Use of ruminococcus gnavus in enhancing immune protection and in the preparation of immunological agents

Abstract

The application relates to the field of biotechnology, and particularly discloses application of a live Ruminococcus gnavus in enhancing immune protection and preparing an immune preparation. The application research finds that the live Ruminococcus gnavus can improve the effect of resisting influenza virus infection of a host, and can be combined with an influenza virus carrier mucosal vaccine to further provide better protection effect, and thus application of the live Ruminococcus gnavus in preparing an influenza virus immune preparation, improving the resistance of a host to influenza virus infection, improving the early protection ability of influenza virus infection, and improving the immune effect of an influenza virus carrier mucosal vaccine is proposed. The application not only provides a new method for preventing and treating respiratory tract infection, but also provides a new way for enhancing the respiratory tract virus carrier mucosal vaccine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the use of active rumenococci in enhancing immune protection and preparing immunomodulatory agents. Background Technology

[0002] The core function of the mucosal immune system is to defend against pathogen invasion and maintain microecological homeostasis. For a long time, the immunological community believed that adaptive immune memory only functions during secondary infections. However, the respiratory tract, as the "front line" of pathogen exposure, urgently needs effective immune defense mechanisms in the early stages of primary infection. The gut immune system co-evolves with a large number of symbiotic microorganisms and maintains a vast and diverse pool of IgA. + B-cell repositories. Whether these immune cells targeting gut symbionts can be mobilized to participate in the immune defense of distant tissues (such as the lungs) has lacked systematic validation. Respiratory infections pose a serious threat to health. Traditional prevention and control methods for respiratory infectious diseases such as influenza mainly rely on vaccination with inactivated or attenuated vaccines based on specific pathogen antigens, providing protection by pre-inducing systemic or local specific antibodies. Probiotic intervention, as an adjunct, is widely used to improve gut microbiota imbalance and assist in enhancing non-specific immunity, but there is a lack of understanding of adaptive immune memory cells, especially the precise regulation of cross-tissue immune memory. Furthermore, current technologies have not recognized that specific gut symbionts can act as "shapers" and "mobilizers" of pulmonary immune memory B cells, providing pre-positioned mucosal protection in the early stages of infection through the "gut-lung axis."

[0003] Active rumen cocci ( Ruminococcus gnavus Also known as Mediterraneibacter gnavus , Rg As a core symbiotic anaerobic bacterium in the gut, research on it has mainly focused on regulating intestinal homeostasis, metabolic function, or general immunomodulatory effects. Its efficacy in the prevention and treatment of respiratory infections or in enhancing the efficacy of mucosal vaccines still needs further development. Summary of the Invention

[0004] One of the objectives of this invention is to provide a new and effective method for preventing and controlling influenza.

[0005] This invention provides the use of active rumen cocci in any of the following aspects: (1) Preparation of influenza virus immunomodulators; (2) To prepare products that prevent influenza virus infection and enhance the host's resistance to influenza virus infection; (3) To develop products that enhance early protection against influenza virus infection; (4) Prepare products that enhance the immune efficacy of influenza virus vector vaccines.

[0006] This invention has found that: (1) Most of the existing prevention and control programs for respiratory viral infections are based on specific antigens, which makes it difficult to provide immediate, broad-spectrum and stable protection in the early stages of initial infection or when pathogens mutate rapidly.

[0007] (2) Most current probiotic applications lack clear effector cell targets and cross-tissue mechanisms of action, and have not yet formed a technology chain that can be directly used for formulation development, namely "specific strain - intestinal memory B cell reservoir - early lung protection".

[0008] (3) The existing technology does not fully recognize that the intestinal mucosal immune system can serve as a memory B cell reserve for lung defense, and there is a lack of application schemes to actively shape this reserve and enhance its lung recruitment capacity by orally administering specific symbiotic bacteria.

[0009] Currently, there is a lack of systematic research on whether various gut bacteria can specifically shape a reservoir of gut memory B cells (MBCs) and migrate to the lungs via the gut-lung axis to form early protective mucosal immunity. Therefore, it is difficult to break through the traditional viral immunization methods.

[0010] Therefore, this invention has found that supplementing with active rumenococci can increase the resident IgA in the lungs after influenza infection. + Memory B cells and IgA + At the plasma cell level, this invention increases IgA secretion and H1N1 neutralizing antibody titers in bronchoalveolar lavage fluid (BALF), reduces viral load in lung tissue, and alleviates pathological damage. Furthermore, this invention successfully combines active rumenococci with IgA derived from *Pennisetum*. + This invention proposes a novel method to induce host immunity against influenza virus by combining the complete mechanism chain of "memory B cell reservoir - lung recruitment - early IgA protection". Furthermore, this invention also found that in the context of influenza virus vector mucosal vaccination, supplementation with active rumenococci can further enhance IgA retention in lung tissue. + Memory B cell reserves and vaccine protective efficacy.

[0011] Specifically, this invention is the first to clearly demonstrate the use of active rumenococci for shaping intestinal IgA with lung migration potential. + It serves as a memory B cell reservoir, rather than simply being used as a general intestinal probiotic. A "lively rumenococcus-Paey's knot IgA" model was also established. + The complete technical pathway of "memory B cell expansion-lung recruitment-early IgA protection" directly links oral bacterial strain intervention with early defense against respiratory infections.

[0012] Furthermore, this invention also verifies that active rumenococci can be used as a preventive intervention component to enhance the host's resistance to primary influenza virus infection, and can also be used as a mucosal vaccine enhancer component to increase the lung resident memory B cell reserve, IgA level and neutralizing antibody level, showing potential for development as a vaccine adjuvant or vaccine enhancer.

[0013] In the application of this invention, the early stage of influenza virus infection refers to the first 1-7 days after influenza virus infection, preferably the third-7 days.

[0014] The protection described in this invention is manifested in the fact that, compared with the control group that did not receive active rumencoccus, the host exhibited reduced viral load in lung tissue, reduced lung pathological damage, increased IgA levels in bronchoalveolar lavage fluid, increased influenza virus neutralizing antibody titers, and increased resident IgA in lung tissue. + Increased number of memory B cells, IgA + At least one of the following: an increase in the number of plasma cells.

[0015] In the application of this invention, the influenza virus vector vaccine is a mucosal vaccine.

[0016] In the application of this invention, the influenza virus is an influenza A virus.

[0017] In the application of this invention, the active rumenococcus is in oral dosage form.

[0018] In the application of this invention, active rumenococci have the advantages of simple administration route, oral administration, combination with existing mucosal vaccines, and suitability for further development into probiotics, functional preparations or vaccine adjuvants.

[0019] The present invention also provides an influenza virus immunization combination comprising active rumenococcus and an influenza virus vector mucosal vaccine.

[0020] In the influenza virus immunization preparation combination of the present invention, the influenza virus vector mucosal vaccine is an influenza A virus vector mucosal vaccine.

[0021] In this invention, without departing from the concept of the invention, active rumenococci can be implemented in the form of live bacteria, inactivated bacteria, bacterial cell components, surface protein extracts, extracellular vesicles, metabolites, or compositions containing their active ingredients.

[0022] Active rumen cocci can be further developed into live bacterial preparations, inactivated bacterial preparations, bacterial component preparations, or other microecological intervention products, showing high potential for transformation and application.

[0023] The administration route of active rumenococci can be adjusted according to the actual application scenario, such as prophylactic oral administration, administration before and after vaccination, administration during the infection risk period, or maintenance administration.

[0024] Active rumenococci can also be used in combination with other beneficial bacteria, prebiotics, immunomodulators, mucosal adjuvants or vaccine antigens. As long as their core is still based on active rumenococci shaping the intestinal memory B cell reservoir and enhancing early respiratory protection, they should all be included in the scope of protection of this invention.

[0025] In addition to influenza viruses, the present invention can also be extended to preventive microecological intervention in other respiratory virus or respiratory mucosa-related infection scenarios.

[0026] The beneficial effects of this invention are at least as follows: This invention is the first to explicitly use active rumenococci to shape a reservoir of intestinal memory B cells and enhance early mucosal protection in the lungs, providing a novel immune intervention approach that differs from traditional specific vaccines.

[0027] This invention provides a new method for enhancing the host's resistance to primary influenza virus infection and improving the protective effect of influenza virus vector mucosal vaccines, which is beneficial for influenza prevention and control. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This study investigates the efficacy of active rumenococci in early defense against primary influenza virus infection in germ-free (GF) mice. A represents a schematic diagram of the experimental procedure and the resident type (ivCD45). - IgA + Memory B cells (MBC) and IgA + B represents the number and proportion of plasma cells (PCs), C represents the results of total IgA and H1N1 neutralizing antibody titers in BALF, D represents the results of virus titer detection in lung tissue, and D represents the results of HE staining of lung tissue from GF mice.

[0030] Figure 2 This study demonstrates the efficacy of active rumenococci in early defense against primary influenza virus infection in SPF mice. A represents a schematic diagram of the experimental procedure and the resident strain (ivCD45). - IgA + Memory B cells (MBC) and IgA + B represents the number and proportion of plasma cells (PCs), C represents the results of total IgA and H1N1 neutralizing antibody titers in BALF, D represents the results of virus titer detection in lung tissue, and D represents the results of HE staining of lung tissue from SPF mice.

[0031] Figure 3 This study aims to assess the efficacy of active Ruminococcus rumeniformis in the immunogenicity of influenza virus vector mucosal vaccines. A represents a schematic diagram of the experimental procedure and the resident type (ivCD45). - IgA + The number of memory B cells, B represents the total IgA and H1N1 neutralizing antibody titers detected in BALF, and C represents the viral titers detected in lung tissue.

[0032] In each diagram (if any), This means P < 0.05. This means P < 0.01. This represents P < 0.001. This means P < 0.0001. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0035] The active rumenococci used in the specific embodiments of this invention are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40452, and have been disclosed in Chinese patent CN116103196A.

[0036] Example 1. Application of active rumenococci in the prevention of influenza virus infection in germ-free mice. Experimental animals: 8-week germ-free (GF) C57BL / 6 mice, 4 mice per group.

[0037] Experimental methods: GF mice were administered live rumen cocci via gavage for 14 consecutive days (1×10⁶ bacteria per mouse per day). 8CFU and the control group (Vehicle) were administered an equal volume of sterile PBS by gavage. After gavage, GF mice were infected with H1N1 influenza virus (A / Puerto Rico / 8 / 1934, 20 TCID50) via nasal drops. 50 / mouse). Continue daily at the original dose after infection (1×1000 mg / mouse per mouse per day). 8 Active rumenococci were administered via gavage (CFU) until the experimental endpoint. Viral titers in lung tissue were measured 3 days after H1N1 infection. Seven days after H1N1 infection, different cell types were analyzed by flow cytometry. Bronchoalveolar lavage fluid (BALF) was collected, and total IgA levels in BALF were detected by ELISA. The titer of H1N1 neutralizing antibodies in BALF was determined using a microneutralization assay; the H1N1 neutralizing antibody titer refers to the highest dilution of BALF sample that still inhibited H1N1 virus infection of MDCK cells. Lung tissue from GF mice was subjected to HE staining to observe pathological damage. Results are shown below. Figure 1 .

[0038] Seven days after H1N1 infection, flow cytometry analysis revealed that active rumenococci could upregulate resident ivCD45 in the lung tissue of GF mice. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs).

[0039] Seven days after H1N1 infection, the detection of total IgA and H1N1 neutralizing antibody titers in BALF revealed that active rumenococci could upregulate IgA secretion and H1N1 neutralizing antibody titers in GF mice BALF.

[0040] Three days after H1N1 infection, detection of viral titers in lung tissue revealed that active rumenococci could downregulate influenza virus titers in the lungs of GF mice.

[0041] Seven days after H1N1 infection, HE staining of lung tissue from GF mice revealed that active rumenococci could alleviate the degree of pathological damage in the lung tissue of GF mice.

[0042] The above results indicate that prophylactic oral administration of active rumenococci can rebuild and enhance the host's early protection against primary influenza virus infection in a sterile environment.

[0043] The data in this embodiment represents at least three independent experiments.

[0044] Example 2. Application of active rumenococci in preventing influenza virus infection in SPF mice Experimental animals: 8-week-old SPF (Specific-pathogen-free) C57BL / 6 mice, 4 mice per group.

[0045] Experimental methods: SPF mice were administered live rumenococci via gavage for 14 consecutive days (1 × 10⁶ bacteria per mouse per day). 8 SPF mice were administered CFU and a control group (Vehicle) via gavage with an equal volume of sterile PBS. Following gavage, SPF mice were then infected with H1N1 influenza virus (A / Puerto Rico / 8 / 1934, 20 TCID50) via nasal drops. 50 / mouse). Continue daily at the original dose after infection (1×1000 mg / mouse per mouse per day). 8 Active rumenococci were administered via gavage (CFU) until the experimental endpoint. Viral titers in lung tissue were measured 3 days after H1N1 infection. Seven days after H1N1 infection, different cell types were analyzed by flow cytometry. Bronchoalveolar lavage fluid (BALF) was collected, and total IgA was detected by ELISA. The titer of H1N1 neutralizing antibodies in BALF was determined using a microneutralization assay (the H1N1 neutralizing antibody titer refers to the highest dilution of BALF sample that still inhibits H1N1 virus infection of MDCK cells). Lung tissue from SPF mice was collected for HE staining to observe pathological damage. Results are shown below. Figure 2 .

[0046] Seven days after H1N1 infection, flow cytometry analysis revealed that active rumenococci could upregulate resident ivCD45 in the lung tissue of SPF mice. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs).

[0047] Seven days after H1N1 infection, the detection of total IgA and H1N1 neutralizing antibody titers in BALF revealed that active rumenococci could upregulate IgA secretion and H1N1 neutralizing antibody titers in the BALF of SPF mice.

[0048] Three days after H1N1 infection, detection of viral titers in lung tissue revealed that active rumenococci could downregulate influenza virus titers in the lungs of SPF mice.

[0049] Seven days after H1N1 infection, HE staining of lung tissue from SPF mice revealed that active rumenococci could alleviate the degree of pathological damage in the lung tissue of SPF mice.

[0050] The above results indicate that prophylactic oral administration of active rumenococci can rebuild and enhance the host's early protection against primary influenza virus infection in the context of SPF.

[0051] The data in this embodiment represents at least three independent experiments.

[0052] Example 3. Application of active rumenococci in enhancing the efficacy of influenza virus vector mucosal vaccines Experimental animals: 8-week-old SPF (Specific-pathogen-free) C57BL / 6 mice, 4 mice per group.

[0053] Experimental methods: SPF mice were administered live rumenococci via gavage for 14 consecutive days (1 × 10⁶ bacteria per mouse per day). 8 CFU and the control group (Vaccine + Ve) were administered an equal volume of sterile PBS by gavage. After gavage, mice were inoculated via nasal drops with an influenza virus vector mucosal vaccine (constructed using a cold-adapted, NS1-deficient attenuated influenza A virus as a vector, with the SARS-CoV-2 spike protein RBD gene fragment inserted; product name: Influenza Virus Vector COVID-19 Vaccine for Intranasal Spray, brand: Beijing Wantai, purchased from: Beijing Wantai Biological Pharmacy Co., Ltd.), 5 × 10⁶ per mouse. 4 PFU. Continue daily administration of the original dose (1×10⁻⁶ PFU per mouse) to mice after vaccination. 8 Mice were administered live rumencocci via gavage (CFU) until the experimental endpoint. Seven days after vaccination, mice were infected with H1N1 influenza virus (A / Puerto Rico / 8 / 1934, 20 TCID50) via intranasal drip. 50 / animal). Viral titer in lung tissue was measured 3 days after H1N1 infection. Resident strain (ivCD45) in mouse lung tissue was analyzed by flow cytometry 7 days after H1N1 infection. - IgA + The number of memory B cells (MBCs) was determined by collecting bronchoalveolar lavage fluid (BALF). Total IgA was detected by ELISA, and the titer of H1N1 neutralizing antibodies in BALF was determined by a microneutralization assay (H1N1 neutralizing antibody titer refers to the highest dilution of BALF sample that still inhibits H1N1 virus infection of MDCK cells). Results are shown below. Figure 3 .

[0054] Seven days after H1N1 infection, flow cytometry analysis revealed that active rumenococci could upregulate resident ivCD45 in mouse lung tissue. - IgA + The number of memory B cells (MBC).

[0055] Seven days after H1N1 infection, the detection of total IgA and H1N1 neutralizing antibody titers in BALF revealed that active rumenococci could upregulate IgA secretion and H1N1 neutralizing antibody titers in mouse BALF.

[0056] Three days after H1N1 infection, detection of viral titers in lung tissue revealed that active rumenococci could downregulate influenza virus titers in the lungs of mice.

[0057] The above results indicate that, in the context of influenza virus vector mucosal vaccination, the combined use of active rumenococci can further enhance resident IgA in mouse lung tissue. + The study of memory B cell count, IgA level in BALF, and H1N1 neutralizing antibody titer reduced influenza virus titer in mouse lungs, suggesting that active rumenococci can be used as a component of mucosal vaccines to enhance the protective effect of humoral immunity in the lungs after mucosal vaccination.

[0058] The data in this embodiment represents at least three independent experiments.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of active rumenococci in the preparation of influenza virus immunomodulators.

2. Application of active rumenococci in the preparation of products for preventing influenza virus infection and improving the host's resistance to influenza virus infection.

3. Application of active rumenococci in the preparation of products that enhance early protection against influenza virus infection.

4. The application according to claim 3, characterized in that, The early stage of influenza virus infection refers to days 1-7 after infection, preferably days 3-7.

5. Application of active rumenococci in the preparation of products that enhance the immunogenicity of influenza virus vector vaccines.

6. The application according to claim 5, characterized in that, The influenza virus vector vaccine is a mucosal vaccine.

7. The application according to any one of claims 1-6, characterized in that, The influenza virus in question is influenza A virus.

8. The application according to any one of claims 1-7, characterized in that, The active rumen cocci are in oral dosage form.

9. A combination of influenza virus immunizing agents, characterized in that, Including live rumen cocci and influenza virus vector mucosal vaccines.

10. The influenza virus immunizing agent combination according to claim 9, characterized in that, The influenza virus vector mucosal vaccine is an influenza A virus vector mucosal vaccine.

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