cancer, and endometrial cancer.

10. The use of the saliva-associated lactobacillus ECON-1 as described in claim 1 in the preparation of products that improve the tumor immunosuppressive microenvironment.

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