DK2190475T3 - PROCEDURES FOR IMPROVING ADJUSTABILITY IN VACCINE COMPOSITIONS - Google Patents

Description

DESCRIPTION

INTRODUCTION

[0001] The fundamental purpose of a vaccine is to provide lasting immunity against a pathological condition. Ideally vaccines provide functionally active antibodies, elicit cell-mediated immunity, and activate T- and B-lymphocytes with highly specific reactivity as well as "memory" to provide protection against further encounters with a pathogen.

[0002] Adjuvants are vaccine additives which nonspecifically augment the immune response. The mechanism by which adjuvants enhance the immune system varies widely. Adjuvants may be classified as "immunomodulatory" or "antigen delivery systems." Immunomodulatory adjuvants prime the immune system by regulating the action of immune cells by altering lymphokine production. Antigen delivery systems, on the other hand, function to deliver the antigen to the appropriate immune cells. In addition, adjuvants may enhance the speed or duration of an immune response, modulate antibody avidity, specificity, isotype or subclass distribution, stimulate cell mediated immunity, promote mucosal immunity, or enhance the immune responses in immunologically immature or senescent individuals. Adjuvants can affect either the humoral or cell-mediated immune response, or a combination of both.

[0003] Immunomodulation refers to the ability of an adjuvant to alter the lymphokine response by activating differential subsets of immune cells. Two major subsets of CD4+ T lymphocytes, Th1 and Th2, play a major role in determining the immune response. Th1 responses typically induce complement fixing antibody and strong delayed-type hypersensitivity (DTH) reactions and are associated with γ-IFN, IL-2, and IL-12, while Th2 responses result in high circulating and secretory antibody levels and are associated with cytokines IL-4, IL-5, IL-6 and IL-10. Activation of Th1 cells also regulates the cellular immune response by activating the proliferation of cytotoxic CD8+ T cells and enhanced cytolysis of target cells.

[0004] Lipid species have been investigated for adjuvant properties. Unlike peptide antigens, lipids are processed and presented to the immune system by the CD1 family of β2 microglobulin-associated molecules. CD1 molecules have evolved to capture and process both foreign and self lipid antigens for display to particular subsets of T cells. The CD1 presentation pathway triggers both innate and adaptive immune response by activating two complementary CD1 restricted T cell subsets; natural killer T (NKT) cells that perform adjuvant functions; and non-NKT T-cells capable of helper or cytolytic functions. NKT cells express both natural killer (NK) cell surface markers and a conserved semi-invariant T-cell receptor (TCR), Va24-Ja18/Vp8 in mice and Va24-Ja18/Vpi1 in humans. Accordingly, NKT cells play an important role in a number of immune functions, including antimicrobial responses, antitumor immunity, and regulation of the balance between tolerance and autoimmunity.

[0005] A number of natural and synthetic lipid molecules are processed by antigen-presenting cells and presented by CD1 molecules to NKT cells. The prototypical compound used to study NKT cell activation in vitro and in vivo is KRN7000, and a-galactosyceramide ("aGalCer") derived from marine sponge Agelas mauritianus. Additional compounds recently identified include isoglobotrihexosylceramide ("iGB3") which is an endogenous glycolipid and PBS-57, a modified 6"amino 6" deoxygalactosyceramide, as described in PCT Application PCT/US07/66250, the disclosure of which is incorporated herein by reference. These compounds activate NKT cells and upregulate cytokine responses in vitro. However, in the context of in vivo vaccinations, little is known regarding the effectiveness of lipid adjuvanticity for these compounds.

[0006] WO 2007/007946 describes the use of alpha-GalCer as adjuvant in a nasal vaccine comprising a virus antigen or a tumor-associated antigens. Using the nasal route, an antigen-specific IgA and IgG immune response is induced. Silk et al. (2004 J. Clin. Invest. 114 :1800-1811 ) describes alpha-GalCer used as adjuvant for inducting CD8 T cell immunity to a coadministered peptide derived from NY-ESO-1. Liu et al. (2006 J. Immun. Methods 312 :34-39) describes the modified alpha-GalCer PBS-57 and its effect on NKT cells compared with that of KRN7000. WO 2007/118234 describes the combined administration of PBS-57 with an antigen in order to stimulate an immune response. Measurement of IL-4 and IFN-gamma levels after 48 hours shows that PBS-57 is able to induce both Th1 and Th2 cytokine secretion. WO 2008/005824 describes the combined use of the modifed alpha-GalCer called "PBS-32" and an antigen in order to induce/enhance an immune response. WO 2008/080926 describes a composition comprising a B subunit of Shiga toxin that is able to bind the Gb3 receptor, which is complexed with an antigens, and at least one ligand of CD1 capable of stimulating NKT cells. The CD1 ligand can in particular be PBS-57.

[0007] Few adjuvants have been approved for use in human vaccines due to toxic side effects. The most common adjuvants are aluminum and oil adjuvants, but they are known to induce side effects such as fever, headache, muscle aches and pains or rash. Other disadvantages include a strong local stimulation of the immune system leading to pain, redness, swelling or a small lump at the site of injection. Adjuvants also have complicated methods of vaccine preparation and may fail to increase immunogenicity of weak antigens. At present, the choice of adjuvants for human vaccination reflects a compromise between the requirement for adjuvanticity and an acceptable level of side effects. There is a need for new adjuvants with fewer side effects that still elicit long-lasting protective immunity.

SUMMARY OF INVENTION

[0008] The inventors have discovered that the addition of a synthetic glycolipid, designated PBS-57, to a vaccine preparation administered to a subject activates both a humoral and cellular immune response when administered to the subject. Accordingly, the disclosure provides methods of enhancing the immunogenicity of a vaccine in a subject, stimulating a humoral immune response, stimulating a CD4+ T cell response and stimulating a CD8+ cytotoxic T cell response in a subject by co-administering PBS-57 and a vaccine to a subject. The present invention provides a vaccine composition comprising: 1. a) a compound PBS-57 of formula:

and 2. b) an antigen, for use as a medicament in a method of enhancing the humoral and/or cell mediated immune response to the antigen of a subject vaccinated with said vaccine composition, relative to a control from a subject vaccinated with said antigen alone, and wherein the antigen is not complexed with a B subunit of Shiga toxin or a functional equivalent thereof which is able to bind the Gb3 receptor.

[0009] The invention is defined by the claims.

BRIEF DESCRIPTION OF FIGURES

[0010] FIG. 1 is a graph showing the percentage of NKT cells on day 7 in a CD3+ T cell population of cultured peripheral blood lymphocytes (PBL) treated with various amounts of adjuvant glycolipids aGalCer, PBS20, PBS25 and PBS57. FIG. 2 is a graph showing the percentage of NKT cells on day 7 in a CD3+ T cell population of cultured PBLs treated with various amounts ofaGalCer, PBS25, PBS57 and PBS83. FIG. 3 is a graph showing an increase in NKT cells in in vitro cultures of PBLs isolated from volunteers treated with aGalCer, PBS20, PBS25, PBS57 and PBS83. The median value is depicted above the graphs. FIG. 4A is a graph depicting the percentage of TCRap cells within the spleen of mice 24 hours after administration of aGalCer, PBS57, and PBS83. FIG. 4B is a graph of the percentage of NKT cells within the TCRap population in the spleen of mice 24 hours after administration of the adjuvant glycolipids. The two-tailed p value was calculated for each adjuvant compared to the control. FIG. 5A is a graph of the percentage of CD40+ cells in the CD1 1c+ CD8a-cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 5B is a graph of the percentage of CD40+ cells in the CD11c+ CD8a+ cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 5C is a graph of the percentage of CD80+ cells in the CD11c+ CD8a- cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 5D is a graph of the percentage of CD80+ cells in the CD1c+ CD8a+ cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 5E is a graph of the percentage of CD86+ cells in the CD11c+ CD8a- cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 5F is a graph of the percentage of CD86+ cells in the CD11c+ CD8a+ cell population from the spleen of mice 24 hours after intravenous administration of adjuvant glycolipids. FIG. 6 is a dot plot and a histogram of results of flow cytometric CFSE staining from an assay to determine the specific lysis of antigen presenting cells for mice immunized with ovalbumin (Ova) alone or Ova and aGalCer. FIG. 7 is a graph depicting the percentage of specific lysis of Ova-specific target cells in the spleen of mice immunized with adjuvant, with or without Ova. FIG. 8 is a graph depicting the percentage of specific lysis of Ova-specific target cells in the blood of mice injected intravenously ("IV") with Ova in combination with different concentrations of aGalCer. FIG. 9 is a graph depicting the percentage of specific lysis of Ova-specific target cells in the blood of mice injected IV with Ova in combination with different concentrations of PBS57. FIG. 10 is a graph depicting the percentage of specific lysis of Ova-specific target cells in the blood of mice injected IV with PBS57 in combination with different concentrations of Ova. FIG. 11 is a graph depicting the percentage of specific lysis of Ova-specific target cells in the blood of mice injected intramuscularly (IM) with Ova in combination with different concentrations of aGalCer. FIG. 12 is a graph depicting of the percentage of specific lysis of Ova-specific target cells in the blood of mice injected IM with Ova in combination with different concentrations of PBS57. FIG. 13 is a graph comparing the percentage of specific lysis of Ova-specific target cells in the blood of mice injected either IV or IM with different Ova and adjuvant combinations. FIG. 14 is a bar graph depicting enhanced antibody production by mice immunized with a tetanus toxoid (TT) vaccine composition containing PBS-57. FIG. 15 is a bar graph depicting enhanced CD8+ T cell responsiveness in mice immunized intramuscularly with ovalbumin (OVA) in combination with PBS57. FIG. 16 is a bar graph depicting enhanced CD8+ T cell responsiveness in mice immunized subcutaneously with OVA in combination with PBS57.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

[0011] Adjuvants enhance the immunogenicity of antigens in vaccine preparations in a variety of ways. In the case of toxins, a good humoral immune response is required. In the case of intracellular bacteria, a cell-mediated response, mediated mainly by cytotoxic T cells and Th1 cells, is important. In the case of viral infections, both humoral and cellular responses are fundamental to control the infection. The ability of an adjuvant to enhance not only the humoral but also the cell-mediated immune response increases the likelihood of developing long-lasting immunity. An effective adjuvant also would be useful for combination with a wide variety of antigens. The inventors have found that a glycosphingolipid, PBS-57, has the ability to stimulate both a cell-mediated and humoral immune response in vivo. In addition, PBS-57 is able to stimulate an immune response against a weak nominal antigen to produce antibodies and simultaneously provide for cell-mediated lysis of cells expressing specific surface antigens.

[0012] The invention provides a vaccine composition comprising: 1. a) a compound PBS-57 of formula:

and 2. b) an antigen, for use as a medicament in a method of enhancing the humoral and/or cell mediated immune response to the antigen of a subject vaccinated with said vaccine composition, relative to a control from a subject vaccinated with said antigen alone, and wherein the antigen is not complexed with a B subunit of Shiga toxin or a functional equivalent thereof which is able to bind the Gb3 receptor.

[0013] The disclosure also provides a method of enhancing the immunogenicity of a vaccine in a subject by co-administering PBS57 and the vaccine. As used herein, a "subject" is a mammal, e.g., a mouse, more suitably a human. "Enhancing the immunogenicity of a vaccine" refers to the ability of PBS-57 to enhance the humoral and/or cell mediated immune response of a subject to a vaccine in relation to a suitable control. For purposes of determining whether immunogenicity is enhanced relative to a control, a quantitative comparison of the signal in a sample from a subject vaccinated with antigen and PBS57 can be compared to the signal in a sample from a subject vaccinated with antigen alone. As used herein, immunogenicity may be measured by any assay used by those of skill in the art to measure the humoral or cell-mediated immune response. For example, the immunogenicity may be measured using an ELISA assay for various cytokine levels, the performance of which is routine to those skilled in the art.

[0014] As described herein, the immune response is enhanced at least 25%, at least 30%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, or at least 400%, relative to a suitable control. A suitable control may be a subject treated with a vaccine composition not including PBS57. Percent enhancement may be calculated using the following formula: [(value representing subject's immune response after treatment with composition containing PBS57)-(value representing immune response of control)//value representing subject's immune response after treatment with composition containing PBS57)]xlOO.

[0015] As used herein the term "co-administration" or "co-administering" refers to administration of at least the adjuvant and the vaccine concurrently, i.e., simultaneously in time, or sequentially, i.e., administration of an adjuvant, followed by administration of the vaccine. That is, after administration of the adjuvant, the vaccine can be administered substantially immediately after the adjuvant or the vaccine can be administered after an effective time period after the adjuvant; the effective time period is the amount of time given for realization of maximum benefit from the administration of the adjuvant. Alternatively, the adjuvant and vaccine may be co-formulated.

[0016] Vaccine compositions are suitably formulated to include PBS-57.

[0017] "Vaccine" refers to a composition which, when administered to a subject, induces cellular or humoral immune responses as described herein. Vaccine compositions may include an antigen or combinations of antigens; the antigen may be a polypeptide or carbohydrate moiety, or combinations thereof, for example, a glycoprotein. The antigen is suitably derived from an infections agent (e.g., a pathogenic microorganism), a tumor, an endogenous molecule (e.g., a "self molecule), or, for purposes of study, a nominal antigen, such as ovalbumin (referred to herein as "Ova"). Vaccine compositions may also include killed or attenuated infectious agents. The vaccine compositions used in conjunction with the invention suitably include PBS57 and an antigen. The structure of PBS-57 is shown below:

[0018] PBS57 activates NKT cells in vitro and in vivo. PBS57 contains an amide group at the C6 position of the galactose and a cis-double bond in the acyl chain of the ceramide portion. Not to be bound by theory, the double bond in the ceramide side-chain is thought to facilitate binding to the groove of the CD1d molecule and increase the solubility of glycosphingolipid. PBS57 has also been shown to induce the release of INF-γ and IL-4 in vitro.

[0019] Vaccine compositions including PBS57 may be formulated using a variety of preparative methods and inactive ingredients known to those of skill in the art. See Remington's Pharmaceutical Sciences, Mack Publishing Co., (2000). The vaccine may also contain a suitable antigen delivery system to target the antigen to immune cells. Suitable antigen delivery systems are known in the art, and include, but are not limited to, MVA (Modified virus ankara), adenovirus, lentivirus, translocated subunit of pertussis or shiga toxin, or antigen encapuslated liposomes. Suitable effective dosage amounts of PBS57 in a vaccine compositions may be determined by those of skill in the art, but typically range from about 1 microgram to about 10,000 micrograms per kilogram of body weight, although they are typically about 1,000 micrograms or less per kilogram of body weight. In some embodiments, the effective dosage amount ranges from about 10 to about 5,000 micrograms per kilogram of body weight. In another embodiment, the effective dosage amount ranges from about 50 to about 1,000 micrograms per kilogram of body weight. In another embodiment, the effective dosage amount ranges from about 75 to about 500 micrograms per kilogram of body weight. For purposes of study, a suitable dosage for a mouse is 1 pg PBS57 per 10ΟμΙ dose. The PBS57 composition can be administered in a single dose, or may be administered in multiple doses over a period of weeks or months.

[0020] One or more antigens may be included in the compositions with PBS57, or may be formulated independently. As used herein, an antigen refers to a molecule that stimulates an immune response. It will be appreciated that the dosage of antigen will depend on the specific antigen, and on the age and immune status of the subject, as well as other relevant factors that may be determined by those skilled in the art.

[0021] Suitably, antigens are derived from attenuated or killed infectious agents. Whole microorganisms or portions thereof (e.g., membrane ghosts; crude membrane preparations, lysates and other preparations of microorganisms) may be utilized. Suitable infectious agents from which an antigen may be derived include, but are not limited to, pathogens and microorganisms such as bacteria, parasites and viruses. In some contexts, suitable antigens are obtained or derived from a viral pathogen that is associated with human disease including, but not limited to, HIV/AIDS (Retroviridae, e.g., gp120 molecules for HIV-1 and HIV-2 isolates, HTLV-I, FITLV-11), influenza viruses ( Orthomyxoviridae, e.g., types A, B and C), herpes (e.g., herpes simplex viruses, FISV-1 and FISV-2 glycoproteins gB, gD and gH), rotavirus infections (Reoviridae), respiratory infections (parainfluenza and respiratory syncytial viruses), Poliomyelitis (Picornaviridae, e.g., polioviruses, rhinoviruses), measles and mumps (Paramyxoviridae), Rubella (Togaviridae, e.g., rubella virus), hepatitis (e.g., hepatitis viruses types A, B, C, D, E and/or G), cytomegalovirus (e.g., gB and gH), gastroenteritis (Caliciviridae), Yellow and West Nile fever (Flaviviridae), Rabies (Rhabdoviridae), Korean hemorrhagic fever (Bunyaviridae), Venezuelan fever (Arenaviridae), warts (Papillomavirus), simian immunodeficiency virus, encephalitis virus, varicella zoster virus, Epstein-Barr virus, and other virus families, including Coronaviridae, Birnaviridae and Filoviridae.

[0022] Suitable bacterial and parasitic antigens can also be obtained or derived from known agents responsible for diseases including, but not limited to, diphtheria, pertussis, tetanus, tuberculosis, bacterial or fungal pneumonia, otitis media, gonorrhea, cholera, typhoid, meningitis, mononucleosis, plague, shigellosis or salmonellosis, Legionnaires' disease, Lyme disease, leprosy, malaria, hookworm, Onchocerciasis, Schistosomiasis, Trypanosomiasis, Leishmaniasis, giardiases, amoebiasis, filariasis, Borrelia, and trichinosis. Still further antigens can be obtained or derived from unconventional pathogens such as the causative agents of kuru, Creutzfeldt-Jakob disease (CJD), scrapie, transmissible mink encephalopathy, and chronic wasting diseases, or from proteinaceous infectious particles such as prions that are associated with mad cow disease.

[0023] Additional specific pathogens from which antigens can be derived include M. tuberculosis, Chlamydia, N. gonorrhoeae, Shigella, Salmonella, Vibrio cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella, Francisella tularensis, Flelicobacter pylori, Leptospira interrogans, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and B), pneumococcus, meningococcus, Haemophilus influenza (type b), Toxoplasma gondii, Moraxella catarrhalis, donovanosis , and actinomycosis; fungal pathogens include candidiasis and aspergillosis; parasitic pathogens include Taenia, flukes, roundworms, amebiasis, giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii, trichomoniasis and trichinosis. The present invention can also be used to provide a suitable immune response against numerous veterinary diseases, such as foot-and-mouth diseases, coronavirus, Pasteurella multocida, Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia, Bovine Viral Diarrhea Virus (BVDV), Klebsiella pneumoniae, E. coli, and Bordetella pertussis, parapertussis and brochiseptica.

[0024] In other embodiments, antigens for inclusion in vaccine compositions that may be used in the present methods are tumor-derived antigens or autologous or allogeneic whole tumor cells. Suitably, the tumor antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA). Several tumor antigens and their expression patterns are known in the art and can be selected based on the tumor type to be treated. Non-limiting examples of tumor antigens include cdk4 (melanoma), β-catenin (melanoma), caspase-8 (squamous cell carcinoma), MAGE-1 and MAGE-3 (melanoma, breast, glioma), tyrosinase (melanoma), surface Ig idiotype (e.g., BCR) (lymphoma), Her-2/neu (breast, ovarian), MUC-1 (breast, pancreatic) and HPV E6 and E7 (cervical carcinoma). Additional suitable tumor antigens include prostate specific antigen (PSA), sialyl Tn (STn), heat shock proteins and associated tumor peptides (e.g., gp96), ganglioside molecules (e.g., GM2, GD2, and GD3), Carcinoembryonic antigen (CEA) and MART-1.

[0025] As appreciated by skilled artisans, vaccines are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, oral (e.g., inhalation), transdermal (topical), intranasal, interperitoneal, transmucosal, and rectal administration. The vaccine may also include a physiologically acceptable vehicle. A "physiologically acceptable" vehicle is any vehicle that is suitable for in vivo administration (e.g., oral, transdermal or parenteral administration) or in vitro use, i.e., cell culture. Suitable physiologically acceptable vehicles for in vivo administration include water, buffered solutions and glucose solutions, among others. Additional components of the compositions may suitably include excipients such as stabilizers, preservatives, diluents, emulsifiers or lubricants, in addition to the physiologically acceptable vehicle and the antigen. In particular, suitable excipients include, but are not limited to, Tween 20, DMSO, sucrose, L-histadine, polysorbate 20 and serum.

[0026] A method of stimulating a humoral immune response to an antigen is also disclosed. The method includes coadministering PBS-57 and the antigen to a subject, as described above. As used herein, a "humoral immune response" is the production of antibodies by B cells, and the accessory process that accompanies it, including, but not limited to, e.g., Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation production and memory cell generation. For purposes of determining whether a humoral immune response is activated, a quantitative comparison of the signal in a sample from a subject vaccinated with antigen and PBS57 can be compared to a sample from a subject vaccinated with antigen alone. The humoral immune response may be evaluated by measuring the effector functions of antibodies, including pathogen or toxin neutralization, classical compliment activation, and opsonin promotion of phagocytosis and pathogen elimination. The antibodies produced in response to co-administering PBS-57 and an antigen may be of any type, e.g., IgM, IgA, or IgG. The humoral immune response may be assayed by any quantitative method known in the art, e.g., ELISA, single radial immunodiffusion assay (SRID), enzyme immunoassay (EIA), or hemagglutination inhibition assay (HAI).

[0027] A method of activating CD4+ T lymphocytes in a subject is also disclosed. As understood in the art, CD4+ T cells, or "T helper cells," are cells that recognize antigens presented by class II major histocompatability marker (MHC) on the surface of antigen presenting cells, and secrete lymphokines to stimulate both cell-mediated and antibody-mediated branches of the immune system. CD4+ T cell activation promotes lymphokine secretion, immunoglobulin isotype switching, affinity maturation of the antibody response, macrophage activation and enhanced activity of natural killer (NK) and cytotoxic T cells (CTL). Lymphokines are proteins secreted by lymphocytes that affect their own activity and/or the activity of other cells. Lymphokines include, but are not limited to, interleukins and cytokines, e.g., IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, or INFy. For purposes of determining whether a CD4+ T lymphocytes are activated, a quantitative comparison of the signal in a sample from a subject vaccinated with antigen and PBS57 can be compared to a sample from a subject vaccinated with antigen alone. Methods to assay activation CD4+ T cells are known in the art.

[0028] A method of activating CD8+ T lymphocytes in a subject is also disclosed. CD8+ T lymphocytes recognize antigens presented by Class I MHC molecules (present on all nucleated cells). Engagement of the MHC class l-peptide complex results in delivery of lytic granules to the target cell causing lysis of the target cell. Methods used to assay the activation of CD8+ T cells are known in the art, including but not limited to ELISPOT, ELISA, and cytotoxicity assays. As used herein, a mouse model is used to monitor the activation of CD8+ T cells using a fluorescent assay to measure cell-mediated cytotoxicity, as described in Hermans et al, 2004, Journal of Immunologic Methods, 285:25-40. In this assay, mice are immunized on day 0 with the vaccine with or without PBS57. Syngeneic target cells are created by isolating splenocytes from a second set of mice and labeling the cells with two separate cell-labeling fluorescent dyes or high and low concentrations of a single fluorescent dye, e.g., CFSE or CMTMR. One set of target cells is loaded with an antigen-specific peptide while a second set of target cells is loaded with an irrelevant peptide. The two target cell populations are mixed in equal amounts and injected into immunized mice. 24 hours later, mice are sacrificed, and splenocytes and blood samples are obtained. The level of each set of target cells is analyzed by flow cytometry. Activation of CD8+ lymphocytes is determined by comparing the number of target cells in a sample vaccinated with antigen and PBS57 to the number of target cells in a sample from a subject vaccinated with antigen alone.

[0029] Other aspects of the invention will become apparent by consideration of the following non-limiting examples and accompanying drawings.

EXAMPLES

Example 1: PBS57 activation of NKT cells in vitro [0030] To determine whether PBS-57 is able to stimulate NKT cells in culture, expansion of NKT cells in culture of peripheral blood lymphocytes (PBL) was measured. PBLs were derived from two healthy donors and grown in RPMI cell culture media with 5% AB serum. Cells were plated on 12-well plates at 2 x 10® PBL/well/2ml. Wells were treated with negative controls (0.05% Tween20 and 1% DMSO in PBS or 0.05% Tween20 and 10% DMSO in PBS) or the test adjuvants (aGalCer, PBS-20, PBS-25, or PBS-57) at a final concentration of 10 ng/ml, 100 ng/ml or 1 pg/ml. The chemical structures for control compounds aGalCer, PBS-20 and PBS25 are shown below

[0031] All cultures were supplemented with recombinant human IL2 (rhlL-2) to obtain final concentration of 100UI/ml by day 1. All cells were cultured for 10 days at 37°C under 5% CO2. On day 7, half the culture medium (1 ml) was removed for NKT analysis. Percentage of NKT cells in the CD3+ population was detected by flow cytometry using fluorescently labeled CD1 tetramers or fluorescently labeled antibodies (anti-CD3, anti-νβΐ 1, and anti NKT). The NKT population was identified by flow cytometry as the \/β11+, Tetramer+ and TCRc$+ population. NKT cell percentage was calculated in each sample by subtracting the \/β11+ PBS-57-tetramer+ ΤΟΡαβ+ from the \/β11+ empty-tetramer+ ΤΟΡαβ+. FIG. 1 shows the increase of NKT cells as after treatment with PBS-57, aGalCer, PBS-25 and PBS-20. As shown, treatment of PBLs with PBS57 or aGalCer leads to an expansion of NKT cells.

[0032] In a separate experiment, the ability of PBS-57 compared to other candidate glycolipids to induce in vitro expansion of NKT cells was measured. Peripheral blood lymphocytes were derived from two healthy donors and cultured in RPMI with 5%AB serum. Cells are plated in a 12-well plate with 2 x 10® cells per well per 2 ml media. One well received carrier (0.05% Tween20 and 1 % DMSO in PBS) as a negative control, and test wells received aGalCer, PBS-25, PBS-57, or PBS-83 at final concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. The structure of PBS-83 is shown below

[0033] All cultures were supplemented with rhlL-2 to obtain a final concentration of 100 Ul/ml by day 1, and cultured for 7 days at 37°C under 5% CO2 conditions. At day 7, the percentage NKT cells was detected by flow cytometry. FIG. 2 demonstrates the percentage of NKT cells on day 7 for the different glycolipid treatment conditions. Treatment with PBS57 resulted in at least a two fold increase in NKT cells over cells treated with aGalCer.

Example 2: Expansion of NKT cells in cultures treated with PBS57 [0034] NKT staining was performed on a panel of fourteen healthy donor peripheral blood lymphocytes (PBL) or peripheral blood monocyte (PBMC) samples. PBLs were isolated from 12 subjects, and PBMCs were isolated from 2 subjects. Each isolated cell sample was split into two samples. The first sample from each donor was stained with 10 pi anti-νβΐΐ FITC(Beckman Coulter), 10 pi anti-TCRc$-PC5 (Beckman Coulter), and 2 μΙ PE-labeled PBS-57-CD1d-tetramer. The second sample from each donor was stained with 10 μΙ \/β11, 10 μΙ anti-TCRc$ -FITC, and 2 μΙ PE-labeled empty-CD1d-tetramer.

[0035] To determine a percentage increase in NKT cells, the number of NKT cells on day 7 was divided by the number of NKT cells on day 0. As shown in FIG. 3, incubation with PBS57 results in a substantial increase in NKT cells in PBL cultures from human volunteers.

Example 3: Analysis of TCRap, NKT cell populations and dendritic cell maturation in mice administered PBS-57 [0036] To test for adjuvant activity of PBS57 in vivo, mice were injected with test compounds and assayed for cell activation and expansion at 24 hours. Four groups of five C57B1/6J mice were each intravenously administered phosphate buffered saline (PBS) with carrier (DMSO alone), 0.97 pg of aGalCer, 1.3 pg of PBS57, or 0.97 pg of PBS83 in a total of 100 pi PBS. 24 hours after administration of the test compound, the mice were sacrificed and blood samples and splenocytes were isolated by standard methods. Percentage of NKT cells in the spleen was evaluated by flow cytometry with staining using anti-TCRc$-FITC and PE-labeled CD1d-tetramer loaded with PBS-57 (PBS57-tetramer) or without lipid (empty-tetramer). FIG. 4A shows the increase in percentage of TCRap cells in mice treated with aGalCer or with PBS57 compared with control mice treated with PBS and carrier alone. The percentage of NKT cells was determined by subtracting the percentage of PBS57-tetramer+ cells from the percentage of empty-tetramer+ cells in the TCRap population. FIG. 4B shows the decrease in percentage of NKT cells in the spleen of mice treated with PBS57 or aGalCer relative to the control. Taken together, the result shown in FIG. 4Aand FIG. 4B demonstrate that PBS57 increased the number of TCRap cells in the spleen of mice as effectively as aGalCer. The loss of NKT cells in the mice injected with aGalCer or PBS57 relative to control demonstrates a down-expression of TCR on the NKT cell surface.

[0037] Dendritic cells (DCs) were detected in the splenocytes by flow cytometry by staining with anti-CD11c R-PE (BD Pharmingen) and anti-CD8a FITC (Beckman Coulter). Two subsets of dendritic cells, CD11c+CD8a- and CD11c+CD8a+ populations, were analyzed further. A decrease of 50% of both subsets of DCs was seen in mice treated with PBS57 or aGalCer adjuvants compared to the control. To determine if administration of glycolipid induced maturation of the two subsets of dendritic cells, the cells were stained with anti-CD40 Biotin/Stepta APC (BD Bioscience), anti-CD80 Biotin (BD Pharmingen) /Stepta APC (BD Bioscience) and anti-CD86 Biotin (BD Pharmingen)/Stepta APC (BD Bioscience) antibodies and analyzed by flow cytometry. FIG. 5A and 5B show the percentage of CD40 positive cells in both the CD11c+CD8a- and CD11c+CD8a+ cells, respectively. FIG. 5C and 5D shows the percentage of CD80 positive cells in both the CD11c+CD8a- and CD11c+CDBa+ cell populations, respectively. FIG. 5E and 5F shows the percentage of CD86-positive cells in both CD11c+CD8a- and CD11c+CD8a+ cells, respectively. Fbrizontal lines represent the averages. Taken collectively, the data show that PBS57 and aGalCer both substantially increased the percentage of CD40+, CD80+ and CD86+ expressing cells in both the CD11c+CD8a- and CD11c+CD8a+ populations.

Example 4: Protocol for testing adjuvanticity of PBS-57 in mouse model [0038] A mouse model was used to test the in vivo specific cytotoxic T cell response (CD8+) elicited by PBS57 in combination with antigen. C57/B1/6J CD45.2 female mice were immunized on day 0 with antigen (Ovalbumin, Ova, grade VII, Sigma, St. Louis, MO) with or without adjuvant, adjuvant alone, or carrier alone (control) in a total of 100 μΙ PBS. Test compounds were 1 *g PBS57, 1 *g aGalCer, 1 pg PBS83 with or without 50 pg Ova. Syngeneic target cells were prepared by isolating splenocytes from a second set of C57/B1/6J CD45.2 female mice and labeling the cells with either low concentration (0.6 pM over 10 min at 37°C) or high concentration (6 pM over 10 minutes at 37°C) of CFSE (fluorescent dye). The population labeled with high concentration CFSE was pre-loaded with 5 *M SIINFEKL peptide (Ova-specific peptide, NeoMPS, Inc, San Diego, CA) over 60 minutes at 37°C. The population labeled with low concentration CFSE was pre-loaded with 5 μΙ LCMV gp33-41 peptide (non-Ova peptide, NeoMPS, Inc,

San Diego, CA) over 60 minutes at 37°C. Equal numbers of both populations of target cells were mixed (1x107 cells of low or high concentration CFSE, 2x107 total per 100 pi) and injected intravenously into each of the immunized mice on day 10. Mice were sacrificed at day 11, and spleen cells and blood samples from the orbital sinus were collected. The mean percentage survival of the peptide-pulsed target cells (CFSE labeled) were calculated relative to the control population by flow cytometric analysis. FIG. 6B depicts typical flow cytometric data showing the loss of Ova-specific peptide loaded target cells. The cytotoxic activity was expressed as a percent specific lysis, calculated by subtracting the mean percent survival of the Ova-specific target cells from 100. FIG. 7 depicts the percentage of specific lysis of target cells in the spleen of immunized mice. Only the combination of Ova and PBS57 produced cytotoxic lysis of Ova-specific target cells in the spleen.

Example 5: Cytotoxic response after immunization with Ova with varying concentrations of aGalCer [0039] The in vivo cytotoxicity induced by the CD8+ T cell response to Ova-peptide loaded target cells was evaluated as described in Example 4 for varying aGalCer concentrations. Eleven groups of three mice were intravenously immunized with 100 μΙ total of PBS alone, 50 pg Ova alone, 1 pg of aGalCer alone, 50 pg of Ova with 1 pg, 100 ng, 10 ng, 1000 pg, 100 pg, 10 pg, or 0.1 pg aGalCer. On day 10, mice were injected intravenously with CFSE-labeled target cells. On day 11, blood samples were collected from the orbital sinus of each mouse. The mean percent survival and cytotoxic activity was determined as described above. FIG. 8 depicts the percent specific lysis for each group of mice for the specified dosages of aGalCer. The results show that aGalCer with antigen is able to generate a dose dependent specific cytotoxic effect when injected intraveneously.

Example 6: Cytotoxic response after immunization with Ova with varying concentrations of PBS57 intraveneously [0040] The in vivo cytotoxicity induced by CD8+ T cell response stimulated with different concentrations of PBS-57 of Ova-peptide loaded CFSE-labeled target cells was evaluated as described in Example 4. Eleven groups of three mice were intravenously immunized with 100 μΙ total of PBS alone, 50 pg Ova alone, 50pg Ova with 10 pg PBS57, 1 pg PBS57, 100 ng PBS57, 10 ng PBS57, 1 ng PBS57, 100 pg PBS57, 10 pg PBS57, 1 pg PBS57, or 0.1 pg PBS57. On day 10, mice were injected intravenously with target cells, and on day 11, blood samples were collected from the orbital sinus. The mean percent survival and cytotoxic activity was determined as described above. FIG. 9 shows the percentage specific lysis in the blood for each group of mice immunized under each dosage of PBS57. The results show that PBS-57 in combination with Ova was able to induce a dose-dependent specific cytotoxic effect when injected intravenously.

Example 7: Cytotoxic response after immunization with PBS57 with varying concentrations of Ova intravenously [0041] The cytotoxic response of CD8+ cells against different concentrations of ovalbumin with constant concentrations of PBS57 was evaluated as described in Example 4. Ten groups of three mice were injected intravenously on day 0 with 100 μΙ total in PBS of 50 pg Ova alone, 1. pg PBS57 alone, 1 pg PBS57 with 50 pg Ova, 10 pg Ova, 1 pg Ova, 100 ng Ova, 10 ng Ova, 1 ng Ova, 100 pg Ova, or 10 pg Ova. On day 10, mice were injected with target cells intravenously and on day 11, mice were sacrificed and blood samples were collected from the orbital sinus and spleen cells were isolated. The mean percentage of survival and cytotoxic activity was calculated as described above. FIG. 10 shows the percentage of specific lysis for each group of mice immunized with each dosage of Ova. The results show the cytotoxic response to vaccination with both antigen and PBS-57 is dependent on antigen concentration.

Example 8: Cytotoxic response after immunization with aGalCer with varying concentrations of Ova intramuscularly [0042] The cytotoxic response of CD8+ cells against different concentrations of ovalbumin with constant concentrations of aGalCer injected intramuscularly was evaluated as described in Example 4. Fourteen groups of three mice were injected intramuscularly in the hind left leg on day 0 with 100 μΙ total in PBS of 400 μg Ova alone, 1 μg aGalCer alone, or 400 μg Ova with 1 μg aGalCer, 0.5 μg aGalCer, 0.1 μg aGalCer, 50 ng aGalCer, 10 ng aGalCer, 5 ng aGalCer, 1 ng aGalCer, 500 pg aGalCer, 100 pg aGalCer, 50 pg aGalCer or 10 pg aGalCer. On day 10, mice were injected intraveneously with target CFSE stained cells, and on day 11 mice were sacrificed and blood samples were collected from the orbital sinus were obtained. Specific lysis of the SIINFEKL-Ioaded target cells was monitored by flow cytometry. The mean percent survival and cytotoxic activity was calculated as described in Example 4. FIG. 11 shows the percentage of specific lysis in the blood for each group of mice immunized under each dosage of aGalCer. The results show that aGalCer is not able to generate a specific cytotoxic T cell response when injected with the Ova antigen intramuscularly, as seen by very low specific lysis over a broad range of aGalCer concentrations from 1 pg-100pg. These concentrations of aGalCer elicited a cytotoxic response when injected intravenously at 1 pg-10 ng, but did not elicit a cytotoxic response upon intramuscular administration.

Example 9: Cytotoxic response after immunization with PBS57 with varying concentrations of Ova intramuscularly [0043] The cytotoxic response of CD8+ cells against intramuscular injection of PBS57 was analyzed as described in Example 4. Fourteen groups of three mice were injected intramuscularly in the hind left leg on day 0 with 100 pi total in PBS of PBS alone, 400 pg Ova alone, 1 pg PBS57 alone, or 400 pg Ova with 1 pg PBS57, 0.5 pg PBS57, 0.1 pg PBS57, 50 ng PBS57, 10 ng PBS57, 5 ng PBS57, 1 ng PBS57, 500 pg PBS57, 100 pg PBS57, 50 pg PBS57, or 10 pg PBS57. On day 10, mice were injected intravenously with target CFSE labeled cells, and blood samples from the orbital sinus on day 11. The mean percentage of survival and cytotoxic activity was determined as described in Example 4. FIG. 12 shows the specific lysis of the dosage curves of mice immunized intramuscularly with PBS57 and Ova. The results show that PBS-57 can elicit a specific cytotoxic response when injected both intravenously and intramuscularly, unlike aGalCer which can only elicit a cytotoxic response when injected intravenously.

Example 10: Comparison of the cytotoxic response after intramuscular or intravenous immunization with Ova and PBS57 or aGalCer [0044] The cytotoxic response to Ova with PBS57 or Ova with aGalCer upon intravenous and intramuscular administration was evaluated as described in Example 4. Eight groups of mice were injected on day 0 as follows: 1. 1)3 mice injected IM with 400 pg Ova in 50 pi PBS; 2. 2) 3 mice injected by IV with 400 pg Ova into 50 pi PBS; 3. 3) 6 mice injected IM with 400 pg Ova and 1 pg aGalCer in 50 pi PBS; 4. 4) 3 mice injected IV with 400 pg Ova and 1 pg aGalCer in 50 pi PBS; 5. 5) 6 mice injected IM with 400 pg Ova and 1 pg aGalCer in 50 pi PBS; 6. 6) 3 mice injected IV with 400 pg Ova and 1 pg aGalCer in 50 pi PBS; 7. 7) 6 mice injected IM with 400 pg Ova and 1 pg PBS57 in 50 μΙ PBS; and 8. 8) 3 mice injected IV with 400 pg Ova and 1 pg PBS57 into 50pl PBS.

[0045] Mice were injected with CFSE labeled target cells on day 10. Mice were sacrificed on day 11 and blood samples were collected. The mean percentage of survival and cytotoxic activity was determined as described in Example 4. FIG. 13 compares the percentage of specific lysis using different routes of administration. The results confirm that aGalCer did not induce a cytotoxic response when injected intramuscularly, while PBS57 caused a cytotoxic response regardless of the route of administration.

Example 11: PBS57 boosts antibody response to tetanus toxoid in vivo [0046] To determine if the addition of PBS57 to tetanus toxoid immunization resulted in an enhanced immune response, a group of six mice were immunized intramuscularly at day 0 and day 15 with 10 pg tetanus toxoid (TT) or 10 pg tetanus toxoid in combination with 1 pg PBS57. IgG titers were determined by standard methods from blood samples drawn on day 0, 15 and 30. The antibody titer was determined by tetanus toxoid speicific ELISA. As seen in FIG. 14, PBS57 enhanced the antibody response to TT.

Example 12: Comparison of the CD8+ T cell response after intramuscular immunization with Ova alone or in combination with PBS57 [0047] A mouse model was used to test the in vivo specific T cell response (CD8+) elicited by PBS57 in combination with antigen. C57B1/6J CD45.2 female mice were immunized intramuscularly on day 0 and 14 with antigen (Ovalbumin, Ova, grade VII, Sigma, St. Louis, MO) with or without adjuvant, adjuvant alone, or carrier alone (control) in a total of 100 pi PBS carrier. Test adjuvant was 1 pg PBS57 with or without 50 pg Ova. Each group included at least three mice. Blood was collected from each mouse at day 21 and isolated cells were subjected to pentamer staining with H2Kb-SIINFEKL pentamers after blocking the Fc receptor. FACS analysis was performed including only CD8+ cells in the analysis (CD19+ cells were excluded from the analysis to exclude any B cells). FIG. 15 depicts typical flow cytometric data showing an increase in Ova-specific CD8+ T cells after intramuscular injection with PBS57 and Ova as compared to Ova alone. The results are reported as mean +/- standard deviation of FI2Kb-SIINFEKL specific CD8+ T cells in the blood.

Example 13: Comparison of the CD8+ T cell response after subcutaneous immunization with Ova alone or in combination with PBS57 or aGalCer [0048] A mouse model was used to test the in vivo specific T cell response (CD8+) elicited by PBS57 in combination with antigen as compared to that of aGalCer. C57/B1/6J CD45.2 female mice were immunized subcutaneously on day 0 and 14 with antigen (Ovalbumin, Ova, grade VII, Sigma, St. Louis, MO) with or without adjuvant, adjuvant alone, or carrier alone (control) in a total of 100 pi PBS carrier. Test adjuvant was 1 pg PBS57 or aGalCer with or without 50 pg Ova. Each group included at least three mice. Blood was collected from each mouse at day 21 and isolated cells were subjected to pentamer staining with FI2Kb-SIINFEKL pentamers after blocking the Fc receptor. FACS analysis was performed including only CD8+ cells in the analysis (CD19+ cells were excluded from the analysis to exclude any B cells). FIG. 16 depicts typical flow cytometric data showing an increase in Ova-specific CD8+ T cells after intramuscular injection with PBS57 and Ova as compared to Ova alone or as compared to immunization with aGalCer and Ova. The results are reported as mean +/- standard deviation of PI2Kb-SIIIVFEKL specific CD8+ T cells in the blood.

[0049] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a polynucleotide" includes a mixture of two or more polynucleotides. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. All publications, patents and patent applications referenced in this specification are indicative of the level of ordinary skill in the art to which this invention pertains.

[0050] It also is specifically understood that any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description • US0766250W [08051 • W02Q07007946A [0086] . VVQ2007118234A [00081 • VV02008005824A [0006] • WG2008080926A [0000]

Non-patent literature cited in the description . SILK et al.J Clin. Invest, 2004, vol. 114, 1800-1811 [00061 . LIU et al.J. Immun. Methods, 2006, vol. 312, 34-39 ΓΟΟΟβΙ • Remington's Pharmaceutical SciencesMack Publishing Co.20000000 [0019] • HERMANS et al.Journal of Immunologic Methods, 2004, vol. 285, 25-40 [0028]

Claims (11)

A vaccine composition comprising: a) a compound PBS-57 of formula: and b) an antigen for use as a medicament in a method of enhancing the humoral and / or cell-mediated immune response to the antigen of a subject vaccinated with the vaccine composition relative to a control of a subject vaccinated with the antigen. alone and where the antigen is not complexed with a B subunit of Shiga toxin or a functional equivalent thereof which can bind the Gb3 receptor. The vaccine composition for use according to claim 1, wherein the humoral and / or cell-mediated immune response to the vaccine is enhanced at least 100% in the subject as compared to a control from a subject vaccinated with the antigen alone. The vaccine composition for use according to claim 1, wherein the vaccine composition is administered intravenously, intradermally, subcutaneously, intramuscularly, orally, transdermally, intranasally, interperitoneally, transmucosally or rectally. The vaccine composition for use according to claim 1, wherein a humoral immune response to the antigen is stimulated in the subject. The vaccine composition for use according to claim 4, wherein the humoral immune response comprises production of IgG antibodies, IgA antibodies or IgM antibodies. The vaccine composition for use according to claim 1, wherein CD4 + T lymphocytes are activated in the subject. The vaccine composition for use according to claim 6, wherein activation of CD4 + T lymphocytes comprises an increase in a Th1 immune response. The vaccine composition for use according to claim 6, wherein activation of the CD4 + T lymphocytes comprises an increase in a Th2 immune response. The vaccine composition for use according to claim 6, wherein activation of the CD4 + T lymphocytes comprises an increase in a Th1 and Th2 immune response. The vaccine composition for use according to claim 1, wherein CD8 + cytotoxic T lymphocytes are activated in the subject. The vaccine composition for use according to claim 10, wherein the vaccine composition is administered intravenously, intradermally, subcutaneously, intramuscularly, orally, transdermally, intranasally, interperitoneally, transmucosally or rectally.