Single vial vaccine formulation

Abstract

The present invention provides heat-stable lyophilized formulations for inducing or enhancing an immune response, including vaccines and pharmaceutical compositions, and methods of use thereof. The lyophilized formulations generally comprise an antigen and / or adjuvant, a metabolizable oil, and a cake-forming excipient.

Description

Invention Field

[0001] This invention generally relates to the field of pharmaceutical and vaccine formulations.

[0002] Cross-reference of related patent applications

[0003] This application claims priority to U.S. Provisional Application No. 61 / 922,761, filed on December 31, 2013, which is incorporated herein by reference in its entirety. Background of the Invention

[0005] The new generation of rationally designed vaccine adjuvants represents a major breakthrough in vaccine development for challenging diseases, including tuberculosis, HIV, and malaria. However, these new adjuvants also require a cold chain process to ensure long-term stability. This highlights the significant financial and technical hurdles to achieving such vaccines globally. Furthermore, maintaining the cold chain may not be possible during natural disasters when power supplies are at risk. Lyophilization of protein-containing pharmaceuticals such as vaccines is a commonly used method for extending shelf life and increasing resistance to heat stress (Kasper et al., 2013, Eur J Pharm Biopharm. Oct 2013; 85(2): 162-9; Wang et al., Int J Pharm, 203: 1-60), and many commercially available vaccines are repackaged as lyophilized products (PATH and Working in Tandem Ltd, 2012, Summary of stability data for licensed vaccines, Seattle, WA). Novel vaccines under development for diseases mediated by complex cell-mediated immunity, such as malaria or tuberculosis, may require adjuvant components to effectively enhance and generate an immune response (Reed et al., 2009, Trends Immunol, 30:23-32). However, adding adjuvants to vaccine antigens results in more complex formulations with potential multiple interactions between components. Therefore, maintaining the long-term stability of adjuvanted vaccines presents a significant challenge for vaccine developers. For this reason, some adjuvanted vaccines are administered bedside-mixed with individual adjuvant vials (FDA, 2012, Advisory Committee Meeting on Vaccines and Related Biologicals). Furthermore, currently available commercially available lyophilized vaccines do not contain adjuvants in their lyophilized formulations (PATH and Working in Tandem Ltd, 2012, Summary of stability data for licensed vaccines, Seattle, WA). In fact, the lyophilization of adjuvant formulations such as aluminum salts or oil-in-water emulsions already used in approved human vaccines can be particularly challenging (Clausi et al., 2008, J Pharm Sci, 97: 2049-2061; Rossi et al., 2007, Role of Lipid Excipients in Modifying Oral and Parenteral Drug Delivery: Basic Principles and Biological Examples, pp 88-123, John Wiley & Sons, Inc., Hoboken NJ).While lyophilization of proteins, attenuated or inactivated viruses, or bacterial vaccines is routine practice, successful lyophilization and thermal stability characterization of adjuvanted clinical vaccine candidates have not yet been reported (PATH and Working in Tandem Ltd, 2012, Summary of stability data for licensed vaccines, Seattle, WA). Lyophilization of anthrax protective antigens in squalene emulsions has been reported; however, thermal stability and biophysical descriptions of this reconstituted system have not been included (Ivins et al., 1995, Vaccine, 13: 1779-1784). The complex properties of adjuvants in clinically approved vaccines (e.g., alum, oil-in-water emulsions, and / or monophospholipid A (MPLA)) pose significant obstacles to the development of lyophilized adjuvanted vaccines.

[0006] The development of adjuvanted vaccines that do not require a cold chain will significantly reduce the cost and technical barriers to achieving new vaccines globally, especially in low-resource settings. Therefore, there is a need for adjuvanted vaccines that are chemically stable at sustained temperatures and remain thermally stable to elicit an immune response against vaccine antigens. As described herein, the present invention addresses these needs and provides other related benefits.

[0007] For all purposes, all publications, patents and patent applications cited herein are incorporated herein by reference in their entirety. Invention Overview

[0009] In one aspect, this document provides a thermostable freeze-dried vaccine composition comprising a metabolizable oil and a cake-forming excipient, wherein the composition is in cake form and, upon remodeling, forms an oil-in-water emulsion, and wherein the cake-forming excipient is (1) a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose; or (2) a sugar selected from the group consisting of trehalose, lactose, raffinose, and lactulose. In some embodiments, the composition further comprises an antigen and / or an adjuvant.

[0010] In one aspect, this document provides a thermostable freeze-dried vaccine composition comprising an effective amount of antigen, a metabolizable oil, and a cake-forming excipient, wherein the composition is in cake form and, upon reconstitution, forms an oil-in-water emulsion, and wherein the cake-forming excipient is (1) a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose; or (2) a sugar selected from the group consisting of trehalose, lactose, raffinose, and lactulose. In some embodiments, the composition further comprises an adjuvant.

[0011] In another aspect, this document provides a thermostable freeze-dried vaccine composition comprising an effective amount of adjuvant, metabolizable oil, and a cake-forming excipient, wherein the composition is in cake form and, upon reconstitution, forms an oil-in-water emulsion, and wherein the cake-forming excipient is (1) a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose; or (2) a sugar selected from the group consisting of trehalose, lactose, raffinose, and lactulose. In some embodiments, the composition further comprises an antigen.

[0012] In some embodiments of the compositions described herein, the cake-forming excipient is a sugar selected from the group consisting of lactose, raffinose, and lactulose.

[0013] In some embodiments of the compositions described herein, the cake-forming excipient is a combination of mannitol and sugar selected from the group consisting of: trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose.

[0014] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, and the oil-in-water emulsion formulation contains less than or about 1% (w / v) glycerol.

[0015] In some embodiments of the compositions described herein, the oil-in-water emulsion formulation contains less than or about 0.5% (w / v) glycerol.

[0016] In some embodiments of the compositions described herein, the oil-in-water emulsion formulation does not contain glycerin.

[0017] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, wherein the cake-forming excipient is trehalose at a concentration of about 10% (w / v) in the oil-in-water emulsion formulation.

[0018] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, wherein the cake-forming excipient is trehalose at a concentration of about 5% (w / v) in the oil-in-water emulsion formulation.

[0019] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, wherein the cake-forming excipient is a combination of mannitol and trehalose, wherein the concentration of mannitol in the oil-in-water emulsion formulation is about 0.1% (w / v) and the concentration of trehalose in the oil-in-water emulsion formulation is about 5% (w / v).

[0020] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, wherein the cake-forming excipient is a combination of mannitol and trehalose, and wherein the concentration of mannitol in the oil-in-water emulsion formulation is about 2.5% (w / v) and the concentration of trehalose in the oil-in-water emulsion formulation is about 2.5% (w / v).

[0021] In some embodiments of the compositions described herein, the oil-in-water emulsion formulation does not contain glycerin.

[0022] In some embodiments of the compositions described herein, the compositions are thermally stable at temperatures between about 8°C and about 60°C for at least one month.

[0023] In some embodiments of the compositions described herein, the compositions are thermally stable at temperatures between about 8°C and about 60°C for at least 3 months.

[0024] In some embodiments of the compositions described herein, the compositions are thermally stable at temperatures between about 8°C and about 60°C for at least 6 months.

[0025] In some embodiments of the compositions described herein, the compositions are thermally stable at temperatures between about 8°C and about 60°C for at least 12 months.

[0026] In some embodiments of the compositions described herein, the compositions are thermally stable at about 25°C for at least 1 day.

[0027] In some embodiments of the compositions described herein, the compositions are thermally stable at about 25°C for at least one week.

[0028] In some embodiments of the compositions described herein, the compositions are thermally stable at about 25°C for at least one month.

[0029] In some embodiments of the compositions described herein, the compositions are thermally stable at about 37°C for at least 1 day.

[0030] In some embodiments of the compositions described herein, the compositions are thermally stable at about 37°C for at least one week.

[0031] In some embodiments of the compositions described herein, the compositions are thermally stable at about 37°C for at least one month.

[0032] In some embodiments of the compositions described herein, the compositions are thermally stable at about 50°C for at least 1 day.

[0033] In some embodiments of the compositions described herein, the compositions are thermally stable at about 50°C for at least one week.

[0034] In some embodiments of the compositions described herein, the compositions are thermally stable at about 50°C for at least one month.

[0035] In some embodiments of the compositions described herein, the compositions are thermally stable at about 30°C to about 50°C for at least 1 day, at least 1 week, or at least 1 month.

[0036] In some embodiments of the compositions described herein, the compositions are in the form of delicate biscuits.

[0037] In some embodiments of the compositions described herein, the compositions are in the form of a cake and do not show browning upon visual inspection when stored under any of the temperature and duration conditions described herein.

[0038] In some embodiments of the compositions described herein, the thermal stability of the compositions is determined prior to reconstructing the lyophilized compositions.

[0039] In some embodiments of the compositions described herein, they are in the form of a cake, and the determination of the thermal stability is made by observing the shrinkage, cracking, and / or browning of the cake.

[0040] In some embodiments of the compositions described herein, the thermal stability is determined after reconstructing the lyophilized composition.

[0041] In some embodiments of the compositions described herein, the determination of thermal stability is performed by examining the layering of the reconstituted oil-in-water emulsion.

[0042] In some embodiments of the compositions described herein, the compositions are formed by lyophilizing an oil-in-water emulsion formulation, wherein the concentration of the antigen or adjuvant in the reconstituted oil-in-water emulsion shows a degradation of no more than or about 25% of the concentration of the antigen or adjuvant in the oil-in-water emulsion formulation prior to lyophilization.

[0043] In some embodiments of the compositions described herein, thermal stability is determined by analyzing the components of the reconstituted oil-in-water emulsion.

[0044] In some embodiments of the compositions described herein, the reconstructed emulsion has a particle size with a Z-average diameter of less than about 200 nm.

[0045] In some embodiments of the compositions described herein, the antigen is a polypeptide, a nucleic acid encoding a polypeptide, or a pathogen.

[0046] In some embodiments of the compositions described herein, the adjuvant is a metabolizable oil. In some embodiments, the metabolizable oil is squalene, synthetic squalene, grape seed oil, olive oil, or synthetic isoprene.

[0047] In some embodiments of the compositions described herein, the adjuvant is a TLR4 agonist. In some embodiments, the TLR4 agonist is MPL, 3d-MPL, or synthetic GLA. In some embodiments, the synthetic GLA adjuvant has the following structure:

[0048]

[0049] Where R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0050] In some embodiments of the compositions described herein, the metabolizable oil is squalene, mineral oil, grape seed oil, synthetic squalene, or synthetic isoprene.

[0051] In some embodiments, the composition further comprises 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), ovite-derived phosphatidylcholine (ovite PC), lecithin, Tween, or combinations thereof.

[0052] In some embodiments, the compositions described herein further comprise a surfactant. In some embodiments, the surfactant is Pluronic F68. In some embodiments, the compositions further comprise an antioxidant. In some embodiments, the antioxidant is vitamin E.

[0053] In another aspect, this document provides a single vial containing the heat-stable lyophilized vaccine composition described herein, wherein the composition is contained in the vial.

[0054] In another aspect, this document provides a method for storing the heat-stable vaccine composition described herein, comprising storing the composition for at least one month between about 8°C and about 60°C or between about 25°C and about 60°C, wherein the vaccine formulation is heat-stable.

[0055] In another aspect, this document provides a method for producing a thermostable lyophilized vaccine composition comprising the step of lyophilizing an oil-in-water emulsion to form a thermostable lyophilized vaccine composition, wherein the oil-in-water emulsion comprises (1) an antigen, (2) a metabolizable oil and (3) a cake-forming excipient, wherein the cake-forming excipient is (a) a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose and lactulose; or (b) a sugar selected from the group consisting of trehalose, lactose, raffinose and lactulose, wherein the vaccine composition is in the form of a cake and is reconstituted into an oil-in-water emulsion.

[0056] In another aspect, this document provides a method for producing a thermostable lyophilized vaccine composition, comprising the step of lyophilizing an oil-in-water emulsion to form a thermostable lyophilized vaccine composition, wherein the oil-in-water emulsion comprises (1) an adjuvant, (2) a metabolizable oil and (3) a cake-forming excipient, wherein the cake-forming excipient is (a) a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose and lactulose; or (b) a sugar selected from the group consisting of trehalose, lactose, raffinose and lactulose, wherein the vaccine composition is in the form of a cake and is reconstituted into an oil-in-water emulsion.

[0057] In some embodiments of the method described herein, the cake-forming excipient is a sugar selected from the group consisting of lactose, raffinose, and lactulose.

[0058] In some embodiments of the method described herein, the cake-forming excipient is a combination of mannitol and sugar, wherein the sugar is selected from the group consisting of: trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose.

[0059] In some embodiments of the method described herein, the oil-in-water emulsion contains less than or about 1% (w / v) glycerol prior to lyophilization.

[0060] In some embodiments of the method described herein, the oil-in-water emulsion contains less than or about 0.5% (w / v) glycerol prior to lyophilization.

[0061] In some embodiments of the method described herein, the oil-in-water emulsion does not contain glycerin prior to freeze-drying.

[0062] In some embodiments of the method described herein, the cake-forming excipient is trehalose at a concentration of about 10% (w / v) in the oil-in-water emulsion prior to freeze-drying.

[0063] In some embodiments of the method described herein, the cake-forming excipient is 5% (w / v) trehalose at a concentration of about 5% (w / v) in the oil-in-water emulsion prior to freeze-drying.

[0064] In some embodiments of the method described herein, the cake-forming excipient is a combination of mannitol and trehalose, wherein the mannitol is present in the oil-in-water emulsion at about 0.1% (w / v) and the trehalose is present in the oil-in-water emulsion at about 5% (w / v) before freeze-drying.

[0065] In some embodiments of the method described herein, the cake-forming excipient in the oil-in-water emulsion prior to freeze-drying is a combination of mannitol and trehalose, wherein the mannitol content in the oil-in-water emulsion prior to freeze-drying is about 2.5% (w / v) and the trehalose content in the oil-in-water emulsion prior to freeze-drying is about 2.5% (w / v).

[0066] In some embodiments of the method described herein, the oil-in-water emulsion does not contain glycerin prior to freeze-drying.

[0067] In some embodiments of the method described herein, the composition is thermally stable at a temperature between about 8°C and about 60°C for at least one month.

[0068] In some embodiments of the method described herein, the composition is thermally stable for at least 3 months.

[0069] In some embodiments of the method described herein, the composition is thermally stable for at least 6 months.

[0070] In some embodiments of the method described herein, the composition is thermally stable for at least 12 months.

[0071] In some embodiments of the method described herein, the composition is thermally stable at about 25°C for at least one month.

[0072] In some embodiments of the method described herein, the composition is thermally stable at about 37°C for at least one month.

[0073] In some embodiments of the method described herein, the composition is thermally stable at about 50°C for at least one month.

[0074] In some embodiments of the method described herein, the freeze-drying step is performed in a single bottle.

[0075] In some embodiments of the method described herein, the reconstructed oil-in-water emulsion has a particle size with a Z-average diameter of less than about 200 nm.

[0076] In some embodiments of the method described herein, the reconstructed oil-in-water emulsion has a particle size with a Z-average diameter of less than about 100 nm.

[0077] In some embodiments of the methods described herein, the concentrations of antigens and / or adjuvants in the reconstructed oil-in-water emulsion show a degradation of no more than or about 25% compared to the concentrations of antigens and / or adjuvants in the oil-in-water emulsion before lyophilization.

[0078] In some embodiments of the method described herein, the freeze-dried composition is in the form of a cake.

[0079] In some embodiments of the method described herein, the thermal stability is determined before reconstructing the lyophilized composition.

[0080] In some embodiments of the method described herein, the freeze-dried composition is in the form of a cake, and the determination of the thermal stability is made by observing the shrinkage or browning of the cake.

[0081] In some embodiments of the method described herein, the thermal stability is determined after the lyophilized composition has been reconstructed.

[0082] In some embodiments of the method described herein, thermal stability is determined by examining the stratification of the reconstructed oil-in-water emulsion.

[0083] In some embodiments of the method described herein, the thermal stability is determined visually.

[0084] In some embodiments of the method described herein, the determination of thermal stability is performed by analyzing the components of the reconstructed oil-in-water emulsion.

[0085] In some embodiments of the method described herein, the emulsion is freeze-dried in a single bottle.

[0086] In some embodiments of the method described herein, the reconstructed emulsion has a particle size with a Z-average diameter of less than about 200 nm.

[0087] In some embodiments of the method described herein, the oil-in-water emulsion contains an antigen and an adjuvant prior to freeze-drying.

[0088] In some embodiments of the methods described herein, the antigen is a polypeptide, a nucleic acid encoding a polypeptide, or a pathogen.

[0089] In some embodiments of the methods described herein, the adjuvant is a metabolizable oil. In some embodiments of the methods described herein, the metabolizable oil adjuvant is squalene, synthetic squalene, grape seed oil, olive oil, or synthetic isoprene.

[0090] In some embodiments of the methods described herein, the antigen is a polypeptide, a nucleic acid encoding a polypeptide, or a pathogen.

[0091] In some embodiments of the method described herein, the adjuvant is a TLR4 agonist. In some embodiments, the TLR4 agonist is MPL, 3d-MPL, or synthetic GLA. In some embodiments, the synthetic GLA adjuvant has the following structure:

[0092]

[0093] Where R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0094] In some embodiments of the method described herein, the metabolizable oil is squalene, mineral oil, grape seed oil, synthetic squalene, or synthetic isoprene.

[0095] In some embodiments, the vaccine composition further comprises 1,2-dimyristic-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), ovum PC, lecithin, Tween, or combinations thereof.

[0096] In some embodiments, the vaccine composition further comprises a surfactant. In some embodiments, the surfactant is Pluronic F68. In some embodiments, the vaccine composition further comprises an antioxidant (e.g., vitamin E).

[0097] In another aspect, this document provides a method for stimulating an immune response in an individual, comprising: (a) reconstituted any of the thermostable lyophilized vaccine compositions described herein into an oil-in-water emulsion; and (b) administering the emulsion to the individual, thereby stimulating an immune response in the individual. In some embodiments, the immune response is a nonspecific immune response. In some embodiments, the immune response is an antigen-specific immune response. In some embodiments, the oil-in-water emulsion is administered intradermally or orally. In some embodiments, the composition comprises GLA. In some embodiments, the mammal is a human, dog, or cow.

[0098] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. Brief description of the attached diagram

[0100] Figure 1 shows that the lyophilized 5% w / v trehalose stable emulsion with 2% v / v retained emulsion properties. Figure 1A The appearance of the emulsion before freeze-drying (left), as a freeze-dried cake (middle), and after reconstructing the freeze-dried cake (right). Figure 1B Particle size and volume distribution were measured by dynamic light scattering (DLS).

[0101] Figure 2 shows that freeze drying significantly improves the physical and pH stability of the emulsion at 37°C and its chemical stability at 90°C. Figure 2A Particle size in the pre-lyophilized liquid emulsion (solid square) and reconstructed lyophilized emulsion (solid round) when stored at 25°C (top) or 37°C (bottom). Figure 2B pH values ​​of liquid emulsion (solid square) and reconstituted lyophilized emulsion (solid circle) when stored at 25°C (top) or 37°C (bottom). Figure 2C The component amounts in the emulsion after storage under accelerated degradation conditions at 90°C. The percentages (%) of follicular phosphatidylcholine (liquid follicular PC; solid round), squalene (liquid squalene; solid triangular), and α-tocopherol (liquid α-tocopherol; solid rhomboid) in the liquid emulsion showed a rapid change in component concentration compared to those in the reconstituted lyophilized emulsion, which contained follicular phosphatidylcholine (lyophilized follicular PC; solid square), squalene (lyophilized squalene; solid inverted triangular), and α-tocopherol (lyophilized α-tocopherol; hollow round).

[0102] Figure 3 shows the physicochemical changes of the lyophilized emulsion after periodic remodeling for 1500 hours during storage at 25°C, 37°C, 50°C, 60°C, and 90°C. Figure 3A Particle size in reconstructed lyophilized emulsions stored at 25°C (solid round), 37°C (solid square), 50°C (solid triangular), 60°C (solid inverted triangular), or 90°C (hollow round). Figure 3B pH of reconstructed lyophilized emulsions stored at 25°C (solid circle), 37°C (solid square), 50°C (solid triangle), 60°C (solid inverted triangle), or 90°C (hollow circle). Figure 3C The width ratio of cake-like particles in freeze-dried emulsions stored at 25°C (solid round), 37°C (solid square), 50°C (solid triangle), 60°C (solid inverted triangle), or 90°C (hollow round). Figure 3DAs measured by the shrinkage of the cake width, melt remelting is inversely correlated with particle size when the lyophilized emulsion is stored under pressure stability conditions at 60°C.

[0103] Figure 4 shows that the choice of excipients affects the maintenance of the reconstituted emulsion. Figure 4A The formulation polydispersity index (PdI) of each excipient. Figure 4B Emulsion particle size (nM) of each excipient. From left to right: stabilized emulsion (matrix material), trehalose (before freeze-drying), trehalose, dextran, lactose, maltose, sucrose, raffinose, mannose, fructose, lactulose, ribose, dextran 40,000, PEG 3350, mannitol, stachyose, sorbitol, pyridinedicarboxylic acid (0.5%), nicotinic acid (0.5%), and proline.

[0104] Figure 5 shows the formulation and cake properties of the reconstituted 2% (v / v) oil-stabilized emulsion (SE) of 5% (w / v) trehalose, ribose, mannitol or proline. Figure 5A Image of a cake-like structure of a freeze-dried emulsion. Figure 5B Comparison of stratification between reconstituted lyophilized formulations of trehalose and mannitol. Arrows indicate stratification. Figure 5C For example, particle size distribution measured by DLS.

[0105] Figure 6 shows how the choice of excipients and chemical structure affect the thermal stability of the cake. Figure 6A Melting phase transition heat analysis diagram of a representative formulation using a melting device. Figure 6B The initial temperature and tm of the cake-like material in each excipient.

[0106] Figure 7 shows that the dimensional stability of the emulsion depends on the morphology of the cake and its sensitivity to thermally induced remelting. Figure 7A Particle size stability depends on the type and concentration of the filler used. Stable emulsions are produced using excipients of 5% dextrose (solid square), 5% sucrose (solid rhombus), 5% maltose (solid inverted triangle), 5% trehalose (solid round), and 15% lactose (solid triangle). Figure 7B The particle size growth rate is related to the transition temperature (a filler property). The dashed line represents the pressure stability temperature of 90°C.

[0107] Figure 8 Formulations containing mannitol and Class 1 excipients show a significant increase in cake thermal stability without disrupting the emulsion. Comparison of cake Tm between 5% (w / v) formulations containing trehalose, dextrose, lactose, maltose, or sucrose with 2% (v / v) oil-stabilized emulsions containing 0.2%, 5% (w / v) mannitol, or none of mannitol.

[0108] Figure 9Representative images of ID93+GLA-SE liquid (left), lyophilized (middle), and reconstructed (right) vials are shown. The vials were stored at 50°C under no pressure (upper) or pressure (lower) for 30 days.

[0109] Figure 10 shows the particle characteristics of liquid and reconstructed lyophilized samples containing ID93 and / or GLA-SE, as indicated in the chart labels. Figure 10A Z-mean diameter from DLS experiment Figure 10B The polydispersity index (PdI) from the DLS experiment. Figure 10C Zeta potential measurements from nanoparticle tracking analysis and Figure 10D Particle concentration measurements from nanoparticle tracking analysis. Solid bars represent unpressurized samples (i.e., stored at 4°C for 30 days), while open bars represent samples subjected to pressure at 50°C for 30 days.

[0110] Figure 11 The images show reduced SDS-PAGE staining with co-bottle ID93+GLA-SE liquid (lanes 1 and 3) and reconstructed lyophilized samples (lanes 2 and 4). Samples were subjected to no pressure (lanes 1 and 2) or pressure at 50°C (lanes 3 and 4) for 30 days. No-pressure GLA-SE and ID93 are shown for comparison (lanes 5 and 6, respectively).

[0111] Figure 12 shows the liquid and reconstructed lyophilized samples containing ID93 and / or GLA-SE. The standard curve was determined from reversed-phase HPLC analysis. Figure 12A GLA Figure 12B DMPC and Figure 12C Squalene concentration. Figure 12D The chromatograms of the components DMPC, squalene, and GLA are displayed. Solid bars represent samples without pressure, while hollow bars represent samples subjected to pressure at 50°C for 30 days.

[0112] Figure 13 shows the lyophilization prevention of ID93+GLA-SE due to loss of biological activity caused by thermal stress. Mice were immunized with ID93+GLA-SE in saline or liquid, liquid-liquid co-bottle, or lyophilized co-bottle after one month of exposure to 4°C or 50°C. Figure 13A B and T cell counts were measured 18 hours after immunization. Figure 13B The titer of ID93-specific serum antibodies was measured three weeks after the first immunization. Figure 13C The frequency of ID93-specific CD4 T cells in the spleen one week after final immunization was assessed by analyzing cytokine production after in vitro ID93 restimulation.

[0113] Figure 14 shows the nebulized tuberculosis challenge and calculations in ID93+GLA-SE immunized mice. Animals were challenged with a low dose of nebulized tuberculosis one month after final immunization. Measurements were performed three weeks later. Figure 14A Lungs and Figure 14B Bacterial burden in the spleen. Data are presented as mean + standard error for N = 5–7 mice / group. Data are presented from one of two groups with similar results. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001 relative to saline, respectively. ns is not significant relative to saline. Statistical comparisons are indicated between samples from 4°C and 50°C.

[0114] Figure 15 Displays lyophilized and reconstructed ID93+SLA-SE co-lyophilized formulations after 30 days at 50°C and pressure. Reproducible samples are shown at day 0 and day 30.

[0115] Figure 16 This shows a reduced SDS-PAGE gel image of the reconstructed ID93+SLA-SE co-lyophilized formulation after Coomassie staining. Each lane was loaded with 1 μg of ID93+SLA-SE.

[0116] Figure 17 shows the DLS particle size of the reconstructed ID93+SLA-SE co-lyophilized formulation. Figure 17A Particle size distribution (diameter) expressed by intensity. Figure 17B Z - Average diameter. Figure 17C Multidispersion index (PdI). The error bars represent 1 standard deviation around the mean, for n = 4 runs.

[0117] Figure 18 Displays the DLS zeta potential (mV) of the reconstructed ID93+SLA-SE co-lyophilized formulation. Error bars represent approximately 1 standard deviation from the mean, n=4 runs.

[0118] Figure 19 This displays the SLA concentration (μg / mL) in the reconstructed ID93+SLA-SE co-lyophilized formulation derived by HPLC. Error bars represent approximately 1 standard deviation from the mean, for n = 3 runs.

[0119] Figure 20 This image shows the cake formation of the double-lyophilized ID93+GLA-SE sample and the appearance of the reconstructed emulsion at zero hour after lyophilization (immediately after lyophilization) and after one month of storage at 4°C, 25°C, and 37°C. The sample stored at 4°C represents the control lyophilized emulsion maintained under normal cold chain storage conditions of 2°C–8°C. The cakes are white and partially wrinkled with no signs of browning. The reconstructed emulsions did not separate at 1 hour or 24 hours after reconstruction.

[0120] Figure 21 shows the stability characteristics of the double-lyophilized ID93+GLA-SE formulation after 3 months of storage at 4°C, 25°C, and 37°C. Figure 21A The cake showed no further signs of collapse or discoloration, and the reconstructed sample maintained the appearance of an emulsion, with no stratification even 24 hours after reconstruction. Figure 21B The table shows the Z-mean diameter, polydispersity index (PDI), pH, and GLA concentration of the reconstituted emulsion from DLS experiments. Error bars represent approximately 1 SD of the mean for pH (2 vials), and approximately 1 SD of the mean for DLS size and PDI measurements (2 vials x 3 dilutions x 3 runs). The HPLC-derived GLA concentration (μg / mL) of the reconstituted ID93+GLA-SE co-lyophilized formulation is also shown. Error bars represent approximately 1 standard deviation from the mean, n = 3. The data confirm that there is no significant increase in particle size, polydispersity, or pH after reconstituted emulsion storage at any temperature. Figure 21C The image shows a Coomassie-stained reduced SDS-PAGE gel image of the reconstructed ID93+SLA-SE co-lyophilized formulation. Each lane was loaded with 1 μg of ID93+SLA-SE. The presence of a 98 kDa band confirms that the ID93 peptide has not undergone significant degradation. Figure 21D HPLC tracking of the emulsion components squalene and DMPC. HPLC analysis confirmed that neither the emulsion components DMPC nor squalene underwent significant degradation at any test temperature, as evidenced by the absence of additional peaks or peak broadening. Figure 21E The figures show the HPLC-derived GLA concentration (μg / mL) in the reconstructed ID93+GLA-SE co-lyophilized formulation. Error bars represent approximately 1 standard deviation from the mean, n = 3 runs. At 3 months, the reconstructed emulsion samples showed considerable GLA loss at 4°C and 25°C (51 μg / mL (102%) in the 4°C sample and 46 μg / mL (92%) in the 25°C sample). However, the sample stored at 37°C showed approximately 50% loss of the initial GLA concentration (25 μg / mL (84%)). The 37°C sample showed some trend toward GLA concentration loss at 3 months (42 μg / mL (84%)).

[0121] Figure 22 shows the stability characteristics of the double-lyophilized ID93+GLA-SE formulation after 6 months of storage at 4°C, 25°C, and 37°C. The sample stored at 4°C represents the control lyophilized emulsion maintained under normal cold chain storage conditions of 2°C–8°C. Figure 22A The cake showed no further signs of collapse or discoloration, and the reconstructed sample maintained the appearance of an emulsion, with no stratification even 24 hours after reconstruction. Figure 22BThe table shows the Z-mean diameter and polydispersity index (PDI), pH, and GLA concentration of the reconstituted emulsion from DLS experiments. Error bars represent approximately 1 SD of the mean for pH (2 vials), and approximately 1 SD of the mean for DLS size and PDI measurements (2 vials x 3 dilutions x 3 runs). The HPLC-derived GLA concentration (μg / mL) of the reconstituted ID93+GLA-SE co-lyophilized formulation is also shown. Error bars represent approximately 1 standard deviation from the mean, n = 3. This table confirms that the reconstituted samples, as well as those stored for 6 months, maintained a physiological pH range of 7.21–7.49. Figure 22C The image shows a Coomassie-stained reduced SDS-PAGE gel image of the reconstructed ID93+SLA-SE co-lyophilized formulation. Each lane was loaded with 1 μg ID93+GLA-SE. The presence of a 98kD band confirms that the ID93 peptide was not observably degraded at 6 months, as evidenced by the absence of additional bands or broadening of the 98kD band. Figure 22D HPLC tracking of the emulsion components squalene and DMPC. HPLC analysis confirmed that the emulsion components did not undergo observable degradation at any test temperature, as evidenced by the absence of additional peaks or peak broadening. Figure 22E The values ​​show the HPLC-derived GLA concentration (μg / mL) of the reconstructed ID93+GLA-SE co-lyophilized formulation. Error bars represent approximately 1 standard deviation from the mean, for n = 3 runs. At 6 months, samples stored at 4°C and 25°C showed no significant loss of GLA (47 μg / mL (94%) in the 4°C sample and 42 μg / mL (84%) in the 25°C sample), but samples stored at 37°C showed approximately 50% loss of the initial GLA concentration (25 μg / mL (84%)).

[0122] Figure 23 shows the stability characteristics of the double-lyophilized ID93+GLA-SE formulation after 9 months of storage at 4°C, 25°C, and 37°C. The sample stored at 4°C represents the control lyophilized emulsion maintained under normal cold chain storage conditions of 2°C–8°C. Figure 23A The cake showed no further signs of collapse or discoloration, and the reconstructed sample maintained the appearance of an emulsion, with no stratification even 24 hours after reconstruction. Figure 23B The table shows the Z-mean diameter and polydispersity index (PDI), pH, and GLA concentration from the DLS experiments. Error bars for pH represent approximately 1 SD of the mean from two vials, while for DLS size and PDI measurements, they represent approximately 1 SD of the mean from two vials x three dilutions x three runs. The HPLC-derived GLA concentration (μg / mL) of the reconstructed ID93+GLA-SE co-lyophilized formulation is also shown. Error bars represent approximately 1 standard deviation from the mean, n=3. This table confirms that the reconstructed samples and those stored for 9 months maintained a pH within the physiological range of 7.19–7.53. Figure 23CThe image shows a Coomassie-stained reduced SDS-PAGE gel image of the reconstructed ID93+GLA-SE co-lyophilized formulation. Each tube was loaded with 1 μg of ID93+GLA-SE. The presence of a 98kD band confirms that the ID93 peptide has not undergone significant degradation at 9 months, as evidenced by the absence of additional bands or broadening of the 98kD band. Figure 23D HPLC tracking of the emulsion components squalene and DMPC. HPLC analysis confirmed that, as demonstrated by the absence of additional peaks or peak broadening, the emulsion components showed no observable degradation at any test temperature. Figure 23E The figures show the HPLC-derived GLA concentration (μg / mL) of the reconstructed ID93+GLA-SE co-lyophilized formulation. Error bars represent approximately 1 standard deviation from the mean, n=3. At 9 months, samples stored at 4°C and 25°C showed no significant GLA loss (40 μg / mL (80%) in the 4°C sample and 40 μg / mL (80%) in the 25°C sample), but samples stored at 37°C after nine months showed a loss of approximately 69% of the initial GLA concentration (15 μg / mL).

[0123] Figure 24 shows the stability characteristics of the double-lyophilized ID93+GLA-SE formulation after 12 months of storage at 4°C, 25°C, and 37°C. The sample stored at 4°C represents the control lyophilized emulsion maintained under normal cold chain storage conditions of 2°C–8°C. Figure 24A The cake showed no further signs of collapse or discoloration, and the reconstructed sample maintained the appearance of an emulsion, with no stratification even 24 hours after reconstruction. Figure 24B The table shows the Z-mean diameter and polydispersity index (PDI), pH, and GLA concentration from the DLS experiments. Error bars for pH represent approximately 1 SD of the mean from two vials, while for DLS size and PDI measurements, they represent approximately 1 SD of the mean from two vials x three dilutions x three runs. The HPLC-derived GLA concentration (μg / mL) of the reconstructed ID93+GLA-SE co-lyophilized formulation is also shown. Error bars represent approximately 1 standard deviation from the mean, n=3. This table confirms that the reconstructed samples and those stored for 12 months maintained a physiological pH range of 7.15–7.48. Figure 24C The image shows a Coomassie-stained reduced SDS-PAGE gel image of the reconstructed ID93+GLA-SE co-lyophilized formulation. Each tube was loaded with 1 μg of ID93+GLA-SE. The presence of a 98kD band confirms that the ID93 peptide has not undergone observable degradation at 12 months, as confirmed by the absence of additional bands or broadening of the 98kD band.

[0124] Figure 25 shows the effects of adding or removing glycerol as a cake-forming excipient, the change in the percentage of oil in the emulsion, and the inclusion of 2% Tris as a tensioning agent on the lyophilized emulsion within the range of GLA concentrations (ng / ml) tested immediately after lyophilization (0 days) or after 30 days of storage at 50°C. Figure 25A The lyophilized emulsion formulations with increased biodegradable squalene concentrations (2-10% v / v) and lacking 0.5% glycerol (labeled as glycerol-free) formed compact cakes after lyophilization. Compared to formulations containing 0.5% v / v glycerol (labeled as glycerol-containing), there was no further shrinkage or visible discoloration of the cakes even after 30 days at 50°C. Cakes containing glycerol showed slight shrinkage and indentation immediately after lyophilization (day 0) and after 30 days of storage at 50°C. Samples stored at 50°C showed further (shrinkage) or collapse after 30 days of storage at 50°C. Figure 25B and Figure 25C This confirms that any formulation, after being stored at 50°C for 30 days, exhibits consistent particle size (Z-mean, nm). Figure 25B ) and polydispersity (PDI) Figure 25C There were no significant differences in the particle size distribution, with all formulations showing a particle size of less than or approximately 200 nm after pie reconstruction. Figure 25D The presence of 0.5% v / v glycerol showed that it affected the stability of GLA adjuvants in lyophilized formulations. Lyophilized formulations containing different concentrations of squalene (2%–10% v / v) contained different concentrations of GLA adjuvant. Zero-time concentrations were compared with GLA concentrations obtained after 30 days of storage at 50°C. Data showed that lyophilized formulations without any glycerol (described as glycerol-free) all demonstrated a greater than 85% percentage of initial GLA concentration, while lyophilized formulations containing glycerol demonstrated a greater than 80% GLA concentration loss after one month of storage at 50°C.

[0125] Figure 26 shows four lyophilized formulations evaluated for their thermal protection against GLA-SE emulsions. All evaluated lyophilized formulations lacked glycerol. For Figures 26-31, the concentration of adjuvant GLA in the GLA-SE emulsion was increased to 100 ng / ml to allow for more reproducible quantification of GLA concentration after reconstructing the lyophilized cake. Cake formation and appearance of the formulations were evaluated, as well as post-reconstruction stratification of samples stored at specified temperatures of 4°C, 25°C, 37°C, and 50°C at 0 hours (immediately after lyophilization), 1 week (1 wk), 2 weeks (2 wk), 1 month (1 mo), and 3 months (3 mo). Figure 26A 5% Trehalose alone (glycerin-free) Figure 26B 5% trehalose w / v, 0.1% w / v mannitol, Figure 26C 2.5% w / v trehalose, 2.5% w / v mannitol, Figure 26D10% w / v trehalose. Data confirm that all lyophilized formulations lacking or without glycerol demonstrated good formation of neat cakes, with no discoloration or browning of the lyophilized cakes at any time (after lyophilization, at least about 1 week, at least about 2 weeks, at least about 1 month, or at least about 3 months) or at any test temperature (at least about 4°C, at least about 25°C, at least about 37°C, and at least about 50°C). The cakes also all showed little or no collapse, shrinkage, or discoloration, and formed a reconstructed emulsion without stratification.

[0126] Figure 27 shows a comparison of the following lyophilized formulations before the addition of the following ingredients: 5% trehalose (without glycerol); 5% trehalose w / v, 0.1% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; and lyophilized GLA-SE emulsions (marked Pre Lyo on the column) before lyophilization of the lyophilized components (cake excipients), the GLA-SE emulsion immediately after the addition of the lyophilized components (marked Lyo on the column), and the reconstituted formulation after lyophilization (marked 0, or the third item for each lyophilized formulation group if not marked). Initial comparisons of the lyophilized formulations confirmed that there were no significant differences between the lyophilized formulations and that they possessed appropriate reconstructed emulsion characteristics (desired characteristics), including a Z-mean diameter of less than about 200 nm, lack of significant aggregation as measured by polydispersity, physiological pH, and no significant loss of GLA (value greater than 90% of the initial content).

[0127] Figure 28 shows the results of various single-bottle GLA-SE lyophilized formulations (emulsions containing cake-forming excipients) stored at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4) for one week (1 wk), as indicated by the strips below: 5% trehalose (glycerol-free); 5% trehalose w / v, 0.1% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol. Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 28A All four lyophilized formulations showed a desired particle size of less than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 28B All four lyophilized formulations showed a lack of significant aggregation, as measured by polydispersity, when stored in a temperature range of 4°C–50°C. Figure 28C All four lyophilized formulations exhibited the desired physiological pH when stored in a temperature range of 4°C–50°C. Figure 28D When stored as lyophilized cakes and reconstituted into GLA-SE emulsions, no significant loss of GLA was observed compared to the initial GLA concentration (the values ​​ranged from approximately 105% to 95%).

[0128] Figure 29 shows the results of specific lyophilized formulations stored at 37°C (strip 3) and 50°C (strip 4) for two weeks (2 weeks), as indicated by the strips below: 5% trehalose (glycerol-free); 5% trehalose w / v, 0.1% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol. Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 29A It was confirmed that all four lyophilized formulations, when stored at 37°C and 50°C for 2 weeks, formed GLA-SE emulsions exhibiting a desired particle size of less than approximately 200 nm upon remodeling. Figure 29B It was confirmed that all four lyophilized formulations, when stored at 37°C and 50°C for 2 weeks, formed GLA-SE emulsions upon reconstitution exhibiting a lack of considerable aggregation, as measured by polydispersity. Figure 29C It was confirmed that all four lyophilized formulations, when stored at 37°C and 50°C for 2 weeks, formed GLA-SE emulsions exhibiting the desired physiological pH of approximately pH 7.0 upon reconstitution. Figure 29D When stored at 37°C or 50°C as lyophilized cakes for 2 weeks and reconstituted to form GLA-SE emulsions, none of the GLA-SE lyophilized formulations showed a significant loss of GLA concentration (ranging from approximately 105% to 95%) of the initial GLA concentration.

[0129] Figure 30 shows the results of various single-bottle GLA-SE lyophilized formulations (emulsions containing cake-forming excipients) stored at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4) for one month (1 month), as indicated by the strips below: 5% trehalose (glycerol-free); 5% trehalose w / v, 0.1% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol. Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 30A It was confirmed that all four lyophilized formulations exhibited a desired particle size of less than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 30B It was confirmed that all four lyophilized formulations showed a lack of significant aggregation, as measured by polydispersity, when stored in a temperature range of 4°C–50°C. Figure 30C It was confirmed that all four lyophilized formulations exhibited the desired physiological pH when stored in a temperature range of 4°C–50°C. Figure 30DWhen stored as lyophilized cakes for one month at temperatures ranging from 4°C to 50°C and then reconstituted to form GLA-SE emulsions, none of the GLA-SE lyophilized formulations showed a significant loss of GLA concentration (ranging from approximately 105% to 94%) of the initial GLA concentration.

[0130] Figure 31 shows the results of various single-bottle GLA-SE lyophilized formulations (emulsions containing cake-forming excipients) stored at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4) for one month (1 month), as indicated by the strips below: 5% trehalose (glycerol-free); 5% trehalose w / v, 0.1% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol; 2.5% w / v trehalose, 2.5% w / v mannitol. Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 31A It was confirmed that all four lyophilized formulations exhibited a desired particle size of less than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 31B It was confirmed that all four lyophilized formulations showed a lack of significant aggregation, as measured by polydispersity, when stored in a temperature range of 4°C–50°C. Figure 31C It was confirmed that all four lyophilized formulations exhibited the desired physiological pH of at least approximately pH 7.0 when stored in a temperature range of 4°C–50°C. Invention Details

[0132] In one aspect, the present invention provides a thermostable lyophilized vaccine composition comprising (1) a metabolizable oil and (2) a cake-forming excipient. The thermostable lyophilized vaccine composition optionally further comprises an antigen and / or an adjuvant. In some embodiments, the cake-forming excipient is a combination of mannitol and a sugar selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose. In some embodiments, the cake-forming excipient is a sugar selected from the group consisting of trehalose, lactose, raffinose, and lactulose. In some embodiments, the cake-forming excipient is a sugar selected from the group consisting of lactose, raffinose, and lactulose. In some embodiments, the composition is formed by lyophilizing an oil-in-water emulsion formulation, wherein the oil-in-water emulsion formulation, prior to lyophilization or after reconstitution, contains less than or equal to 1% (w / v) glycerol, less than or equal to 0.5% (w / v) glycerol, or contains no glycerol. In some embodiments, the composition is in the form of a cake and, upon reconfiguration, forms an oil-in-water emulsion. In some embodiments, the composition is stored in a single bottle.

[0133] As will be understood by those skilled in the art, the terms thermostable lyophilized vaccine composition, lyophilized vaccine composition, lyophilized thermostable cake, and lyophilized cake are used interchangeably herein. The term generally refers to a lyophilized oil-in-water stable emulsion comprising a biodegradable or metabolizable oil, and / or one or more antigens, and / or one or more adjuvants, and a cake-forming excipient for producing the cake of the present invention. Once reconstituted into a liquid oil-in-water emulsion, the thermostable lyophilized vaccine composition possesses the desired characteristics of the present invention: an average particle size of less than or about 200 nm, a physiological pH of about 7.4, and a loss of concentration of each active ingredient (e.g., antigen or adjuvant) of no more than or about 25% compared to the initial concentration of each active ingredient in the oil-in-water formulation before lyophilization, or no significant degradation or alteration of the active ingredients (e.g., antigens, adjuvants) suitable for inducing or stimulating an immune response in an individual.

[0134] As provided herein, the lyophilized vaccine compositions are heat-stable. For example, the compositions are stable between about 8°C and about 60°C. Such compositions may additionally contain suitable excipients, such as pharmaceutically acceptable excipients (carriers), including buffers, acids, bases, sugars, diluents, preservatives, etc., which are well known in the art and described herein. In another aspect, the present invention provides a method for producing the heat-stable lyophilized vaccine compositions described herein.

[0135] In some aspects, the present invention provides a method for stimulating an immune response in an individual, comprising reconstituted the thermostable lyophilized vaccine composition described herein into an emulsion and administering the emulsion to the individual. In some embodiments, the emulsion is an oil-in-water emulsion. In some embodiments, the immune response is a nonspecific immune response. In some embodiments, the immune response is an antigen-specific immune response. The method for stimulating an immune response described herein, or the reconstituted thermostable lyophilized vaccine composition described herein, can be used alone or in combination with other conventional treatment methods (e.g., chemotherapy agents).

[0136] In some implementations, a reference to "about" a value or parameter in this document includes (and describes) variations of that value or parameter itself. For example, a description of a reference to "about X" includes a description of "X".

[0137] definition

[0138] It should be understood that the various aspects and embodiments of the present invention described herein include “comprising aspects and embodiments”, “consisting of aspects and embodiments”, and “substantially consisting of aspects and embodiments”.

[0139] "Individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sporting animals, pets (e.g., cats, dogs, horses), primates, mice, and rats.

[0140] Unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” as used herein and in the appended claims include plural references.

[0141] The terms "bulking agent" and "bulking agent" are used interchangeably herein. A bulking agent is a substance added to a stable liquid oil-in-water emulsion formulation prior to lyophilization, which, upon lyophilization, produces a cake-like substance. This lyophilized cake-like substance is reconstructed to form a stable emulsion suitable for delivering pharmacologically active pharmaceutical ingredients, including the vaccines described herein. As used herein, a bulking agent is a substance that reconstructs the lyophilized cake-like substance without disrupting the emulsion.

[0142] As used herein, excipients refer to substances that are different from pharmacologically active pharmaceutical ingredients, including those used in the manufacturing process, or in the fill-finish process (including but not limited to lyophilization) for the storage or shipment of pharmacologically active pharmaceutical ingredients, and are included in the completed pharmaceutical process.

[0143] As used herein, lyophilized excipients may refer to substances other than pharmacologically active pharmaceutical ingredients, including those that promote the formation or formulation of a suitable cake structure during the lyophilization process. Lyophilized excipients may include fillers, buffers, or solubilizers.

[0144] General technology

[0145] Unless otherwise stated, the practice of this invention will utilize conventional techniques of molecular biology, recombinant DNA, biochemistry, and chemistry, which are within the scope of the art to which this invention pertains. Such techniques are well explained in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (edited by DN Glover, 1985); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Mullis et al., U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (edited by B.D. Hames & S.J. Higgins, 1984); B. Perbal, A Practical Guide to Molecular Cloning (1984); Methods in Enzymology (Academic Press, Inc., NY); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[0146] Characteristics of freeze-dried vaccine compositions

[0147] This invention provides a thermostable lyophilized vaccine composition comprising antigen and / or adjuvant. The invention describes lyophilized and stored oil-in-water emulsion formulations, or those exposed to temperatures between about 8°C and about 60°C, wherein the emulsion formed upon reconstitution may have one or more of the following characteristics: (1) not exhibiting stratification; (2) maintaining a desired pH at approximately physiological pH 7.4; (3) maintaining a Z-mean diameter of particles less than about 200 nm with little or no aggregation; (4) a loss of active ingredient concentration not exceeding or approximately equal to 25% of the initial oil-in-water emulsion formulation before lyophilization, or exhibiting any significant degradation or alteration of the various active ingredients (e.g., antigens, adjuvants); and (5) being suitable for inducing or stimulating an immune response in an individual.

[0148] These freeze-dried formulations contain cake-forming excipients comprising: (a) a combination of mannitol and sugars selected from the group consisting of trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose; (b) sugars selected from the group consisting of trehalose, lactose, raffinose, and lactulose; or (c) sugars selected from the group consisting of lactose, raffinose, and lactulose.

[0149] These heat-stable formulations represent an improvement over existing technologies and can significantly reduce vaccine losses by more than 50% annually due to the inability to maintain cold chain storage in many developed countries. Cold chain storage is described by the Centers for Disease Control and Prevention (CDC) and the United States Drug Administration (UAFDA) at http: / / www.vac-storagecdcpDF.pdf. Furthermore, ambient temperatures exceeding 25°C occur in many parts of developing countries; therefore, the heat-stable vaccine formulations described herein can be stored, exposed to, or maintained at temperatures 25°C above ambient temperature.

[0150] In one aspect, the desired thermal stability characteristics of a thermostable lyophilized vaccine composition are that the lyophilized composition should possess certain desired properties, including: long-term stability; short remodeling time; maintaining a pie appearance after storage equivalent to the pie appearance immediately after lyophilization; maintaining the remodeled properties of the original dosage form, including solution properties, protein structure or conformation; and particle size and distribution (Frank Kofi Bedu-Addo in Understanding Lyophilization Development. Pharmaceutical Technology). Additional desired properties of the thermostable lyophilized vaccine composition may include one or more of the following: long-term stability at temperatures above 8°C typical of cold chain products; short remodeling time; maintaining a pie appearance substantially similar to the pie appearance immediately after lyophilization after storage, exposure, or maintenance at about 8°C or above; maintaining the original dosage form properties of the vaccine active ingredient (including, but not limited to, antigen concentration and / or structure and adjuvant concentration) (with an increase or decrease of 25% of the original concentration or function) and maintaining solution properties, protein structure or conformation (if included) after remodeling; and particle size and distribution not greater than an average particle size of at least or about 200 nm.

[0151] In one embodiment, as used herein, a heat-stable cake refers to a single-bottle lyophilized cake produced from the oil-in-water stable emulsion (SE) of the present invention, which may contain additional active ingredients of the present invention, including antigens and / or adjuvants in the presence of suitable cake-forming excipients of the present invention, and demonstrates the desired vaccine oil-in-water emulsion characteristics when stored or exposed to or transported at temperatures 2°–8°C above typical cold chain storage temperatures.

[0152] In one embodiment, a thermostable vaccine, as used herein, refers to a reconstructed vaccine composition derived from the thermostable cake / thermally stable lyophilized vaccine composition of the present invention. Furthermore, as used herein, a thermostable vaccine may also refer to a thermostable lyophilized / cake composition reconstructed as a thermostable vaccine.

[0153] Thermal stability assessment

[0154] The thermal stability of the lyophilized vaccine compositions provided herein can be assessed in the lyophilized state, before or after reconstitution. The thermal stability of the lyophilized vaccine compositions provided herein can be assessed visually and / or by means of one or more of the tests provided herein. These tests can provide an assessment of the integrity of the emulsion, antigen, and / or adjuvant after lyophilization and reconstitution.

[0155] The tests or observations described herein can be performed immediately after lyophilization, at 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, or 12 months after lyophilization, or at other times. Prior to conducting the tests or observations, the lyophilized composition can be maintained, stored, or exposed to temperatures greater than or equal to 8°C, for example, greater than or equal to 25°C, greater than or equal to 37°C, or greater than or equal to 50°C or about 60°C.

[0156] The thermal stability tests and observations described herein can be performed on the lyophilized composition immediately after reconstruction, 1 hour after reconstruction, 6 hours after reconstruction, 12 hours after reconstruction, 24 hours after reconstruction, 36 hours after reconstruction, 36 hours after reconstruction, 48 hours after reconstruction, or 1 week after reconstruction. Prior to reconstruction and the testing and observation, the lyophilized composition can be maintained, stored, or exposed to temperatures greater than or equal to 8°C, for example, greater than or equal to 25°C, greater than or equal to 37°C, or greater than or equal to 50°C or about 60°C.

[0157] Those skilled in the art will understand that the present invention is designed to provide freeze-dried vaccine compositions that can be stored and / or shipped at temperatures closer to the ambient temperatures of developed or developing countries. Therefore, in some embodiments, the freeze-dried compositions are maintained, stored, or exposed to temperatures or combinations thereof greater than 8°C, for example greater than or equal to 25°C, greater than or equal to 37°C, greater than or equal to 50°C, or about 60°C.

[0158] In some embodiments, the thermal stability of the freeze-dried vaccine compositions provided herein is assessed visually before reconstitution. In other embodiments, the thermal stability of the freeze-dried vaccine compositions provided herein is assessed after reconstitution by means of one or more tests (e.g., biophysical and biochemical tests).

[0159] In some embodiments, the thermal stability of the freeze-dried vaccine compositions provided herein is assessed visually after reconstitution. In other embodiments, the thermal stability of the freeze-dried vaccine compositions provided herein is assessed after reconstitution by means of one or more tests (e.g., biophysical and biochemical tests).

[0160] In one embodiment, the color and consistency of the lyophilized cake produced by lyophilizing an oil-in-water emulsion formulation can be observed. In some embodiments, the cake referred to herein is a porous and sponge-like structure material produced by the lyophilization process; or the cake is a solid retained after the freeze-drying process. In some embodiments, the appearance of the cake can be described as a sponge-like cake, a cute cake, and a delicate cake. In some embodiments, the cake can be visually inspected for cracks, collapses (which can also be described as wrinkling or pulling apart from the bottle edge, a depression or slight indentation on the top of the cake, or a reduction in the overall volume of the cake) and / or changes in color, discoloration, or browning. In some embodiments, the cake can be classified as a delicate cake, a white cake, a delicate white cake, a sponge-like white cake, a white cake with increased volume, a brown cake, a browned cake, or a wrinkled / shriveled cake. In some embodiments, as used herein, discoloration or browning refers to the Maillard reaction or sugar reduction that occurs when a preparation containing reducing sugars (e.g., lactose and maltose) is lyophilized and stored in a cake at 8°C or higher (e.g., 25°C, 37°C, and / or 60°C), resulting in a visual change from yellow to brown in the original cake. In some embodiments, if no cake is formed after lyophilization, the resulting composition may be characterized as a clear film, a thick film, a thick white film, or a solidified foam. In some embodiments, the desired cake of the present invention refers to a cake that exhibits the desired characteristics of a lyophilized vaccine preparation after exposure, storage, or maintenance at the typical cold chain storage temperature of 2°-8°C, or at a temperature above or about 8°C (“Excipients used in lyophilization of small molecules” Ankit Bahetia, Lokesh Kumarb, Arvind K. Bansal, J. Excipients and Food Chem. 1(1) 2010; 41-54).

[0161] In some implementations, the melting temperature (Tm) of the freeze-dried cake is measured.

[0162] In some embodiments, the emulsion particle size is evaluated after reconstructing the lyophilized composition. For example, dynamic light scattering (DLS) can be used to evaluate the emulsion particle size. In some embodiments, it is compared to the particle size of the emulsion before lyophilization (e.g., the stable emulsion state before lyophilization). In some embodiments, the emulsion particle size is not compared to the particle size before lyophilization. In some embodiments herein, the particle size is determined by measuring the Z-mean diameter (Z-Aved) of the liquid lyophilized composition. In a particular embodiment, a reconstructed emulsion of a lyophilized composition maintained, stored, or exposed to temperatures greater than or equal to 8°C is considered a thermally stable composition when it has a Z-average diameter of less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, or less than about 90 nm, less than about 80 nm, less than about 70 nm, or less than about 60 nm. In a particular embodiment, the reconstructed emulsion has a Z-average diameter ranging from about 100 nm to about 200 nm.

[0163] In some embodiments, the polydispersity index (PdI) is evaluated after reconstructing the lyophilized composition. For example, dynamic light scattering (DLS) can be used to evaluate the PdI. In some embodiments, it is compared with the PDI of the pre-lyophilized emulsion (e.g., the pre-lyophilized stable emulsion state).

[0164] In one embodiment, the zeta potential is evaluated after reconstructing the lyophilized composition. For example, dynamic light scattering (DLS) can be used to evaluate the zeta potential. In some embodiments, it is compared with the zeta potential before lyophilization (e.g., before lyophilization in a stable emulsion state).

[0165] In some embodiments, the pH of the emulsion is evaluated after reconstructing the lyophilized composition. In some embodiments, it is compared with the pH before lyophilization (e.g., before lyophilization in a stable emulsion state).

[0166] In some embodiments, the emulsion creaming is evaluated after reconstructing the lyophilized composition.

[0167] In some embodiments, the percentage of deterioration or degradation of the antigen, adjuvant, and / or other components in the reconstructed lyophilized composition is evaluated. In some embodiments, reversed-phase high-performance liquid chromatography (RP-HPLC) is used to evaluate the chemical degradation of components (if any). In one exemplary embodiment, the chemical degradation of squalene, DMPC, and GLA is monitored by RP-HPLC. In other embodiments, gel-based Coomassie brilliant blue staining is used to evaluate the degradation of the vaccine protein antigen (if any) in the reconstructed lyophilized composition. The thermostable compositions provided herein are compositions exhibiting no more than or about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% degradation, loss, or decomposition of the antigen and / or adjuvant, or other components, after reconstructing a thermostable lyophilized composition maintained at a temperature greater than or equal to 8°C.

[0168] Thermal stability characteristics

[0169] In one aspect, the freeze-dried vaccine compositions provided herein are thermally stable at 8°C or higher. In some embodiments, the freeze-dried vaccine compositions provided herein are thermally stable at 9°C or higher, at 10°C or higher, at 11°C or higher, at 12°C or higher, at 13°C or higher, at 14°C or higher, at 15°C or higher, at 16°C or higher, at 17°C or higher, at 18°C ​​or higher, at 19°C or higher, at 20°C or higher, at 25°C or higher, at 30°C or higher, at 32°C or higher, at 35°C or higher, at 37°C or higher, at 40°C or higher, at 42°C or higher, at 45°C or higher, at 50°C or higher, and at 60°C or higher. In other embodiments, the freeze-dried vaccine compositions provided herein are thermally stable at about 8°C to about 25°C, about 25°C to about 37°C, about 37°C to about 50°C, about 25°C to about 50°C, about 8°C to about 37°C, about 8°C to about 50°C, or about 8°C to about 60°C. In one exemplary embodiment, the freeze-dried vaccine compositions provided herein are thermally stable at or about 25°C. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are thermally stable at or about 37°C. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are thermally stable at or about 50°C. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are thermally stable at or about 60°C.

[0170] In some embodiments, the freeze-dried vaccine compositions provided herein are heat-stable at or above 8°C for at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, and at least 5 years. In one exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 8°C. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 8°C for at least three months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 8°C for at least six months. In yet another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 8°C for at least twelve months. In one embodiment, the freeze-dried vaccine composition provided herein is thermally stable indefinitely at a temperature greater than or approximately equal to 8°C.

[0171] In some embodiments, the freeze-dried vaccine compositions provided herein are heat-stable at or above 25°C for at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, and at least 5 years. In one exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 25°C for at least 1 month. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 25°C for at least 3 months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 25°C for at least 6 months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 25°C for at least 12 months. In one embodiment, the freeze-dried vaccine compositions provided herein are heat-stable indefinitely at or above 25°C.

[0172] In some embodiments, the freeze-dried vaccine compositions provided herein are heat-stable at or above 37°C for at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, and at least 5 years. In one exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 37°C for at least one month. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 37°C for at least three months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 37°C for at least six months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 37°C for at least twelve months. In one embodiment, the freeze-dried vaccine compositions provided herein are heat-stable indefinitely at or above 37°C.

[0173] In some embodiments, the freeze-dried vaccine compositions provided herein are heat-stable at or above 50°C for at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, and at least 5 years. In one exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 50°C for at least one month. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 50°C for at least three months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 50°C for at least six months. In another exemplary embodiment, the freeze-dried vaccine compositions provided herein are heat-stable at or above 50°C for at least twelve months. In one embodiment, the freeze-dried vaccine compositions provided herein are heat-stable indefinitely at or above 50°C.

[0174] Excipients and reagents for use in heat-stable vaccine compositions

[0175] This document provides a thermostable lyophilized vaccine composition comprising antigens and / or adjuvants. In some embodiments, the composition comprises additional reagents and / or excipients such as cake-forming excipients, cake-forming fillers, buffers, solubilizers, isotonic agents, surfactants, and / or emulsifiers.

[0176] excipient

[0177] The excipients of this invention can be used alone or in combination with other excipients, including but not limited to cake excipients, cake fillers, fillers, buffers, solubilizers, isotonic agents, tension agents, surfactants, emulsifiers, antibacterial agents and / or collapse temperature modifiers.

[0178] In some embodiments, the excipient is a substance different from the pharmacologically active pharmaceutical ingredient, which is included in the manufacturing process, or in the fill-finish process (including but not limited to lyophilization) for storing or shipping the pharmacologically active pharmaceutical ingredient, and is included in the completed pharmaceutical process.

[0179] In some embodiments, the excipient is a substance added to a liquid-stable oil-in-water emulsion formulation before freeze-drying, which produces a cake-like substance after freeze-drying.

[0180] Excipients suitable for vaccine formulations and / or lyophilization are known in the art (see, for example, Bahetia et al., 2010: J. Excipients and Food Chem.: 1(1) 41-54, Grabenstein JD. ImmunoFacts: Vaccines and Immunologic Drugs-2012 (37th edition). St Louis, MO: Wolters Kluwer Health, 2011 and by Vaccine) and include cake-forming excipients, cake-forming fillers, fillers, buffers, solubilizers, isotonic agents, tension agents, surfactants, emulsifiers, antimicrobial agents and / or collapse temperature regulators. The list of excipients currently approved in vaccines can be found at the Centers for Disease Control and Prevention (see global website cdc.gov / vaccines / pubs / pinkbook / downloads / appendices / B / excipient-table-2.pdf, September 2013, "Vaccine Excipient & Media Summary. Excipients Included in US Vaccines, by Vaccine"), and includes, but is not limited to, sucrose, D-mannose, D-fructose, dextrose, potassium phosphate, povidone-iodine C, anhydrous lactose, microcrystalline cellulose, polacolin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, castor oil, FD&C Yellow #6 aluminum lake dye, human serum albumin, fetal bovine serum, sodium bicarbonate, human diploid fibroblast culture (WI-38), Dalberg modified Eagle medium, aluminum hydroxide, benzyl chloride, formaldehyde, glutaraldehyde, amino acids, vitamins, Inorganic salts, sugars, glycerol, asparagine, citric acid, potassium phosphate, magnesium sulfate, ferric ammonium citrate, lactose, potassium aluminum sulfate, aluminum hydroxyphosphate, potassium aluminum sulfate, peptone, bovine extract, thimerosal (trace amounts), modified Mueller and Miller media, β-propiolactone, thimerosal (multi-dose vials only), sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, potassium glutamate, calcium chloride, sodium taurodeoxycholate, neomycin sulfate, polymyxin B, ovalbumin, hydrolyzed whey protein, and neomycin sulfate.

[0181] Pie excipients / pie fillers

[0182] In some embodiments, the cake-forming excipient is a substance added to a stable oil-in-water emulsion liquid formulation prior to lyophilization, resulting in a cake-like form after lyophilization. The lyophilized cake-like form is then reconstituted into a stable oil-in-water emulsion suitable for delivering pharmacologically active pharmaceutical ingredients, including the vaccines of this invention.

[0183] In some implementations, the cake-forming excipients are those substances that do not disrupt the reconstructed emulsion of the cake.

[0184] In some embodiments, the agents used as cake-forming excipients (also referred to as fillers) in this invention comprise sugars / sugars or combinations of sugars / sugars and sugar alcohols. In some embodiments disclosed herein, the sugars / sugars or combinations of sugars / sugars and sugar alcohols are used as fillers or cake-forming excipients. These include, but are not limited to, trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, stachyose, fructose, lactulose, glucose, and optionally glycerol, sorbitol, and / or mannitol.

[0185] In some embodiments, the cake-forming excipient is a combination of mannitol and sugar, wherein the sugar is selected from the group consisting of: trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, stachyose, fructose, and lactulose.

[0186] In some embodiments, the cake-forming excipient is a combination of sorbitol and sugar, wherein the sugar is selected from the group consisting of: trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, stachyose, fructose, and lactulose.

[0187] In some embodiments, the cake-forming excipient is a sugar selected from the group consisting of trehalose, lactose, raffinose, and lactulose.

[0188] In some embodiments, the cake-forming excipient is a sugar selected from the group consisting of lactose, raffinose, and lactulose.

[0189] In some embodiments, the cake-forming excipient is a sugar or a combination of sugar and sugar alcohol in the absence of glycerol. In other embodiments, the cake-forming excipient is a sugar or a combination of sugar and sugar alcohol in the presence of less than about 1% w / v glycerol, less than about 0.5% glycerol, or less than about 0.1% glycerol.

[0190] In some embodiments, the cake-forming excipient is sugar, and the sugar is present in the oil-in-water emulsion formulation prior to lyophilization or in the reconstituted oil-in-water emulsion at concentrations ranging from 0.01% w / v to about 20% w / v, from about 0.05% w / v to about 10% w / v, from about 0.05% w / v to about 5% w / v, from about 0.5% w / v to about 10% w / v, from about 0.5% w / v to about 7.5% w / v, from about 0.5% w / v to... The concentrations range from about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 2.5% w / v to about 10% w / v, about 2.5% w / v to about 7.5% w / v, about 2.5% w / v to about 5% w / v, about 1% w / v to about 2.5% w / v, about 5% w / v to about 10% w / v, or a concentration range of about 5% w / v to about 7.5% w / v. In some embodiments, the concentration of the cake-forming excipient in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion is about 5% w / v. In some embodiments, the concentration of the cake-forming excipient is about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0. Approximately 0.8% w / v, about 0.9% w / v, about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, or about 20% w / v. In some embodiments, the cake-forming excipient is provided in the presence of less than about 1% w / v glycerol, less than about 0.5% glycerol, less than about 0.1% glycerol, or in the absence of glycerol (% glycerol refers to the concentration of glycerol in the oil-in-water emulsion formulation prior to lyophilization).

[0191] In some exemplary embodiments, the cake-forming excipient is trehalose, and the concentration of trehalose in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion ranges from about 0.01% w / v to about 20% w / v, about 0.05% w / v to about 10% w / v, about 0.05% w / v to about 5% w / v, about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 7.5% w / v, and about 0.5%. w / v to about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 2.5% w / v to about 10% w / v, about 2.5% w / v to about 7.5% w / v, about 2.5% w / v to about 5% w / v, about 1% w / v to about 2.5% w / v, about 5% w / v to about 10% w / v, or a concentration range of about 5% w / v to about 7.5% w / v. In some embodiments, the concentration of trehalose in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion is about 5% w / v. In some embodiments, the concentration of trehalose is about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, and about 0. 8% w / v, about 0.9% w / v, about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, or about 20% w / v. In some embodiments where the cake-forming excipient is trehalose, trehalose is provided in the presence of less than about 1% w / v glycerol, less than about 0.5% glycerol, less than about 0.1% glycerol, or in the absence of glycerol (% glycerol refers to the concentration of glycerol in the oil-in-water emulsion formulation prior to lyophilization).

[0192] In some exemplary embodiments, the cake-forming excipient is a combination of sugar and sugar alcohol. In such embodiments, the sugar is present in a concentration ranging from about 0.01% w / v to about 20% w / v in the pre-lyophilized oil-in-water emulsion formulation or in the reconstituted oil-in-water emulsion, and the sugar alcohol is present in a concentration ranging from about 0.01% w / v to about 20% w / v. In some embodiments, sugars are present in combination with sugar alcohols, and the concentration range of sugars in the oil-in-water emulsion formulation before lyophilization or in the reconstructed oil-in-water emulsion is about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 7.5% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 2.5% w / v to about 10% w / v, about 2.5% w / v to about 7.5% w / v, about 2.5% w / v to about 5% w / v, etc. / v, about 1% w / v to about 2.5% w / v, about 5% w / v to about 10% w / v, about 5% w / v to about 7.5% w / v, or a concentration of about 5% w / v where the sugar alcohol is present in concentrations ranging from about 0.01% w / v to about 10% w / v, about 0.01% w / v to about 7.5% w / v, about 0.01% w / v to about 5% w / v, about 0.01% w / v to about 2.5% w / v, about 0.01% w / v to about 1% w / v, about 0.01% w / v to about 0.1% w / v, about 0.0 1% w / v to about 0.05% w / v, about 0.05% w / v to about 10% w / v, about 0.05% w / v to about 7.5% w / v, about 0.05% w / v to about 5% w / v, about 0.05% w / v to about 2.5% w / v, about 0.05% w / v to about 1% w / v, about 0.05% w / v to about 0.1% w / v, about 0.1% w / v to about 10% w / v, about 0.1% w / v to about 7.5% w / v, about 0.1% w / v to about 5% w / v, about 0.1% w / v About 2.5% w / v, about 0.1% w / v to about 1% w / v, about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 7.5% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 7.5% w / v, about 1% w / v to about 5% w / v, about 1% w / v to about 2.5% w / v or a concentration of about 0.1% w / v.In some embodiments, sugars are present in combination with sugar alcohols, wherein the sugar concentration is approximately 0.01% w / v, approximately 0.02% w / v, approximately 0.03% w / v, approximately 0.04% w / v, approximately 0.05% w / v, approximately 0.06% w / v, approximately 0.07% w / v, approximately 0.08% w / v, approximately 0.09% w / v, approximately 0.1% w / v, approximately 0.2% w / v, approximately 0.3% w / v, approximately 0.4% w / v, approximately 0.5% w / v, approximately 0.6% w / v, or approximately 0.7% w / v. / v, approximately 0.8% w / v, approximately 0.9% w / v, approximately 1% w / v, approximately 2% w / v, approximately 3% w / v, approximately 4% w / v, approximately 5% w / v, approximately 6% w / v, approximately 7% w / v, approximately 7.5% w / v, approximately 8% w / v, approximately 9% w / v, approximately 10% w / v, approximately 11% w / v, approximately 12% w / v, approximately 13% w / v, approximately 14% w / v, approximately 15% w / v, approximately 16% w / v, approximately 17% w / v, approximately 18% w / v, approximately 19% w / v or approximately 20% w / v / v, and the concentrations of sugar alcohol present are approximately 0.01% w / v, approximately 0.02% w / v, approximately 0.03% w / v, approximately 0.04% w / v, approximately 0.05% w / v, approximately 0.06% w / v, approximately 0.07% w / v, approximately 0.08% w / v, approximately 0.09% w / v, approximately 0.1% w / v, approximately 0.2% w / v, approximately 0.3% w / v, approximately 0.4% w / v, approximately 0.5% w / v, approximately 0.6% w / v, approximately 0.7% w / v, approximately 0.8% w / v, and approximately 0. 9% w / v, about 1% w / v, about 1.5%, about 2% w / v, about 2.5%, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, or about 20% w / v. In some embodiments, a combination of sugar and sugar alcohol is provided in the presence of less than about 1% w / v glycerol, less than about 0.5% glycerol, less than about 0.1% glycerol, or in the absence of glycerol (% glycerol refers to the concentration of glycerol in the oil-in-water emulsion formulation before lyophilization).

[0193] In some exemplary embodiments, the cake-forming excipient is a combination of trehalose and mannitol. In such embodiments, the concentration of trehalose in the pre-lyophilized oil-in-water emulsion formulation ranges from about 0.01% w / v to about 20% w / v, and the concentration of mannitol in the pre-lyophilized oil-in-water emulsion formulation or in the reconstituted oil-in-water emulsion ranges from about 0.01% w / v to about 20% w / v. In some embodiments, trehalose is present in combination with mannitol, and the concentration range of trehalose in the oil-in-water emulsion formulation before lyophilization or in the reconstructed oil-in-water emulsion is about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 7.5% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 2.5% w / v to about 10% w / v, about 2.5% w / v to about 7.5% w / v, and about 2.5% w / v to about 5% w / v. The concentrations are approximately 1% w / v to approximately 2.5% w / v, approximately 5% w / v to approximately 10% w / v, approximately 5% w / v to approximately 7.5% w / v, or a concentration of approximately 5% w / v where mannitol is present in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion at concentrations ranging from approximately 0.01% w / v to approximately 10% w / v, approximately 0.01% w / v to approximately 7.5% w / v, approximately 0.01% w / v to approximately 5% w / v, approximately 0.01% w / v to approximately 2.5% w / v, approximately 0.01% w / v to approximately 1% w / v, approximately 0.01% w / v to approximately 1% w / v, approximately 0.01% w / v to approximately 2.5 ... v to about 0.1% w / v, about 0.01% w / v to about 0.05% w / v, about 0.05% w / v to about 10% w / v, about 0.05% w / v to about 7.5% w / v, about 0.05% w / v to about 5% w / v, about 0.05% w / v to about 2.5% w / v, about 0.05% w / v to about 1% w / v, about 0.05% w / v to about 0.1% w / v, about 0.1% w / v to about 10% w / v, about 0.1% w / v to about 7.5% w / v, about 0.1% w / v to about 5% w / v, About 0.1% w / v to about 2.5% w / v, about 0.1% w / v to about 1% w / v, about 0.5% w / v to about 10% w / v, about 0.5% w / v to about 7.5% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 1% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 7.5% w / v, about 1% w / v to about 5% w / v, about 1% w / v to about 2.5% w / v, or a concentration of about 0.1% w / v.In some embodiments, trehalose is present in combination with mannitol, wherein the concentration of trehalose in the oil-in-water emulsion formulation before lyophilization or in the reconstructed oil-in-water emulsion is about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, Approximately 0.6% w / v, approximately 0.7% w / v, approximately 0.8% w / v, approximately 0.9% w / v, approximately 1% w / v, approximately 2% w / v, approximately 3% w / v, approximately 4% w / v, approximately 5% w / v, approximately 6% w / v, approximately 7% w / v, approximately 7.5% w / v, approximately 8% w / v, approximately 9% w / v, approximately 10% w / v, approximately 11% w / v, approximately 12% w / v, approximately 13% w / v, approximately 14% w / v, approximately 15% w / v, approximately 16% w / v, approximately 17% w / v, approximately 18% w / v, approximately 19% w / v, or approximately 20%. The concentrations of mannitol in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion are approximately 0.01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, 0.1% w / v, 0.2% w / v, 0.3% w / v, 0.4% w / v, 0.5% w / v, 0.6% w / v, and 0.7% w / v. Approximately 0.8% w / v, approximately 0.9% w / v, approximately 1% w / v, approximately 1.5%, approximately 2% w / v, approximately 2.5%, approximately 3% w / v, approximately 4% w / v, approximately 5% w / v, approximately 6% w / v, approximately 7% w / v, approximately 7.5% w / v, approximately 8% w / v, approximately 9% w / v, approximately 10% w / v, approximately 11% w / v, approximately 12% w / v, approximately 13% w / v, approximately 14% w / v, approximately 15% w / v, approximately 16% w / v, approximately 17% w / v, approximately 18% w / v, approximately 19% w / v, or approximately 20% w / v. In some embodiments, a combination of trehalose and mannitol is provided in the presence of less than about 1% w / v glycerol, less than about 0.5% glycerol, less than about 0.1% glycerol, or in the absence of glycerol (% glycerol refers to the concentration of glycerol in the oil-in-water emulsion formulation before lyophilization).

[0194] In other embodiments, the reagents used as cake-forming excipients for the present invention include any amino acids. Exemplary amino acids used as fillers in the present invention include arginine, glycine, proline, glutamic acid, methionine, cysteine, proline, and histidine, either individually or in combination, in pure molecular form or in formulated form.

[0195] In other embodiments, the filler includes polymers such as dextran and polyethylene glycol.

[0196] buffer

[0197] In some embodiments, the compositions of the present invention comprise a buffer. Buffers used as excipients in the present invention include Tris acetic acid, Tris base, Tris HCl, ammonium phosphate, citric acid, sodium citrate, potassium citrate, tartaric acid, sodium phosphate, zinc chloride, arginine, and histidine. In some embodiments, the buffer includes pH adjusters such as hydrochloric acid, sodium hydroxide, and meglumine.

[0198] Solubilizer

[0199] In some embodiments, suitable solubilizers include complexing excipients such as ethylenediaminetetraacetic acid (EDTA), α-cyclodextrin, and hydroxypropyl-β-cyclodextrin (HP-β-CD). Surfactants including polysorbate 80 and Tween may also be included as solubilizing excipients. Other cosolvents known in the art as solubilizers may be used, including tert-butanol, isopropanol, dichloromethane, ethanol, and acetone.

[0200] The tonicifying agents used as excipients in this invention include glycerol, sodium chloride, sucrose, mannitol, and dextran. Collapse temperature regulators include dextran, hydroxyethyl starch, ficoll, and gelatin. Antimicrobial agents include benzyl alcohol, phenol, m-cresol, methylparaben, ethylparaben, and thimerosal.

[0201] isotonic agent

[0202] In some embodiments, the compositions of the present invention comprise an isotonic agent. In some embodiments, the isotonic agent is glycerol. In a particular embodiment, the isotonic agent is present at a concentration of about 0.36% v / v in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion.

[0203] surfactants

[0204] In some embodiments, the compositions of the present invention comprise a surfactant. In some embodiments, the surfactant is Pluronic F68. In some embodiments, the surfactant content is about 100:1 (oil:surfactant). In some embodiments, the surfactant concentration is about 0.018% w / v. In some embodiments, the surfactant concentration is about 0.0001% w / v, about 0.0005% w / v, about 0.001% w / v, about 0.005% w / v, about 0.01% w / v, about 0.011% w / v, about 0.012% w / v, about 0.013% w / v, about 0.014% w / v, about 0.015% w / v, about 0.016% w / v, about 0.017% w / v, about 0.018% w / v, or about 0.019%. w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, or about 1% w / v. The percentages and ratios mentioned herein refer to the ratios and percentages in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion.

[0205] emulsifier

[0206] In some embodiments, the compositions of the present invention comprise an emulsifier. In some embodiments, the emulsifier is 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC). In some embodiments, the emulsifier is lecithin. In some embodiments, the emulsifier is present in a ratio of about 1:5 (emulsifier: oil). In some embodiments, the concentration of the emulsifier is about 0.38% w / v. In some embodiments, the concentration of the emulsifier is about 0.002% w / v, about 0.005% w / v, about 0.010% w / v, about 0.015% w / v, about 0.020% w / v, about 0.025% w / v, about 0.030% w / v, about 0.035% w / v, about 0.040% w / v, about 0.045% w / v, about 0.050% w / v, about 0.055% w / v, about 0.060% w / v, about 0.065% w / v, about 0.070% w / v, about 0.075% w / v, about 0.080% w / v, about 0.085% w / v, about 0.090% w / v, about 0.095% w / v, about 0.10% w / v, about 0.15% w / v, approximately 0.20% w / v, approximately 0.25% w / v, approximately 0.30% w / v, approximately 0.35% w / v, approximately 0.40% w / v, approximately 0.45% w / v, approximately 0.50% w / v, approximately 0.55% w / v, approximately 0.60% w / v, approximately 0.65% w / v, approximately 0.70% w / v, approximately 0.75% w / v % w / v, about 0.80% w / v, about 0.85% w / v, about 0.90% w / v, about 0.95% w / v, about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 9% w / v, or about 10% w / v. The percentages and ratios mentioned herein refer to the ratios and percentages in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion.

[0207] Adjuvants used in thermostable freeze-dried vaccine compositions

[0208] In some aspects of the invention provided herein, the compositions described herein (e.g., thermostable lyophilized vaccines) comprise an adjuvant. In some embodiments, the adjuvant is provided alone, for example, for use as a therapeutic agent. In other embodiments, the adjuvant is provided in combination with an antigen. Adjuvants used to modify the immune response in the composition are well known in the art. For example, adjuvants used in the compositions described herein may include one or more immunostimulatory adjuvants, delivery adjuvants, inorganic adjuvants, or organic adjuvants. Non-limiting examples of adjuvants used in the compositions described herein can be found in particular Barouch DH, 2008, Nature, 455(7213): 613-9; Morrow et al., 2008, AIDS, 22(3): 333-8; and McGeary et al., 2003, Peptide Sci., 9(7): 405-181.

[0209] In some embodiments, the adjuvant used in the compositions described herein (e.g., thermostable lyophilized vaccines) is an immunostimulatory adjuvant. An immunostimulatory adjuvant may be an adjuvant that acts directly on the immune system, such as cytokines, TLR ligands, or microbial toxins. In some embodiments herein, the adjuvant is a cytokine adjuvant. One or more cytokines may be used alone as adjuvants or in combination with one or more additional adjuvants in the compositions described herein. Suitable cytokines include interferon (IFN), interleukin (IL), chemokines, colony-stimulating factors, or tumor necrosis factor. In some embodiments, the interferon is type I, type II, or type III IFN. In some embodiments, the interferon is IFN-α, IFN-β, IFN-γ, or IFN-λ, and subtypes derived from these (e.g., IFN-λ, IFN-λ2, and IFN-λ3). In some embodiments, the cytokine is an interleukin. Non-limiting examples of interleukins that can be used as adjuvants in the compositions described herein include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36. In some embodiments, the cytokine is a chemokine. In some embodiments, the chemokine is a Cx chemokine, a CXxC chemokine, a Cx chemokine, or a CXx3C chemokine. Non-limiting examples of CC chemokines that can be used as adjuvants in the compositions described herein include CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28. Non-limiting examples of CXC chemokines that can be used in the compositions described herein include CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17. In some implementations, cytokines are colony-stimulating factors.In some embodiments, the colony-stimulating factor is granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or macrophage colony-stimulating factor (M-CSF). In some embodiments, the cytokine is tumor necrosis factor. Non-limiting examples of tumor necrosis factor family proteins that can be used as adjuvants in the compositions described herein include TNF-α and 4-1BBL.

[0210] In some embodiments, the immunostimulatory adjuvant is a Toll-like receptor (TLR) ligand (e.g., a TLR agonist). One or more TLR ligands may be used alone as adjuvants or in combination with one or more additional adjuvants in the compositions described herein. TLRs include transmembrane receptors on the cell surface of the innate immune system, which endow host cells with the ability to recognize a variety of conserved microbial molecular structures at an early stage. These molecular structures can be present in or on the surface of numerous infectious pathogens (e.g., 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; Kaiso et al., 2004 Microbes Infect. 6: 1388; Datta et al., 2003 J. Immunol. 170: 4102).

[0211] Inducing TLR-mediated signal transduction to promote the initiation of an immune response through the innate immune system can be achieved by TLR agonists (i.e., TLR ligands) that bind to TLRs on the cell surface. For example, lipopolysaccharide (LPS) can be a TLR agonist acting via TLR2 or TLR4 (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(inosine-cytidine) (polyI:C) can be a TLR agonist acting via TLR3 (Salem et al., 2006 Vaccine 24: 5119); CpG sequences (oligodeoxynucleotides containing unmethylated cytosine-guanine or “CpG” dinucleotide motifs, such as CpG7909, 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) can be TLR agonists acting through TLR9 (Andaloussi et al., 2006 Glia 54:526; Chen et al., 2006 J. Immunol. 177:2373); peptidoglycan can be TLR2 and / or TLR6 agonists (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-imidazol[4,5-C]quinoline-1-ethanol hydrate, molecular weight 318 Da, from 3M Pharmaceuticals, St. Paul, MN, which is also the source of related compounds 3M001 and 3M002; Gorden et al., 2005 J. Immunol. 174: 1259) may be a TLR7 agonist (Johansen 2005 Clin. Exp. Allerg. 35: 1591) and / or a TLR8 agonist (Johansen 2005); flagellin may be a TLR5 agonist (Feuillet et al., 2006 Proc. Nat. Acad. Sci.).USA 103:12487); the inhibitory protein may be a TLR11 agonist (Hedhli et al., 2009, Vaccine, 27(16):2274-87); the lipopeptide may be a TLR1, TLR2, and / or TLR6 agonist (Gao et al., 2013, Vaccine, 31(26):2796-803); and hepatitis C antigen may be a TLR agonist acting 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 antigens are known (e.g., Schirmbeck et al., 2003 J.Immunol.171:5198) and may be used according to some of the embodiments described herein.

[0212] For example, based on background knowledge (see, for example, U.S. Patent No. 6,544,518), it is known that immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides (“CpG”) can serve as adjuvants for administration via both systemic and mucosal routes (WO 96 / 02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2): 870-876; McCluskie and Davis, J. Immunol., 1998, 161(9): 4463-6). CpG is an abbreviation for cytosine-guanine dinucleotide motifs present in DNA. The important role of CG motifs in immunostimulation was elucidated by Krieg, Nature 374, p546, 1995. Detailed analysis has shown that CG motifs must be within a specific sequence context, and these sequences are common in bacterial DNA but rare in vertebrate DNA. The immunostimulatory sequence is typically: purine, purine, C, G, pyrimidine, pyrimidine; wherein the dinucleotide CG motif is not methylated, but other unmethylated CpG sequences are known to be immunostimulatory and can be used in some embodiments of the present invention. When CpG is formulated in a vaccine, it can be administered in a free solution with a free antigen (WO 96 / 02555; McCluskie and Davis, see above), or covalently coupled with an antigen (PCT Publication No. WO 98 / 16247), or formulated with a carrier such as aluminum hydroxide (e.g., Davis et al., see above, Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

[0213] In some embodiments, the preferred oligonucleotide used as an adjuvant of the present invention comprises two or more dinucleotide CpG motifs separated by at least three, more preferably at least six or more nucleotides. The oligonucleotides of the present invention are typically deoxynucleotides. In a preferred embodiment, the internucleotide bonds in the oligonucleotide are dithiophosphate or more preferably thiophosphate bonds, although phosphodiester and other internucleotide bonds are within the scope of the present invention, which includes oligonucleotides with mixed internucleotide bonds. Methods for producing thiophosphate oligonucleotides or dithiophosphates are described in U.S. Patent Nos. 5,666,153, 5,278,302, and WO95 / 26204.

[0214] Examples of preferred oligonucleotides have sequences disclosed in the following publications; for some embodiments disclosed herein, the sequences preferably contain phosphate-thiolated internucleotide bonds: (1) CPG 7909: Cooper et al., “CPG 7909 adjuvant improves hepatitis B virus vaccines eroprotection in antiretroviral-treated HIV-infected adults.” AIDS, Sep 23, 2005; 19(14): 1473-9; (2) CpG 10101: Bayes et al., “Gateways to clinical trials.” MethodsFind. Exp. Clin. Pharmacol. Apr 2005; 27(3): 193-219; and (3) Vollmer J., “Progress indrug development of immunostimulatory CpG oligodeoxynucleotide ligands for TLR9.” Expert Opinion on Biological Therapy. May 2005; 5(5): 673-682.

[0215] Optional CpG oligonucleotides may include preferred sequence variants described in the cited publications, differing only in having inconsequential nucleotide sequence substitutions, insertions, deletions, and / or additions. CpG oligonucleotides used in certain embodiments of the invention can be synthesized by any method known in the art (e.g., EP468520). Such oligonucleotides can be conveniently synthesized using automated synthesizers. The oligonucleotides are typically deoxynucleotides. In a preferred embodiment, the internucleotide bonds in the oligonucleotide are dithiophosphate bonds, or more preferably thiophosphate bonds, although phosphate diesters are also within the scope of embodiments of the invention. Oligonucleotides comprising different internucleotide bonds are also involved, for example, mixed thiophosphate phosphate diesters. Other internucleotide bonds in stable oligonucleotides may also be used.

[0216] In some embodiments, the adjuvant is a TLR4 agonist. In some embodiments, the TLR4 agonist used in the compositions of the present invention comprises a glucopyranoside adjuvant (GLA), such as those described in U.S. Patent Publications US2007 / 021017, US2009 / 045033, US2010 / 037466, and US2010 / 0310602, the contents of which are incorporated herein by reference in their entirety.

[0217] For example, in some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0218]

[0219] Or its pharmaceutically acceptable salt, wherein:

[0220] L1, L2, L3, L4, L5, and L6 may be the same or different, and are independently -O-, -NH-, or -(CH2)-;

[0221] L7, L8, L9 and L 10 Same or different, and independently non-existent or -C(=O)-;

[0222] Y1 is an acidic functional group;

[0223] Y2 and Y3 may be the same or different, and are independently -OH, -SH or acid functional groups;

[0224] Y4 is -OH or -SH;

[0225] R1, R3, R5, and R6 may be the same or different, and each is independently C. 8-13 Alkyl; and

[0226] R2 and R4 may be the same or different, and are independently C. 6-11 alkyl.

[0227] In some embodiments of the synthetic GLA structure, R 1 R 3 R 5 and R 6 It is C 10 Alkyl; and R 2 and R 4 It is a C8 alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0228] For example, in some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0229]

[0230] In one particular implementation, R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C 12 -C 20 alkyl.

[0231] In another specific embodiment, the GLA has the structure shown above, wherein R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is C 13 alkyl.

[0232] In another specific embodiment, the GLA has the structure shown above, wherein R 1 R 3 R 5 and R 6 It is C 10 Alkyl; and R 2 and R 4 It is a C8 alkyl group.

[0233] In another specific embodiment, the GLA has the structure shown above, wherein R 1 R 3 R 5 and R6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0234] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0235]

[0236] In some embodiments of the GLA structure described above, R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0237] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0238]

[0239] In some embodiments of the GLA structure described above, R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R2 and R 4 It is a C9 alkyl group.

[0240] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0241]

[0242] In some embodiments of the GLA structure described above, R 1 R 3 R 5 and R 6 It is C 11 -C 20 Alkyl; and R 2 and R 4 It is C9-C 20 Alkyl group. In some embodiments, R 1 R 3 R 5 and R 6 It is C 11 Alkyl; and R 2 and R 4 It is a C9 alkyl group.

[0243] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0244]

[0245] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0246]

[0247] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0248]

[0249] In another embodiment, the adjuvant used in the composition of the present invention is an attenuated lipid A derivative (ALD). An ALD is a lipid A-like molecule that has been modified or constructed to exhibit fewer or different side effects from lipid A. These side effects include pyrogenicity, local Shwarzman reactivity, and toxicity, which is determined at a 50% lethal dose in chicken embryos (CELD). 50The ALDs available according to the present invention, as evaluated, include monophosphoryl lipid A (MLA) and 3-deacylated monophosphoryl lipid A (3D-MLA). MLA and 3D-MLA are known and need not be detailed herein. See, for example, U.S. Patent No. 4,436,727, assigned to Ribi ImmunoChem Research, Inc., dated March 13, 1984, which discloses monophosphoryl lipid A and its manufacture. U.S. Patent No. 4,912,094 and Reexamination Certificate B1, also assigned to Ribi ImmunoChem Research, Inc., implement 3-deacylated monophosphoryl lipid A and a method for its manufacture.

[0250] In some embodiments, other immunomodulators, such as imidazoquinoline immunomodulators, are also known in the art and may be included as adjuvants in some embodiments disclosed herein. Certain preferred imidazoquinoline immunomodulators include (as non-limiting examples) resimimod (R848), imiquimod, and gademod (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 imidazoquinoline immunomodulators may also have TLR agonist activity as described herein under appropriate conditions. Other immunomodulators are nucleic acid-based bistem-ring immunomodulators (dSLIMs). See Schmidt et al., 2006 Allergy 61:56; Weihrauch et al., 2005 ClinCancer Res.11(16):5993-6001; Modern Biopharmaceuticals, J. (Editor). John Wiley & Sons, December 6, 2005 (pages 183 to 200 discuss dSLIM) and specific examples of dSLIM used in certain embodiments of the present invention were found in Mologen AG (Berlin, FRG: [retrieved online 8 / 18 / 06, see global network mologen.com / English / 04.20-dSLIM.shtml]).

[0251] In some embodiments, the adjuvant used in the compositions described herein is a polysaccharide derived from bacteria or plants. Non-limiting examples of polysaccharide-based adjuvants that may be used alone or in combination with one or more additional adjuvants in the compositions described herein include dextran (e.g., β-glucan), dextran (e.g., sulfated and diethylaminoethyl-dextran), glucomannan, galactomannan, levanan, xylan, fructan (e.g., inulin), chitosan, endotoxins (e.g., lipopolysaccharide), polysaccharide MGN-3, polysaccharides derived from Actinidis aestivum, edesomurin and variants thereof.

[0252] In some embodiments, the adjuvant used in the compositions described herein is a protein body or a subunit thereof. In some embodiments, the adjuvant used in the compositions described herein comprises the same or different antigenic peptide sequences assembled around a lysine core. In some embodiments, the adjuvant used in the compositions described herein is a toxin (e.g., a bacterial toxin). In some embodiments, the toxin is derived from one or more bacteria selected from the group consisting of: *Escherichia coli*, *Vibrio cholerae*, *Bordetella pertussis*, and *Bordetella parapertussis*.

[0253] In some embodiments, the adjuvant used in the compositions described herein (e.g., thermostable lyophilized vaccines) is a delivery adjuvant. Delivery adjuvants can serve as both adjuvants and / or deliverable antigens. Non-limiting examples of adjuvants that can be used alone or in combination with one or more additional adjuvants in the compositions described herein include mineral salts (e.g., calcium phosphate), emulsions (e.g., squalene in water), liposomes (e.g., DPPC:cholesterol liposomes), virions (e.g., immune-enhancing recombinant influenza virions), and microspheres.

[0254] Other adjuvants used according to certain embodiments disclosed herein include block copolymers or biodegradable polymers, which relate to a class of polymeric compounds familiar to those skilled in the art. Examples of block copolymers or biodegradable polymers that may be included in the compositions described herein include L121 (BASF Corp., Mount Olive, NJ; see, for example, Yeh et al., 1996 Pharm. Res. 13: 1693; U.S. Patent No. 5,565,209), CRL1005 (for example, Triozzi et al., 1997 Clin Canc. Res. 3: 2355), poly(lactic acid-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly-(D,L-lactide-co-glycolic acid) (PLG), and polyI:C (see, for example, Powell and Newman, “Vaccine design - The Subunit and Adjuvant Approach”, 1995, Plenum Press, New York).

[0255] In some embodiments, the adjuvant used in the compositions described herein (e.g., thermostable freeze-dried vaccines) is an organic adjuvant. Organic adjuvants can be biologically derived or chemically carbon-containing adjuvants. In some embodiments, the adjuvant is a peptide derived from the cell wall of a microorganism (e.g., muramyl dipeptide and its variants). In some embodiments, the adjuvant is trehalose 6,6'-dimycinate or a variant thereof. See Schweneker et al., 2013, Immunobiology, 218(4): 664-73. In some embodiments, the adjuvant is stearoyltyrosine.

[0256] According to certain embodiments described herein, saponins and saponin analogues, including QS21 and structure-related compounds that produce similar effects and are referred to herein as QS21 analogues (see, for example, U.S. Patent No. 5,057,540; EP 0362 279 B1; WO 95 / 17210), alkaloids such as tomatine, detergents such as (but not limited to) saponins, polysorbate 80, Span 85 and stearoyl tyrosine, imidazoquinoline immunomodulators, and bicyclic immunomodulators (dSLIM, for example, Weeratna et al., 2005 Vaccine 23: 5263) may be used as adjuvants.

[0257] In some embodiments, the adjuvant used in the compositions described herein is a saponin or a saponin analogue. Cleansers comprising saponins are taught, for example, in U.S. Patent 6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine 2: 363-386), U.S. Patent No. 5,057,540, Kensil, Crit Rev Ther DrugCarrier Syst, 1996, 12(1-2): 1-55, and EP 0 362 279 B1. Microparticle structures comprising the Quil A moiety (saponin), known as immunostimulatory complexes (ISCOMS), are hemolytic and have been used in vaccine preparation (Morein, B., EP0 109 942 B1). These structures have been reported to have adjuvant activity (EP 0 109 942 B1; WO 96 / 11711). These hemolytic saponins QS21 and QS17 (HPLC-purified fractions of QuilA) have been described as effective systemic adjuvants, and their preparation methods are disclosed in U.S. Patent Nos. 5,057,540 and EP 0 362 279 B1. QS21 may comprise a non-toxic fraction purified by HPLC from the bark of the soapberry tree. The production of QS21 is disclosed in U.S. Patent No. 5,057,540 (see also U.S. Patent Nos. 6,936,255, 7,029,678, and 6,932,972). The use of QS7 (the non-hemolytic fraction of Quil-A) as an effective adjuvant for systemic vaccines is also described in these references. The use of QS21 is further described in Kensil et al. (1991. J. Immunology 146: 431-437). Combinations of QS21 with polysorbates or cyclodextrins are also known (WO 99 / 10008). Microparticle adjuvant systems containing QuilA moieties such as QS21 and QS7 are described in WO96 / 33739 and WO 96 / 11711. Other saponins that have been used in systemic vaccine studies include those from other plant species such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10(9): 572-577, 1992).

[0258] In some embodiments, the adjuvant is an "immunostimulatory complex" known as ISCOMS (e.g., U.S. Patent Nos. 6,869,607, 6,846,489, 6,027,732, 4,981,684), including those derived from saponins. It can be purchased from companies such as Iscotec (Stockholm, Sweden) and CSL Ltd. (Parkville, Victoria, Australia).

[0259] Aescin is another detergent associated with saponins in the adjuvant compositions used in the embodiments disclosed herein. Aescin is described in the Merck Index (12th edition: entry 3737) as a mixture of saponins found in the seeds of the horse chestnut tree, Aesculus hippocastanum. It is described by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)) and separation by ion exchange resin (Erbring et al., U.S. Patent No. 3,238,190). Aescin fractions (also known as aescin glycosides) have been purified and shown to have biological activity (Yoshikawa M et al. (Chem Pharm Bull (Tokyo) August 1996; 44(8): 1454-1464)). Digitalis saponins are another type of detergent, also described in the Merck Index (12th edition, entry 3204) as saponins derived from the seeds of digitalis (Digitalis purpurea) and purified according to the methods described in Gisvold et al., J. Am. Pharm. Assoc., 1934, 23, 664; and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

[0260] In some embodiments, the adjuvants used in the compositions described herein (e.g., heat-stable lyophilized vaccines) are inorganic adjuvants. Inorganic adjuvants can be adjuvants that are not typically carbon-based, such as mineral salts, emulsions, and calcium phosphate. Mineral salt adjuvants of interest herein include, but are not limited to, aluminum-based compounds such as aluminum phosphate and aluminum hydroxide. As used herein, calcium phosphate adjuvants include, but are not limited to, calcium ions (Ca2+) along with orthophosphate (PO43-), metaphosphate (PO3-), or pyrophosphate (P2O74-).

[0261] Additionally, as mentioned above, one type of adjuvant used in the compositions described herein can be an aluminum adjuvant, commonly referred to as "alum." Alum adjuvants are based on aluminum hydroxyaluminate, aluminum hydroxyphosphate, or various proprietary salts. Vaccines using alum adjuvants may include vaccines for tetanus strains, HPV, hepatitis A virus, poliovirus, and other antigens described herein. Aluminum adjuvants are advantageous because they have a good safety record, amplify antibody responses, stabilize antigens, and are relatively simple for large-scale production (Edelman 2002 Mol. Biotechnol. 21: 129-148; Edelman, R. 1980 Rev. Infect. Dis. 2: 370-383).

[0262] In some embodiments, the compositions of the present invention comprise an adjuvant. In some embodiments, the adjuvant is a TLR4 agonist. In some embodiments, the adjuvant is present at a concentration of about 0.5 μg / mL to about 12 mg / mL. In some embodiments, the adjuvant is present at a concentration of about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, or about 100 μg / mL. In some embodiments, the adjuvant is MPL or GLA as described herein. In some embodiments, the adjuvant is present at a concentration of about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, or about 12 mg / mL.

[0263] Suitable adjuvants used in some of the compositions described herein (e.g., thermostable lyophilized vaccine compositions) include commercially available adjuvants such as Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 and its derivatives (SmithKlineBeecham, Philadelphia, Pa.); AddaVax (InvivoGen); MF59 (Norvartis); AS03 (GlaxoSmithKline); AS01B (GlaxoSmithKline); AS02A (GlaxoSmithKline).

[0264] Some embodiments provided herein include compositions (e.g., thermostable freeze-dried vaccine compositions) that contain an adjuvant and at least one more adjuvant different from the first adjuvant. For example, compositions provided herein may contain GLA and a second adjuvant different from GLA. In some embodiments, compositions provided herein contain two, three, four, or five adjuvants. In some embodiments, compositions provided herein contain two adjuvants.

[0265] Adjuvants as described herein include an adjuvant that, when administered to an individual such as a human (e.g., a human patient), a non-human primate, a mammal, or another higher eukaryote with a recognized immune system, is capable of altering (i.e., statistically significantly increasing or decreasing; and in some embodiments, enhancing or increasing) the immune response and / or its lifespan (see, for example, Powell and Newman, “Vaccine design—The Subunit and Adjuvant Approach,” 1995, Plenum Press, New York). In some embodiments disclosed herein, the GLA and the desired antigen, and optionally one or more adjuvants, can thus be altered, for example, to induce or enhance an immune response against the desired antigen.

[0266] Antigen for use in heat-stable freeze-dried vaccine compositions

[0267] In some embodiments, the thermostable vaccine composition is used to induce or enhance the immune reactivity or immune response against the antigen in the host.

[0268] In some embodiments, the antigen may already be present in the host, such as an autoantigen, allergen, or cancer antigen, and the vaccine composition may contain only a stabilizing emulsion and optional adjuvant to induce or enhance an immune response against an antigen already present in the individual upon administration. Administration of a vaccine composition comprising a heat-stable lyophilized emulsion and an adjuvant to induce an immune response against an antigen already present in the host, as used herein, is a monotherapy.

[0269] In some embodiments, the vaccine compositions described herein contain one or more antigens.

[0270] In certain embodiments of the compositions described herein and methods for producing and using such compositions, the antigen may be any target antigenic epitope, molecule (including biomolecules), molecular complex (including molecular complexes containing biomolecules), subcellular cluster, cell, or tissue against which a desired immune reactivity is induced or enhanced in an individual. Generally, the term antigen refers to the polypeptide antigen of interest. However, as used herein, antigen may also refer to a recombinant construct (e.g., an expression construct) encoding the polypeptide antigen of interest. In some preferred embodiments, the antigen may be, or may be derived from, an infectious pathogen and / or epitope, biomolecule, cell, or tissue associated with infection, cancer, autoimmune disease, allergy, asthma, or any other state (where activation of an antigen-specific immune response would be desirable or beneficial), or immunologically cross-reactive with it.

[0271] In some embodiments, the antigen may be present at any concentration sufficient to induce or enhance a desired level of immune reactivity in an individual. In some embodiments, the antigen may be present at concentrations of about 0.1 μg / mL to about 50 μg / mL, about 1 μg / mL to about 50 μg / mL, about 2.5 μg / mL to about 50 μg / mL, about 5 μg / mL to about 50 μg / mL, about 10 μg / mL to about 50 μg / mL, 0.1 μg / mL to about 25 μg / mL, about 1 μg / mL to about 25 μg / mL, about 2.5 μg / mL to about 25 μg / mL, about 5 μg / mL to about 25 μg / mL, or about 10 μg / mL to about 25 μg / mL. The concentrations are present in the range of g / mL, from 0.1 μg / mL to about 10 μg / mL, from about 1 μg / mL to about 10 μg / mL, from about 2.5 μg / mL to about 10 μg / mL, from about 5 μg / mL to about 10 μg / mL, from 0.1 μg / mL to about 5 μg / mL, from about 1 μg / mL to about 5 μg / mL, from about 2.5 μg / mL to about 5 μg / mL, from 0.1 μg / mL to about 2.5 μg / mL, from about 1 μg / mL to about 2.5 μg / mL, or from about 0.1 μg / mL to about 1 μg / mL. The concentrations provided refer to the antigen concentration in the oil-in-water emulsion formulation before lyophilization or in the reconstructed oil-in-water emulsion.

[0272] In some embodiments, the compositions described herein (e.g., thermostable lyophilized vaccine compositions) comprise antigens or antigenic components capable of inducing an immune response against human or other mammalian pathogens. These antigens or antigenic components may include viral components, such as those from HIV-1 (e.g., tat, nef, gp120, or gp160), human herpesviruses (e.g., gD or derivatives thereof, or the immediate early protein of ICP27 from HSV1 or HSV2), cytomegaloviruses (particularly human) (e.g., gB or derivatives thereof), and rotaviruses (including attenuated rotaviruses). Hepatitis viruses, such as Epstein-Barr virus (e.g., gp350 or its derivatives), varicella-zoster virus (e.g., gp1, II, and IE63), or hepatitis viruses such as hepatitis B virus (e.g., hepatitis B surface antigen or its derivatives), hepatitis A virus, hepatitis C virus, and hepatitis E virus, or other viral pathogens such as paramyxoviruses: respiratory syncytial virus (e.g., F and G proteins or their derivatives), parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, 11, 16, 18, etc.), and arboviruses (e.g., yellow fever virus). Fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, or influenza virus (whole or inactivated virus, lysed influenza virus inoculated into oocytes or MDCK cells, or intact influenza virion (as described by Gluck, Vaccine, 1992, 10, 915-920) or its purified or recombinant proteins, such as HA, NP, NA, or M proteins or combinations thereof).

[0273] In some embodiments, the compositions described herein (e.g., thermostable lyophilized vaccine compositions) comprise antigens or antigenic components capable of inducing an immune response against human or other mammalian pathogens. These antigens or antigenic components may include components derived from one or more bacterial pathogens, such as certain species of the genus *Neisseria*, including *Neisseria gonorrhoeae* and *Neisseria meningitides* (e.g., capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, PilC, adhesins); *Streptococcus pyogenes* (e.g., M protein or fragments thereof, C5A protease, lipoteichoic acid), *Streptococcus agalactiae*, *Streptococcus mutans*, *Haemophilus ducreyi*; and certain species of the genus *Moraxella*, including *Moraxella catarrhalis*, also known as *Branhamella*. Catarrhalis (e.g., high and low molecular weight adhesins and infiltrates); certain species of Bordetella spp., including Bordetella pertussis (e.g., Bordetella pertussis adhesins, pertussis toxin or its derivatives, filamentous hemagglutinin, adenylate cyclase, fimbriae), Bordetella parapertussis, and Bordetella bronchiseptica; certain species of Mycobacterium spp. The genera *Mycobacterium tuberculosis* (e.g., ESAT6, antigen 85A, -B, or -C), *Mycobacterium bovis*, *Mycobacterium leprae*, *Mycobacterium avium*, *Mycobacterium paratuberculosis*, and *Mycobacterium smegmatis*; certain species of *Legionella*, including *L. pneumophila*; certain species of *Escherichia*, including enterotoxic *E. coli* (e.g., colonization factor, heat-labile toxin or its derivatives, heat-stable toxin or its derivatives), enterohemorrhagic *E. coli*, and enteropathogenic *E. coli* (e.g., Shiga toxin-like toxin or its derivatives); and certain species of *Vibrio*. spp), including Vibrio cholerae (V).* *Cholera* (e.g., cholera toxin or its derivatives); certain species of *Shigella* spp., including *Shigella sonnei*, *Shigella dysenteriae*, and *Shigella flexnerii*; certain species of *Yersinia* spp., including *Yersinia enterocolitica* (e.g., Yop protein), *Yersinia pestis*, and *Yersinia pseudotuberculosis*; certain species of *Campylobacter* spp., including *Campylobacter jejuni* (e.g., toxins, adhesins, and infiltrates) and *Campylobacter coli*; certain species of *Salmonella* spp. The genus *Salmonella* includes *Salmonella typhi*, *Salmonella paratyphi*, *Salmonella choleraesuis*, and *Salmonella enteritidis*; certain species of *Listeria*, including *Listeria monocytogenes*; certain species of *Helicobacter*, including *Helicobacter pylori* (e.g., urease, catalase, vacuolation enzymes); certain species of *Pseudomonas*, including *Pseudomonas aeruginosa*; certain species of *Staphylococcus*, including *Staphylococcus aureus* and *Staphylococcus epidermidis*; and certain species of *Enterococcus*. The genera *Clostridium* spp. include *E. faecalis* and *E. faecium*; certain species of *Clostridium* spp., including *C. tetani* (e.g., tetanus toxin and its derivatives), *C. botulinum* (e.g., botulinum toxin and its derivatives), and *C. difficile* (e.g., clostridium toxin class A or B and its derivatives); certain species of *Bacillus* spp., including *B. anthracis* (e.g., botulinum toxin and its derivatives); certain species of *Corynebacterium* spp., including *C. diphtheriae* (e.g., diphtheria toxin and its derivatives); and certain species of *Borrelia* spp., including *B. burgdorferi*.* *Borrelia burgdorferi* (e.g., OspA, OspC, DbpA, DbpB), *Borrelia garinii* (e.g., OspA, OspC, DbpA, DbpB), *Borrelia afzelii* (e.g., OspA, OspC, DbpA, DbpB), *Borrelia Andersonii* (e.g., OspA, OspC, DbpA, DbpB), *Borrelia hermsii*; certain species of *Ehrlichias* pp., including *E. equi* and *Human Granulocytic Ehrlichiosis*, are vectors of these diseases; certain species of *Rickettsia* spp., including *R. rickettsii*; certain species of *Chlamydia* spp. The spp. include *Chlamydia trachomatis* (e.g., MOMP, heparin-binding protein), *Chlamydia pneumoniae* (e.g., MOMP, heparin-binding protein), and *Chlamydia psittaci*; certain species of *Leptospira* spp., including *Leptospira interrogans*; certain species of *Treponema* spp., including *Treponema pallidum* (e.g., rare outer membrane protein), *Treponema denticola*, and *Treponema hyodysenteriae*; or other bacterial pathogens.

[0274] In some embodiments, the compositions described herein (e.g., thermostable lyophilized vaccine compositions) comprise an antigen or antigenic component capable of inducing an immune response against human or other mammalian pathogens. The antigen or antigenic component may include components derived from one or more parasites (see, for example, John, DT and Petri, WA., Markell and Voge's Medical Parasitology—9th edition, 2006, WB Saunders, Philadelphia; Bowman, DD, Georges' Parasitology for Veterinarians—8th edition, 2002, WB Saunders, Philadelphia). These parasites may include species of *Plasmodium* spp., including *Plasmodium falciparum*; species of *Toxoplasma* spp., including *T. gondii* (e.g., SAG2, SAG3, Tg34); and species of *Entamoeba*. The genera *Baberia* include *E. histolytica*, *Babesia* species, *B. microti*, *Trypanosoma* species, *T. cruzi*, *Giardia* species, *G. lamblia*, *Leishmania* species, *L. major*, *Pneumocystis* species, *P. carinii*, and *Trichomonas* species, *T. spp.*.(vaginalis); or from worms capable of infecting mammals, such as: (i) nematode infections (including but not limited to Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necatora Americanus, Ancylostoma duodenale, Wucheereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, and Strongyloides stercoralis)); (ii) trematode infections (including but not limited to Schistosoma mansoni and Schistosoma haematobium). (iii) Pathogenic worm infection (including but not limited to beef tapeworm and pork tapeworm). Therefore, certain implementations may involve vaccine compositions comprising antigens from certain species of the genus *Schistosoma* or from yeast, such as *Schistosoma mansoni*, *Schistosoma haematobium*, and / or *Schistosoma japonicum*, and yeasts such as *Candida* spp., including *C. albicans*; and *Cryptococcus* spp., including *C. neoformans*.

[0275] In some embodiments, the compositions described herein comprise at least two heterologous polypeptides of the Mycobacterium tuberculosis complex. The Mycobacterium tuberculosis complex includes those species conventionally considered to cause tuberculosis, as well as environmental and opportunistic mycobacterial species that cause tuberculosis and lung disease in immunocompromised patients, such as those with AIDS, for example, *Mycobacterium tuberculosis* (Mtb), *Mycobacterium bovis* or *Mycobacterium africanum*, BCG, *Mycobacterium avium*, intracellular mycobacteria, *Mycobacterium cryptidis*, *Mycobacterium Genevai*, *Mycobacterium haemophilus*, *Mycobacterium kansasii*, *Mycobacterium simianum*, *Mycobacterium bovis*, *Mycobacterium occulta*, and *Mycobacterium scrofula* (see, for example, Harrison's Principles of Internal Medicine, Vol. 1, pp. 1004-1014 and 1019-1020). Sequences of antigens from mycobacterial species are readily available. For example, Mycobacterium tuberculosis sequences can be found in Cole et al., Nature 393:537 (1998), and on sites maintained, for example, by the Wellcome Trust, the Sanger Institute, and the Institut Pasteur.

[0276] Other specific antigens for Mycobacterium tuberculosis that can be used in the compositions described herein are, for example, ThRa12, Tb H9, Tb Ra35, Tb38-1, Erd14, DPV, MTI, MSL, mTTC2, and hTCC1 (WO 99 / 51748). Proteins targeting Mycobacterium tuberculosis also include fusion proteins and their variants, wherein at least two, preferably three, polypeptides of Mycobacterium tuberculosis are fused into a larger protein. In some embodiments, the fusion proteins include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, and TbH9-DPV-MTI (WO 99151748). Other antigens that may be used include antigens, antigen conjugates, and fusion proteins described in US2010 / 0129391 and WO 2008 / 124647.

[0277] In some embodiments, the compositions described herein comprise an isolated fusion protein comprising a combination of two or more covalently linked Mycobacterium tuberculosis antigens or immunogenic fragments thereof, wherein the antigens are selected from the group consisting of: Rv0164, Rv0496, Rv2608, Rv3020, Rv3478, Rv3619, Rv3620, Rv1738, Rv1813, Rv3810, Rv2389, Rv2866, Rv3876, Rv0054, Rv0410, Rv0655, Rv0831, Rv1009, Rv1099, Rv1 240, Rv1288, Rv1410, Rv1569, Rv1789, Rv1818, Rv1860, Rv1886, Rv1908, Rv2220, Rv2032, Rv2623, Rv2875, Rv3044, Rv3310, Rv3881, Rv0577, Rv1626, Rv0733, Rv2520, Rv1253, Rv1980, Rv3628, Rv1884, Rv3872, Rv3873, Rv1511 and Rv3875, and antigens having at least 90% identity with any of the aforementioned sequences.

[0278] In some embodiments, the compositions described herein comprise an ID93 fusion protein comprising antigens Rv2608, Rv3619, Rv3620, and Rv1813, or sequences having at least 90% identity with the antigen combination. In another embodiment, the composition comprises an ID93 fusion protein comprising antigens Rv2608, Rv3619, Rv3620, and Rv1813, wherein the antigen sequences are derived from Mycobacterium tuberculosis. In another embodiment, the ID93 fusion protein comprises the sequence shown in SEQ ID NO: 1, or a sequence having at least 90% identity with it. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 2, or a sequence having at least 90% identity with it. In some embodiments, the therapeutic vaccine comprises a fusion protein comprising a combination of mycobacterial antigens Rv2608, Rv3620, and Rv1813, or a sequence having at least 90% identity with the antigen combination. In some embodiments, the mycobacterial antigens Rv2608, Rv3620, and Rv1813 are Mycobacterium tuberculosis antigens Rv2608, Rv3620, and Rv1813. In some embodiments, the fusion protein comprises the sequence shown in SEQ ID NO: 3 or 4, or a sequence having at least 90% identity with SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, antigen Rv1813 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, antigen Rv3620 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, antigen Rv2608 comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, antigen Rv3619 comprises the amino acid sequence of SEQ ID NO: 8. Those skilled in the art will understand that one or more N-terminal amino acids (e.g., signal sequences) may be removed. These sequences are described in US 8,486,414, which is incorporated herein by reference.

[0279] In some embodiments, the composition comprises an ID93 fusion protein or a polynucleotide encoding thereon, which contains four antigens belonging to the Mycobacterium tuberculosis protein family and associated with virulence (Rv2608, Rv3619, Rv3620) or latency (Rv1813), as described in U.S. Patent Application Publication No. 2010 / 0129391 (which is incorporated herein by reference in its entirety).

[0280] In some embodiments, the compositions described herein comprise chlamydia antigens. Chlamydia antigens include, for example, high molecular weight protein (HWMP) (WO 99 / 17741), ORF3 (EP 366412), and putative membrane proteins (Pmps). Other chlamydia antigens of the compositions may be selected from the group described in WO 99128475. In some embodiments, the compositions described herein comprise antigens from certain species of Streptococcus, including Streptococcus pneumoniae (e.g., capsular polysaccharides and their conjugates, PsaA, PspA, streptococcal hemolysin, choline-binding protein) and the protein antigen streptococcal hemolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and their mutant detoxified derivatives (WO 90 / 06951; WO 99 / 03884). Other bacterial vaccines are antigens derived from the genus Haemophilus spp., including Haemophilus influenzae type B (e.g., PRP and its conjugates), untyped Haemophilus influenzae such as OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, as well as filamentin and filamentin-derived peptides (US Patent No. 5,843,464) or multiple copy variants or fusion proteins thereof.

[0281] Hepatitis B surface antigen derivatives are well known in the art and particularly include those PreS1 and Pars2S antigens described in European patent applications EP-A414374, EP-A-0304578, and EP198474. In some embodiments, the compositions described herein comprise HIV-1 antigen, gp120, particularly when expressed in CHO cells. In other embodiments, the compositions comprise gD2t as defined herein.

[0282] In some embodiments, the compositions described herein contain antigens derived from human papillomaviruses (HPV) (HPV6 or HPV11 and others) considered to be the cause of genital warts, and HPV viruses (HPV16, HPV18 and others) considered to be the cause of cervical cancer. In some embodiments, the compositions are prophylactic or therapeutic vaccines for genital warts, comprising L1 particles or capsomeres, and a fusion protein comprising one or more antigens selected from HPV6 and HPV11 proteins E6, E7, L1, and L2. Certain forms of fusion proteins include L2E7 as disclosed in WO 96 / 26277, and protein D(1 / 3)-E7 as disclosed in GB 9717953.5 (PCT / EP98 / 05285). In some embodiments, the compositions are prophylactic or therapeutic vaccines for HPV cervical infection or cancer, comprising antigens of HPV 16 or 18. For example, L1 or L2 antigen monomers, or L1 or L2 antigens presented together as virus-like particles (VLPs), or L1 proteins presented separately within VLPs or capsomeres. These antigens, virus-like particles, and capsomeres are known in themselves. See, for example, WO94 / 00152, WO94 / 20137, WO94 / 05792, and WO93 / 02184.

[0283] Other early proteins may be included individually or as fusion proteins such as E7, E2, or preferably, for example, F5; some embodiments include a VLP containing the L1E7 fusion protein (WO 96 / 11272). In some embodiments, the HPV16 antigen contains an early protein E6 or E7 fused to a protein D vector to form a protein D-E6 or E7 fusion from HPV16, or a combination thereof; or contains a combination of E6 or E7 with L2 (WO 96 / 26277). Alternatively, the early proteins E6 and E7 of HPV16 or 18 may be presented as single molecules, preferably protein D-E6 / E7 fusions. The composition (e.g., a thermostable lyophilized vaccine composition) may optionally contain either or both of the E6 and E7 proteins from HPV18, preferably in the form of a protein D-E6 or protein D-E7 fusion protein or a protein DE6 / E7 fusion protein. The compositions of the present invention may additionally contain antigens from other HPV strains, preferably from HPV31 or 33 strains.

[0284] The compositions of this invention may additionally contain antigens from parasites that cause malaria. For example, antigens from Plasmodium falciparum include RTS,S and TRAP. RTS is a hybrid protein containing substantially the entire C-terminal portion of the cyclosporin (CS) protein of Plasmodium falciparum, which is linked to the hepatitis B virus surface (S) antigen via four amino acids of the preS2 portion of the hepatitis B surface antigen. Its complete structure is disclosed in International Patent Application No. PCT / EP92 / 02591, Publication No. WO 93 / 10152, claiming priority to UK Patent Application No. 9124390.7. When expressed in yeast, RTS is produced as lipoprotein particles, and when it is co-expressed with the S antigen from HBV, it produces mixed particles called RTS,S.

[0285] The TRAP antigen is described in International Patent Application No. PCT / GB89 / 00895, published under WO 90 / 01496. One embodiment of the invention is a malaria vaccine in which the antigen formulation comprises a combination of RTS,S and TRAP antigens. Other Plasmodium antigens that may be candidates for components of a multi-stage malaria vaccine are Plasmodium falciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16, Pfs48 / 45, Pfs230, and their analogues in certain species of the Plasmodium genus.

[0286] Some embodiments disclosed herein relate to antigens from at least one infectious pathogen, such as bacteria, viruses, or fungi, including actinomycetes such as Mycobacterium tuberculosis or Mycobacterium leprae or other mycobacteria; bacteria such as members of the genus Salmonella, Neisseria, Treponema, Chlamydia, or Bordetella; viruses such as herpes simplex virus, human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), cytomegalovirus, varicella-zoster virus, hepatitis virus, Epstein-Barr virus (EBV), respiratory syncytial virus, human papillomavirus (HPV), and cytomegalovirus; HIV such as HIV-1 or HIV-2; fungi such as Aspergillus, Blastomyces, Coccidioides and Pneumocystis, or yeasts, including Candida species such as Candida albicans, Candida glabrata, Candida krusei, Candida lucida, Candida tropicalis, and Candida parapsilosis; parasites such as protozoa, such as Plasmodium species including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale; or other parasites such as Acanthamoeba, Entamoeba histolytica, Angiostrongylus cantonensis, Schistosoma mansoni, Schistosoma japonicum, Cryptosporidium, Schistosoma haematobium, hookworm, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmannii, Entamoeba bosne, Wucet's nematode, Giardia lamblia, and one or more of Leishmania.

[0287] For example, in embodiments of compositions containing antigens from the genus *Treponema*, the antigen may include nucleic acids, pathogen-derived antigens or antigenic preparations, recombinant-generated proteins or peptides, and chimeric fusion proteins. One such antigen is OspA. OspA can be a fully mature protein in an apolipoprotein form (Lipo-OspA) through its biosynthesis within host cells, or alternatively, a non-apolipoprotein derivative. Such non-apolipoprotein derivatives include a non-apolipoprotein NS1-OspA fusion protein having the first 81 amino acids of the N-terminus of the non-structural protein (NS1) of influenza virus, and the complete OspA protein, and another MDP-OspA is a non-apolipoprotein form of OspA carrying three additional N-terminal amino acids.

[0288] Compositions and methods for identifying individuals who are at risk of infection with infectious pathogens as described herein are known in the art.

[0289] For example, the bacterium Mycobacterium tuberculosis causes tuberculosis (TB). This bacterium typically attacks the lungs, but can also invade the kidneys, spine, and brain. TB can be fatal if left untreated. The disease can spread from person to person when an infected person sneezes or coughs. In 2003, more than 14,000 cases of TB were reported in the United States.

[0290] Although tuberculosis can often be controlled with long-term antibiotic treatment, this treatment is insufficient to prevent the spread of the disease and raises concerns about the potential selection of antibiotic-resistant strains. Infected individuals may be asymptomatic for a period of time but remain infectious. Furthermore, while adherence to treatment protocols is crucial, patient behavior is difficult to monitor. Some patients do not complete the treatment course, leading to ineffective treatment and the development of drug resistance (e.g., U.S. Patent 7,087,713).

[0291] Currently, live bacterial vaccines are the most effective method for inducing protective immunity against tuberculosis. The most common mycobacterium used for this purpose is Bacillus Calmette-Guerin (BCG), a nonvirulent strain of a bovine mycobacterium. However, the safety and efficacy of BCG are controversial, and it is not available to the general public in some countries, such as the United States. Diagnosis is usually accomplished using a skin test, which involves intradermal exposure to tuberculin PPD (a purified protein derivative). An antigen-specific T-cell response results in a measurable induration at the injection site 48 and 72 hours post-injection, indicating exposure to the mycobacterial antigen. However, sensitivity and specificity have been challenges with this test, and individuals vaccinated with BCG cannot be distinguished from infected patients (e.g., U.S. Patent 7,087,713).

[0292] Although macrophages have long been considered the primary effector cells in the immunity against Mycobacterium tuberculosis, T cells are the dominant inducing cells for this immunity. The crucial role of T cells in protecting against Mycobacterium tuberculosis infection is exemplified in AIDS patients: Mycobacterium tuberculosis is commonly found in AIDS patients due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-responsive CD4 T cells have been shown to be potent producers of interferon-γ (IFN-γ), which has also been further shown to trigger the anti-mycobacterial effect of macrophages in mice. While the role of IFN-γ in humans is unclear, studies have shown that 1,25-dihydroxyvitamin D3, alone or in combination with IFN-γ or tumor necrosis factor-α, activates human macrophages to suppress Mycobacterium tuberculosis infection. Furthermore, IFN-γ is known to activate human macrophages to produce 1,25-dihydroxyvitamin D3. Similarly, IL-12 has been shown to play a role in inducing resistance to Mycobacterium tuberculosis infection. For an immunological review of Mycobacterium tuberculosis infection, see Chan and Kaufmann, in Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, DC (1994).

[0293] Existing compounds and methods for diagnosing tuberculosis or inducing protective immunity against tuberculosis include the use of peptides and DNA molecules encoding such peptides, said peptides containing at least one immunogenic moiety of one or more mycobacterial proteins. Diagnostic kits containing such peptides or DNA sequences and appropriate detection reagents can be used to detect mycobacterial infections in patients and biological samples. Antibiotics targeting such peptides are also provided. Furthermore, such compounds can be formulated in the compositions described herein for immunization against mycobacterial infections (US Patent Nos. 6,949,246 and 6,555,653).

[0294] Malaria was eradicated in many parts of the world in the 1960s, but the disease persists, and new strains resistant to existing drugs have emerged. Malaria is a major public health problem in more than 90 countries. Nine out of ten cases of malaria occur in sub-Saharan Africa. More than one-third of the world's population is at risk, and 350 to 500 million people are infected with malaria each year. This year, 45 million pregnant women are at risk of contracting malaria. Of those already infected, more than one million die each year from preventable disease. The majority of those deaths are among children in Africa.

[0295] Malaria is typically transmitted when a person is bitten by an infected female Anopheles mosquito. The mosquito must become infected by feeding on the blood of an infected person to transmit the disease. Malaria is caused by a parasite, and clinical symptoms include fever and flu-like symptoms such as chills, headache, muscle aches, and fatigue. These symptoms may be accompanied by nausea, vomiting, and diarrhea. Malaria can also cause anemia and jaundice due to the loss of red blood cells. One type of malaria, falciparum malaria, if left untreated, can lead to kidney failure, seizures, confusion, coma, and death.

[0296] In vitro diagnostic methods for malaria in individuals are known, which involve contacting a tissue or biological fluid collected from an individual with a molecular or polypeptide composition (under conditions that allow an in vitro immune response to occur between the composition and antibodies present in the tissue or biological fluid), and in vitro detection of the resulting antigen-antibody complex (see, for example, U.S. Patent 7,087,231), wherein the molecular or polypeptide composition comprises one or more polypeptide sequences carrying all or part of one or more T epitopes of a protein resulting from the infectious activity of Plasmodium falciparum.

[0297] The expression and purification of the extracellular region of recombinant Plasmodium falciparum (3D7) AMA-1 have been described. Previous methods have yielded highly purified proteins that retain the folding and disulfide bonds of the native molecule. Recombinant AMA-1 can be used as a diagnostic reagent, as well as for antibody preparation, and as a protein alone or as part of a vaccine for malaria prevention (US Patent 7,029,685).

[0298] Species-specific Plasmodium vivax malaria peptide antigens encoding species-specific antigens have been described in the art, said antigens being proteins or protein fragments secreted into the plasma of susceptible mammalian hosts after infection, thus generating monoclonal or polyclonal antibodies against these antigens. These peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are used in assays for diagnosing malaria and for determining whether Plasmodium vivax is the causative species (US Patent 6,706,872). Species-specific Plasmodium vivax malaria peptide antigens, which are proteins or protein fragments secreted into the plasma of susceptible mammalian hosts after infection, thus generating monoclonal or polyclonal antibodies against these antigens, have also been reported. These peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are used in assays for diagnosing malaria and for determining whether Plasmodium vivax is the causative species (see, for example, US Patent 6,231,861).

[0299] The extracellular region of recombinant Plasmodium falciparum (3D7) AHA-1 has also been expressed using methods to produce high-purity proteins, retaining the folding and disulfide bonds of the native molecule. This recombinant AMA-1 is used as a diagnostic reagent and as a vaccine for antibody production (US Patent 7,060,276). Also known is recombinant Plasmodium falciparum (3D7) MSP-1. 42 The expression and purification of recombinant MSP-1 preserved the folding and disulfide bonds of the natural molecule. 42 Used as a diagnostic reagent and as a vaccine for antibody production (US Patent 6,855,322).

[0300] Therefore, based on these and related disclosures, diagnostic methods for detecting human malaria infection are known to identify individuals who are at risk of or suspected of being infected with the infectious malaria pathogen. Specifically, for example, a blood sample is combined with a reagent containing 3-acetylpyrimidine adenine dinucleotide (APAD), a substrate (e.g., lactate or lactate), and a buffer. This reagent is designed to detect the presence of a unique glycolytic enzyme produced by the malaria parasite. This enzyme is known to be parasitic lactate dehydrogenase (PLDH). Using the aforementioned reagent, PLDH can be easily distinguished from the host's LDH. The combination of the reagent with the parasite-infected blood sample results in the reduction of APAD. However, APAD is not reduced by the host's LDH. The reduced APAD can then be detected using various techniques, including spectroscopy, fluorescence assays, electrophoresis, or colorimetric analysis. Detection of the reduced APAD in the manner described above provides a positive indication of malaria infection (e.g., U.S. Patent 5,124,141). In another methodology for diagnosing malaria, a polypeptide containing a characteristic amino acid sequence is derived from the Plasmodium falciparum antigen GLURP and can be recognized in a test sample by a specific antibody against or reacting with the polypeptide (US Patent 5,231,168).

[0301] Leishmaniasis is a worldwide parasitic disease, prevalent in the Indian subcontinent, Africa, and Latin America, and is a priority for vaccine development by the World Health Organization. Combining various diseases, leishmaniasis causes fatal infections of internal organs as well as severe skin diseases. One of the most devastating forms of leishmaniasis is a disfiguring infection of the nose and mouth. The number of leishmaniasis cases is increasing and currently difficult to control in many areas. As a consequence of HIV infection, leishmaniasis is also on the rise in some developed regions, particularly in Southern Europe. Available medications are toxic, expensive, and require long-term daily injections.

[0302] Leishmania are protozoan parasites that reside in macrophages or white blood cells of the immune system. These parasites are transmitted through the bites of small blood-sucking insects (sand flies), which are difficult to control due to their widespread distribution across the globe.

[0303] Visceral leishmaniasis is the most dangerous of the three manifestations of the disease. An estimated 500,000 new cases of the visceral form (kala-azar, or "deadly disease") occur annually. More than 20 million people are currently at risk of contracting visceral leishmaniasis. Over 90% of visceral leishmaniasis cases occur in India, Bangladesh, Sudan, Brazil, and Nepal. Most deaths occur in children. Those with the cutaneous form often suffer permanent disfigurement.

[0304] Leishmaniasis is difficult to diagnose and usually involves histopathological analysis of tissue biopsy samples. However, several serological and immunological diagnostic assays have been developed (US Patent 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). Proflaviformes release metabolites into the culture medium to produce conditioned medium. These metabolites are host immunogenic. See Schnur, LF et al., (1972) lsrl. J. Med. Sci. 8: 932942; Sergeiev, VP et al., (1969) Med. Parasitol. 38: 208212; El-On, J. et al., (1979) Exper. Parasitol. 47: 254269; and Bray, RS et al., (1966) Trans. R. Soc. Trop. Med. Hyg. 60: 605609; U.S. Patent Nos. 6,846,648, 5,912,166, 5,719,263, and 5,411,865.

[0305] In some embodiments, the antigen is a Leishmania antigen described in US 2009 / 0041798, US 2009 / 0291099, US 8,410,258, US 8,231,881, and WO 2012 / 064659, all of which are incorporated herein by reference. In some embodiments, the antigen is a fusion polypeptide comprising at least a Leishmania sterol 24-c-methyltransferase (SMT) polypeptide sequence and a Leishmania nonspecific nucleoside hydrolase (NH) polypeptide sequence. In some embodiments, the Leishmania NH polypeptide sequence comprises an immunogenic portion of a sequence having at least 90% identity with the Leishmania NH sequences of Leishmania donovani, Leishmania infantum, and Leishmania major. In some embodiments, the leishmaniasis NH polypeptide sequence comprises an immunogenic portion of a sequence selected from at least the group consisting of SEQ ID NOs: 1, 3, and 5, or a sequence having at least 90% identity with the above sequences. In some embodiments, the leishmaniasis SMT polypeptide sequence comprises an immunogenic portion of a sequence having at least 90% identity with the SMT sequences of *L. donovani*, *L. infantum*, and *L. major*. In some embodiments, the leishmaniasis SMT polypeptide sequence comprises an immunogenic portion of a sequence selected from at least the group consisting of SEQ ID NOs: 7, 9, and 11, or a sequence having at least 90% identity with the above sequences. In some embodiments, the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13 or a sequence having at least 90% identity with that sequence. The sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13 are provided in WO 2012 / 064659 and US20120114688, which are incorporated herein by reference.

[0306] Approximately 40 million people worldwide are living with HIV, the virus that causes AIDS. About 3 million people die from the disease each year, 95% of whom die in developing countries. Nearly 5 million people are infected with HIV annually. The burden of the disease is currently heaviest in sub-Saharan Africa, but it is spreading rapidly to other countries such as India, China, and Russia. The epidemic is spreading fastest among minority populations. In the United States, more than 950,000 cases of AIDS have been reported since 1981. AIDS attacks people of reproductive age. Women are at increased risk of contracting HIV / AIDS due to both biological and social factors.

[0307] AIDS is caused by the human immunodeficiency virus (HIV), which kills and destroys cells in the human immune system and progressively impairs the body's ability to fight infections and certain cancers. The most common transmission of HIV is through unprotected sex with an infected partner. The most effective solution to this problem is preventing the spread of the virus. One way to achieve this is to develop a safe, effective, and affordable HIV vaccine. Worldwide, less than one in five people at high risk of HIV infection take effective preventative measures.

[0308] Methods for diagnosing HIV infection are known, including viral culture from patient samples, PCR of identified nucleic acid sequences, and antibody tests that detect the presence of anti-HIV antibodies in patient serum (see, for example, U.S. Patent Nos. 6,979,535, 6,544,728, 6,316,183, 6,261,762, 4,743,540).

[0309] According to certain other embodiments disclosed herein, the composition and methods of application may include antigens from cancer cells that can be used for immunotherapeutic treatment of cancer. For example, the composition may utilize tumor rejection antigens, such as rejection antigens for prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, or melanoma cancer. Exemplary cancer or cancer cell-derived antigens include MAGE1, 3, 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, pp. 628-636; Van den Eynde et al., International Journal of Clinical & Laboratory Research (1997 & 1998); Correale et al. (1997), Journal of the National Cancer Institute 89, p. 293. These non-limiting examples of cancer antigens are expressed in a variety of tumor types such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518.

[0310] Other tumor-specific antigens suitable for use in the compositions described herein include, but are not limited to, tumor-specific or tumor-associated gangliosides, such as GM2 and GM3 or their conjugates with carrier proteins; or antigens used in GLA vaccine compositions to induce or enhance anti-cancer immune responses may be autopeptide hormones, such as full-length gonadotropin-releasing hormone (GnRH, WO 95 / 20600), a short peptide of 10 amino acids used to treat many cancers. In another embodiment, prostate antigens such as prostate-specific antigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA 95(4) 1735-1740 1998), PSMA, or in a preferred embodiment, an antigen called prostase (e.g., 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; U.S. Patent No. 5,955,306; WO 98 / 20117; U.S. Patent Nos. 5,840,871 and 5,786,148; WO 00 / 04149. Other prostate-specific antigens can be identified from WO98 / 137418 and WO / 004149. Another one is STEAP (PNAS 96 14523 145287-12 1999).

[0311] Other tumor-associated antigens used in the context of 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, U.S. Patent No. 5,654,140), and Criptin (U.S. Patent No. 5,981,215). Furthermore, antigens particularly relevant to cancer treatment vaccines include tyrosinases and survivin.

[0312] The embodiments of compositions containing cancer antigens disclosed herein can be used to combat any cancer characterized by tumor-associated antigen expression, such as HER-2 / neu expression or other cancer-specific or cancer-associated antigens.

[0313] Diagnosing cancer in individuals who are at or suspected of having cancer can be accomplished using any of a number of well-established methodologies in the art, which can vary depending on factors including clinical presentation, degree of cancer progression, cancer type, or other factors. Examples of cancer diagnostics include histopathological, histocytological, immunohistochemical, and immunohistopathological examinations of patient samples (e.g., blood, skin biopsy, other tissue biopsy, surgical samples, etc.), PCR detection of defined genetic (e.g., nucleic acid) markers, serological detection of circulating cancer-associated antigens or cells carrying such antigens, or serological detection of defined specific antibodies, or other methodologies familiar to those skilled in the art. See, for example, U.S. Patent 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; 7,070,931; JP5-328975; Waslylyk et al., 1993 Eur. J Bioch. 211(7): 18.

[0314] Compositions and methods according to certain embodiments of the present invention can be used to prevent or treat autoimmune diseases, including diseases, states, or conditions in which the host or individual's immune system adversely mediates an immune response against "self" tissues, cells, biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins, lipoproteins, proteolipids, lipids, glycolipids, nucleic acids such as RNA and DNA, oligosaccharides, polysaccharides, proteoglycans, glycosaminoglycans, etc., and other molecular components of individual cells and tissues) or epitopes (e.g., specific immune-defined recognition structures, such as those recognized by antibody variable region complementarity-determining regions (CDRs) or T-cell receptor CDRs).

[0315] Therefore, autoimmune diseases are characterized by abnormal immune responses involving cells or antibodies, which in either case target normal self-organization. Autoimmune diseases in mammals are generally classified into one of two distinct types: cell-mediated diseases (i.e., T-cell) or antibody-mediated conditions. Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes (juvenile diabetes), and autoimmune uveitis. Antibody-mediated autoimmune conditions include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (SLE), hyperthyroidism, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, autoimmune asthma, cryoglobulinemia, thrombotic thrombocytopenic purpura, primary biliary fibrosis, and pernicious anemia. Antigens associated with systemic lupus erythematosus (SLE) include small nucleonucleotide protein (snRNP); antigens associated with hyperthyroidism include thyroid-stimulating hormone receptor, thyroglobulin, and other components of thyroid epithelial cells (Akamizu et al., 1996; Kellerman et al., 1995; Raju et al., 1997; and Texier et al., 1992); antigens associated with pemphigus include cadherin-like pemphigus antigens such as desmosome core glycoprotein 3 and other adhesion molecules (Memar et al., 1996; Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993); and antigens associated with thrombotic thrombocytopenic purpura include platelet antigens (see, for example, U.S. Patent 6,929,796; Gorski et al. (eds.), Autoimmunity, 2001, Kluwer Academic Publishers, Norwell, MA; Radbruch and Lipsky, PE (eds.) Current Concepts in Autoimmunity and Chronic Inflammation (Curr. Top. Microbiol. and Immunol.) 2001, Springer, NY.).

[0316] Autoimmune diseases are involved in more than 80 different conditions, including type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, scleroderma, and thyroid disorders. For most autoimmune diseases, robust quantitative assessments of incidence are lacking. The most recent research, completed in the late 1990s, indicates that autoimmune diseases are the third most common major disease in the United States, affecting more than 85 million Americans. The current estimate of the prevalence of these diseases is 5% to 8% of the U.S. population. Most autoimmune diseases tend to affect women. Women are more than 2.7 times more likely to develop autoimmune diseases than men. Women are more susceptible to autoimmune diseases; men appear to have higher levels of natural killer cell activity than women (Jacobsen et al., Clinical Immunology and Immunopathology, 84: 223-243, 1997).

[0317] Autoimmune diseases occur when the immune system mistakes its own tissues for foreign substances and launches an inappropriate attack. The body can be attacked in various ways by autoimmune diseases, including, for example, the gut (Crohn's disease) and the brain (multiple sclerosis). Autoantibodies are known to attack the body's own cells or tissues, impairing their function and thus leading to autoimmune diseases. Furthermore, autoantibodies are known to be detectable in a patient's serum before autoimmune diseases actually develop (e.g., the appearance of clinical signs and symptoms). Therefore, the detection of autoantibodies can help detect or identify the presence of autoimmune diseases or the risk of developing them at an early stage. Based on these findings, a variety of autoantibodies against self-antigens have been identified and measured in clinical testing (e.g., U.S. Patents 6,919,210, 6,596,501, 7,012,134, 6,919,078). Other autoimmune diagnostics may involve detecting related metabolites (e.g., U.S. Patent No. 4,659,659) or immunological activity (e.g., U.S. Patents 4,614,722 and 5,147,785, 4,420,558, 5,298,396, 5,162,990, 4,420,461, 4,595,654, 5,846,758, 6,660,487).

[0318] In some embodiments, the compositions of the present invention will be particularly suitable for treating elderly and / or immunocompromised patients, including individuals undergoing kidney dialysis, chemotherapy and / or radiation therapy, transplant recipients, etc. Such individuals typically exhibit a weakened immune response to vaccines, and therefore, the use of the compositions of the present invention can enhance the immune response achieved in these individuals.

[0319] In other embodiments, one or more antigens used in the compositions of the present invention include antigens associated with respiratory diseases, such as those caused or exacerbated by bacterial infections (e.g., pneumococcal infection), for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD). The physiological definition of COPD is the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Am J Respir Crit Care Med. November 1995; 152(5 Pt 2): S77-121). Exacerbations of COPD are often caused by bacterial (e.g., pneumococcal) infection (Clin Microbiol Rev. April 2001; 14(2): 336-63).

[0320] Oils used in thermally stable compositions

[0321] Some embodiments involve compositions described herein comprising oils that, in some such embodiments, may enhance adjuvant activity, and in other such embodiments may additionally or selectively provide a pharmaceutically acceptable carrier or excipient. Many suitable oils are known and may be selectively included in compositions based on the disclosure of this invention. Examples of such oils include, but are not limited to, squalene, synthetic squalene, mineral oil, grapeseed oil, synthetic isoprene, olive oil, cholesterol, and dimannitol monooleate.

[0322] The oils discussed herein can be used in emulsion systems, and such emulsion systems are referred to as emulsion adjuvants. Emulsion adjuvants include oil-in-water, water-in-oil, or mixtures of water and oil-in-water. Without being limited by theory, such emulsion adjuvants can be used to provide sustained stimulation of the immune system by allowing antigen release. Certain emulsion adjuvants can also be used as delivery systems for other adjuvants, including, but not limited to, CpG oligodeoxynucleotides (CpG ODN), glucopyranose ester adjuvants (GLA), monophosphoryl lipid A (MLA), and 3-deacylated monophosphoryl lipid A (3D-MLA). Certain emulsion systems, including monophase or multiphase emulsion systems, have been described for formulating adjuvant compositions. Oil-in-water emulsion adjuvants have been proposed for use in adjuvant compositions (EP 0 399 843B), and combinations of oil-in-water emulsions with other active agents have been described as vaccine adjuvants (WO 95 / 17210; WO 98 / 56414; WO 99 / 12565; WO 99 / 11241). Other oil emulsion adjuvants have been described, such as water-in-oil emulsions (US Patent No. 5,422,109; EP 0 480 982 B2) and water-in-oil-in-water emulsions (US Patent No. 5,424,067; EP 0 480 981 B).

[0323] The oil emulsion adjuvant used in this invention can be natural or synthetic, and can be mineral or organic. Examples of mineral oils and organic oils will be apparent to those skilled in the art. In a particular embodiment, the compositions of this invention (e.g., heat-stable lyophilized vaccines) comprise an oil-in-water emulsion in which the adjuvant is incorporated into the oil phase. To make the oil-in-water composition suitable for human administration, the oil phase of the emulsion system preferably comprises a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "capable of being metabolized" (Dorland's illustrated Medical Dictionary, WBSaunders Company, 25th edition (1974)). The oil can be any vegetable oil, fish oil, animal oil, or synthetic oil that is non-toxic to the recipient and capable of being metabolized. Nuts (e.g., peanut oil), seeds, and grains are common sources of vegetable oils. Synthetic oils may also be used.

[0324] For example, squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-docosahexaene) is an unsaturated oil, abundantly obtained in shark liver oil and in small amounts in olive oil, wheat germ oil, rice bran oil, and yeast, and is a preferred oil for use in this invention. Squalene is considered a metabolizable oil because it is an intermediate in cholesterol biosynthesis (Merck index, 10th edition, registration number 8619). Exemplary metabolizable oils usable according to this invention include, but are not limited to, squalene, soybean oil, sesame oil, and caprylic / capric triglyceride (MIGLYCOL 810 oil). In one embodiment, the metabolizable oil comprises squalene. In another embodiment, the metabolizable oil comprises one or more yeast-derived isoprene-like structures, such as yeast-derived squalene or yeast-derived related isoprene structures.

[0325] In some embodiments, the compositions of the present invention comprise metabolizable oils present at concentrations of about 0.01%-5% v / v, about 0.01%-4% v / v, about 0.01%-3% v / v, about 0.01%-2% v / v, about 0.01%-1% v / v, or about 0.01%-0.5% v / v. In some embodiments, the metabolizable oil is present at the following concentrations: about 0.01% v / v, about 0.05% v / v, about 0.1% v / v, about 0.5% v / v, about 1% v / v, about 1.5% v / v, about 2% v / v, about 2.5% v / v, about 3% v / v, about 3.5% v / v, about 4% v / v, about 4.5% v / v, about 5% v / v, about 6% v / v, about 7% v / v, about 8% v / v, about 9% v / v, about 10% v / v, about 11% v / v, about 12% v / v, about 13% v / v, about 14% v / v, about 15% v / v, about 16% v / v, about 17% v / v, about 18% v / v, about 19% v / v, or about 20% v / v. In some embodiments, the metabolizable oil is present at a concentration of about 2% v / v. In some embodiments, the metabolizable oil is present at a concentration of less than 1% v / v. The percentage refers to the percentage in the oil-in-water emulsion formulation before lyophilization or in the reconstituted oil-in-water emulsion.

[0326] The size of the oil droplets obtained in a stable oil-in-water emulsion is preferably less than 1 micrometer, and their diameter can be approximately 30-600 nm, preferably approximately 30-500 nm, and most preferably approximately 150-500 nm, and particularly approximately 150 nm, as measured by photon correlation spectroscopy. In this regard, 80% of the oil droplets (by number) should be within the preferred range, more preferably greater than 90%, and most preferably greater than 95% of the oil droplets (by number) are within the defined size range.

[0327] The hydrophilic-lipophilic balance (HLB) value of an emulsion allows for the assessment of the hydrophilic or lipophilic properties of a surfactant. The HLB of amphiphilic molecules is typically calculated as follows:

[0328] HLB = (2OX of hydrophilic portion weight) / (weight of amphiphilic molecule)

[0329] HLB values ​​can range from 0 (for the most lipophilic molecules) to 20 (for the most hydrophilic molecules). This assessment can be modified and the HLB value range can increase depending on the chemical composition of the surfactant (particularly the addition of ethoxy or olefin oxides, for example, LUTROL). The HLB value is 29). For surfactant mixtures, the HLB of the mixture is the HLB addition of each surfactant, balanced by their weight ratios:

[0330] HLB = (HLB of surfactant X × weight of surfactant X) + (HLB of surfactant Y × weight of surfactant Y) / (weight of surfactant X + weight of surfactant Y)

[0331] In one embodiment of the emulsion prepared according to the present invention, the final HLB of the emulsion is about 9 to about 12, preferably about 9.5 to about 11.5 and more preferably about 10 to about 11.5. In some embodiments, the HLB of the emulsion is about 10.5 to about 11.0. Methods for preparing oil-in-water emulsions are well known to those skilled in the art. Typically, the method involves reacting the oil phase with a suitable surfactant such as... The solution is mixed and then homogenized using a homogenizer. For example, this involves passing the mixture through a syringe needle once, twice, or multiple times to homogenize a small volume of liquid. Similarly, the emulsification process in a microfluidic (M110S microfluidic machine, up to 50 channels, continuous for 2 minutes at a maximum input pressure of 6 bar (output pressure approximately 850 bar)) can be modified to prepare emulsions of smaller or larger volumes. This modification can be achieved through routine experiments, including measuring the resulting emulsion until a formulation with oil droplets of the desired diameter is obtained.

[0332] Uses of heat-stable pharmaceutical compositions

[0333] In another aspect, this document provides a method for stimulating an immune response in an individual, comprising administering the reconstituted thermostable vaccine composition described herein to the individual. This method may further include the step of reconstituted the thermostable lyophilized vaccine composition into an oil-in-water emulsion prior to administration.

[0334] Therefore, this invention is used to enhance or induce an immune response in a host, patient, or individual, or in a cell culture. The patient may have an infectious disease, cancer such as breast cancer, or an autoimmune disease, or may be normal (i.e., without detectable disease and / or infection). A “cell culture” is any preparation containing isolated cells of the immune system (including, but not limited to, T cells, macrophages, monocytes, B cells, and dendritic cells). Such cells can be isolated using any of the various techniques known to those skilled in the art (e.g., Ficoll-hypaque density centrifugation). Cells can (but must) be isolated from patients with cancer and can be reintroduced into post-treatment patients.

[0335] route of administration

[0336] This invention relates to methods and compositions for vaccination, treatment, and prevention of conditions such as infectious diseases, cancer, or autoimmune diseases. The methods of this invention include routes of administration comprising parenteral and non-parenteral administration. Non-parenteral administration routes include, but are not limited to, oral, oral, sublingual, topical, transdermal, ocular, ear, nasal, rectal, and vaginal routes. Injectable methods include, but are not limited to, parenteral administration, intravenous, intramuscular, subcutaneous, intraperitoneal, intracystic, intravenous, intra-arterial, and other injection routes. These inventions relate to compositions that provide controlled, sustained, or continuous release of antigens and / or adjuvants over a predetermined time period.

[0337] preparation

[0338] The formulation is known to those skilled in the art and includes, but is not limited to, formulations such as tablets, coated tablets, chewable tablets, effervescent tablets, pills, capsules, syrups, suppositories, injectable formulations, and dispersions of active agents in physiological fluids or in media in which antigens and / or adjuvants are released after degradation of the formulation due to mechanical, chemical, or enzymatic activity.

[0339] It should be understood that the present invention is not limited to the specific formulations, method steps and materials disclosed herein, and such formulations, method steps and materials may vary.

[0340] reagent kits and drug packaging

[0341] Some embodiments also involve kits comprising the vaccine compositions described herein, which may be provided in one or more containers. In one embodiment, all components of the vaccine composition are co-present in a single container, but embodiments of the invention are not intended to be limited thereto and may also involve two or more containers.

[0342] The container according to embodiments of such kits can be any suitable container, vessel, vial, ampoule, tube, cup, box, bottle, flask, jar, tray, hole, slot, barrel, etc., of a single-hole or multi-hole instrument, or other device in which the compositions disclosed herein can be placed, stored, and / or transported, and from which the contents can be easily removed. Typically, this container can be made of a material compatible with the intended use, from which the recovery of the contained contents can be easily achieved. Preferred examples of such containers include glass and / or plastic-sealed or resealable tubes and ampoules, including those with rubber diaphragms or other sealed devices compatible with the retraction of contents using needles and syringes. For example, such containers can be made of glass or a chemically compatible plastic or resin, which can be made of a material or coated with such a material, allowing for the effective recovery of the substance in the container and / or protecting such substance from degradation conditions such as ultraviolet light or temperature limits, or preventing the entry of unwanted contaminants, including microbial contaminants. The container is preferably sterile or sterilizable and made of a material compatible with any carrier, excipient, solvent, medium, etc., so that it can be used to suspend or dissolve the vaccine compositions and / or adjuvant compositions and / or antigens and / or recombinant expression constructs described herein.

[0343] The invention will be more fully understood by referring to the following embodiments. However, they should not be construed as limiting the scope of the invention. It should be understood that the embodiments and implementations described herein are for illustrative purposes only. The various embodiments described above can be combined to provide other implementations. Various modifications or changes thereto will become apparent to those skilled in the art from the foregoing description, and are contained within the spirit and scope of this application and fall within the scope of the appended claims. Example

[0344] Example 1: Stability of freeze-dried vaccine emulsion formulations in single and multi-excipient systems

[0345] Lyophilization of oil-in-water stabilized emulsions (SEs) for use in vaccines is desirable. The ability of excipients to form thermostable, compact cakes through lyophilization and / or to maintain emulsion integrity through remodeling is investigated to develop thermostable vaccine systems that reduce or eliminate the need for cold chain maintenance at all georelevant temperatures.

[0346] Materials and methods

[0347] Formulation and freeze-drying

[0348] The above-mentioned squalene-containing stabilized emulsion (SE) (EM001; Fox et al., 2012, Influenza and Other Respiratory Viruses, in press) was lyophilized at a final concentration of 2.0% squalene (v / v) (diluted from the initial 10% formulation to the target final injection concentration) for a total filling volume of 1.0 mL. Excipient concentrations are reported as mass percentages (%w / v) and emulsion concentrations as volume percentages of the squalene oil phase (%v / v). Maltose, D-(-)-ribose, D-(-)-fructose, lactose, polyethylene glycol (PEG; molecular weight 3,350 Da), lactulose, nicotinic acid, D(+)-raffinose pentahydrate, 2,6-pyridinedicarboxylic acid, and L-proline were purchased from Sigma-Aldrich (St. Louis, Mo). USP grade D(+)-mannitol and D(+)-trehalose dihydrate, NF grade sucrose and lactose monohydrate, and dextran (molecular weight 40,000 Da) were purchased from Spectrum Chemical (New Brunswick, NJ). D(+)-mannose, stachyose hydrate, and USP grade dextran monohydrate were purchased from Thermo Scientific (Waltham, MA). Samples were prepared using different concentrations of the above excipients dissolved in deionized water and filtered sequentially through a Barnstead / Thermolyne (Dubuque IA) E-Pure D4631 filtration system and a 20nm Whatman (Maidstone, Kent, UK) Anotop plus filter. Formulations that had undergone remodeling resulting in extreme pH shifts (below 5.0 or above 7.0) were reconstituted to pH 5.5 before lyophilization using NaCl and NaOH. Lyophilization was performed using a VirTis (Gardiner, NY) AdVantage 2.0EL-85 benchtop freeze dryer. The lyophilization method employed a heat treatment arrangement consisting of a 10-hour freezing step from 4 to -40°C, followed by an annealing step at -15°C. The primary drying stage (at 100 mTorr) lasted 18.3 hours from -40°C to 25°C. Finally, a secondary drying stage of 9 hours was performed at 25°C at 50 mTorr. All samples were plugged in atmospheric gas at 500 mTorr, sealed with aluminum caps, and stored at 4°C until use.

[0349] Refactoring

[0350] Prior to reconstruction, the pie was visually characterized as a fine white pie (meaning the pie was white, approximately the same volume as when filled, and appeared to have a uniform lattice), a film, or any other form different from a fine white pie. All samples were reconstructed using 20 nm filtered water (as described above) and manually vortexed gently until all components were dissolved or until three minutes had elapsed, whichever came first. After reconstruction, the formulation was described as a milky white emulsion (appearing similar to the pre-lyophilized formulation), or alternatively, any differences from a milky white emulsion were recorded. A small amount of each formulation was then allowed to stand at room temperature for 1 hour, and the presence of thick, white phases on the surface, termed stratification, was visually assessed. The pH of each formulation was also tested using a Mettler-Toledo (Columbus, OH) MP225 pH meter.

[0351] Melting point determination

[0352] The melting point of each lyophilized formulation was triple-evaluated using the Stanford Research System (Sunnyvale, CA) OptiMelt automated melting system. Each melting capillary was inserted into the cake once and pressed down until the material was fixed to the bottom. Melting was performed from 27°C to 200°C at a heating rate of 1°C / min (typically stopped once melting was complete).

[0353] One alternative method of measuring melting point involves using an imaging melting point apparatus. As described above, vials of lyophilized stable emulsions with different excipients are triple-melted. The melting phase transition is observed as an opaque cake melting into a transparent liquid, which reduces the light intensity of the sample in the image. The initial point for visual evaluation is the first significant decrease in the volume of the cake at the onset of the melting phase transition (i.e., the beginning of the change in the cake before melting), and the melting point (T0) at the midpoint of the cake. m The temperature at which the disc has lost half the pixel intensity between completely solid and completely melted is determined. Similarly, the temperatures at which the disc has lost 25% and 75% intensity (normalized to the start and end of the transition) are calculated as melting range measures.

[0354] Humidity measurement

[0355] Following two independent lyophilization runs, the moisture content of lyophilized cakes containing 5% trehalose was characterized using duplicate flasks of the lyophilized stabilized emulsions (5% trehalose and 2% oil stabilized emulsions) in a Denver Instruments (Bohemia, NT) Coulometric Karl Fischer titrator (Model 270). Titrations were performed at moderate stirring speeds, with an endpoint duration of 15 seconds and an endpoint slope of 0.05. Samples were reconstructed using Riedel-de-Haens Hydranal AG solution (Sigma-Aldrich, St. Louis, Mo), and the weighed solution was injected into the titrator. After determining the water content by mass using the instrument, the percentage water content was calculated as %w / w.

[0356] Particle size and zeta potential

[0357] Emulsion particle size, polydispersity (PdI), and zeta potential were evaluated on Malvern (Worcestershire, UK) Nano-ZS using dynamic light scattering (DLS). Measurements were typically performed as previously described (Fox et al., 2011, Pharmaceutical Development and Technology, 16(5): 511-519). In short, at 10... -2 All DLS size measurements were performed in 20 nm filtered water (as described above), and a single sample was triple-evaluated. For zeta potential measurements, the same sample was evaluated using triple measurements and automated software with measurement times ranging from 20 to 40 runs.

[0358] High performance liquid chromatography

[0359] Chemical degradation of squalene, DMPC, and GLA was monitored by reversed-phase high-performance liquid chromatography (RP-HPLC). An Agilent 1200 (Santa Clara, CA) and an ESA Biosciences Corona charged electrosol detector (CAD; Chelmsford, MA) were used with a Waters Atlantics C18 μm column (4.6 mm x 250 mm; Milford, MA). See Fox et al., 2008, Colloids and Sufaces B: Biointerfaces, 65:98-105. Mobile phase A contained 75:15:10 (v / v / v) methanol:chloroform:water and 20 mM ammonium acetate with 1% acetic acid, and mobile phase B contained 50:50 (v / v) methanol:chloroform with 20 mM ammonium acetate and 1% acetic acid. Samples were prepared by diluting the reconstituted sample 1:20 into mobile phase B. Using 9 μl of injection solution, a linear gradient was applied over 45 minutes from 100% to 10% mobile phase A, with the column temperature at 30°C. All solvents used were HPLC grade.

[0360] Reconstructing the filter

[0361] As described in 3.1, formulations were prepared containing 2% SE and each of the following: trehalose, dextran, lactose, maltose, sucrose, raffinose, mannose, fructose, lactulose, ribose, dextran, PEG, mannitol, stachyose, sorbitol, and proline. Formulations containing pyridinedicarboxylic acid and nicotinic acid were also prepared, but were formulated at 0.5% due to solubility limitations. Each formulation was reconstructed as described above. The samples were characterized as described above based on pie appearance, layering, particle size, zeta potential, PdI, and pH.

[0362] Cake stability screening

[0363] The melting points of a subset of compounds at 5% were evaluated as described above. These compounds included trehalose, dextrose, lactose, maltose, sucrose, mannose, fructose, lactulose, raffinose, ribose, stachyose, mannitol, and proline. As a follow-up study, formulations containing 5% trehalose, dextrose, lactose, maltose, or sucrose were prepared using 0.2% or 5% mannitol and 2% stable emulsion. These formulations were evaluated as described above based on cake melting point, separation, pH, PdI, and particle size.

[0364] Accelerated stability characterization

[0365] 5% trehalose in SE vials were stored at 25, 37, 50, 60, and 90 °C and periodically reconstituted over 1500 hours, or until complete failure was observed. Samples were characterized at each time point by DLS, pH, HPLC, and cake appearance. Similar characterization of undried stable emulsions was used for comparison. Formulations identified as having high cake melting temperatures were selected for additional accelerated stability characterization to develop more stable systems. Formulations contained 5% dextrose, maltose, sucrose, and trehalose. An experimental formulation containing 15% lactose was also included. Accelerated stability of these formulations was characterized at 90 °C as described above to obtain changes in all of these formulations.

[0366] result

[0367] freeze-dried SE in trehalose

[0368] Due to the widespread use of trehalose in lyophilized formulations, the feasibility of using trehalose-containing formulations for emulsion lyophilization was evaluated. After lyophilization, a cake containing 5% trehalose and 2% v / v squalene SE produced a slightly wrinkled white cake with a water content of 0.3% w / w and good reconstitution solubility. Figure 1A The reconstructed emulsion visually appeared similar to the un-lyophilized formulation. An increase in average particle size of 21 nm was observed after reconstruction. Figure 1B The pH increased by 0.02, along with the PdI. Lyophilization and reconstitution did not alter the pH. These results provide a preliminary assessment of emulsion maintenance after lyophilization and reconstitution.

[0369] Accelerated stability characterization of SE in trehalose

[0370] After successful freeze-drying and reconstructing of SE in trehalose, the stability of freeze-dried and non-freeze-dried stable emulsions was evaluated to determine whether freeze-drying improved emulsion stability. The stability was determined based on the particle size of the reconstructed freeze-dried stable emulsion. Figure 2A ) and pH ( Figure 2B Lyophilized formulations were more stable than liquid formulations at 25°C and 37°C. No significant changes in the chemical composition of the stable emulsions, whether liquid or reconstituted lyophilized, were observed at these temperatures. Lyophilization also increased the chemical stability of the emulsion at the elevated temperatures necessary to induce changes in the composition of liquid formulations. Figure 2C In particular, α-tocopherol, squalene, and ovite-derived phosphatidylcholine (ovite PC) were all protected in the lyophilized formulation compared to the liquid formulation. Furthermore, no separated oil phase indicated by oiling-off was detected. Because the oil did not appear to redistribute or enter different phases in this system, the particles remained an emulsion after reconstruction.

[0371] Determination of the main failure mechanism

[0372] To improve the stability of lyophilized stable emulsions of trehalose, a study was conducted to determine the main failure mechanism, aiming to select future formulations that inhibit this mechanism. The lyophilized stable emulsions were stored at temperatures ranging from 25, 37, 50, 60, and 90 °C for up to 1500 hours, or until the formulation had failed outside its characterization range (Figure 3). The lyophilized emulsions were significantly less stable above 50 °C. The first observed change was particle size increase, which occurred simultaneously with the remelting of the cake (Figure 3). Figure 3A and Figure 3C Large pH changes only begin to occur when the particles have already failed due to particle size. Figure 3B Therefore, it is not considered a major failure mechanism. This is because the concentration of the emulsion component ( Figure 2C ) and pH ( Figure 3B The structure did not change initially, so the proposed mechanism is not based on covalent bonding. However, the remelting of the cake-like material is directly related to the increase in particle size over time. Figure 3D Since the remelting of the cake appears to occur faster than the increase in particle size, the resistance of the cake to remelting is a key property for maintaining the particle size stability of the emulsion under pressure.

[0373] Excipient screening for SE reconstruction

[0374] Based on the assumed failure mechanism, excipients were screened to identify compounds that maintain emulsion properties and resist remelting (Table 1, Figure 4). Excipients were categorized into four classes based on their pie structure and the maintenance of emulsion properties after reconstruction. Representative data for these categories are shown in Figure 5.

[0375] Table 1. Comparison of formulation characteristics between 2% (v / v) oil-stabilized emulsions in reconstructed 5% (w / v) formulations.

[0376]

[0377]

[0378] Class 1 excipients form white cake-like substances with a cake-like morphology range, and the emulsion properties are not significantly changed after lyophilization (Table 1). Figure 4A -B). This category includes trehalose, dextrose, lactose, maltose, sucrose, raffinose, mannose, fructose, and lactulose. The reconstructed emulsion had a particle size less than 200 nm, no stratification was observed after one hour, and the PdI was less than 0.25. The Zeta potential decreased from -9 mV to -15 to -25 mV. The pH varied from 5.4 to 6.2. The cake-like appearance of the stable emulsion in lyophilized 5% trehalose (a representative Class 1 formulation) was acceptable, although slightly wrinkled. Figure 5A No stratification was observed within 24 hours. Figure 5B And the particle size distribution remains uniform, less than 150 nm. Figure 5C ).

[0379] A type 2 excipient ribose, when freeze-dried, does not form a cake but rather a film. Figure 5A However, the emulsion can be reconstructed to have acceptable particle size, PdI, zeta potential, and pH (Table 1, ...). Figure 4A -B).

[0380] The three types of excipients formed good cake-like structures, but disrupted the emulsion after remodeling (Table 1, Figures 4 and 5). The remodeled particles had sizes greater than 200 nm and high PdI values, ranging from 0.59 to 0.93. The pH and consequently zeta potentials within this group varied considerably. The observed large pH changes in formulations containing PEG and mannitol occurred during lyophilization. The pH was adjusted to 5.5 before lyophilization. As expected from the large particle size and high polydispersity, the three types of excipients caused stratification after remodeling (Table 1, Figures 4 and 5). Figure 5B ).

[0381] The four classes of excipients did not form cakes and disrupted the emulsion in the same manner as the three classes of compounds, although these formulations were not completely reconstituted rather than stratified (Table 1, Figures 4 and 5). Each of these excipients resulted in large SE particles with high PdI values ​​and pH values ​​between 4.3 and 5.1. Pyridine dicarboxylic acid and nicotinic acid are quite insoluble in water, meaning that these formulations were produced at 10% of the mass concentration of the other formulations. This may be a contributing factor to the poor emulsion maintenance of these excipients.

[0382] Screening of single excipient structures for thermal stability of cake-like materials

[0383] After identifying available lyophilization excipients, the thermal stability of the cakes produced using the selected excipients was evaluated. This was used as a method for identifying excipients with higher melting points and greater resistance to remelting (Table 2). Class 1 excipients had the lowest onset point range, from instantaneous (meaning at or below 27°C onset temperature) to 78°C. The midpoint melting point ranged from 36°C to 94°C. Class 3 excipients had onset points above 90°C and all had midpoint melting points above 100°C. Mannitol exhibited the greatest cake stability, with a narrow melting point range centered at 160.1°C. Representative cake melting heat analysis plots are shown in... Figure 6A As shown in the figure. Comparison of excipients based on the starting point and melting point of the cake-like material. Figure 6B As shown in the figure, monosaccharides appear to have a significantly lower melting point compared to polysaccharide excipients.

[0384] Table 2. Comparison of cake melting characteristics between 2% (v / v) oil-stabilized emulsions in 5% (w / v) formulations

[0385]

[0386] The effect of the thermal stability of the cake on the stability of the formulation

[0387] To assess the correlation between melting point and thermal stability of cake-like products, the particle size growth rate (SE) of various sugars with different melting points was evaluated at 90 °C. The stability differences between 5% and 15% lactose formulations of dextrose, sucrose, maltose, and trehalose, which have a wide range of melting points, were investigated (Figure 7). 15% lactose was identified as having a higher melting point than 5% lactose and was selected because its melting point was significantly higher than the pressure storage temperature of 90 °C. Dextrose was observed to have the lowest melting point and experienced the fastest particle size growth. Figure 7A Sucrose, maltose, and trehalose have intermediate melting points and experience moderate melting rates. As the melting point of the cake increases across formulations, the particle size increase decreases, with 15% lactose exhibiting the highest melting point and the lowest particle size increase rate. Furthermore, a bifurcation of the particle size increase rate was observed between samples with melting points above and below storage temperatures. Figure 7B This provides further evidence that the remelting of the cake is the main thermal degradation failure mechanism.

[0388] Screening of excipient combinations for thermal stability of cake-like materials

[0389] Because mannitol was observed to increase the thermal stability of cake emulsions (Table 2), various Class 1 excipients were combined with mannitol in an attempt to produce formulations that maintained the emulsion but exhibited excellent cake stability (Table 3). Figure 8 In the presence of mannitol, Class 1 excipients reduced emulsion particle size growth. Adding 0.2% w / v mannitol did not increase particle size or Tm. Adding 5% w / v mannitol significantly increased cake Tm but also increased emulsion particle size and PdI, while simultaneously decreasing pH.

[0390] Table 3. The relationship between 2% (v / v) oil-stabilized emulsions in 5% (w / v) formulations containing 0.2% or 0.5% (w / v) mannitol. Comparison of characteristics of cake-shaped products

[0391]

[0392] Example 2: Stability of adjuvant-containing freeze-dried anti-tuberculosis vaccine

[0393] The only approved tuberculosis vaccine (TB), BCG, was first used in humans in 1921 and has been effective in reducing the incidence of disseminated TB in children. However, BCG has been shown to be ineffective in the prevention of pulmonary TB in adolescents and adults (Checkley et al., 2011, Trends Pharmacol Sci, 32:601-606; Rowland et al., 2011, Expert Rev Vaccines, 10:645-658; Anderson et al., 2005, Nat Rev Microbiol, 3:656-662). Mathematical modeling of the impact of a new TB vaccine assuming 60% efficacy predicts an 80% reduction in incidence by 2050 (Abu-Raddad et al., 2009, PNAS, 106:13980-13985). Therefore, there is an urgent need for new TB vaccines that promote immunization sensitized by BCG or that replace BCG. Protective immunity against Mycobacterium tuberculosis (Mtb) requires the production of TNF and IFN-γ by CD4 T cells (Flynn et al., 2001, Annu Rev Immunol, 19: 93-129; Cooper AM, Annu Rev Immunol; 27: 393-422). A recombinant fusion protein antigen designated ID93, consisting of four Mtb proteins: Rv3619, Rv1813, Rv3260, and RV2608, has been developed. These constituent proteins have been confirmed to be recognized by human T cells derived from TB-infected or BCG-immune donors and to protect against Mtb challenge in mouse and guinea pig models when conjugated with adjuvants that induce a potent TH1 response (Bertholet et al., 2010, SciTransl Med, 2:53ra74; Bertholet et al., 2008, J Immunol, 181:7948-7947), such as the synthetic TLR4 agonist glucopyranose liposide adjuvant formulated in a water-encapsulated squalene-stabilized emulsion (GLA-SE). ID93+GLA-SE is currently undergoing Phase I safety testing in human volunteers.

[0394] Both ID93 and GLA-SE were stable for over one year when stored under sustained cold chain conditions. No changes in protein concentration, GLA concentration, particle size, or physical appearance were observed under these conditions. Stability observations are ongoing, with no estimated degradation time at 4°C. While the stability of ID93+GLA-SE is comparable to other vaccines, increased vaccine thermal stability is desirable to reduce the need for sustained cold chain maintenance. One approach to improving vaccine stability by increasing temperature is to freeze-dry the antigenic components of the vaccine and then mix them with the adjuvant at the time of use. However, this requires a cold chain to maintain the adjuvant and increases the technical burden of administration. To overcome this problem, single-vial (covialed) versions of both the ID93 antigen and the GLA-SE adjuvant have been developed.

[0395] Materials and methods

[0396] Sample preparation and freeze drying

[0397] The construction, expression, and purification of the ID93 tandem fusion protein containing the Mtb genes Rv3619, Rv1813, Rv3620, and Rv2608 have been previously described (Bertholet et al., 2010, Sci Transl Med, 2:53ra74). In summary, the ID93 fusion protein was expressed in *E. coli*, purified by chromatography on DEAE and Q agarose gel columns under denaturing conditions, and analyzed by SDS-PAGE on 4–20% Tris-glycine gels (Invitrogen). GLA (also known as PHAD) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). GLA-SE containing 1,2-dimyristic-sn-glycerol-3-phosphocholine (DMPC) was prepared according to the previously described method (Orr et al., 2013 J Control Relea SE 172: 190-200; Anderson et al., 2010 Colloids Surf B: Biointerfaces 75: 123-32). In short, the GLA-SE emulsion was produced by mixing a buffered aqueous phase (poloxamer 188 and glycerol in pH 5.1 ammonium phosphate buffer) and an oil phase (DMPC and GLA dispersed in squalene by sonication at 70°C) followed by microfluidization of the mixture 12 times at 30,000 psi using a Microfluidics M110P (Newton, MA). The emulsion contains the following components: 10% v / v squalene, 1.9% w / v phosphatidylcholine, 0.1% w / v poloxamer 188, 2.3% w / v glycerol, and 25 mM ammonium phosphate buffer. Dilute GLA-SE to the specified concentration.

[0398] For the analysis of vaccines containing GLA-SE adjuvant, liquid and lyophilized samples were prepared using 1.5 mL filler volumes in 3 mL glass vials. Co-vial samples containing ID93 (5 μg / mL) + GLA-SE (50 μg / mL, 2% total oil) were prepared in 20 mM tromethorphanol (Tris) at pH 8.0 (Coler et al., 2011, PLoS One, 6, e16333). Individually vials of ID93 or GLA-SE (10 μg / mL ID93 or 100 μg / mL GLA, 4% total oil GLA-SE) were prepared at twice the concentration of the co-vial samples and mixed 1:1 prior to injection. SDS-PAGE samples were prepared at 100 μg / mL ID93 for analysis. The lyophilized samples also contained 5% (w / v) D-trehalose dehydrate as a stabilizer and were lyophilized using a VirTis (Gardiner, NY) AdVantage 2.0EL-85 benchtop lyophilizer. The lyophilization method employed a heat treatment arrangement consisting of a 10-hour freezing step from 4 to -40°C, followed by an annealing step at -15°C. The primary drying stage (at 100 mTorr) lasted 18 hours from -40°C to 25°C. Finally, a secondary drying stage of 9 hours was performed at 25°C at 50 mTorr. All samples were plugged in atmospheric gas at 500 mTorr, sealed with aluminum caps, and stored at 4°C until use. Thermo-stressed samples were incubated at 50°C for 30 days, while unstressed samples were stored at 4°C before injection.

[0399] For the analysis of vaccines containing SLA-SE adjuvant, lyophilized samples were prepared in 1.5 mL filler volumes of 3 mL glass vials. Co-flask samples containing 2% (v / v) oil-stabilized emulsion, 50 μg / mL SLA, 100 μg / mL ID93, 20 mM Tris pH 8.0, and 5% (w / v) trehalose were prepared. Lyophilization was performed using a VirTis (Gardiner, NY) AdVantage 2.0EL-85 benchtop lyophilizer. The lyophilization method employed a heat treatment arrangement consisting of a 10-hour freezing step from 4 to -40 °C, followed by an annealing step at -15 °C. The primary drying stage (at 100 mTorr) lasted 18 hours from -40 °C to 25 °C. Finally, a secondary drying stage of 9 hours was performed at 25 °C at 50 mTorr. All samples were plugged in atmospheric gas at 500 mTorr, sealed with aluminum caps, and stored at 4 °C until use. Thermo-stressed samples were incubated at 50°C for 30 days, while unstressed samples were stored at 4°C. Characterization replicates (A and B) were used. Sample reconstruction was performed using 1.5 mL of filtered H₂O.

[0400] Restore SDS-PAGE

[0401] SDS-PAGE was reduced using Life Technologies (Grand Island, NY) NuPAGE LDS sample buffer containing 1.25% β-mercaptoethanol and incubated at 90°C for 15 min. Samples were run for 65 min at 180 V using 1 μg ID93 / channel in a Life Technologies Novex 4-20% acrylamide tris-glycine pre-cast gel cartridge. The gels were stained overnight using Life Technologies SimplyBlue SafeStain, then destained, dried, and imaged. Band intensities were compared using ImageJ software (NIH) (Schneider et al., 2012, Nat Methods, 9: 671-675).

[0402] Particle analysis

[0403] As previously described, particle size, polydispersity, and zeta potential measurements were performed (Fox et al., 2008, Colloids and Surfaces B: Biointerfaces, 65: 98-105). Measurements were taken using a Malvern (Worcestershire, UK) Nano-ZS camera after a 100-fold dilution in ultrapure water filtered through a 20 nm Whatman (Maidstone, Kent, UK) Anotop+ filter. Nanoparticle tracking analysis was performed using a NanoSight LM10 (Amesbury, UK) with a 405 nm laser and a Hamamatsu Orca Flash 2.8 CMOS camera (Hamamatsu, JP). Samples were diluted 1:105 in 20 nm filtered ultrapure water in three steps. Each sample was independently diluted and analyzed four times to account for dilution errors. 90-second video recordings of each sample were taken using optimized shutter and gain settings. The camera histogram gating was set to maximum sensitivity. Data analysis was performed using the standard NanoSight NTA2.3 software (Wiltshire, UK).

[0404] Chemical integrity of adjuvants

[0405] As previously described, reversed-phase HPLC (RP-HPLC) was used to monitor the concentrations of squalene, DMPC, GLA, and SLA (Fox et al., 2008, Colloids and Surfaces B: Biointerfaces, 65:98-105). An Agilent 1200 (Santa Clara, CA) and an ESA Biosciences Corona charged electrosol detector (CAD; Chelmsford, MA) equipped with a Waters (Milford, MA) Atlantic C18 5 μm column (4.6 mm x 250 mm) were used. Mobile phase A consisted of 75:15:10 (v / v / v) methanol, chloroform, and water, along with 20 mM ammonium acetate and 1% acetic acid. Mobile phase B consisted of 50:50 (v / v) methanol and chloroform, 20 mM ammonium acetate, and 1% acetic acid. Samples were prepared by diluting (1:20) into mobile phase B, and 9 μl was injected onto a column at 30 °C. Gradient elution was performed using 100% to 10% mobile phase A for 45 minutes. A quadratic polynomial-matched standard curve was used as recommended by the detector manufacturer, and sample concentration was determined by interpolation.

[0406] Animals and Immunization

[0407] Female C57BL / 6 mice aged 6–8 weeks were purchased from Charles River and kept in a specific pathogen-free environment. Following infection, the animals were kept in biosafety level 3 control. Mice were immunized three times over three weeks by intramuscular injection of 100 μL of the specified vaccine formulation. For BCG immunization, 5 x 10⁴ CFU (Pasteurstrain, Sanofi Pasteur) was administered intradermally once during the first subunit immunization.

[0408] Blood cell count

[0409] Peripheral blood was collected from mice 18 hours after immunization (N=5 / group). Whole blood was stained for CD90.2 (clone 53-2.1) and CD19 (clone 6D5). Sphero AccuCount rainbow particles (Spherotech, LakeForest, IL) were added according to the manufacturer's instructions. Cells were washed and resuspended in PBS. Cells were collected up to 10⁶ pieces using a four-laser LSR Fortessa flow cytometer (BDBiosciences). Data were analyzed using FlowJo. The absolute number of CD19+ B cells and CD90.2+ T cells per μL of blood was calculated according to the manufacturer's instructions.

[0410] antibody response

[0411] Twenty-one days after immunization, mouse serum was prepared by collecting retroorbital blood into Microtainer serum collection tubes (VWR International, West Chester, PA) and then centrifuging (N=5 / group). Each serum sample was then analyzed by antibody capture ELISA. Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 1 μg / ml recombinant antigen in 0.1M bicarbonate buffer and blocked with 1% BSA-PBS. Then, after sequential washing in PBS / Tween 20, serially diluted serum samples, anti-mouse IgG, IgG1 or IgG2 c-HRP (all from Southern Biotech, Birmingham, AL), and ABTS-H2O2 (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added to the plates. The plates were analyzed at 405 nm (ELX808, Bio-Tek Instruments Inc, Winooski, VT).

[0412] Intracellular cytokine staining

[0413] One month after the final immunization, spleen cells were isolated from 5 animals in each group. Red blood cells were lysed using erythrocyte lysis buffer (eBioscience) and resuspended in RPMI 1640 and 10% FBS. Cells were placed in 96-well plates at 2 x 10⁶ cells / well and stimulated at 37°C for 1 h with a medium or ID93 (10 μg / mL). GolgiPlug (BDBiosciences) was added, and cells were cultured for an additional 7 h at 37°C. Cells were washed and surface-stained for 20 min at 4°C with fluorescently labeled antibodies against CD4 (clone GK1.5), CD8 (clone 53-6.7), and CD44 (clone IM7) (BioLegend and eBioscience) in the presence of CD16 / 32 antibody (clone 2.4G2). Cells were washed and permeabilized with Cytofix / Cytoperm (BDBiosciences) at room temperature for 20 min. Cells were washed twice with Perm / Wash (BD Biosciences) and stained intracellularly for 20 minutes at room temperature with antibodies (BioLegend and eBioscience) labeled with fluorescent dyes for IFN-γ (clone XMG-1.2), IL-2 (JES6-5H4), TNF (MP6-XT22), CD154 (clone MR1), IL-5 (clone TRFK5), and IL-17A (clone TC11-18H10.1). Cells were washed and resuspended in PBS. Cells were collected up to 10⁶ pieces using a four-laser LFR Tortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo. Cell gating was performed as follows: singlet cells > lymphocytes > CD4+CD8- > CD44+ > cytokine-positive.

[0414] Tuberculosis aerosol attack and computation

[0415] Four weeks after the final immunization, mice (n=7 / group) were infected with Mycobacterium tuberculosis H37Rv (ATCC number 35718; American Center for Type Culture Collection) using a GlasCol aerosol generator calibrated to deliver 50-100 bacteria into the lungs. To confirm the delivered bacterial load, three additional unimmunized animals from each infection group were euthanized one day later, and the bacterial burden in the lungs was calculated. Three weeks after challenge, protection was determined by harvesting lungs from infected mice, homogenizing the tissue in 0.1% PBS-Tween 80, and serially spotting the tissue onto 7H10 agar plates (Molecular Toxicology) at 5-fold dilutions. Bacterial colonies were counted after incubation at 37°C with 5% CO2 for 14-21 days.

[0416] Statistical methods

[0417] Bacterial burden was normalized using log10 transformation. Statistical significance of differences in bacterial burden, cytokine production, blood cell count, and antibody titer was determined using one-way ANOVA with Bonferroni multiple comparison assays in Prism 5 (GraphPad software).

[0418] result

[0419] Physicochemical characterization of vaccine formulations containing GLA-SE adjuvant

[0420] A lyophilization method was developed for this co-channel adjuvanted vaccine. Upon lyophilization, a white, partially wrinkled cake-like substance is formed, which, after being reconstituted with water, forms an emulsion that appears similar to the emulsion before lyophilization. Figure 9 (Upward). The potential for increased stability against thermal stress by lyophilization was evaluated by incubating the liquid at 50°C or by lyophilizing ID93+GLA-SE for 30 days. After thermal stress, when compared with unstressed samples ( Figure 9 No visible change in sample quality was observed during the upward comparison. Figure 9 (Downstream). The reconstructed sample maintained the emulsion appearance and the lyophilized cake showed no further signs of collapse or discoloration.

[0421] Particle characteristics are critical for effective vaccine development because particle size determines the rate and mechanism of in vivo vaccine delivery. Maintaining a particle size of less than 200 nm is desirable to allow for terminal aseptic filtration of the product; furthermore, particles <200 nm can rapidly enter lymph nodes (Bachmann et al., 2010, Nature Rev Immunol, 10: 787-796). To assess whether lyophilization and reconstitution of co-bottle or co-bottle ID93+GLA-SE alters biophysical properties, the particle size, concentration, polydispersity, and overall zeta potential of ID93, GLA-SE, co-bottle ID93+GLA-SE, and lyophilized co-bottle ID93+GLA-SE were examined. Because the particle concentration was five orders of magnitude higher than that of ID93, the measured particle characteristics after mixing primarily reflected the contribution of GLA-SE. Co-bottle ID93 and GLA-SE did not affect the particle size (80 nm in both cases) compared to GLA-SE alone. Figure 10A Lyophilization and subsequent reconstruction of ID93+GLA-SE resulted in a small increase of approximately 10 nm, within the measurement error range. ID93 formed polydisperse aggregates with a Z-average diameter of approximately 70 nm. Thermal stress of ID93 alone reduced the observed average particle size; however, this was not statistically significant (p > 0.05). Figure 10A The thermal stress of GLA-SE alone or in combination with ID93 did not affect the particle size or concentration across any test platform. Figure 10A and Figure 10D ). For example, as reflected by low polydispersity values ​​( Figure 10B GLA-SE is a highly homogenized solution. Although the polydispersity observed for ID93 is significantly higher, the mixture of ID93 and GLA-SE retains the overall low polydispersity of GLA-SE, which reflects the relative proportions of ID93 and GLA-SE particles. Importantly, lyophilized and reconstituted ID93+GLA-SE retain this consistent particle size. Exposure to thermal stress did not affect the polydispersity of ID93, GLA-SE, or co-flask ID93+GLA-SE in liquid or lyophilized formats. Both ID93 and GLA-SE have an overall negative zeta potential in the current configuration. The mixture of the two results in an average zeta potential of -13 mV, which is unaffected by lyophilization. Figure 10C Upon thermal stress, all GLA-SE-containing samples exhibited a more negative zeta potential; however, this change was statistically significant only for lyophilized ID93+GLA_SE (p < 0.025). Overall lyophilization and reconstitution of ID93+GLA-SE did not alter its physicochemical properties compared to unlyophilized ID93+GLA-SE. Prolonged exposure to elevated temperatures significantly affected the physicochemical properties of ID93, but not GLA-SE.

[0422] To assess how co-bottle formation, lyophilization, and thermo-stressing affect the chemical integrity of ID93+GLA-SE, ID93 concentration was evaluated by SDS-PAGE, and GLA, squalene, and DMPC (the latter two being the main components of the stabilizing emulsion) concentrations were evaluated by RP-HPLC. Since ID93 cannot be detected by SDS-PAGE at 5 μg / mL, samples containing 100 μg / mL ID93 were evaluated. Co-bottle samples containing 100 μg / mL and 5 μg / mL ID93 showed similar particle size, particle concentration, zeta potential, and GLA degradation profile under liquid, lyophilization, and thermo-stressing conditions. It is likely that the GLA-SE reaction was disrupted by SDS-PAGE particles, resulting in a disintegration smear that was visible after staining. Lyophilization and remodeling of co-bottle ID93+GLA-SE resulted in a 5-10% decrease in ID93 concentration, expected to be due to dilution after remodeling, suggesting that substantial hydrolysis of ID93 did not occur. Figure 11 When exposed to 50°C heat stress for one month, the ID93 present in ID93+GLA-SE showed a sharp decrease. Lyophilization of ID93+GLA-SE induced protein resistance to this degradation; liquid and lyophilized samples showed 6% and 90% of the ID93 band intensity, respectively, after heat stress compared to unstressed samples. Figure 11 Therefore, lyophilized ID93+GLA-SE protects the ID93 protein from thermal stress-induced degradation.

[0423] As expected, after mixing GLA-SE with ID93 at a 1:1 ratio, approximately half of the original concentrations of GLA, DMPC, and squalene were measured, and no substances were lost after lyophilization and remodeling. Figure 12A Exposure of liquid GLA-SE to thermal pressure resulted in a 50% loss of GLA concentration (p < 0.001). No detectable amount of GLA was observed after co-bottle pressing with ID93 to thermal pressure. Figure 12A This enhanced sensitivity is likely due to the more alkaline pH of the co-flask ID93+GLA-SE compared to GLA-SE alone. This GLA loss was mitigated by lyophilizing the co-flask ID93+GLA-SE, and approximately 50% of the GLA was recovered after reconstructing the lyophilized ID93+GLA-SE under thermal stress. Figure 12D GLA is the main thermally unstable component of GLA-SE because the concentrations of DMPC or squalene are not affected by thermal stress. Figure 12B -D). In summary, these data demonstrate that freeze-drying protects the two active components of ID93+GLA-SE from heat-induced degradation.

[0424] In summary, lyophilization of ID93+GLA-SE results in a white to off-white disc-like substance that, upon remodeling, retains the chemical and biophysical properties of the co-vase ID93+GLA-SE. Exposure of ID93+GLA-SE to thermal stress leads to significant loss of both ID93 and GLA. Lyophilization of co-vase ID93+GLA-SE largely mitigates these losses due to thermal stress, suggesting that this approach could reduce or eliminate the need for cold chain maintenance for this vaccine candidate.

[0425] Immunogenicity and efficacy of vaccines containing GLA-SE adjuvant

[0426] To determine how thermostress affects the biological activity of ID93+GLA-SE and whether lyophilization mitigates any adverse effects, mice were immunized with saline or ID93+GLA-SE stored as a liquid antigen and adjuvant in separate vials (liquid), a mixture of antigen and adjuvant (liquid co-vial), or co-lyophilized antigen and adjuvant (lyophilized co-vial). Immunization materials were stored at 4°C (i.e., no pressure) or 50°C (i.e., thermostress) for one month prior to immunization. Immunization resulted in a transient loss of circulating B and T cells from the blood as these cells homed to draining lymph nodes, where they encountered the antigen (Shiow et al., 2006, Nature, 440:540-544). This transient lymphocytopenia is termed lymph node closure, as lymphocytes transiently enter the lymph nodes during the process required for effective interaction between antigen-presenting cells and homologous lymphocytes. GLA-SE adjuvant amplifies this effect, which may partially explain its superior adjuvant activity. Immunization with pressureless liquid ID93+GLA-SE caused a rapid loss of both B and T cells from the bloodstream. Figure 13A Stressing the liquid ID93+GLA-SE at 50°C for one month reduced this effect, suggesting that thermal stress damages GLA activity. Unstressed liquid co-bottle ID93+GLA-SE induced lymph node closure as effectively as the liquid vaccine; however, this co-bottle material was more affected by thermal stress because the degree of lymph node closure was significantly reduced, which likely reflects detectable GLA loss. Figure 13A Lyophilized co-cultured ID93+GLA-SE induced transient lymphopenia to a level similar to that of liquid materials; however, unlike liquid co-cultured materials, this effect was not impaired by the thermal stress of the lyophilized co-cultured vaccine. These data suggest that the biological activity of the GLA-SE adjuvant is sensitive to thermal stress, and that this sensitivity is exacerbated by co-culture with the ID93 antigen. Importantly, lyophilization made the co-cultured ID93+GLA-SE resistant to the damage caused by thermal stress, as read from this parameter.

[0427] To more fully examine the effects of heat stress and lyophilization on the biological activity of ID93+GLA-SE, the titer of ID93-specific antibodies after immunization was assessed. Immunotherapy with ID93+GLA-SE induced a mixed IgG1 / IgG2c response, predominantly producing IgG2c. This reflects the IFN-γ-dominated CD4 T cell response induced by ID93+GLA-SE. Exposure to heat stress significantly impaired the ability of liquid ID93+GLA-SE to induce measurable antibody titers. Figure 13B This is most likely due to the degradation of the ID93 protein under thermal stress. Figure 11When the vaccine was stored at 4°C, although the magnitude of the antibody response was not altered by the co-channel ID93+GLA-SE, this was insufficient to prevent the loss of antibody induction potential caused by thermal stress. Conversely, the lyophilized co-channel ID93+GLA-SE induced a strong antibody response similar in magnitude and IgG1 / IgG2c propensity to that of the liquid, stress-free material, and it was not impaired by thermal stress. Figure 13B ).

[0428] ID93+GLA-SE protects against Mycobacterium tuberculosis by inducing the production of ID93-specific CD4 T cells (i.e., TH1 cells) that produce IFN-γ, TNF, and IL-2. Following a third immunization with liquid ID93+GLA-SE, exposure to heat stress reduced the frequency of ID93-specific TH1 cells by almost 50%, as measured by the production of any of these cytokines. Figure 13C Despite ID93 protein degradation, the TH1 response to stress-exposed liquid ID93+GLA-SE was maintained, likely reflecting the presence of immunogenic peptides and residual GLA after heat exposure. When stored at 4°C, co-flasking with liquid ID93+GLA-SE slightly increased the magnitude of the TH1 response; however, exposure to heat stress completely eliminated the ability of liquid co-flask ID93+GLA-SE to induce this response. Lyophilized co-flask ID93+GLA-SE induced the TH1 response to levels similar to those produced using liquid ID93+GLA-SE. Crucially, unlike liquid or liquid co-flask ID93+GLA-SE, lyophilized co-flask ID93+GLA-SE completely retained its ability to induce ID93-specific TH1 cells after heat stress. Figure 13C Previously, it was determined that only TH1 cells, which are immune to natural ID93+GLA-SE-induced immunization, are the only ID93-specific CD4 T cells that cannot produce IL-5 (TH2) or IL-17 (TH17) upon restimulation (Orr et al., 2013, Eur J Immunol.). Co-flasking, lyophilization, and / or exposure to heat stress did not enhance the induction of TH2 or TH17 cells by ID93+GLA-SE as measured by detectable IL-5 or IL-17 production. CD154 production after stimulation has been proposed as a generalized marker of CD4 T cell activation regardless of cytokine production (Frentsch et al., 2005, Nat Med, 11:1118:1124). In all cases, CD154 expression levels closely correlated with both IFN-γ and TNF, further indicating no deviation from TH1 programming. Overall, early injury lymphocytes are expelled from the blood ( Figure 13A ) and antibodies against ID93+GLA-SE vaccine ( Figure 13B ) and CD4 T cell response ( Figure 13C The subsequent losses of the two are closely related.

[0429] To assess how heat stress, co-bottle formation, and lyophilization affect the protective efficacy of ID93+GLA-SE, mice were challenged with a low-dose nebulized Mycobacterium tuberculosis. As measured by reduction in bacterial burden in the lungs and spleen, animals immunized with liquid ID93+GLA-SE showed significantly greater protection against Mycobacterium tuberculosis at three weeks compared to saline-immunized animals. Figure 14A and Figure 14B Thermal stress-induced liquid ID93+GLA-SE alone did not impair this protective efficacy, which likely reflects a residual ID93-specific TH1 response induced by this immune response. Figure 13C When stored at 4°C, co-channel ID93+GLA-SE did not compromise protective efficacy, but the liquid co-channel vaccine lost all protective efficacy upon exposure to heat stress. The lyophilized co-channel ID93+GLA-SE maintained protective efficacy, and most importantly, it eliminated the loss of protective efficacy caused by heat stress. Figure 14A and Figure 14B ).

[0430] Co-lyophilization and reconstructing the antigen and nanoemulsion adjuvant did not significantly alter the physicochemical properties of the vaccine. The reconstructed concentrations of the antigen, TLR4 agonist GLA, and squalene oil were not substantially different from those of the starting materials. Prolonged exposure to thermal stress had little effect on the physical properties of the liquid co-flask or co-lyophilized vaccine; however, thermal exposure led to chemical degradation of both the antigen and the TLR4 agonist, but the squalene or phospholipid components of the adjuvant remained undegraded. Co-lyophilization partially protected against this loss of the TLR4 agonist.

[0431] GLA loss due to heat stress is a strong predictor of impaired immune responses and vaccine efficacy when administered to test animals. While this correlation is not perfectly linear, reduction in GLA in liquid samples leads to a decrease in the frequency of ID93-specific CD4 T cells after immunization. Complete loss of GLA in heat-stressed liquid co-bottle ID93+GLA-SE corresponds to CD4 T cell-induced loss. Conversely, degradation of ID93 protein in liquid samples has a small effect on the magnitude of the CD4 T cell response. This may be attributed to the retention of immunodominant peptides required for T cell response preparation in heat-stressed samples. On the other hand, heat-induced degradation of ID93 significantly impairs the magnitude of antibody responses against vaccines. Many ID93-specific antibodies can be conformation-dependent. Furthermore, the residual antibody response is IgG1-preferred, indicating a loss of GLA-driven IgG2c bias. Co-lyophilized ID93+GLA-SE largely prevents heat-stress-induced loss of ID93-specific antibody responses, suggesting protection of protein structure through this process. This suggests that, compared to T-cell-dependent vaccines such as ID93+GLA-SE, protection against heat stress antigens is a more critical parameter in antibody-dependent vaccines. In fact, individually bottled liquid vaccines subjected to heat stress retain a certain degree of protective efficacy against experimental challenges using nebulized Mtb. There is a clear correlation between the ability to retain GLA concentration through lyophilization in the face of heat stress, TH1-induced retention, and the maintenance of protective efficacy.

[0432] Physicochemical properties of vaccine formulations containing SLA-SE adjuvant

[0433] A freeze-drying method was developed for ID93 vaccines containing SLA-SE adjuvant in co-bottles. Upon freeze-drying, the product forms a white, partially wrinkled cake-like substance, which can be reconstituted with water to form an emulsion. Figure 15 The potential for increased stability against thermal stress by lyophilization was evaluated by incubating lyophilized ID93+SLA-SE replicates (A and B) at 50 °C for 30 days. After thermal stress, compared with unstressed samples (… Figure 15 No visible changes in sample quality were observed when comparing samples A and B on day 0. Figure 15 (30 days, A and 30 days, B). The reconstructed samples maintained the emulsion appearance, showed no stratification 24 hours after reconstruction, and the lyophilized cakes showed no further signs of collapse or discoloration.

[0434] To assess how co-bottle formation, lyophilization, and thermal stress affect the chemical integrity of ID93+SLA-SE, the concentration of ID93 in the reconstituted formulation was evaluated by SDS-PAGE. Figure 16Each load contained 1 μg / mL of reconstituted ID93. ID93 was observed as a 98 kDa band in all tested samples. Lyophilization and reconstitution of ID93+SLA-SE under pressure did not result in substantial hydrolysis of ID93 compared to pressure-treated co-flask ID93+SLA-SE. Figure 16 Therefore, lyophilization of ID93+SLA-SE protects the ID93 protein from thermal stress-induced degradation.

[0435] To assess whether the thermostress of reconstituted lyophilized co-vial ID93+SLA-SE alters the biophysical properties of adjuvanted vaccines, the particle size, concentration, polydispersity, and overall zeta potential of the lyophilized co-vial ID93+SLA-SE were examined under both stressed and unstressed conditions. The thermostress of the reconstituted lyophilized co-vial ID93+SLA-SE did not significantly alter the particle size or polydispersity aggregation compared to unstressed co-vial ID93+SLA-SE. Figure 17A -C) or zeta potential ( Figure 18 Similar to the reconstructed lyophilized co-flask ID93+GLA-SE, the thermal stress of the reconstructed lyophilized co-flask ID93+SLA-SE resulted in approximately 50% SLA recovery. Figure 19 ).

[0436] Example 3: Lyophilized vaccine emulsion formulation and long-term stability

[0437] A lyophilization method was developed for a co-bottled ID93 vaccine containing GLA-SE adjuvant. The formulation used to assess long-term stability was the same as described in Example 1, with the GLA-SE formulation consisting of 2% v / v squalene, 0.4% w / v DMPC, 0.02% w / v poloxamer 188, 0.5% w / v glycerol, and 5 mM ammonium phosphate, plus the ID93 peptide, lyophilized in the presence of 20 mM tromethorphanol and 5% w / v trehalose. Upon lyophilization, a white, partially wrinkled cake-like substance was formed, which, upon reconstitution with water, formed an emulsion without visible stratification. Figure 20 The potential to increase stability against thermal stress by lyophilization was evaluated by culturing lyophilized ID93+GLA-SE replicates (A and B) at 4 °C, 25 °C, and 37 °C for one year.

[0438] 3-month stability data

[0439] Storage at 4°C, 25°C, and 37°C for three months confirmed no change compared to zero in terms of pie appearance or ability to be reconstructed into a suitable emulsion. Figure 21A and Figure 21B The freeze-dried cakes showed no further signs of collapse or discoloration, and the reconstructed samples maintained the emulsion appearance without stratification up to 24 hours after reconstruction.

[0440] The biophysical properties of the lyophilized adjuvant-containing vaccine ID93+GLA-SE were examined based on particle size (Z-mean, nm) and polydispersity (PDI) after three months of storage at 4°C, 25°C, and 37°C. No significant changes in particle size or aggregation were observed in the formulation after three months at any storage temperature up to 37°C. Figure 21B ).

[0441] To assess how co-bottle formation, lyophilization, and thermal stress affect the chemical integrity of the ID93 peptide in lyophilized emulsions stored at 4°C, 25°C, and 37°C, the reconstituted formulation was evaluated by SDS-PAGE. Figure 21C Each loading contained 1 μg / mL of reconstituted ID93. ID93 was observed as a 98 kDa band in all tested samples. Lyophilization and reconstitution of ID93+GLA-SE did not result in substantial hydrolysis of the ID93 peptide. Therefore, lyophilization of ID93+GLA-SE protects the ID93 protein from thermal stress-induced degradation.

[0442] The chemical integrity of the SE formulation was assessed by HPLC and DMPC and squalene were analyzed. Figure 21D The results confirmed that no component in the oil-in-water emulsion was lost or degraded.

[0443] The adjuvant concentration in lyophilized ID93+GLA-SE formulations stored at 4°C, 25°C, and 37°C was evaluated. Figure 21E Data confirm that GLA did not experience significant loss from its initial concentration of 50 μg / ml after 3 months at any storage temperature.

[0444] 6-month stability data

[0445] Storage at 4°C, 25°C, or 37°C for six months confirmed no change compared to zero in terms of pie appearance or ability to be reconstituted into a suitable emulsion. Figure 22A and Figure 22B The freeze-dried cakes showed no further signs of collapse or discoloration, and the reconstructed samples maintained the emulsion appearance without stratification up to 24 hours after reconstruction.

[0446] The biophysical properties of the lyophilized adjuvanted vaccine ID93+GLA-SE were examined for particle size (Z-mean, nm) and polydispersity (PDI) after six months of storage at 4°C, 25°C, and 37°C. No significant changes in particle size or aggregation were observed in the formulation after six months of storage at any temperature up to 37°C. Figure 22B ).

[0447] To assess how co-bottle formation, lyophilization, and thermal stress affect the chemical integrity of the ID93 peptide in the ID93+GLA-SE lyophilized formulation after six months of storage at 4°C, 25°C, and 37°C, the reconstituted formulation was evaluated by SDS-PAGE. Figure 22C Each loading contained 1 μg / mL of reconstituted ID93. ID93 was observed as a 98 kDa band in all tested samples. Lyophilization and reconstitution of ID93+GLA-SE did not result in substantial hydrolysis of the ID93 peptide. Therefore, lyophilization of ID93+GLA-SE protects the ID93 protein from thermal stress-induced degradation.

[0448] The chemical integrity of the SE formulation was assessed by HPLC and DMPC and squalene were analyzed. Figure 22D The results confirmed that no component in the oil-in-water emulsion was lost or degraded.

[0449] The adjuvant concentration in lyophilized ID93+GLA-SE formulations stored at 4°C, 25°C, and 37°C was evaluated. Figure 22E Data confirmed that there was no significant loss of GLA compared to the initial 50 μg / ml concentration after 6 months of storage at 4°C or 25°C, but approximately 50% loss of GLA was confirmed after 6 months of storage at 37°C.

[0450] 9-month stability data

[0451] Storage at 4°C, 25°C, or 37°C for nine months confirmed no change compared to zero in terms of the appearance of the cake or the ability to be reconstituted into a suitable emulsion. Figure 23A and Figure 23B The freeze-dried cakes showed no further signs of collapse or discoloration, and the reconstructed samples maintained the emulsion appearance without stratification up to 24 hours after reconstruction.

[0452] The biophysical properties of the lyophilized adjuvanted vaccine ID93+GLA-SE were examined at nine months for samples stored at 4°C, 25°C, and 37°C, based on particle size (Z-mean, nm) and polydispersity (PDI). No significant changes in particle size or aggregation were observed in the formulation at any storage temperature up to 37°C after nine months. Figure 23B ).

[0453] To assess how co-bottle formation, lyophilization, and thermal stress affect the chemical integrity of ID93 peptides stored at 4°C, 25°C, and 37°C for nine months, the reconstituted formulation was evaluated by SDS-PAGE. Figure 23CEach loading contained 1 μg / mL of reconstituted ID93. ID93 was observed as a 98 kDa band in all tested samples. Lyophilization and reconstitution of ID93+GLA-SE did not result in substantial hydrolysis of the ID93 peptide. Therefore, lyophilization of ID93+GLA-SE protects the ID93 protein from thermal stress-induced degradation.

[0454] The chemical integrity of the SE formulation was assessed by HPLC and DMPC and squalene were analyzed. Figure 23D The results confirmed that none of the components in the oil-in-water emulsion were lost or degraded after nine months of storage.

[0455] The adjuvant concentration in lyophilized ID93+GLA-SE formulations stored at 4°C, 25°C, and 37°C was evaluated. Figure 23E Data confirmed that there was no significant loss of GLA at 4°C or 25°C compared to the initial 50 μg / ml concentration, but at 37°C for nine months, the same approximately 69% loss of GLA as seen at six months was confirmed.

[0456] 12-month stability data

[0457] Storage at 4°C, 25°C, or 37°C for twelve months confirmed no change compared to zero in terms of the appearance of the cake or the ability to be reconstituted into a suitable emulsion. Figure 24A and Figure 24B The freeze-dried cakes showed no further signs of collapse or discoloration, and the reconstructed samples maintained the emulsion appearance without stratification up to 24 hours after reconstruction.

[0458] The biophysical properties of the lyophilized adjuvanted vaccine ID93+GLA-SE were examined at twelve months for samples stored at 4°C, 25°C, and 37°C, based on particle size (Z-mean, nm) and polydispersity (PDI). No significant changes in particle size or aggregation were observed in the formulation at any storage temperature up to 37°C after twelve months. Figure 24B ).

[0459] To assess how co-bottle formation, lyophilization, and thermal stress affect the chemical integrity of ID93 peptides stored at 4°C, 25°C, and 37°C for twelve months, the reconstituted formulation was evaluated by SDS-PAGE. Figure 24C Each loading contained 1 μg / mL of reconstituted ID93. ID93 was observed as a 98 kDa band in all tested samples. Lyophilization and reconstitution of ID93+GLA-SE did not result in substantial hydrolysis of the ID93 peptide. Therefore, lyophilization of ID93+GLA-SE at twelve months protected the ID93 protein from heat stress-induced degradation.

[0460] The adjuvant concentration in lyophilized ID93+GLA-SE formulations stored at 4°C, 25°C, and 37°C was evaluated. Figure 23E Data confirmed that there was no significant loss of GLA at 4°C or 25°C compared to the initial 50 μg / ml concentration, but a 69% loss of GLA was confirmed at 37°C for twelve months.

[0461] Example 4: Lyophilized vaccine emulsion formulations and their application in single and multi-excipient systems with improved thermal stability It exhibits stability and exhibits no adjuvant loss at higher temperatures.

[0462] The freeze-dried and formulated oil-in-water stable emulsions were prepared using the materials and methods described in Example 1 and then freeze-dried. The properties of the reconstructed freeze-dried oil-in-water stable emulsion (SE) formulations were characterized as described in Example 1, including melting point, humidity determination, particle size and zeta potential, chemical degradation as determined by high performance liquid chromatography, reconstruction screening, cake stability screening, and accelerated stability characterization.

[0463] Based on preliminary experiments, it is hypothesized that the use of glycerol as a tonic agent may increase the thermal instability of the formulations of the present invention at temperatures above 25°C. Further investigation is needed to determine whether the removal of glycerol as a tonic agent in the formulations of the present invention would provide greater thermal stability at temperatures above 25°C for more than three months. In addition to removing glycerol, the effect of the percentage of biodegradable oil (in this example, squalene) on thermal stability and cake properties was evaluated when freeze-dried in the 2.5% trehalose and 2.5% mannitol lyophilized formulation of the present invention with improved 50°C thermal stability by the method described in Example 1.

[0464] The sample as described in Example 1 represents a specified percentage of squalene (as shown, 2% to 10% v / v squalene), 0.4% w / v DMPC, 0.02% w / v poloxamer 188, 0.5% w / v glycerol, and 5 mM ammonium phosphate, formulated with or without 0.5% w / v glycerol, with 2% v / v Tris added or subtracted as an additional tensioning agent. Figure 25A Data confirmed that emulsion formulations with increased squalene concentrations (2-10% v / v) and lacking 0.5% glycerol (labeled as glycerol-free) formed fine cake-like structures after lyophilization. These cakes did not shrink or discolor even after 30 days of storage at 50°C. In contrast, cake-like structures containing 0.5% v / v glycerol (labeled as glycerol-containing) showed slight shrinkage and indentation immediately after lyophilization (0 days) and after 30 days of storage at 50°C. The comparison of cake-like structure reconstruction for formulation stratification showed no significant difference.

[0465] Figure 25B and Figure 25C It was confirmed that after storage at 50°C for 30 days, no significant difference was observed in particle size (Z-mean, nm) or polydispersity (PDI) for any formulation. Figure 25D Evidence was provided that the presence of 0.5% v / v glycerol did not affect the stability of the adjuvant GLA in the formulation. Emulsion formulations prepared with increased biodegradable oil content (25-10%) showed no loss of GLA concentration compared to the initial concentration (described at zero vial 0) when reconstituted from lyophilized vials stored at 50°C for 30 days. Lyophilized emulsions in the presence of glycerol showed a 30-40% loss of GLA.

[0466] Based on this data, the vaccine formulation of the present invention can likely be freeze-dried to withstand temperatures of 50°C, which will produce a more refined cake-like structure and thus, as will be recognized by those skilled in the art, result in greater thermal stability.

[0467] Example 5: Development and characterization of four freeze-dried vaccine emulsion formulations and their improved thermal stability in single-component formulations. Stability in multi-excipient systems, with no adjuvant loss at higher temperatures.

[0468] The ability of four lyophilized formulations to heat-protect the GLA-SE emulsion described herein was evaluated; all formulations lacked glycerol as a tonic agent. The formulation developed and evaluated was 5% trehalose alone (glycerol-free). Figure 26A ), 5% trehalose w / v, 0.1% w / v mannitol ( Figure 26B ), 2.5% w / v trehalose, 2.5% w / v mannitol ( Figure 26C ) and 10% w / v trehalose ( Figure 26D The formation and appearance of the cakes were evaluated, as well as the stratification after reconstruction at 0 hours (immediately after lyophilization), 1 week (1 wk), 2 weeks (2 wk), 1 month (1 mo), and 3 months (3 mo) after lyophilization at 4°C, 25°C, 37°C, and 50°C for specified times, as shown. Data comparison showed that all samples formed a good white cake, with 5% trehalose w / v and 0.1% w / v mannitol ( Figure 26B ) and 2.5% w / v trehalose, 2.5% w / v mannitol ( Figure 26C It forms the most delicate cake-like shape at all storage temperatures. This is achieved with 5% trehalose w / v and 0.1% w / v mannitol (…). Figure 26B (and 2.5% w / v trehalose, 2.5% w / v mannitol) Figure 26C The resulting delicate cake-like structure confirms the sophisticated structure known in the prior art. Therefore, removing glycerol as a tensioning agent from the lyophilized GLA-SE emulsion of this invention produces an even more sophisticated cake-like structure, maintaining its structural integrity over a temperature range of 4°C, 25°C, 37°C, and 50°C when stored for 1 week (1 wk), 2 weeks (2 wk), 1 month (1 mo), and 3 months (3 mo). Figure 26A -D).

[0469] As instructed, all lyophilized formulations were stored at 4°C, 25°C, 37°C, and 50°C for 1 week (1 week), 2 weeks (2 weeks), 1 month (1 month), and 3 months (3 months). Samples were removed from storage, reconstructed, and compared as shown in Figures 27-31 for particle size (Z-mean, nm), polydispersity (PDI), pH, and GLA content.

[0470] Figure 27 illustrates a comparison of the pre-lyophilized emulsions (labeled "Pre-lyophilized" on the column) before the addition of the lyophilized component, the pre-lyophilized GLA-SE formulation (labeled "lyophilized" on the column) and the reconstituted formulation after lyophilization (labeled 0). Initial comparisons of the formulations confirmed no significant differences between the lyophilized formulations and that they possessed appropriate reconstituted emulsion characteristics, including a Z-mean diameter of less than approximately 200 nm, a lack of significant aggregation as measured by polydispersity, physiological pH, and no significant loss of GLA.

[0471] Figure 28 illustrates various single-bottle lyophilized formulations stored for one week (1 wk) at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4). Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 28A Data from the study confirmed that all four lyophilized formulations exhibited a particle size smaller than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 28A ), such as the lack of observable aggregation as measured by polydispersity ( Figure 28B ) and physiological pH ( Figure 28C It is worth noting that although the average particle size of the lyophilized cakes of all formulations at 50°C increased by approximately 40% compared to other samples, this particle size was still within the expected range of less than approximately 200 nm. Importantly, all glycerol-deficient formulations tested confirmed no GLA loss after one week of storage at any test temperature.

[0472] Figure 29 illustrates various single-bottle lyophilized formulations stored at 37°C (strip 3) and 50°C (strip 4) for two weeks (2 weeks). Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 29A Data from the study confirmed that all four lyophilized formulations exhibited a particle size smaller than approximately 200 nm when stored in a temperature range of 37°C–50°C. Figure 28A ), such as the lack of observable aggregation as measured by polydispersity ( Figure 29B ), and physiological pH ( Figure 29CIt is noteworthy that while the average particle size of the lyophilized cakes after one week at 50°C increased by approximately 40% compared to other samples, this particle size appeared to remain unchanged in the second week and remained within the expected range of less than approximately 200 nm. Importantly, all glycerol-deficient formulations tested confirmed no GLA loss after one week of storage at any test temperature.

[0473] Figure 30 illustrates various single-bottle lyophilized formulations stored for one month (1 month) at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4). Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 30A Data from the study confirmed that all four lyophilized formulations exhibited a particle size smaller than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 30A ), such as the lack of observable aggregation as measured by polydispersity ( Figure 30B ), and physiological pH ( Figure 30C It was noted that the average particle size of the 2.5% trehalose and 2.5% mannitol formulations increased from 120 nm to 175 nm, but the average particle size remained below 200 nm and the formulations did not show any GLA loss.

[0474] Figure 31 illustrates various single-bottle lyophilized formulations stored for one month (1 month) at 4°C (strip 1), 25°C (strip 2), 37°C (strip 3), and 50°C (strip 4). Samples were reconstructed and analyzed for particle size (Z-mean diameter, nm), polydispersity as a function of aggregation (PDI), pH, and GLA concentration (mg / ml). Figure 31A Data from the study confirmed that all four lyophilized formulations exhibited a particle size smaller than approximately 200 nm when stored in a temperature range of 4°C–50°C. Figure 31A ), such as the lack of observable aggregation as measured by polydispersity ( Figure 31B ) and physiological pH ( Figure 31C The data in Example 5 provide additional leading candidate formulations for single-bottle lyophilized oil-in-water emulsions (stable emulsions (SE)) containing adjuvants (GLA), which exhibit enhanced thermal stability at 50°C when stored at temperatures up to 50°C for more than or equal to one month.

[0475] This invention provides a variety of formulations for single-bottle lyophilized oil-in-water emulsions, suitable for vaccine delivery of antigens, single adjuvants, multiple adjuvants, or any combination thereof, with the particular benefit of reducing or eliminating the need for cold chain storage, making them improved formulations on top of the prior art.

[0476] sequence

[0477] ID93 fusion peptide with optional His tag (SEQ ID NO: 1)

[0478]

[0479] ID93 fusion peptide (SEQ ID NO: 2)

[0480]

[0481] ID83 fusion peptide with optional His tag (SEQ ID NO: 3)

[0482]

[0483] ID83 fusion peptide (SEQ ID NO: 4)

[0484]

[0485] Rv1813 (SEQ ID NO: 5)

[0486]

[0487] Rv3620 (SEQ ID NO: 6)

[0488]

[0489] Rv2608 (SEQ ID NO: 7)

[0490]

[0491] Rv3619 (SEQ ID NO: 8)

[0492]

Claims

1. A thermostable lyophilized vaccine composition comprising squalene present at 2% - 10% v / v, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), poloxamer 188, vitamin E, an adjuvant, an antigen, and a cake-forming excipient present at 0.01% - 20% w / v, wherein the adjuvant is synthetic GLA present at 0.5 pg / mL - 12 mg / mL, wherein the composition is in the form of a cake and forms an oil-in-water emulsion upon reconstitution, wherein the cake-forming excipient is trehalose or a combination of trehalose and mannitol, wherein the composition is formed by lyophilization of an oil-in-water emulsion formulation that is free of glycerol and free of liposomes, wherein DMPC is present at a DMPC:squalene ratio of 1:5, and wherein poloxamer 188 is present at a squalene:poloxamer 188 ratio of 100:

1.

2. The composition of claim 1, wherein: (a) the cake-forming excipient is trehalose at a concentration of 10% w / v in the oil-in-water emulsion formulation; (b) the cake-forming excipient is trehalose at a concentration of 5% w / v in the oil-in-water emulsion formulation; (c) the cake-forming excipient is a combination of mannitol and trehalose, wherein the mannitol is at a concentration of 0.1% w / v and trehalose is at a concentration of 5% w / v in the oil-in-water emulsion formulation; or (d) the cake-forming excipient is a combination of mannitol and trehalose, wherein the mannitol is at a concentration of 2.5% w / v and trehalose is at a concentration of 2.5% w / v in the oil-in-water emulsion formulation.

3. The composition of claim 1, wherein the composition is thermostable for at least 1 month at a temperature between 8°C and 60°C.

4. The composition of claim 3, wherein: (a) the composition is thermostable for at least 3 months, 6 months, or 12 months; (b) the composition is thermostable for at least 1 month at 25°C; (c) the composition is thermostable for at least 1 month at 37°C; or (d) the composition is thermostable for at least 1 month at 50°C.

5. The composition of claim 1, wherein the composition (i) is in the form of a precision cake; or (ii) is in the form of a cake that does not show browning by visual inspection when stored under any of the conditions in claims 3 or 4.

6. The composition of claim 1, wherein: (a) the thermostability of the composition is measured prior to reconstitution of the composition, optionally wherein the composition is in the form of a cake and wherein the thermostability is measured by observing shrinkage, cracking, and / or browning of the cake; (b) the thermostability is measured after reconstitution of the composition, optionally wherein the thermostability is measured by inspecting the separation of the oil-in-water emulsion formed upon reconstitution; (c) the thermostability is measured by analyzing the components of the oil-in-water emulsion formed upon reconstitution; or (d) the oil-in-water emulsion upon reconstitution has a particle size with a Z-average diameter of less than 200 nm.

7. The composition of claim 1, wherein the antigen is a polypeptide, a nucleic acid encoding a polypeptide, or a pathogen.

8. The composition of claim 1, wherein the concentration of antigen in the oil-in-water emulsion formed upon reconstitution exhibits no more than 25% degradation compared to the concentration of antigen in the oil-in-water emulsion formulation prior to lyophilization.

9. The composition of claim 1, wherein the synthetic GLA has the following structure: wherein R 1 , R 3 , R 5 and R 6 are C 11 -C 20 alkyl; and R 2 and R 4 are C9-C 20 alkyl, optionally wherein R 1 , R 3 , R 5 and R 6 are C 11 alkyl; and R 2 and R 4 are C9 alkyl.

10. The composition of claim 1, wherein the concentration of adjuvant in the oil-in-water emulsion formed upon reconstitution exhibits no more than 25% degradation compared to the concentration of adjuvant in the oil-in-water emulsion formulation prior to lyophilization.

11. A single vial comprising the composition of any one of claims 1-10, wherein the composition is contained in the vial.

12. A method of storing a vaccine composition comprising storing the thermostable lyophilized vaccine composition of any one of claims 1-10 for at least 1 month between 25 °C and 60 °C.

13. A method of producing a thermostable lyophilized vaccine composition comprising the step of lyophilizing an oil-in-water emulsion formulation to form the thermostable lyophilized vaccine composition, wherein the oil-in-water emulsion formulation prior to lyophilization comprises squalene present at 2% - 10% v / v, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), poloxamer 188, vitamin E, an adjuvant, an antigen, and a cake-forming excipient present at 0.01% - 20% w / v, wherein the adjuvant is a synthetic GLA present at 0.5 pg / mL - 12 mg / mL, wherein the cake-forming excipient is trehalose or a combination of trehalose and mannitol, wherein the composition is in the form of a cake and forms an oil-in-water emulsion upon reconstitution, wherein the oil-in-water emulsion formulation is free of glycerol and free of liposomes, wherein DMPC is present at a DMPC:squalene ratio of 1 :5, and wherein poloxamer 188 is present at a squalene:poloxamer 188 ratio of 100:

1.

14. The method of claim 13, wherein (a) the cake-forming excipient is trehalose at a concentration of 10% w / v in the oil-in-water emulsion formulation; (b) the cake-forming excipient is trehalose at a concentration of 5% w / v in the oil-in-water emulsion formulation; (c) the cake-forming excipient is a combination of mannitol and trehalose, wherein the mannitol is at a concentration of 0.1% w / v and trehalose is at a concentration of 5% w / v in the oil-in-water emulsion formulation; or (d) the cake-forming excipient is a combination of mannitol and trehalose, wherein the mannitol is at a concentration of 2.5% w / v and trehalose is at a concentration of 2.5% w / v in the oil-in-water emulsion formulation.

15. The thermostable lyophilized vaccine composition of any one of claims 1-10 for use in a method of stimulating an immune response in a subject, the method comprising: (a) reconstituting the composition into an oil-in-water emulsion; and (b) administering the emulsion to the subject, thereby stimulating an immune response in the subject.

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