Application of ackermann muciniphile Akk-101

Abstract

The invention relates to the technical field of microbial preparations, and particularly provides application of Ackermann muciniphile Akk-101. According to the application, the ackermann mucilaginosus Akk-101 is used for treating the HIV patient for the first time. Research finds that the Ackerman muciniphila Akk-101 can significantly improve the immune function of an HIV patient, regulate the balance of intestinal flora, increase the abundance of beneficial bacteria and inhibit the growth of pathogenic bacteria; the intestinal barrier function is repaired, the intestinal permeability is reduced, and endotoxin translocation is reduced; the CD4 + T cell activity is enhanced, the Th1 / Th2 immune balance is regulated, and the antiviral immune response is improved. A brand-new, safe and effective adjuvant therapy means is provided for HIV treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, and specifically provides an application of Akkermansia muciniphila Akk-101. BACKGROUND

[0002] Acquired Immune Deficiency Syndrome (AIDS) is a chronic immune system disease caused by Human Immunodeficiency Virus (HIV) infection. HIV virus is a virus that attacks the human immune system, especially CD4+ T cells, leading to gradual immune dysfunction. Although Antiretroviral Therapy (ART) can effectively inhibit viral replication, it cannot completely restore the immune function of patients, and long-term use may lead to drug resistance and side effects. In addition, HIV infection is often accompanied by intestinal flora imbalance, especially intestinal barrier dysfunction and immune regulation imbalance, further exacerbating disease progression.

[0003] Akkermansia muciniphila is a probiotic bacteria that colonizes in the intestinal mucus layer, and has the effects of regulating intestinal barrier function, improving metabolic disorders and enhancing immune response. In recent years, studies have shown that Akkermansia bacteria exhibit significant immune regulation function in diseases such as obesity, diabetes and inflammatory bowel disease. However, its application in HIV infection has not been fully explored.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] One of the purposes of the present application is to provide an application of Akkermansia muciniphila Akk-101, and to provide a safe and effective adjuvant therapy for HIV treatment.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] The application of Akkermansia muciniphila Akk-101 in any of the following:

[0008] (1) preparing a medicine for preventing and / or treating HIV infection;

[0009] (2) application in preparing a medical raw material for preventing and / or treating HIV infection;

[0010] (3) preparing a medicine for improving the immune function of HIV patients;

[0011] The preservation number of the Akkermansia muciniphila Akk-101 is CGMCC No. 40786.

[0012] Further, the drug in (3) regulates intestinal flora, repairs intestinal barrier and enhances immune cell function.

[0013] Further, the amount of Akkermansia muciniphila Akk-101 in the drug is at least 1 x 10 8 CFU / g, preferably 1 x 10 8 CFU / g to 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g to 1 x 10 9 CFU / g.

[0014] Further, the drug comprises fermentation broth, live bacteria and / or freeze-dried powder of Akkermansia muciniphila Akk-101.

[0015] Further, the drug further comprises pharmaceutically acceptable excipients;

[0016] Preferably, the drug is administered orally or rectally.

[0017] Further, the drug comprises other drugs for treating and / or preventing HIV infection, preferably antiretroviral drugs.

[0018] A drug containing Akkermansia muciniphila Akk-101, the preservation number of which is CGMCC No. 40786.

[0019] Further, the amount of Akkermansia muciniphila Akk-101 in the drug is at least 1 x 10 8 CFU / g, preferably 1 x 10 8 CFU / g to 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g to 1 x 10 9 CFU / g.

[0020] Further, the drug comprises fermentation broth, live bacteria and / or freeze-dried powder of Akkermansia muciniphila Akk-101.

[0021] Further, the drug further comprises pharmaceutically acceptable excipients;

[0022] Preferably, the drug is administered orally or rectally.

[0023] Preferably, the drug comprises other drugs for treating and / or preventing HIV infection.

[0024] Preferably, the other drug for treating and / or preventing HIV infection is an antiretroviral drug.

[0025] Compared with the prior art, the technical effects of the present application are:

[0026] The present application first proposes to use Akkermansia muciniphila Akk-101 for the treatment of HIV patients. Studies have found that Akkermansia muciniphila Akk-101 can significantly improve the immune function of HIV patients, regulate the balance of intestinal flora, increase the abundance of beneficial bacteria, and inhibit the growth of pathogenic bacteria; repair the intestinal barrier function, reduce the intestinal permeability, and reduce the translocation of endotoxin; enhance the activity of CD4+ T cells, regulate the Th1 / Th2 immune balance, and improve the antiviral immune response. It provides a new safe and effective adjuvant therapy for HIV treatment. BRIEF DESCRIPTION OF DRAWINGS

[0027] The various technical features of the present application and the relationship between them will be further described below with reference to the accompanying drawings. The drawings are exemplary, some technical features are not shown in actual proportion, and some technical features in the drawings can be omitted, which are conventional in the technical field to which the present application belongs and are not essential for understanding and implementing the present application, or additional technical features are shown, which are not essential for understanding and implementing the present application, that is, the combination of various technical features shown in the drawings is not used to limit the present application. In addition, the same reference signs refer to the same contents throughout the present application. The specific drawings are as follows:

[0028] Figure 1 is a comparison chart of intestinal flora structure of HIV infected persons (PLWH) and healthy controls (HC) in Example 2;

[0029] Figure 2 is a comparison chart of intestinal flora structure of HIV infected persons (PLWH) and healthy controls (HC) in Example 2;

[0030] Figure 3 is a comparison chart of intestinal flora structure of HIV infected persons (PLWH) and healthy controls (HC) in Example 2;

[0031] Figure 4 is a comparison chart of intestinal flora structure of HIV infected persons (PLWH) and healthy controls (HC) in Example 2;

[0032] Figure 5 is the change of intestinal flora of HIV patients after Akk intervention in Example 2;

[0033] Figure 6 is the change of intestinal flora of HIV patients after Akk intervention in Example 2;

[0034] Figure 7 is the change of immune cell content in the blood of HIV patients after Akk intervention in Example 2;

[0035] Figure 8 is the change of immune cell content in the blood of HIV patients after Akk intervention in Example 2;

[0036] Figure 9 is the change of lipopolysaccharide, Th2 type cytokine, Th1 type cytokine and CD4+ T cell content in the blood of HIV patients after Akk intervention in Example 2;

[0037] Figure 10 is the change of enhanced CD4+ T cell function after Akk intervention in Example 2;

[0038] Figure 11 is the evaluation result of plasma markers of inflammation and bacterial translocation after Akk intervention in Example 2. DETAILED DESCRIPTION

[0039] In order for those skilled in the art to better understand the technical solutions of the present application, the technical solutions described in the present application will be further described in detail in combination with specific embodiments.

[0040] In the present application, "further", "further", "especially" and the like are used for description purposes, indicating differences in content, but should not be understood as limiting the scope of protection of the present application.

[0041] In the present application, "optionally", "optional" and "optional" mean optional, that is, selected from any one of the two parallel schemes of "yes" or "no". If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each "option" is independent.

[0042] In the present application, "multiple", "multiple", "multiple", "multiple" and the like are referred to, unless otherwise specified, which means greater than or equal to two in quantity. For example, "one or more" means one or more than two.

[0043] The mucinophilic Akkermansia Akk-101 (CN117701423A) used in the present application was preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2023, with the preservation number being CGMCC No: 40786, and the preservation address being No. 3, Beichen West Road, Chaoyang District, Beijing.

[0044] The mucinophilic Akkermansia with the preservation number CGMCC No: 40786 was applied in the present application, and the results showed that it could significantly improve the immune function of HIV patients, specifically:

[0045] Regulate the balance of intestinal flora, increase the abundance of beneficial bacteria, and inhibit the growth of pathogenic bacteria;

[0046] Repair intestinal barrier function, reduce intestinal permeability, and reduce endotoxin translocation;

[0047] Enhance CD4+ T cell activity, regulate Th1 / Th2 immune balance, and improve anti-viral immune response.

[0048] In addition, the strain can be used in combination with other antiretroviral drugs (ART) to enhance the therapeutic effect.

[0049] Therefore, the application provides the use of the mucinophilic Akkermansia in the preparation of a medicine for preventing and / or treating HIV infection or a medical raw material. The use of the mucinophilic Akkermansia in the preparation of a medicine for improving the immune function of HIV patients is also provided. A safe and effective adjuvant therapy is provided for HIV patients.

[0050] The application provides a medicine containing the mucinophilic Akkermansia.

[0051] In some embodiments, the medicine includes a fermentation broth, a fermentation broth supernatant, a fermentation broth precipitate, live bacteria, dead bacteria, a freeze-dried powder, and / or a cell lysate of the mucinophilic Akkermansia.

[0052] The fermentation broth refers to a liquid obtained by inoculating a strain into a culture medium and culturing for a period of time.

[0053] The fermentation broth supernatant refers to the clear liquid in the upper layer after centrifugation of the fermentation broth; it contains rich metabolic products and a part of bacterial fragments during bacterial growth and reproduction, acid substances and bacteriocins secreted by bacteria have antagonistic and killing effects on harmful bacteria; amino acids after bacterial decomposition of food, as well as synthesized vitamins are in the culture broth, and enzymes secreted by bacteria are also in the culture broth; and part of the bacterial components also have immune promoting effects on the human body.

[0054] The fermentation broth precipitate refers to the liquid precipitate obtained by centrifugation, which includes free proteins, residual bacterial bodies, broken cells, and residues of culture medium, mainly proteins and intracellular substrates.

[0055] Live bacteria, also known as active bacterial flora, can colonize and reproduce in the intestinal tract, which is beneficial to increasing the number of beneficial bacteria.

[0056] Dead bacteria are microorganisms that have lost their life activity and cannot grow and reproduce, resulting in loss of activity of probiotics caused by the production process.

[0057] The freeze-dried powder is obtained by freeze-drying the aforementioned culture solution. The freeze-dried powder generally further comprises a freeze-drying protective agent. The freeze-drying protective agent includes, but is not limited to, any one or more of a pH buffer, a bulking agent, a sugar, a non-ionic surfactant, a ligand, etc. The pH buffer includes, but is not limited to, any one or more of Tris, an amino acid or a salt thereof, citric acid or a salt thereof, acetic acid or a salt thereof. The bulking agent includes, but is not limited to, any one or more of mannitol, glycine, bovine serum albumin. The sugar can be a disaccharide, such as any one or more of sucrose or trehalose. The non-ionic surfactant includes, but is not limited to, Tween, which can be Tween-20, Tween-60, Tween-80, etc. The freeze-drying protective agent can further comprise an antioxidant, etc. The freeze-drying protective agent can further comprise albumin, polyethylene glycol, etc.

[0058] The cell lysate can be obtained by lysing the bacterial cells of the aforementioned culture. The lysis can be physical lysis or chemical lysis. The physical lysis includes, but is not limited to, grinding, ultrasonic disruption, etc. The chemical lysis includes, but is not limited to, chemical reagent lysis, enzymatic lysis, which can be hydrolytic enzyme or oxidase. The lysis can also be self-rupture of the cells by increasing the intracellular pressure.

[0059] In some embodiments, the amount of the Akkermansia muciniphila in the medicament is at least 1 x 10 8 CFU / g, preferably 1 x 10 8 CFU / g to 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g to 1 x 10 9 CFU / g. The amount of the Akkermansia muciniphila can be, but is not limited to, 1 x 10 8 CFU / g, 1 x 10 9 CFU / g, 1 x 10 10 CFU / g, or 1 x 10 11 CFU / g.

[0060] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient includes, but is not limited to, any one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, a lubricant, an antioxidant, etc.

[0061] In preferred embodiments, the pharmaceutically acceptable excipient can be selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0062] In some embodiments, the administration form of the medicament can be oral or rectal administration, or other known administration forms for microorganisms.

[0063] In some embodiments, the effective component of the drug, in addition to the mucus-hormone Akkermansia Akk-101 of the present application, can also include other drugs for treating and / or preventing HIV infection, preferably antiretroviral drugs.

[0064] ART is usually used in combination with multiple drugs to enhance efficacy and reduce drug resistance. Antiretroviral drugs include: nucleoside reverse transcriptase inhibitors (NRTIs): such as Zidovudine, Tenofovir. Non-nucleoside reverse transcriptase inhibitors (NNRTIs): such as Efavirenz, Nevirapine. Protease inhibitors (PIs): such as Lopinavir, Darunavir. Integrase inhibitors (INSTIs): such as Raltegravir, Dolutegravir. Entry inhibitors: such as Maraviroc. Fusion inhibitors: such as Enfuvirtide.

[0065] The present application provides a method for preparing the above-mentioned drug, which comprises culturing the mucus-hormone Akkermansia Akk-101 of the present application.

[0066] The present application will be further described in conjunction with specific examples, and the advantages and characteristics of the present application will be clear with the description. The examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0067] The chemical reagents used in the examples of the present application are all of analytical purity and purchased from the National Pharmaceutical Group.

[0068] In order to make the present application easier to understand, the present application will be further described in conjunction with specific examples. The experimental methods described in the present application are all conventional methods if not specifically stated, and the biological materials described in the present application can be obtained from commercial channels if not specifically stated.

[0069] Example 1 Preparation of Akkermansia preparation

[0070] The activated mucus-hormone Akkermansia Akk-101 (with the preservation number of CGMCC No. 40786) is prepared into a freeze-dried powder or a capsule preparation after anaerobic culture expansion.

[0071] The concentration of Akkermansia in the preparation is 1×10 8 CFU / g to 1×1011 CFU / g.

[0072] Example 2 Clinical Application

[0073] HIV patients were given the Akk bacteria preparation from Example 1 orally daily at a dose of 1 × 10⁻⁶. 9 CFU / day, taken continuously for 4 to 12 weeks.

[0074] Monitor changes in the patient's gut microbiota, serum inflammatory factor levels, and CD4+ T cell count to assess the improvement in immune function.

[0075] Changes in gut microbiota

[0076] Intestinal fecal samples were collected from patients every 5 to 15 days, and metagenomic sequencing was used on the Illumina HiSeq X Ten sequencing platform to detect changes in the patients' gut microbiota.

[0077] Results: The abundance of Akk bacteria in the gut of HIV patients was significantly lower than that in healthy individuals. After Akk bacteria intervention, the gut microbiota diversity of patients increased, the abundance of Akk bacteria significantly increased, and the abundance of pathogenic bacteria decreased.

[0078] like Figures 1 to 4 As shown, the gut microbiota structure of HIV-infected individuals (PLWH) and healthy controls (HC) is compared. Figure 1 It is evident that, in terms of both the number of species and the evenness of their distribution, HIV-infected individuals have lower alpha diversity (alpha diversity measures the richness and evenness of bacterial taxa) than HC individuals. HC individuals have a more stable gut ecosystem, while HIV infection can lead to microbiome imbalance, resulting in a decrease in their alpha diversity. Figure 2 and Figure 3 It is evident that the two groups exhibited significant compositional separation, with statistically significant differences. This indicates that HIV infection not only affects the diversity of HIV-related genes but also alters their overall structure and composition.

[0079] Changes in gut microbiota in HIV patients after intervention with FMTakk bacteria, such as Figure 5 As shown, we found that bacterial richness (OTU) gradually increased from 250 OTUs at week 0 to 287 at week 6 in the FMT group compared to the placebo group, while in the placebo group, bacterial richness remained stable from 252 OTUs at week 0 to 254 at week 6. Although this effect diminished during follow-up after the FMT intervention, the intervention appears to have had a long-term effect at this structural level. Similar results were observed for the Chao1 estimator and the Shannon index. The increase in diversity was most pronounced among participants who received FMT from donor A. No differences were found between the four subjects who had recently taken antibiotics and the remaining ten patients in the FMT group.

[0080] Figure 5 Changes in three alpha diversity metrics for OTUs (bacterial richness) by level in each FMT (red) and placebo (blue) group. Bacterial richness (number of OTUs), Chao1 and Shannon indices increased gradually in the FMT group. Black dots represent individual measurements, blue lines represent the mean, horizontal dashed lines indicate the baseline level, and vertical lines indicate the 95% confidence interval. Gray areas indicate the induction period when subjects received FMT or placebo.

[0081] As shown in Figure 6 A, changes in three alpha diversity metrics for OTUs by level in each study group according to donor, B, changes in three alpha diversity metrics for OTUs by level in the FMT-Akk arm according to prior antibiotic exposure (representing only subjects in FMT-Akk), and C, changes in beta diversity for OTUs by level in the FMT arm according to prior antibiotic exposure. Weighted Unifrac distance between subjects and donors was separated according to recent antibiotic exposure in 14 subjects who received FMT-Akk. Horizontal dashed lines indicate the baseline level, black dots represent individual measurements, blue lines represent smoothed means, vertical lines indicate the 95% confidence interval, and gray areas indicate the induction period when subjects received FMT-Akk or placebo. Two-sided P values estimated using mixed model with unadjusted multiple comparisons, n = 361, biologically independent samples from 14 individuals in the FMT-Akk group and 15 individuals in the placebo group.

[0082] Intestinal barrier repair

[0083] Serum samples were collected from patients, and the levels of serum inflammatory factors in the samples were detected using enzyme-linked immunosorbent assay (ELISA).

[0084] Results: As shown in Figures 7 to 9 , after Akkermansia intervention, the level of lipopolysaccharide (LPS) in the serum of patients was significantly reduced, indicating that the intestinal barrier function was repaired.

[0085] Immune function enhancement

[0086] Blood samples were collected from patients, and the number of CD4+ T lymphocytes was detected using a cd4 flow cytometer.

[0087] Results: After Akkermansia intervention, the number of CD4+ T cells in the peripheral blood of patients increased significantly, the level of Th1 type cytokines (such as IFN-γ) increased, and the level of Th2 type cytokines (such as IL-4) decreased, indicating that the immune function was significantly improved.

[0088] As shown in Figure 10As shown, the impact of FMT-AKK intervention on T cells and inflammatory plasma biomarkers, CD4+ and CD8+ T cells in circulation, CD4 / CD8, immune competency markers (CD4+ CD8+ T cell counts, CD4 / CD8), immune activation (% HLADR+ CD38 on CD8+ T cells) and senescence (CD28- and PD-1+ on CD8+ T cells), and as Figure 11 As shown, plasma markers of inflammation and bacterial translocation were assessed. IFABP was significantly reduced by 0.5-fold early, with a clear reduction at week 1 (FMT vs. placebo, P=0.063), a statistical difference at week 4 (P=0.040), and remained stable until week 48 (FMT vs. placebo comparison, P=0.013) Figure 11 H).

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions of terms in this disclosure and that of a related art, the definitions provided in this disclosure are intended to control. In addition, the terminology used in the present specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0090] It should be noted that the above only represents the preferred embodiments of the present application and the applied technical principles. It should be understood by those skilled in the art that the present application is not limited to the specific embodiments described herein, and various obvious changes, modifications and substitutions can be made by those skilled in the art without departing from the scope of the present application. Therefore, although the present application has been described in detail through the above embodiments, the present application is not limited to the above embodiments, and can include more other equivalent embodiments without departing from the technical concept of the present application, and all of them belong to the protection scope of the present application.

Claims

1. Use of Akkermansia muciniphila Akk-101 in any of the following: (1) preparation of a medicament for preventing and / or treating HIV infection; (2) use in a pharmaceutical raw material for preventing and / or treating HIV infection; (3) preparation of a medicament for improving immune function of HIV patients; The preservation number of the Akkermansia muciniphila Akk-101 is CGMCC No. 40786.

2. Use according to claim 1, characterized in that, The medicament in (3) modulates intestinal flora, repairs intestinal barrier and enhances immune cell function.

3. Use according to claim 1, characterized in that, Akk-101 in the medicament is at least 1 x 10 8 CFU / g, preferably 1 x 10 8 CFU / g to 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g to 1 x 10 9 CFU / g.

4. Use according to claim 1, characterized in that, The medicament comprises fermentation broth, live bacteria and / or freeze-dried powder of Akkermansia muciniphila Akk-101.

5. The use according to claim 1, characterized in that, The medicament further comprises pharmaceutically acceptable excipients. Preferably, the administration mode of the medicament comprises oral or rectal administration.

6. Use according to claim 1, characterized in that, The medicament comprises other drugs for treating and / or preventing HIV infection, preferably antiretroviral drugs.

7. A medicament comprising Akkermansia muciniphila Akk-101, the preservation number of which is CGMCC No. 40786.

8. The medicament according to claim 7, characterized in that, Akk-101 in said medicament is at least 1 x 10 8 CFU / g, preferably 1 x 10 8 CFU / g to 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g to 1 x 10 9 CFU / g.

9. The medicament according to claim 7, characterized in that, The medicament comprises fermentation broth, live bacteria and / or freeze-dried powder of Akkermansia muciniphila Akk-101.

10. The medicament according to any one of claims 7 to 9, characterized in that, The medicament further comprises pharmaceutically acceptable excipients. Preferably, the administration mode of the medicament comprises oral or rectal administration. Preferably, the medicament comprises other drugs for treating and / or preventing HIV infection. Preferably, the other drugs for treating and / or preventing HIV infection are antiretroviral drugs.

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