Mesenchymal stem cells for improving immune response to vaccination in elderly or animal subjects

Abstract

The present invention relates to the use of mesenchymal stem cells (MSCs) for enhancing the immune response of elderly or animal subjects to vaccination. According to the application, the defects of spleen stromal cell networks and lymphocyte structures of old subjects can be repaired by applying the MSC in advance, and the immune level of organisms is improved; and the vaccine is injected after a period of time, so that the specific antibody level after vaccine immunization can be improved. The effect can also significantly prevent the elderly subjects from being infected by influenza. Therefore, the MSC has a good application prospect in solving related immune decline of old people.

Description

[0001] TECHNICAL FIELD The present application belongs to the field of biological medicine, and specifically relates to mesenchymal stem cells for improving the immune response of elderly or animal subjects to vaccination. BACKGROUND

[0002] The immune function of the elderly population declines, which significantly increases their susceptibility to infection. The mortality rate of elderly people aged 65 and above after suffering from severe influenza and pneumonia increases significantly. Among patients hospitalized due to influenza infection at this age, the mortality rate is more than 60%. Given the limited effect of antiviral treatment after infection in reducing mortality, vaccination has become the preferred solution to prevent influenza in the elderly. However, due to the aging-related immune deficiency characterized by progressive decline in immune function, the efficacy of vaccines in the elderly population and the actual protective effect are significantly reduced. How to enhance the vaccine response of the elderly is still a difficult problem to be solved. SUMMARY

[0003] The present inventors have confirmed through animal experiments that exogenous MSCs can enhance the immune response and specific antibody production after vaccination in elderly mice and elderly cynomolgus monkeys, and confirmed that activating spleen MRC is an effective strategy for repairing the spleen tissue structure of elderly individuals and enhancing immune response.

[0004] Therefore, the first aspect of the present application provides the use of mesenchymal stem cells (MSCs) in the manufacture of a medicament for improving the immune response of elderly or animal subjects to vaccination.

[0005] The second aspect of the present application provides the use of a combination of MSCs and a vaccine in the manufacture of a medicament for preventing infectious diseases in elderly or animal subjects.

[0006] The third aspect of the present application provides a method for improving the immune response of elderly or animal subjects to vaccination, which comprises administering an effective amount of MSCs to a subject in need thereof, preferably administering an effective amount of MSCs to a subject in need thereof 2 days to 16 weeks before vaccination.

[0007] The fourth aspect of the present application provides a method of vaccination, which comprises administering an effective amount of MSCs and a vaccine to a subject in need thereof, preferably administering the vaccine after administering an effective amount of MSCs 2 days to 16 weeks.

[0008] The fifth aspect of the present application provides a method for preventing infectious diseases in elderly or animal subjects, comprising administering an effective amount of MSCs and a vaccine to the subject in need thereof; preferably, an effective amount of MSCs is first administered to the subject in need thereof, and then an effective amount of the vaccine is administered to the subject 2 days to 16 weeks later, the vaccine being a vaccine having a preventive effect on the infectious disease.

[0009] The sixth aspect of the present application provides a pharmaceutical composition comprising MSC and a vaccine product in separate packages or separate containers, respectively, the vaccine product comprising an antigen component, an adjuvant, and an auxiliary agent.

[0010] The seventh aspect of the present application provides a pharmaceutical kit comprising MSC in a first container, a vaccine product in a second container, and an instruction, the vaccine product comprising an antigen component, an adjuvant, and an auxiliary agent, the instruction indicating to administer the MSC first and then the vaccine product.

[0011] In the above first to seventh aspects, the MSC can be replaced by an agent for activating marginal zone reticular cells (MRCs) of the spleen, and the same effects can be achieved.

[0012] In some embodiments of the present application, improving the immune response of the elderly or animal subjects to vaccination comprises one or more of repairing splenic stromal cells, promoting germinal center formation, and enhancing specific antibody production. In some embodiments, the MSCs can remodel the stromal cell network of the spleen of the elderly mice, thereby restoring the homeostasis of the immune cell population and the integrity of the spleen tissue structure; the MSCs can rejuvenate the spleen of the elderly mice by regulating the splenic stromal cells, thereby promoting germinal center formation and enhancing the production of specific antibodies; or the MSCs can improve the age-related decline of humoral immunity, enhance the vaccine response capacity of the elderly population, and provide effective protection for the elderly against pathogen infection. The MSCs can better improve the immune response of the elderly to vaccines by promoting the proliferation of MRCs in the spleen of the elderly mice, thereby promoting the expansion of the follicular dendritic cell population.

[0013] In some embodiments of the present application, the vaccine is against an infectious disease. Preferably, the vaccine is against a pathogen infection. Preferably, the pathogen is selected from one or more of a bacterium, a virus, or a fungus.

[0014] In some embodiments of the present application, the vaccine is against an infectious disease caused by a virus. Preferably, the virus is selected from one or more of a rhinovirus, a coronavirus, an influenza virus, or a respiratory syncytial virus. Preferably, the coronavirus is selected from one or more of avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV. Preferably, the influenza virus is selected from one or more of influenza A virus and / or influenza B virus. Preferably, the influenza virus is selected from one or more of influenza A H1N1, H3N2, influenza B Victoria lineage, and influenza B Yamagata lineage.

[0015] In some embodiments of the application, the vaccine comprises an influenza vaccine, a pneumococcal vaccine, a shingles vaccine, a DPT vaccine, a COVID-19 vaccine, an HPV vaccine, and the like. The vaccine comprises an inactivated vaccine, a live attenuated vaccine, a recombinant protein vaccine, an mRNA vaccine, and the like.

[0016] In some embodiments of the application, the MSCs are administered to the subject by intravenous, intra-arterial, intraperitoneal, intrapleural, intraparenchymal, intraventricular, or intra-articular injection.

[0017] In some embodiments of the application, the MSCs are VEGFA-positive MSCs. The inventors found that MSCs regulate and repair the splenic stromal cell network through VEGFA, and thus, VEGFA-expressing MSCs (i.e., VEGFA-positive MSCs) are preferred.

[0018] In some embodiments of the application, the MSCs are autologous, allogeneic, or xenogeneic to the subject. In some embodiments of the application, the MSCs are human MSCs.

[0019] In some embodiments of the application, the subject is a human, a domesticated animal, a companion animal, or a canine.

[0020] Vaccination remains a core means of preventing infectious diseases in the elderly, but the age-related impairment of immune system function leads to decreased vaccine response efficiency. The inventors confirmed that the deterioration of the splenic microenvironment caused by aging is a key factor restricting germinal center formation and specific antibody production. The present application shows that infusion of MSCs before vaccination can effectively remodel the splenic MAdCAM-1 / α4β7 axis, and thus significantly enhance the immune response level and antibody production capacity after immunization in aged mice and aged cynomolgus monkeys. In addition, the MSC-mediated enhancement of specific antibody production has a significant spleen-dependent effect, which is completely eliminated in spleen-removed mice. The above research results show that infusion of MSCs can be used as a new strategy for regulating the splenic microenvironment of the aged and improving the efficacy of vaccines. Notably, the inventors found that the germinal center formation and OVA-specific antibody titers did not increase after infusion of MSCs in young mice (data not shown), which strongly confirmed that the core role of MSCs is to repair the damaged lymphoid tissue microenvironment due to aging, rather than excessive stimulation of the functional intact immune system.

[0021] The production of specific antibodies is a key mechanism for the host to defend against pathogen invasion. Currently, various strategies have been used to enhance vaccine response, such as high-dose vaccines, multivalent vaccine formulations, and the application of adjuvants. Although these methods have achieved some success, the aging microenvironment makes it difficult to maintain plasma cell survival and the production of long-acting antibodies. Increasing evidence suggests that the aging microenvironment is a key factor limiting vaccine efficacy. This application demonstrates that infused exogenous MSCs can effectively colonize the spleen of aged mice and remodel the splenic microenvironment, thereby promoting germinal center formation and increasing specific antibody production. Furthermore, this application shows that the enhancing effect of MSCs on specific antibody production is likely achieved through the regulation of stromal cells rather than immune cells, further highlighting the central role of stromal cell regulation in the remodeling of the splenic microenvironment.

[0022] Splenic stromal cells, as key "guardians" of the lymphoid tissue microenvironment, play a crucial role in constructing a supportive microenvironment, regulating immune cell migration, and promoting immune cell development and differentiation. However, with aging, the number of lymphoid tissue stromal cells gradually decreases, which is a significant reason for the decline in immune response in the elderly. This application also observed a similar phenomenon, with a significant reduction in the number of mature stromal cell populations in the spleen of aged mice. Notably, the inventors demonstrated that exogenous MSCs can regulate spleen MAdCAM- Activation of the MRC facilitates the repair of the stromal cell network in the spleen of aged mice. This finding confirms PDGFR MAdCAM- Stromal cells are key progenitor cells in the development of the splenic stromal cell network. MRCs are considered a type of intermediate mesenchymal lymphoid tissue-inducing cell (mLTo) capable of differentiating into follicular dendritic cells, which are crucial for supporting B cell development and germinal center formation. This application further validates that MAdCAM- was detected in the white pulp region of the mouse spleen 3-4 weeks after MSC infusion. The presence of cells, and at the same time FDC-M The number of follicular dendritic cells also showed a gradual upward trend. In summary, these research results indicate that targeted delivery of MSCs to tissue-resident stromal cells or their precursor cell populations holds promise as a novel strategy for restoring tissue homeostasis and achieving long-term therapeutic effects.

[0023] Furthermore, this application reveals that VEGFA is a key functional molecule in MSC-regulated MRC proliferation and repair of the stromal cell network, while VEGFR3 is a functional receptor on MRC mediating this effect. VEGFA is closely related to the aging process—the activity of the VEGF signaling pathway decreases with age, and VEGF supplementation can effectively delay the healthy aging process. Notably, both MSC-derived VEGFA and recombinant VEGFA can effectively repair the integrity of the spleen stromal network in aged mice. Therefore, it is speculated that the decreased VEGFA level in aged individuals may impair the proliferation and differentiation capacity of spleen stromal cells, similar to the mechanism by which insufficient VEGFA levels inhibit myoblast differentiation, leading to age-related muscle loss. These findings confirm that MSCs or VEGFA have therapeutic potential in improving age-related immune decline, especially in the fields of immune microenvironment remodeling, immune homeostasis regulation, and vaccine development. The mechanism of action revealed in this application not only elucidates the key signaling pathways of stromal cell interactions but also highlights the therapeutic potential for repairing the basic structure of immune tissues. Although VEGFA administration can partially mimic the effects of MSCs, VEGFA is only one component of the many biological activities of MSCs, and its full therapeutic effect requires the synergistic effect of multiple factors.

[0024] In summary, this application provides ample evidence that exogenous MSCs can enhance the immune response and specific antibody production following vaccine immunization in elderly subjects, and demonstrates that activating splenic MRCs is an effective strategy for repairing splenic tissue structure and enhancing immune responses in elderly individuals. These results greatly expand our understanding of the mechanism of action of exogenous MSCs—their effects are not limited to directly influencing immune cells, but also include maintaining immune homeostasis by regulating the number and function of endogenous stromal cells. Therefore, this opens new avenues for the clinical translation of cell therapy and provides new solutions for improving age-related immune decline and enhancing vaccine efficacy in the elderly population. Attached Figure Description

[0025] Figure 1 A- FIG. IK This illustrates the structure of MSCs repairing damaged spleen matrix tissue in aged mice, in which... FIG. 1A The experimental procedure diagram shows that aged BALB / c mice (≥18 months old) were divided into three groups: the Vehicle group (Vehicle group, sample size...). n =5), HDF administration group (HDF group, sample size) n =3) and the MSC-treated group (MSC group, sample size) n =5).

[0026] FIG. IBRepresentative images of the spleens of mice in the Vehicle, HDF, and MSC groups.

[0027] FIG. 1C Statistical analysis results of spleen length and spleen weight / body weight ratio in mice from the Vehicle, HDF, and MSC groups.

[0028] FIG. ID Total number of spleen cells in mice in the Vehicle group, HDF group and MSC group.

[0029] FIG. IE-FIG. IF CD4 in the spleen of mice in the Vehicle, HDF, and MSC groups CD3 Ter11 PDGFR Representative scatter plot of stromal cell population and cell count results (G).

[0030] FIG. IH Motor stromal cells (PDGFR) from the spleen of mice in the Vehicle and MSC groups (1-4 weeks) A representative immunofluorescence staining image (magenta), scale bar: 100 μm.

[0031] FIG. II Statistical analysis results of spleen stromal cell area and mean fluorescence intensity (MFI) in mice in the Vehicle group and MSC-treated group (1-4 weeks); 5 replicate tissue samples were taken from each group, and 3 fields of view were selected from each tissue sample for quantitative analysis.

[0032] FIG. 1J : Splenic T lymphocytes (CD3+) in mice in the Vehicle group and MSC-treated group (1-4 weeks) (green) and B lymphocytes (B22) A representative immunofluorescence staining image (red), scale bar: 200 μm.

[0033] FIG. IK Statistical analysis results of B lymphocyte and T lymphocyte area in mice in the Vehicle group and MSC treatment group (1-4 weeks); 5 replicate tissue samples were taken from each group, and 3 fields of view were selected from each tissue sample for quantitative analysis.

[0034] Experimental data are expressed as mean ± standard error of the mean (mean ± SEM) and are derived from at least three independent replicates. Statistical significance analysis was performed using one-way ANOVA combined with multiple comparison tests. P< 0.05 indicates a statistically significant difference; P < 0.01 indicates a statistically significant difference; *P < 0.001 indicates a highly statistically significant difference; **P < 0.0001 indicates a highly statistically significant difference. ns This indicates that the difference is not statistically significant.

[0035] Figure 2A- FIG. 2L The study showed that MSCs promote the formation of germinal centers in the spleen of aged mice and increase the level of specific antibodies. FIG. 2A : Schematic diagram of experimental procedure and ovalbumin (OVA) immunization protocol for aged BALB / C mice.

[0036] FIG. 2B Representative images of spleens from aged BALB / c mice in the Sham, MSC, Sham + OVA, and MSC + OVA groups.

[0037] FIG. 2C Statistical analysis results of spleen length and spleen weight / body weight ratio in mice of the Sham group, MSC group, Sham + OVA group, and MSC + OVA group, with sample size of mice in each group. n =5.

[0038] FIG. 2D Statistical results of total spleen cell count in mice in the Sham group, MSC group, Sham + OVA group, and MSC + OVA group, and sample size of mice in each group. n =5.

[0039] FIG. 2E Statistical results of total splenic lymphocyte count in mice in the Sham group, MSC group, Sham + OVA group, and MSC + OVA group, and sample size of mice in each group. n =5.

[0040] FIG. 2F-FIG. 2G B cell subsets (B22) in the splenic germinal center of mice in the Sham group, MSC group, Sham + OVA group, and MSC + OVA group Ig GL Flow cytometry analysis results, sample size of each group of mice n =5.

[0041] FIG. 2H : Splenic B lymphocytes (B22) of mice in the Sham group, MSC group, Sham + OVA group and MSC + OVA group (red) and hair growth center (PN) A representative immunofluorescence staining image (green), scale bar: 50 μm.

[0042] FIG. 21 Germinal centers (PN) of the spleen in mice of the Sham group, MSC group, Sham + OVA group, and MSC + OVA group Statistical analysis results of the region area, sample size of mice in each group n =5.

[0043] FIG. 2J Serum total immunoglobulin G1 (IgG1) concentration 28 days after immunization in young (2-month-old) mice, Sham group, MSC group, Sham + OVA group, and MSC + OVA group, and sample size of mice in each group. n =5.

[0044] FIG. 2K-FIG. 2L Serum soluble OVA-specific IgG1 antibody concentration and titer were measured 28 days after immunization in young (2-month-old) mice, Sham group, MSC group, Sham + OVA group, and MSC + OVA group. Sample size for each group was also recorded. n =5.

[0045] Experimental data are expressed as mean ± standard error of the mean (mean ± SEM) and are derived from at least three independent replicates. Statistical significance analysis was performed using one-way ANOVA combined with multiple comparison tests. P < 0.05 indicates a statistically significant difference; P < 0.01 indicates a statistically significant difference; *P < 0.001 indicates a highly statistically significant difference; **P < 0.0001 indicates a highly statistically significant difference. ns This indicates that the difference is not statistically significant.

[0046] Figure 3 A- FIG. 3R The results show that MSCs enhance the efficacy of vaccines in aged mice and exert a protective effect against influenza infection. FIG. 3A : Quadrivalent inactivated influenza vaccine immunization program.

[0047] FIG. 3B-FIG. 3C Statistical results of spleen length and total number of spleen cells in mice in the non-vaccinated group (Non vac), the vaccine-immunized control group (Ctrl vac), and the MSC+Vac group (MSC+Vac), with sample size for each group. n =4.

[0048] FIG. 3D The total number of splenic lymphocytes in mice in the Non-vac, Ctrl-vac, and MSC+Vac groups, and the sample size of each group. n =3.

[0049] FIG. 3E : B cell subsets (B22) in the splenic germinal center of mice in the Non-vac, Ctrl-vac, and MSC+Vac groups Ig GL Flow cytometry analysis results, sample size of each group of mice n =3.

[0050] FIG. 3F Statistical results of B cell counts in the splenic germinal centers of mice in the Non-vac, Ctrl-vac, and MSC+Vac groups, with sample size for each group. n =3.

[0051] FIG. 3G : Splenic B lymphocytes (B22) of mice in the Non-vac group, Ctrl-vac group, and MSC+Vac group (red) and hair growth center (PN) A representative immunofluorescence staining image (green) showing the sample size of each mouse group. n =4. Scale: 30 μm.

[0052] FIG. 3H Serum soluble IgG antibody concentration and titer were measured 28 days after immunization in mice in the Non-vac, Ctrl-vac, and MSC+Vac groups. Sample size for each group was [not specified]. n =4.

[0053] FIG. 31 The serum vaccine-specific antibody titers of mice in the Non-vac, Ctrl-vac, and MSC+Vac groups were measured using a hemagglutination inhibition (HI) test 28 days after immunization. Sample size for each group was [not specified]. n =5.

[0054] FIG. 3J The process of constructing an H1N1 influenza virus (A / PR8 strain) infection model in aged mice after vaccination / non-vaccination with a tetravalent split influenza vaccine.

[0055] FIG. 3K The results of monitoring weight changes in uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (CtrlPR8 group and MSC+PR8 group) over 14 days.

[0056] FIG. 3L Survival analysis results of uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (CtrlPR8 group and MSC+PR8 group) within 14 days, with sample size of mice in each group.n =5.

[0057] FIG. 3M The results of lung wet weight analysis on day 14 post-influenza virus infection in uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group), with sample size for each group. n =3.

[0058] FIG. 3N The results of viral titer analysis in bronchoalveolar lavage fluid (BALF) of uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group) on day 14 post-infection with influenza virus, with sample size for each group. n =3.

[0059] FIG. 3O Representative hematoxylin-eosin (HE) pathological staining images of lung tissue from uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group) on day 14 after influenza virus infection.

[0060] FIG. 3P Clinical score analysis results of mice on day 14 post-infection with influenza virus in uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group). Scale bar: 250 μm.

[0061] FIG. 3Q Analysis results of inflammatory cytokines [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-1β (IL-1β)] in bronchoalveolar lavage fluid of uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group) on day 14 post-infection with influenza virus, with sample size of mice in each group. n =3.

[0062] FIG. 3R Analysis of serum inflammatory cytokine (TNF-α, IFN-γ, IL-1β) levels in mice from uninfected aged mice (Vehicle group, MSC Vehicle group) and infected aged mice (Ctrl PR8 group and MSC+PR8 group) on day 14 post-infection with influenza virus. Sample size for each group. n =3.

[0063] Experimental data are expressed as mean ± standard error of the mean (mean ± SEM) and are derived from at least three independent replicates. Statistical significance analysis was performed using one-way ANOVA combined with multiple comparison tests. P < 0.05 indicates a statistically significant difference; P < 0.01 indicates a statistically significant difference; *P < 0.001 indicates a highly statistically significant difference; **P < 0.0001 indicates a highly statistically significant difference. ns This indicates that the difference is not statistically significant.

[0064] FIG. 4A-FIG. 4N This study demonstrates that MSCs promote the proliferation of reticular cells in the marginal zone of the spleen in aged mice. FIG. 4A MSCs were infused into aged BALB / c mice (≥18 months old), and the results were analyzed 3 days later (see Figure 4B-). FIG. 4J ).

[0065] FIG. 4B MSCs (green) and MRCs (MAdCAM-) in mouse spleen Magenta), FDC (FDC-M) (blue), FRC (PDP) Representative immunofluorescence staining images of DAPI (orange) and DAPI (gray), and results of intercellular distance analysis, scale bar: 50 μm.

[0066] FIG. 4C Statistical analysis results of the distance between MSCs and various stromal cell populations in the spleen.

[0067] FIG. 4D Splenic MRC (CD4+) levels were found in the control group (Sham group) and the MSC infusion group (MSC group). CD3 Ter11 PDGFR MAdCAM- A representative flow cytometry image.

[0068] FIG. 4E Statistical analysis results of the proportion of spleen MRC cells in the Sham and MSC groups, with sample size of mice in each group. n =3.

[0069] FIG. 4F Statistical analysis results of spleen MRC counts in the Sham and MSC groups, with sample size of mice in each group. n =3.

[0070] FIG. 4G: Splenic MRC proliferation (CD4+) in the Sham and MSC groups CD3 Ter11 MAdCAM- KI-6 A representative flow cytometry image.

[0071] FIG. 4H Statistical analysis results of spleen MRC proliferation levels in the Sham and MSC groups, with sample size of mice in each group. n =3.

[0072] FIG. 41 Spleen MRC (MAdCAM- Representative immunofluorescence staining images of magenta, KI-67 (green) and DAPI (gray), scale bar: 20 μm.

[0073] FIG. 4J Statistical analysis results of spleen MRC counts in the Sham and MSC groups, with sample size of mice in each group. n =3; and MAdCAM- in the Sham group and MSC group KI-6 in cells The statistical analysis results showed that the sample size of each group of mice was... n ==5.

[0074] FIG. 4K : 1, 2, 3, and 4 weeks after administration of vehicle (Sham) or MSCs, MRC (MAdCAM-) was found in the spleen of aged mice. Representative immunofluorescence staining images of magenta and DAPI (gray), scale bar: 30 μm.

[0075] FIG. 4L : 1, 2, 3, and 4 weeks after administration of vehicle (Sham) or MSCs, MRC (MAdCAM-) was found in the spleen of aged mice. Statistical analysis results of the region area, sample size of mice in each group n =5.

[0076] FIG. 4M : 1, 2, 3, and 4 weeks after administration of vehicle (Sham) or MSC, FDC (FDC-M) in the spleen of aged mice Representative immunofluorescence staining images of magenta and DAPI (gray), scale bar: 30 μm.

[0077] FIG. 4N : 1, 2, 3, and 4 weeks after administration of vehicle (Sham) or MSC, FDC (FDC-M) in the spleen of aged mice Statistical analysis results of the region area, sample size of mice in each group n =5.

[0078] Experimental data are expressed as mean ± standard error of mean (mean ± SEM) and are derived from at least three independent replicate experiments. FIG. 4E , FIG. 4F , FIG. 4H , FIG. 4J The statistical significance analysis in the data was performed using a two-tailed unpaired t-test; FIG. 4C , FIG. 4L , FIG. 4N The statistical significance analysis was performed using one-way ANOVA combined with multiple comparison tests. P <0.05 indicates a statistically significant difference, P <0.01 indicates a statistically significant difference, *P <0.001 indicates a highly statistically significant difference, and **P <0.0001 indicates a highly statistically significant difference. ns This indicates that the difference is not statistically significant.

[0079] Figure 5A- FIG. 5K The results showed that MSCs enhanced antibody levels in elderly cynomolgus monkeys vaccinated against influenza. FIG. 5A : Elderly cynomolgus monkeys (18-21 years old) were given vehicle (Ctrl vac) n =3) or MSC (MSC vac, n =5) Nine weeks later, the procedure for administering the quadrivalent inactivated influenza vaccine will be carried out.

[0080] FIG. 5B Representative spleen ultrasound images of cynomolgus monkeys at weeks 4, 6, and 8 post-vaccination in the Ctrl vac and MSC vac groups.

[0081] FIG. 5C Ultrasound measurement and analysis results of spleen length changes in cynomolgus monkeys at weeks 4, 6, and 8 post-vaccination in the Ctrl vac and MSC vac groups.

[0082] FIG. 5D-FIG. 5G In the Ctrl vac group and the MSC vac group, cynomolgus monkeys showed elevated serum levels of vaccine-specific antigen (anti-recombinant H1N1) at weeks 4, 6, and 8 post-vaccination. FIG. 5D ), anti-recombination H3N2 ( FIG. 5E ), anti-recombinant B / PHUKET ( FIG. 5F ) and anti-recombinant B / Austria ( FIG. 5G The results of the soluble IgG antibody titer test.

[0083] FIG. 5H Analysis of changes in white blood cell count in cynomolgus monkeys in the aged Ctrl group and the aged +MSC group.

[0084] FIG. 51 Analysis of changes in blood lymphocyte counts in cynomolgus monkeys at weeks 4, 6, and 8 after vaccination in the Ctrl vac and MSC vac groups.

[0085] FIG. 5J In the Ctrl vac group and the MSC vac group, cynomolgus monkeys showed CD4 counts at weeks 4, 6, and 8 post-vaccination. / CD Results of peripheral blood lymphocyte population analysis of T cell ratio.

[0086] FIG. 5K In the Ctrl vac group and the MSC vac group, cynomolgus monkeys showed elevated peripheral blood plasma B cells (CD1) levels at weeks 4, 6, and 8 post-vaccination. CD2 CD3 Group analysis results.

[0087] Experimental data are expressed as mean ± standard error of the mean (mean ± SEM) and are derived from at least three independent replicates. Statistical significance was analyzed using a two-tailed unpaired t-test. P < 0.05 indicates a statistically significant difference, P < 0.01 indicates a statistically significant difference, *P < 0.001 indicates a highly statistically significant difference, and **P < 0.0001 indicates a highly statistically significant difference. ns This indicates that the difference is not statistically significant. Detailed Implementation

[0088] It should be understood that different applications of the disclosed substances and methods may be adapted to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0089] All publications, patents, and patent applications cited in this article—whether above or below—are incorporated herein by reference in their entirety.

[0090] Definitions To more clearly explain the embodiments of the present invention, some scientific terms and proper nouns are used herein. Unless explicitly defined herein, all such terms and nouns should be understood to have the meanings commonly understood by those skilled in the art. For clarity, some terms used herein are defined as follows.

[0091] The core objective of vaccination is to establish long-lasting protective immunity, a process primarily driven by germinal center (GC) responses occurring in the spleen and other secondary lymphoid organs. However, these GC responses significantly weaken with aging, leading to a decline in immune system function. The activation and maturation of GCs largely depend on B cells and the interactions between B cells and regulatory support cells within the microenvironment—especially stromal cells in secondary lymphoid organs.

[0092] The microenvironment of lymphoid organs is established and maintained by lymphosplenic stromal cells. Aging leads to a decrease in the number of stromal cells in the spleen, resulting in tissue structural abnormalities. Furthermore, the regulation of follicular dendritic cells (FDCs), a specialized subset of stromal cells in the microenvironment, has been shown to significantly remodel germinal center responses. The elimination of lymphosplenic cells severely impairs immune responses and antibody production, highlighting the central role of lymphosplenic cells as key regulators of the immune response.

[0093] As used herein, the term "mesenchymal stem cells (MSCs)," also known as "mesenchymal stromal cells," refers to a type of pluripotent cell capable of self-renewal and differentiation into various mesenchymal lineages. These cells are widely distributed in almost all tissues of the human body, playing a crucial role in the development, maintenance, function, and regeneration of most tissues. The low immunogenicity of MSCs gives them a unique advantage in allogeneic therapy. After infusion, MSCs can exert multi-target therapeutic effects, promoting tissue repair and maintaining homeostasis. However, in the context of age-related immunosenescence, the core issue is not excessive inflammatory response, but rather the degenerative changes and dysfunction of the immune system. In this application, MSCs can exert an immune-rejuvenating effect by repairing the health of the thymus and lymphoid tissues, thereby rebuilding a fully functional immune microenvironment. Therefore, MSC-based treatment regimens are an ideal strategy for regulating the spleen microenvironment in the elderly and improving adaptive immune responses.

[0094] MSCs are the most commonly used in cell therapy because they are relatively easy to obtain from a range of tissues and rarely undergo spontaneous differentiation during in vitro expansion. Human MSCs can be obtained from a variety of sources, including bone marrow and systemic vascularized tissues, including adipose tissue, placenta, amniotic fluid, dental pulp, synovium, peripheral blood periodontal ligament, endometrium, and umbilical cord blood. However, the homogeneity, efficacy, and differentiation potential of MSCs can vary depending on their source.

[0095] As used herein, the term 'antigen' refers to a protein, subunit, peptide, or other compound that triggers an immune response when recognized by a subject as something other than themselves. Such an immune response can be mobilized through innate and / or adaptive mechanisms to react against invading pathogens, toxins, or allergens. Antigens can typically originate from pathogenic organisms, such as pathogenic bacteria or pathogenic viruses.

[0096] As used in this article, the term 'subject' refers to an elderly person or an animal, including humans, domesticated animals, companion animals, or canines, such as humans, cattle, cats, dogs, donkeys, pigs, horses, mice, rats, etc., especially humans.

[0097] As used in this article, an older person is a person who is over 65 years of age, for example, over 70 years of age, for example, over 75 years of age, for example, over 80 years of age; for example, between 65 and 80 years of age, for example, between 65 and 90 years of age, for example, between 90 and 100 years of age, for example, over 100 years of age.

[0098] As used herein, the term 'vaccine' refers to an immunogenic composition intended for administration to animals or humans to confer protection against diseases, particularly viral, bacterial, or fungal diseases.

[0099] According to some embodiments of the invention, the vaccine can be delivered to the subject in any form that enables the vaccine to elicit an immune response. Examples of administration routes include: parenteral, subcutaneous, intradermal, oral, intranasal, sublingual, inhalation, local, rectal, buccal, and vaginal. Therefore, according to some embodiments of the invention, a method or application of the invention is provided in which the vaccine is administered via one or more of the following routes: parenteral, subcutaneous, intradermal, oral, intranasal, sublingual, inhalation, local, rectal, buccal, and vaginal.

[0100] According to some embodiments of the present invention, MSCs are administered to the subject via intravenous, intra-arterial, intraperitoneal, intrapleural, intraparenchymal, intraventricular, or intra-articular injection.

[0101] According to some embodiments of the invention, the vaccine is administered some time after the administration of MSCs. Optionally, the administration of MSCs may be stopped at the time of vaccination, or it may continue simultaneously with and after vaccination. Vaccines can be administered several hours to several weeks after MSC administration, for example, at 2 days (48 hours), 3 days, 4 days, 5 days, 6 days, 1 week (7 days), 2 weeks (14 days), 3 weeks (21 days), 4 weeks (28 days), 5 weeks (35 days), 6 weeks (42 days), 7 weeks (49 days), 8 weeks (56 days), 9 weeks (63 days), 10 weeks (70 days), 11 weeks (77 days), 12 weeks (84 days), 13 weeks (91 days), 14 weeks (98 days), 15 weeks (105 days), 16 weeks (112 days), 17 weeks (119 days), 18 weeks (126 days), and any interval between any two adjacent values ​​mentioned above, such as 2 days to 16 weeks.

[0102] As used herein, the term "effective amount" refers to the amount of MSCs sufficient to enhance the immune response in a subject, for example, sufficient to repair splenic stromal cells, promote germinal center formation, and enhance the production of specific antibodies.

[0103] According to some embodiments of the present invention, the effective amount of MSC is 1 × 10⁻⁶ per kilogram of body weight. 5 One cell to 9×10 7 Cells, for example 2×1 Cells, for example, 1 × 10⁻⁶ per kilogram of body weight per day during the period from 2 days prior to inoculation to 16 weeks prior. 5 One cell to 9×10 7 Individual cells.

[0104] In some embodiments, MSCs are administered once daily, meaning at least once every 24 hours. In other embodiments, MSCs are administered multiple times daily, such as twice daily, or three or four times daily. In this case, the daily dose is distributed among these multiple doses. In one embodiment, administration is once daily; in a second embodiment, administration is twice daily; and in a third embodiment, administration is three times daily.

[0105] In some embodiments, the mesenchymal stem cells are autologous stem cells or allogeneic stem cells, or preferably treated autologous stem cells or allogeneic stem cells. VEGFA-positive MSCs are preferred.

[0106] In some implementation plans, vaccines suitable for the elderly include: influenza vaccines, pneumococcal vaccines, shingles vaccines, DPT vaccines, COVID-19 vaccines, and HPV vaccines. Vaccines include inactivated vaccines, live attenuated vaccines, recombinant protein vaccines, and mRNA vaccines.

[0107] The “vaccine” or “vaccine product” in this application includes therapeutic vaccines or preventive vaccines, but preferably preventive vaccines, that is, pharmaceutical products that can induce a specific immune response in the body and form immune memory after vaccination (giving to a subject), thereby preventing a specific disease.

[0108] As used in this article, the term "vaccine adjuvant," also known as immune adjuvant, refers to a class of substances or complexes (including adjuvant systems) added to vaccine formulations that typically do not possess specific antigenicity. Their core function is to activate the body's innate immunity and regulate or prolong adaptive immune responses in a non-specific manner. Specifically, this can be achieved through mechanisms such as activating antigen-presenting cells (APCs), promoting the activation and proliferation of T / B lymphocytes, regulating the type of immune response (humoral immunity / cellular immunity), and prolonging the retention and presentation efficiency of antigens in the body. These mechanisms result in enhanced vaccine immunogenicity, reduced effective antigen dosage, increased immune protection efficacy, and prolonged duration of immune memory, making them particularly suitable for low-immunogenic vaccine systems such as recombinant proteins, subunits, and nucleic acids. The vaccine adjuvants described in this application mainly include: 1. Mineralized salt adjuvants: including traditional aluminum salt adjuvants such as aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate, as well as modified mineralized salt substances such as nano-aluminum salt adjuvants. These are currently the most widely used classic adjuvants and can effectively induce humoral immune responses; 2. Oil-water emulsion adjuvants: including oil-in-water emulsions (such as MF59, whose main components are squalene, Span 85, and Tween 80; AS03, whose main components are squalene, Tween 80, and α-tocopherol) and water-in-oil emulsions (such as Freund's adjuvant, which contains mineral oil and surfactants. The complete Freund's adjuvant also contains inactivated Mycobacterium tuberculosis, while the incomplete Freund's adjuvant does not contain inactivated bacteria. Both are mostly used in animal vaccines); 3. Liposomes and lipid-based complex adjuvant systems: including liposomal adjuvants composed of phospholipid and cholesterol bilayers, and complex adjuvants developed based on liposomes (such as AS01, containing monophosphoacyllipid A (MPLA) and saponin QS-21; AS04, formed by the adsorption of TLR4 agonist molecules onto aluminum salts); 4. Protein adjuvants: including cytokines (such as interleukin-2 (IL-2), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF)) and natural proteins (such as heat shock proteins, complement, CD40 ligands), which can regulate T cell differentiation and enhance immune cell function; 5. Nucleic acid adjuvants: including CpG oligodeoxynucleotides (CpG ODN), which can activate innate immunity and enhance immune responses by targeting model recognition receptors such as TLR9; 6. Polymer adjuvants: These include adjuvants formed from biodegradable polymer materials such as poly(D,L-lactide-glycolic acid) (PLG), polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol, and polystyrene. They have good biocompatibility and can induce a wide range of immune cell responses. 7. Other novel adjuvants: Such as pica adjuvants (targeting TLR3, RIG-I, and MDA5 receptors) and saponin adjuvants (such as QS-21), which can simultaneously promote humoral and cellular immune responses.

[0109] As used in this article, the term "vaccine adjuvant," also known as vaccine excipient, refers to functional components added to vaccine formulations, excluding antigens and adjuvants, to ensure the stability of physicochemical properties, optimize the feasibility of formulation processes, and improve drug safety and clinical compliance throughout the entire process of vaccine production, storage, and use. Their core characteristic is that they do not directly trigger or enhance the body's immune response; they only maintain the stability and performance of the vaccine formulation through physical or chemical actions. The vaccine adjuvants described in this application mainly include: 1. Preservatives: used to prevent contamination of vaccine preparations by bacteria and fungi during production and storage, including thimerosal, 2-phenoxyethanol, etc.; single-dose vaccines usually do not require preservatives due to their sealed packaging, while preservatives are more widely used in multi-dose vaccines; 2. Stabilizers: used to maintain the natural conformation of antigens and prevent them from being degraded, aggregated, or inactivated during storage and transportation, including sugars (sucrose, trehalose, lactose), proteins (human serum albumin), gelatin, etc.; 3. Buffer solutions: used to adjust and stabilize the pH value of vaccine preparations to avoid inactivation due to fluctuations in the acid-base environment, including phosphate buffer solutions (such as disodium hydrogen phosphate, potassium dihydrogen phosphate systems), citrate buffer solutions, Tris buffer solutions, etc.; 4. Osmotic pressure regulators: used to adjust the osmotic pressure of vaccine preparations to match the physiological osmotic pressure of the human body and reduce local irritation after vaccination, including electrolytes such as sodium chloride and potassium chloride; 5. Emulsifiers / suspending agents: used to improve the dispersibility of vaccine formulations and prevent component stratification, including surfactants such as Tween 80 and Span 85 (Note: if such substances are part of the adjuvant system, they should be classified as vaccine adjuvants; if they are only used as formulation dispersants, they are classified as this class); 6. Diluents: used to dilute vaccines before use to ensure the accuracy of the dosage, including sterile saline and sterile water for injection.

[0110] In addition, those skilled in the art can select appropriate conventional vaccine adjuvants and vaccine excipients from the above limitations based on the specific vaccine type and usage requirements.

[0111] Example The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0112] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0113] Materials and methods 1. Animal models mice Young female BALB / c mice (8-12 weeks old) and aged female mice (≥18 months old) were purchased from Guangdong Yaokang Biotechnology Co., Ltd. (Guangdong, China). Female Pdgfrb- Cre mice (≥10 months old) were donated by Professor Tang Yamei's research group at Sun Yat-sen University. Female NDG mice (NOD.CB17-Prkdc scid Il2rg tm1 Bcgen / Bcgen) were purchased from Biocytogen Biosciences Co., Ltd. (Beijing, China). Madcam1 - CreERT2 mice were purchased from Shanghai Model Organisms Research Center Co., Ltd. (Shanghai, China). ROSA26-iDTR Mice were purchased from Jackson Laboratory (Maine, USA). All mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of Sun Yat-sen University. The environmental conditions were: controlled temperature of 24℃±1℃, relative humidity of 50%-60%, and a 12-hour light / 12-hour dark cycle. Mice had free access to standard rodent feed and water. Mice were randomly assigned to their respective experimental groups.

[0114] Crab-eating macaques Senior cynomolgus macaques (17-20 years old) were purchased from HZ-Bio Biotechnology Co., Ltd. They were housed in an air-conditioned environment with a temperature maintained between 16℃ and 26℃, a relative humidity of 40% to 70%, and a 12-hour light / 12-hour dark cycle. The macaques had free access to water and were fed commercial primate feed, fresh fruit, and other nutritional supplements. In addition, the researchers provided the macaques with toys, music, and other forms of entertainment and educational activities, and a veterinarian with over 20 years of experience continuously monitored their health. Detailed information about the macaques is shown in Table 1.

[0115] Table 1. Information on Crab-eating Mammals 2. Ethics Approval Instructions All experimental procedures were conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and relevant regulations of Sun Yat-sen University, and were approved by the Laboratory Animal Ethics Committee of Sun Yat-sen University (Approval No.: SYSU-IACUC-2024-B1562) and Guangzhou Huazhen Biotechnology Co., Ltd. (Approval No.: HZ-EXF-001).

[0116] 3. MSC separation methods The MSCs used in this study were isolated from the bone marrow of healthy donors. The experimental procedures followed the relevant guidelines of the Declaration of Helsinki, and informed consent was obtained from the donors. The specific methods were described in reference (Bao, Y). et al.Ex vivo-generatedhuman CD1c+ regulatory B cells by a chemically defined system suppress immune responses and alleviate graft-versus-host disease. Molecular Therapy 32,4372–4382 (2024); Huang, J. et al. Mesenchymal stromal cells alleviate depressive and anxiety-like behaviors via a lung vagal-to-brain axis in malemice. Nat Commun 14, 7406 (2023); Zhang, X. et al. Mesenchymal stromal cells alleviate acute respiratory distress syndrome through the cholinergic anti-inflammatory pathway. Signal Transduct Target Ther 7, 307 (2022); Zhang, X. et al. CXCR5-Overexpressing Mesenchymal Stromal Cells Exhibit Enhanced Homingand Can Decrease Contact Hypersensitivity. Mol Ther 25, 1434–1447 (2017)). The simplified procedure is as follows: Mononuclear cells were obtained by density gradient centrifugation using a Ficoll-Hypaque (Amersham Biosciences, Cat#:17544202), followed by centrifugation at 1×10⁻⁶ cells / cells. pcs / cm 2 Inoculate at a density of 75 cm 2 Cell culture flasks (Corning, CellBIND); when cell confluence reached approximately 80%, cells were digested using trypsin-EDTA, at which point the cells were defined as generation 1 (P1). Well-identified generation 5-8 (P5-P8) MSCs were used in the experiment.

[0117] The animal dosing regimen is as follows: Take 1×1 One cell line (P5-P8) was suspended in 0.1 ml PBS and transplanted into mice via tail vein injection for MSC administration. For cynomolgus monkeys, MSCs were suspended in 3-5 ml of physiological saline and administered at a dose of 2 × 10⁻⁶ cells per kilogram of body weight. The drug is administered at a dose per cell.

[0118] 4. Immunization methods 100 ng of OVA (egg white albumin, grade II, Sigma, Cat# A5253) was dissolved in sterile ddH2O and then emulsified with Freund's complete adjuvant (Sigma, Cat# SA-F5506) at a 1:1 ratio. Mice were immunized via intraperitoneal injection (ip). A booster immunization was performed 14 days later by intraperitoneal injection of an emulsion of 100 ng of OVA and Freund's incomplete adjuvant (Sigma, Cat# SA-F5881). Serum antibody levels were measured by ELISA on day 28 post-immunization, and germinal center responses were analyzed.

[0119] Mice were immunized with an influenza vaccine (Hualan Biological, quadrivalent / inactivated / lysed virus), with each mouse receiving an intraperitoneal (ip) dose of 7.5 μg. A booster immunization was administered 14 days after the initial immunization at the same dose (7.5 μg). On day 28 post-immunization, serum vaccine-specific antibody titers were measured using a hemagglutination inhibition (HI) test, total serum secretory immunoglobulin G (sIgG) levels were measured using an enzyme-linked immunosorbent assay (ELISA), and germinal center responses were analyzed by flow cytometry.

[0120] Crab-eating macaques were immunized with an influenza vaccine (Hualan Biological, quadrivalent / inactivated / lysed virus), with each macaque receiving an intramuscular (im) dose of 15 μg. A booster immunization was administered 28 days after the initial immunization with the same dose (15 μg). Serum levels of vaccine-specific antibodies and total secretory immunoglobulin G (sIgG) were measured using enzyme-linked immunosorbent assay (ELISA).

[0121] 5. Virus infection and sample collection methods Experimental animals were initially housed in an SPF environment with a 12-hour light / 12-hour dark cycle, and then transferred to an animal biosafety level 2 (ABSL2) laboratory for infection experiments. The influenza A virus strain A / Puerto Rico / 8 / 34 (A / PR8 / 34, H1N1) was kindly provided by Professor Zhao Jincun's research group at the State Key Laboratory of Respiratory Diseases and the National Clinical Research Center for Respiratory Diseases. Mice were anesthetized with isoflurane inhalation and then nasally inoculated with 50 PFU of influenza A virus A / PR8 / 34 (H1N1), with a total dose of 50 μL per mouse, 25 μL instilled into each nostril. Mouse weight was monitored daily post-infection, and mice were euthanized when their weight decreased by 25%. Mice were sacrificed on day 14 post-infection, and lung, spleen, and blood samples were collected. Blood samples were centrifuged at 2600 rpm for 25 minutes at 4°C in the presence of heparin sodium, and the supernatant was collected as plasma. Lung and spleen tissues were fixed in 4% paraformaldehyde solution for further processing.

[0122] The clinical scoring criteria include the presence and extent of inflammatory infiltration, edema, congestion and ecchymosis, and focal necrosis of alveolar epithelium. Specific scoring rules are as follows: 0 points = no abnormalities; 1 point = mild changes; 2 points = moderate changes; 3 points = significant changes; 4 points = severe changes (refer to Barman, TK). et al. Viral PB1-F2and host IFN-γ guide ILC2 and T cell activity during influenza virusinfection. Proc. Natl. Acad. Sci. U.S.A. 119, e2118535119 (2022)). The feeding, use and all experimental procedures of the laboratory animals comply with relevant national regulations and have been approved by the Laboratory Animal Feeding and Use Management Committee.

[0123] 6. Diphtheria toxin (DT)-mediated ablation of spleen stromal cells To achieve PDGFR in the spleen Cell ablation, while ensuring the specificity of the experimental system, to Pdgfrb -Cre mice were directly injected with AAV-DTR (PAV-CAG-DIO-DTR-P2A-mCherry) at a dose of 1 × 10¹¹ viral genome copies (vg) per mouse. Ten days after viral injection, mice were treated with intraperitoneal injection: each mouse in the experimental group received an intraperitoneal injection of 100 ng diphtheria toxin (Millipore, Cat# 322326), while the control group received an equal volume of saline; the drug was administered once daily for three consecutive days.

[0124] 7. Methods for separating spleen cells The enzymatic separation method for stromal cells was basically based on the literature (Denton, AE, Roberts, EW, Linterman, MA & Fearon, DT). Fibroblastic reticular cells of the lymph node are required for retention of resting but not activated CD8+ T cells. Proc Natl Acad Sci U S A 111, 12139–12144 (2014)). Freshly isolated mouse spleens were treated with the following enzyme combination: type IV collagenase (2 mg / ml, Gibco, Cat# 17104019), DNase I (0.1 mg / ml, Thermo Fisher Scientific, Cat# EN0521), Liberase (0.2 mg / ml, Roche, Cat# 5401119001), and Dispase® II (0.8 mg / ml, Roche, Cat# 4942078001). Spleen tissue was efficiently and gently processed using a gentle MACS tissue dissociator (Miltenyi Biotec, Cat# 130-093-235) to prepare a spleen cell suspension; subsequently, the suspension was digested in a 37°C water bath with frequent agitation during digestion. Cells were collected and resuspended in a-MEM medium containing 1% FBS (PAN-Biotech, Cat# P30-3033) and 2 mM EDTA (Sigma-Aldrich, Cat# 4008-M). After filtration through a 70 μm cell filter, single cells were collected and washed three times with PBS. Red blood cells were removed using erythrocyte lysis buffer (Roche), and spleen cells were then resuspended in a-MEM medium (Hyclone, Cat# SH30265.FS) containing 10% FBS and 1% penicillin / streptomycin (Hyclone, Cat# SV30010) to maintain cell viability. Finally, spleen stromal cells (CD4+) were sorted by flow cytometry. CD3 Ter11 PDGFR ).

[0125] 8. Flow cytometry detection Cells were collected into flow cytometry tubes and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded to obtain a cell pellet. Cells were washed twice with phosphate-buffered saline (PBS, Gibco, Cat# 10010023). Cells were resuspended in permeabilization buffer (perm buffer):antibody = 50:1 (volume ratio) and incubated at 4°C in the dark for 30 minutes. After incubation, cells were washed twice with PBS and centrifuged at 1500 rpm for 5 minutes. Cells were resuspended in PBS, filtered through a 70 μm cell filter (Corning, Cat# 352350), and analyzed by flow cytometry. Antibody concentrations were used according to the manufacturer's recommended protocol; details are shown in Table 2. Experimental data were acquired using a CytoFLEX flow cytometer (Beckman) and analyzed using FlowJo 10.6.2 software (FlowJo LLC).

[0126] 9. Immunofluorescence staining Freshly isolated mouse spleens were fixed overnight at 4°C with 4% paraformaldehyde (PFA, PHYGENE, Cat# PH0427), followed by dehydration in 30% sucrose solution (MilliporeSigma, Cat# 57-50-1) at 4°C until the tissue was completely solidified. The treated spleen tissue was embedded in a Tissue-Tek optimal cutting temperature complex (Sakura, Cat# 4583) and stored at -80°C. Subsequently, 15 μm thick frozen sections were prepared using a cryostat (RWD FS800). Spleen tissue sections were first incubated at room temperature with 0.5% Triton X-100 (Sigma Aldrich, Cat# 9036-19-5) for 20 minutes, followed by blocking with goat serum (BOSTER, Cat# AR0009) for 30 minutes. The staining procedure was as follows: The slides were incubated overnight at 4°C with primary antibody (diluted in PBS containing 0.1% BSA); after washing five times, the slides were incubated at room temperature for 2 hours with secondary antibody (diluted in PBS containing 0.1% BSA). The concentrations of both primary and secondary antibodies were used according to the manufacturer's recommended protocols; detailed information is provided in Table 2. In addition, nuclear staining was performed using DAPI (Sigma Aldrich, Cat# D9542). The stained slides were mounted with fluorescent mounting medium (Dako, Cat# 302380-2), covered with coverslips (CITOTEST, Cat# 0212450C), and finally imaged using a confocal microscope (Dragonfly, CR-DFLY-202 2540).

[0127] Table 2. Antibodies 10. Histopathological examination Mouse spleen, lymph nodes, and lung tissues were prepared using the formalin fixation-paraffin embedding-sectioning method. The simplified steps are as follows: Formalin-fixed and paraffin-embedded tissue blocks were used to prepare tissue sections with a thickness of 4 μm. After dewaxing and rehydration, the sections were boiled in citrate buffer (pH 6.0) at 97°C for 20 minutes to extract antigenic epitopes. Subsequently, the sections were incubated with 3% H₂O₂ solution for 15 minutes to block endogenous peroxidase activity. The sections were then treated with blocking solution at room temperature for 10 minutes. Finally, the sections were stained with hematoxylin (Sigma Aldrich, Cat# H9627) and eosin (Sigma Aldrich, Cat# E4009), respectively.

[0128] 11. Enzyme-linked immunosorbent assay (ELISA) Plasma or bronchoalveolar lavage fluid from normal control BALB / c mice and model mice were collected, placed in centrifuge tubes, and centrifuged at 2500 rpm for 20 minutes at 4°C. The supernatant was collected for testing. The experiments used enzyme-linked immunosorbent assay (ELISA) kits for mouse immunoglobulin G (IgG), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) (Neobioscience, Cat# EMC116, EMC101g, and EMC102a); the detection of ovalbumin-specific secretory immunoglobulin G1 (OVA-sIgG1) in mice was performed using a mouse anti-OVA IgG1 antibody detection kit (Chondrex, Cat# 3013). All experimental procedures were strictly performed according to the manufacturer's instructions. The steps are briefly as follows: The collected supernatant is added to a 96-well plate pre-coated with TNF-α, interleukin-1β (IL-1β), IFN-γ, soluble OVA-IgG1 and IgG antibodies, and the detection is performed using the enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method; finally, the absorbance value of each well is measured by an ELISA reader (Thermo Fisher Scientific).

[0129] For the detection of specific antibodies against influenza vaccines (Hualan Biological, quadrivalent / inactivated / lysed virus), the procedure is as follows: Four recombinant antigen HA proteins—influenza A H1N1 (A / Victoria / 4897 / 2022 strain), influenza A H3N2 (A / Darwin / 9 / 2021 strain), influenza B (B / Austria / 1359417 / 2021 strain), and influenza B (B / PHUKET / 3073 / 2013 strain) hemagglutinin (all purchased from Shenzhou Cell Engineering Co., Ltd.)—were diluted separately to a final concentration of 1 μg / ml in coating buffer (pH 9, 60.05 M carbonate buffer, 1.59 g Na2CO3, 2.93 g NaHCO3, distilled water to 1000 ml). The solutions were then added to a 96-well clear flat bottom polystyrene microplate. Bind Microplate (Corning, Cat# 9018) was incubated at 4°C for 16 hours. 300 μL of washing buffer was added to each well, and the plate was washed 5 times. Then, it was blocked with 200 μL PBST + 5.0% skim milk powder at room temperature for 2 hours. After washing the plate 3 times with washing buffer, horseradish peroxidase (HRP)-conjugated goat anti-monkey IgG antibody (1:10000, Invitrogen) was added, and the plate was incubated at 37°C for 60 minutes. The plate was washed 5 times again with washing buffer, and the chromogenic solution was added. The plate was incubated at 37°C for 15 minutes. The absorbance (450 / 630 nm) was read using a BioTek microplate reader. The antibody endpoint titer was determined according to the kit manufacturer's instructions.

[0130] 12. Western Blot To investigate the effect of VEGFA siRNA on VEGFA protein expression levels in MSCs, a Western blot assay was performed. MSCs were transfected with siRNA for 72 hours, and the culture medium was collected. Adherent cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer (Sigma-Aldrich, Cat# R0278) containing benzyl sulfonyl fluoride (PMSF, Roche, Cat# 329-98-6) to extract total protein. Protein concentration was determined using the Pierce™ BCA protein quantification kit (Thermo Fisher Scientific, Cat# 23227). After denaturation at 100°C, the expression levels of VEGFA and GAPDH proteins were detected according to the standard Western blot procedure provided by Abcam. 10 μg of total protein from the supernatant and total protein from cell lysate were sequentially subjected to SDS-PAGE gel electrophoresis, transferred to a membrane, and blocked. Then, primary antibodies [anti-human VEGFA antibody (Abogen Biosciences, Cat# ab46154), anti-GAPDH antibody (Cell signaling, Cat# 97166)] and secondary antibodies [anti-rabbit IgG HRP conjugate antibody (Cell signaling, Cat# 7076), anti-mouse IgG HRP conjugate antibody (Cellsignaling, Cat# 7076)] were added and incubated. Finally, protein signals were detected by chemiluminescence immunoassay, and images were acquired using a ChemiDoc imaging system (Bio-Rad).

[0131] 13. Statistical Analysis and Experimental Reproducibility Verification All experiments in this study were independently repeated at least three times to ensure the reliability and reproducibility of the results. The legends in each figure indicate that the experimental data are summaries of multiple independent experiments. Statistical analysis and visualization of the experimental data were performed using GraphPad Prism 9 software. Statistical analysis was conducted using one-way ANOVA, Kruskal-Wallis multiple comparison test, Mann-Whitney rank-sum test, or unpaired t-test, according to the annotations in the figure legends. P A value <0.05 was used as the criterion for statistical significance. Each data point in the attached figure represents one biological replicate, and the fitted line represents the median of each group.

[0132] Example 1: MSCs repair age-related defects in spleen stromal cells of aged mice First, changes in the spleen during aging were examined. Comparison of the spleens of young and aged BALB / c mice revealed mild spleen atrophy in aged mice (18-21 months old) compared to young mice (2-3 months old). The proportion and number of stromal cells in the spleen of aged mice were significantly reduced, a conclusion confirmed by immunofluorescence staining. Furthermore, immunofluorescence staining and hematoxylin and eosin (H&E) staining showed structural disorder in the spleen of aged mice, with a significant reduction in the area of ​​the white pulp region (data not shown). In summary, these results indicate that aging leads to a decrease in the number of stromal cells in the spleen and disrupts its microstructure.

[0133] To validate the unique translational application potential of MSCs at the functional level, aged mice were intravenously infused with human MSCs (rather than non-human MSCs) because human skin fibroblasts (HDF) and MSCs share the same mesodermal origin and are similar in cell morphology, in vitro behavior, and some surface markers. Therefore, mice infused with HDF served as the control group, while mice infused with the same volume of saline served as the Vehicle group. FIG. 1A Twenty-eight days after MSC infusion, compared with the aged Vehicle group, the MSC group mice showed significantly increased spleen volume, significantly higher spleen-to-body ratio (also known as spleen index in mouse experiments), and an increased total number of spleen cells. FIG. IB , FIG. 1C Mice in the HDF group did not exhibit the aforementioned changes. Furthermore, MSC infusion significantly increased the proportion and number of splenic stromal cells, while HDF infusion did not induce significant changes. FIG. IE-FIG. IG These pieces of evidence directly demonstrate that the therapeutic effect of MSCs does not stem from the body's nonspecific response to exogenous cells, but rather depends on their unique regulatory mechanisms.

[0134] Immunofluorescence staining further confirmed that MSCs could repair the stromal cell network of the spleen in aged mice. Within one week after MSC infusion, signs of recovery in splenic stromal cells began to appear, and this repair was well maintained until week four. FIG. IH , FIG. II Simultaneously, the spleen's microstructure was reconstructed, and the distribution areas of B lymphocytes and T lymphocytes significantly expanded; this effect began to appear in the 3rd week after MSC infusion and further improved in the 4th week (Figure 1J). FIG. IK By this time, the infused MSCs had been largely cleared from the spleen. In summary, these results indicate that exogenous MSCs can remodel the stromal cell network of the spleen in aged mice, thereby restoring the homeostasis of the immune cell population and the integrity of the spleen's tissue structure.

[0135] Example 2: MSC-mediated spleen rejuvenation promotes germinal center formation and enhances specific antibody production in aged mice. Given that MSCs can repair spleen stromal cells and reconstruct their microstructure in aged mice, this study further explored the potential of MSCs to improve age-related humoral immune decline. The experimental design was as follows: MSCs were infused into aged BALB / c mice (18-21 months old). FIG. 2A After 28 days of treatment, mice were intraperitoneally immunized with ovalbumin (OVA) combined with complete Freund's adjuvant (CFA), followed by a booster immunization with OVA combined with incomplete Freund's adjuvant (IFA) 14 days later. Mice that received only MSCs without OVA immunization were assigned to the MSC group; mice that received an equal volume of saline without OVA immunization were assigned to the Sham group; mice that received a combination of MSC infusion and OVA immunization were assigned to the MSC+OVA group; and mice that received an equal volume of saline and OVA immunization were assigned to the Sham+OVA group. Results showed that the spleen of mice in the Sham group did not change significantly after OVA immunization, while the spleen volume of mice in the MSC-treated group increased significantly, especially after OVA immunization. FIG. 2B-FIG. 2E The production of high-affinity antibodies typically depends on antibody affinity maturation within the germinal center, a process often impaired during aging. Therefore, the formation of germinal centers was further examined. Interestingly, CD4+ was found in the spleen of mice treated with MSCs. B22 Ig GL The proportion of B cell subsets in germinal centers was significantly increased ( FIG. 2F , FIG. 2G Furthermore, compared to the Sham group, the area of ​​peanut agglutinin (PNA) positive fluorescent regions (i.e., germinal center regions) in the spleen of mice in the treatment group was significantly larger, further confirming that MSCs can promote germinal center formation. FIG. 2H , FIG. 21 Furthermore, it was observed that follicular helper T cells (Tfh) were localized within germinal centers, providing support for germinal center responses. This result further corroborates that MSC treatment can enhance the germinal center formation capacity of the spleen in mice immunized with OVA.

[0136] After immunization with OVA in young mice, aged mice (sham mice), and MSC mice, serum total immunoglobulin G (IgG) levels were significantly elevated, indicating that the immune systems of all three groups of mice were activated. Consistent with germinal center formation, serum total IgG levels in aged mice were significantly lower than in young mice, while MSC intervention significantly promoted IgG synthesis in aged mice. FIG. 2J Most importantly, OVA-specific IgG level analysis showed that the antibody response level after immunization in young mice was significantly stronger than that in older mice. FIG. 2K ,FIG. 2L MSC treatment significantly increased the concentration and titer of OVA-specific IgG in the serum of aged mice, highlighting the therapeutic potential of MSC cells in enhancing antigen-specific antibody responses in aged populations. FIG. 2K , FIG. 2L In summary, these results indicate that MSCs rejuvenate the spleen in aged mice by regulating splenic stromal cells, thereby promoting germinal center formation and enhancing the production of specific antibodies.

[0137] Example 3: MSC-mediated vaccine efficacy is enhanced in aged mice and can effectively prevent influenza infection in aged mice. Most vaccines targeting the elderly offer limited protection, leading to significantly higher morbidity and mortality rates after infection with pathogens such as influenza A virus. To address this issue, we evaluated the effect of MSC infusion on the efficacy of vaccines and protection against influenza infection in aged BALB / c mice. Four weeks prior to vaccination, aged mice underwent MSC infusion (…). FIG. 3A To bridge the gap between preclinical research and clinical application, mice were immunized with a quadrivalent influenza vaccine (QIV, currently the standard clinical vaccine for human influenza vaccination) using a two-dose regimen, with a 14-day interval between the two doses. The unvaccinated group was designated Nonvac; the group receiving the same volume of saline without MSC infusion and then vaccinated was designated Ctrlvac; and the group receiving MSC infusion followed by vaccination was designated MSC+Vac. Consistent with the results of the OVA immunization experiment, the spleen volume of mice in the MSC-treated group was significantly increased, and the number of splenic lymphocytes and germinal center B cells were also increased. FIG. 3B-FIG. 3F This indicates that the vaccine-induced immune response in this group of mice was significantly stronger than that in the control group. Immunofluorescence staining further confirmed the enhanced germinal center formation capacity of the spleen in the treated group mice. FIG. 3G Furthermore, after vaccination, the increase in serum total IgG levels in the MSC-treated group was significantly higher than that in the control group. FIG. 3H Subsequent hemagglutination inhibition (HI) assays showed that the titer of specific antibodies against the influenza vaccine in the serum of mice treated with MSCs was significantly higher than that in the control group (Figure 3I). These results indicate that MSC infusion can significantly enhance the humoral immune response to the influenza vaccine in aged mice.

[0138] To comprehensively evaluate the immunoprotective potential conferred by MSC-mediated humoral immunity enhancement, mice were challenged with the H1N1 influenza A virus strain A / PR8 / 34. FIG. 31This strain is a commonly used adapted virus model in laboratories, and studies have confirmed that antibodies induced by quadrivalent influenza vaccines can provide cross-protective humoral immunity against this subtype of virus. Among them, the uninfected aged mouse MSC Vehicle group consisted of mice infused with MSCs but not infected with the strain; the uninfected aged mouse Vehicle group consisted of mice infused with the same dose of PBS but not infected with the strain; the Ctrl PR8 group consisted of mice infused with the same dose of PBS but not infected with the strain; and the MSC+PR8 group consisted of mice infused with MSCs and infected with the strain. After challenge, the aged control mice experienced persistent weight loss and all died within two weeks. FIG. 3J , FIG. 3K Mice in the MSC-treated group began to gradually regain their body weight on day 7 post-infection, and their survival rate after viral challenge reached 90%. FIG. 3L , FIG. 3K Furthermore, the lung wet weight of infected mice in the control group was significantly increased, and the viral titer in lung tissue was significantly elevated, while the above indicators were significantly reduced in infected mice in the treatment group. FIG. 3L , FIG. 3M ). Lung tissue pathological analysis and clinical scoring results further confirmed the therapeutic effect of MSCs, with reduced lung inflammation and alleviated clinical symptoms in the treated mice. FIG. 3N , FIG. 3O Meanwhile, the levels of pro-inflammatory factors in the lung tissue and peripheral blood of mice in the post-infection treatment group were significantly lower than those in the control group. FIG. 3P , FIG. 3Q These results suggest that MSCs have the potential to improve age-related humoral immune decline, enhance vaccine response in older adults, and provide effective protection against pathogen infections.

[0139] Example 4: MSCs reconstruct the splenic stromal cell network by promoting the proliferation of reticular cells in the splenic marginal zone. Following systemic infusion of MSCs, the cells initially remain in the lungs, then redistribute and colonize the liver, while accumulating significantly in the spleen. In vivo two-photon confocal microscopy confirmed that MSCs can efficiently colonize the spleen, are present in very small quantities in the liver, and are undetectable in the kidneys and other organs. Furthermore, splenectomy experiments were conducted to rule out the effects of non-splenic targets. Results showed that the MSC-enhanced vaccine response effect was completely eliminated after splenectomy (data not shown). These findings mechanistically confirm that the spleen is an indispensable lymphoid organ microenvironment mediating the immune-enhancing effects of MSCs.

[0140] Subsequently, the mechanism of MSC remodeling in aged spleens was investigated. NDG immunodeficient mice, lacking T cells, B cells, and natural killer (NK) cells, were used in the experiment. It was found that MSCs could still effectively repair the spleen's matrix structure, suggesting that this repair process may not depend on the participation of immune cells. To further verify this conclusion, [further details were needed]. FIG. 3R -Cre mice underwent targeted ablation of splenic stromal cells via administration of diphtheria toxin followed by delivery of a Cre-dependent adeno-associated virus construct (PAV-CAG-DIO-DTR-P2A-mCherry). This conditional ablation system specifically removed almost all splenic stromal cells without affecting PDGFR in other organs, including the liver, which is closely related to the spleen. Cells. Interestingly, in these stromal cell ablation mice, MSCs were unable to reconstruct the survival microenvironment of T and B lymphocytes, and consequently, could not increase antibody levels after OVA immunization. Furthermore, in the early stages 3 days after MSC infusion into aged BALB / c mice ( PDGFRβ The study found that the proliferative activity of mouse spleen cells (expressed as Ki-67) was significantly enhanced, and this proliferative effect was observed in CD4+ cells. This is particularly evident in cell subsets. Remodeling of spleen stromal cells precedes changes in T and B lymphocytes. FIG. 4A This result further corroborates the above observations. In summary, these results suggest that spleen stromal cells may be a key target cell population for MSCs to exert their interventional effects.

[0141] Splenic stromal cells are mainly divided into three types: marginal zone reticular cells (MRCs), follicular dendritic cells (FDCs), and fibroblastic reticular cells (FRCs). It was found that a large number of infused MSCs could be distributed in the mouse spleen, and mainly colonized the splenic marginal zone composed of MRCs expressing the adhesion molecule MAdCAM-1 (Figure 4B). FIG. 1J-FIG. IK Notably, after MSC infusion (MSC group), MRC (CD4+) levels in the spleen of mice were significantly increased. CD3 Ter11 PDGFR MAdCAM- The proportion and absolute number of MSCs were significantly higher than those in the control group (sham group, receiving the same volume of saline) compared to the control group (sham group, receiving the same volume of saline). FIG. 4C ), prompt MAdCAM- MRC is likely the primary target of MSCs. Results from MSC-mediated cell proliferation experiments further validate this hypothesis: after MSC infusion, MAdCAM- Ki-6 in MRC The proportion of proliferating cells was significantly higher than that of the control group that did not receive MSC infusion. FIG. 4D-FIG. 4F The proliferation levels of FDC and FRC were not significantly affected. Furthermore, quantitative spatial analysis showed that, compared to Ki-6... Compared to the MRC, the Ki-6 The distance between MRC and MSC has been significantly shortened. p <0.01), the distance between the two decreased by about 40%. Extending the observation period, analysis of the spleens of aged mice 1-4 weeks after MSC infusion (MSC group) revealed that in week 1 after infusion, MAdCAM- The number of MRCs was significantly increased compared to the sham group (which received the same dose of normal saline), and this level was well maintained from week 2 to week 4. FIG. 4G-FIG. 4J , FIG. 4K Interestingly, MAdCAM- was also detected in the white pulp region of the mouse spleen 3-4 weeks after infusion. Cells, at the same time, FDC-M The number of follicular dendritic cells also gradually increased. FIG. 4L , FIG. 4M ).

[0142] In addition, select FIG. 4N -Cre / ERT2 Madcam1 Mice, by administration of diphtheria toxin to specifically clear MAdCAM- Cells. This method selectively targets reticular cells (MRCs) in the marginal zone of the spleen without affecting the follicular dendritic cell (FDC) population or the overall lymphoid tissue structure of the spleen. Crucially, after MRC clearance, the effects of MSCs promoting germinal center responses and the production of antigen-specific antibodies after OVA immunization were completely eliminated. These results provide conclusive evidence confirming the effectiveness of MAdCAM- MRCs are the core mediators of MSCs' immune regulation in the spleen. Based on the above findings, we conclude that MSCs promote the proliferation of MRCs in the spleen of aged mice, thereby driving the expansion of the follicular dendritic cell population. This process is crucial for promoting germinal center formation and enhancing vaccine response.

[0143] Example 5: MSC-mediated enhanced vaccine efficacy observed in an aged cynomolgus monkey model To evaluate the translational application potential of MSC intervention strategies in human vaccination, an influenza vaccination model was constructed using elderly cynomolgus monkeys (17-20 years old, Table 1). The modeling method was based on the following references: (1. Clemens E, Angeletti D, Holbrook BC, Kanekiyo M, Jorgensen MJ, Graham BS, Yewdell J, Alexander-Miller MA. Influenza-infected newborn and adult monkeys exhibit a strong primary antibody response to hemagglutinin stem. JCI Insight. 2020 Mar 12;5(5):e135449. doi: 10.1172 / jci.insight.135449. PMID: 32078584; PMCID: PMC7141390;2. Carroll TD, Matzinger SR, Barry PA, McChesney MB, Fairman J, Miller CJ. Efficacy of influenza vaccination of elderly rhesus macaques is Dramatically improved by addition of a cationic lipid / DNA adjuvant. J Infect Dis. 2014 Jan1;209(1):24-33. doi: 10.1093 / infdis / jit540. Epub 2013 Oct 17. PMID: 24141979; PMCID: PMC3864389; 3. Nakayama, M., Kitagawa, N., Nguyen, CT x ROSA26-iDTR Long-term efficacy of an inactivated H5N1 whole-particle influenza vaccine in nonhuman primates. et al. 10, 164 (2025). https: / / doi.org / 10.1038 / s41541-025-01221-x; 4. Crofts, KF, Holbrook, BC, Page, CL npj VaccinesAntibody function predicts viral control in newborn monkeys immunized with aninfluenza virus HA stem nanoparticle. et al. 16, 3785 (2025). https: / / doi.org / 10.1038 / s41467-025-59149-8. The cynomolgus monkey's immune system is highly similar to that of humans, making it an ideal animal model for preclinical research.

[0144] Prior to administration of MSCs, blood samples were collected from all experimental animals, and abdominal spleen ultrasound was performed to obtain baseline data. Subsequently, aged cynomolgus monkeys were intravenously infused with either MSCs or an equivalent dose of phosphate-buffered saline (PBS, vehicle control group), and their health status was assessed daily. Nat Commun Nine weeks after infusion, all experimental animals received primary immunization with a quadrivalent influenza vaccine (QIV) at a dose of 0.5 mL, administered intramuscularly. This vaccine contains hemagglutinin antigens from four influenza virus strains (H1N1, H3N2, Victoria strain B, and Yamagata strain B), with each strain containing 15 μg of HA. Following immunization, blood samples were collected periodically, and abdominal spleen ultrasound was performed to monitor the immune response. Four weeks after primary immunization, all experimental animals received a booster immunization. FIG. 5A ).

[0145] Abdominal ultrasound imaging showed a positive correlation between enhanced immune response and increased spleen length: MSC infusion group (MSCvac, n =5) The spleen length of cynomolgus monkeys increased significantly in weeks 2 and 4 after booster immunization. FIG. 5A ); while the vehicle control group (Ctrl vac, n =3) No significant changes were observed. Furthermore, the levels of vaccine-specific antibodies in cynomolgus monkey serum before and after booster doses were detected using ELISA after coating with the antigen proteins of the four major viral strains contained in the vaccine, including specific IgG antibodies against influenza A H1N1, H3N2, and influenza B Victoria and Yamagata strains. Consistent with expectations, after primary immunization, the titers of all four specific IgG antibodies in the MSC infusion group were significantly higher than those in the Ctrl vac group ( FIG. 5B-FIG. 5C After increasing the dosage, the antibody titers in both groups increased further, with the increase being more significant in the MSC infusion group. FIG. 5D-FIG. 5G Of particular importance, at all corresponding time points, the antibody levels in the MSC infusion group were consistently higher than those in the Ctrl vac group. FIG. 5D-FIG. 5G This confirms that MSC-mediated enhancement of vaccine efficacy.

[0146] Furthermore, hematological analysis showed that the relative proportions of white blood cells and lymphocytes in cynomolgus monkeys were significantly increased after immunization, and this effect could persist until week 8. FIG. 5D-FIG. 5G Lymphocyte count, CD4 / CD8 ratio, and CD1+ were measured by flow cytometry in the MSC infusion group. CD2 CD3 B cells (plasma blasts) were also significantly elevated, further supporting the conclusion that this group of animals produced a superior vaccine response. FIG. 5H-FIG. 51 FIG. 5J-FIG. 5K ).

[0147] These results demonstrate that MSC therapy significantly enhances the immune response of older cynomolgus monkeys to the quadrivalent influenza vaccine, highlighting the great potential of this therapy in improving vaccine efficacy in older populations.

[0148] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of mesenchymal stem cells (MSCs) in the manufacture of drugs for improving the immune response to vaccination in elderly or animal subjects.

2. The application according to claim 1, wherein the enhancement of the immune response to vaccination in elderly or animal subjects comprises one or more of repairing splenic stromal cells, promoting germinal center formation, and enhancing the production of specific antibodies.

3. The use of the combination of MSCs and vaccines in the manufacture of drugs for the prevention of infectious diseases in elderly people or animal subjects.

4. The application according to any one of claims 1 to 3, wherein the vaccine targets an infectious disease, preferably a pathogen infection, said pathogen preferably selected from one or more of bacteria, viruses or fungi.

5. The application according to any one of claims 1 to 4, wherein the vaccine targets an infectious disease caused by a virus, preferably the virus being selected from one or more of rhinovirus, coronavirus, influenza virus, or respiratory syncytial virus; preferably the coronavirus being selected from one or more of avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV, and the influenza virus being selected from influenza A virus and / or influenza B virus; preferably the influenza virus being selected from one or more of influenza A H1N1, H3N2, influenza B Victoria lineage, and influenza B Yamagata lineage.

6. The application according to any one of claims 1 to 5, wherein the MSC is administered to the subject by intravenous, intra-arterial, intraperitoneal, intrapleural, intraparenchymal, intraventricular, or intra-articular injection.

7. The application according to any one of claims 1 to 6, wherein the MSC is a VEGFA-positive MSC, preferably, the MSC is autologous, allogeneic or xenogeneic to the subject, and more preferably, the MSC is human MSC.

8. The application according to any one of claims 1 to 7, wherein the subject is a human, a domesticated animal, a companion animal, or a canine.

9. A pharmaceutical composition comprising MSC and a vaccine product, each in separate packaging or container, said vaccine product comprising an antigenic component, an adjuvant, and an excipient.

10. A pharmaceutical kit comprising MSCs in a first container, a vaccine product in a second container, and instructions for use, wherein the vaccine product comprises an antigen component, an adjuvant, and an adjuvant, and the instructions for use include instructions to administer the MSCs first, followed by the vaccine product.

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