Vaccine composition containing synthetic adjuvant

<u style="single">Statement on government rights</u> The present invention has been partially achieved with government support under Grant No. Al-25038 awarded by the National Institute of Health. Government has specific rights to the invention.

<u style="single">Field of invention</u> The present invention relates to the fields of pharmaceutical and vaccine compositions. More specifically, the embodiments described herein relate to pharmaceutical and vaccine compositions comprising a glucopyranosyl lipid adjuvant (GLA), and related prophylactic and therapeutic methods.

<u style="single">Description of related technology</u> The immune system of higher organisms identifies foreign (or "non-self") substances from familiar or "self" components, so that the foreign substances induce an immune response while the self components are ignored or tolerated. It is characterized as being done. The immune response is traditionally a humoral response in which antigen-specific antibodies are produced by differentiated B lymphocytes known as plasma cells, or by multiple types of action mechanisms in which various types of T lymphocytes act. It is characterized as one of the cellular responses that eliminates the antigen. For example, CD4 + helper T cells capable of recognizing specific antigens can respond by releasing soluble mediators such as cytokines and recruiting other cells of the immune system to participate in the immune response. .. In addition, CD8 + cytotoxic T cells capable of recognizing specific antigens can respond by binding to, destroying or damaging antigen-bearing cells or particles. In the field of immunology, it is known to provide a particular vaccine by various formulations, usually for the purpose of inducing a desired immune response in the host.

Immunization with heat-killed or attenuated viable infectious agents (eg, viruses, bacteria or certain eukaryotic pathogens) as several strategies to induce a specific immune response by immunizing the host. Immunization with a non-toxic infectious agent capable of directing the expression of a genetic material encoding an antigen for which an immune response is desired; and an immunogen isolated from a particular pathogen to induce immunity to the pathogen (eg, Examples include immunization with a subunit vaccine containing (protein) (see, eg, Liu, 1998 Nature Medicine 4 (5 suppl.): 515). For certain antigens, one or more desirable immunities are believed to be present, in which case these techniques are against the human immunodeficiency virus or other infectious agents, cancer, autoimmune diseases, or other clinical conditions. Neither is particularly effective, including the development of vaccines that are effective in immunologically protecting the host.

The gut flora lipopolysaccharide (LPS) is known to be a potent stimulant of the immune system, but its use in adjuvants has been omitted due to its toxic effects. Ribi et al. Have described monophosphoryl lipid A (MPL), a non-toxic derivative of LPS prepared by removing core carbohydrate groups and phosphates from reducing terminal glucosamine (1986, Immunology and Immunopharmacology of Bacterial Endotoxins,). Plenum Publ. Corp., NY, p407-419).

By removing the acyl chain from the 3-position of the disaccharide skeleton, yet another detoxified form of MPL is obtained, which is called 3-O-deacylated monophosphoryl lipid A (3D-MPL). It can be purified and prepared by the method taught in GB 2122204B, and this reference also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variant. For example, 3D-MPL is prepared in the form of an emulsion with a small particle size less than 0.2 μm in diameter, the method of which is disclosed in WO 94/21292. An aqueous formulation containing monophosphoryl lipid A and a surfactant is described in WO 9843670A2.

The bacterial lipopolysaccharide-derived adjuvant formulated in the adjuvant combination may be purified and processed from a bacterial source or may be a synthetic. For example, purified monophosphoryl lipid A was described by Ribi et al., 1986 (supra), and 3-O-deacylated monophosphoryl or diphosphoryl lipid A from Salmonella sp. Is GB. It is described in 2220211 and US Pat. No. 4,912,094. 3D-MPL and β (1-6) glucosamine disaccharides, as well as other purified and synthetic lipopolysaccharides are described (WO 98/01139; US Pat. No. 6,005,099 and EP 0 729 473 B1, Hilgers et al., 1986 Int. . Arch. Allergy Immunol., 79 (4): 392-6; Hilgers et al., 1987, Immunology, 60 (1); 141-6; and EP 0 549 074 B1). A combination of 3D-MPL and a saponin adjuvant derived from the bark of the Rosaceae Quillaja Saponaria Molina is described in EP 0 761 231B. WO 95/17210 is based on squalene, α-tocopherol, and polyoxyethiresorbitan monooleate (TWEEN -80), formulated with the immunostimulatory substance QS21 and optionally containing 3D-MPL. Adjuvant emulsion systems are disclosed. Despite the availability of such combinations, the use of natural product-derived adjuvants has high production costs, lot-to-lot variability, difficulties associated with large-scale production, and any preparation given. There is uncertainty about the presence of impurities in the composition.

Obviously, improved vaccines, especially high purity, that are consistent across lots and can be efficiently produced on an industrial scale without the inclusion of unwanted or unstructured contaminants. There is a need for vaccines that beneficially contain adjuvant components of chemically clear composition. The present invention provides compositions and methods for such vaccines and provides other related benefits.

<u style="single">A brief overview of the invention</u> The present invention relates to compositions and methods that, in some embodiments thereof, favorably use synthetic glucopyranosyl lipid adjuvant (GLA) as an adjuvant and vaccine component. According to one embodiment of the invention described herein, a vaccine composition comprising an antigen and a glucopyranosyl lipid adjuvant (GLA) is provided.

In another embodiment, a vaccine composition comprising (a) an antigen, a glucopyranosyl lipid adjuvant (GLA), and a Toll-like receptor (TLR) agonist is provided, and in some still yet another embodiments, the TLR described above. Agonists selected from lipopolysaccharide, peptide glycans, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and Toll-like mania homologues (LeIF) of initiator 4a, and at least one hepatitis C antigen, vaccine composition Things are provided. In another embodiment, a vaccine composition comprising an antigen; a glucopyranosyl lipid adjuvant (GLA); and at least one co-adjuvant selected from saponins and saponin mimetics is provided. In another embodiment, a vaccine composition comprising an antigen; a glucopyranosyl lipid adjuvant (GLA); and a carrier comprising at least one of oil and ISCOMATRIX is provided. In another embodiment, an antigen and a glucopyranosyl lipid adjuvant (GLA) and (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier. , And (iv) a vaccine composition comprising at least one of at least one dual stem loop immunomodulator (dSLIM). In yet another particular embodiment, (i) if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, where the plant alkaloids are selected from tomatin and the surfactant is saponin. , Polysorbate 80, Span 85 and stearyl tyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptidoglycan, polyl: Selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue of initiation factor 4a (LeIF), and at least one hepatitis C antigen (iii) in the presence of imiquimodinoline immune response modifiers , It is selected from Leishmania (R848), Imiquimod and gardiquimod. In another embodiment, a vaccine composition comprising an antigen, a glucopyranosyl lipid adjuvant (GLA), a co-adjuvant and at least one of a pharmaceutically acceptable carrier, wherein the co-adjuvant is a cytokine, surfactant. Agents and carriers selected from block copolymers or biodegradable polymers, said pharmaceutically acceptable carriers, are selected from calcium phosphate, oil-in-water emulsions, water-in-oil emulsions, liposomes, novosomes, niosomes and microparticles. The vaccine compositions comprising: In certain embodiments, when liposomes or similar carriers are used, GLA is present in or encapsulated in the laminated structure of liposomes. In other specific embodiments, when microparticles are used, the microparticles are based on or include polymeric lipid lipids. It is present in or encapsulated in the laminated structure of the membrane. In other specific embodiments, when microparticles are used, the microparticles are based on or include polymeric lipid lipids. It is present in or encapsulated in the laminated structure of the membrane. In other specific embodiments, when microparticles are used, the microparticles are based on or include polymeric lipid lipids.

In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic L121, It is selected from CRL1005, PLGA, PLA, PLG, and polymer: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In other embodiments, a vaccine composition comprising at least one recombinant expression construct comprising a promoter operably linked to an antigen-encoding nucleic acid sequence and a glucopyranosyl lipid adjuvant (GLA) is provided. In one embodiment, the recombinant expression construct is present in the viral vector, and in another particular embodiment, the virus is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Exists in.

According to any of the particular embodiments, GLA is not 3'-O-deacylated. According to any of the specific embodiments, the GLA is (i) a reducing end bound to the non-reducing terminal glucosamine by an ether bond between the hexosamine 1 position of the non-reducing terminal glucosamine and the hexosamine 6 position of the reducing terminal glucosamine. It contains a diglucosamine skeleton having glucosamine, (ii) an O-phosphoryl group bonded to the hexosamine 4-position of the non-reducing terminal glucosamine, and (iii) 6 or less aliphatic acyl chains. One of them is bonded to 3-hydroxy of the reducing terminal glucosamine by an ester bond, and one of the aliphatic acyl chains is bonded to 2-amino of the non-reducing terminal glucosamine by an amide bond and alkanoyl having 13 or more carbon atoms by an ester bond. A tetra that contains a tetradecanoyl chain attached to the chain, and one of the aliphatic acyl chains is attached to the 3-hydroxy of the non-reducing terminal glucosamine by an ester bond and to an alkanoyl chain having 13 carbon atoms or more by an ester bond. Contains decanoyle chains.

According to any of the above specific embodiments, including TLR agonists, the TLR agonists are TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR- Biosignals can be delivered by interacting with at least one TLR selected from nine. In another particular embodiment, the TLR agonists are lipopolysaccharide, peptidoglycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and initiation factor 4a Leishmania homologue (LeIF), and at least one type C. Selected from hepatitis antigens. In certain embodiments, when a TLR-7 and / or TLR-8 agonist is used, the TLR-7 and / or TLR-8 agonist is encapsulated within the vesicle.

According to any of the particular embodiments, the GLA is:<chemistry num="1"><img id="000003" he="61" wi="93" file="JP2018024656A_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

[In the formula, R¹, R³, R⁵And R⁶Is C₁₁-C₂₀Alkyl; R²And R⁴Is C₁₂-C₂₀Alkyl] Have.

According to any of the particular embodiments, the vaccine composition is capable of inducing an immune response in the host. In another particular embodiment, this immune response is specific for said antigen. According to any of the particular embodiments, the antigen is capable of inducing an immune response selected from a humoral and cellular response in a host. According to any of the particular embodiments, the vaccine composition is T in the host._HType 1 T lymphocyte response, T_HIt can induce at least one immune response selected from type 2 T lymphocyte response, cytotoxic T lymphocyte (CTL) response, antibody response, cytokine response, lymphokine response, chemokine response, and inflammatory response. .. According to any of the particular embodiments, the vaccine composition is (a) the production of one or more cytokines in the host, wherein the cytokines are interferon-γ (IFN-γ), tumor necrosis. The above production selected from factor-α (TNF-α), (b) production of one or more interleukins, the interleukins being IL-1, IL-2, IL-3, IL-4. , IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23, the above production, (c) production of one or more chemokines The chemokines are selected from MIP-1α, MIP-1β, RANTES, CCL4 and CCL5, and (d) memory T cell response, memory B cell response, effector T cell response, cytotoxicity. It is possible to induce at least one immune response selected from the sex T cell response and the lymphocyte response selected from the effector B cell response.

According to any of the particular embodiments, the antigen is derived from at least one infectious agent selected from bacteria, viruses, and fungi.

In another particular embodiment, the bacterium is an actinobacteria, and in yet another embodiment, the actinobacteria are mycobacteria. In other related specific embodiments, the mycobacteria are selected from M. tuberculosis and M. leprae. In other related specific embodiments, the bacterium is selected from the genus Salmonella, Neisseria, Borrelia, Chlamydia and Bordetella.

In other related specific embodiments, the virus is a simple herpesvirus, a human immunodeficiency virus (HIV), a feline immunodeficiency virus (FIV), a cytomegalovirus, a varicella-zoster virus, a hepatitis virus, an Epsteiner virus ( EBV), respiratory symptom virus, human papillomavirus (HPV) and cytomegalovirus are selected. According to any of the particular embodiments, the antigen is derived from a human immunodeficiency virus, which virus is selected from HIV-1 and HIV-2 in another particular embodiment.

In other related specific embodiments, the fungus is selected from the genus Aspergillus, the genus Blastomyces, the genus Coccidioides and the genus Pneumocystis. In other related specific embodiments, the fungus is yeast, in another particular embodiment, the genus Candida, and in yet another embodiment, the genus Candida is C. albicans (C. albicans). Choose from albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis Will be done.

According to any of the particular embodiments, the antigen is derived from a parasite, in another particular embodiment is a protozoa, and in another particular embodiment, Plasmodium. In yet another embodiment, Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Quartan malaria (P. malariae) and Plasmodium falciparum (P. malariae). ovale) is selected. In other particular embodiments, the parasites are Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium. , Japanese blood-flukes (Schistosoma japonicum), Mekon blood-flukes (Schistosoma mekongi), Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli, Entamoeba coli dispar), Hartmanni amoeba (Entamoeba hartmanni), Polek amoeba (Entamoeba polecki), Bancroft filamentous worm (Wuchereria) bancrofti), Giardia, Leishmania, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Ancylostoma duo Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Threadworm (Strongyloides stercoralis), Fasciola gigantis (Opisthorchis sinensis), Paragonimus It is selected from sp), hepatic worm (Fasciola hepatica), fasciola magna, giant hepatic worm (Fasciola gigantica), taenia saginata, and taenia solium.

According to any of the particular embodiments, the antigen is derived from at least one type of cancer cell. In another particular embodiment, the cancer cell originates from a primary solid tumor, in another particular embodiment, the cancer cell originates from a metastatic or secondary solid tumor, and in another particular embodiment. In, cancer cells originate from cancers that are circulating tumors or ascites tumors. In a particular embodiment, the cancer cells are cervical cancer, ovarian cancer, breast cancer, prostate cancer, fibrosarcoma, mucinosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, spondyloma, angiosarcoma, endothelial sarcoma, lymphatic vessels Sarcoma, peritoneal pseudomyeloma, lymphatic endothelial sarcoma, osteosarcoma, mesopharia, Ewing's tumor, smooth myoma, horizontal print myoma, colon cancer, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, Selected from sweat adenocarcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, sac adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, spermatoma, embryonic cancer, and Wilms tumor Originates from sarcoma. In other related specific embodiments, the cancer cells are testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, stellate cell tumor, medulloblastoma, cranial pharyngeal tumor, coat cell tumor, pine fruit. Body tumor, hemangioblastoma, auditory neuroma, dilute glioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenström macroglobulinemia, And originate from cancers selected from H-chain disease.

According to any of the particular embodiments, the antigen is derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with an autoimmune disease. .. In another particular embodiment, the epitope, biomolecule, cell or tissue associated with the autoimmune disease is selected from snRNP when the autoimmune disease is systemic erythematosus and the autoimmune disease is Graves' disease. When the autoimmune disease is thrombocytopenic purpura, when the autoimmune disease is thrombocytopenia, and when the autoimmune disease is nephrosis. It is at least one of the vesicle antigen, desmograin-3, desmoprakin, emboplakin and bullous vesicle antigen 1, and is a myelin basic protein when the autoimmune disease is multiple sclerosis. When the autoimmune disease is type 1 diabetes, it is pancreatic islet β cells, and when the autoimmune disease is severe asthenia, it is an acetylcholine receptor.

In another embodiment, the pharmaceutical composition provided is a pharmaceutical composition for inducing or enhancing an immune response, comprising a glucopyranosyl lipid adjuvant (GLA) and a pharmaceutically acceptable carrier or excipient. Will be done. In another embodiment, said medicament comprising a pharmaceutical composition for inducing or enhancing an immune response, comprising an antigen, a glucopyranosyl lipid adjuvant (GLA), and a pharmaceutically acceptable carrier or excipient. The composition is provided. In another embodiment, a pharmaceutical composition for inducing or enhancing an immune response, the antigen, a glucopyranosyl lipid adjuvant (GLA), a Toll-like receptor (TLR) agonist, and a pharmaceutically acceptable carrier. Alternatively, the above-mentioned pharmaceutical composition containing an excipient is provided. In another embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl: It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. In another embodiment, a pharmaceutical composition for inducing or enhancing an immune response, comprising an antigen, a glucopyranosyl lipid adjuvant (GLA), and at least one co-adjuvant selected from saponins and saponin mimetics. The pharmaceutical compositions are provided that include a pharmaceutically acceptable carrier or excipient. In another embodiment, a pharmaceutical composition for inducing or enhancing an immune response, pharmaceutically including an antigen, a glucopyranosyl lipid adjuvant (GLA), an oil and at least one of ISCOMATRIX . The pharmaceutical compositions are provided that include with an acceptable carrier. In another embodiment, a pharmaceutical composition for inducing or enhancing an immune response, which is (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) (i) at least one co. One or more of an adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier, and (iv) at least one dual stem loop immunomodulator (dSLIM) And (d) the pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. In another particular embodiment, (i) if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is saponin, Selected from Polysorbate 80, Span 85 and stearyl tyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptidoglycan, polyl:

In another embodiment, a pharmaceutical composition for inducing or enhancing an immune response, comprising an antigen, a glucopyranosyl lipid adjuvant (GLA), at least one co-adjuvant, and a pharmaceutically acceptable carrier. Including, the co-adjuvant is selected from cytokines, block copolymers or biodegradable polymers, and surfactants, and the pharmaceutically acceptable carriers are calcium phosphate, oil-in-water emulsions, water-in-oil emulsions. , Liposomes, and carriers selected from microparticles are provided. In another particular embodiment, cytokines are selected from GM-CSF, IL-2, IL-7, IL-12, TNF and IFN-γ, and block copolymers or biodegradable polymers are Pluralonic®. It is selected from L121, CRL1005, PLGA, PLA, PLG, and polymer: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In another embodiment, at least one recombinant expression construct comprising a promoter operably linked to an antigen-encoding nucleic acid sequence, a glucopyranosyl lipid adjuvant (GLA), and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising and is provided. In another particular embodiment, the recombinant expression construct is present in the viral vector, and in another particular embodiment, it is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Present in the virus.

According to another particular embodiment of the pharmaceutical composition, the antigen and GLA are in contact with each other, and according to another particular embodiment of the pharmaceutical composition, the antigen and GLA are. Not in contact with each other. In another particular embodiment where the antigen and GLA are not in contact with each other, both are present in separate containers. In other embodiments, a pharmaceutical composition for inducing or enhancing an immune response, the first combination comprising an antigen and a first pharmaceutically acceptable carrier or excipient, and a glucopyranosyl lipid adjuvant. Provided is the pharmaceutical composition comprising a second combination comprising (GLA) and a second pharmaceutically acceptable carrier or excipient, wherein the antigen and GLA are not in contact with each other. To. In another embodiment, the antigen and GLA are in separate containers. In certain relevant embodiments, the first pharmaceutically acceptable carrier or excipient is different from the second pharmaceutically acceptable carrier or excipient. In other related embodiments, the first pharmaceutically acceptable carrier or excipient is not different from the second pharmaceutically acceptable carrier or excipient.

In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) is administered to the subject, wherein the antigen is derived from or immunologically cross-reacts with at least one infectious pathogen associated with the infectious disease. Being sexual, this provides the methods, including treating or preventing the infection. In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) a tall-like receptor (TLR) agonist is administered to the subject, wherein the antigen is derived from at least one infectious pathogen associated with the infectious disease. Or immunologically cross-reactive with the pathogen, which provides the method comprising treating or preventing the infection. In yet another embodiment, the TLR agonists are lipopolysaccharide, peptidoglycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and initiation factor 4a Leishmania homologue (LeIF), and at least one type C. Selected from hepatitis antigens. In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) at least one co-adjuvant selected from the group consisting of saponin and saponin mimetics was administered to the subject, in which the antigen contributed to the infection. Derived from or immunologically cross-reactive with at least one related infectious agentAnd this provides the above methods, including treating or preventing the above infections. In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) a carrier comprising at least one of (c) oil and ISCOMATRIX is administered to the subject, where the antigen is at least associated with the infection. The method is provided that comprises treating or preventing the infectious disease, which is derived from or immunologically cross-reactive with the infectious agent. In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. (GLA) and (c) (i) at least one co-antigen, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier, and (iv) at least one A vaccine composition containing one or more of the dual stem loop immunomodulators (dSLIM) is administered to the subject, wherein the antigen is derived from at least one infectious pathogen associated with the infectious disease. Or immunologically cross-reactive with the pathogen, which provides the method comprising treating or preventing the infection. In another particular embodiment, (i) if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is saponin, Selected from Polysorbate 80, Span 85 and stearyl tyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptidoglycan, polyl:

In another embodiment, a method of treating or preventing an infection in a subject who has or is suspected of having an infection, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) a co-adjuvant and at least one of a pharmaceutically acceptable carrier is administered to the subject, wherein the co-adjuvant is a cytokine, block copolymer or organism. The pharmaceutically acceptable carrier selected from degradable polymers and surfactants comprises a carrier selected from the group consisting of calcium phosphate, oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles. The antigen is derived from or immunologically cross-reactive with at least one infectious agent associated with the infectious disease, thereby treating or preventing the infectious disease. The above methods are provided, including. In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic. It is selected from L121, CRL1005, PLGA, PLA, PLG, and polyl: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In another embodiment, it is a method of treating or preventing an infection in a subject who has or is suspected of having an infection and is functional in (a) the nucleic acid sequence encoding the antigen. A vaccine composition comprising at least one recombinant expression construct comprising a promoter bound to the above and (b) glucopyranosyl lipid adjuvant (GLA) was administered to the subject, where the antigen was associated with the infection. The method is provided, comprising treating or preventing the infectious disease, which is derived from or immunologically cross-reactive with the at least one infectious agent. In another embodiment, the recombinant expression construct is present in a viral vector, and in yet another particular embodiment, it is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. It is present in the virus. According to certain embodiments of the aforementioned method, the antigen is derived from at least one infectious agent selected from bacteria, viruses, and fungi.

In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. A vaccine composition comprising a lipid adjuvant (GLA) is administered to the subject, wherein the antigen is derived from or from at least one epitope, biomolecule, cell or tissue associated with the autoimmune disease. It is immunologically cross-reactive with these, which provides the methods, including treating or preventing the autoimmune disease. In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. A vaccine composition comprising a lipid adjuvant (GLA) and (c) a Toll-like receptor (TLR) agonist is administered to the subject, wherein the antigen is at least one epitope associated with the autoimmune disease. , Derived from or immunologically cross-reactive with biomolecules, cells or tissues, thereby providing the methods comprising treating or preventing the autoimmune disease. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :. It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. A vaccine composition comprising a lipid adjuvant (GLA) and (c) at least one co-adjuvant selected from the group consisting of saponin and saponin mimetics is administered to the subject, wherein the antigen is the autoimmune. Derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with an immune disease, which comprises treating or preventing the autoimmune disease. , The above method is provided. In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. A vaccine composition comprising a lipid adjuvant (GLA) and (c) a carrier containing at least one of oil and ISCOMATRIX was administered to the subject, wherein the antigen was added to the autoimmune disease. The method comprising treating or preventing the autoimmune disease, which is derived from or immunologically cross-reactive with at least one related epitope, biomolecule, cell or tissue. Is provided. In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. Lipid adjuvant (GLA) and (c) (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier, and (iv) at least one Seed double stem loop A vaccine composition containing one or more of the immunomodulators (dSLIM) is administered to the subject, wherein the antigen is at least one epitope, biomolecule, cell associated with the autoimmune disease. Alternatively, the methods are provided that are derived from tissue or immunologically cross-reactive with them, comprising treating or preventing the autoimmune disease. In another particular embodiment, (i) if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is saponin, Selected from Polysorbate 80, Span 85 and stearyl tyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptide glycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leash of initiator 4a. Selected from the group consisting of mania homologues (LeIF), as well as at least one hepatitis C antigen, and (iii) imidazoquinolin immune response modifiers, if present, this consists of reshikimod (R848), imikimod and gardikimod. Selected from the group.

In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) an antigen and (b) glucopyranosyl. A vaccine composition comprising a lipid adjuvant (GLA) and (c) a co-adjuvant and at least one of a pharmaceutically acceptable carrier is administered to the subject, wherein the co-adjuvant is a cytokine, block copolymer. Alternatively, the carrier selected from the biodegradable polymer and the surfactant, and the pharmaceutically acceptable carrier is selected from the group consisting of calcium phosphate, oil-in-water emulsion, water-in-oil emulsion, liposome, and fine particles. Included and the antigen is derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with the autoimmune disease, thereby. The methods are provided, including treating or preventing the autoimmune disease. In another embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic L121, CRL1005. , PLGA, PLA, PLG, and polyl: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In another embodiment, a method of treating or preventing an autoimmune disease in a subject who has or is suspected of having an autoimmune disease, wherein (a) the nucleic acid sequence encoding the antigen. A vaccine composition comprising at least one recombinant expression construct comprising a functionally bound promoter and (b) a glucopyranosyl lipid adjuvant (GLA) was administered to the subject, wherein the antigen was autoimmune. Derived from, or immunologically cross-reactive with, at least one epitope, biomolecule, cell or tissue associated with the disease, comprising treating or preventing the autoimmune disease. A method is provided. In another embodiment, the recombinant expression construct is present in a viral vector and in another particular embodiment, it is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Present in the virus.

In the particular embodiment relating to a method of treating or preventing an autoimmune disease, the autoimmune disease is type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, severe asthenia, Crohn's disease, Graves' disease, Selected from thrombocytopenic purpura and lupus. In the other particular embodiment described above with respect to a method of treating or preventing an autoimmune disease, the epitope, biomolecule, cell or tissue associated with the autoimmune disease is snRNP when the autoimmune disease is systemic erythematosus. Selected from, when the autoimmune disease is Graves' disease, it is at least one of tyroglobulin, tyrotropin receptor and thyroid epithelial cells, and when the autoimmune disease is thrombocytopenic purpura, it is platelets. , The autoimmune disease is at least one of the vesicle antigen, desmograin-3, desmoprakin, emboplakin and vesicular asthma antigen 1 when the autoimmune disease is polysclerosis. When the autoimmune disease is type 1 diabetes, it is a pancreatic islet β cell, and when the autoimmune disease is severe asthenia, it is an acetylcholine receptor.

According to another embodiment, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) is administered to the subject, wherein the antigen is derived from or immunology with at least one epitope, biomolecule, cell or tissue associated with the cancer. It is cross-reactive, which provides the methods, including treating or preventing the cancer. According to other embodiments, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) a Toll-like receptor (TLR) agonist is administered to the subject, wherein the antigen is the at least one epitope, biomolecule, associated with the cancer. The methods are provided that are derived from or immunologically cross-reactive with cells or tissues, comprising treating or preventing the cancer. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :. It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. According to other embodiments, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) at least one co-adjuvant selected from the group consisting of saponin and saponin mimetics was administered to the subject, where the antigen was associated with the cancer. The methods are provided that are derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue that comprises treating or preventing the cancer. To.

According to other embodiments, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen; (b) a glucopyranosyl lipid adjuvant ( A vaccine composition comprising a carrier containing at least one of GLA); and (c) oil and ISCOMATRIX is administered to the subject, wherein the antigen is at least one of the cancer-related species. The methods are provided that are derived from or immunologically cross-reactive with epitopes, biomolecules, cells or tissues, comprising treating or preventing the cancer. According to another embodiment, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. (GLA) and (c) (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier, and (iv) at least one A vaccine composition containing one or more of the dual stem loop immunomodulators (dSLIM) is administered to the subject, wherein the antigen is the at least one epitope, biomolecule, associated with the cancer. The methods are provided that are derived from or immunologically cross-reactive with cells or tissues, comprising treating or preventing the cancer. In another particular embodiment, (i) if a co-adjuvant is present, it is selected from the group consisting of alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is , Saponin, Polysorbate 80, Span 85 and stearyltyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptidoglycan, polyl: Selected from the group consisting of C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen, and (iii) imiquimodinoline immune response modifier. If present, it is selected from the group consisting of leishmania (R848), imiquimod and gardiquimod. According to other embodiments, a method of treating or preventing cancer in a subject who has or is suspected of having cancer, wherein (a) an antigen and (b) a glucopyranosyl lipid adjuvant. A vaccine composition comprising (GLA) and (c) a co-adjuvant and at least one of a pharmaceutically acceptable carrier is administered to the subject, wherein the co-adjuvant is a cytokine, block copolymer or organism. The pharmaceutically acceptable carrier selected from the group consisting of degradable polymers and surfactants is a carrier selected from the group consisting of calcium phosphate, oil-in-water emulsion, water-in-oil emulsion, liposomes, and fine particles. The antigen is derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with the cancer, thereby treating the cancer. Alternatively, the above methods are provided, including prevention. In another embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic L121, CRL1005. , PLGA, PLA, PLG, and polyl: Selected from C, and the surfactant is selected from saponin, Polysorbate 80, Span 85 and stearyl tyrosine. According to other embodiments, a method of treating or preventing cancer in a subject who has or is suspected of having cancer and is functional in (a) an antigen-encoding nucleic acid sequence. A vaccine composition comprising at least one recombinant expression construct comprising a promoter bound to the above and (b) a glucopyranosyl lipid adjuvant (GLA) was administered to the subject, where the antigen is associated with the cancer. The methods are provided that are derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue, which comprises treating or preventing the cancer. In another embodiment, the recombinant expression construct is present in a viral vector and in another particular embodiment, it is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Present in the virus.

In another particular embodiment of the method of treating or preventing cancer, the antigen is derived from at least one cancer cell, and in another particular embodiment, is derived from a primary solid tumor. In other specific embodiments, the cancer cells are derived from a cancer that is a metastatic or secondary solid tumor, and in other specific embodiments, the cancer cells are circulating tumors or ascites tumors. Derived from cancer. In certain embodiments, the cancer cells are cervical cancer, ovarian cancer, breast cancer, prostate cancer, fibrosarcoma, mucinosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, spondyloma, angiosarcoma, endothelial sarcoma, lymphangisarcoma. , Peritoneal pseudomyeloma, lymphatic endothelial sarcoma, osteosarcoma, mesopharyngeal sarcoma, Ewing's tumor, smooth myoma, horizontal print myoma, colon cancer, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland Selected from cancer, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, sac adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, chorionic villi cancer, sperm epithelioma, embryonic cancer, and Wilms tumor Derived from cancer. In other specific embodiments, the cancer cells are testicular tumors, lung cancers, small cell lung cancers, bladder cancers, epithelial cancers, gliomas, stellate cell tumors, medulloblastomas, cranial pharyngeal tumors, coat cell tumors, pine cones. Tumors, hemangioblastomas, auditory neuromas, dilute gliomas, meningiomas, melanomas, neuroblastomas, retinoblastomas, leukemias, lymphomas, multiple myeloma, Waldenström macroglobulinemia, and Derived from cancer selected from H-chain disease.

According to another particular embodiment of any one of the aforementioned methods of treating or preventing an infectious disease or autoimmune disease or cancer, the administration step is performed once, but yet another particular practice of such method. In one embodiment, the dosing step is performed at least twice, in another particular embodiment the dosing step is performed at least 3 times, and in another particular embodiment the dosing step is performed 4 or more times. According to yet another specific embodiment of any one of the aforementioned methods of treating or preventing an infectious disease or autoimmune disease or cancer, prior to the administration step, a bacterial extract, a live viral vaccine, a nucleic acid encoding an antigen. Subjects are first presented with a primary immunogen selected from at least one recombinant expression construct containing a promoter functionally bound to a sequence and a viral vector containing a promoter functionally bound to an antigen-encoding nucleic acid sequence. Immunize. In another embodiment, the bacterial extract is derived from Bacillus Calmette-Guerlain (BCG).

In another embodiment, the subject is provided with a vaccine composition comprising (a) an antigen and (b) a glucopyranosyl lipid adjuvant (GLA), which is a method of inducing or enhancing a desired antigen-specific immune response in the subject. The above methods are provided, including administration. In another embodiment, a method of inducing or enhancing a desired antigen-specific immune response in a subject: (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a Toll-like receptor (c). The above method is provided, comprising administering to the subject a vaccine composition comprising a TLR) agonist. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :. It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. In another embodiment, a method of inducing or enhancing a desired antigen-specific immune response in a subject, wherein (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a saponin and a saponin mimetic. The method is provided comprising administering to the subject a vaccine composition comprising at least one co-adjuvant selected from the group consisting of. In another embodiment, a method of inducing or enhancing a desired antigen-specific immune response in a subject, wherein (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), (c) an oil and ISCOMATRIX . ) Is provided, the method comprising administering to the subject a vaccine composition comprising a carrier comprising at least one of). According to another embodiment, a method of inducing or enhancing a desired antigen-specific immune response in a subject, wherein (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) (i). Of at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinolin immune response modifier, and (iv) at least one dual stem loop immunomodulator (dSLIM) The method is provided, comprising administering to the subject a vaccine composition comprising one or more of them. In another particular embodiment, if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, in which case plant alkaloids. De is selected from tomatin, detergent is selected from saponin, Polysorbate 80, Span 85 and stearyltyrosine, and (ii) if TLR agonists are present, this is lipopolysaccharide, peptidoglycan, polyl: C, CpG, Selected from the group consisting of 3M003, flagellin, eukaryotic ribosome elongation and the Leishmania homologue (LeIF) of initiator 4a, and at least one hepatitis C antigen, (iii) imiquimodinoline immune response modifier, if present. It is selected from Reshikimod (R848), Imiquimod and Garjikimod.

In another embodiment, the method of inducing or enhancing the desired antigen-specific immune response in the subject is (a) antigen, (b) glucopyranosyl lipid adjuvant (GLA), (c) co-adjuvant and pharmaceutical. The subject comprises administering a vaccine composition comprising at least one of the carriers allowed in the subject, wherein the coadjuvant is selected from cytokines, block copolymers, biodegradable polymers, and surfactants. The method is provided, wherein the pharmaceutically acceptable carrier comprises a carrier selected from calcium phosphate, oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles. In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Plluronic. It is selected from L121, CRL1005, PLGA, PLA, PLG, and polyl: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In another embodiment, a method of inducing or enhancing a desired antigen-specific immune response in a subject, wherein (a) at least one recombinant expression comprising a promoter operably linked to a nucleic acid sequence encoding an antigen. The method is provided, comprising administering to the subject a vaccine composition comprising the construct and (b) a glucopyranosyl lipid adjuvant (GLA). In another particular embodiment, the recombinant expression construct is present in a viral vector, and in another particular embodiment, from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Present in the selected virus.

In another particular embodiment of the method described above inducing or enhancing the desired antigen-specific response in a subject, GLA is not 3'-O-deacylated. In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, GLA is (i) between the hexosamine 1 position of the non-reducing terminal glucosamine and the hexosamine 6 position of the reducing terminal glucosamine. A diglucosamine skeleton having a reducing terminal glucosamine bound to a non-reducing terminal glucosamine by an ether bond, (ii) an O-phosphoryl group bonded to the hexosamine 4-position of the non-reducing terminal glucosamine, and (iii) 6 or less aliphatic acyls. It contains a chain, in which case one of the aliphatic acyl chains is bound to 3-hydroxy of the reducing terminal glucosamine by an ester bond, and one of the aliphatic acyl chains is 2-hydroxy of the non-reducing terminal glucosamine by an amide bond. It contains a tetradecanoyl chain that is bound to an amino and is bound to an alkanoyl chain of 13 carbon atoms or more by an ester bond, and one of the above aliphatic acyl chains is bound to 3-hydroxy of the non-reducing terminal glucosamine by an ester bond. Moreover, it contains a tetradecanoyl chain bonded to an alkanoyl chain having 13 or more carbon atoms by an ester bond. In another particular embodiment related, if a TLR agonist is present, it will be TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR- Biosignals can be delivered by interacting with at least one TLR selected from nine. In another particular embodiment, the TLR agonists are lipopolysaccharide, peptidoglycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and initiation factor 4a Leishmania homologue (LeIF), and at least one C. Selected from hepatitis antigens.

In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, the GLA is expressed by the formula:<chemistry num="2"><img id="000004" he="63" wi="93" file="JP2018024656A_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

[In the formula, R¹, R³, R⁵And R⁶Is C₁₁-C₂₀Alkyl; R²And R⁴Is C₁₂-C₂₀Alkyl] Have.

In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, the vaccine composition can elicit an immune response in the host. In another particular embodiment, this immune response is specific for said antigen. In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, the antigen can induce an immune response in the host selected from a humoral and cellular response. .. In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, the vaccine composition is T in the host._HType 1 T lymphocyte response, T_HInduces at least one immune response selected from the group consisting of type 2 T lymphocyte response, cytotoxic T lymphocyte (CTL) response, antibody response, cytokine response, lymphokine response, chemokine response, and inflammatory response. be able to. In another particular embodiment of the method of inducing or enhancing a desired antigen-specific response in a subject, the vaccine composition is (a) the production of one or more cytokines in the host, wherein the cytokines. , The above production selected from the group consisting of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), (b) production of one or more interleukins, wherein the interleukin is Selected from IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23 The above production, (c) the production of one or more chemokines, wherein the chemokine is selected from MIP-1α, MIP-1β, RANTES, CCL4 and CCL5, the above production, and (d) memory T. Inducing at least one immune response selected from the group consisting of cell response, memory B cell response, effector T cell response, cytotoxic T cell response, and lymphocyte response selected from effector B cell response. Can be done.

According to other specific embodiments, there is provided a method of preparing a vaccine composition comprising (a) mixing an antigen with (b) a glucopyranosyl lipid adjuvant (GLA). According to other specific embodiments, a vaccine composition comprising mixing (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a Toll-like receptor (TLR) agonist. Preparation methods are provided. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :. It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. According to other specific embodiments, (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) at least one co-adjuvant selected from the group consisting of saponins and saponin mimetics. A method for preparing a vaccine composition, including mixing, is provided. According to another particular embodiment, mixing (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA) with a carrier containing (c) an oil and at least one of ISCOMATRIX . A method for preparing a vaccine composition, which comprises, is provided. According to other specific embodiments, (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), (c) (i) at least one co-adjuvant, (ii) at least one TLR agonist, A vaccine composition comprising mixing (iii) at least one imidazoquinolin immune response modifier and (iv) at least one of at least one dual stem loop immunomodulator (dSLIM). A preparation method is provided. In another particular embodiment, (i) if a co-adjuvant is present, it is selected from the group consisting of alum, plant alkaloids and surfactants, in which the plant alkaloids are selected from tomatin and the surfactant. Is selected from saponins, Polysorbate 80, Span 85 and stearyl tyrosine, and (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptidoglycan, polyl: Selected from the group consisting of C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen, and (iii) imiquimodinoline immune response modifier. If present, it is selected from the group consisting of leishmania (R848), imiquimod and gardiquimod. According to other specific embodiments, (a) an antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a co-adjuvant and at least one of a pharmaceutically acceptable carrier are mixed. A method for preparing a vaccine, wherein the co-adjuvant is selected from cytokines, block copolymers or biodegradable polymers, and surfactants, and the pharmaceutically acceptable carrier is calcium phosphate, oil-in-water emulsion. The above method comprises a carrier selected from the group consisting of water-in-oil emulsions, liposomes, and microparticles. In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Plluronic. It is selected from L121, CRL1005, PLGA, PLA, PLG, and polymer: C, and the surfactant is selected from saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

According to other specific embodiments, (a) at least one recombinant expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an antigen and (b) a glucopyranosyl lipid adjuvant (GLA) are mixed. A method of preparing a vaccine composition is provided, including the preparation of the vaccine composition. In another particular embodiment, the recombinant expression construct is present in a viral vector, and in another particular embodiment, it is selected from adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, and retrovirus. Present in the virus. In certain embodiments, GLA is not 3'-O-deacylated. In certain embodiments, GLA is a diglucosamine backbone having (i) a reducing terminal glucosamine bound to the non-reducing terminal glucosamine by an ether bond between the hexosamine 1 position of the non-reducing terminal glucosamine and the hexasamine 6 position of the reducing terminal glucosamine. And (ii) an O-phosphoryl group attached to the hexosamine 4-position of the non-reducing terminal glucosamine, and (iii) 6 or less aliphatic acyl chains, in which case one of the aliphatic acyl chains is It binds to 3-hydroxy of the reducing terminal glucosamine by an ester bond, and one of the above aliphatic acyl chains is bonded to 2-amino of the non-reducing terminal glucosamine by an amide bond and becomes an alkanoyl chain having 13 carbon atoms or more by an ester bond. Tetra containing a bound tetradecanoyl chain, and one of the above aliphatic acyl chains was bound to 3-hydroxy of non-reducing terminal glucosamine by an ester bond and to an alkanoyl chain having 13 carbon atoms or more by an ester bond. Includes decanoyle chains. In certain embodiments, the TLR agonist is at least one selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. By interacting with TLRs, biological signals can be delivered. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :.

According to a particular embodiment of the method of preparing a vaccine composition, GLA is expressed by the formula:<chemistry num="3"><img id="000005" he="62" wi="93" file="JP2018024656A_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

[In the formula, R¹, R³, R⁵And R⁶Is C₁₁-C₂₀Alkyl; R²And R⁴Is C₁₂-C₂₀Alkyl] Have.

In another particular embodiment, the mixing step comprises emulsifying, in another particular embodiment the mixing step comprises forming particles, and in another particular embodiment, the particles are Contains fine particles. In another particular embodiment, the mixing step comprises forming a precipitate containing all or part of the antigen or all or part of the GLA.

In other specific embodiments, an immunoadjuvant pharmaceutical composition comprising a glucopyranosyl lipid adjuvant (GLA) and a pharmaceutically acceptable carrier or excipient is provided. According to other specific embodiments, an immunoadjuvant composition comprising a glucopyranosyl lipid adjuvant (GLA) and a Toll-like receptor (TLR) agonist is provided. In another particular embodiment, the TLR agonists are lipopolysaccharide, peptidoglycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and initiation factor 4a Leishmania homologue (LeIF), and at least one C. Selected from hepatitis antigens. In other specific embodiments, an immunoadjuvant composition is provided that comprises a glucopyranosyl lipid adjuvant (GLA) and at least one co-adjuvant selected from saponins and saponin mimetics. In another particular embodiment, an immunoadjuvant pharmaceutical composition comprising a glucopyranosyl lipid adjuvant (GLA) and a pharmaceutically acceptable carrier comprising at least one of oil and ISCOMATRIX is provided. .. In other specific embodiments, (a) glucopyranosyl lipid adjuvant (GLA) and (b) (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazo. An immunoadjuvant composition comprising a quinoline immune response modifier and (iv) one or more of at least one dual stem loop immunomodifier (dSLIM) is provided.

In another particular embodiment, (i) if a co-adjuvant is present, it is selected from the group consisting of alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is , Saponin, Polysorbate 80, Span 85 and stearyl tyrosine, (ii) in the presence of TLR agonists, this is lipopolysaccharide, peptide glycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and initiator Selected from the group consisting of the Leishmania homologue (LeIF) of 4a, as well as at least one hepatitis C antigen, and (iii) imidazoquinolin immune response modifiers, if present, this is leshikimod (R848), imikimod and Selected from the group consisting of gardiquimods.

In another particular embodiment, an immunoadjuvant composition comprising a glucopyranosyl lipid adjuvant (GLA) and at least one of a co-adjuvant and a pharmaceutically acceptable carrier, wherein the co-adjuvant is a cytokine. Selected from the group consisting of block copolymers or biodegradable polymers, and surfactants, the pharmaceutically acceptable carrier is selected from calcium phosphate, oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles. The above composition comprising a carrier is provided. In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic L121, It is selected from CRL1005, PLGA, PLA, PLG, and polymer: C, and the surfactant is selected from the group consisting of saponin, Polysorbate 80, Span 85 and stearyl tyrosine.

In another particular embodiment, a method of modifying immune responsiveness in a host, wherein an immunoadjuvant pharmaceutical composition comprising a glucopyranosyl lipid adjuvant (GLA) and a pharmaceutically acceptable carrier or excipient is described above. The above methods are provided, comprising administering to the host, thereby altering the immune responsiveness of the host. In another particular embodiment, a method of modifying immune responsiveness in a host, the host comprising an immunoadjuvant composition comprising a glucopyranosyl lipid adjuvant (GLA) and (b) a Toll-like receptor (TLR) agonist. The above methods are provided, comprising administering to, thereby altering the immune responsiveness of the host. In another particular embodiment, the TLR agonist is lipopolysaccharide, peptidoglycan, polyl :. It is selected from C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and leishmania homologue (LeIF) of initiation factor 4a, and at least one hepatitis C antigen. In another particular embodiment, a method of modifying immune responsiveness in a host comprises a glucopyranosyl lipid adjuvant (GLA) and at least one co-adjuvant selected from the group consisting of saponins and saponin mimetics. Provided are the methods described above comprising administering the immunoadjuvant composition to the host, thereby modifying the immune responsiveness of the host. In another particular embodiment, a method of modifying immune responsiveness in a host, a pharmaceutically acceptable carrier comprising a glucopyranosyl lipid adjuvant (GLA) and at least one of oil and ISCOMATRIX . The above method is provided comprising administering to the host an immunoadjuvant composition comprising, thereby altering the immune responsiveness of the host. In another particular embodiment, a method of modifying immune responsiveness in a host, the glucopyranosyl lipid adjuvant (GLA) and (i) at least one co-adjuvant, (ii) at least one TLR agonist, ( iii) An immunoadjuvant composition comprising at least one imidazoquinolin immune response modifier and (iv) at least one dual stem loop immunomodulator (dSLIM) is administered to the host. This provides the above methods, including modifying the immune responsiveness of the host.

In another particular embodiment, if a co-adjuvant is present, it is selected from alum, plant alkaloids and surfactants, where the plant alkaloid is tomatin and the surfactant is saponin, Polysorbate 80, Selected from Span 85 and stearyl tyrosine, in the presence of TLR agonists, this is lipopolysaccharide, peptide glycan, polyl: C, CpG, 3M003, flagellin, eukaryotic ribosome elongation and Leishmania homologue of initiator 4a (LeIF), Also selected from at least one hepatitis C antigen, if an imidazoquinolin immune response modifier is present, it is selected from the group consisting of reshikimod (R848), imikimod and gardiquimod.

In another particular embodiment, a method of modifying immune responsiveness in a host, an immunoadjuvant composition comprising a glucopyranosyl lipid adjuvant (GLA) and at least one of a co-adjuvant and a pharmaceutically acceptable carrier. The coadjuvant is selected from the group consisting of cytokines, block copolymers or biodegradable polymers, and surfactants, and the pharmaceutically acceptable carriers are calcium phosphate, water. The above method is provided that comprises a carrier selected from the group consisting of oil emulsions, water emulsions in oil, liposomes, and microparticles, which comprises modifying the immune responsiveness in the host. In another particular embodiment, the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-γ, and the block copolymer or biodegradable polymer is Pluronic. Selected from L121, CRL1005, PLGA, PLA, PLG, and polyl: C, and said surfactants are saponin, Polysorbate 80, Span. Selected from the group consisting of 85 and stearyltyrosine.

In another particular embodiment of the method of modifying immune responsiveness in a host, the dosing step is performed once, twice, three times, four or more times. In another particular embodiment of the method of modifying an immune response in a host, modifying the immune response in the host comprises inducing or enhancing an immune response. In another particular embodiment of the method of modifying an immune response in a host, the modification of the immune response in the host comprises downregulating the immune response. In another particular embodiment of the method of modifying immunoresponsiveness in a host, the method is derived from at least one infectious agent associated with an infectious disease for which induced or enhanced immunoresponsiveness is desired. Alternatively, it further comprises administering the antigen, which is immunologically cross-reactive, simultaneously or continuously and in either order. In another such specific embodiment, the antigen administration step is performed once, twice, three times, four times or more. In another particular embodiment of the method of modifying the immune responsiveness in a host, the method comprises at least one epitope, biomolecule, cell or associated with an autoimmune disease in which a down-regulated immune responsiveness is desired. It comprises administering antigens derived from tissues or immunologically cross-reactive with them simultaneously or sequentially and in either order. In another such specific embodiment, the antigen administration step is performed once, twice, three times, four times or more. In another particular embodiment of the method of modifying immunoresponsiveness in a host, the method comprises at least one epitope, biomolecule, cell or tissue associated with a cancer for which an induced or enhanced immunoresponsiveness is desired. Includes administration of antigens derived from or immunologically cross-reactive with the antigens simultaneously or sequentially and in either order. In another such specific embodiment, the antigen administration step is performed once, twice, three times, four times or more.

In another embodiment, the above-mentioned immunoadjuvant composition is in contact with the above-mentioned antigen in a kit containing the above-mentioned immunoadjuvant composition in a first container and an antigen in a second container. A kit is provided. In another embodiment, at least one recombinant expression construct comprising the immunoadjuvant composition described above in a first container and a promoter operably linked to an antigen-encoding nucleic acid sequence in a second container. The kit is provided, wherein the immunoadjuvant composition is not in contact with the recombinant expression construct. In another particular embodiment of the kit, the antigen is derived from at least one infectious agent selected from bacteria, viruses, yeasts and protozoa. In other specific embodiments of the kit, the antigen is derived from at least one type of cancer cell. In another particular embodiment of the kit, the antigen is derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with an autoimmune disease. is there.

The above or other aspects of the invention will become apparent with reference to the following detailed description and accompanying drawings. In addition, various references are described herein, but these references describe in more detail some aspects of the invention, and are therefore referred to herein in full. Be incorporated.

<figref num="1">Figure 1 shows HPLC data demonstrating the number and amount of contaminants in MPL-AF and GLA-AF. These chromatograms were collected using the Agilent 1100 system and the ESA Corona CAD detector. This method was performed on a Waters Atlantis C18 column with a methanol / chloroform gradient. The injectate contained 2.5 μg of GLA and MPL, respectively, and 0.27 μg of synthetic phosphocholine (POPC) used as a solubilizer.</figref><figref num="2">Figure 2 shows ELISA data demonstrating the levels of cytokines and chemokines expressed by human macrophages (panels a-e) of the Mono Mac6 cell line and DCs derived from PBMC (panels f-h) in response to GLA stimulation. Shown. 1x10 using an aqueous formulation of GSK Biologicals MPL® (MPL-AF), GLA (GLA-AF), or AF vehicle only⁵Cells were cultured in cells / wells for 24 hours. MIP-1b, IP-10, IL-6, IL-23 and IL-1b levels in the supernatant were measured by sandwich ELISA.</figref><figref num="3">Figure 3 shows one week after each immunization with two different doses of Fluzone vaccine formulated with GLA-AF, or GLA-SE (ie, panel A on day 7; panel B). The 28th day) shows the ELISA data showing the level of anti-Fluzone antibody production induced in mice by comparison with Fluzone alone. Panels A and B are 20 ml (1.8 μg) or 2 ml (0.18 mg) Fluzone (Flu) vaccines in formulations containing GLA-AF, GLA-SE, or no adjuvant, 2 every 3 weeks. The ELISA Ab titers of the immunized mice one week after the first (A) or second (B) injection are shown. Panel C shows the titers of neutralizing antibody (HAI) in mouse sera after the second immunization.</figref><figref num="4">FIG. 4 shows ELISA data demonstrating the level of anti-SMT antibody production induced in mice 1 week after the third immunization with SMT formulated with SMT antigen alone or with GLA-SE. Shown. C57BL / 6 mice with SMT antigen (10 μg per animal per immunization) formulated in a stable emulsion containing GLA (GLA-SE; 20 μg per animal per immunization) for 3 weeks Immunized 3 times every other time or injected with SMT protein only. Serum was collected by collecting blood one week after each immunization, and the serum levels of SMT-specific IgG1 and IgG2c antibodies were tested by ELISA. Shows the reciprocal average of endpoint titers and SEM.</figref><figref num="5">Figure 5 shows an anti-Leish-110f antibody induced in mice one week after initial immunization with Leish-110f antigen formulated with various amounts (40, 20, 5, or 1 μg) of GLA. Shown are ELISA data showing the level of production by comparison with saline control. Balb / c mice with Leish-110f antigen (10 μg per animal for each immunization) formulated in a stable emulsion (GLA-SE) containing 40, 20, 5, or 1 mg of GLA. Immunized 3 times every 2 weeks or injected with saline only. Serum was collected by blood sampling 1 week after each immunization, and serum levels of Leish-110f-specific IgG1 and IgG2c antibodies were tested by ELISA. For sera collected 7 days after initial immunization, the mean reciprocal of endpoint titers and SEM are shown.</figref><figref num="6">Figure 6 shows the levels of anti-Leish-110f IFN-γ cytokine production induced in mice 1 week after the third immunization with Leish-110f antigen formulated with various amounts of GLA. The ELISA data is shown by comparison with a water control. Balb / c immunized 3 times every 2 weeks with Leish-110f antigen (10 μg) formulated in a stable emulsion containing 40, 5 or 1 μg MPL (MPL-SE) or GLA (GLA-SE). Spleen cells obtained from mice or saline-injected mice, in vitro 3 in medium alone or in medium containing 10 mg / ml Leish-110f or 3 mg / ml Concanabalin A (ConA). Incubated for days. IFN-γ levels in the supernatant were measured by ELISA. Shows average and SEM.</figref><figref num="7">Figure 7 shows ID83 with ID83 alone or supplemented with a formulation containing GLA (GLA-SE), GLA + CpG (GLA / CpG-SE), or GLA + GDQ (GLA / GDQ-SE). ICS data demonstrating the frequency of ID83-specific IFN-γ, IL-2, and TNF cytokine-producing CD4 + and CD8 + T cells induced in mice 1 week after the third immunization. Immunize with human M. tuberculosis ID83 fusion protein (8 μg) formulated with GLA-SE, GLA / CpG-SE, GLA / Gardiquimod (GDQ) -SE three times every three weeks, or Alternatively, splenocytes obtained from C57BL / 6 mice injected with saline were cultured in vitro for 12 hours in a medium containing 10 mg / ml ID83. Cellular levels of IL-2, TNF, and IFN-γ in CD3 + CD4 + or CD3 + CD8 + T cells were detected by intracellular staining and measured by flow cytometry with BD LSRII FACS.</figref><figref num="8">Panel A is a third dose using an aqueous formulation containing CpG or imikimod (IMQ), or a stable oil emulsion (GLA-SE) containing GLA, or the ML0276 antigen formulated with a mixture of these three. ICS data are shown showing the frequency of ML0276-specific IFN-γ cytokine-producing CD4 + T cells induced in mice 1 week after immunization by comparison with saline and naive controls. Immunized with bacillus (M.leprea) ML0276 antigen (10 μg) formulated with CpG, imiquimod (IMQ), GLA-SE, a mixture of these three, or injected with saline three times every 3 weeks. , C57BL / 6 mice were cultured in vitro for 12 hours in medium containing 10 mg / ml ML0276. Panel A shows that cellular levels of IFN-γ in CD3 + CD4 + T cells were detected by intracellular staining and measured by flow cytometry with BD LSRII FACS. Panel B shows cell enrichment of inflow region lymph nodes as a correlation of defense.</figref>

<u style="single">Detailed description of the invention</u> The present invention provides vaccine compositions, adjuvant compositions, and related methods thereof, including the use of synthetic glucopyranosyl lipid adjuvants (GLA) in some embodiments thereof. GLA provides a synthetic immunoadjuvant that can be prepared in a substantially homogeneous form in an advantageous manner as compared to prior art adjuvants, specifically as compared to natural adjuvants. Moreover, GLA, unlike adjuvants derived from natural substances, can be prepared efficiently and economically by large-scale synthetic chemical production. As a synthetic adjuvant that is chemically synthesized from a defined starting material to obtain a chemically defined product with consistent quality and quantity across batches, GLA includes improved product quality control, etc. Brings unprecedented benefits. Surprisingly, 3-acylated monophosphoryl lipid A was found to have a certain toxicity, but when the amine 2-position contained a single acyl chain, the molecule retained an acceptable safety profile. .. In addition, the specific deacylation at the 3-position presents technical challenges, simplifying the synthesis of such compounds. Therefore, the present invention provides additional advantages in terms of safety and ease of synthesis.

As described herein, GLA-containing compositions and methods of their use are pharmaceutically acceptable due to immunoadjuvant activity, including "adjuvanting" in some embodiments. Containing the use of GLA itself with a carrier or excipient, administration of GLA to a subject is one in which it is desired to induce or enhance an immune response (eg, an antigen-specific response) in the subject. Administration of the above antigens to a subject may be completely independent and / or temporally and / or spatially isolated. Other embodiments include the use of GLA in a vaccine composition further comprising one or more antigens for which the vaccine is desired to induce or enhance an immune response. As described herein, these vaccine compositions also, in certain relevant embodiments, are one or more Toll-like receptor (TLR) agonists and / or one or more co-adjuvants, imidazoquinolin. It may contain one or more of an immune response modifier and a dual stem loop immunomodulator (dSLIM). In other related embodiments, the vaccine compositions described herein are nucleic acid sequences encoding a GLA and an antigen, each of which is desired to induce or enhance an immune response (eg, an antigen-specific response) in a subject. It may include one or more recombinant expression constructs, including a promoter operably linked to.

<u style="single">GLA</u> As already mentioned, GLA, which is a chemically synthesized adjuvant, can be prepared in a substantially homogeneous form, so that this is at least 80%, preferably at least 85%, more preferably with respect to the GLA molecule. Refers to at least 90%, more preferably 95%, even more preferably 96%, 97%, 98% or 99% pure GLA preparations, (i) hexosamine 1 of non-reducing terminal glucosamine and hexosamine of reducing terminal glucosamine. A diglucosamine skeleton having a reducing terminal glucosamine bound to a non-reducing terminal glucosamine by an ether bond with the 6-position, (ii) an O-phosphoryl group bonded to the hexosamine 4-position of the non-reducing terminal glucosamine, and (iii) 6 It contains one or less aliphatic acyl chains, in which case one of the aliphatic acyl chains is bonded to 3-hydroxy of the reducing terminal glucosamine by an ester bond, and one of the aliphatic acyl chains is an amide bond. Containing a tetradecanoyl chain bound to the 2-amino of the non-reducing end glucosamines and to an alkanoyl chain of 13 carbon atoms or more by an ester bond, and one of the above aliphatic acyl chains is a non-reducing end by an ester bond. It contains a tetradecanoyl chain that is bound to 3-hydroxy of glucosamine and is bound to an alkanoyl chain of 13 carbon atoms or more by an ester bond. Determining the purity of a given GLA preparation can be easily carried out by anyone familiar with suitable analytical chemistry methods, such as gas chromatography, liquid chromatography, mass spectrometry. Analysis and / or nuclear magnetic resonance analysis and so on.

The GLA used herein is the following general structural formula:<chemistry num="4"><img id="000006" he="61" wi="95" file="JP2018024656A_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

[In the formula, R¹, R³, R⁵And R⁶Is C₁₁-C₂₀Alkyl; R²And R⁴Is C₁₂-C₂₀Alkyl] Have.

GLA is, for example, Avanti Polar Lipids, Inc. (Alabaster, AL; product number 699800, where R¹, R³, R⁵And R⁶Is undecylic and R²And R⁴Can be obtained from Dodecyl).

"Alkyl" is a linear or branched, acyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing 1 to 20 carbon atoms, in certain preferred embodiments 11 to 20 carbon atoms. Means. Typical saturated linear alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc., such as undecyl, dodecyl, tridecylic, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc. As the saturated branched alkyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl and the like can be mentioned. Typical saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, and unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl and the like. Cyclic alkyls are also referred to herein as "homocycles" or "monoprime rings." Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (each referred to as an "alkenyl" or "alkynyl"). Typical linear and branched alkynes include ethylenyl, propyrenyl, 1-butenyl, 2-butenyl, isobutyrenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, Examples include 3-dimethyl-2-butenyl; typical linear and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butenyl and the like. Can be mentioned.

Thus, in a particular embodiment as intended herein, the GLA may have any of the structures described above, and in the particular embodiment the GLA is disclosed in one or more of the following documents: It is expressly intended to include any structure of the lipid adjuvant, and in other embodiments, it is expressly intended to include none of such structures: US Pat. No. 6,544,518, EP 1531158. , WO 2001/036433, WO 97/11708, WO 95/14026, US Pat. No. 4,987,237, Japanese Patent No. 63010728, Japanese Patent No. 07055906, WO 2000/013029, US Pat. No. 5,530,113, US Patent No. 5,612,476, US Patent No. 5,756,718, US Patent No. 5,843,918, WO 96/09310, US Publication Number: 2004/161776, US Publication Number: 2003/170249, US Publication Number: 2002/176867, WO 2002/032450, WO 2002/028424, WO 2002/016560, WO 2000/042994, WO 2000/025815, WO 2000/018929, Japanese Patent No. 10131046, WO 93/12778, EP 324455, DE 3833319, US Pat. No. 4,844,894, US Pat. No. 4,629,722. According to certain embodiments, GLA is not 3'-O-deacylated.

<u style="single">antigen</u> The vaccine compositions described herein, and the antigens used in some embodiments of methods using GLA, are any target epitopes, molecules (biomolecules) for which it is desired to induce and enhance immunoreactivity in the subject. Etc.), molecular complexes (including molecular complexes containing biomolecules), intracellular assemblies, cells or tissues. The term antigen often refers to the polypeptide antigen of interest. However, as used herein, the antigen may also refer to a recombinant construct (eg, an expression construct) that encodes the polypeptide antigen of interest. In certain preferred embodiments, the antigen is an infectious agent and / or associated with any other condition in which stimulation of an infection, cancer, autoimmune disease, allergy, asthma, or antigen-specific immune response is desirable or beneficial. It may be an epitope, a biomolecule, a cell or tissue, or is derived from them, or may be immunologically cross-reactive with them.

Preferably, in certain embodiments, the viral preparation of the invention comprises an antigen or antigen composition capable of inducing an immune response against human or other mammalian pathogens, such antigen or antigen composition. The object may include compositions derived from viruses such as: HIV-1 (eg tat, nef, gp120 or gp160), human herpesviruses such as gD or derivatives thereof, or HSV1. Alternatively, pre-early proteins such as HSV2-derived ICP27, cytomegalovirus ((especially human), eg gB or derivatives thereof), rotavirus (attenuated viable virus, etc.), Epsteinver virus (eg, gp350 or a derivative thereof), Spout-herpes zoster virus (eg, gpl, II and IE63), or hepatitis B virus (eg, hepatitis B surface antigen or derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus. Hepatitis virus, or viral pathogens other than those listed above, such as paramyxovirus: respiratory symptom virus (eg, F and G proteins or derivatives thereof), parainfluenza virus, measles virus, epidemic parotid inflammation virus, human papillomavirus (For example, HPV6, 11, 16, 18, etc.), Flavivirus (eg, yellow fever virus, dengue fever virus, tick-borne encephalitis virus, Japanese encephalitis virus) or influenza virus (total survival or inactivating virus, egg or MDCK) Split influenza virus propagated in cells, or whole influenza virusome (described by Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant protein thereof (eg, HA, NP, NA, or M protein, or a combination thereof).

In another particular preferred embodiment, the vaccine formulation of the invention comprises an antigen or antigen composition capable of inducing an immune response against human or other mammalian pathogens, which antigen or antigen composition. It may contain compositions derived from one or more bacterial pathogens such as those listed below: Neisseria spp., For example, N. gonorrhea and N. meningitidis (. Examples: Capsular polysaccharide and its conjugates, transferase-binding protein, lactoferrin-binding protein, PilC, Adhesin); S. pyogenes (eg, M protein or fragment thereof, C5A protease, lipoteiconic acid), B group S. agalactiae, S. mutans: Soft Streptococcus (H. mutans) ducreyi); Moraxella spp, for example Moraxella catarrhalis, also known as Branhamella catarrhalis (eg, high and low molecular weight adhesin and inbeicin): Bordetella spp, eg B. pertussis (eg, pertustin, B. pertussis toxin or a derivative thereof, fibrous blood cell agglutinin, adenylate cyclase, fimbrier), B. parapertussis and B. bronchiseptica; myco Mycobacterium spp., For example, human tuberculosis (eg ESAT6, Antigen 85A, -85B or -85C), bovine tuberculosis (M. bovis), leprosy (M. leprae) , Mycobacterium tuberculosis (M. avium), Mycobacterium tuberculosis (M. paratuberculosis), Smeguma (M. smegmatis; Legionella spp., For example, L. pneumophila; Escherichia spp., Enterotoxic E. coli (eg, colonization factor, heat-labile toxin) Or its derivatives, thermostable toxins or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (eg, Shigella toxin-like toxins or derivatives thereof); Vibrio spp.), For example, Vibrio cholerae (eg, Vibrio cholerae or derivatives thereof); Shigella spp., For example, S. sonnei, S. dysenteriae. , S. flexnerii; Yersinia spp., For example, Y. enterocolitica (eg, Yop protein), Pest (Y. pestis), Pseudo-tuberculosis Ersina (Y. pestis) Y. Pseudotuberculosis); Campylobacter spp., For example, C. jejuni (eg, toxins, adhesin and invasin) and C. coli; Salmonella spp., For example. S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., For example, L. monocytogenes; Helicobacter spp., For example, H. pylori (eg, urease, catalase, vacuolar toxin); Pseudomonas spp., For example, P. aeruginosa; grapes The genus Staphylococcus spp., For example, Salmonella japonicum (S. aureus), Salmonella epidermidis (S. epidermidis); Enterococcus spp., For example, stool Lenza sphere (E. faecalis), Hesium (E. faecium); Clostridium spp., For example, Clostridium tetani (eg, C. tetani). Clostridium tetani and its derivatives), C. botulinum (eg, botulinum toxin and its derivatives), C. difficile (eg, Clostridium toxin A or B and its derivatives); Bacillus spp .), For example, B. anthracis (eg, botulinum toxin and its derivatives); Corynebacterium spp., For example, C. diphtheriae (eg, diphtheria toxin and its derivatives). Borrelia spp., For example, Lime's disease Borrelia (B. burgdorferi) (eg OspA, OspC, DbpA, DbpB), B. Garini (B. garinii) (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA, DbpB), goose spirochete (eg B. andersonii) (eg OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., For example, E. equi and factors of human granular ehrlichia; Rickettsia spp., For example, spots R. rickettsii; Chlamydia spp., For example, C. trachomatis (eg MOMP, heparin binding protein), Chlamydia pneumoniae (eg MOMP, heparin binding) Protein), parrot disease Chlamydia (C. psittaci); Leptospira spp., For example, L. interrogans; Treponema spp., For example, syphilis treponema (T. pallidum) (eg, rare outer membrane proteins), T. denticola, T. hyodysenteriae; or other bacterial pathogens.

In a preferred other specific embodiment, the vaccine composition of the invention comprises an antigen or antigen composition capable of inducing an immune response against human or other mammalian pathogens, which antigen or antigen composition. , Plasmodium spp., For example, Plasmodium falciparum; Toxoplasma spp., For example, Toxoplasma protozoa (T. gondii) (eg SAG2, SAG3, Tg34); Entamoeba spp., For example, E. histolytica; Babesia spp., For example, Babesia protozoa (B. microti); Trypanosoma spp., For example, Cruise tripanosoma ( T. cruzi); Giardia spp., For example, Giardia spp.; G. lamblia; Leishmania spp., For example, forest-type tropical Leishmania (L. major); Pneumocystis spp., For example, P. carinii; Trichomonas spp., For example, vaginal trichomonas (T. One or more parasites such as vaginalis) (eg John, DT and Petri, WA, Markell and Voge's Medical Parasitology-9th Edition, 2006, WB Saunders, Philadelphia; Bowman, DD, Georgis' Parasitology for Veterinarians-8th Edition, 2002, Compositions derived from WB Saunders, Philadelphia; or: (i) Necator infections (eg, but not limited to, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria) , American worm (Necator americanus), Zubini worm (Ancylostoma duodenale), Bacroft filamentous worm (Wuchereria bancrofti), Malay filamentous worm (Brugiamalayi), circumcision filamentous worm (Onchocerca volvulus) ), And fecal nematodes (Strongyloides stercoralis); (ii) Blood-flukes (eg, but not limited to, Schistosoma mansoni, Schistosoma haematobium, Japanese blood-flukes (Schistosoma) japonicum), Mekon resident blood sucker (Schistosoma mekongi), liver sucker (Opisthorchis sinensis), lung sucker (Paragonimus sp.), Hepatica (Fasciola hepatica), Fasciola magna (Fasciola gigantica) And (iii) to asphyxia that can infect mammals, such as, but not limited to, Taenia saginata and Taenia solium. It may contain the derived composition. Thus, certain embodiments are derived from the genus Schisostoma spp., Such as the genus Schistosoma mansonii, the Schistosoma haematobium, and / or the Japanese blood-flukes (Schistosoma japonicum). , Or Candida spp., For example, Candida albicans (C. albicans); May be intended as a vaccine composition comprising an antigen derived from a yeast such as Cryptococcus spp., For example, C. neoformans.

Other specific antigens preferred for human M. tuberculosis include, for example, Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO 99/51748). ). In addition, the protein of M. tuberculosis includes a fusion protein in which at least 2 kinds, preferably 3 kinds of polypeptides of M. tuberculosis are fused to a larger protein, and a mutant thereof. Preferred fusions are Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9- DPV-MTI (WO 99151748) and the like can be mentioned.

The most preferred antigens for clazimia include, for example, ultra high molecular weight proteins (HWMP) (WO 99/17741), ORF3 (EP 366 412), and putative membrane proteins (Pmps). Other Chlamydia antigens in the vaccine formulation can be selected from the group described in WO 99128475. Preferred bacterial vaccines are Streptococcus spp., For example, Streptococcus pneumoniae (S. pneumoniae). Antigens derived from pneumoniae) (eg, capsular polysaccharides and their conjugates, PsaA, PspA, streptolysin, choline-binding proteins), and the protein antigen neumorisine (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Includes Pathogenesis, 25, 337-342) and its mutant detoxified derivatives (WO 90/06951; WO 99/03884). Other preferred bacterial vaccines are Haemophilus spp., Such as Haemophilus spp., Such as Haemophilus influenzae (eg, PRP and its conjugates), antigens derived from Haemophilus influenzae, such as OMP26, high molecular weight adhesin, P5, P6. , Protein D and lipoprotein D, as well as Haemophilus and Haemophilus-derived peptides (US Pat. No. 5,843,464) or multicopy variants or fusion proteins thereof.

Derivatives of hepatitis B surface antigens are also well known in the art, including the PreS1 and Pars2 S antigens described in European patent applications EP-A414 374; EP-A-0304 578 and EP 198474. In a preferred embodiment, the vaccine formulation of the invention comprises the HIV-1 antigen, gp120, especially when expressed in CHO cells. In another embodiment, the vaccine formulation of the present invention comprises gD2t already defined herein.

In a preferred embodiment of the invention, the vaccine comprising the adjuvant according to claim is a human papillomavirus (HPV) (such as HPV 6 or HPV 11) that is believed to cause genital warts, and an HPV virus that causes cervical cancer (such as HPV 6 or HPV 11). Contains antigens derived from (such as HPV16 or HPV18). Particularly preferred embodiments of the prophylactic or therapeutic vaccine for genital warts include L1 particles or capsomers, and fusion proteins containing one or more antigens selected from the HPV6 and HPV11 proteins E6, E7, L1 and L2. Certain preferred embodiments of the fusion protein are L2E7, and GB disclosed in WO 96/26277. Contains protein D (1/3) -E7 disclosed in 9717953.5 (PCT / EP98 / 05285). Preferred HPV cervical infection or cancer prophylactic or therapeutic vaccine compositions can include HPV 16 or 18 antigens. For example, the L1 or L2 antigen monomer, or the L1 or L2 antigen may be provided together as a virus-like particle (VLP), or the L1-only protein may be provided alone in the VLP or capsomer structure. Such antigens, virus-like particles and capsomers are known in their own right. See, for example, WO94 / 00152, WO94 / 20137, WO94 / 05792, and WO93 / 02184.

Additional initial proteins may be contained alone or as a fusion protein such as, for example, E7, E2 or preferably F5, with particularly preferred embodiments comprising VLPs comprising the L1E7 fusion protein (WO 96/11272). ). A particularly preferred HPV 16 antigen is a protein D-E6 or E7 fusion derived from HPV 16 or a combination thereof; or a combination of L2 and E6 or E7 by including the initial protein E6 or E7 fused with a protein D carrier. Form (WO 96/26 277). Alternatively, the HPV 16 or HPV 18 early proteins E6 and E7 may be provided as a single molecule, preferably as a protein D-E6 / E7 fusion. Such vaccines optionally include either or both of E6 and E7 protein front HPV 18, preferably in the form of protein D-E6 or protein D-E7 fusion protein or protein D-E6 / E7 fusion protein. And may be included. The vaccine of the present invention may contain antigens from other HPV strains, preferably HPV 31 strains or HPV 33 strains.

The vaccines of the present invention further contain antigens derived from parasites that cause malaria. For example, preferred antigens from Plasmodia falciparum include RTS, S and TRAP. RTS provides virtually all of the Plasmodium falciparum sporozoite peri (CS) proteins bound to the hepatitis B virus surface (S) antigen via the four amino acids in the pre-S2 portion of the hepatitis B surface antigen. It is a hybrid protein containing the C-terminal part. Its entire structure is disclosed in International Patent Application No. PCT / EP 92/02591 published as WO 93/10152 claiming priority from UK Patent Application No. 9124390.7. When expressed in yeast, RTS is produced as lipoprotein particles, and when co-expressed with HBV-derived S antigen, it produces mixed particles known as RTS, S.

The TRAP antigen is described in International Patent Application No. PCT / GB89 / 00895 published as WO 90/01496. A preferred embodiment of the invention is a malaria vaccine in which the antigen preparation comprises a combination of RTS, S and TRAP antigens. Other potential malaria protozoan antigens for the components of the multistage malaria vaccine are Plasmodium falciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, These are analogs of SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16, Pfs48 / 45, Pfs230, and the genus Plasmodium spp.

Accordingly, the particular embodiments disclosed herein are intended for antigens derived from at least one infectious agent, such as a bacterium, virus, or fungus, such pathogens include: Actinobacteria, such as human tuberculosis or leprosy or other mycobacteria; bacteria, such as members of the genus Salmonella, Nyceria, Borrelia, Chlamydia or Bordetella; viruses, such as simple herpesvirus, human immunodeficiency. Virus (HIV), feline immunodeficiency virus (FIV), cytomegalovirus, varicella-herpes zoster virus, hepatitis virus, Epsteinver virus (EBV), respiratory follicles virus, human papillomavirus (HPV) and cytomegalovirus; HIV HIV such as -1 or HIV-2; fungi, such as Aspergillus, Blast Mrs, Coccidioides or Pneumocystis, or yeast, such as Candida species, such as C. albicans, C. glabrata, C. crusei, C. Lucitaniae, C. tropicalis and C. parapsillosis; parasites such as protozoa, such as Plasmodium species (including falciparum malaria protozoa, vivax malaria protozoa, quadruple fever malaria protozoa and oval malaria protozoa) ); Or another parasite, such as Acant amoeba, Red diarrhea amoeba, Sumi blood nematode, Manson Sumi blood sucker, Birhardz Sumi blood sucker, Japanese Sumi blood sucker, Cryptosporidium, Sprout, Red diarrhea amoeba, Colon amoeba, Ent amoeba -Disper, Hartmann amoeba, Polek amoeba, Bancroft filamentous insect, one or more of the genus Giardia, the genus Leishmania, etc.

For example, in the GLA-containing vaccine embodiment containing an antigen derived from Borrelia species, the antigen may include nucleic acid, pathogen-derived antigen or antigen preparation, recombinantly prepared protein or peptide, chimeric fusion protein, and the like. .. One such antigen is OspA. OspA may be a fully mature protein (Lipo-OspA) in a form lipidized by its biosynthesis in a host cell, or it may be a non-lipidized derivative. Such non-lipidized derivatives include non-lipidized NS1-OspA fusion proteins with the first 81 amino acids at the N-terminus of the influenza virus non-structural protein (NS1) and the complete OspA protein. MDP-OspA is a non-lipidized form of OspA that retains an additional 3 amino acids at the N-terminus.

Compositions and methods for identifying subjects who have or are suspected of being infected with an infectious pathogen described herein are known in the art.

For example, M. tuberculosis causes tuberculosis (TB). The bacterium usually attacks the lungs, but it can also attack the kidneys, spinal cord, and brain. Without proper treatment, TB disease can be fatal. The disease spreads from person to person by airborne infection when an infected person sneezes or coughs. In 2003, more than 14,000 TB cases were reported in the United States.

Tuberculosis can generally be controlled with long-term antibiotic treatment, but such treatment is not sufficient to prevent the spread of the disease, raising concerns about potential selection of antibiotic-resistant strains. To. Even if the infected individual is asymptomatic, it can be contagious for some time. In addition, compliance with the treatment regimen is important, but it is difficult to monitor patient behavior. Some patients do not complete the treatment process, which can invalidate the treatment and lead to the development of drug resistance (eg, US Pat. No. 7,087,713).

At present, vaccination with live bacteria is the most effective way to induce protective immunity against tuberculosis. The most common mycobacterium used for this purpose is Bacillus carmet-geran (BCG), a non-toxic strain of Mycobacterium bovis. However, the safety and efficacy of BCG has been a source of controversy, and some countries, such as the United States, have not vaccinated the general public. Diagnosis is generally made using a skin test, which involves intradermal exposure to tuberculin PPD (a purified protein derivative). The antigen-specific T cell response results in measurable sclerosis at the injection site within 48, 72 hours after injection, indicating exposure to mycobacterium antigens. However, there are problems with sensitivity and specificity in this study, and BCG-inoculated individuals cannot be distinguished from infected individuals (eg, US Pat. No. 7,087,713).

Macrophages have been shown to act as major effectors of human M. tuberculosis immunity, and T cells are the major inducers of such immunity. The primary role of T cells in defense against M. tuberculosis infection is due to the frequent occurrence of M. tuberculosis in AIDS patients due to the deletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Be explained. Mycobacterium reactive CD4 T cells have been shown to be potent producer cells of γ-interferon (IFN-γ), which have been shown to induce macrophage anti-mycobacterial activity in mice. Although the role of IFN-γ in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3 activates human macrophages alone or in combination with IFN-γ or tumor necrosis factor α in humans. It has been shown to inhibit M. tuberculosis infection. In addition, IFN-γ has been shown to stimulate human macrophages to produce 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to human M. tuberculosis infection. For an overview of immunity to human M. tuberculosis infection, see Chan and Kaufmann, Tuberculosis: Pathogenesis, Protection and Control, Bloom (eds.), ASM Press. Washington DC (1994).

Existing compounds and methods for diagnosing tuberculosis or inducing protective immunity against tuberculosis include polypeptides containing at least one immunogenic portion of one or more mycobacterial proteins, and such polypeptides. Includes the use of the encoding DNA molecule. Diagnostic kits containing such polypeptides or DNA sequences and suitable detection reagents can be used to detect mycobacterium infections in patients and biological samples. Antibodies to such polypeptides are also provided. In addition, such compounds can be formulated into vaccines and / or pharmaceutical compositions for immunization against mycobacterium infection (US Pat. Nos. 6,949,246 and 6,555,653).

Malaria was eliminated from many parts of the world in the 1960s, but it still remains, and new strains of malaria resistant to existing drugs are emerging. Malaria is a major public health challenge in more than 90 countries. Nine out of ten malaria cases occur in sub-Saharan Africa. More than one-third of the world's population is at risk of illness, with 350-500 million people infected with malaria each year. This year, 45 million pregnant women are at risk of developing malaria. Of the already infected individuals, more than one million infected each year die from preventable disease. The majority of dead patients are African children.

Malaria is commonly transmitted by human bites in infected female Anopheles mosquitoes. In order to be transmitted, the mosquito must be infected by sucking blood from a person who has already been infected with malaria. Malaria is caused by parasites, and clinical symptoms of the disease include fever and flu-like symptoms such as chills, headache, myalgia, and fatigue. These symptoms may also be accompanied by nausea, vomiting, and diarrhea. Malaria can also cause anemia and jaundice due to the loss of red blood cells. Infection with Plasmodium falciparum, a type of malaria, can cause renal failure, seizures, mental confusion, coma and death if not treated promptly.

Methods for in vitro diagnosis of malaria in an individual are well known, in which the tissue or biofluid collected from the individual is brought into contact with the molecule or polypeptide composition, wherein the molecule or polypeptide composition is tropical. It comprises one or more peptide sequences that retain all or part of one or more epitopes of a protein obtained from the infectious activity of a thermomalaria protozoa, and the contact is to the composition and the tissue or biofluid. It involves performing in vitro immune reactions with possible antibodies and performing in vitro detection of the formed antigen-antibody complex (eg, US Pat. No. 7,087,231). See).

Expression and purification of the recombinant Plasmodium falciparum (3D7) AMA-1 extracellular domain has been described. Conventional methods have produced highly purified proteins that retain the folding and disulfide bridges of natural molecules. Recombinant AMA-1 is equally useful as a diagnostic reagent, in antibody production, and as a protein used alone for malaria prophylaxis or as part of a vaccine (US Pat. No. 7,029,685). ..

Conventional techniques include species-specific Plasmodium vivax (P.), which is a protein or fragment thereof secreted into the plasma of a susceptible mammalian host after infection. vivax) Polynucleotides encoding malaria peptide antigens are described, as are monoclonal or polyclonal antibodies against these antigens. Peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are used in assays used to diagnose malaria and to determine whether Plasmodium vivax is the causative species of infection. (US Patent No. 6,706,872). In addition, peptide antigens of species-specific vivax Plasmodium malaria, which are proteins or fragments thereof secreted into the plasma of susceptible mammalian hosts after infection, have also been reported, as well as monoclonals against these antigens. Alternatively, polyclonal antibodies have also been reported. Peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are used in assays used to diagnose malaria and to determine if Plasmodium vivax is the causative species of infection. (See, for example, US Pat. No. 6,231,861).

Recombinant Plasmodium falciparum (3D7) AMA-1 extracellular domain is also expressed by methods that produce highly purified proteins that retain the folding and disulfide bridges of natural molecules. Recombinant AMA-1 is useful as a diagnostic reagent, for use in antibody production, and as a vaccine (US Pat. No. 7,060,276). Similarly, recombinant Plasmodium falciparum (3D7) MSP-1 carrying natural molecule folding and disulfide bridges₄₂Expression and purification of is also known. Recombinant MSP-1₄₂Is useful as a diagnostic reagent, for use in antibody production, and as a vaccine (US Pat. No. 6,855,322).

Thus, diagnostic methods for detecting human malaria infection and identifying subjects infected with or suspected of being infected with a malaria-infecting pathogen are known from these and related literature. Specifically, the blood sample is mixed with, for example, a reagent, substrate (eg, lactate or lactate) and buffer containing 3-acetylpyridine adenine dinucleotide (APAD). This reagent is designed to detect the presence of unique glycolytic enzymes produced by malaria parasites. This enzyme is known as the parasite lactate dehydrogenase (PLDH). PLDH can be easily identified from host LDH using the reagents described above. Mixing reagents with parasitic blood samples results in a reduction in APAD. However, APAD is not reduced by host LDH. Reduced APAD can then be detected by a variety of techniques such as spectrum, fluorescence, electrophoresis, or colorimetry. Detection of reduced APAD as described above provides a positive indicator of malaria infection (eg, US Pat. No. 5,124,141). Another method of diagnosing malaria is to test a polypeptide containing a characteristic amino acid sequence derived from the Plasmodium falciparum antigen GLURP with a specific antibody produced or reactive against this polypeptide. Recognized in samples (eg, US Pat. No. 5,231,168).

Leishmaniasis is a widespread parasitic disease that is prevalent in the Indian subcontinent, Africa and Latin America, making vaccine development a priority for the World Health Organization. Leishmania protozoan, a complex of various diseases, causes fatal infections of internal organs as well as serious skin diseases. One of the most devastating forms of leishmaniasis is a serious infection of the nose and mouth. The number of cases of leishmaniasis is increasing and is now out of control in many areas. Leishmaniasis is also increasing in some developed countries, specifically in Southern Europe as a result of HIV infection. The available drugs are toxic and expensive, and require daily injections for extended periods of time.

Leishmania is a protist parasite that parasitizes macrophages or leukocytes of the immune system. This parasite is transmitted by the bite of a small blood-sucking insect (Drain fly), but it is difficult to control because it inhabits a large area of the earth.

Visceral leishmaniasis is the most dangerous of the three manifestations of the disease. For the visceral type (Kala Azar, or "killing disease"), it is estimated that about 500,000 new cases occur each year. Currently, over 200 million people are at risk of developing visceral leishmaniasis. Over 90% of cases of visceral leishmaniasis occur in India, Baclades, Sudan, Brazil and Nepal. Most of the dead patients are children. Skin-type leishmaniasis often remains impaired for life.

Leishmania infections are difficult to diagnose and typically involve histopathological analysis of tissue biopsy material. However, several serological and immunological diagnostic assays have been developed (US Pat. No. 7,008,774; Senaldi et al., (1996) J. Immunol. Methods 193: 95; Zijlstra et al., (1997) Trans. R. Soc. Trop. Med. Hyg. 91: 671 673; Badaro et al., (1996) J. Inf. Dis. 173: 758 761; Choudhary, S. et al., (1992) J. Comm. Dis. 24:32 36; Badaro, R. et al., (1986) Am. J. Trop. Med. Hyg. 35:72 78; Choudhary, A. et al., (1990) Trans. R. Soc. Trop. Med. Hyg. 84: 363 366; and Reed, SG et al., (1990) Am. J. Trop. Med. Hyg. 43: 632 639). Promastigote releases metabolites into culture medium to produce acclimatization medium. These metabolites are immunogenic to the host (Schnur, LF, et al., (1972) Isrl. J. Med. Sci. 8: 932 942; Sergeiev, VP et al., (1969) Med. Parasitol. 38: 208 212; El-On, J. et al., (1979) Exper. Parasitol. 47: 254 269; and Bray, RS et al., (1966) Trans. R. Soc. Trop. Med. Hyg. 60: 605 609 See US Pat. No. 6,846,648, US Pat. No. 5,912,166; US Pat. No. 5,719,263; US Pat. No. 5,411,865).

About 40 million people worldwide are infected with HIV, the virus that causes AIDS. About 3 million people die each year from the disease, 95% of whom live in developing countries. Nearly five million people are infected with HIV each year. Currently, sub-Saharan Africans bear the largest burden of illness, but are rapidly spreading to other countries such as India, China and Russia. This infectious disease spreads fastest among a minority population. In the United States, more than 950,000 cases have been reported since 1981. AIDS attacks people during their most working age. Women are at increased risk of HIV / AIDS for biological and social reasons.

AIDS is caused by the human immunodeficiency virus (HIV), which kills and damages cells in the body's immune system, gradually destroying the body's ability to fight infections and certain cancers. HIV is most commonly transmitted by unprotected sexual intercourse with an infected partner. The most robust solution to this problem is to stop the spread of the virus. Creating an HIV vaccine that is safe, effective, and affordable is one way to achieve this goal. In the world, less than one in five people at high risk of HIV infection can get effective prevention.

Methods for diagnosing HIV infection are known and include by viral culture, PCR of definitive nucleic acid sequences from patient specimens, and antibody testing for the presence of anti-HIV antibodies in patient serum (eg, US Pat. No. 6,979,535). No., US Pat. No. 6,544,728, US Pat. No. 6,316,183, US Pat. No. 6,261,762, US Pat. No. 4,743,540).

According to other specific embodiments disclosed herein, vaccine compositions and related formulations, as well as methods of use, can include antigens derived from cancer cells, which are also immunotherapeutic for cancer. Can be useful for treatment. For example, the adjuvant preparation can be found to be useful with tumor-rejecting antigens such as prostate, breast, rectal colon, lung, pancreas, kidney or melanoma cancer antigens. Examples of antigens derived from cancer or cancer cells are MAGE1, MAGE3 and MAGE4 or other MAGE antigens, such as those disclosed in WO99 / 40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE (Robbins and Kawakami, 1996 Current Opinions in Immunology 8, pps 628-636; Van den Eynde et al., International Journal of Clinical &amp; Laboratory Research (1997 and 1998); Correale et al., (1997), Journal of the National Cancer Institute 89, p. 293). Non-limiting examples of these cancer antigens are expressed in various types of tumors, such as melanoma, lung cancer, sarcoma and bladder cancer. See, for example, US Pat. No. 6,544,518.

According to the particular embodiments disclosed herein, other tumor-specific antigens suitable for use with GLA include, but are not limited to, tumor-specific or tumor-related gangliosides, such as GM.₂And GM₃, Or a conjugate with the carrier protein thereof. Alternatively, the antigen used in the GLA vaccine composition for inducing or enhancing the anticancer immune response may be an autopeptide hormone, such as a full-length gonadotropin-releasing hormone (GnRH, WO 95/20600). Is a short 10-aminogen-long peptide useful in the treatment of many cancers. In another embodiment, prostate antigens such as Prostate Specific Antigen (PSA), PAP, PSCA (eg, Proc. Nat. Acad. Sci. USA 95 (4) 1735-1740 1998), PSMA are used, or In a preferred embodiment, an antigen known as Prostase is used (eg, Nelson et al., Proc. Natl. Acad. Sci. USA (1999) 96: 3114-3119; Ferguson et al., Proc. Natl. Acad. Sci. USA 1999. 96, 3114-3119; WO 98/12302; US Pat. No. 5,955,306; WO 98/20117; US Pat. Nos. 5,840,871 and 5,786,148; WO 00/04149). Other prostate-specific antigens are known from WO 98/137418, and WO / 004149. Another antigen is STEAP (PNAS 96 14523 14528 7-12 1999).

Other tumor-related antigens useful in this invention include Plu-1 (J Biol. Chem 274 (22) 15633 -15645, 1999), HASH-1, HasH-2, Cripto (Salomon et al., Bioessays 199, 21:61). -70, US Pat. No. 5,654,140) and Criptin (US Pat. No. 5,981,215). Furthermore, antigens particularly related to vaccines in the treatment of cancer include tyrosinase and sulbibin.

The embodiments disclosed herein for a GLA-containing vaccine composition comprising a cancer antigen are tumor-related antigen expression, eg, HER-2 / neu expression, or any cancer characterized by other cancer-specific or cancer-related antigens. It is useful for.

Diagnosis of cancer in subjects with or suspected of being at risk of developing cancer may be achieved by any of the various methods accepted in the art, such methods are clinical. It can vary depending on a variety of factors, including findings, cancer progression, cancer type, and other factors. Examples of cancer diagnosis are histopathological, histopathological, immunohistocytochemical and immune of patient samples (eg, blood, skin biopsy materials, other tissue biopsy materials, surgical specimens, etc.) Histopathological tests, PCR tests for defined gene (eg, nucleic acid) markers, serological tests for circulating cancer-related antigens or cells carrying such antigens, or antibodies of a given specificity, or those skilled in the art. Includes other well-known methods. For example, U.S. Pat. Nos. 6,734,172; 6,770,445; 6,893,820; 6,979,730; 7,060,802; 7,030,232; 6,933,123; 6,682,901; 6,587,792; 6,512,102; 7,078,180; Issue; JP 5-328975; Waslylyk et al., 1993 See Eur. J Bioch. 211 (7):18.

Vaccine compositions and methods according to specific embodiments of the invention can also be used to prevent or treat autoimmune diseases, such autoimmune diseases in which the immune system of the host or subject is "self". Tissues, cells, biomolecules (eg peptides, polypeptides, proteins, glycoproteins, lipoproteins, proteolipids, lipids, glycolipides, nucleic acids such as RNA and DNA, oligosaccharides, polysaccharides, proteoglycans, glycosaminoglycans, etc., as well as , Other molecular components of the subject's cells and tissues), or epitopes (eg, recognized by a particular immunologically apparent cognitive structure, eg, antibody variable region complementarity determination region (CDR) or T cell receptor CDR). Includes diseases, symptoms or disorders that adversely mediate an immune response to (such as those that are).

Thus, autoimmune diseases are characterized by an abnormal immune response involving either cells or antibodies, in each case such an immune response to normal autologous tissue. Autoimmune diseases in mammals can generally be divided into two different categories: cell-mediated diseases (ie, T cells) or antibody-mediated diseases. Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes (juvenile diabetes) and autoimmune uveal retinitis. Antibody-mediated autoimmune diseases include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (or SLE), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, and autoimmune asthma. , Cold globulinemia, thrombocytopenic purpura, primary sclerosing cholangitis, and pernicious anemia. The antigen associated with systemic lupus erythematosus is the nuclear low molecular weight ribonucleic acid protein (snRNP); Graves' disease is a tyrotropin receptor, tyroglobulin, and other components of thyroid epithelial cells (Akamizu et al., 1996; Kellerman). Et al. 1995; Raju et al., 1997; and Texier et al., 1992); Pemphigus is a cadherin-like pemphigus antigen and other adherent molecules such as desmograin 3 (Memar et al., 1996: Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993); and thrombotic thrombocytopenic purpura are antigens of platelets (eg, US Pat. No. 6,929,796; Gorski et al., (E.), Autoimmunity, 2001, Kluwer Academic Publishers, Norwell, MA; Radbruch and Lipsky, PE (ed.) Current Concepts in Autoimmunity and Chronic Inflammation (Curr. Top. Microbiol.

Autoimmunity plays a role in more than 80 different diseases, including type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, bark disease, and thyroid disease. There is a lack of valid quantitative estimates of morbidity for most autoimmune diseases. Most recent studies conducted in the late 1990s have shown that autoimmune diseases are the third most common and most common serious illness in the United States, affecting more than 8.5 million Americans. It is clear. Current estimates of the prevalence of this disease are 5-8% of the US population. Most autoimmune diseases have an imbalanced effect on women. Women are 2.7 times more likely to develop autoimmune diseases than men. Women are more susceptible to autoimmune diseases, and men appear to have higher levels of natural killer cell activity than women (Jacobsen et al., Clinical Immunology and Immunopathology, 84: 223-243, 1997).

Autoimmune diseases occur when the immune system mistakes self-organization for non-self and carries out inappropriate attacks. The body, such as the digestive system (Crohn's disease) and the brain (multiple sclerosis), can be affected in various ways from autoimmune diseases. Autoantibodies cause autoimmune diseases by attacking autologous cells or tissues and damaging their function, and autoantibodies cause the actual onset of autoimmune diseases (eg, the appearance of clinical signs and symptoms). ) Previously known to be detectable in patient serum. Therefore, by detecting an autoantibody, it becomes possible to detect an autoimmune disease at an early stage, recognize its existence, or recognize its risk of developing it. Based on these findings, various autoantibodies against self-antigens have been discovered, and autoantibodies against self-antigens have been measured in clinical trials (eg, US Pat. Nos. 6,919,210, 6,596,501, 7,012,134, No. 6,919,078), but other autoimmune diagnostic methods include detection of related antigens (eg, US Pat. No. 4,659,659) or immunological reactivity (eg, US Pat. Nos. 4,614,722 and 5,147,785, No. 6,919,078). It can also include 4,420,558, 5,298,396, 5,162,990, 4,420,461, 4,595,654, 5,846,758, 6,660,487).

In certain embodiments, the compositions of the invention are particularly elderly and / or immunosuppressed subjects, such as subjects undergoing renal dialysis, subjects undergoing chemotherapy and / or radiation therapy. It can be applied to the treatment of graft recipients and the like. Since such individuals generally exhibit only a weak immune response to the vaccine, the compositions of the invention can be used to enhance the immune response achieved in such subjects.

In other embodiments, one or more antigens used in the compositions of the invention are caused by a bacterial infection (eg, pneumococcus) for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD). Contains antigens associated with respiratory illnesses such as exacerbating illnesses. COPD is physiologically defined by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Am J Respir Crit Care Med. 1995 Nov; 152 (Am J Respir Crit Care Med. 1995 Nov; 152) 5 Pt 2): S77-121). Bacterial (eg, Streptococcus pneumoniae) infections often exacerbate COPD (Clin Microbiol Rev. 2001 Apr; 14 (2): 336-63).

<u style="single">TLR</u> As described herein, certain embodiments of the invention are intended to be vaccine compositions and immunoadjuvant compositions (such as pharmaceutical compositions) that include one or more Toll-like receptor agonists (TLR agonists). doing. Toll-like receptors (TLRs) are cells of the congenital immune system that confer early recognition of various conserved microbial molecular structures, such as those present in or on various infectious pathogens. Surface transmembrane receptors include (eg, Armant et al., 2002 Genome Biol. 3 (8): reviews 3011.1-3011.6; Fearon et al., 1996 Science 272: 50; Medzhitov et al., 1997 Curr. Opin. Immunol. 9: 4 Luster 2002 Curr. Opin. Immunol. 14: 129; Lien et al., 2003 Nat. Immunol. 4:1162; Medzhitov, 2001 Nat. Rev. Immunol. 1:135; Takeda et al., 2003 Ann Rev Immunol. 21:335; Takeda et al., 2005 Int. Immunol. 17: 1; Kaisho et al., 2004 Microbes Infect. 6: 1388; Datta et al., 2003 J. Immunol. 170: 4102).

Induction of TLR-mediated signaling that enhances the initiation of the immune response by the innate immune system is thought to be caused by TLR agonists that bind to cell surface TLRs. For example, lipopolysaccharide (LPS) can be a TLR2- or TLR4-mediated TLR agonist (Tsan et al., 2004 J. Leuk. Biol. 76: 514; Tsan et al., 2004 Am. J. Physiol. Cell Phsiol. 286: C739; Lin et al., 2005 Shock 24: 206); Poly (inosin-cytidine) (polyl: C) can be a TLR3 mediated TLR agonist (Salem et al., 2006 Vaccine 24: 5119); CpG Sequences (oligodeoxynucleotides containing unmethylated cytidine-guanosine) or "CpG" dinucleotide motifs, such as CpG 7909 (Cooper et al., 2005 AIDS 19: 1473; CpG 10101, Bayes et al., Methods Find Exp Clin Pharmacol 27:193 Vollmer et al., Expert Opinion on Biological Therapy 5: 673; Vollmer et al., 2004 Antimicrob. Agents Chemother. 48: 2314; Deng et al., 2004 J. Immunol. 173: 5148) may be TLR9-mediated TLR agonists. Done (Andaloussi et al., 2006 Glia 54: 526; Chen et al., 2006 J. Immunol. 177: 2373); Peptidoglycan can be a TLR2 and / or TLR6 agonist (Soboll et al., 2006 Biol. Reprod. 75: 131; Nakao et al., 2005 J. Immunol. 174: 1566); 3M003 (4-amino). -2- (ethoxymethyl) -α, α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo [4,5-c] quinoline-1-ethanol hydrate (molecular weight 318 Da; related compound 3M001 And 3M Pharmaceuticals (St. Paul, MN), which is also the source of 3M002; Gorden et al., 2005 J. Immunol. 174: 1259), TLR7 agonist (Johansen 2005 Clin. Exp. Allerg. 35: 1591) and / Or it can be a TLR8 agonist (Johansen 2005); Flagerin is a TLR5 agonist (Feuillet et al., 2006 Proc. Nat. Acad. Sci.). USA 103: 12487); hepatitis C antigens can also act as TLR agonists via TLR7 and / or TLR9 (Lee et al., 2006 Proc. Nat. Acad. Sci. USA 103: 1828). Horsmans et al., 2005 Hepatol. 42: 724). Other TLR agonists are known (eg, Schirmbeck et al., 2003 J. Immunol. 171: 5198) and they may be used according to the particular embodiments described herein.

For example, as a background technique (see, eg, US Pat. No. 6,544,518), immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides (CpG) are adjuvants when administered by both systemic and mucosal pathways. Known as (WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998. 160 (2): 870-876; McCluskie and Davis, J. et al. Immunol., 1998, 161 (9): 4461-6). CpG is an abbreviation for cytosine-guanosine dinucleotide motif present in DNA. The major role of CG motifs in immune stimulation was elucidated by Krieg, Nature 374, p546 1995. Detailed analysis shows that CG motifs need to be in a particular sequence situation, and that such sequences are common in bacterial DNA but rare in vertebrate DNA. know. Often, the immunostimulatory sequences are purines, purines, C, G, pyrimidines, pyrimidines, where the dinucleotide CG motif is unmethylated, but other unmethylated CpG sequences are immunostimulatory. These are known to be used in certain embodiments of the invention. When formulating CpG into a vaccine, in free solution with free antigen (WO 96/02555; McCluskie and Davis, supra), or covalently conjugated to an antigen (PCT Publication No. WO 98/16247), or formulated with a carrier such as aluminum hydroxide (eg, Daivis et al., Supra, Brazolot-Millan). Et al., Proc. Natl. Acad. Sci., USA, 1998, 95 (26), 15553-8), can be administered.

Preferred oligonucleotides for use in the adjuvants or vaccines of the invention preferably include at least three, more preferably at least six, or more nucleotide-separated two or more dinucleotide CpG motifs. The oligonucleotides of the present invention are typically deoxynucleotides. In a preferred embodiment, the internucleotide bond in the oligonucleotide is a phosphorodithioate, or more preferably a phosphorothioate bond, but phosphodiester and other internucleotide bonds are also included within the scope of the invention, as such. , Oligonucleotides containing mixed nucleotide linkages and the like. Methods for making phosphorothioate oligonucleotides or phosphorodithioates are described in US Pat. Nos. 5,666,153, 5,278,302 and WO 95/26204.

Examples of preferred oligonucleotides have the sequences disclosed in the following publications, and for certain embodiments disclosed herein, this sequence preferably comprises phosphorothioate-modified internucleotide linkages: CPG 7909: Cooper et al., "CPG 7909 adjuvant improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults." AIDS, September 23, 2005; 19 (14): 1473-9.

CpG 10101: Bayes et al., "Gateways to clinical trials." Methods Find. Exp. Clin. Pharmacol. April 2005; 27 (3): 193-219.

Vollmer J., "Progress in drug development of immunostimula-tory CpG oligodeoxynucleotide ligands for TLR9." Expert Opinion on Biological Therapy. May 2005; 5 (5): 673-682.

Other CpG oligonucleotides include variants of the above sequences that differ from the preferred sequences described in the above references in that they have substitutions, insertions, deletions and / or additions of nucleotide sequences that are not significant. .. The CpG oligonucleotides used in certain embodiments of the invention can be synthesized by any method known in the art (eg, EP 468520). Preferably, such oligonucleotides can be synthesized using an automated synthesizer. Oligonucleotides are typically deoxynucleotides. In a preferred embodiment, the internucleotide bond in the oligonucleotide is a phosphorodithioate, or more preferably a phosphorothioate bond, but phosphodiesters are also included within the scope of the embodiments intended in the present invention. Oligonucleotides containing various internucleotide bonds, such as phosphorothioate-phosphodiester mixed forms, are also contemplated. In addition, other internucleotide bonds that stabilize the oligonucleotide may be used.

<u style="single">Coadjuvant</u> In addition to GLA, the particular embodiments described herein include at least one core juban. including including bets, vaccines and immunological adjuvant compositions (pharmaceutical composition, etc.). The co-adjuvant is a component of the above composition and means a component having an adjuvant activity other than GLA. Co-adjuvants with such adjuvant activity are administered to subjects such as humans (eg, human patients), non-human primates, mammals, or other higher eukaryotes with a recognized immune system. Includes compositions that can modify the potency and / or persistence of an immune response (ie, increase or decrease in a statistically significant manner, and, in certain preferred embodiments, increase or increase). (For example, Powell and Newman, "Vaccine design-The Subunit and Adjuvant Approach", 1995, Plenum Press, See New York). In certain embodiments disclosed herein, the GLA and the desired antigen, and optionally one or more co-adjuvants, are administered at the same time as the GLA, or temporally and / or spatially (eg, another dissection). The immune response to a desired antigen that can be administered in a scholarly manner) can be modified, eg, induced or enhanced, as described above, but certain embodiments of the invention are so limited. Unintentionally, the GLA in a composition that does not contain the designated antigen but may contain one or more of TLR agonists, coadjuvants, imidazoquinolin immune response modifiers, and dual stem loop immunomodulators (dSLIM). Administration is also intended.

Thus, as mentioned above, co-adjuvants include compositions other than GLA that have an adjuvant effect, such as saponins and saponin mimetics, such as QS21 and QS21 mimetics (eg, US Pat. No. 5,057,540). EP 0 362 279 B1; see WO 95/17210), alum, plant alkaliids such as tomatin, surfactants such as (but not limited to), saponins, polysorbate 80, Span 85 and stearyl tyrosine, One or more cytokines (eg, GM-CSF, IL-2, IL-7, IL-12, TNF-α, IFN-γ), imidazoquinolin immune response modifiers, and dual stem loop immunomodifiers (dSLIM) , For example, Weeratna et al., 2005 Vaccine 23: 5263).

Surfactants containing saponins are described, for example, in US Pat. No. 6,544,518; Lacaille-Dubois, M and Wagner H. et al. (1996 Phytomedicine 2: 363-386), US Pat. No. 5,057,540, Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55, and EP 0 362 279 B1. A particulate structure called the immunostimulatory complex (ISCOMS), which contains a fraction of Quil A (saponin), is hemolytic and is used in the production of vaccines (Morein, B., EP 0 109 942 B1). .. These structures have been reported to have adjuvant activity (EP 0 109 942 B1; WO 96/11711). Hemolytic saponins QS21 and QS17 (Quil A's HPLC purified fractions) have been described as potent systemic adjuvants and methods of their preparation are disclosed in US Pat. No. 5,057,540 and EP 0 362 279 B1. .. These references also describe the use of QS7 (a non-hemolytic fraction of Quil-A), which acts as a potent adjuvant for systemic vaccines. The use of QS21 was described by Kensil et al. (1991. J. It is also described in Immunology 146: 431-437). Combinations of QS21 with polysorbate or cyclodextrin are also known (WO 99/10008). Particulate adjuvant systems containing Quil A fractions such as QS21 and QS17 are described in WO 96/33739 and WO 96/11711. Other saponins that have been used in systemic vaccination studies include those derived from other plant species such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10 (9): 572). -577, 1992).

Essin is another saponin-related surfactant for use in the adjuvant compositions of embodiments disclosed herein. Essin is listed in the Merck Index as a mixture of saponins in the seeds of horse chestnut tree (Aesculus hippocastanum) (12th edition: entry 3737). Its isolation is by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and those with ion exchange resins (Erbring et al., US Pat. No. 3,238,190) are described. A fraction of escin (known as aescin) has been purified and has been shown to be biologically active (Yoshikawa M et al., (Chem Pharm Bull (Tokyo) August 1996; 44). 8): 1454-1464)). Digitonin is another surfactant, which is also listed in the Merck Index as a saponin (12th edition: entry 3204), which is derived from the seeds of digitalis purpurea, Gisvold et al., Purified according to the methods described by J. Am. Pharm. Assoc., 1934, 23, 664; and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

Other co-adjuvants used in accordance with the particular embodiments disclosed herein include block copolymers or biodegradable polymers, which fall into the class of polymer compounds well known to those of skill in the art. As an example of a block copolymer or biodegradable polymer that can be contained in a GLA vaccine composition or GLA immunoadjuvant, Pluronic® L121 (BASF Corp., Mount Olive, NJ; eg Yeh et al., 1996 Pharm. Res 13: 1693; US Pat. No. 5,565,209), CRL1005 (eg, Triozzi et al., 1997 Clin Canc. Res. 3: 2355), Poly (lactic acid-co-glycolic acid) (PLGA), Polylactic acid (PLA), Poly -(D, L-lactide-co-glycolide) (PLG), and polymer: C (see, eg, Powell and Newman, "Vaccine design-The Subunit and Adjuvant Approach", 1995, Plenum Press, New York. ).

Certain embodiments are intended for oil-containing GLA vaccines and GLA immunoadjuvants, which can contribute to co-adjuvant activity in certain such embodiments, and in other such embodiments, to this. In addition, or in place of this, pharmaceutically acceptable carriers or excipients are provided. A large number of suitable oils are known and can be selected for inclusion in vaccine compositions and immunoadjuvant compositions according to the disclosure of the present invention. Examples of such oils include, but are not limited to, squalene, squalene, mineral oil, olive oil, cholesterol, and mannide monooleate.

Immune response modifiers, such as the imidazole quinoline immune response modifier, are also known in the art and may be included as co-adjuvants in certain embodiments disclosed herein. Non-limiting examples of certain preferred imiquimodinoline immune response modifiers include ricikimod (R848), imiquimod and gardiquimod (Hemmi et al., 2002 Nat. Immunol. 3:196; Gibson et al., 2002 Cell. Immunol. 218: 74; Gorden et al., 2005 J. Immunol. 174: 1259); These and other imiquimodinoline immune response modifiers also have TLR agonist activity as described herein under appropriate conditions. there is a possibility. Another immune response modifier is a nucleic acid-based double-stem-loop immunomodifier (dSLIM). Specific examples of dSLIM intended for use in the particular embodiments disclosed herein are Schmidt et al., 2006 Allergy 61:56; Weihrauch et al., 2005 Clin Cancer Res. 11 (16): 599-6001; Modern Biopharmaceuticals, J. Knablein (supervised). John Wiley &amp; Sons, on December 6, 2005 (see pages 183-200 for dSLIM), and Mologen AG (Berlin,). It can be found from FRG: [Search online at http://www.mologen.com/English/04.20-dSLIM.shtml on 8/18/06]).

As already mentioned, one type of co-adjuvant used with GLA described herein may be an aluminum co-adjuvant, commonly referred to as "alum". The alum co-adjuvant is based on: aluminum oxyhydroxide; aluminum hydroxyphosphate; or various suitable salts. Vaccines that use alum co-adjuvants include tetanus strains, HPV, hepatitis A, inactivated poliovirus, and vaccines for other antigens described herein. The alum co-adjuvant is advantageous because it has excellent safety records, enhances antibody response, stabilizes antigens, and has relatively simple large scale production (Edelman 2002 Mol. Biotechnol. 21: 129-). 148; Edelman, R. 1980 Rev. Infect. Dis. 2: 370-383).

Other co-adjuvants that can be combined with GLA for effective immune stimulation include saponins and saponin mimetics, which are QS21 and structurally related compounds that have similar effects. , Includes what are referred to herein as QS21 mimetics. QS21 is recognized as the preferred co-adjuvant. QS21 can contain an HPLC purified non-toxic fraction obtained from the bark of Quillaja Saponaria Molina. The manufacture of QS21 is described in US Pat. No. 5,057,540 (see also US Pat. Nos. 6,936,255, 7,029,678 and 6,932,972).

GLA may also, in certain embodiments, be combined with an "immunostimulatory complex" known as ISCOMS (eg, US Pat. Nos. 6,869,607, 6,846,489, 6,027,732, 4,981,684). Such are, for example, ISCOMATRIX® derived from saponin marketed by Iscotec (Stockholm, Sweden) and CSL Ltd. (Parkville, Victoria, Australia).

<u style="single">Recombinant expression construct</u> According to the specific embodiments disclosed in the present invention, the GLA vaccine composition may comprise at least one recombinant expression construct comprising a promoter operably linked to a nucleic acid sequence encoding an antigen. In another particular embodiment, the recombinant expression construct is present in a viral vector such as an adenovirus, adeno-related virus, herpesvirus, lentivirus, poxvirus, or retroviral vector. Compositions and methods for making and using such expression constructs and vectors are intended for the expression of the polypeptide antigens described herein, eg, Ausubel et al. (E.), Current Protocols in Molecular Biology,. 2006 John Wiley &amp; Sons, Well known in the field according to NY. Non-limiting examples of recombinant expression constructs generally include, for example, US Pat. Nos. 6,844,192; 7,037,712; 7,052,904; 7,001,770; 6,106,824; 5,693,531; 6,613,892; 6,875,610; No. 7,067,310; No. 6,218,186; No. 6,783,981; No. 7,052,904; No. 6,783,981; No. 6,734,172; No. 6,713,068; No. 5,795,577; No. 6,770,445 and other documents. For use in the particular embodiments disclosed herein, it can be adapted to the expression of the polypeptide antigens described herein.

<u style="single">Immune response</u> Thus, the present invention modifies an immune response in a host capable of eliciting an immune response (ie, statistically significant compared to suitable controls such as those familiar to those skilled in the art). Compositions for (increase or decrease in style) are provided. As is known to those skilled in the art, the immune response may be any active modification of the host's immune status, including one or more that participates in the maintenance and / or regulation of the host's immune status. It can include any modification of the structure or function of a tissue, organ, cell or molecule of. Typically, the immune response can be detected by any of a variety of well-known parameters, such as, but not limited to, in vivo or in. In vitro measurements include: soluble immunoglobulins or antibodies; soluble mediators such as cytokines, lymphocaines, chemokines, hormones, growth factors, and other soluble small peptides, carbohydrates, nucleotides and / or lipid mediators; Changes in the activation state of cells as determined by changes in the functional or structural properties of cells of the immune system, such as cell proliferation, changes in motility, specific gene expression or specializations such as cytolytic behavior. Induction of activity; differentiation of cells by cells of the immune system, eg, alteration of the surface antigen expression profile or initiation of apoptosis (programmed cell death); or any other criterion capable of detecting the presence of an immune response. ..

The immune response can often be viewed, for example, at the molecular and cellular level as a distinction between self-structuring and non-self-structuring by cells and tissues of the host's immune system, but thus limits the invention. Should not be. For example, an immune response can also include changes in the immune system state caused by immune recognition of its own molecules, cells or tissues, which leads to numerous normal states such as the typical regulation of immune system components. It may be associated and may be present in pathological conditions such as the inappropriate autoimmune response found in autoimmune and degenerative diseases. As another example, in addition to induction by upregulation of specific immune system activity (eg, antibody and / or cytokine production, or activation of cell-mediated immunity), the immune response is a detectable immune suppression, attenuation, etc. Or other down-regulation, which may be the result of the antigen of choice, the route of antigen administration, the induction of specific tolerance, or other factors.

Measurement of the induction of an immune response by the vaccine of the present invention can be confirmed by any of a number of well-known immunological assays that are readily recognized by those of skill in the art. Such assays include, but are not limited to, in vivo or in vitro measurements of: soluble antibodies; soluble mediators such as cytokines, lymphocaines, chemokines, hormones, growth factors, etc., as well as , Other soluble small peptides, carbohydrates, nucleotides and / or lipid mediators; changes in the activation state of cells as determined by changes in the functional or structural properties of cells of the immune system, such as cell proliferation, motility. Induction of specialized activities such as sexual changes, specific gene expression or cell lysis behavior; cell differentiation by cells of the immune system, eg, alteration of surface antigen expression profile or initiation of apoptosis (programmed cell death). Methods for performing these and similar assays are well known, for example Lefkovits (Immunology Methods Manual: See also The Comprehensive Sourcebook of Techniques, 1998; Current Protocols in Immunology; Also, for example, Weir, Handbook of Experimental Immunology, 1986 Blackwell Scientific, Boston, MA; Michel and Shigii (eds.) Selected Methods in Cellular Immunology, 1979 Freeman It can be found in Publishing, San Francisco, CA; Green and Reed, 1998 Science 281: 1309, as well as the references cited therein).

Detection of the proliferation of antigen-reactive T cells can be achieved by a variety of known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis, and antigen-presenting cells pulsed with a stimulus (eg, a particular desired antigen or control antigen) to which a candidate antigen-reactive T cell is exposed. ) Can be controlled to determine antigen specificity. T cells stimulated to proliferate exhibit an increased rate of DNA synthesis. A typical method for measuring the rate of DNA synthesis is, for example, by pulse labeling a T cell culture with thymidine tritylated, a nucleoside precursor that integrates into newly synthesized DNA. A liquid scintillation spectrophotometer can be used to measure the amount of incorporated thymidine tritium. Another way to detect T cell proliferation is increased interleukin-2 (IL-2) production, Ca²⁺Includes measuring flux, or dye uptake, such as 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl-tetrazolium. Alternatively, the synthesis of lymphokines (eg, interferon gamma) may be measured, or the relative number of T cells capable of responding to a particular antigen may be quantified.

Detection of antigen-specific antibody production is performed using in vitro methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis or solid phase immunoblotting (such as Western blotting). This can be achieved by assaying samples from hosts treated with the vaccines of the invention (eg, immunoglobulin-containing samples such as serum, plasma or blood). In a preferred embodiment, the ELISA assay may further include antigen capture and immobilization of the target antigen with, for example, an antigen-specific solid phase monoclonal antibody to increase the sensitivity of the assay. The production of soluble mediators (eg, cytokines, chemokines, lymphokines, prostaglandins, etc.) can also be produced, for example, by readily available commercially available products (eg, Sigma, St. Louis, MO; and R &amp; D Systems 2006 Catalog, R &amp; It can be easily measured by enzyme-linked immunosorbent assay (ELISA) using the methods, equipment and reagents of D Systems, Minneapolis, MN).

Many other immunological parameters may be monitored using conventional assays well known in the art. Such assays include, for example, antibody-dependent cellular cytotoxicity (ADCC) assays of various peripheral blood or lymphoid mononuclear cell subpopulations using a well-established marker antigen system, secondary in vitro antibodies. Responses, cellular cytotoxicity assays, immunohistochemistry or other related assays can be mentioned. These and other assays can be found, for example, in Rose et al. (Ed.), Manual of Clinical Laboratory Immunology, 5th Edition, 1997 American Society of Microbiology (Washington, DC).

Therefore, the vaccine and adjuvant compositions provided herein are T in the host._HType 1 T lymphocyte response, T_HInducing or enhancing at least one immune response selected from type 2 T lymphocyte response, cytotoxic T lymphocyte (CTL) response, antibody response, cytokine response, lymphokine response, chemokine response, and inflammatory response. Is intended to be possible. In certain embodiments, the immune response is the production of one or more cytokines selected from interferon gamma (IFN-γ), tumor necrosis factor α (TNF-α), IL-1, IL-2, IL-3. , IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and production of one or more interleukins selected from IL-23, MIP Production of one or more chemokines selected from -1α, MIP-1β, RANTES, CCL4 and CCL5, as well as memory T cell response, memory B cell response, effector T cell response, cytotoxic T cell response, and effector It can include at least one of the lymphocyte responses selected from the B cell responses. For example, see the following references: WO 94/00153; WO 95/17209; WO 96/02555; US 6,692,752; US 7,084,256; US 6,977,073; US 6,749,856; US 6,733,763; US 6,797,276; US 6,752,995; US 6,057,427; US 6,472,515; US 6,309,847; US 6,969,704; US 6,120,769; US 5,993,800; US 5,595,888; Smith et al., 1987 J Biol Chem. 6951; Kriegler et al., 1988 Cell 53:45 53; Beutler et al., 1986 Nature 320: 584; US 6,991,791; US 6,654,462; US 6,375,944.

<u style="single">Pharmaceutical composition</u> Pharmaceutical compositions generally include GLA (Avanti Polar Lipids, Inc., Alabaster, AL; product number 699800) and, as described herein, pharmaceutically acceptable carriers, excipients or In combination with a diluent, an additional component selected from antigens, TLR agonists, co-adjuvants (optionally including cytokines, imidazoquinolin immune response modifiers and / or dSLIM), and / or recombinant expression constructs. It may be included.

Thus, in certain aspects, the invention relates to GLA "monotherapy", where the GLA is formulated in a composition substantially devoid of other antigens, as described herein. It is administered to a subject to stimulate an immune response, eg, a non-specific immune response, for the purpose of treating or preventing a disease or other condition, such as an immune response. In one embodiment, for example, the compositions and methods of the invention use monophosphorylated disaccharides to stimulate an immune response in a subject. In another specific embodiment, the composition and method use the 2-monoacyl form of Lipid A to stimulate an immune response in a subject. In another specific embodiment, the GLA is in the form of a spray, which is optionally provided in the kit.

GLA can preferably be formulated in a stable emulsion. In one specific embodiment, for example, a composition comprising a Lipid A derivative in a stable emulsion substantially devoid of other antigens is provided. In another specific embodiment, a composition comprising a derivative of 3-acylated monophosphoryl lipid A suitable for use in mammals is provided, wherein the amine 2-position has a single acyl chain. , Substantially lacking other antigens.

In other specific embodiments, the pharmaceutical composition comprises both a GLA and an antigen and is further combined with a pharmaceutically acceptable carrier, excipient or diluent as described herein. , TLR agonists, co-adjuvants (eg, cytokines, imidazoquinolin immune response modifiers and / or dSLIM), and / or vaccine compositions that may contain one or more components selected from recombinant expression constructs.

The exemplary carrier is non-toxic to the recipient at the dose and concentration used. For GLA + nucleic acid-based vaccines, or vaccines containing GLA + antigens, typically about 0.1 μg to about 100 mg / kg body weight by intradermal, subcutaneous, intramuscular or intravenous route, or by other routes. Administer.

Preferred doses are from about 1 μg / kg to about 1 mg / kg, particularly preferably from about 5 μg / kg to about 200 μg / kg. It will be apparent to those skilled in the art that the frequency and frequency of administration will vary depending on the response of the host. "Pharmaceutically acceptable carriers" for therapeutic applications are well known in the pharmaceutical field, eg,<u style="single">Remingtons Pharmaceutical Sciences</u>, Mack Publishing Co. (AR Gennaro ed. 1985). For example, sterile saline solution and phosphate buffered saline solution having physiological pH can be used. Preservatives, stabilizers, dyes and even flavors may be included in the pharmaceutical composition. For example, esters of sodium benzoate, sorbic acid and p-hydroxybenzoic acid can be added as preservatives (ibid., 1449). In addition, antioxidants and suspending agents may be used (ibid.).

A "pharmaceutically acceptable salt" is a salt of the compound of the present invention obtained from a combination of the compound of the present invention and an organic or inorganic acid (acid addition salt) or an organic or inorganic base (base addition salt). means. The compositions of the present invention may be used in either form of free base or salt, both forms of which are considered to be within the scope of the invention.

The pharmaceutical composition may be in any form that allows the composition to be administered to the patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, but are not limited to, oral, topical, parenteral (eg, sublingual or oral), sublingual, rectal, vaginal, and intranasal (eg, as a spray). Can be mentioned. As used herein, the term "parenteral" refers to ion migration (eg, US 7,033,598; 7,018,345; 6,970,739), ultrasound (eg, US 4,780,212; 4,767,402; 4,948,587; 5,618,275; 5,656,016; 5,722,397; 6,322,532; 6,018,678), Fever (eg, US 5,885,211; 6,685,699), passive transdermal (eg, US 3,598,122; 3,598,123; 4,286,592; 4,314,557; 4,379,454; 4,568,343; 5,464,387; UK Patent Specification No2232892; US 6,871,477; 6,974,588; 6,676,961) , US 6,908,453; 5,457,041; 5,591,139; 6,033,928) Administration, including subcutaneous injection, intravenous, intramuscular, intrasternal, intraspongeic, intrasheath, intradural, intraurethral injection or infusion. In a specific embodiment, the compositions described herein (such as vaccines and pharmaceutical compositions) are administered intradermally by a technique selected from iontophoresis, microcavitation, ultrasound, or microneedle.

The pharmaceutical composition is formulated so that the active ingredient contained therein becomes bioavailable when the composition is administered to a patient. The composition administered to the patient is in the form of one or more units of administration, for example, one tablet may be in the form of a single dose, and one or more compositions of the invention in the form of an aerosol. A container of goods can hold multiple dosage units.

For oral administration, excipients and / or binders may be present. Examples include sucrose, kaolin, glycerin, starch dextrin, sodium alginate, carboxymethyl cellulose and ethyl cellulose. Colorants and / or flavors may be included. Moreover, you may use a coating shell.

The composition may be in liquid form, eg, an elixir, syrup, solvent, emulsion or suspension. The liquid can be given as two examples for oral administration or for delivery by injection. When intended for oral administration, preferred compositions include one or more sweeteners, preservatives, pigments / colorants and flavor enhancers. If intended for administration by injection, one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be contained.

Liquid pharmaceutical compositions used herein, in any form of solution, suspension or other, may contain one or more of the carriers or excipients listed below: sterile diluents such as. , Injectable water, saline, preferably saline, Ringer's solution, isotonic saline, fixed oils such as squalane, squalane, mineral oil, monooleic acid mannide, cholesterol, and / or as a solvent or suspension medium. Synthetic mono or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium hydrogen sulfite; chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate, and substances for regulating tonicity, such as sodium chloride or dextrose. Parenteral preparations can be encapsulated in glass or plastic ampoules, disposable syringes or multi-dose vials. The pharmaceutical composition for injection is preferably sterile.

In a specific embodiment, the pharmaceutical or vaccine composition of the invention comprises a stable aqueous suspension of less than 0.2 μm, plus phospholipids, fatty acids, surface active agents, surfactants, saponins, fluorodated. Contains lipids and the like.

In another embodiment, the compositions of the invention are formulated for aerosol administration.

It may be desirable to include other ingredients, such as delivery vehicles, in the vaccine or pharmaceutical composition, such as, but not limited to, aluminum salts, water-in-oil emulsions, biodegradable. Examples include oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Other immunostimulatory substances (coadjuvants) used in such vehicles have also been described, including N-acetylmuramil-L-alanine-D-isogramin (MDP), glucan, IL-12, GM-CSF, Examples include γ interferon and IL-12.

Any suitable carrier known to those of skill in the art can be used in the pharmaceutical compositions of the present invention, but the type of carrier may vary depending on the mode of administration and whether sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax, or buffer. In the case of oral administration, any of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate may be used. In addition, biodegradable microspheres (for example, polylactic acid galactide) may be used as a carrier for the pharmaceutical composition of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4,897,268 and 5,075,109. In this regard, the microspheres are preferably larger than about 25 microns.

Pharmaceutical compositions (such as GLA vaccines and GLA immunoadjuvants) also include diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, glucose, etc. Carbohydrates such as glucose or dextrin, chelating agents such as EDTA, glutathione, and other stabilizers and excipients may be included. Neutral buffered saline solution, or saline solution mixed with non-specific serum albumin, is an example of a suitable diluent. Preferably, the composition may be formulated as a lyophilized product using a suitable excipient solution (eg, sucrose) as the diluent.

As mentioned above, in certain embodiments, the invention comprises a composition capable of delivering a nucleic acid molecule encoding a desired antigen. Such compositions include recombinant viral vectors (see, eg, retroviruses (see, eg, WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenoviruses (Berkner,). Biotechniques 6: 616-627, 1988; Li et al., Hum. Gene Ther. 4: 403-409, 1993; Vincent et al., Nat. Genet. 5: 130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994), Poxvirus (see US Pat. No. 4,769,330; US Pat. No. 5,017,487; and WO 89/01973)), recombinant expression construct nucleic acid complexed with polycation molecule Molecules (see WO 93/03709) and nucleic acids associated with lipolomes (Wang et al., Proc. Natl. Acad. Sci. USA 84: 7851, 1987). In certain embodiments, DNA may be bound to a dead or inactivated adenovirus (Curiel et al., Hum. Gene Ther. 3: 147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA. 89: 6094, 1992). Another suitable composition is a DNA-ligand (see Wu et al., J. Biol. Chem. 264: 16985-16987, 1989) and a lipid-DNA combination (Felgner et al., Proc. Natl. Acad. Sci. USA 84). : 7413-7417, see 1989).

In addition to the direct in vivo method, an ex vivo method may be used, in which cells are removed from the host, modified and then introduced into the same or another host animal. It will be clear that any of the compositions described above may be used to introduce the antigen-encoding nucleic acid molecule into histiocytes in ex vivo. Protocols for viral or physical or chemical uptake methods are well known in the art.

Therefore, the present invention is useful for enhancing or inducing an immune response in a host, patient or cell culture. As used herein, the term "patient" refers to any warm-blooded animal, preferably human. The patient may have an infection, cancer such as breast cancer, or an autoimmune disease, or may be normal (ie, no detectable disease and / or infection). A "cell culture" is any preparation that includes immunocompetent cells or isolated cells of the immune system, including, but not limited to, T cells, macrophages, monocytes, B cells and / or dendritic cells. is there. Such cells can be isolated by any of a variety of techniques well known to those of skill in the art (eg, Ficoll-hypaque density centrifugation). Cells can also be isolated (but not always) from patients with cancer and reintroduced into post-treatment patients.

In certain embodiments, the liquid composition intended for either parenteral or oral administration must contain an amount of GLA vaccine composition that provides a suitable dose. Typically, this amount is at least 0.01% by weight of antigen in the composition. If intended for oral administration, this amount can vary between 0.1% and about 70% of the weight of the composition. Preferred oral compositions contain from about 4% to about 50% antigen. Preferred compositions and preparations are prepared such that the parenteral dosing unit comprises 0.01 to 1% by weight of the active composition.

The pharmaceutical composition may be intended for topical administration, where the carrier can appropriately comprise a solvent, emulsion, ointment, or gel substrate. The substrate can include, for example, one or more of the following: diluents such as petrolatum, lanolin, polyethylene glycol, wax, mineral oil, water and alcohol, as well as emulsifiers and stabilizers. A thickener may be contained in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or iontophoresis device. Topical formulations include antigens (eg, GLA-antigen vaccine compositions) or GLA (eg, immunoadjuvant compositions; GLA, Avanti Polar Lipids,) at concentrations of about 0.1 to about 10% w / v (weight / unit volume). It is available from Inc., Alabaster, AL; eg, product number 699800) can be contained.

The composition may be intended for rectal administration, for example, in the form of a suppository that dissolves in the rectum and releases the drug. Compositions for rectal administration may include a fatty substrate as a suitable non-irritating excipient. Examples of such a substrate include, but are not limited to, lanolin, cocoa butter, polyethylene glycol and the like. In the methods of the invention, the vaccine composition / adjuvant can be administered using inserts, beads, timed release formulations, patches, or rapid release formulations.

Kits containing the GLA vaccine compositions and / or GLA immunoadjuvant compositions described herein are also intended in certain embodiments, even if such compositions are provided in one or more containers. Good. In one embodiment, all components of the GLA vaccine composition and / or the GLA immunoadjuvant composition are present together in a single container, but it is not intended that embodiments of the invention are so limited. Instead, two or more containers are also intended, where, for example, the GLA immunoadjuvant composition is separated from the antigenic components and not in contact. As a non-limiting theory, in some cases it may be beneficial to administer only the GLA immunoadjuvant composition, or such administration may be temporal and / or spatial (eg, another anatomical) from the administration of the antigen. Sites) may be beneficial, and also subject to the GLA vaccine composition described herein, which also comprises both the antigen and GLA and optionally the other components described herein. It may be beneficial to administer to the examiner.

The containers of such kit embodiments are any suitable container, vessel, vial, ampoule, tube, cup, box, bottle, flask, jar, dish, single-well or multi-well device well, reservoir, tank. Etc., or other devices, wherein the compositions disclosed herein can be placed, stored, and / or transported and utilized to transfer the contents. Typically, such containers may be made from materials that are suitable for the intended use and whose contents can be easily recovered. Preferred examples of such containers include glass and / or plastic sealed or resealable tubes and ampoules, for example, rubber diaphragms, or others compatible with removal of contents using needles and syringes. Some are equipped with a sealing means. Such containers can be made of, for example, glass, or chemically compatible plastic or resin, which allows efficient recovery of material from the container and / or, for example. It may be manufactured from or coated with a material that protects the material from degradable conditions such as ultraviolet rays or extreme temperatures, or from contamination with unwanted contaminants such as microbial contaminants. May be. The container is preferably sterile or sterile, suspending or dissolving vaccine compositions and / or immunoadjuvant compositions and / or antigens and / or recombinant expression constructs described herein. It consists of materials compatible with any carrier, excipient, solvent, vehicle, etc. that can be used to make it.

In addition, an emulsion system may be used to formulate the composition of the present invention. For example, many single-phase or polyphase emulsion systems have been described. Oil-in-water emulsion adjuvants have been suggested to be useful as adjuvant compositions in their own right (EP 0 399 843B), but combinations of oil-in-water emulsions with other active substances have also been described as adjuvants for vaccines. (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Oil-based emulsion adjuvants other than the above are also described, for example, water-in-oil emulsions (US Pat. No. 5,422,109; EP 0 480 982 B2) and water-in-water emulsions (US Pat. No. 5,424,067; EP 0 480 981). B) and the like. The oily emulsion adjuvant used in the present invention may be either natural or synthetic and may be inorganic or organic. Examples of mineral or organic oils will be readily apparent to those skilled in the art.

In a specific embodiment, the composition of the invention comprises an oil-in-water emulsion, in which GLA is contained in the oil phase. In another embodiment, the composition of the invention comprises an oil-in-water emulsion, wherein the GLA is contained in the oil phase and, as described herein, a co-adjuvant, TLR agonist, etc. There are also additional ingredients of.

The emulsion-based oil phase preferably contains a metabolizable oil so that any oil-in-water composition is suitable for human administration. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "can be converted by metabolism" (Dorland's illustrated Medical Dictionary, WB Saunders Company, 25th edition (1974)). The oil may be any of vegetable oils, fish oils, animal oils or synthetic oils that are non-toxic to the recipient and can be converted by metabolism. Nuts (such as peanut oil), seeds, and grains are common sources of vegetable oil. Synthetic oil is also a part of the present invention, and examples thereof include commercially available oils such as NEOBEE (registered trademark).

For example, squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is abundant and less abundant in shark liver oil. However, it is an unsaturated oil that is also present in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferable oil for use in the present invention. Squalene is a metabolizable oil because it is an intermediate in cholesterol biosynthesis (Merck index, 10th edition, entry no.8619). A particularly preferable oil-based emulsion is an oil-in-water emulsion, specifically, an emulsion of squalene in water. In addition, the most preferred oily emulsion adjuvants of the present invention include antioxidants, which are preferably oil α-tocopherols (vitamin E, EP 0 382 271 B1). WO 95/17210 and WO 99/11241 are based on squalene, α-tocopherol, and TWEEN® 80 and optionally with the immunostimulators QS21 and / or 3D-MPL (which has already been mentioned). It discloses an emulsion adjuvant that is formulated together. WO 99/12565 discloses improvements to these squalene emulsions, including the addition of sterols to the oil phase. In addition to this, tricapriline (C) to stabilize the emulsion₂₇H₅₀0₆) May be added to the oil phase (WO 98/56414).

The size of the oil droplets found in a stable oil-in-water emulsion is preferably less than 1 micron and may be in the range of substantially 30-600 nm, preferably substantially about 30-500 in diameter. It is nm, most preferably the diameter is substantially 150-500 nm, specifically about 150 nm, which is measured by photon correlation spectroscopy. In this regard, 80% of the oil droplets should be in the preferred range, more preferably more than 90% of the oil droplets, most preferably more than 95% of the oil droplets within the size range specified above. .. The amount of ingredients present in the oily emulsions of the present invention is typically 2-10% in oils such as squalene; and 2-10% in the presence of α-tocopherols; polyoxyethylene sorbitan monooleate. Surfactants such as are in the range of 0.3 to 3%. Preferably, the oil: α-tocopherol ratio is less than or equal to 1 as it provides a more stable emulsion. Span 85 may also be present at a level of about 1%. In some cases, it may be advantageous for the vaccines of the invention to further contain stabilizers.

Methods of preparing oil-in-water emulsions are well known to those of skill in the art. Generally, this method involves mixing the oil phase with a surfactant such as PBS / TWEEN80® solution and then homogenizing with a homogenizer. For example, a method involving passing the mixture through the needle once, twice or more may be considered suitable for homogenizing a small amount of liquid. Similarly, emulsification steps with a microfluidizer (M110S microfluidix machine, 6 bar maximum pressure input (output pressure about 850 bar) for 2 minutes, up to 50 passes) to produce smaller or larger emulsions. It can also be adapted. This conformance can be achieved by routine experiments involving measuring the resulting emulsion until a preparation containing oil droplets of the required diameter is achieved.

The examples described below are provided by way of example and are not for limitation.

<u style="single">Example</u>

<p num="0173"><u style="single">GLA aqueous formulation</u> This example describes the preparation of a GLA-containing adjuvant aqueous formulation. Aqueous formulation of GLA (GLA-AF) is water for injection (WFI), GLA (Avanti Polar Lipids, Inc., Alabaster, AL; product number 699800), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). This formulation was prepared by adding a solution of ethanol and POPC to a pre-measured amount of GLA. The wet GLA was sonicated for 10 minutes to disperse the GLA as much as possible. The GLA was then dried under nitrogen gas. Dried GLA and POPC were reconstituted with WFI to the proper amount. The solution was sonicated at 60 ° C. for 15-30 minutes until all GLA and POPC were dissolved. For long-term storage, the GLA-AF formulation must be lyophilized. The lyophilization step consisted of adding glycerol to the solution to a total volume of 2%. The solution was then placed in a vial in an amount of 1-10 mL. The vials were subjected to a lyophilization step, which consisted of freezing the solution and then placing it under reduced pressure to remove frozen water by sublimation.</p>

<p num="0174"><u style="single">GLA HPLC analysis</u> This example describes an HPLC analysis of a GLA-containing adjuvant aqueous formulation. After manufacturing the formulation (see Example 1 above), several release and stability tests were performed to confirm the quality and reproducibility of the formulation. All formulations were tested for release and long-term stability using high performance liquid chromatography (HPLC), dynamic light scattering (DLS) and visual inspection. HPLC chromatograms were collected using the Agilent 1100 system and the ESA Corona CAD detector. This method was performed on a Waters Atlantis C18 column with a methanol / chloroform gradient. Injectables include GLA (Avanti Polar Lipids, Inc., Alabaster, AL; product number 699800, GLA-AF) or MPL® (GSK Biologicals, Rixensart, Belgium, MPL-AF), 2.5 μg each, and acceptable. It contained 0.27 μg of synthetic phosphocholine (POPC) used as a solubilizer.</p><p num="0175"> Figure 1 shows PLC data showing the number and amount of contaminants in MPL-AF and GLA-AF.</p><p num="0176"> The HPLC profile showed that GLA-AF was substantially purer than MPL-AF. That is, GLA-AF had fewer pollutant peaks than MPL-AF adjuvant preparation. A purer starting material is extremely beneficial to researchers, as the resulting biological response is from a single key ingredient used in GLA formulations.</p>

<p num="0177"><u style="single">GLA oily preparation</u> This example describes the preparation of a 1 ml GLA-containing adjuvant oil formulation. Using 0.5 mg of D, L-α-tocopherol as an antioxidant, glycerol (22.7 mg) in 25 mmol ammonia ammonia buffer (pH = 5.1), phosphothidylcholine or lecithin (7.64 mg), Pluronic ( GLA (100 μg; Avanti Polar Lipids, Inc., Alabaster, AL) in Squalene (34.3 mg) with F-68 (BASF Corp., Mount Olive, NJ) or a similar block copolymer (0.364 mg). Product number 699800) was emulsified. The mixture was treated under high pressure until an emulsion was formed that did not separate and had an average particle size of less than 180 nm. The emulsion was then sterile filtered into single-dose glass vials and capped for long-term storage. This preparation can be used for at least 3 years when stored at 2-8 ° C.</p>

<p num="0178"><u style="single">GLA stimulation of mouse macrophages and dendritic cells</u> This example describes an in vitro model that reveals the adjuvant effect of GLA. Standard tissue culture methods and reagents were used. Macrophage cell lines of mouse J774 and RAW 267.4 (American Type Culture Collection, Manassas, VA) were maintained according to supplier recommendations and cultured as adherent cell monolayers in a multi-weldish. Dendritic cells were obtained from bone marrow progenitor cells according to a protocol by Xiong et al. (J. Biol. Chem 2004, 279, pp10776-83). Diluting the aqueous adjuvant preparation in cell culture medium (DMEM containing 10% fetal bovine serum) gives synthetic GLA (Avanti Polar Lipids, Inc., Alabaster, AL; product number 699800) at various adjuvant concentrations. 5% CO₂The cells were maintained at 37 ° C. for 24 hours in a humidified atmosphere containing the cells, and then the cell-free culture supernatant was collected. Using a specific sandwich ELISA assay kit (eBiosciences, San Diego, CA for cytokines, and R &amp; D Systems, Minneapolis, MN for chemokines) and following product instructions, such as IL-12, IL-6, and TNF. The supernatant was assayed for soluble mouse cytokines, as well as chemokines such as RANTES.</p><p num="0179"> GLA-AF is a dose-dependent immune response characterized by the secretion of cytokines such as IL-12p40, IL-6, and TNF, and chemokines such as RANTES in mouse macrophage cell lines and primary mouse DCs. Was induced.</p>

<p num="0180"><u style="single">GLA stimulation of human macrophages and dendritic cells</u> This example describes an in vitro model that reveals the adjuvant effect of GLA. Standard tissue culture methods and reagents were used.</p><p num="0181"> Cells from the human Mono Mac 6 macrophage cell line (American Type Culture Collection, Manassas, VA) were maintained according to supplier recommendations and cultured as adherent cell monolayers in multiwell plates. Dendritic cells were obtained from peripheral blood mononuclear cells (PBMCs) according to standard protocols. Synthetic GLA (Avanti Polar Lipids, Inc.) at various adjuvant concentrations by diluting the aqueous adjuvant preparation in cell culture medium (DMEM with 10% fetal bovine serum for MonoMac6 and 10% human serum for DC) , Alabaster, AL; product number 699800) or natural product MPL® (GSK Biologicals, Rixensart, Belgium), 5% CO₂The cells were maintained at 37 ° C. for 24 hours in a humidified atmosphere containing the cells, and then the cell-free culture supernatant was collected. Using a specific sandwich ELISA assay kit (eBiosciences, San Diego, CA for cytokines, and Invitrogen, Carlsbad, CA for chemokines), according to product instructions, IL-1β, IL-23, and IL- The supernatant was assayed for soluble human cytokines such as 6 and chemokines such as IP-10, RANTES and MIP-1β.</p><p num="0182"> Figure 2 shows ELISA data demonstrating the levels of cytokines and chemokines expressed by human macrophages (panels a-e) of the Mono Mac6 cell line and monocyte-derived DCs (panels f-h) in response to GLA stimulation. Is shown.</p><p num="0183"> GLA-AF is a cytokine such as IL-1β, IL-6, IL-23 in the human macrophage cell line Mono Mac6 (Fig. 2, panels a to e), and primary D (Fig. 2, panels f to h). , And induced a dose-dependent immune response characterized by the secretion of chemokines such as RANTES, IP-10, MIP-1β. GLA-AF was active at concentrations 5 to 500-fold lower than MPL-AF for all cytokines and chemokines tested.</p>

<p num="0184"><u style="single">GLA stimulation of human blood cells</u> This example describes an in vitro model that reveals the adjuvant effect of GLA. Standard tissue culture methods and reagents were used.</p><p num="0185"> Synthetic GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800) at various adjuvant concentrations obtained by diluting aqueous adjuvant preparations in cell culture medium (DMEM containing 10% fetal bovine serum). Alternatively, whole human blood cells were cultured with any of the natural products MPL® (GSK Biologicals, Rixensart, Belgium). 5% CO₂Blood cells were maintained at 37 ° C. for 16 hours in a humidified atmosphere containing the cells, and then the cell-free culture supernatant was collected. The supernatant was assayed for the soluble human cytokine IL-1β using a specific sandwich ELISA assay kit (eBiosciences, San Diego, CA) according to the product instructions.</p><p num="0186"> GLA-AF induced a dose-dependent immune response characterized by the secretion of IL-1β cytokines in whole human blood cells. In this assay, 92 nM GLA was comparable in efficacy to 57,000 nM MPL-AF.</p>

<p num="0187"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines against influenza. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0188"> Human doses of 1/25 (20 μl) and 1/250 (2 μl) Fluzone vaccines (Sanofi-Aventis, Swiftwater, PA) alone or (i) according to the procedure used in Example 1 above, GLA (Avanti Polar) Lipids, Inc., Alabaster, AL; Product No. 699800; Aqueous emulsion (GLA-AF) containing 20 μg per animal for each immunization, or (ii) GLA (ii) according to the procedure used in Example 3 above. Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; Mice (3 per group) using the above vaccine formulated in a stable emulsion (GLA-SE) containing 20 μg per animal for each immunization. Balb / c mice) were immunized twice every 3 weeks. Serum was collected by collecting blood from mice one week after each immunization and examined by ELISA according to the published method (ibid.) For Fluzone-specific total IgG antibody. Serum levels of virus-neutralizing antibodies were also tested by hemagglutination inhibition assay (HAI) according to published methods.</p><p num="0189"> Figure 3 shows one week after each immunization with two different amounts of Fluzone vaccine formulated with GLA-AF or GLA-SE (ie, panel A on day 7; panel B on day 7; 28th) shows the ELISA data showing the level of anti-Fluzone antibody production induced in mice by comparison with Fluzone alone. Shows the reciprocal of endpoint titers and SEM for each group / time point. Panel C in Figure 3 shows mouse-induced virus-neutralizing antibody production one week after the second immunization with two different doses of Fluzone vaccine formulated with GLA-AF or GLA-SE. HAI data that clearly indicate the level of is shown by comparison with Fluzone alone. Shows the reciprocal of endpoint titers and SEM for each group / time point.</p><p num="0190"> Total IgG and neutralizing antibody titers in response to Fluzone vaccination were enhanced by the addition of GLA to either aqueous or stable oily formulations. The effect of GLA as an adjuvant is more pronounced with the 2 μl dose of Fluzone vaccine, which is equivalent to (GLA-AF) or higher (GLA-SE) antigen-specific humoral response than 20 μl of Fluzone vaccine alone. Was induced. These results indicate that the addition of a GLA-containing formulation as an adjuvant to the Fluzone vaccine can reduce the dose of the Fluzone vaccine and still induce high levels of IgG and neutralizing antibody titers. It is suggested. This is especially important for global epidemics such as avian influenza.</p>

<p num="0191"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Leishmania antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0192"> GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; Each Immunization) using SMT antigen (10 μg per animal per immunization) alone or according to the procedure used in Example 3 above. Mice (3 C57BL / 6 mice per group) 3 times every 3 weeks using the above antigens formulated in a stable emulsion (GLA-SE) containing (20 μg per animal) per animal. I was immunized. Serum was collected by blood sampling from mice 1 week after the third immunization and examined by ELISA according to published methods for SMT antigen-specific IgG1 and IgG2c antibody serum levels.</p><p num="0193"> Figure 4 shows ELISA data showing the level of anti-SMT antibody production induced in mice one week after the third immunization with SMT antigen formulated with SMT antigen alone or with GLA-SE. Is shown. The average reciprocal of the endpoint titer and SEM for each group are shown.</p><p num="0194"> Which isotype of IgG1 or IgG2c antibody predominates is associated with a TH2 or TH1 response, respectively. TH1 responses have been shown to be necessary to protect against leishmania infections. Vaccination with SMT alone induced predominantly SMT-specific IgG1 antibodies. SMT + GLA-SE vaccination induced higher antibody titers, yet the phenotype was reversed to the predominant IgG2c antibody response associated with protection against the disease.</p>

<p num="0195"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Leishmania antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0196"> Various amounts of GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; 40, 20, 5, or 1 μg per animal for each immunization) according to the procedure used in Example 3 above. 2 mice (3 Balb / c mice per group) using the Leish-110f antigen (10 μg per animal per immunization) formulated with a stable emulsion containing (GLA-SE). Immunized 3 times every week. Serum was collected from mice one week after initial immunization and serum levels of Leish-110f-specific IgG1 and IgG2c antibodies were tested by ELISA according to published methods (ibid.).</p><p num="0197"> Figure 5 shows an anti-Leish-110f antibody induced in mice one week after initial immunization with Leish-110f antigen formulated with various amounts (40, 20, 5, or 1 μg) of GLA. Shown are ELISA data showing the level of production by comparison with saline control. The average reciprocal of the endpoint titer and SEM for each group are shown.</p><p num="0198"> Leish-110f-specific IgG1 and IgG2c antibody titers were GLA dose-dependent. The predominance of TH1-related IgG2c antibodies was observed at all concentrations of GLA tested.</p>

<p num="0199"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Leishmania antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0200"> Stable emulsion with saline or with GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; 20 μg per animal for each immunization according to the procedure used in Example 3 above). Using Leish-111f antigen (10 μg per animal per immunization) formulated in (GLA-SE), mice (3 Balb / c mice per group) were administered 3 times every 3 weeks. I was immunized. Two weeks after the last injection, mice were sacrificed and spleens were harvested and analyzed by ELISA according to published methods for T cell-dependent IFN-γ and IL-4 cytokine responses to in vitro antigen stimulation.</p><p num="0201"> Which of the IL-4 or IFN-γ cytokines predominates is associated with a TH2 or TH1 response, respectively. We have demonstrated that a TH1 response is necessary for protection against leishmania infection. All mice responded well to the powerful mitogen ConA. Leish-111f + GLA-SE vaccination induced a Leish-111f antigen-specific cytokine response, but no such response was observed in the saline control group. Compared to ConA, Leish-111f + GLA-SE vaccination induced much higher IFN-γ cytokines, TH1: TH2 ratios, or phenotypes associated with protection against the disease than IL-4.</p>

<p num="0202"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Leishmania antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0203"> GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; 40 per animal for each immunization, using saline or according to (i) the procedure used in Example 3 above. 5 or 1 μg) (GLA-SE), or (ii) MPL in emulsion form supplied by the manufacturer (GSK Biologicals, Rixensart, Belgium) (40, 5 or 40 per animal for each immunization) Mice (3 Balb / c mice per group) using Leish-110f antigen (10 μg per animal per immunization) formulated in a stable emulsion containing 1 μg) (MPL-SE). ) Was immunized 3 times every 2 weeks. Mice were sacrificed one week after the last immunization and the spleen was harvested and analyzed by ELISA according to the published method (ibid.) For T cell-dependent IFN-γ cytokine response to in vitro antigen stimulation. The IFN-γ cytokine response is associated with the TH1 defense phenotype against leishmania infection.</p><p num="0204"> Figure 6 shows the levels of anti-Leish-110f IFN-γ cytokine production induced in mice 1 week after the third immunization with Leish-110f antigens formulated with various amounts of GLA. The ELISA data specified by comparison with the water control is shown. The mean and SEM for each group are shown.</p><p num="0205"> All mice responded well to the potent cell activator and mitogen ConA. Leish-110f + GLA-SE vaccination induced a dose-dependent Leish-110f antigen-specific cytokine response, but no such response was observed in the saline control group. At all concentrations tested, GLA-SE was more potent than MPL-SE in inducing higher levels of IFN-γ secreted by antigen-specific T cells.</p><p num="0206"> In conclusion, the addition of a stable oily formulation of GLA to Leish-110f, a candidate for the Leishmania vaccine antigen, primarily induces an antigen-specific immune response of cell types (T cells) associated with the protective TH1 phenotype. It was. In addition, GLA-SE was more potent than MPL-SE in inducing defense-related cytokines such as IFN-γ.</p>

<p num="0207"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Leishmania antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0208"> GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; 20 or 20 per animal per immunization, using saline solution or according to (i) the procedure used in Example 3 above. 5 μg) (GLA-SE), or (ii) Manufacturer (GSK Biologicals, Rixensart, Leish-110f antigen (each) formulated in a stable emulsion containing the emulsion form MPL® (MPL-SE) (20 or 5 μg per animal per immunization) supplied by Belgium). For immunization, mice (3 Balb / c mice per group) were immunized 3 times every 2 weeks using 10 μg per animal. One week after the last immunization, the mice were sacrificed, the spleen was harvested, and T cell-dependent IFN for in vitro antigen stimulation by intracellular staining (ICS) and flow cytometry according to published methods (same as above). -γ, IL-2, and TNF cytokine responses were analyzed. These three cytokine responses are associated with the TH1 defense phenotype against leishmania infection.</p><p num="0209"> When analyzed at the single cell level, the frequency of CD4 + T cells expressing all three cytokines IFN-γ, IL-2, and TNF or a combination of IFN-γ and IL-2 was Leish-110f + GLA- The SE group was higher than the Leish-110f + MPL-SE group, which was observed at both 20 μg and 5 μg doses. High frequencies of CD4 + T cells expressing all three cytokines IFN-γ, IL-2, and TNF have been reported to correlate with protection against leishmania infection (Seder et al.).</p><p num="0210"> In conclusion, the addition of a stable oily formulation of GLA to Leish-110f, a candidate for the Leishmania vaccine antigen, predominantly induced an antigen-specific immune response of cell types (T cells) associated with the protective TH1 phenotype. .. In addition, GLA-SE was more potent than MPL-SE in inducing defense-related cytokines such as IFN-γ, IL-2, and TNF.</p>

<p num="0211"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Mycobacterium tuberculosis antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0212"> GLA (Avanti Polar Lipids, Inc., Alabaster, AL; Product No. 699800; Each Immunization) using ID83 antigen (8 μg per animal per immunization) alone or according to the procedure used in Example 3 above. Mice (3 C57BL / 6 mice per group) 3 times every 3 weeks using the above antigens formulated in a stable emulsion (GLA-SE) containing (20 μg per animal) per animal. I was immunized. Serum was collected by collecting blood from mice one week after the third immunization, and serum levels of ID83-specific IgG1 and IgG2c antibodies were tested by ELISA according to the published method (ibid.). Which isotype of IgG1 or IgG2c antibody predominates is associated with a TH2 or TH1 response, respectively. TH1 responses have been shown to be necessary for protection against human M. tuberculosis infection.</p><p num="0213"> Vaccination with ID83 alone induced predominantly antigen-specific IgG1 antibodies. In contrast, ID83 + GLA-SE vaccination induced higher antibody titers and reversed the phenotype to the predominant IgG2c antibody response associated with protection against the disease.</p>

<p num="0214"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on vaccines containing a particular Mycobacterium tuberculosis antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0215"> ID83 antigen (8 μg per animal for each immunization) alone, or GLA (GLA-SE), GLA + CpG (CpG)₁₈₂₆, Coley Pharmaceuticals, 25 μg) (GLA / CpG-SE), or GLA + Gardiquimod (GDQ) (Invivogen, 20 μg) (GLA / GDQ-SE) in a stable emulsion containing the above antigens. The mice (3 C57BL / 6 mice per group) were immunized 3 times every 3 weeks. Three weeks after the last injection, the mice were sacrificed, the spleen was harvested, and according to published methods, CD4 + and CD8 + T cell-dependent IFN-γ, IL- for in vitro ID83 antigen stimulation by ICS and flow cytometry. 2, and TNF cytokine response was analyzed. Expression of IFN-γ, IL-2, and TNF cytokines is associated with a protective TH1 response to human M. tuberculosis infection.</p><p num="0216"> Figure 7 shows ID83 using ID83 alone or with an adjuvant containing a formulation containing GLA (GLA-SE), GLA + CpG (GLA / CpG-SE) or GLA + GDQ (GLA / GDQ-SE). ICS data are shown showing the frequency of ID83-specific IFN-γ, IL-2, and TNF cytokine-producing CD4 + and CD8 + T cells induced in mice 1 week after the third immunization used.</p><p num="0217"> The frequency of ID83-specific cytokine-producing CD4 + or CD8 + T cells was at background levels in the saline and ID83-only vaccine groups. ID83 antigen-specific cytokine-producing T cells (both CD4 + and CD8 +) are induced by ID83 + GLA-SE vaccination, the frequency of which is second, such as CDG (TLR7 / 8) or CpG (TLR9). It was further increased by the addition of the ligand of. T cells expressing IFN-γ + TNF or IFN-γ + IL-2 were the predominant population.</p><p num="0218"> In conclusion, when GLA-SE is added as an adjuvant to an antigen against human tuberculosis infection, the antigen-specific cellular response (T) is measured by the frequency of T cells expressing IFN-γ, IL-2, and / or TNF cytokines. (Cells) were significantly enhanced. In addition, combining GLA-SE with another TLR ligand further increased the frequency of antigen-specific cytokine-producing cells, a phenotype associated with defense against the disease.</p>

<p num="0219"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on a vaccine containing a specific Mycobacterium leprae antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0220"> CpG (CpG₁₈₂₆, Coley Pharmaceuticals, 25 μg per animal per immunization), or Imikimod (IMQ) (3M Pharma, 25 μg per animal per immunization), or GLA (Avanti Polar Lipids, Inc., Alabaster, AL) Product No. 699800; ML0276 antigen (GLA-SE), an aqueous formulation containing a mixture of these three, added as an adjuvant, 25 μg per animal for each immunization, according to the procedure used in Example 3 above). For each immunization, mice (3 C57BL / 6 mice per group) were immunized 3 times every 3 weeks using (10 μg per animal) or saline as a negative control. Serum was collected by collecting blood from mice 3 weeks after the second immunization and examined by ELISA according to the published method (ibid.) For ML0276-specific IgG antibody.</p><p num="0221"> Mice from the saline control group did not exhibit ML0276-specific IgG, and mice from the ML0276 + CpG and ML0276 + IMQ groups exhibited very low levels of antigen-specific antibody. In contrast, ML0276 + GLA-SE induced significant levels of ML0276-specific IgG, which was further increased when the three adjuvants were used together.</p><p num="0222"> In conclusion, the above data support the adjuvant effect of GLA-SE and / or the combination of GLA-SE and another TLR ligand when used with antigen ML0276 for the purpose of inducing antigen-specific antibodies. To do.</p>

<p num="0223"><u style="single">In vivo use of GLA-containing vaccines</u> This example describes an in vivo model that reveals the adjuvant effect of GLA on a vaccine containing a specific Mycobacterium leprae antigen. Standard immunological methods and reagents were used (Current Protocols in Immunology, Coligan et al., 2006 John Wiley &amp; Sons, NY).</p><p num="0224"> CpG (CpG₁₈₂₆, Coley Pharmaceuticals, 25 μg per animal for each immunization), or Imikimod (IMQ) (3M Pharma, 25 μg per animal for each immunization), or GLA (Avanti Polar Lipids, Inc., Alabaster, AL) Product number 699800; ML0276 antigen (each) supplemented with an aqueous formulation containing 25 μg per animal for each immunization, following the procedure used in Example 3 above) (GLA-SE), a mixture of these three. For immunization, mice (3 C57BL / 6 mice per group) were immunized 3 times every 3 weeks using (10 μg per animal) or saline as a negative control. Mice were sacrificed 3 weeks after the last injection and the spleen was harvested and analyzed for CD4 + T cell-dependent IFN-γ cytokine response to in vitro ML0276 antigen stimulation by ICS and flow cytometry according to published methods. Expression of IFN-γ cytokines is associated with a protective TH1 response against Mycobacterium leprae infection.</p><p num="0225"> Figure 8, Panel A uses an aqueous formulation containing CpG or imiquimod (IMQ), a stable oily emulsion containing GLA (GLA-SE), or the ML0276 antigen formulated with a mixture of these three 3 ICS data are shown showing the frequency of ML0276-specific IFN-γ cytokine-producing CD4 + T cells induced in mice 1 week after the second immunization by comparison with saline and naive controls. The average of each group is shown. Figure 8, panel B shows mice immunized with an aqueous formulation containing CpG or imikimod (IMQ), or a stable oily emulsion containing GLA (GLA-SE), or the ML0276 antigen formulated with a mixture of these three. Data showing the cell integrity of the influx region lymph nodes of Mycobacterium leprae infection in comparison with saline and naive controls are shown. The average and SEM of each group are shown.</p><p num="0226"> Mice from the saline control group did not exhibit an ML0276-specific IFN-γ response and had a background frequency of 0.04% positive cells. Mice from the CpG and IMQ groups showed a slight increase in antigen-specific cytokine-producing cells, 0.17% and 0.11%, respectively. In contrast, a significantly higher number of ML0276-specific IFN-γ + CD4 + T cells (0.66%) was observed when GLA-SE was used as an adjuvant, and the frequency was further increased when the three adjuvants were mixed. (2.14%).</p><p num="0227"> A subset of mice was then challenged with Mycobacterium leprae and protected by ML0276 + GE-SE as measured by a reduction in the number of cells in the influx area lymph nodes of the challenge compared to infected saline control. I found out. Vaccination with ML0276 + CpG and ML0276 + IMQ induced only a slight reduction in cell number compared to saline.</p><p num="0228"> In conclusion, the above data show the effect of GLA-SE and / or the combination of GLA-SE and another TLR ligand as an adjuvant when used with antigen ML0276 for the purpose of inducing an antigen-specific cellular response. Is to support.</p><p num="0229"> All U.S. patents, U.S. patent application gazettes, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to herein and / or described in the application datasheet are hereby referenced in their entirety. It shall be incorporated into the specification.</p><p num="0230"> From the above, although specific embodiments of the present invention have been described in the present specification for the purpose of explanation, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited except according to the appended claims.</p>