Mesenchymal stem cells as vaccine adjuvants and methods for using same

Abstract

The present invention provides a method for enhancing an immune response to a vaccine by administering the vaccine and a population of isolated allogeneic human mesenchymal stem cells. The present invention also provides a kit comprising a vaccine in a first container and a population of isolated allogeneic human mesenchymal stem cells in a second container. [Selected Figure] Figure 5D

Description

[Technical Field]

[0001]

[0001] The present invention relates to vaccines and vaccine adjuvants, methods of using same, and kits containing same. In particular, the present invention relates to vaccine adjuvants comprising isolated allogeneic human mesenchymal stem cells. [Background technology]

[0002] Age-related frailty and inflammaging

[0002] Age-related frailty poses very serious problems to an individual's overall health and well-being. Age-related frailty is a geriatric syndrome characterized by weakness, low physical activity, slow motor activity, extreme fatigue, and unintentional weight loss. See Yao, X. et al., Clinics in Geriatric Medicine 27(1):79-87 (2011). Furthermore, there are many studies showing a direct correlation between age-related frailty and inflammation. See Hubbard, RE et al., Biogerontology 11(5):635-641 (2010).

[0003]

[0003] Immunosenescence is characterized by a state of low-grade chronic systemic inflammation known as inflammaging. See Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000). This state of high inflammation or chronic inflammation seen in aging and age-related frailty leads to immune dysregulation and complex remodeling of both innate and adaptive immunity. In immunosenescence, the T cell and B cell repertoire becomes overexpressed by CD8 IgG1-dependent inflammatory cytokines that re-express CD45ra (TEMRA). + Effector memory T cells and CD19 + Increase in late / exhausted memory B cells and CD8 + Naive T cells and switched memory B cells (CD27 +) as a result of a decrease in immune responses. See Blomberg, B.B. et al., Immunologic Research 57(1-3):354-360 (2013); Colonna-Romano, G. et al., Mechanisms of Ageing and Development 130(10):681-690 (2009); and Koch S. et al., Immunity & Ageing: 5:6 (2008). This shift in T cell and B cell repertoire results in a refractory or inefficient immune state. This immune system deterioration leads to high susceptibility to infectious diseases and a poor response to vaccination. Optimal B cell function is critical for providing effective antibody responses to vaccines and protection from infectious pathogens. It is well known that age-related increases in systemic inflammation (TNF-α, IL-6, IL-8, INF-γ, and CRP) cause a decline in B cell function, leading to inadequate antibody responses and poor vaccine efficacy.

[0004]

[0004] Inflammation has attracted considerable attention because it suggests a link between immune changes and many diseases and conditions common in aging, such as age-related frailty. Circulating inflammatory mediators, such as cytokines and acute-phase proteins, are markers of low-grade inflammation that have been shown to increase with age. These proinflammatory cytokines (e.g., TNF-α, IL-6) reduce the ability of B cells to produce protective antibodies against foreign antigens and vaccines. This reduced B cell response is measured by a decrease in class switch recombination (CSR), the ability of immunoglobulins to switch isotype from IgM to a secondary isotype (IgG, IgA, or IgE). Immunoglobulin isotype switching is crucial for appropriate immune responses because effector functions differ for each isotype. A key participant in CSR and somatic hypermutation (SHM) is the enzyme activation-induced cytidine deaminase (AID), encoded by the Aicda gene. The essential function of AID in CSR and SHM is to initiate DNA cleavage by converting cytosines to uracils in immunoglobulin switch and variable regions. E47, encoded by the Tcfe2a (E2A) gene, is a transcription factor belonging to the class I basic helix-loop-helix (bHLH) protein family, also known as E proteins. Absence of E47 expression results in the absence of expression of the B cell-specific transcription factors EBF1 (early B cell factor) and Pax-5 (paired box protein). Both E47 and Pax-5 are important transcription factors in the early development of the B cell lineage and the function of mature B cells. See Hagman J. et al., Immunity 27(1):8-10 (2007); Horcher M. et al., Immunity 14(6):779-790 (2001); Riley RL et al., Seminars in Immunology 17(5):330-336 (2005). The Pax-5 gene encodes the B cell lineage-specific activating protein (BSAP), which is expressed at all stages of B cell differentiation but not in terminally differentiated B cells.Pax-5 regulates B cell commitment by suppressing inappropriate B lineage genes and activating B cell-specific genes to produce the B cell gatekeeper Pax-5, which is expressed exclusively in the B lymphocyte lineage from committed pro-B cells to mature B cells. The B cell-specific transcription factor Pax-5 is not only crucial for early B cell development and B cell lineage commitment, but also participates in CSR.

[0005]

[0005] The amount of TNF-α produced (1) depends on the amount of inflammation in the system, and (2) has also been shown in humans to reduce the ability of the same B cells to be stimulated by mitogens or antigens. See Frasca, D. et al., Journal of Immunology 188(1):279-286 (2012). Thus, immune responses in subjects suffering from aging frailty are reduced for many reasons.

[0006] Vaccination of the elderly

[0006] Vaccination against influenza is strongly recommended for individuals over 65 years of age to protect against infection. Commercially available influenza vaccines provide protection and ensure continued immunological memory in children and adults, but are less effective in elderly and frail individuals. See Frasca D. et al., Current Opinion in Immunology 29:112-118 (2014) and Yao X. et al., Vaccine 29(31):5015-5021 (2011). Despite receiving regular influenza vaccinations, elderly individuals are at higher risk of influenza infection, which can lead to secondary complications, hospitalization, physical debilitation, and ultimately death. See Gross, P. et al., Annals of Internal Medicine 123(7):518-527 (1995); Simonsen L. et al., The Journal of Infectious Diseases 178(1):53-60 (1998); and Vu T. et al., Vaccine 20(13-14):1831-1836 (2002). Influenza vaccines also prevent other complications resulting from influenza infection (e.g., pneumonia) in most elderly individuals and reduce hospitalization rates to a certain extent. Nichol KL et al., The New England Journal of Medicine 331(12):778-784 (1994). However, hospitalization rates for influenza-related illnesses remain very high. See Thompson, WW et al., JAMA 292(11):1333-1340 (2004). Thus, there remains a need to enhance vaccine immune responses in the elderly.

[0007] Previously published results have shown that the specific B cell response to influenza vaccines in vitro (as measured by AIDs) and the in vivo serum response (as measured by HAI assay and ELISA) decline with age and are significantly correlated. See Frasca, D. et al., Vaccine 28(51):8077-8084 (2010). The percentage of switched memory B cells and CpG-induced AIDs, both measured before vaccination (t0), were also found to decline with age and significantly correlated with in vivo responses. Thus, these markers predict in vivo responses.

[0008] Mesenchymal stem cells

[0008] Mesenchymal stem cells are multipotent cells capable of migrating to sites of injury and are also immune privileged by expressing undetectable major histocompatibility complex class II (MHC-II) molecules and low levels of MHC-I molecules. See Le Blanc, K. et al., Lancet 371(9624):1579-1586 (2008) and Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005). Thus, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine and have repeatedly been shown to have a high safety and efficacy profile in clinical trials for multiple disease processes. See Hare, JM et al., Journal of the American College of Cardiology 54(24):2277-2286 (2009); Hare, JM et al., Tex. Heart Inst. J. 36(2):145-147 (2009); and Lalu, MM et al., PloS One 7(10):e47559 (2012). Mesenchymal stem cells have also been shown not to undergo malignant transformation after transplantation into patients. See Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005). Treatment with mesenchymal stem cells has been shown to reverse severe graft-versus-host disease, prevent acute ischemic renal failure, contribute to pancreatic islet and renal glomerular repair in diabetes, reverse fulminant liver failure, regenerate damaged lung tissue, attenuate sepsis, and reverse remodeling and improve cardiac function after myocardial infarction.Le Blanc K. et al., Lancet 371(9624):1579-1586 (2008); Hare, JM et al., Journal of the American College of Cardiology 54(24):2277-2286 (2009); Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005); Lee RH et al., PNAS 103(46):17438-17442 (2006); Parekkadan, B. et al., PloS One 2(9):e941 (2007); Ishizawa K. et al., FEBS Letters 556(1-3):249-252 (2004); Nemeth K. et al., Nature Medicine 15(1):42-49 (2009); Iso See Y. et al., Biochem. Biophys. Res. Comm. 354(3):700-706 (2007); Schuleri KH et al., Eur. Hearth J. 30(22):2722-2732 (2009); and Heldman AW et al., JAMA 311(1):62-73 (2014). Furthermore, mesenchymal stem cells are also a potential source of multiple cell types for use in tissue engineering. See Gong Z. et al., Methods in Mol. Bio. 698:279-294 (2011); Price, AP et al., Tissue Engineering Part A 16(8):2581-2591 (2010); and Togel F. et al., Organogenesis 7(2):96-100 (2011).

[0009]

[0009] Mesenchymal stem cells have immunomodulatory capabilities. Mesenchymal stem cells control inflammation and cytokine production in lymphocytes and myeloid-derived immune cells without evidence of immunosuppressive toxicity, and are low immunogenic. See Bernardo ME et al., Cell Stem Cell 13(4):392-402 (2013).

[0010]

[0010] Mesenchymal stem cells also have the ability to differentiate not only into cells of mesodermal origin, but also into cells of endodermal and ectodermal origin. See Le Blanc K. et al., Exp. Hematol. 31(10):890-896 (2003). For example, mesenchymal stem cells cultured in vitro in airway growth medium differentiate to express lung-specific epithelial markers, such as surfactant protein C, Clara cell secretory protein, and thyroid transcription factor 1. See Jiang Y. et al., Nature 418(6893):41-49 (2002) and Kotton DN et al., Development 128(24):5181-5188 (2001).

[0011]

[0011] In vivo studies have shown that human mesenchymal stem cells, when transplanted into fetal sheep, undergo site-specific differentiation into various cell types, including myocytes and cardiomyocytes. See Airey JA et al., Circulation 109(11):1401-1407 (2004). These mesenchymal stem cells can persist in multiple tissues for as long as 13 months after transplantation into non-immunosuppressed, immunocompetent hosts. Other in vivo studies using rodents, dogs, goats, and baboons have similarly shown that human mesenchymal stem cell xenografts do not induce lymphocyte proliferation or systemic alloantibody production in recipients.Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005); Aggarwal S. et al., Blood 105(4):1815-22 (2005); Augello A. et al., Arthritis and Rheumatism 56(4):1175-86 (2007); Bartholomew A. et al., Exp Hematol. 30(1):42-48 (2002); Dokic J. et al., European Journal of Immunology 43(7):1862-72 (2013); Gerdoni E. et al., Annals of Neurology 61(3):219-227 (2007); Lee SH et al., Respiratory Research 11:16 (2010); Urban VS et al., Stem Cells 26(1):244-253 (2008); Yang H. et al., PloS One 8(7):e69129 (2013); Zappia E. et al., Blood 106(5):1755-1761 (2005); Bonfield TL et al., American Journal of Physiology Lung Cellular and Molecular Physiology 299(6):L760-70 (2010); Glenn JD et al., World Journal of Stem Cells. 6(5):526-39 (2014); Guo K. et al., Frontiers in Cell and Developmental Biology 2:8 (2014); Puissant B. et al., British Journal of Haematology 129(1):118-129 (2005); and Sun L. et al., Stem Cells 27(6):1421-32 (2009). Overall, these repeated findings of allogeneic safety and efficacy are consistent with the use of mesenchymal stem cells as allografts for successful tissue regeneration.

[0012]

[0012] However, despite being a safe therapeutic agent, mesenchymal stem cells have been reported in the literature to exert a suppressive effect on antibody production and B cell proliferation and maturation. See Uccelli, A. et al., Trends in Immunology 28(5):219-226 (2007). Mesenchymal stem cells have also been reported to suppress the generation and function of antigen-presenting cells. See Hoogduijn MJ et al., Int. Immunopharmacology 10(12):1496-1500 (2010). Finally, mesenchymal stem cells have been reported to suppress the generation and function of CD4 + and CD8 + It has been reported to suppress T cell proliferation. See Ghannam S. et al., Stem Cell Res. & Ther. 1:2 (2010). Summary of the Invention

[0013]

[0013] Surprisingly, despite reports that mesenchymal stem cells have a suppressive effect on aspects of the immune system, the inventors have discovered a method of enhancing a subject's immune response to a vaccine or inducing an immune response in a non-responsive subject, which method comprises administering to the subject, simultaneously or sequentially, a vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and related kits and uses.

[0014]

[0014] In one embodiment of the present invention, the subject is a human. In another embodiment of the present invention, the subject is a human exhibiting symptoms of age-related frailty. In another embodiment of the present invention, the subject is a human exhibiting inflammaging.

[0015]

[0015] In one embodiment of the present invention, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. In one embodiment of the present invention, the mesenchymal stem cells do not express STRO-1. In another embodiment of the present invention, the mesenchymal stem cells do not express CD45. In another embodiment of the present invention, the mesenchymal stem cells do not express fibroblast surface markers or have fibroblast morphology. In another embodiment of the present invention, the mesenchymal stem cells are not genetically engineered.

[0016] In one embodiment of the present invention, the vaccine is monovalent. In another embodiment of the present invention, the vaccine is multivalent. In one embodiment of the present invention, the vaccine comprises one or more inactivated viruses. In another embodiment, the one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus. In another embodiment, the vaccine comprises an inactivated orthomyxovirus. In another embodiment, the vaccine comprises an inactivated influenza virus. In one embodiment of the invention, the vaccine comprises one or more live attenuated viruses, hi another embodiment, the one or more attenuated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus.

[0017] In one embodiment of the present invention, an adjuvant is administered before the vaccine. In another embodiment, the adjuvant is administered at least one week before the vaccine is administered. In another embodiment, the adjuvant is administered at least two weeks before the vaccine is administered. In another embodiment, the adjuvant is administered at least three weeks before the vaccine is administered. In another embodiment, the adjuvant is administered at least four weeks before the vaccine is administered. In another embodiment of the invention, the adjuvant is , long To enhance vaccine response during At least annually It is administered.

[0018] In one embodiment of the invention, the adjuvant and vaccine are administered parenterally. In one embodiment, the adjuvant is administered systemically. In one embodiment, the adjuvant is administered by infusion or direct injection. In one embodiment, the adjuvant is administered intravenously, intraarterially, or intraperitoneally. In another embodiment, the adjuvant is administered intravenously. In one embodiment of the invention, the vaccine is administered intramuscularly, intravenously, intraarterially, intraperitoneally, subcutaneously, intradermally, orally, or intranasally. In another embodiment, the vaccine is administered intramuscularly.

[0019] In one embodiment of the present invention, the adjuvant is about 20×10 6 In another embodiment of the invention, the adjuvant is administered at a dose of about 100×10 6 In another embodiment of the invention, the adjuvant is administered at a dose of about 200×10 6 The mesenchymal stem cells are administered at a dose of 100 mg / kg.

[0020] In one embodiment of the present invention, the mesenchymal stem cells are obtained from a human donor and no step of MHC matching of the human donor to the subject prior to administration of the vaccine and adjuvant to the subject is utilized.

[0021] In one embodiment of the present invention, a method of enhancing a subject's immune response to the disclosed vaccine or inducing an immune response in a non-responsive subject comprises administering to the subject, simultaneously or sequentially, an immunoprotective amount of the vaccine and an adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, resulting in at least a two-fold decrease in intracellular TNF-α expression in the subject's B cells compared to the TNF-α expression level in the subject's B cells prior to administration of the adjuvant. + :CD8 + T cell ratio in the subject prior to administration of the adjuvant + :CD8 + In another embodiment of the invention, the number of switched memory B cells in the subject is increased by at least two-fold compared to the number of switched memory B cells in the subject before administration of the adjuvant. In another embodiment of the invention, the number of exhausted B cells in the subject is reduced by at least two-fold compared to the number of exhausted B cells in the subject before administration of the adjuvant. In another embodiment, the invention relates to a method of inducing an immune response in a subject, comprising administering to the subject, simultaneously or sequentially, an immunoprotective amount of a vaccine and an adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and further wherein intracellular TNF-α expression in the subject's B cells is reduced by at least two-fold compared to the TNF-α expression level in the subject's B cells before administration of the adjuvant. The invention also relates to a method of inducing an immune response in a subject, comprising administering to the subject, simultaneously or sequentially, an immunoprotective amount of a vaccine and an adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and further wherein intracellular TNF-α expression in the subject's B cells is reduced by at least two-fold compared to the TNF-α expression level in the subject's B cells before administration of the adjuvant. + :CD8 + T cell ratio in the subject prior to administration of the adjuvant + :CD8 +The present invention further relates to a method of inducing an immune response in a subject, comprising administering to the subject, simultaneously or sequentially, a vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and wherein the number of switched memory B cells in the subject is increased by at least two-fold compared to the number of switched memory B cells in the subject prior to administration of the adjuvant.The present invention further relates to a method of inducing an immune response in a subject, comprising administering to the subject, simultaneously or sequentially, a vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and wherein the number of exhausted B cells in the subject is reduced by at least two-fold compared to the number of exhausted B cells in the subject prior to administration of the adjuvant.

[0022]

[0022] In another aspect, the present invention relates to a kit having at least two containers, comprising a vaccine in a first container and an adjuvant in a second container, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells. In one embodiment of the present invention, the mesenchymal stem cells of the kit are cryopreserved. In one embodiment of the present invention, the kit further comprises a dilution buffer in a third container. The vaccine and mesenchymal stem cells present in the kit may be any of the vaccines or allogeneic human mesenchymal stem cells disclosed herein. [Brief explanation of the drawings]

[0023] [Figure 1A] FIG. 1 shows RT-PCR for E47 and GAPDH in five subjects. [Figure 1B] FIG. 1 shows RT-PCR for E47 and GAPDH in five subjects. [Figure 2] Graph showing AID mRNA expression correlated with E47 expression. Reproduced from Figure 3 in Frasca, D. et al., J. Immunol. 180(8):5283-5290 (2008). [Figure 3]1 is a graph showing the % switched B cells as measured by flow cytometry. An increase in the % switched memory B cells was observed 6 months after mesenchymal stem cell infusion in subjects who received 20×10, 100×10, or 200×10 mesenchymal stem cells compared to baseline measurements. [Figure 4] 1 is a graph showing the % of switched memory B cells and exhausted B cells measured by flow cytometry. The % of switched memory B cells and exhausted B cells were measured in subjects who received a second infusion of mesenchymal stem cells one year after the first infusion of mesenchymal stem cells. [Figure 5A] 5A-5D are graphs showing T cell concentrations measured by flow cytometry. Figures 5A-5D show that allogeneic mesenchymal stem cells at any dose do not induce T cell activation (rejection). CD69 is an early marker of T cell activation. CD25 is a late / long-term marker of T cell activation. [Figure 5B] 5A-5D are graphs showing T cell concentrations measured by flow cytometry. Figures 5A-5D show that allogeneic mesenchymal stem cells at any dose do not induce T cell activation (rejection). CD69 is an early marker of T cell activation. CD25 is a late / long-term marker of T cell activation. [Figure 5C] 5A-5D are graphs showing T cell concentrations measured by flow cytometry. Figures 5A-5D show that allogeneic mesenchymal stem cells at any dose do not induce T cell activation (rejection). CD69 is an early marker of T cell activation. CD25 is a late / long-term marker of T cell activation. [Figure 5D] 5A-5D are graphs showing T cell concentrations measured by flow cytometry. Figures 5A-5D show that allogeneic mesenchymal stem cells at any dose do not induce T cell activation (rejection). CD69 is an early marker of T cell activation. CD25 is a late / long-term marker of T cell activation. [Figure 6]1 is a graph showing CD4+:CD8+ T cell ratios calculated from flow cytometry measurements. Improvement in CD4+:CD8+ T cell ratios (immune risk phenotype) was observed in subjects who received a second mesenchymal stem cell infusion one year after the first mesenchymal stem cell infusion. [Figure 7A] Figure 7A shows a graph depicting the downregulation of intracellular TNF-α in B cells after mesenchymal stem cell infusion by flow cytometry. "Baseline" refers to flow cytometry measurements taken at baseline. "3 months" refers to flow cytometry measurements taken 3 months after a subject received a second mesenchymal stem cell infusion one year after the first. "FSC-A" is forward light scatter and is proportional to cell surface area or size. "SSC-A" is side light scatter and is proportional to cell granularity or internal complexity. Thus, correlated measurements of FSC-A and SSC-A allow for the differentiation of cell types (e.g., lymphocytes) in heterogeneous populations. "PE-A" is a measurement of the fluorescent dye phycoerythrin. "APC-A" is a measurement of another fluorescent dye, allophycocyanin. Figure 7A shows flow cytometry measurements from one CRATUS subject. [Figure 7B] Figure 7B shows the downregulation of intracellular TNF-α in B cells after mesenchymal stem cell infusion by flow cytometry. "Baseline" refers to flow cytometry measurements taken at baseline. "3 months" refers to flow cytometry measurements taken 3 months later in a subject who received a second mesenchymal stem cell infusion 1 year after the first infusion. "FSC-A" is forward light scatter and is proportional to cell surface area or size. "SSC-A" is side light scatter and is proportional to cell granularity or internal complexity. Thus, correlated measurements of FSC-A and SSC-A allow for the differentiation of cell types (e.g., lymphocytes) in heterogeneous populations. "PE-A" is a measurement of the fluorescent dye phycoerythrin. "APC-A" is a measurement of another fluorescent dye, allophycocyanin. Figure 7B shows flow cytometry measurements from a second CRATUS subject. DETAILED DESCRIPTION OF THE INVENTION

[0024]

[0030] In a specific embodiment, the present invention is directed to a method for improving immune responses to vaccines in elderly patients. This example shows that administration of isolated allogeneic human mesenchymal stem cells in vivo results in an increase in the percentage of switched memory B cells and a decrease in exhausted B cells in the subject. This example shows that administration of isolated allogeneic human mesenchymal stem cells in vivo results in an increase in CD4+ in the subject. + :CD8 + The study also showed that the T cell ratio improved. Furthermore, as shown in this example, intracellular TNF-α was reduced in subjects receiving allogeneic human mesenchymal stem cell infusions. Based on these unexpected results, the inventors confirmed that isolated allogeneic human mesenchymal stem cells are effective in reducing inflammaging, a common feature of age-related frailty. Furthermore, because isolated allogeneic human mesenchymal stem cells were shown to reduce inflammaging, these mesenchymal stem cells enhance the immune response to vaccination.

[0025] definition

[0031] The embodiments may be practiced without using the theoretical aspects presented. Further, the theoretical aspects are presented with the understanding that the embodiments are not bound by any presented theory.

[0026]

[0032] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. Terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with the meaning of those terms in the context of the relevant art, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0027]

[0033] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0028]

[0034] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually recited herein. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0029]

[0035] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will be determined in part by how the value is measured or determined, i.e., by the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of ±10% of the referenced value.

[0030] Dosage, duration and subjects

[0036] An "immunoprotective amount" refers to an amount that stimulates a T cell-dependent (TD) immune response. Such a response is characterized by the ability to generate significant levels of IgG and opsonic activity. Immunological memory is generated against an immunogenic antigen such that the antibodies produced ameliorate infections and disease states associated with the pathogen and / or prevent infection by the pathogen. The dosage and number of doses (e.g., single dose or multiple doses) administered to a subject will vary depending on various factors, including the route of administration, patient condition and characteristics (sex, age, weight, health, size), severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect.

[0031]

[0037] As used herein, an "immunoprotective amount" of a vaccine and adjuvant is determined based on the combined effectiveness of the adjuvant and vaccine. For example, if an adjuvant is shown to significantly enhance the immune response to a vaccine, a smaller amount of vaccine will be required than if an adjuvant is shown to only weakly enhance the immune response to a vaccine. Effective amounts of adjuvant and vaccine can be determined by one of skill in the art using known dosage development techniques. In one embodiment, the present invention includes a method for enhancing a subject's immune response to a vaccine or for inducing an immune response in a non-responsive subject, comprising administering to the subject, simultaneously or sequentially, an immunoprotective amount of a vaccine and an adjuvant, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and further wherein the amount of vaccine required for immunoprotection is less than half the amount required for immunoprotection in the absence of the adjuvant. In other embodiments, the amount of vaccine required for immunoprotection is less than 0.1%, less than 0.5%, less than 1.0%, less than 10%, less than 20%, or less than 30% of the amount required for immunoprotection without the use of an adjuvant.

[0032]

[0038] In one embodiment of the invention, the adjuvant is administered simultaneously with the vaccine. In another embodiment of the invention, the adjuvant is administered sequentially with the vaccine. In another embodiment, the adjuvant is administered at least one week before the vaccine is administered. In another embodiment, the adjuvant is administered at least two weeks before the vaccine is administered. In another embodiment, the adjuvant is administered at least three weeks before the vaccine is administered. In another embodiment, the adjuvant is administered at least four weeks before the vaccine is administered. In other embodiments, the adjuvant is administered about 1 to 8 weeks, 1 to 12 weeks, 1 to 36 weeks, 2 to 8 weeks, 2 to 12 weeks, 2 to 26 weeks, 2 to 36 weeks, 2 to 48 weeks, 3 to 4 weeks, 3 to 12 weeks, 3 to 8 weeks, 3 to 26 weeks, 3 to 36 weeks, 3 to 48 weeks, or 4 to 12 weeks before the administration of the vaccine. In other embodiments, the adjuvant is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months prior to administration of the vaccine, or 1-2 months, 1-3 months, 1-4 months, 1-5 months, 1-6 months, 2-3 months, 2-4 months, 2-6 months, or 3-6 months prior to administration of the vaccine. , long For enhancing the vaccine response of subjects over a period of time At least annually The present disclosure also relates to methods in which the vaccine is administered prior to the adjuvant, including, but not limited to, about 1 to 8 weeks, 1 to 12 weeks, 1 to 36 weeks, 2 to 8 weeks, 2 to 12 weeks, 3 to 4 weeks, 3 to 12 weeks, 3 to 8 weeks, or 4 to 12 weeks prior to administration of the adjuvant.

[0033]

[0039] In another embodiment of the invention, administration of the adjuvant and vaccine is repeated, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months after the initial administration of the vaccine, or between 2-4, 2-6, 2-8, 2-10, 3-4, 3-6, 3-8, 3-10, 4-6, 4-8, 4-10, 6-8, 6-10, 6-12, or 12-18 months after the initial administration of the vaccine. Repeated administration of a "vaccine" includes, but is not limited to, administration of a vaccine against influenza virus followed by a vaccine against an influenza virus that is not identical to the initial vaccine but is recognized to provide vaccination against influenza virus types of a serotype that is circulating at the time of the second or repeat administration. In other embodiments, the simultaneous or sequential administration of the adjuvant and vaccine is repeated 3, 4, 5, 6, or 5-10 times. For example, but not by way of limitation, the present invention includes administering an adjuvant on day 0, followed by administration of a vaccine on day 7, followed by administration of an adjuvant on day 180, and then administration of a vaccine on day 187. In certain embodiments, the actual vaccine content may vary over the course of this repeated therapy, but the vaccine administered at each time point in the repeated therapy will be directed against the same class of pathogen.

[0034]

[0040] In one embodiment of the invention, the adjuvant is about 1 x 10 6 , 2 × 10 6 , 5×10 6 , 10×10 6 , 20×10 6 , 30×10 6 , 40×10 6 , 50×10 6 , 60×10 6 , 70×10 6 , 80×10 6 , 90×10 6 , 100×10 6 , 110×10 6 , 120×10 6 , 130×10 6 , 140×10 6 , 150×106 , 160×10 6 , 170×10 6 , 180×10 6 , 190×10 6 , 200×10 6 , 300×10 6 , 400×10 6 , 500×10 6 , or 10 x 10 7 In another embodiment, the adjuvant is administered at a dose of about 20×10 mesenchymal stem cells. 6 In another embodiment, the adjuvant is administered at a dose of about 100×10 6 In yet another embodiment, the adjuvant is administered at a dose of about 200×10 mesenchymal stem cells. 6 In yet another embodiment, the adjuvant is administered at a dose of about 1 to 400 x 10 mesenchymal stem cells. 6 , 10 to 400 × 10 6 , 100~400×10 6 , 20~200×10 6 , 20~400×10 6 , 0.1 to 5 × 10 6 , 0.1 to 10 × 10 6 , 0.1 to 100 × 10 6 , 1 to 50 × 10 6 , 1 to 100 × 10 6 , 0.01 to 10 × 10 6 or 0.01 to 100 x 10 6 The mesenchymal stem cells are administered at a dose of 100 mg / kg.

[0035]

[0041] In some embodiments, an immunoprotective amount of an adjuvant is administered to a subject in an amount sufficient to induce CD4 + :CD8 + increasing the ratio of CD4 T cells, e.g., by at least 2, 3, 4, 5, or 6 fold compared to the ratio before administration of the adjuvant + :CD8 +In some embodiments, an immunoprotective amount of an adjuvant is sufficient to increase the number of switched memory B cells in a subject, e.g., to increase the number of switched memory B cells by at least 2-, 3-, 4-, or 5-fold compared to the number before administration of the adjuvant. In some embodiments, an immunoprotective amount is sufficient to reduce intracellular TNF-α expression in B cells of the subject, e.g., to reduce the amount of intracellular TNF-α in B cells by at least 2-, 3-, 4-, 5-, or 6-fold compared to the amount in B cells before administration of the adjuvant. In some embodiments, an immunoprotective amount is sufficient to upregulate activation-induced cytidine deaminase (AID) in a subject. In some embodiments, an immunoprotective amount is sufficient to reduce the number of exhausted B cells in a subject, e.g., to reduce the number of exhausted B cells by at least 2- or 3-fold compared to the number before administration of the adjuvant.

[0036]

[0042] In another embodiment, the use of an adjuvant allows for the administration of a lower level of vaccine than would be recommended in the absence of an adjuvant for the patient population in question, while still obtaining an immune response. For example, current recommendations call for a single 0.5 mL dose of Fluzone high-dose vaccine (Sanofi Pasteur) intramuscularly for patients 65 years of age and older. In the methods of the present invention, the amount of Fluzone vaccine may be reduced to a smaller amount, such as 0.3 or 0.2 mL, for patients 65 years of age and older.

[0037]

[0043] "Administering" a composition may be accomplished by oral administration, injection, infusion, parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, intranasal, intraperitoneal, intraarterial, subcutaneous absorption, or by any method in combination with other known techniques. In one embodiment of the invention, the adjuvant is administered systemically. In another embodiment of the invention, the adjuvant is administered by infusion or direct injection. In another embodiment of the invention, the adjuvant is administered intravenously, intraarterially, or intraperitoneally. In another embodiment, the adjuvant is administered intravenously. In one embodiment of the invention, the vaccine is administered intramuscularly, intravenously, intraarterially, intraperitoneally, subcutaneously, intradermal, orally, or intranasally. In another embodiment, the vaccine is administered intramuscularly.

[0038]

[0044] The term "subject," as used herein, includes, but is not limited to, humans and non-human vertebrates, such as wild, domestic, and livestock animals. In some embodiments, the term refers to non-human animals, such as dogs, cats, birds, mice, rats, rabbits, guinea pigs, hamsters, gerbils, goats, sheep, cows, horses, camels, and non-human primates. In some embodiments, the term refers to humans, such as elderly humans aged 65 years or older, or elderly humans aged 60-95 years. In some embodiments, the human subject exhibits symptoms of age-related frailty. In some embodiments, the human subject exhibits inflammaging.

[0039]

[0045] In one embodiment of the present invention, the subject is a non-responder. It is known that when a population of individuals is vaccinated against a disease, many do not "respond" to the vaccination, i.e., their immune system does not appear to react to the administered antigen. This problem exists to a greater or lesser extent depending on the disease and the population involved, but vaccine manufacturers are still trying to reduce the number of potential "non-responder" subjects with each vaccine made available to physicians. This problem is thought to be particularly important for vaccines containing purified antigens, such as subunit vaccines produced by genetic engineering.

[0040]

[0046] The term "allogeneic" refers to cells that are of the same animal species as the animal that will become the "recipient host," but that differ genetically at one or more loci. This typically applies to cells transplanted from one animal to another animal of the same, but non-identical, species.

[0041]

[0047] As used herein, the phrase "in need thereof" means that a subject has been identified as having a need for a particular method or treatment. In some embodiments, identification can be by any diagnostic means. A subject may be in need of any of the methods and treatments described herein. In some embodiments, the subject is in or will be going to an environment where a particular disease, disorder, or condition is prevalent.

[0042]

[0048] Cells are referred to herein as being positive or negative for a particular marker. For example, cells may be negative for CD45, and are referred to as CD45 - It can also be called " - " refers to cells that are negative for the marker associated with the superscript. In contrast, " + A marker with " " refers to cells that are positive for that marker. For example, "CD8 + " are positive for CD8. A "+" can also be used to refer to a marker being positive. A "-" can also be used to refer to a marker being negative.

[0043]

[0049] As used herein, the term "stem cell" refers to a cell from an embryo, fetus, or adult that, under certain conditions, has the ability to renew itself over long periods of time, or, in the case of adult stem cells, throughout the life of the organism. Stem cells can also give rise to specialized cells that make up the tissues and organs of the body.

[0044]

[0050] Mesenchymal stem cells are found in bone marrow, blood, dermis, and periosteum, among others, and are formative pluripotent blast cells that can differentiate into any kind of specific type of mesenchymal or connective tissue (i.e., specialized element-bearing body tissue; in particular, adipose tissue, bone tissue, cartilage tissue, elastic tissue, and fibrous connective tissue) in response to various influences from bioactive factors such as cytokines.

[0045]

[0051] Certain methods for isolating and / or purifying mesenchymal stem cells are described herein and known in the art. In some embodiments, mesenchymal stem cells are isolated from adult human bone marrow. In some embodiments, the cells are passed through a density gradient to remove undesired cell types. The cells can be plated and cultured in an appropriate medium. In some embodiments, the cells are cultured for at least 1 day or for about 3 to about 7 days, and non-adherent cells are removed. The adherent cells can then be plated and expanded.

[0046]

[0052] Other methods for isolating and culturing stem cells are also known. The placenta is an excellent and readily available source of mesenchymal stem cells. Furthermore, mesenchymal stem cells can be derived from adipose tissue, and bone marrow stromal cells are presumed to exist in other tissues. Although there are significant qualitative and quantitative differences between the organs from which adult stem cells can be derived, the initial differences between the cells are relatively superficial and are balanced by the similar range of plasticity that the cells exhibit.

[0047]

[0053] Homogeneous human mesenchymal stem cell compositions are provided that serve as progenitors for all mesenchymal cell lineages. The mesenchymal stem cells are identified by specific cell surface markers identified using specific monoclonal antibodies. The homogeneous mesenchymal stem cell compositions are obtained by positive selection of adherent bone marrow or periosteal cells, and the compositions do not contain markers associated with either hematopoietic cells or differentiated mesenchymal cells. These isolated mesenchymal cell populations exhibit epitope characteristics unique to mesenchymal stem cells, have the ability to regenerate in culture without differentiation, and have the ability to differentiate into specific mesenchymal lineages when induced in vitro or placed at a site of inflammation in vivo.

[0048]

[0054] To obtain human mesenchymal stem cells for the compositions, methods, and kits disclosed herein, pluripotent mesenchymal stem cells are separated from other cells in bone marrow or other mesenchymal stem cell sources. Bone marrow cells can be obtained from the iliac crest, femur, tibia, vertebrae, ribs, or other medullary cavities. Other locations for human mesenchymal stem cells include the embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, and blood.

[0049]

[0055] In some embodiments, human mesenchymal stem cells are identified by the absence of markers. For example, human mesenchymal stem cells useful in the present invention include STRO-1 negative and / or CD45 negative cells. Similarly, human mesenchymal stem cells useful in the present invention include cells that do not express fibroblast surface markers or do not express fibroblast morphology.

[0050] Method for enhancing immune response and kit therefor

[0056] As noted above, the present invention is directed to methods of enhancing a subject's immune response to a vaccine or inducing an immune response in a non-responsive subject, comprising administering to the subject, simultaneously or sequentially, an immunoprotective amount of a vaccine and an adjuvant, wherein the adjuvant comprises a population of isolated allogeneic human mesenchymal stem cells, and kits related to such methods. In some embodiments of the present invention, the mesenchymal stem cells are not genetically engineered. In some embodiments of the present invention, the mesenchymal stem cells are obtained from a human donor, and no step of MHC matching of the human donor to the subject is utilized prior to administering the vaccine and adjuvant to the subject.

[0051]

[0057] In one embodiment of the invention, the vaccine is monovalent. In another embodiment of the invention, the vaccine is multivalent.

[0052]

[0058] In one embodiment of the present invention, the vaccine comprises one or more inactivated viruses. In another embodiment, the one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus. In another embodiment, the vaccine comprises an inactivated orthomyxovirus. In another embodiment, the vaccine comprises an inactivated influenza virus.

[0053]

[0059] In one embodiment of the invention, the vaccine comprises one or more live attenuated viruses, hi another embodiment, the one or more attenuated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus.

[0054]

[0060] In another embodiment of the invention, the vaccine comprises an antigen from a bacterial pathogen. In another embodiment, the bacterial pathogen is selected from the group consisting of Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter The bacterial strain is selected from the group consisting of Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

[0055]

[0061] In another embodiment of the invention, the vaccine comprises an antigen from a parasitic pathogen. In another embodiment, the parasitic pathogen is selected from the group consisting of Acanthamoeba, Anisakis, Ascaris lumbricoides, Balantidium coli, Cestoda, Tsutsugamushi, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworms, Leishmania, Linguatula serrata, liver fluke, Loa loa, Paragonimus, pinworms, and Plasmodium falciparum. falciparum, Schistosoma, Strongyloides stercoralis, Tapeworm, Toxoplasma gondii, Trypanosoma, Trichuris, and Wuchereria bancrofti.

[0056]

[0062] In another embodiment of the invention, the vaccine comprises an influenza hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), influenza neuraminidase (NA), Lassa virus (LASV) glycoprotein 1 (gp1), LASV glycoprotein 2 (gp2), LASV nucleocapsid-associated protein (NP), LASV L protein, LASV Z protein, SARS virus S protein, Ebola virus GP2, measles virus fusion 1 (F1) protein, HIV-1 transmembrane (TM) protein, HIV-1 glycoprotein 41 (gp41), HIV-1 glycoprotein 120 (gp120), hepatitis C virus (HCV) envelope glycoprotein 1 (E1), HCV envelope glycoprotein 2 (E2), HCV nucleocapsid protein (p22), West Nile virus (WNV) envelope glycoprotein (E), Japanese encephalitis virus (JEV) envelope glycoprotein (E), yellow fever virus (YFV) envelope glycoprotein (E), tick-borne encephalitis virus (TBEV) envelope glycoprotein (E), hepatitis G virus (HGV) envelope glycoprotein 1 (E1), respiratory syncytial virus (RSV) fusion (F) protein, herpes simplex virus 1 (HSV-1) gD protein, HSV-1 gG protein, HSV-2 gD protein, HSV-2 gG protein, Hepatitis B virus (HBV) core protein, Epstein-Barr virus (EBV) glycoprotein 125 (gp125), bacterial outer membrane protein assembly factor BamA, bacterial translocation assembly module protein TamA, bacterial polypeptide transport-associated protein domain protein, bacterial surface antigen D15, Bacillus anthracis protective protein, Bacillus anthracis lethal factor, Bacillus anthracis edema factor, Salmonella typhi S1Da, Salmonella typhi S1Db, cholera toxin, cholera heat shock protein, Clostridium botulinum antigen S, botulinum toxin, Yersinia pestispestis F1, Yersinia pestis V antigen, Yersinia pestis Yoph, Yersinia pestis YopM, Yersinia pestis YopD, Yersinia pestis plasminogen activator (Pla), Plasmodium circumsporozoite protein (CSP), Plasmodium sporozoite surface protein (SSP2 / TRAP), Plasmodium liver stage antigen 1 (LSAT), Plasmodium export protein 1 (EXP 1), Plasmodium erythrocyte-associated antigen 175 (EBA-175), Plasmodium cysteine-rich protective antigen (cyRPA), Plasmodium heat shock protein 70 (hsp70), Schistosoma mansoni Sm29, and Schistosoma mansoni signaling protein 14-3-3.

[0057]

[0063] Compositions for use in the present invention can be formulated using any suitable method. Formulation of cells using standard pharmaceutically acceptable carriers and / or excipients can be carried out using conventional methods in the pharmaceutical field. The exact nature of the formulation will depend on several factors, including the cells to be administered and the desired route of administration. Suitable types of formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0058]

[0064] The composition can be prepared with a physiologically acceptable carrier or diluent. Generally, such a composition is prepared as a liquid suspension of cells. The cells may be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc., and combinations thereof.

[0059]

[0065] In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, and / or adjuvants to enhance effectiveness. In one embodiment of the invention, the adjuvant comprises human serum albumin (HSA).

[0060]

[0066] One suitable carrier or diluent is PlasmaLyte A™. PlasmaLyte A™ is a sterile, non-pyrogenic, isotonic solution for intravenous administration. Each 100 mL contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11 NaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO 23 HO); 37 mg of potassium chloride, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl 6H 0). Plasmalite A™ does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0061]

[0067] As described above, the present invention also relates to a kit having at least two containers, the kit comprising a vaccine in a first container and an adjuvant in a second container, the adjuvant comprising an isolated allogeneic human mesenchymal stem cell population. Any adjuvant or vaccine described herein can be formulated as a kit according to the present disclosure. In one embodiment of the present invention, the mesenchymal stem cells are not genetically engineered. In another embodiment of the present invention, the mesenchymal stem cells are cryopreserved in the second container. For example, the mesenchymal stem cells can be suspended in a cryoprotectant consisting of Hespan® (6% hetastarch in 0.9% sodium chloride) supplemented with 2% HSA and 5% DMSO, and then placed in a cryopreservation container for placement in a vapor-phase nitrogen freezer. In another embodiment, the mesenchymal stem cells in the second container can be placed in Plasmalyte A™ supplemented with 1% HSA. In another embodiment, the kit has at least three containers, and the third container contains a dilution buffer for suspending and diluting the mesenchymal stem cells. In another embodiment, the dilution buffer comprises Plasmalyte A™ with 1% HSA.

[0062]

[0068] In one embodiment, the present invention is a composition comprising an immunoprotective amount of an adjuvant and a vaccine. One skilled in the art can formulate a suitable composition from among the adjuvants and vaccines disclosed herein. [Example]

[0063] Example 1 Effect of aging on E47 mRNA expression in human peripheral blood-derived B cells

[0069] As mentioned above, both E47 and Pax-5 are important transcription factors in the early development of the B cell lineage and the function of mature B cells. It has been demonstrated that the putative regulatory region of the Aicda gene contains both E47 and Pax-5 binding sites essential for AID gene expression. The following experiments demonstrate that E47 expression declines with age and that E47 expression in B cells is positively correlated with AID expression. See Figures 1 and 2.

[0064]

[0070] Forty-six subjects, ranging in age from 20 to 85 years, were recruited and peripheral blood samples were collected. + B cells (10 6 Cells / mL) were cultured with anti-CD40 (1 μg / mL) and IL-4 (10 ng / mL) for 24 hours. PCR was performed for E47 and GAPDH. E47 was normalized to GAPDH. Figure 1(A) shows undiluted and 1 / 4 diluted RT-PCR of five representative subjects. In Figure 1(B), the graph shows densitometric analysis of CT normalized to GAPDH. The numbers shown for each sample are percentages, with the highest value set at 100. The human r value for the linear curve representing the correlation between age and E47 expression is r = -0.84, p = 0.00001. The solid line represents linear regression, and the dashed line represents quadratic regression. Thus, this data shows that E47 expression declines with age.

[0065]

[0071] Blood from these subjects was also subjected to PCR for AID and GAPDH, and the results are shown in Figure 2. E47 (24-hour stimulation) and AID (5-day stimulation) were individually normalized to their respective GAPDH values. The numbers shown for each sample are percentages, with the highest value set at 100. The E47 and AID PCR data were positively correlated. The correlation is significant at the 0.01 level (two-sided). The solid line represents a linear regression. As can be seen in Figure 2, E47 expression in B cells is positively correlated with AID expression (r=0.80, p=0.01 level, two-sided).

[0066] Example 2 Switched memory B cells

[0072] The following experiment demonstrates that switched memory B cells are increased in elderly subjects after treatment with allogeneic mesenchymal stem cells (MSCs). Switched memory B cells were measured in human patients in the CRATUS trial at baseline (before intravenous infusion of MSCs) and 6 months after MSC infusion. The results show that MSC infusion upregulates the switched memory B cell compartment, a biomarker predictive of improved antibody responses. See Figure 3. This was demonstrated with the three doses of MSCs tested (20 x 10 6 , 100×10 6 , or 200 x 10 6 The data showed that intravenous administration of allogeneic mesenchymal stem cells increased switched memory B cells by twofold in some patients.

[0067]

[0073] Figure 4 shows the percentage of switched memory and exhausted B cells in subjects who received a second infusion of mesenchymal stem cells one year after the first infusion. This graph suggests that a second injection at 12 months may be necessary to maintain the improvements to immune cells induced from the first mesenchymal stem cell treatment.

[0068] Example 3 T cell activation

[0074] The following experiments demonstrate that both early and late / long-term T cell activation are reduced after allogeneic MSC treatment. Early markers of T cell activation (CD69) and late / long-term markers of T cell activation (CD25) were measured in human patients from CRATUS study samples. As shown in Figures 5A and 5B, at any dose (20x10 6 , 100×10 6 , or 200 x 10 6 There is no T cell activation (rejection) induced by allogeneic mesenchymal stem cells (MSCs). See also Figures 5C and 5D, which show the absolute difference 6 months after MSC infusion for CD69 cells % or CD25 cells %.

[0069] Example 4 Immune Risk Phenotypes

[0075] The immune risk phenotype was measured in the same CRATUS subjects receiving a second MSC infusion one year after the first MSC infusion. See Figure 6. The results show that MSC infusion increases the risk of developing CD4 + :CD8 + These results suggest that MSCs improve T cell ratios. Furthermore, by 12 months after the first infusion, the effect begins to fade, at which point a second MSC infusion provides further improvement. A second injection at 12 months may be necessary to maintain the immune cell improvement induced from the initial mesenchymal stem cell treatment.

[0070] Example 5 TNF-alpha

[0076] TNF-α reduces AID. TNF-α was measured in samples from two CRATUS subjects by flow cytometry using intracellular staining of B cells and confirmed by qPCR. Results are shown at baseline (12 months after the first infusion of MSCs) and 3 months (3 months after the second infusion) in Figures 7A and 7B. Flow cytometry results demonstrate downregulation of intracellular TNF-α in B cells after mesenchymal stem cell infusion. These results were confirmed by qPCR.

[0071] Example 6 Effect of intravenous delivery of allogeneic human mesenchymal stem cells on vaccine-specific antibody responses in elderly patients with frailty - HERA( H uman Mesenchymal Stem Cells on Vaccine E -Specific Antibody R Responses in Patients with A Ginger Frailty Study (Phase I / II)

[0077] The HERA trial is a phase I / II randomized, double-blind, placebo-controlled trial whose primary goal is to demonstrate that intravenous administration of mesenchymal stem cells can improve adaptive immunity to influenza vaccines and improve primary B-cell responses in elderly, frail subjects.

[0072]

[0078] Forty-three (43) frail age-related subjects will be enrolled. The adjuvant (allogeneic mesenchymal stem cells) will be administered by peripheral intravenous infusion. The total duration for each subject after infusion will be 12 months plus up to an additional 2 months for screening and baseline visits. A safety run-in period will be followed by a double-blind randomization phase. All subjects must meet inclusion / exclusion criteria, and all subjects will be evaluated prior to the planned infusion to establish a baseline.

[0073] Safety introduction period

[0079] A safety run-in period will include 23 subjects in three cohorts and will be conducted to determine the optimal time point for administering the influenza vaccine after the mesenchymal stem cell infusion.

[0074]

[0080] Cohort A (3 subjects): 20 x 10 6 Each subject will receive a single peripheral intravenous infusion of mesenchymal stem cells. One week after the infusion, subjects will receive a single 0.5 mL intramuscular injection of the high-dose Fluzone vaccine (Sanofi Pasteur), which is recommended for patients 65 years of age or older. These subjects will be infused first (i.e., prior to any subjects in Cohorts B and C), with no gap of more than five days between infusions.

[0075]

[0081] Cohort B (10 subjects): 100 x 10 6 Each subject will receive a single peripheral intravenous infusion of mesenchymal stem cells. One week after the infusion, subjects will receive a single 0.5 mL dose of the Fluzone high-dose vaccine intramuscularly.

[0076]

[0082] Cohort C (10 subjects): 100 x 10 6 Each subject will receive a single peripheral intravenous infusion of mesenchymal stem cells. Four weeks after the infusion, subjects will receive a single 0.5 mL dose of the Fluzone high-dose vaccine intramuscularly.

[0077]

[0083] Subjects in Cohorts B and C will be randomized, with the first three subjects receiving injections no more than five days apart. Follow-up visits will be conducted one and four weeks after vaccination to determine the safety and efficacy of vaccination after mesenchymal stem cell injection. After all 23 subjects have been injected and vaccinated, a 30-day follow-up will be conducted to evaluate all safety data. The double-blind randomization phase, which took place after the successful completion of the safety run-in period, was reviewed and accepted. After the safety run-in period data is analyzed, standard vaccination timepoints at both one and four weeks after vaccination will be utilized for the double-blind randomized portion of the study.

[0078] Double-blind randomized trial

[0084] The double-blind, randomized study will include 20 subjects in two cohorts. All subjects must meet the inclusion / exclusion criteria and will be evaluated prior to the planned infusion to establish a baseline. Subjects will be randomized into the two cohorts in a 1:1 ratio as follows:

[0079]

[0085] Cohort 1 (10 subjects): 100 x 10 6 Each subject will receive a single peripheral intravenous infusion of mesenchymal stem cells. At the optimal time point after the infusion (1 or 4 weeks as determined by the safety run-in period), subjects will receive a single 0.5 mL dose of the Fluzone high-dose vaccine intramuscularly.

[0080]

[0086] Cohort 2 (10 subjects): Each subject will receive a single peripheral intravenous infusion of placebo (Plasmalite A™ with 1% HSA). At the optimal time point after the infusion (1 or 4 weeks as determined during the safety run-in period), subjects will receive a single 0.5 mL dose of the Fluzone high-dose vaccine intramuscularly.

[0081]

[0087] Telephone follow-up will occur one day after injection and vaccination. In-clinic follow-up visits will occur at weeks 1 and 4 post-vaccination, and at months 6 and 12 post-vaccination to complete all safety and efficacy assessments. Any subject who develops influenza-type symptoms during the first 7 months post-vaccination should be scheduled for an immediate clinic visit so that evaluation of the likely influenza strain can be determined.

[0082] Primary endpoint

[0088] The primary efficacy endpoints are (1) the ability of B cells to upregulate activation-induced cytidine deaminase (AID) in response to CpG or influenza vaccine by qPCR and (2) B cell function as measured by HAI and influenza-specific antibody production by ELISA. Data for the primary efficacy endpoint will be obtained from a baseline visit, a vaccination visit (performed 1 or 4 weeks after mesenchymal stem cell infusion), follow-up visits at 1 and 4 weeks post-vaccination, and follow-up visits at 6 and 12 months post-infusion.

[0083] Selection Criteria

[0089] All subjects enrolled in this study must provide written informed consent, must be 65-95 years old at the time of signing the informed consent form, and must have been diagnosed with frailty using the Canadian Frailty Scale with a score of 4-7, ≤5% switched memory B cells, ≥10% late / exhausted memory, ≤20% CD8 + Naive cells and ≥40% CD8 +Patients must demonstrate immunosenescence as measured by whole blood flow cytometry staining resulting in TEMRA cells and have a total bilirubin between 0.3 and 1.9 mg / dL.

[0084] Adjuvant and placebo

[0090] 100×10 6 The final mesenchymal stem cell formulation for subjects receiving 100 mg of mesenchymal stem cells is 2.5 x 10 cells suspended in 80 mL of Plasmalyte A™ containing 1.0% HSA. 6 mesenchymal stem cells / mL: 20 x 10 6 For induction phase subjects receiving 0.5 x 10 mesenchymal stem cells, the final formulation is 0.5 x 10 suspended in 80 mL of Plasmalyte A™ containing 1.0% HSA. 6 The adjuvant was purchased from Longeveron LLC (Life Sciences and Technology Park, 1951 NW 7 th Manufactured by the company's cell processing facility at 1000 W. Ave., Miami, FL 33136.

[0085]

[0091] Dilution buffer will be used for the placebo. The final formulation of the placebo is Plasmalite A™ containing 1.0% HSA (80 mL total).

[0086] Adjuvant dosage and rate

[0092] The subject of this test is 20 x 10 6 mesenchymal stem cells, 100 × 10 6 The maximum infusion rate was 2.5 × 10 6 of mesenchymal stem cells / minute, which is much lower than the maximum reported dosing rate. Table 3 details the infusion parameters for the mesenchymal stem cell and placebo infusions. A total of 80 mL is delivered intravenously to each subject.

[0087] [Table 1]

Claims

1. 1. A kit for enhancing or inducing an immune response to a vaccine in an elderly human subject 65 years of age or older, comprising an immunoprotective amount of a vaccine and an adjuvant, the adjuvant is a population of isolated, allogeneic, non-genetically engineered human bone marrow-derived mesenchymal stem cells; the vaccine comprises one or more inactivated viruses; the kit is for sequential use of the vaccine and the adjuvant, such that the adjuvant is administered before the vaccine; The kit, wherein the adjuvant is used to enhance or induce an immune response to the vaccine in elderly human subjects 65 years of age or older.

2. The kit of claim 1 , wherein the elderly human subject exhibits symptoms of age-related frailty.

3. 3. The kit of claim 1 or 2, wherein the elderly human subject exhibits inflammaging.

4. (i) the mesenchymal stem cells do not express STRO-1, and / or (ii) the mesenchymal stem cells do not express CD45, and / or (iii) the mesenchymal stem cells do not express fibroblast surface markers or have fibroblast morphology; The kit according to any one of claims 1 to 3.

5. The kit of any one of claims 1 to 4, wherein the vaccine is multivalent.

6. 6. The kit of any one of claims 1 to 5, wherein the one or more inactivated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus.

7. 7. The kit of claim 6, wherein the one or more inactivated viruses comprise an inactivated orthomyxovirus.

8. 8. The kit of claim 7, wherein the one or more inactivated viruses comprise an inactivated influenza virus.

9. The kit of any one of claims 1 to 5, wherein the vaccine comprises one or more live attenuated viruses.

10. 10. The kit of claim 9, wherein the one or more attenuated viruses are selected from the group consisting of adenovirus, picornavirus, papillomavirus, polyomavirus, hepadnavirus, parvovirus, poxvirus, Epstein-Barr virus, cytomegalovirus (CMV), herpesvirus, roseolovirus, varicella-zoster virus, filovirus, paramyxovirus, orthomyxovirus, rhabdovirus, arenavirus, coronavirus, human enterovirus, hepatitis A virus, human rhinovirus, poliovirus, retrovirus, rotavirus, flavivirus, hepacivirus, togavirus, and rubella virus.

11. (i) the adjuvant is administered at least one week before the vaccine is administered; (ii) the adjuvant is administered at least two weeks before the vaccine is administered; (iii) the adjuvant is administered at least three weeks before the vaccine is administered; (iv) the adjuvant is administered at least 4 weeks before the vaccine is administered; or (v) the adjuvant is administered at least annually for long-term enhancement of vaccine response; The kit according to any one of claims 1 to 10.

12. (i) the adjuvant is about 20 x 10 ６ or for administration at a dose of mesenchymal stem cells of (ii) the adjuvant is about 100 x 10 ６ or (iii) the adjuvant is about 200 x 10 ６ and a dose of mesenchymal stem cells of The kit according to any one of claims 1 to 11.

13. 13. The kit of any one of claims 1 to 12, wherein the mesenchymal stem cells are obtained from a human donor and no step of MHC matching of the human donor to the human subject is utilized prior to administration of the vaccine and adjuvant to the human subject.

14. The kit of any one of claims 1 to 13, for repeating the administration of the vaccine and the adjuvant at least six months after the first administration of the vaccine.

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