Application of lactobacillus reuteri in improving immune effect of mucosal vaccine

Abstract

The present application relates to the field of biotechnology, and specifically discloses application of lactobacillus reuteri in improving immune effect of mucosal vaccine. The present application research finds that lactobacillus reuteri has excellent mucosal vaccine adjuvant / potentiator potential, can be combined with existing respiratory mucosal vaccine, significantly expands lung resident memory B cell library, improves the onset speed and protection upper limit of the vaccine, and further proposes application of lactobacillus reuteri in preparing products for improving immune effect of influenza virus vector mucosal vaccine. The present application directly relates oral lactobacillus reuteri intervention to early defense of respiratory infection for the first time, provides a new type of immune intervention idea different from traditional specific vaccine, improves the immune effect of the original influenza virus vector mucosal vaccine, and provides a new effective method for preventing and treating influenza virus infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of Lactobacillus reuteri in enhancing the immune efficacy of mucosal vaccines. Background Technology

[0002] Respiratory infections, such as those caused by influenza viruses, coronaviruses (e.g., SARS-CoV-2), and respiratory bacteria (e.g., Streptococcus pneumoniae, Klebsiella pneumoniae), are serious health threats. The mucosal immune system is a crucial line of defense for the body in responding to the external environment, fighting infection, and maintaining homeostasis. Immune memory generated by the adaptive immune system typically plays a role in secondary immune responses and is the theoretical basis for current vaccine design. However, whether adaptive memory cells play a role in primary immune responses or in the face of unknown pathogens remains unclear. The respiratory and intestinal mucosa contain a large number of mucosal immune cells and symbiotic microorganisms, and metabolites produced by intestinal symbiotic flora have been shown to regulate immune homeostasis. The intestinal mucosa contains a large number of IgA antibodies targeting symbiotic bacteria, and the intestinal mucosal B cell receptor (BCR) repertoire exhibits high diversity. However, whether memory B cells (MBCs) generated against intestinal flora can participate in immune responses to other mucosal tissues (such as the lungs) and provide cross-protection lacks systematic research and mature application protocols in current technologies.

[0003] Current respiratory infection prevention primarily relies on vaccines based on specific pathogen strains. While these strategies can induce specific antibodies and a certain level of mucosal immune response, they lack early, broad-spectrum immune interventions to provide protection against initial infection or exposure to unknown pathogens. On the other hand, current probiotic interventions largely focus on improving gut microbiota composition, promoting metabolic homeostasis, or enhancing general immune function, lacking clear and verifiable effector cell targets and cross-tissue mechanisms of action, making it difficult to establish a direct technological chain from strain to immune protection. Furthermore, current technologies do not recognize the gut mucosal immune system as a "reservoir of memory B cells" for pulmonary immune defense. Particularly in cases of initial infection or exposure to unknown pathogens, how to mobilize gut-derived immune memory cells to provide early protection for the lungs remains a technological gap. Moreover, current technologies have not clarified whether specific gut commensal bacteria can selectively enhance the local IgA immune response in the lungs induced by influenza virus vector mucosal vaccines, and there is a particular lack of technical solutions for combining oral probiotic interventions with nasal mucosal vaccines to enhance the host's resistance to primary influenza virus infection.

[0004] Further research is needed on how to enhance the effectiveness of existing immunization methods. Summary of the Invention

[0005] One of the objectives of this invention is to provide a new method for enhancing the immune protection effect of mucosal vaccines.

[0006] This invention has found that, in the context of mucosal vaccination, oral supplementation with Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus reuteri Also known as Limosilactobacillus reuteri , Lr It can increase lung-resident IgA + The levels of IgA in memory B-cell reservoirs and bronchoalveolar lavage fluid (BALF) were compared, but oral supplementation with *Lactobacillus reuteri* did not show a significant synergistic effect in the context of inactivated vaccine vaccination. Oral administration of a specific gut commensal bacterium—*Lactobacillus reuteri*—can specifically amplify IgA levels derived from Pausch's knot. + It can enhance the memory of B cell reservoirs, increase their recruitment to the lungs, and improve early defense against respiratory infections. It also preferentially enhances the lung mucosa-associated IgA immune response, making it more suitable for use in combination with mucosal vaccines such as those administered via the nose.

[0007] Continuous oral administration of Lactobacillus reuteri before and after vaccination, and continued administration after influenza virus challenge, can further enhance the protective effect of mucosal vaccines. Specifically, continuous oral supplementation with Lactobacillus reuteri before and after vaccination, and continued administration after influenza virus challenge, can increase lung-resident IgA levels. + Memory B cells and IgA + At the plasma cell level, *Lactobacillus reuteri* enhances IgA secretion and H1N1 neutralizing antibody titers in BALF, reduces viral load in lung tissue, and alleviates pathological damage, thereby improving the host's resistance to primary influenza virus infection. This finding indicates that *Lactobacillus reuteri* has a selective synergistic effect on mucosal vaccines. This selectivity suggests that *Lactobacillus reuteri* does not function as a typical non-specific immune enhancer, but is more suitable for enhancing vaccine types that rely on mucosal-mediated local immunity. This helps to determine its applicable boundaries and product positioning as an oral synergist for mucosal vaccines.

[0008] Therefore, this invention provides the application of Lactobacillus reuteri in the preparation of products that enhance the immunogenicity of influenza virus vector mucosal vaccines.

[0009] This invention also provides the application of the combined use of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of products for the prevention of influenza virus infection.

[0010] This invention also provides the application of using influenza virus vector mucosal vaccines and Lactobacillus reuteri in combination in the preparation of products that enhance the host's resistance to influenza virus infection.

[0011] This invention also provides the application of the combined use of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of products that enhance early protection against influenza virus infection.

[0012] 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.

[0013] The protection described in this invention is manifested in the fact that, compared with the control group not treated with Lactobacillus reuteri, the host exhibits reduced viral load in lung tissue, lessened 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.

[0014] This invention also provides the application of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of influenza virus immunomodulators.

[0015] In the application of this invention, the influenza virus is an influenza A virus.

[0016] 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.

[0017] In the application of this invention, the Lactobacillus reuteri is an oral dosage form.

[0018] In practical applications, the administration route of this invention is simple, allowing for oral administration of Lactobacillus reuteri, which can then be used in combination with existing mucosal vaccines. This makes it suitable for further developing Lactobacillus reuteri into a mucosal vaccine combination preparation, an oral mucosal vaccine enhancer, or a microecological enhancement preparation targeting local IgA immune responses in the lungs.

[0019] The present invention also provides an influenza virus immunization combination comprising Lactobacillus reuteri 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] Without departing from the concept of the present invention, Lactobacillus reuteri can be implemented in the form of live bacteria, inactivated bacteria, bacterial lysates, surface protein extracts, extracellular vesicles, bacterial metabolites, or compositions containing their active ingredients.

[0022] The administration method 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.

[0023] The formulations of this invention can be used in combination with other beneficial bacteria, prebiotics, immunomodulators, mucosal adjuvants, or mucosal vaccine antigens. As long as their core is still based on Lactobacillus reuteri shaping an intestinal memory B cell reservoir and enhancing early respiratory protection, they should all be included within the scope of this invention.

[0024] The beneficial effects of this invention are at least as follows: This invention is the first to explicitly demonstrate the use of Lactobacillus reuteri in shaping intestinal IgA with lung migration potential. + It establishes a "Lactobacillus reuteri-Paecilomyces IgA" reservoir of memory B cells (rather than simply using it as a general gut probiotic) and enhances early mucosal protection of the lungs. + The complete technical pathway of "memory B cell expansion-lung recruitment-early IgA protection" directly links oral Lactobacillus reuteri intervention with early defense against respiratory infections, providing a novel immune intervention approach that differs from traditional specific vaccines.

[0025] This invention demonstrates that *Lactobacillus reuteri* possesses excellent potential as a mucosal vaccine adjuvant / synergist. It can be used as a prophylactic intervention component to enhance the host's resistance to primary influenza virus infection, and also as an oral non-invasive formulation used in combination with existing respiratory mucosal vaccines to significantly expand the resident lung memory B cell pool, improve the onset speed and upper limit of protection of the vaccine, thus possessing significant clinical application value.

[0026] The present invention demonstrates high safety and feasibility. *Lactobacillus reuteri* is a recognized safe food-grade microorganism widely found in the intestines of humans and animals. Interventions based on *Lactobacillus reuteri* offer significant advantages such as high safety, mature preparation processes, and convenient administration (oral). It is suitable as a selective synergistic component for use in combination with vaccines administered via nasal, nebulized, or oral mucosal immunization routes, and its translational application prospects are broad. Attached Figure Description

[0027] 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.

[0028] Figure 1 Lactobacillus reuteri ( Lr The application effect of this novel mucosal vaccine in selective synergistic effects. A represents the flowchart of Experiment 1 and the effects of the resident type (ivCD45). - IgA + The number of memory B cells (MBC), where B represents the total IgA level in BALF from Experiment 1, and C represents the flowchart of Experiment 2 and the resident type (ivCD45). - IgA + The number of memory B cells (MBC), and D is the total IgA level in BALF from Experiment 2.

[0029] Figure 2 Lactobacillus reuteri ( LrThe application effect of ) in enhancing the immunogenicity of novel mucosal vaccines. A is a schematic diagram of the experimental procedure and the resident type (ivCD45) - IgA + The number of memory B cells (MBC), where B represents the total IgA and H1N1 neutralizing antibody titers detected in BALF.

[0030] Figure 3 This study aimed to enhance the early defense against primary influenza virus infection in germ-free (GF) mice using *Lactobacillus reuteri*. 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.

[0031] Figure 4 This study demonstrates the efficacy of *Lactobacillus reuteri* 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.

[0032] In each diagram (if any), P < 0.05; P < 0.01; P < 0.001; P < 0.0001; ns, not significant. 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] In the accompanying drawings of this invention, the data are presented as mean ± SEM, and pairwise comparisons are performed using unpaired t-tests for statistical analysis.

[0036] The *Lactobacillus reuteri* used in the specific embodiments of this invention was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC 1.3222.

[0037] Example 1. Application of Lactobacillus reuteri in selectively enhancing novel mucosal vaccines Experimental animals: 8-week-old SPF (Specific-pathogen-free) C57BL / 6 mice, 4 mice per group.

[0038] The method of Experiment 1: SPF mice were administered live Lactobacillus reuteri via gavage for 14 consecutive days (1 × 10⁻⁶ bacteria per mouse per day). 8 Mice were administered an equal volume of sterile PBS via gavage (CFU) to the control group (Vehicle). Following gavage, mice were inoculated intranasally 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.) at a dose of 5 × 10⁶ per mouse. 4 PFU. Seven days after inoculation, the resident type (ivCD45) in mouse lung tissue was analyzed by flow cytometry. - IgA + The number of memory B cells (MBCs) was determined by collecting bronchoalveolar lavage fluid (BALF) and detecting total IgA levels using ELISA. Results are shown below. Figure 1 A and B in the example.

[0039] Flow cytometry analysis revealed that *Lactobacillus reuteri* upregulated the resident type (ivCD45) in mouse lung tissue. - IgA + The number of memory B cells (MBC).

[0040] Bronchoalveolar lavage fluid (BALF) was collected and the total IgA level was measured. It was found that Lactobacillus reuteri could upregulate the secretion of IgA in mouse BALF.

[0041] The method of Experiment 2: SPF mice were administered live Lactobacillus reuteri via gavage for 14 consecutive days (1 × 10⁻⁶ bacteria per mouse per day). 8 Mice were administered an equal volume of sterile PBS via gavage (CFU) to the control group (Vehicle). Following gavage, mice were then injected intramuscularly with an inactivated influenza vaccine (product name: Influenza Virus Split Vaccine, brand: Anerlaifu). ® (Purchased from Beijing Sinovac Biotech Co., Ltd.), specifically, each mouse was inoculated with 7.5 μg H1N1 hemagglutinin, 7.5 μg H3N2 hemagglutinin, and 7.5 μg β-hemagglutinin. Seven days after inoculation, the resident phenotype (ivCD45) in the mouse lung tissue was analyzed by flow cytometry. - IgA + The number of memory B cells (MBCs) was determined by collecting bronchoalveolar lavage fluid (BALF) and detecting total IgA levels using ELISA. Results are shown below. Figure 1 C and D in the example.

[0042] Flow cytometry analysis revealed that *Lactobacillus reuteri* could not significantly upregulate resident *ivCD45* in mouse lung tissue. - IgA + The number of memory B cells (MBC).

[0043] Seven days after inactivated vaccine administration, bronchoalveolar lavage fluid (BALF) was collected to detect the level of total IgA. It was found that Lactobacillus reuteri could not significantly upregulate IgA secretion in mouse BALF.

[0044] This result indicates that *Lactobacillus reuteri* (… Lr It exhibits selective synergistic effects against novel mucosal vaccines, primarily manifested in increased lung-resident IgA. + Memory B cell reservoirs and IgA levels in BALF, while Lactobacillus reuteri ( Lr It does not have a significant synergistic effect on inactivated vaccines.

[0045] The data in this embodiment represents at least three independent experiments.

[0046] Example 2. Application of Lactobacillus reuteri in enhancing the anti-influenza virus infection effect of novel mucosal vaccines Experimental animals: 8-week-old SPF (Specific-pathogen-free) C57BL / 6 mice, 4 mice per group.

[0047] Experimental methods: SPF mice were administered live Lactobacillus reuteri via gavage for 14 consecutive days (1 × 10⁻⁶ bacteria per mouse per day). 8CFU and the control group (Vehicle+Ve) were administered an equal volume of sterile PBS by gavage. After gavage, mice were inoculated via nasal drops with the influenza virus vector COVID-19 Vaccine for Intranasal Spray (Beijing Wantai Biological Pharmacy Co., Ltd.), with each mouse receiving 5 × 10⁶ doses. 4 PFU. Continue daily administration of the original dose (1×10⁻⁶ PFU per mouse) to mice after vaccination. 8 Mice were administered CFU (carbohydrate, lactobacillus reuteri) via gavage 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). Seven days after H1N1 infection, the resident type (ivCD45) in mouse lung tissue was analyzed by flow cytometry. - IgA + The number of memory B cells (MBCs) was determined by collecting bronchoalveolar lavage fluid (BALF). Total IgA levels 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 the BALF sample that still inhibits H1N1 virus infection of MDCK cells after dilution. Results are shown below. Figure 2 .

[0048] Flow cytometry analysis revealed that *Lactobacillus reuteri* upregulated the resident type (ivCD45) in mouse lung tissue. - IgA + The number of memory B cells (MBC).

[0049] Seven days after H1N1 infection, bronchoalveolar lavage fluid (BALF) was collected to detect total IgA and H1N1 neutralizing antibody titers. The results showed that *Lactobacillus reuteri* upregulated IgA secretion and H1N1 neutralizing antibody titers in mouse BALF. These results indicate that *Lactobacillus reuteri* can be used as a component in mucosal vaccine combination therapy to enhance the protective effect of mucosal vaccine-induced pulmonary humoral immunity. The data in this example represent at least three independent experiments.

[0050] Example 3. Application of Lactobacillus reuteri 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.

[0051] Experimental methods: GF mice were administered Lactobacillus reuteri via gavage for 14 consecutive days (1 × 10⁻⁶ bacteria per mouse per day).8 CFU 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 Lactobacillus reuteri was administered CFU via gavage until the experimental endpoint. Lung tissue viral titers were measured 3 days after H1N1 infection. Seven days after H1N1 infection, the resident virulence type (ivCD45) in mouse lung tissue was analyzed by flow cytometry. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs) were determined, and bronchoalveolar lavage fluid (BALF) was collected for analysis. Total IgA levels were detected by ELISA, and 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 after dilution). Lung tissue from GF mice was collected for HE staining to observe pathological damage. Results are shown below. Figure 3 .

[0052] Seven days after H1N1 infection, flow cytometry analysis revealed that *Lactobacillus reuteri* upregulated resident *ivCD45* in the lung tissue of GF mice. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs).

[0053] Seven days after H1N1 infection, bronchoalveolar lavage fluid (BALF) was collected to detect total IgA and H1N1 neutralizing antibody titers. It was found that Lactobacillus reuteri could upregulate IgA secretion in BALF of GF mice and upregulate H1N1 neutralizing antibody titers.

[0054] Three days after H1N1 infection, the viral titer in lung tissue was measured, and it was found that Lactobacillus reuteri could downregulate the influenza virus titer in the lungs of GF mice.

[0055] Seven days after H1N1 infection, lung tissue from GF mice was stained with hematoxylin and eosin (HE) to observe pathological damage. The results showed that *Lactobacillus reuteri* could alleviate the degree of pathological damage in the lung tissue of GF mice. These results indicate that prophylactic oral administration of *Lactobacillus reuteri* can rebuild and enhance the host's early protection against primary influenza virus infection in a sterile environment. The data in this example represent at least three independent experiments.

[0056] Example 4. Application of Lactobacillus reuteri in the prevention of influenza virus infection in SPF mice. Experimental animals: 8-week-old SPF (Specific-pathogen-free) C57BL / 6 mice, 4 mice per group.

[0057] Experimental methods: SPF mice were administered Lactobacillus reuteri via gavage for 14 consecutive days (1 × 10⁻⁶ 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 Lactobacillus reuteri was administered CFU via gavage until the experimental endpoint. Lung tissue viral titers were measured 3 days after H1N1 infection. Seven days after H1N1 infection, the resident virulence type (ivCD45) in mouse lung tissue was analyzed by flow cytometry. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs) were determined, and bronchoalveolar lavage fluid (BALF) was collected for analysis. Total IgA levels were detected by ELISA, and 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 4 .

[0058] Seven days after H1N1 infection, flow cytometry analysis revealed that *Lactobacillus reuteri* upregulated resident *ivCD45* in the lung tissue of SPF mice. - IgA + Memory B cells (MBC) and IgA + The number and proportion of plasma cells (PCs).

[0059] Seven days after H1N1 infection, bronchoalveolar lavage fluid (BALF) was collected to detect total IgA and H1N1 neutralizing antibody titers. It was found that Lactobacillus reuteri could upregulate IgA secretion in BALF of SPF mice and upregulate H1N1 neutralizing antibody titers.

[0060] Three days after H1N1 infection, the viral titer in lung tissue was measured, and it was found that Lactobacillus reuteri could downregulate the influenza virus titer in the lungs of SPF mice.

[0061] Seven days after H1N1 infection, lung tissue from SPF mice was examined using hematoxylin and eosin (HE) staining to observe pathological damage. The results showed that *Lactobacillus reuteri* could alleviate the degree of pathological damage in the lung tissue of SPF mice. These results indicate that prophylactic oral administration of *Lactobacillus reuteri* can rebuild and enhance the host's early protection against primary influenza virus infection in the context of SPF. The data in this example represent at least three independent experiments.

[0062] 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 Lactobacillus reuteri in the preparation of products that enhance the immunogenicity of influenza virus vector mucosal vaccines.

2. Application of the combined use of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of products for the prevention of influenza virus infection.

3. Application of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of products that enhance the host's resistance to influenza virus infection.

4. Application of the combined use of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of products that enhance early protection against influenza virus infection.

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

6. Application of influenza virus vector mucosal vaccine and Lactobacillus reuteri in the preparation of influenza virus immunizing agents.

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

8. The application according to any one of claims 1-7, characterized in that, The Lactobacillus reuteri mentioned is an oral dosage form.

9. A combination of influenza virus immunizing agents, characterized in that, This includes Lactobacillus reuteri 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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