MESENCHYMAL STEM CELLS AS VACCINE ADJUVANTS AND METHODS FOR USING THE SAME

Abstract

The present invention provides a method of enhancing an immune response to a vaccine by administering a vaccine and a population of isolated allogeneic human mesenchymal stem cells. The present invention also provides kits comprising a vaccine in a first container and a population of isolated allogeneic human mesenchymal stem cells in a second container.

Claims

1. 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 mesenchymal stem cells which are not genetically manipulated, for use in enhancing an elderly human subject's immune response to a vaccine or for use in inducing an immune response to a vaccine in a non-responding elderly human subject, wherein the elderly human subject is ?65 years of age, and wherein the immunoprotective amounts of the vaccine and the adjuvant are administered to the subject concurrently, or sequentially such that the adjuvant is administered before the vaccine.

2. The kit of claim 1, wherein the mesenchymal stem cells are cryopreserved.

3. The kit of claim 1, further comprising dilution buffer in a third container.

4. The kit of claim 1, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

5. The kit of claim 1, wherein: (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 a fibroblast morphology.

6. The kit of claim 1, wherein the vaccine is multivalent.

7. The kit of claim 1, wherein the vaccine comprises one or more inactivated viruses, preferably wherein the one or more inactivated viruses are selected from the group consisting of adenoviruses, picornaviruses, papillomaviruses, polyomaviruses, hepadnaviruses, parvoviruses, pox viruses, Epstein-Barr virus, cytomegalovirus (CMV), herpes viruses, roseolovirus, varicella zoster virus, filoviruses, paramyxoviruses, orthomyxoviruses, rhabdoviruses, arenaviruses, coronaviruses, human enteroviruses, hepatitis A virus, human rhinoviruses, polio virus, retroviruses, rotaviruses, flaviviruses, hepaciviruses, togaviruses, and rubella virus.

8. The kit of claim 7, wherein the vaccine comprises an inactivated orthomyxovirus.

9. The kit of claim 8, wherein the vaccine comprises an inactivated influenza virus.

10. The kit of claim 1, wherein the vaccine comprises one or more live, attenuated viruses, preferably wherein the one or more attenuated viruses are selected from the group consisting of adenoviruses, picornaviruses, papillomaviruses, polyomaviruses, hepadnaviruses, parvoviruses, pox viruses, Epstein-Barr virus, cytomegalovirus (CMV), herpes viruses, roseolovirus, varicella zoster virus, filoviruses, paramyxoviruses, orthomyxoviruses, rhabdoviruses, arenaviruses, coronaviruses, human enteroviruses, hepatitis A virus, human rhinoviruses, polio virus, retroviruses, rotaviruses, flaviviruses, hepaciviruses, togaviruses, and rubella virus.

11. The kit of claim 1, wherein: (i) the adjuvant is administered at least 1 week before the vaccine is administered; (ii) the adjuvant is administered at least 2 weeks before the vaccine is administered; (iii) (iii) the adjuvant is administered at least 3 weeks before the vaccine is administered; (iv) (iv) the adjuvant is administered at least 4 weeks before the vaccine is administered; or (v) (v) the adjuvant is administered at least yearly for long-term enhancement of vaccine response.

12. The kit of claim 1, wherein: (i) the adjuvant is administered at a dose of about 20?10.sup.6 mesenchymal stem cells; or (ii) the adjuvant is administered at a dose of about 100?10.sup.6 mesenchymal stem cells; or (iii) the adjuvant is administered at a dose of about 200?10.sup.6 mesenchymal stem cells.

13. The kit of claim 1, wherein the mesenchymal stem cells are obtained from a human donor and wherein a step of MHC matching of the human donor to the subject is not employed prior to the administration of the vaccine and adjuvant to the human subject.

14. The kit of claim 1, further comprising repeating administration of the vaccine and the adjuvant at least six months after the first administration of the vaccine.

15. The kit of claim 1, wherein: (i) the intracellular TNF-? expression in B cells of the subject decreases by at least two-fold as compared to the TNF-? expression levels in B cells of the subject prior to administration of the adjuvant; or (ii) the CD4.sup.+:CD8.sup.+ T cell ratio in the subject increases by at least two-fold as compared to the CD4.sup.+:CD8.sup.+ T cell ratio in the subject prior to administration of the adjuvant; or (iii) the number of switched memory B cells in the subject increases by at least two-fold as compared to the number of switched memory B cells in the subject prior to administration of the adjuvant; or (iv) the number of exhausted B cells in the subject decreases by at least two-fold as compared to the number of exhausted B cells in the subject prior to administration of the adjuvant.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0024] FIG. 1A shows RT-PCR for E47 and GAPDH from five subjects. FIG. 1B shows the densitometry analyses of CT's normalized to GAPDH.

[0025] FIG. 2 shows AID mRNA expression correlated to E47 expression.

[0026] FIG. 3 shows the % of switched B cells as measured by flow cytometry. An increase in the % of switched memory B cells was observed in subjects who received 20?10.sup.6, 100?10.sup.6, or 200?10.sup.6 mesenchymal stem cells at 6 months post-mesenchymal stem cell infusion as compared to the baseline measurement.

[0027] FIG. 4 shows the % of switched memory B cells and exhausted B cells as measured by flow cytometry. The % of switched memory B cells and exhausted B cells was measured in subjects who received a second mesenchymal stem cell infusion one year after their first mesenchymal stem cell infusion.

[0028] FIGS. 5A-5D show T cell concentrations as measured by flow cytometry. FIGS. 5A-5D show that there is no T cell activation (rejection) induced by the allogeneic mesenchymal stem cells at any dose. CD69 is an early marker of T cell activation. CD25 is a late/chronic marker of T cell activation.

[0029] FIG. 6 shows CD4.sup.+:CD8.sup.+ T cell ratios, calculated from flow cytometry measurements. An improvement in the CD4.sup.+:CD8.sup.+ T cell ratio (Immune Risk Phenotype) was observed in subjects who received a second mesenchymal stem cell infusion one year after their first mesenchymal stem cell infusion.

[0030] FIGS. 7A and 7B show downregulation of intracellular TNF-? in B cells by flow cytometry after mesenchymal stem cell infusion. Base line refers to flow cytometry measurements taken at baseline. 3 months refers to flow cytometry measurements taken 3 months after subjects received a second mesenchymal stem cell infusion one year after their first mesenchymal stem cell infusion. FSC-A is forward-scattered light and is proportional to cell-surface area or size. SSC-A is side-scattered light and is proportional to cell granularity or internal complexity. Thus, correlated measurements of FSC-A and SSC-A allow for differentiation of cell types in a heterogeneous population (e.g., lymphocytes). PE-A is measurement of phycoerythrin, a fluorochrome. APC-A is measurement of allphycocyanin, another fluorochrome. FIG. 7A shows the flow cytometry measurements from one CRATUS subject. FIG. 7B shows the flow cytometry measurements from a second CRATUS subject.

DETAILED DESCRIPTION

[0031] In certain embodiments, the present invention is directed to methods of improving the immune response to vaccines in elderly patients. The examples demonstrate that in vivo administration of isolated allogeneic human mesenchymal stem cells result in an increase in the percentage of switched memory B cells and a decrease in exhausted B cells in subjects. The examples also demonstrate that in vivo administration of isolated allogeneic human mesenchymal stem cells results in an improvement in the CD4.sup.+:CD8.sup.+ T cell ratio in subjects. Also, as shown in the examples, intracellular TNF-? is reduced in subjects having received infusions of allogeneic human mesenchymal stem cells. From these unexpected results, the present inventors determined that isolated allogeneic human mesenchymal stem cells are effective at reducing inflammaging, a prevalent feature in aging frailty. And because isolated allogeneic human mesenchymal stem cells were shown to reduce inflammaging, these mesenchymal stem cells enhance the immune response to vaccination.

Definitions

[0032] Embodiments may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that the embodiments are not bound by any theory presented.

[0033] 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. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0034] 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 indicates otherwise. Furthermore, to the extent that the terms including, includes, having, has, with, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term comprising.

[0035] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate 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., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0036] The term about or approximately means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, about can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, about can mean a range of ?10% of the referenced value.

Dosage, Duration and Subjects

[0037] Immunoprotective amount means an amount that stimulates a T cell dependent (TD) immune response. Such a response is characterized by the ability to elicit significant levels of IgG and opsonic activity. An immunological memory is developed to the immunogenic antigen such that the antibodies produced ameliorate the infection and disease condition mediated by the pathogen and/or prevent infection by the pathogen. The dosage and number of doses (e.g., single or multiple dose) administered to the subject will vary depending upon a variety of factors, including the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired, and the like.

[0038] As used herein, the immunoprotective amount of a vaccine and adjuvant is determined based on the effect of the combination of the adjuvant and vaccine. For example, if the adjuvant is shown to enhance the immune response of a vaccine significantly, the amount of vaccine needed is less than if the adjuvant enhances the immune response of the vaccine less. Useful amounts of adjuvant and vaccine can be determined by a person of skill in the field using known dosage development techniques. In one embodiment, the invention comprises a method of enhancing a subject's immune response to a vaccine or inducing an immune response in a non-responding subject, comprising administering to the subject concurrently or sequentially the vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant is a population of isolated allogeneic human mesenchymal stem cells, and further wherein the amount of vaccine required for immunoprotective effect is less than half that required for immunoprotective effect in the absence of use of the adjuvant. In other embodiments, the amount of vaccine required for immunoprotective effect is less than 0.1%, less than 0.5%, less than 1.0%, less than 10%, less than 20%, or less than 30% that required for immunoprotective effect in the absence of use of the adjuvant.

[0039] In one embodiment of the invention, the adjuvant is administered concurrently with the vaccine. In another embodiment of the invention, the adjuvant is administered sequentially with the vaccine. In a further embodiment, the adjuvant is administered at least 1 week before the vaccine is administered. In a further embodiment, the adjuvant is administered as least 2 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 3 weeks before the vaccine is administered. In a further embodiment, the adjuvant is administered at least 4 weeks before the vaccine is administered. In other embodiments, the adjuvant is administered from about 1-8 weeks, 1-12 weeks, 1-36 weeks, 2-8 weeks, 2-12 weeks, 2-26 weeks, 2-36 weeks, 2-48 weeks, 3-4 weeks, 3-12 weeks, 3-8 weeks, 3-26 weeks, 3-36 weeks, 3-48 weeks, or 4-12 weeks prior to 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 from 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. In certain embodiments of the invention, the adjuvant is administered at least yearly for long-term enhancement of the subject's vaccine response. This disclosure also concerns methods in which the vaccine is administered in advance of the adjuvant, such as, without limitation, from about 1-8 weeks, 1-12 weeks, 1-36 weeks, 2-8 weeks, 2-12 weeks, 3-4 weeks, 3-12 weeks, 3-8 weeks, or 4-12 weeks prior to administration of the adjuvant.

[0040] In another embodiment of the invention, the 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 first administration of the vaccine, or repeated 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 first administration of the vaccine. Repeated administration of the the vaccine includes without limitation an administration of a vaccine for an influenza virus that is then followed by a vaccine for influenza virus that is not identical to the first vaccine but is recognized to provide vaccination for the type of influenza virus in its prevalent serotypes at the time of the second or repeated administration. In other embodiments, the administration of adjuvant and vaccine, concurrently or sequentially, is repeated three times, four times, five times, six times, or 5-10 times. For example, without limitation, the invention includes administering the adjuvant at day 0, then at day 7 administering the vaccine, then at day 180 administering the adjuvant, and then at day 187 administering the vaccine. In certain embodiments the actual vaccine contents may differ during this repeated therapy, however, the vaccine administered at each point in the repeated therapy would be directed against the same class of pathogen.

[0041] In one embodiment of the invention, the adjuvant is administered at a dose of about 1?10.sup.6, 2?10.sup.6, 5?10.sup.6, 10?10.sup.6, 20?10.sup.6, 30?10.sup.6, 40?10.sup.6, 50?10.sup.6, 60?10.sup.6, 70?10.sup.6, 80?10.sup.6, 90?10.sup.6, 100?10.sup.6, 110?10.sup.6, 120?10.sup.6, 130?10.sup.6, 140?10.sup.6, 150?10.sup.6, 160?10.sup.6, 170?10.sup.6, 180?10.sup.6, 190?10.sup.6, 200?10.sup.6, 300?10.sup.6, 400?10.sup.6, 500?10.sup.6, or 10?10.sup.7 mesenchymal stem cells. In a further embodiment, the adjuvant is administered at a dose of about 20?10.sup.6 mesenchymal stem cells. In a further embodiment, the adjuvant is administered at a dose of about 100?10.sup.6 mesenchymal stem cells. In yet a further embodiment, the adjuvant is administered at a dose of about 200?10.sup.6 mesenchymal stem cells. In further embodiments, the adjuvant is administered at a dose of from about 1-400?10.sup.6, 10-400?10.sup.6, 100-400?10.sup.6, 20-200?10.sup.6, 20-400?10.sup.6, 0.1-5?10.sup.6, 0.1-10?10.sup.6,0.1-100?10.sup.6, 1-50?10.sup.6, 1-100?10.sup.6, 0.01-10?10.sup.6 or 0.01-100?10.sup.6 mesenchymal stem cells.

[0042] In some embodiments, the immunoprotective amount of adjuvant is sufficient to increase the ratio of CD4.sup.+:CD8.sup.+ T cells in a subject, such as to increase the ratio of CD4.sup.+:CD8.sup.+ T cells by at least two-, three, four-, five-, or six-fold as compared to the ratio prior to administration of the adjuvant. In some embodiments, the immunoprotective amount of adjuvant is sufficient to increase the number of switched memory B cells in a subject, such as to increase the number of switched memory B cells by at least two-, three-, four-, or five-fold as compared to the number prior to administration of the adjuvant. In some embodiments, the immunoprotective amount is sufficient to decrease the intracellular TNF-? expression in the B cells of a subject, such as to decrease the intracellular TNF-? amounts in B cells by at least two-, three-, four-, five-, or six-fold as compared to the amounts in B cells prior to administration of the adjuvant. In some embodiments, the immunoprotective amount is sufficient to upregulate activation-induced cytidine deaminase (AID) in a subject. In some embodiments, the immunoprotective amount is sufficient to decrease the number of exhausted B cells in a subject, such as to decrease the number of exhausted B cells by at least two- or three-fold as compared to the number prior to administration of the adjuvant.

[0043] In a further embodiment, the use of an adjuvant allows for administration of lower levels of vaccine than those advised in the absence of adjuvant for the patient population at issue and still obtains an immune response. For example, current recommendations call for a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine (Sanofi Pasteur) for patients ?65 years of age. In the methods of the present invention, the amount of Fluzone vaccine may be reduced to less than that, such as 0.3 or 0.2 mL for a patient ?65 years of age.

[0044] 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 a further 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 a further embodiment, the vaccine is administered intramuscularly.

[0045] The term subject as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. In some embodiments, the term refers to humans, such as elderly humans ?65 years of age, or elderly humans 60-95 years of age. In some embodiments, the human subject exhibits symptoms of aging frailty. In some embodiments, the human subject exhibits inflammaging.

[0046] In one embodiment of the invention, the subject is a non-responder. It is known that, when a population of individuals is vaccinated against a disease, a number of them do not respond to the vaccination, that is to say that their immune system does not appear to react to the antigen administered. This problem is substantial to a greater or lesser degree depending on the diseases and the populations involved, but vaccine manufacturers are still trying to reduce, for each of the vaccines which they make available to doctors, the number of subjects likely to be non-responders. This problem is considered particularly important for vaccines comprising purified antigens such as subunit vaccines produced by genetic engineering.

[0047] The term allogeneic refers to a cell that is of the same animal species but genetically different in one or more genetic loci as the animal that becomes the recipient host. This usually applies to cells transplanted from one animal to another non-identical animal of the same species.

[0048] As used herein, the phrase in need thereof means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.

[0049] Cells are referred to herein as being positive or negative for certain markers. For example, a .sup.? refers cell can be negative for CD45, which can also be referred to as CD45.sup.?. The superscript notation .sup.? to a cell that is negative for the marker linked to the superscript. In contrast a marker with the .sup.+ refers to a cell that is positive for that marker. For example, a cell that is referenced as CD8.sup.+ is positive for CD8. A + can also be used to reference the marker as positive. A ? can also be used to reference the marker as negative.

[0050] As used herein, the term stem cell refers to a cell from the embryo, fetus, or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body.

[0051] Mesenchymal stem cells are the formative pluripotent blast cells found inter alia in bone marrow, blood, dermis, and periosteum that are capable of differentiating into any kind of the specific types of mesenchymal or connective tissues (i.e., the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines.

[0052] Certain methods of isolating and/or purifying mesenchymal stem cells have been described herein and are known in the art. In some embodiments, mesenchymal stem cells are isolated from bone marrow of adult humans. In some embodiments, the cells are passed through a density gradient to eliminate undesired cell types. The cells can be plated and cultured in appropriate media. In some embodiments, the cells are cultured for at least one day or about three to about seven days, and removing non-adherent cells. The adherent cells can then be plated and expanded.

[0053] Other methods for isolating and culturing stem cells are also known. Placenta is an excellent readily available source for mesenchymal stem cells. Moreover, mesenchymal stem cells can be derivable from adipose tissue and bone marrow stromal cells are speculated to be present in other tissues. While there are dramatic qualitative and quantitative differences in the organs from which adult stem cells can be derived, the initial differences between the cells may be relatively superficial and balanced by the similar range of plasticity they exhibit.

[0054] Homogeneous human mesenchymal stem cell compositions are provided which serve as the progenitors for all mesenchymal cell lineages. Mesenchymal stem cells are identified by specific cell surface markers which are identified with unique monoclonal antibodies. The homogeneous mesenchymal stem cell compositions are obtained by positive selection of adherent marrow or periosteal cells which are free of markers associated with either hematopoietic or differentiated mesenchymal cells. These isolated mesenchymal cell populations display epitopic characteristics associated with only mesenchymal stem cells, have the ability to regenerate in culture without differentiating, and have the ability to differentiate into specific mesenchymal lineages when either induced in vitro or placed in vivo at a site of inflammation.

[0055] In order to obtain the human mesenchymal stem cells for the compositions, methods, and kits disclosed herein, pluripotent mesenchymal stem cells are separated from other cells in the bone marrow or other mesenchymal stem cell source. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib, or other medullary spaces. Other spaces of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, and blood.

[0056] In some embodiments, the human mesenchymal stem cells are identified by the absence of markers. For example, human mesenchymal stem cells useful in the invention include those that are negative for STRO-1 and/or negative for CD45. Similarly, human mesenchymal stem cells useful in the invention include those that do not express fibroblast surface markers or have a fibroblast morphology.

Methods of Enhancing Immune Responses and Kits Therefor

[0057] As discussed above, the present invention is directed to a method of enhancing a subject's immune response to a vaccine or inducing an immune response in a non-responding subject, comprising administering to the subject concurrently or sequentially the vaccine and an adjuvant in immunoprotective amounts, wherein the adjuvant comprises a population of isolated allogeneic human mesenchymal stem cells, and kits associated with such methods. In some embodiments of the invention, the mesenchymal stem cells are not genetically manipulated. In some embodiments of the invention, the mesenchymal stem cells are obtained from a human donor and wherein a step of MHC matching of the human donor to the subject is not employed prior to the administration of the vaccine and adjuvant to the subject.

[0058] In one embodiment of the invention, the vaccine is monovalent. In another embodiment of the invention, the vaccine is multivalent.

[0059] In one embodiment of the invention, the vaccine comprises one or more inactivated viruses. In a further embodiment, the one or more inactivated viruses are selected from the group consisting of adenoviruses, picornaviruses, papillomaviruses, polyomaviruses, hepadnaviruses, parvoviruses, pox viruses, Epstein-Barr virus, cytomegalovirus (CMV), herpes viruses, roseolovirus, varicella zoster virus, filoviruses, paramyxoviruses, orthomyxoviruses, rhabdoviruses, arenaviruses, coronaviruses, human enteroviruses, hepatitis A virus, human rhinoviruses, polio virus, retroviruses, rotaviruses, flaviviruses, hepaciviruses, togaviruses, and rubella virus. In a further embodiment, the vaccine comprises an inactivated orthomyxovirus. In a further embodiment, the vaccine comprises an inactivated influenza virus.

[0060] In one embodiment of the invention, the vaccine comprises one or more live, attenuated viruses. In a further embodiment, the one or more attenuated viruses are selected from the group consisting of adenoviruses, picornaviruses, papillomaviruses, polyomaviruses, hepadnaviruses, parvoviruses, pox viruses, Epstein-Barr virus, cytomegalovirus (CMV), herpes viruses, roseolovirus, varicella zoster virus, filoviruses, paramyxoviruses, orthomyxoviruses, rhabdoviruses, arenaviruses, coronaviruses, human enteroviruses, hepatitis A virus, human rhinoviruses, polio virus, retroviruses, rotaviruses, flaviviruses, hepaciviruses, togaviruses, and rubella virus.

[0061] In another embodiment of the invention, the vaccine comprises an antigen from a bacterial pathogen. In a further 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, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

[0062] In another embodiment of the invention, the vaccine comprises an antigen from a parasitic pathogen. In a further embodiment, the parasitic pathogen is selected from the group consisting of: Acanthamoeba, Anisakis, Ascaris lumbricoides, Balantidium coli, Cestoda, Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrate, Liver fluke, Loa boa, Paragonimus, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Tapeworm, Toxoplasma gondii, Trypanasoma, Whipworm, and Wuchereria bancrofti.

[0063] In another embodiment of the invention, the vaccine comprises one or more antigenic polypeptides selected from the group consisting of 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 virus1 (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, anthrax protective protein, anthrax lethal factor, anthrax edema factor, Salmonella typhii S1 Da, Salmonella typhii S1db, cholera toxin, cholera heat shock protein, Clostridium botulinum antigen S, botulinum toxin, Yersinia pestis F1, Yersinia pestis V antigen, Yersinia pestis YopH, Yersinia pestis YopM, Yersinia pestis YopD, Yersinia pestis plasminogen activation factor (Pla), Plasmodium circumsporozoite protein (CSP), Plasmodium sporozoite surface protein (SSP2/TRAP), Plasmodium liver stage antigen 1 (LSAT), Plasmodium exported protein 1 (EXP 1), Plasmodium erythrocyte binding antigen 175 (EBA-175), Plasmodium cysteine-rich protective antigen (cyRPA), Plasmodium heat shock protein 70 (hsp70), Schistosoma Sm29, and Schistosoma signal transduction protein 14-3-3.

[0064] Compositions for use in the invention may be formulated using any suitable method. Formulation of cells with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0065] Compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of cells. The cells may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.

[0066] In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. In one embodiment of the invention, the adjuvant comprises human serum albumin (HSA).

[0067] One suitable carrier or diluent is PlasmaLyte A?. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C.sub.6H.sub.11NaO.sub.7); 368 mg of Sodium Acetate Trihydrate, USP (C.sub.2H.sub.3NaO.sub.23H.sub.2O); 37 mg of Potassium Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCl.sub.26H.sub.2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0068] As discussed above, the present invention also 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 comprises a population of isolated allogeneic human mesenchymal stem cells. Any of the adjuvants or vaccines discussed herein may be formulated in a kit according to the present disclosure. In one embodiment of the invention the mesenchymal stem cells are not genetically manipulated. In another embodiment of the invention, the mesenchymal stem cells are cryopreserved in the second container. For example, the mesenchymal stem cells can be suspended in cryoprotectant consisting of Hespan? (6% hetastarch in 0.9% sodium chloride) supplemented with 2% HSA and 5% DMSO and then aliquoted into cryopreservation containers for placement in vapor phase nitrogen freezers. In another embodiment, the mesenchymal stem cells may be provided in the second container in PlasmaLyte ATM supplemented with 1% HSA. In another embodiment, the kit has at least three containers, wherein the third container comprises dilution buffer for mesenchymal stem cell suspension and dilution. In a further embodiment, the dilution buffer contains PlasmaLyte ATM supplemented with 1% HSA.

[0069] In one embodiment, the invention is a composition comprising an adjuvant and a vaccine in immunoprotective amounts. One of skill in the art may formulate suitable compositions from among the adjuvants and vaccines disclosed herein.

EXAMPLES

Example 1

[0070] Effects of Aging on E47 mRNA Expression in Human Peripheral Blood Derived B Cells

[0071] As discussed above, both E47 and Pax-5 are important transcription factors in early development for the B-cell lineage and mature B cell function. It has been demonstrated that a putative regulatory region in the Aicda gene contains both E47 and Pax-5 binding sites, both indispensable for AID gene expression. The following experiment demonstrates that E47 expression decreases as a function of age and that E47 expression in B cells is positively correlated with AID expression. See FIGS. 1 and 2.

[0072] Forty-six subjects ranging in age from 20 to 85 years of age were recruited and peripheral blood was taken. CD19.sup.+ B cells (10.sup.6 cells/mL) were cultured with anti-CD40 (1 ?g/mL) and IL-4 (10 ng/mL) for 24 hours. PCR was performed for E47 and GAPDH. E47 was normalized to GAPDH. FIG. 1 (A) shows undiluted and ? diluted RT-PCR from 5 representative subjects. In FIG. 1 (B), the graph shows densitometry analyses of CT's normalized to GAPDH. Numbers shown for each sample are percentages of the highest value taken as 100. Pearson's r value for the linear curve expressing correlation between age and E47 expression is r=?0.84, p=0.00001. Solid line refers to linear regression and dashed line refers to quadratic regression. Thus, the data shows that E47 expression decreases as a function of age.

[0073] The blood from these subjects was also subjected to PCR for AID and GAPDH, and the results are shown in FIG. 2. E47 (from 24 hour stimulation) and AID (from 5 day stimulation) were individually normalized to their respective GAPDH value. Numbers shown for each sample are percentages of the highest value taken as 100. Data for E47 and AID PCR were positively correlated. Correlation is significant at the 0.01 level (2-tailed). Solid line refers to linear regression. E47 expression in B cells is positively correlated with AID expression as seen in FIG. 2. (r=0.80, p=0.01 level, 2-tailed).

Example 2

Switched Memory B Cells

[0074] The following experiment shows that switched memory B cells increase in elderly subjects after treatment with allogeneic mesenchymal stem cells (MSC). Switched memory B cells have been measured at base line (prior to intravenous infusion of MSC) and 6 month post MSC infusion in human patients from the CRATUS test. The results show that MSC infusion upregulates the Switch memory B cell compartment which is a predictive biomarker for improved antibody response. See FIG. 3. This was true for the 3 doses of MSCs tested (20?10.sup.6, 100?10.sup.6, or 200?10.sup.6 mesenchymal stem cells). The data shows that intravenous administration of allogeneic mesenchymal stem cells augment by two-fold the Switch memory of B cells in some patients.

[0075] FIG. 4 shows the % of switched memory B cells and exhausted B cells in subjects who received a second mesenchymal stem cell infusion one year after their first mesenchymal stem cell infusion. The graphs suggest that a second injection at 12 months may be necessary to maintain the improvements on the immune cells derived from the initial mesenchymal stem cell treatment.

Example 3

T Cell Activation

[0076] The following experiment shows that both early and late/chronic T cell activation decrease after allogeneic MSC treatment. The early marker of T cell activation (CD69) and the late/chronic marker of T cell activation (CD25) have been measured in human patients from the CRATUS test sample. As shown in FIGS. 5A and 5B, there is no T cell activation (rejection) induced by the allogeneic mesenchymal stem cells at any dose (20?10.sup.6, 100?10.sup.6, or 200?10.sup.6 mesenchymal stem cells). See also FIGS. 5C and 5D, which show the absolute difference at six months post MSC infusion against the % CD69 or % CD25 cells.

Example 4

Immune Risk Phenotype

[0077] The Immune Risk Phenotype was measured on the same CRATUS subjects undergoing a second MSC infusion one year post the first MSC infusion. See FIG. 6. The results show MSC infusion improves the CD4.sup.+:CD8.sup.+ T cell ratio. Furthermore, by 12 months post the first infusion the effects begin to regress. At that time, a second MSC infusion yields additional improvement. A second injection at 12 months may be necessary to maintain the improvements on the immune cells derived from the initial mesenchymal stem cell treatment.

Example 5

TNF-Alpha

[0078] TNF-? reduces AID. TNF-? was measured in samples from two CRATUS subjects by intracellular staining of B cells by flow cytometry and confirmed by qPCR. Results are shown at baseline (12 months post-first infusion of MSCs) and three months (three months post 2.sup.nd infusion) as shown in FIGS. 7A and 7B. The results show downregulation of intracellular TNF-? in B cells by flow cytometry after mesenchymal stem cell infusion. These results were confirmed by qPCR.

Example 6

[0079] Effects of Intravenous Delivery of Allogeneic Human Mesenchymal Stem Cells on VaccinE-Specific Antibody {right arrow over (R)}esponses in Patients with {right arrow over (A)}ging Frailtythe HERA Trial (Phase I/II)

[0080] The HERA Trial is a phase I/II randomized, double-blinded, and placebo-controlled study. The primary objective of the study is to demonstrate that intravenous administration of mesenchymal stem cells can improve adaptive immunity and can improve primary B cell response to the influenza vaccine in subjects with aging frailty.

[0081] Forty-three (43) subjects with aging frailty are enrolled. The adjuvant (allogeneic mesenchymal stem cells) are administered by peripheral intravenous infusion. The total duration for each subject after infusion is 12 months, plus up to an additional 2 months for the Screening and Baseline Visits. A Safety Run-In is followed by a Double-Blinded Randomized Phase. All subjects must meet the inclusion/exclusion criteria, and are evaluated prior to the scheduled infusion to establish the baseline.

The Safety Run-In

[0082] The Safety Run-In includes 23 subjects in 3 cohorts and is performed to determine the optimal time point to administer the influenza vaccine after mesenchymal stem cell infusion.

[0083] Cohort A (3 subjects): A single peripheral intravenous infusion of 20?10.sup.6 mesenchymal stem cells is administered to teach subject. At 1 week post-infusion, the subjects receive a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine (Sanofi Pasteur), which is recommended for patients ?65 years of age. These subjects are infused first (i.e., prior to any subjects of Cohorts B and C), and are infused no less than 5 days apart.

[0084] Cohort B (10 subjects): A single peripheral intravenous infusion of 100?10.sup.6 mesenchymal stem cells is administered to each subject. At 1 week post-infusion, the subjects receive a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine.

[0085] Cohort C (10 subjects): A single peripheral intravenous infusion of 100?10.sup.6 mesenchymal stem cells is administered to each subject. At 4 weeks post-infusion, the subjects receive a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine.

[0086] The subjects of Cohorts B and C are randomized, and the first 3 subjects are infused no less than 5 days apart. Follow-up visits occur at 1 and 4 weeks post-vaccination, to determine safety and efficacy of the vaccination after mesenchymal stem cell infusion. After all 23 subjects are infused and vaccinated, a 30-day review is conducted to assess all safety data. The Double-Blinded Randomized Phase is conducted after the successful completion of the Safety Run-In has been reviewed and approved. After the Safety Run-In data has been analyzed, both at 1 week and 4 weeks post-vaccination, a standard vaccination time point is utilized for the Double-Blinded Randomized portion of the trial.

The Double-Blinded Randomized Trial

[0087] The Double-Blinded Randomized Trial includes 20 subjects in 2 cohorts. All subjects must meet the inclusion/exclusion criteria, and are evaluated prior to the scheduled infusion to establish baseline. The subjects are randomized at a ratio of 1:1 into 2 cohorts as follows:

[0088] Cohort 1 (10 subjects): A single peripheral intravenous infusion of 100?10.sup.6 mesenchymal stem cells are administered to each subject. At the optimal time-point post-infusion (1 or 4 weeks, as determined in the Safety Run-In), the subjects receive a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine.

[0089] Cohort 2 (10 subjects): A single peripheral intravenous infusion of placebo (PlasmaLyte ATM with 1% HSA) is administered to each subject. At the optimal time-point post-infusion (1 or 4 weeks, as determined in the Safety Run-In), the subjects will receive a single 0.5 mL dose intramuscular injection of the Fluzone High-Dose vaccine.

[0090] A phone-call follow-up is done at 1 day post-infusion and post-vaccination. In-office follow-up visits are conducted at Week 1 and Week 4 post-vaccination, and Month 6 and Month 12 post-infusion, to complete all safety and efficacy assessments. Any subjects that develop influenza-type symptoms during the first 7 months after the vaccination must immediately schedule an office visit, so that evaluation of the potential influenza strain can be assessed.

Primary Endpoint

[0091] The primary efficacy endpoint is B cell function as measured by (1) the ability of B cells to upregulate activation-induced cytidine deaminase (AID) in response to CpG or Influenza Vaccine through qPCR and (2) Influenza specific antibody production through HAI and ELISA. The data for the primary efficacy endpoint is obtained from the Baseline Visit, Vaccination Visit (performed at 1 or 4 weeks post-mesenchymal stem cell infusion), the Week 1 and Week 4 post-vaccination Follow-Up Visits, and the Month 6 and Month 12 post-infusion Follow-Up Visits.

Inclusion Criteria

[0092] All subjects enrolled in this trial must: provide written informed consent; be 65-95 years of age at the time of signing the Informed Consent Form; have a diagnosis of frailty, with a score of 4 to 7 using the Canadian Frailty Scale; show immunosenescence as measured by whole blood flow cytometry staining resulting in ?5% switched memory B cells, late/exhausted memory at ?10%, CD8.sup.+ na?ve cells ?20%, and CD8.sup.+ TEMRA cells ?40%; and have total bilirubin between 0.3-1.9 mg/dL.

Adjuvant and Placebo

[0093] The final mesenchymal stem cell formulation is 2.5?10.sup.6 mesenchymal stem cells/mL suspended in 80 mL of PlasmaLyte ATM containing 1.0% HSA for subjects to receive 100?10.sup.6 mesenchymal stem cells. For subjects in the Run-In Phase who receive 20?10.sup.6 mesenchymal stem cells, the final formulation is 0.5?10.sup.6 mesenchymal stem cells/mL suspended in 80 mL PlasmaLyte ATM containing 1.0% HSA. The adjuvant is manufactured by Cell Processing Facility of Longeveron LLC (Life Sciences and Technology Park, 1951 NW 7th Ave., Miami, FL 33136).

[0094] Dilution buffer is used for placebo. The final formulation of placebo is PlasmaLyte ATM containing 1.0% HSA (total 80 mL).

Adjuvant Administration Dosage and Rate

[0095] Subjects in this trial receive a single infusion of 20?10.sup.6 mesenchymal stem cells, 100?10.sup.6 mesenchymal stem cells, or placebo. The maximum infusion rate is 2.5?10.sup.6 mesenchymal stem cells/min, which is far below the maximum reported dosing rate. Table 3 provides a breakdown of the infusion parameters for the mesenchymal stem cell and placebo infusions. A total of 80 mL is delivered intravenously to each subject.

TABLE-US-00001 TABLE 3 Mesenchymal Stem Cell Dosing for the Randomized Study Total Volume Total (cells + Delivery Delivery Rate Total Mesenchymal PlasmaLyte Concentration (Mesenchymal Delivery Delivery Stem Cells A? with (Mesenchymal Stem Rate Time Delivered 1% HSA) Stem Cells/mL) Cells/min) (mL/min) (min) 0 (placebo) 80 mL N/A N/A 2 40 20 ? 10.sup.6 cells 80 mL 0.25 ? 10.sup.6 0.5 ? 10.sup.6 2 40 100 ? 10.sup.6 cells 80 mL 1.25 ? 10.sup.6 2.5 ? 10.sup.6 2 40