RU2796539C2 - Pegylated liposomes and their application methods - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: in the invention, pegylated liposomes and methods for their production and application are proposed. Liposome contains cholesterol; unpegylated neutral lipid; pegylated lipid, wherein an average molecular weight of PEG in pegylated lipid is about 2000 Dalton; and TLR4 agonist and TLR7/8 agonist, wherein the specified TLR4 agonist is GLA (glucopyranosyl-lipid adjuvant), and TLR7/8 agonist is 3M-052. Compositions and methods related to the production of pegylated liposomes and use of pegylated liposomes for the stimulation of an immune response are also proposed.

EFFECT: pegylated liposomes are stable and capable of delivering an agent for the formation of an immune response, for example an agent for use for the production of vaccines, therapeutic or diagnostical use.

16 cl, 15 dwg, 7 tbl, 2 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from U.S. Provisional Application No. 62/337,328, filed May 16, 2016, the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING RESEARCH OR DEVELOPMENT FINANCED FROM THE FEDERAL BUDGET

[0002] The present invention was made with government support under Contract No. HHSO100201000039C, placed by the Biomedical Advanced Research and Development Administration (BARDA) within the Office of the Assistant Secretary for Preparedness and Response (ASPR) of the U.S. Department of Health and Human Services, and under Grant No. 5R21AI109118 and contract no. HHSN272200800045C, hosted by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Human Services and Health. The government has certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] Liposomal delivery systems have been developed for biomolecules such as drugs and imaging agents. Such liposomes offer advantages such as longer circulating stability leading to improved presentation to the immune system, controlled release of the biomolecule, and versatility to enclose both hydrophobic and hydrophilic biomolecules. In order to prolong the circulating half-life of liposomes, they must be shielded from normal clearance mechanisms, including interactions with serum proteins and phagocytes. Many approaches have been developed to improve the shielding of liposomes from the immune system, including PEGylation of liposomes (polyethylene glycol (PEG) containing liposomes). Liposomes containing PEG and other copolymers designed to shield the liposome from macrophages have been referred to in the art as "stealth liposomes". In addition to screening properties, pegylation can affect the stability of a liposome preparation by limiting, but not eliminating, fusion of liposomes during long-term storage. In order to avoid such fusions, the PEG halo must create a thick enough layer to sterically shield the surface of the liposome.

[0004] PEG molecular weight has traditionally been considered an important factor for effective surface shielding. PEGylated liposomes coated with lower molecular weight PEGs were not effectively shielded. For example, when 750 dalton PEGylated liposomes were compared with non-pegylated liposomes, no difference was observed (Mori et al., FEBS Lett, 1991). A longer presence in the bloodstream and a decrease in uptake by phagocytes was observed only when the PEG molecular weight was raised to over 5000 daltons.

[0005] Liposomes have also been evaluated as delivery vehicles for subunit protein vaccines and adjuvants. Liposomes are attractive vehicles for vaccine delivery due to the ability to tailor the composition of liposomes to achieve the desired concentration, charge, size and distribution of lipids or antigen and adjuvant targeting. Many liposome-based systems have been evaluated, including anionic, cationic, and neutral liposomes, however, not all liposomes are produced equally. Many formulations with liposomes based on cationic lipids are toxic. Many neutral liposomes are unstable ex vivo and either increase in size over time or disintegrate immediately upon receipt. The limitations of anionic liposomes are related to their charge, which affects their ability to deliver various antigens and fuse with cell membranes. Other liposomes contain synthetic lipids or block copolymers that may affect the immune system itself. Cholesterol-based liposomal formulations are ideal in that cholesterol is a natural component of human cell membranes and when combined with other colipids such as N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate ( DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol forms stable cationic nanoparticles with a diameter of approximately 100-200 nm with suitable polydispersity coefficients of approximately 0.3. Various strategies are being used to improve liposomal formulations for use as vaccine formulations, including pegylation strategies. To date, no precise formulation has been developed that would overcome the problems associated with the stability and toxicity of known liposomes, and could also effectively improve the quality of the immune response (for a review, see Carstens et al. Vaccine 29, 2011; Kaur et al. Journal of Controlled Release, 2012; Schwendener, Ther Adv Vaccines, 2014; Nag and Awasthi, Pharmaceutics, 2013; Vartak and Sucheck, Vaccines, 2016; Fan et al., Journal of Controlled Release, 2015; Schmidt et al., Pharmaceutics, 2016).

[0006] Thus, there is a need to develop pegylated liposomes that are stable, suitable for production, compatible with antigens and lipid-based adjuvants, and have a small size for vaccines, drugs and diagnostics. There is a particular need in the field of vaccines to combine such liposomes with adjuvants that can enhance the immune response, and the development of such liposomes may be useful, for example, for vaccinations against influenza, intestinal diseases (such as amoebiasis), tuberculosis, HIV, cancer, and hepatitis. This application provides compositions and related methods.

[0007] All sources cited in this application, including patent applications and patent publications, are hereby incorporated by reference in their entirety, as if each individual source were specifically and separately indicated as being incorporated by reference.

BRIEF DESCRIPTION OF THE INVENTION

[0008] The present invention provides pegylated liposomes, formulations and compositions containing pegylated liposomes, and methods for making and using pegylated liposomes. PEGylated liposomes are suitable for the production of vaccines, drugs and diagnostics.

[0009] PEGylated liposomes contain at least cholesterol, a non-PEGylated neutral lipid, and a PEGylated lipid, wherein the average molecular weight of the PEGylated lipid component is about 5,000 daltons or less. PEGylated liposomes are stable and capable of delivering an agent to generate an immune response. Pegylation of liposomes, in which the molecular weight of the pegylated lipid is approximately 5000 daltons or less, ensures the stability of the liposome and allows its delivery to immune cells to generate an immune response. In this application, the size of pegylated liposomes present in said composition ranges from about 1 nm to about 450 nm, and said size remains stable over varying temperatures and over time. PEGylated liposomes are stable and show little or no aggregation, or show reduced aggregation, and can be subjected to a final sterilization step before dispensing into vials. The pegylated liposomes provided herein may additionally contain a TLR agonist and/or some agent, such as an agent for use in vaccines, therapy or diagnostics. Also provided are compositions and methods related to the preparation and use of PEGylated liposomes to stimulate an immune response.

[0010] In one aspect, the present invention provides a liposome comprising: (a) cholesterol; (b) a non-pegylated neutral lipid; and (c) a pegylated lipid, wherein the average molecular weight of the PEG in the pegylated lipid is about 5,000 daltons or less. In some embodiments, the average molecular weight of the PEG in the pegylated lipid is in the range of about 750 daltons to about 5000 daltons. In some embodiments, the PEGylated lipid has an average molecular weight of about 2000 daltons or less. In some embodiments, the average molecular weight of the PEG in the pegylated lipid is approximately 750 daltons. In some embodiments, the lipid component of said pegylated lipid comprises a neutral lipid. In some embodiments, the lipid component of said pegylated lipid comprises a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain. In some embodiments, the lipid component of said pegylated lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DOPC, dilinoleoylphosphatidylcholine (DLPC ), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or dimyristoylphosphatidylethanolamine (DMPE) . In some embodiments, the lipid component of said pegylated lipid is DSPE. In some embodiments, the lipid component of said pegylated lipid is DPPE. In some embodiments, the non-pegylated neutral lipid comprises a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain. In some embodiments, the non-pegylated neutral lipid is DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE, or DMPE. In some embodiments, the non-pegylated neutral lipid is DPPC. In some embodiments, said liposome is stable. In some embodiments, said liposome is stable for at least 1 month at about 2°C to about 8°C. In some embodiments, said liposome is stable for at least 1 month at a temperature of approximately 25°C. In some embodiments, said liposome is stable for at least 1 month at a temperature of about 37°C. In some embodiments, the polydispersity index of the liposome is maintained at about 0.3 or less. In some embodiments, the size of the liposome is less than or approximately equal to 450 nm. In some embodiments, the size of the liposome is maintained at less than or approximately equal to 450 nm. In some embodiments, the size of the liposome is in the range from about 50 nm to about 300 nm. In some embodiments, the polydispersity index of the liposome is less than or approximately equal to 0.3. In some embodiments, the mole percentage (mol %) of pegylated lipid in the liposome ranges from about 1 mol. % to about 25 mol. %. In some embodiments, they say. % pegylated lipid in the liposome ranges from about 1 mol. % to about 10 mol. %. In some embodiments, they say. % pegylated lipid in the liposome is approximately 5 mol. %. In some embodiments, they say. % cholesterol in the liposome is in the range from about 1 mol. % to about 50 mol. %. In some embodiments, they say. % cholesterol in the liposome is approximately 50 mol. %. In some embodiments, they say. % non-pegylated lipid in the liposome ranges from about 45 mol. % to about 98 mol. %. In some embodiments, they say. % non-pegylated lipid in the liposome is approximately 45 mol. %. In some embodiments, the lipid non-pegylated neutral lipid:cholesterol:pegylated lipid molar ratio is about 9.8:5.7:0.8. In some embodiments, the lipid non-pegylated neutral lipid:cholesterol:pegylated lipid molar ratio is approximately 18:5.5:3. In some embodiments, the liposome further comprises at least one TLR agonist. In some embodiments, a TLR agonist includes a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7/8 agonist, or a TLR9 agonist. In some embodiments, the TLR agonist includes TLR4, SLA, GLA, 3D-MPL, R837, or R848. In some embodiments, the TLR agonist includes a hydrophobic tail. In some embodiments, the TLR agonist includes a TLR7/8 agonist. In some embodiments, the TLR agonist includes a TLR7 agonist. In some embodiments, the TLR agonist includes a TLR8 agonist. In some embodiments, the TLR7/8 agonist comprises an imidazoquinoline or an imidazoquinoline-containing compound. In some embodiments, the TLR7/8 agonist includes 3M-052. In some embodiments, the TLR7/8 agonist includes R848. In some embodiments, the TLR agonist includes a TLR4 agonist. In some embodiments, the TLR4 agonist includes 3D-MPL. In some embodiments, the TLR4 agonist includes GLA. In some embodiments, the TLR4 agonist comprises a synthetic glucopyranosyl lipid adjuvant (GLA) of formula (V). In some embodiments, the TLR4 agonist comprises a synthetic GLA of formula (VI).

[0011] In some embodiments, the TLR4 agonist includes a synthetic GLA of the formula:

or a pharmaceutically acceptable salt of said compound. In some embodiments, the liposome contains a TLR4 agonist and a TLR7/8 agonist. In some embodiments, the liposome contains GLA and 3M-052. In some embodiments, the liposome further comprises at least one agent. In some embodiments, said agent includes a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent, or organism. In some embodiments, the agent includes an antigen. In some embodiments, the antigen is an amoebiasis-associated antigen. In some embodiments, the antigen includes LecA. In some embodiments, the antigen includes an influenza-associated antigen. In some embodiments, the antigen includes H5N1. In some embodiments, the antigen includes a tuberculosis-associated antigen. In some embodiments, the antigen includes ID91. In some embodiments, the antigen includes ID93. In some embodiments, the antigen includes an antigen from BCG. In some embodiments, the antigen includes a hepatitis virus-related antigen. In some embodiments, the antigen includes a hepatitis B antigen. In some embodiments, the antigen includes a hepatitis C antigen. In some embodiments, the antigen includes an HIV-associated antigen. In some embodiments, the antigen includes a cancer-associated antigen.

[0012] In a related aspect of the present invention, a composition is provided comprising any of the liposomes provided herein. In some embodiments, said composition contains a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the composition is a vaccine. In some embodiments, the composition is therapeutic. In some embodiments, the composition is diagnostic.

[0013] In another aspect of the present invention, there is provided a method for stimulating an immune response in a subject, comprising administering to said subject any of the liposomes described herein, or any of the compositions containing liposomes described herein, whereby an immune response is stimulated in the subject. In another aspect, the present invention provides a method of inducing a Th1 cell response in a subject, comprising administering to said subject any of the liposomes described herein, or any of the compositions containing liposomes described herein, whereby a Th1 cell response is elicited in the subject . In some embodiments, the immune response is a non-specific immune response. In some embodiments, the immune response is an antigen-specific immune response. In some embodiments, the immune response includes a systemic immune response. In some embodiments, the immune response includes a mucosal immune response (mucosal immune response). In some embodiments, the mucosal immune response includes an immune response from the intestinal, fecal, or vaginal mucosa. In some embodiments, the composition is used to treat or prevent cancer. In some embodiments, the composition is used as a vaccine. In some embodiments, the composition is used to enhance protective immunity against an influenza-causing virus. In some embodiments, the composition is used to enhance protective immunity against an amoebiasis-causing organism. In some embodiments, the composition is used to enhance protective immunity against Entamoeba histolytica. In some embodiments, the composition is used to enhance protective immunity against influenza. In some embodiments, the composition is used to enhance protective immunity against amebiasis. In some embodiments, the route of administration of said composition is oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, or subcutaneous. In some embodiments, the route of administration is intranasal.

[0014] In another aspect, the present invention provides a method of stimulating a systemic immune response and a mucosal immune response in a subject, comprising intranasally administering to the subject any of the liposomes proposed in this application, or any of the compositions containing liposomes proposed in this application. In some embodiments, said method comprises administering a liposome that contains a TLR4 agonist and a TLR7/8 agonist. In some embodiments, said method includes administering a liposome that contains GLA and 3M-052. In some embodiments, the mucosal immune response includes an immune response from the intestinal, fecal, or vaginal mucosa. In some embodiments, the mucosal immune response is distant from the nasal cavity.

[0015] In any of the methods described in this application, the liposome or composition can be administered together with a coadjuvant - retinoic acid.

[0016] In any of the methods described in this application, the liposome or composition is administered to a human.

[0017] In any of the methods described in this application, the liposome or composition is administered to a non-human mammal. In some embodiments, said non-human mammal is a dog, cow, or horse.

[0018] In another aspect of the present invention, there is provided a process for preparing any of the pegylated liposomes provided herein, comprising: (a) mixing a non-pegylated neutral lipid, a pegylated lipid, and cholesterol in an organic solvent; (b) evaporating said organic solvent, whereby a lipid film is obtained; (c) rehydration of the lipid film in buffer; and (d) sonicating, microfluidizing, or extruding the rehydrated product from step (c). In some embodiments, step (a) further comprises mixing with a TLR agonist. In some embodiments, the organic solvent is chloroform. In some embodiments, the rehydrated product from step (c) is sonicated and then subjected to microfluidization. In some embodiments, said method further comprises mixing the agent with the pegylated liposome. In some embodiments, the agent includes an antigen.

[0019] These and other aspects of the present invention will become apparent upon reading the following detailed description and the attached figures. In addition, various contrasting materials are presented in this application, which describe some aspects of the present invention in more detail, and, therefore, their descriptions are fully incorporated into this application by reference.

BRIEF DESCRIPTION OF THE FIGURES

[0020] Figures 1A-1D depict the physical and stability properties of various formulations. In FIG. 1A shows the particle diameter over time at 5°C. In FIG. 1B shows the polydispersity ratio over time at 5°C. In FIG. 1C shows the particle diameter over time at 37°C. In FIG. 1D shows the coefficient of polydispersity over time at 37°C.

[0021] In FIG. 2 shows the response of IgG1 and IgG2a to various formulations, which was determined using enzyme-linked immunosorbent assay (ELISA). The figure shows a comparison of plasma IgG1 and IgG2a titers following immunization of mice with different formulations of LecA antigen mixed with the appropriate adjuvant.

[0022] Figures 3A-D depict the response of LecA-specific cytokines.

[0023] Figures 4A-B depict the mucosal IgA response and ability to inhibit adhesion prior to antigen challenge. Mice were immunized with LecA adjuvanted with GLA-3M-052-liposome using a mixed regimen of intranasal (weeks 0 and 4) and subcutaneous (week 2) administration. Mice in these groups received a weekly intraperitoneal injection of 150 mg of all-trans retinoic acid (RA). Fecal samples were collected three weeks after the third immunization. In FIG. 4A, stool supernatants are diluted 120-fold and IgA titer to lectin before antigen challenge determined by ELISA; in fig. 4B, the ability of fecal IgA to inhibit trophozoite adherence to mammalian cells was determined in vitro using the adhesion inhibition assay described.

[0024] Figures 5A-B depict vaccine-mediated protection using a murine intestinal amebiasis model. The adjuvant GLA-3M-052-liposome provided moderate protection in the caecal challenge test. Mice immunized using the heterologous regimen were challenged with intracaecal E. histolytica four weeks after the final immunization. Mice were sacrificed one week after challenge and the caecal contents (FIG. 5A) were analyzed for antigenic load using ELISA and (FIG. 5B) live amoebae by culture as a measure of sterile immunity. The number of infected mice out of all challenged is indicated above each column. The results of two independent but identical trials were combined.

[0025] FIG. 6 depicts the plasma IgG response to these formulations.

[0026] FIG. 7 depicts the IgA response in feces to these formulations. This figure shows that increasing the length of the PEG enhances the IgA response in the mucosa.

[0027] Figures 8A-B show the effect of the delivery route on immunization, with the highest titers of IgG2a and IgA obtained with an I/N (nasal) only regimen. The liposome + adjuvant elicited a strong mucosal and systemic immune response of Th1 cells: IgA to LecA in the intestine (Fig. 8B).

[0028] Figures 9A-B show the effect of the delivery route on immunization. The I/N-only regimen resulted in equivalent or higher titers of IFN-γ and IL-17 compared to other regimens. The liposome + adjuvant elicited a strong mucosal and systemic Th1 immune response: IFN-γ (FIG. 9A) and IL-17 (FIG. 9B).

[0029] Figures 10A-B show (a) particle diameter and (b) size polydispersity index for typical 3M-052 formulations at 5°C for 6 months. Error bars represent the standard deviation of three separate particle size analyzes for one batch of formulation at each time point. The content of 3M-052 was not measured in these batches, but was estimated to be 0.04 mg/ml based on subsequent batches produced using the same process.

[0030] Figures 11A-H show that mice immunized with 3M-052 showed increased survival after H5N1 challenge. Animals were immunized once with rHA protein (A/VN/1203/04) in combination with adjuvants as indicated. Twenty-one days after immunization, animals were challenged with 10 ⁶ plaque forming units (PFU) A/VN/1203/04 (H5N1, clade 1). Survival (FIG. 11A) and body weight loss of the animals (FIGS. 11B, 11C) were monitored daily. After 3 days (FIG. 11D) and 7 days (FIG. 11E) after challenge, mice were sacrificed and virus titers in their lungs were determined by plaque analysis. In addition, intact lungs were weighed on day 7 post-challenge for pooling (11F) and assessed for their occurrence of macropathology (Fig. 11G). Significance was determined using the Cox-Mantel log-rank test (A) or using one-way ANOVA (Figures 11D-11G) (**p<0.005, ***p<0.0005, ****p<0.0001 ). In FIG. 11H shows the virus titer in the lungs.

[0031] Figures 12A-D show CD4 T cell responses in mice immunized with 3M-052 adjuvanted formulations. Animals were immunized once with rHA protein (A/VN/1203/04) in combination with adjuvants as indicated. Seven days after immunization, splenocytes from sacrificed mice (n=5/group) were analyzed for cytokine stimulation after rGA stimulation. The secretion patterns of the cytokines IFN-γ (I), TNFα (T), and IL-2 (2) were determined to investigate the induction of a Th1 CD4+ T cell response. The relative percentage of polyfunctional T cells was also determined. Significance between groups was determined using one-way analysis of variance (*p<0.05, **p<0.005).

[0032] Figures 13A-H show the protection of ferrets from homologous H5N1 challenge after a single immunization. Male Fitch ferrets were immunized once with split H5N1 vaccine (H5N1, Sanofi Pasteur) in combination with 3M-052 adjuvant formulations and challenged 21 days after immunization with 10 ⁶ PFU A/VN/1203. Animal survival (FIGS. A, E), body weight loss (FIGS. B, F), and clinical performance (FIGS. C, G) were monitored for up to 14 days. In addition, nasal swabs were collected to assess the virus titer (FIGS. D, H). For both formulations, 3M-052-ES and 3M-052-liposomal adjuvant showed effective protection after a single application in this model, characterized by faster viral clearance from nasal swabs, reduced body weight loss and clinical parameters, and 100% survival.

[0033] Figures 14A-F depict the induction of a virus-neutralizing titer with 3M-052 adjuvant formulations. Male Fitch ferrets were immunized once with a split H5N1 vaccine (SP-H5, Sanofi Pasteur) in combination with 3M-052 adjuvant formulations. Twenty-one days after immunization, blood was collected from all animals and analyzed for the presence of virus-neutralizing antibodies using a retrovirus pseudotype neutralization assay. Incorporation of 3M-052 into adjuvant formulations resulted in a significant (one-way analysis of variance) increase in neutralizing titer against both homologous clade 1 virus (Fig. A, D), clade 2 virus strain (Fig. B, E) and clade 2.2 strain ( Fig. C, F).

Figures 15A-B show the protection of ferrets from heterologous H5N1 challenge after a single immunization. Male Fitch ferrets were immunized once with split H5N1 vaccine (H5N1, Sanofi Pasteur) in combination with 3M-052 adjuvant formulations and challenged 21 days after immunization with 10 ⁶ PFU A/whooper swan/Mongolia/244/05. Nasal swabs were collected to assess virus titer. 3M-052-ES adjuvants produced faster viral clearance with no titers detected on day 5. For 3M-052-liposomal adjuvant formulations, titers were detected on day 3 and clearance occurred by day 5.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Provided herein are, inter alia, formulations of pegylated liposomes, compositions containing pegylated liposomes, and methods for making and using pegylated liposomes. The present inventors, in formulating an insoluble Toll-like receptor (TLR) agonist, developed a liposome formulation that was unexpectedly versatile to incorporate various TLR ligands and/or antigens while maintaining stability, and that effectively increased the immune response when mixed with an antigen. The present inventors have found that mixing a variety of different antigens with these liposomes results in antigen-liposome formulations that can be administered to a mammal. Said liposome formulations comprise cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the average molecular weight of the PEG in the pegylated lipid is about 5000 Daltons or less, preferably about 750 to about 5000 Daltons, or from 750 to about 2000 Daltons. The use of pegylated lipids provides stability and allows the delivery and production/stimulation/modification of an immune response. In some preferred embodiments, the non-pegylated neutral lipid is DPPC and the pegylated lipid is pegylated DSPE or DPPE. In some particularly preferred embodiments, the liposomes further comprise a Toll-like receptor (TLR) agonist and optionally an antigen.

[0035] Also provided are compositions (such as vaccine compositions, pharmaceutical compositions) containing pegylated liposomes as described herein. Said compositions are suitable for generating/stimulating/modifying an immune response in a subject. In some embodiments, the implementation of the composition described in this application, additionally contains one or more antigens and/or TLR agonists.

[0036] The present inventors also found, when formulating oil-in-water emulsions based on squalene of the insoluble Toll-like receptor 3M-052, that emulsions containing egg phosphatidylcholine or POPC instead of DMPC produced a higher yield of Toll-like receptor after treatment. In addition, the addition of an initial mixing step to combine the Toll-like receptor with egg phosphatidylcholine or POPC in chloroform further increased the yield of the Toll-like receptor. The present application also provides, inter alia, squalene-based oil-in-water emulsions containing squalene, unsaturated phosphatidylcholine, and a Toll-like receptor (e.g., an insoluble Toll-like receptor), and methods for preparing such emulsions by mixing the Toll-like receptor with unsaturated phosphatidylcholine in chloroform.

I. Definitions

[0037] The following terms have the following meanings, unless otherwise indicated. Any terms for which no definitions are given have the meanings known in the art.

[0038] In the present description, the terms "approximately" and "consisting essentially of" mean ± 20% of the specified range, value or structure, unless otherwise indicated.

[0039] The use of an alternative (eg, "or") should be understood to mean either one, or both, or any combination of the listed alternatives.

[0040] In this application, the terms "comprise", "have" and "include" are used synonymously, these terms and their variations are to be construed as non-limiting.

[0041] In this application and in the appended claims, the singular forms include reference to the plural, unless the context clearly indicates otherwise.

[0042] The term "macromolecule" in this application refers to large molecules, examples of which, without limitation, are peptides, proteins, oligonucleotides and polynucleotides of biological or synthetic origin.

[0043] The terms "polypeptide", "peptide", and "protein" are used interchangeably in this application to refer to polymers of amino acids of any length. Said polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid molecules. The scope of these terms also includes a polymer of amino acids that has been modified in nature or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling moiety. Also included within the scope of this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0044] The term "isolated" means a molecule that has been removed from its natural environment.

[0045] "Purified" means that the purity of the specified molecule has been increased so that it is in a purer form than it is in the natural environment and/or after the initial synthesis and/or amplification in the laboratory. Purity is a relative term and does not necessarily mean absolute purity.

[0046] The terms "polynucleotide" or "nucleic acid", used interchangeably in this application, refer to polymers of nucleotides of any length, including DNA and RNA. Said nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by synthetic reaction. The polynucleotide may contain modified nucleotides such as methylated nucleotides and their analogs. Modification of the nucleotide structure, if present, can be imparted before or after polymer assembly.

[0047] "Oligonucleotide" as used herein generally refers to short, typically single-stranded, typically synthetic polynucleotides that are typically, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

[0048] The term "individual" or "subject" is any mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, pets (such as cats, dogs, horses), and rodents.

[0049] "Alkyl" is a linear or branched saturated hydrocarbon. For example, an alkyl group may contain 1 to 30 carbon atoms (i.e., (C ₁ -C ₃₀ )alkyl) or 1 to 20 carbon atoms (i.e., (C ₁ -C ₂₀ )alkyl) , or from 1 to 10 carbon atoms (i.e., (C ₁ -C ₁₀ )alkyl), or from 1 to 8 carbon atoms (i.e., (C ₁ -C ₈ )alkyl), or from 1 up to 6 carbon atoms (ie, (C ₁ -C ₆ )alkyl), or from 1 to 4 carbon atoms (ie, (C ₁ -C ₄ )alkyl). This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH ₃ -), ethyl (CH ₃ CH ₂ -), n-propyl (CH ₃ CH ₂ CH ₂ -), isopropyl (( CH ₃ ) ₂ CH-), n-butyl (CH ₃ CH ₂ CH ₂ CH ₂ -), isobutyl ((CH ₃ ) ₂ CHCH ₂ -), sec-butyl ((CH ₃ ) (CH ₃ CH ₂ )CH -), tert-butyl ((CH ₃ ) ₃ C-), n-pentyl (CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ -), neopentyl ((CH ₃ ) ₃ CCH ₂ -) and n-hexyl (CH ₃ (CH ₂ ) ₅ -).

[0050] "Halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0051] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0052] "Alkoxyl" refers to the group -O-alkyl, where alkyl is as described in this application. Alkoxy includes, by way of example, methoxyl, ethoxyl, n-propoxyl, isopropoxyl, n-butoxyl, t-butoxyl, sec-butoxyl, n-pentoxyl, and the like.

[0053] The terms "carboxylic acid ester" or "carboxylic ester" refer to the groups -C(O)O-alkyl and substituted -C(O)O-alkyl, where alkyl and substituted alkyl are as described in this application.

[0054] For the implementation of the present invention will use, unless otherwise indicated, the usual techniques of molecular biology, recombinant DNA, biochemistry and chemistry, which are within the competence in this field. Such techniques are fully explained in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd ed., Sambrook et al., eds., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Mullis and others, US patent number 4683195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, eds. 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); scientific work, Methods in Enzymology (Academic Press, Inc., New York); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1989).

[0055] As used herein, "water-insoluble" refers to a compound that does not dissolve, or only slightly dissolves, when said compound is mixed with water, such as when mixed with water at room temperature, such as between or between about 25°C. and 50°C.

II. pegylated liposomes.

[0056] The present invention provides pegylated liposomes comprising at least cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the average molecular weight of the PEGylated lipid component is about 5,000 daltons or less. The PEGylated liposomes provided herein may additionally contain an agent, such as an agent for use in vaccines, therapy, or diagnostics. The pegylated liposomes provided herein may additionally contain a TLR agonist, for example, for use in a vaccine, therapy or diagnostic as mentioned above. Description of each individual component of PEGylated liposomes and properties of PEGylated liposomes are given below.

A. PEGylated lipids.

[0057] The PEGylated liposomes provided herein contain PEGylated lipids (polyethylene glycol (PEG)-linked lipids) wherein the PEGylated lipid has an average molecular weight of about 5,000 daltons or less. It was concluded that the use of such PEG-linked lipids in combination with a neutral non-pegylated lipid and cholesterol conferred stability on an otherwise neutral liposome that did not contain pegylated lipids. The use of such pegylated lipids ensures long-term stability of the structure of the pegylated liposome and allows effective stimulation of the immune response.

Properties of PEG in PEGylated Lipid

[0058] In the embodiments of this application, the average molecular weight of PEG in pegylated lipid is approximately 5000 daltons or less. In some embodiments, the PEGylated lipid has an average molecular weight of about 2000 daltons or less. In specific embodiments, the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons. In specific embodiments, the average molecular weight of the PEG is in the range from about 750 daltons to about 2000 daltons. In some embodiments, the average molecular weight of the PEG is in the range from about 750 to 1000; from 750 to 1500; from 750 to 2000; from 750 to 2500; from 750 to 3000; from 750 to 3500; from 750 to 4000; from 750 to 4500; or 750 to 5000 daltons. In some embodiments, the average molecular weight of the PEG is in the range of about 4500 to 5000; from 4000 to 5000; from 3500 to 5000; from 3000 to 5000; from 2500 to 5000; from 2000 to 5000; from 1500 to 5000; from 1000 to 5000; or from 750 to 5000 daltons. In some embodiments, the average molecular weight of the PEG is in the range from about 500 to 1000; from 500 to 750; or from 750 to 1000 daltons. In some embodiments, the average molecular weight of the PEG is in the range from about 1500 to 2500; from 1500 to 2000; or from 2000 to 2500 daltons. In some embodiments, the average molecular weight of the PEG is in the range of about 4500 to 5500; from 4500 to 5000; or from 2000 to 5000 daltons.

[0059] In one exemplary embodiment, the pegylated lipid comprises PEG-750. In another exemplary embodiment, the pegylated lipid comprises PEG-1000. In another exemplary embodiment, the pegylated lipid comprises PEG-1500. In another exemplary embodiment, the pegylated lipid comprises PEG-2000. In another exemplary embodiment, the pegylated lipid comprises PEG-2500. In another exemplary embodiment, the pegylated lipid comprises PEG-3000. In another exemplary embodiment, the pegylated lipid comprises PEG-3500. In another exemplary embodiment, the pegylated lipid comprises PEG-4000. In another exemplary embodiment, the pegylated lipid comprises PEG-4500. In another exemplary embodiment, the pegylated lipid comprises PEG-5000.

Lipid Properties in PEGylated Lipid

[0060] This application contemplates that the lipid component of said pegylated lipid may include any lipid (which includes phospholipids) that can bind to the cholesterol and non-pegylated neutral lipid components to form a stable liposome structure.

[0061] Table 1 shows just some of the list of typical lipids that can be combined with PEG for use in the present invention.

[0062] In some embodiments, the lipid component of said pegylated lipid is a phospholipid or a quaternary ammonium salt type lipid. In some embodiments, the lipid component of said pegylated lipid is a phospholipid, which is phosphatidylcholine or phosphoglyceride. In some embodiments, the lipid component of said pegylated lipid comprises any of the following molecules:

where X ^- is an alkali metal counterion and Y ⁺ is a halide counterion.

[0063] In some embodiments, the lipid component of said pegylated lipid contains a C _10-20 alkyl chain. In some embodiments, the lipid component of said pegylated lipid contains a C _2-18 alkyl chain. In some embodiments, the lipid component of said pegylated lipid comprises a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain.

[0064] In some embodiments, the lipid component of said pegylated lipid is anionic. In some embodiments, the lipid component of said pegylated lipid is cationic. In some embodiments, the lipid component of said pegylated lipid is generally neutrally charged. In some embodiments, the lipid component of said pegylated lipid is a zwitterion.

[0065] In some exemplary embodiments, the lipid component of said pegylated lipid is DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DSPE, DPPE, or DMPE. In one exemplary embodiment, the lipid component of said pegylated lipid is DSPE. In another exemplary embodiment, the lipid component of said pegylated lipid is DPPE. In another exemplary embodiment, the lipid component of said pegylated lipid is DMPE.

[0066] In any of the embodiments described herein, the lipid component of said pegylated lipid may be DLPE.

[0067] In some embodiments, the mole percentage (mol. %) of pegylated lipid in the liposome is in the range of about 1 mol. % to about 25 mol. %. In some embodiments, the mole percentage (mole %) of pegylated lipid in the liposome is about 1 mole. %, 2 mol. %, 3 mol. %, 4 mol. %, 5 mol. %, 6 mol. %, 7 mol. %, 8 mol. %, 9 mol. %, 10 mol. %, 11 mol. %, 12 mol. %, 13 mol. %, 14 mol. %, 15 mol. %, 16 mol. %, 17 mol. %, 18 mol. %, 19 mol. %, 20 mol. %, 21 mol. %, 22 mol. %, 23 mol. %, 24 mol. % or even about 25 mol. %. In a typical implementation pier. % pegylated lipid in the liposome is approximately 5 mol. %. In another typical implementation pier. % pegylated lipid in the liposome is approximately 10 mol. %. In another typical implementation pier. % pegylated lipid in the liposome is approximately 15 mol. %. In another typical implementation pier. % pegylated lipid in the liposome is approximately 20 mol. %. In another typical implementation pier. % pegylated lipid in the liposome is approximately 25 mol. %.

Typical pegylated lipids

[0068] In some embodiments, the pegylated lipid in the pegylated liposome is DSPE-PEG-750, DSPE-PEG-1000, DSPE-PEG-1500, DSPE-PEG-2000, DSPE-PEG-2500, DSPE-PEG-3000, DSPE-PEG-3500, DSPE-PEG-4000, DSPE-PEG-4500 or DSPE-PEG-5000.

[0069] In some embodiments, the pegylated lipid in the pegylated liposome is DPPE-PEG-750, DPPE-PEG-1000, DPPE-PEG-1500, DPPE-PEG-2000, DPPE-PEG-2500, DPPE-PEG-3000, DPPE-PEG-3500, DPPE-PEG-4000, DPPE-PEG-4500 or DPPE-PEG-5000.

[0070] In some embodiments, the pegylated lipid in the pegylated liposome is DMPE-PEG-750, DMPE-PEG-1000, DMPE-PEG-1500, DMPE-PEG-2000, DMPE-PEG-2500, DMPE-PEG-3000, DMPE-PEG-3500, DMPE-PEG-4000, DMPE-PEG-4500 or DMPE-PEG-5000.

[0071] In some embodiments, the pegylated lipid in the pegylated liposome is DPPC-PEG-750, DPPC-PEG-1000, DPPC-PEG-1500, DPPC-PEG-2000, DPPC-PEG-2500, DPPC-PEG-3000, DPPC-PEG-3500, DPPC-PEG-4000, DPPC-PEG-4500 or DPPC-PEG-5000.

[0072] In some embodiments, the pegylated lipid in the pegylated liposome is DOPC-PEG-750, DOPC-PEG-1000, DOPC-PEG-1500, DOPC-PEG-2000, DOPC-PEG-2500, DOPC-PEG-3000, DOPC-PEG-3500, DOPC-PEG-4000, DOPC-PEG-4500 or DOPC-PEG-5000.

[0073] In some embodiments, the pegylated lipid in the pegylated liposome is DLPC-PEG-750, DLPC-PEG-1000, DLPC-PEG-1500, DLPC-PEG-2000, DLPC-PEG-2500, DLPC-PEG-3000, DLPC-PEG-3500, DLPC-PEG-4000, DLPC-PEG-4500 or DLPC-PEG-5000.

[0074] In some embodiments, the pegylated lipid in the pegylated liposome is DMPC-PEG-750, DMPC-PEG-1000, DMPC-PEG-1500, DMPC-PEG-2000, DMPC-PEG-2500, DMPC-PEG-3000, DMPC-PEG-3500, DMPC-PEG-4000, DMPC-PEG-4500 or DMPC-PEG-5000.

[0075] In some embodiments, the pegylated lipid in the pegylated liposome is POPC-PEG-750, POPC-PEG-1000, POPC-PEG-1500, POPC-PEG-2000, POPC-PEG-2500, POPC-PEG-3000, RORS-PEG-3500, RORS-PEG-4000, RORS-PEG-4500 or RORS-PEG-5000.

[0076] In some embodiments, the pegylated lipid in the pegylated liposome is DSPC-PEG-750, DSPC-PEG-1000, DSPC-PEG-1500, DSPC-PEG-2000, DSPC-PEG-2500, DSPC-PEG-3000, DSPC-PEG-3500, DSPC-PEG-4000, DSPC-PEG-4500 or DSPC-PEG-5000.

B. Non-pegylated neutral lipids

[0077] The pegylated liposomes provided herein also contain non-pegylated neutral lipids (neutral lipids not bound to polyethylene glycol (PEG)). In this application, the non-pegylated neutral lipid component of a liposome is contemplated to include any neutral lipid (which includes neutral phospholipids) that carries a generally neutral charge, or is a zwitterion that can bind to the pegylated lipid component to form a stable liposome structure.

[0078] In this application, the non-pegylated neutral lipid is generally neutrally charged. In some embodiments, the non-pegylated neutral lipid is a zwitterion. The neutral non-pegylated lipid component of a pegylated liposome, in some embodiments, may make the pegylated liposome generally neutrally charged.

[0079] In some embodiments, the non-pegylated neutral lipid component of the pegylated liposome contains a C _10-20 alkyl chain. In some embodiments, the non-pegylated neutral lipid component contains a C _12-18 alkyl chain. In some embodiments, the non-pegylated neutral lipid component comprises a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain.

[0080] In some embodiments, the non-pegylated neutral lipid is DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE, or DMPE.

[0081] In some embodiments, the mole percentage (mol. %) of non-pegylated neutral lipid in the liposome is in the range of about 45 mol. % to about 98 mol. %. In some embodiments, the mole percentage (mol %) of non-pegylated neutral lipid in the liposome is approximately 45 mol. %, 50 mol. %, 55 mol. %, 60 mol. %, 65 mol. %, 70 mol. %, 75 mol. %, 80 mol. %, 85 mol. %, 90 mol. %, 95 mol. %, or even about 98 mol. %. In a typical implementation pier. % non-pegylated neutral lipid in the liposome is approximately 45 mol. %.

[0082] In a typical embodiment, the lipid molar ratio of non-pegylated neutral lipid to pegylated lipid is 9.8:0.8. In a typical implementation, the molar ratio between non-pegylated neutral lipid and pegylated lipid is 18:3.

[0083] In a typical embodiment, the lipid molar ratio between non-pegylated DPPC neutral lipid and DPPE-PEG-2000 pegylated lipid is 9.8:0.8. In a typical embodiment, the lipid molar ratio between non-pegylated DPPC neutral lipid and DPPE-PEG-750 pegylated lipid is 9.8:0.8. In a typical embodiment, the lipid molar ratio between non-pegylated DPPC neutral lipid and DPPE-PEG-750 pegylated lipid is 18:3.

C. Cholesterol

[0084] The PEGylated liposomes provided herein contain cholesterol (which includes cholesterol-containing compounds).

[0085] In some embodiments, they say. % cholesterol in pegylated liposome is in the range from about 1 mol. % to about 50 mol. %, from about 5 mol. % to about 50 mol. %, from about 10 mol. % to about 50 mol. %, from about 20 mol. % to about 50 mol. %, from about 25 mol. % to about 50 mol. %, from about 5 mol. % to about 10 mol. %, from about 5 mol. % to about 20 mol. %, from about 5 mol. % to about 25 mol. %, from about 10 mol. % to about 20 mol. %, from about 10 mol. % to about 25 mol. %, from about 20 mol. % to about 30 mol. %, from about 20 mol. % to about 40 mol. %, or even from about 1 mol. % to about 10 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 50 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 45 mol. %. In some embodiments, they say. % cholesterol in the pegylated liposome is approximately 40 mol. %. In some embodiments, they say. % cholesterol in the pegylated liposome is approximately 35 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 30 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 25 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 20 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 15 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 10 mol. %. In some embodiments, they say. % cholesterol in pegylated liposome is approximately 5 mol. %.

[0086] In some embodiments, the molar ratio between cholesterol lipid, non-pegylated neutral lipid, and pegylated lipid is approximately 5.7:9.8:0.8. In some embodiments, the molar ratio between cholesterol lipid, non-pegylated neutral lipid, and pegylated lipid is approximately 5.5:18:3.

[0087] In some embodiments, the molar ratio between cholesterol lipid, non-pegylated DPPC neutral lipid, and DPPE-PEG-2000 pegylated lipid is approximately 5.7:9.8:0.8. In some embodiments, the molar ratio between cholesterol lipid, non-pegylated DPPC neutral lipid, and DPPE-PEG-750 pegylated lipid is approximately 5.7:9.8:0.8. In some embodiments, the molar ratio between cholesterol lipid, non-pegylated DPPC neutral lipid, and DPPE-PEG-750 pegylated lipid is approximately 5.5:18:3.

D. TLR agonists

[0088] This application describes that the pegylated liposomes described in this application may contain one or more Toll-like receptor agonists (TLR agonists). Toll-like receptors (TLRs) include transmembrane receptors on the surface of cells of the innate immune system that confer the ability of host cells at an early phase to recognize various conserved microbial molecular structures, for example, that may be present in or on a wide variety of infectious pathogens. Induction of TLR-mediated signaling to stimulate the initiation of immune responses by the innate immune system can be accomplished with TLR agonists that involve TLRs on the cell surface. For example, lipopolysaccharide (LPS) may be a TLR agonist acting through 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) (poly(I:C)) may be a TLR agonist acting through TLR3 (Salem et al., 2006 Vaccine 24:5119); CpG sequences (oligodeoxynucleotides containing unmethylated cytosine-guanosine or "CpG" dinucleotide motifs, e.g. CpG 7909, Cooper et al., 2005 AIDS 19:1473; CpG 10101 Bayes et al. Methods Find Exp Clin Pharmacol 27:193; Vollmer and Expert Opinion on Biological Therapy 5:673; Vollmer et al., 2004 Antimicrob Agents Chemother. 48:2314; Deng et al., 2004 J. Immonol, 173:5148) may be TLR agonists acting through TLR9 ( Andaloussi et al., 2006 Glia 54:526; Chen et al., 2006 J. Immonol, 177:2373); the peptidoglycans may be a TLR2 and/or TLR6 agonist (Soboll et al., 2006 Biol. Reprod, 75:131; Nakao et al., 2005 J. Immonol, 174:1566); 3M003 (4-amino-2-(ethoxymethyl)-α,α-dimethyl-6,7,8,9-tetrahydro-1Н-imidazo(4,5-с)quinoline-1-ethanol hydrate, mol. wt. 318 Da from 3M Pharmaceuticals, St. Paul, MN, which is also the source of related compounds 3M001 and 3M002; Gorden et al., 2005 J. Immonol, 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); and hepatitis C antigens can act as TLR agonists via TLR7 and/or TLR9 (Lee et al., 2006 Proc. Nat. Acad. Sci. USA 103:1828; Horsmans et al., 2005 Hepatol, 42:724). Other TLR agonists are known (eg, Schirmbeck et al., 2003 J. Immonol, 171:5198) and may be used according to some of the embodiments described herein.

[0089] In various embodiments, a TLR agonist can be a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7/8 agonist, a TLR9 agonist, combinations of these agonists.

TLR7/8 agonists

[0090] This application provides TLR7/8 agonists that can be used in the compositions described in this application. In this application, "TLR7/8 agonist" refers to an agonist that affects its biological activities through interaction with TLR7, TLR8, or both. Such biological activities include, but are not limited to, the induction of TLR7 and/or TLR8 mediated signaling to enhance immune responses by the innate immune system. In some embodiments, the TLR is an imidazoquinoline, an imidazoquinoline containing compound, or an imidazoquinoline amine derivative (see, for example, US Pat. No. 4,689,338 (Gerster)), but other classes of compounds are also known (see, for example, US Pat. others); US patent number 6194425 (Gerster and others); and US patent number 6110929 (Gerster and others); and international publication number WO 2005/079195 (Hays and others)).

[0091] In some embodiments, the TLR7/8 agonist that is used in the compositions described herein comprises an imidazoquinoline derivative such as that described in Shi et al. (ACS Med. Chem. Lett., 2012, 3(6), pp. 501-504), the contents of which are fully incorporated into this application by reference.

[0092] In some embodiments, the TLR7/8 agonist comprises an imidazoquinoline or an imidazoquinoline-containing compound. In some embodiments, the imidazoquinoline is imiquimod (denoted by IMQ or R837), an immune response modifier. In some embodiments, the imidazoquinoline is resiquimod (R848), a drug that acts as an immune response modifier (Tomai MA, Miller RL, Lipson KE, Kieper WC, Zarraga IE, Vasilakos JP (October 2007). "Resiquimod and other immune response modifiers as vaccine adjuvants". Expert Review of Vaccines 6 (5): 835-47.) In some embodiments, the TLR 7/8 agonist is CL075.

[0093] For example, in some embodiments, the TLR7/8 agonist is a compound with the following structure of formula (I):

or a pharmaceutically acceptable salt of said compound, wherein:

R ¹¹ is selected from the group consisting of hydrogen and C _1-6 alkyl, where the specified C _1-6 alkyl optionally contains as substituents one or more groups selected from the group consisting of halogen, hydroxyl and C _1-6 alkoxy;

R ¹² is selected from the group consisting of hydrogen and a carboxylic acid ester; And

R ¹³ is selected from the group consisting of hydrogen and C _1-6 alkyl, where the specified C _1-6 alkyl optionally contains as substituents one or more groups selected from the group consisting of halogen, hydroxyl and C _1-6 alkoxy.

[0094] In some embodiments of formula (I), R ¹¹ is hydrogen. In some embodiments, R ¹¹ is C _1-6 alkyl. In some embodiments, R ¹¹ is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R ¹¹ is n-butyl. In some embodiments, R ¹¹ is C _1-6 alkyl which is substituted with C _1-6 alkoxy. In some embodiments, R ¹¹ is -CH ₂ -O-CH ₂ -CH ₃ .

[0095] In some embodiments of formula (I), R ¹² is hydrogen. In some embodiments, R ¹² is a carboxylic acid ester. In some embodiments, R ¹² is —C(O)OC _1-4 alkyl. In some embodiments, R ¹² is -C(O)O-CH ₃ .

[0096] In some embodiments of formula (I), R ¹³ is C _1-6 alkyl. In some embodiments, R ¹³ is C _2-4 alkyl. In some embodiments, R ¹³ is -CH ₂ -CH(CH ₃ ) ₂ .

[0097] In some embodiments, R ¹³ is C _1-6 alkyl, which is substituted with halogen, hydroxyl, or C _1-6 alkoxy. In some embodiments, R ¹³ is C _1-6 alkyl which contains hydroxyl as a substituent. In some embodiments, R ¹³ is C _2-4 alkyl which contains hydroxyl as a substituent. In some embodiments, R ¹³ is -CH ₂ -C(CH ₃ ) ₂ OH.

[0098] In some embodiments, the TLR7/8 agonist is a compound with the following structure of formula (II):

or a pharmaceutically acceptable salt of said compound, wherein:

R ¹¹ is selected from the group consisting of hydrogen and C _1-6 alkyl, where the specified C _1-6 alkyl optionally contains as substituents one or more groups selected from the group consisting of halogen, hydroxyl and C _1-6 alkoxy;

R ¹² is selected from the group consisting of hydrogen and a carboxylic acid ester;

And

R ^13a is selected from the group consisting of hydrogen and hydroxyl.

[0099] In some embodiments of formula (II), R ¹¹ is hydrogen. In some embodiments, R ¹¹ is C _1-6 alkyl. In some embodiments, R ¹¹ is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R ¹¹ is n-butyl. In some embodiments, R ¹¹ is C _1-6 alkyl which is substituted with C _1-6 alkoxy. In some embodiments, R ¹¹ is -CH ₂ -O-CH ₂ -CH ₃ .

[00100] In some embodiments of formula (II), R ¹² is hydrogen. In some embodiments, R ¹² is a carboxylic acid ester. In some embodiments, R ¹² is —C(O)O—C _1-4 alkyl. In some embodiments, R ¹² is -C(O)O-CH ₃

[00101] In some embodiments of formula (II), R ^13a is hydroxyl. In some embodiments, R ^13a is hydrogen.

[00102] In some embodiments, the TLR7/8 agonist is a compound with the following structure of formula (III):

or a pharmaceutically acceptable salt of said compound, wherein:

R ¹¹ is selected from the group consisting of hydrogen and C _1-6 alkyl, where the specified C _1-6 alkyl optionally contains as substituents one or more groups selected from the group consisting of halogen, hydroxyl and C _1-6 alkoxy;

R ¹² is selected from the group consisting of hydrogen and a carboxylic acid ester;

And

R ^13b is C _1-6 alkyl optionally substituted with one or more groups selected from the group consisting of halogen, hydroxyl, C _1-6 alkoxy and acylamine.

[00103] In some embodiments of formula (III), R ¹¹ is hydrogen. In some embodiments, R ¹¹ is C _1-6 alkyl. In some embodiments, R ¹¹ is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R ¹¹ is n-butyl. In some embodiments, R ¹¹ is C _1-6 alkyl which is substituted with C _1-6 alkoxy. In some embodiments, R ₁₁ is -CH ₂ -O-CH ₂ -CH ₃ .

[00104] In some embodiments of formula (III), R ¹² is hydrogen. In some embodiments, R ¹² is a carboxylic acid ester. In some embodiments, R ¹² is —C(O)O—C _1-4 alkyl. In some embodiments, R ¹² is -C(O)O-CH ₃ .

[00105] In some embodiments of formula (III), R ^13b is C _2-4 alkyl which is substituted with acylamine. In some embodiments, R ^13b is -(CH ₂ ) ₄ -acylamine. In some embodiments, R ^13b is -(CH ₂ ) ₄ -NH-C(O)-C _1-25 alkyl. In some embodiments, R ^13b is -(CH ₂ ) ₄ -NH-C(O)-C _15-25 alkyl. In some embodiments, R ^13b is -(CH ₂ ) ₄ -NH-C(O)-C _15-20 alkyl. In some embodiments, R ^13b is -(CH ₂ ) ₄ -NH-C(O)-C ₁₇ alkyl.

[00106] In some embodiments, the TLR7/8 agonist is a compound with the following structure of formula (IV):

or a pharmaceutically acceptable salt of said compound, wherein:

R ¹⁰ is selected from the group consisting of hydrogen and C _1-6 alkyl; And

R ^11b is C _1-6 alkyl optionally substituted with one or more groups selected from the group consisting of halogen, hydroxyl, C _1-6 alkoxy and acylamine.

[00107] In some embodiments of formula (IV), R ¹⁰ is hydrogen. In some embodiments, R ¹⁰ is C _1-6 alkyl. In some embodiments, R ¹⁰ is methyl, ethyl, n-propyl, or n-butyl. In some embodiments, R ¹⁰ is n-butyl.

[00108] In some embodiments of formula (IV), R ^11b is C _2-4 alkyl which is substituted with acylamine. In some embodiments, R ^11b is -(CH ₂ ) ₄ -acylamine. In some embodiments, R ^11b is -(CH ₂ ) ₄ -NH-C(O)-C _1-25 alkyl. In some embodiments, R ^11b is -(CH ₂ ) ₄ -NH-C(O)-C _15-25 alkyl. In some embodiments, R ^11b is -(CH ₂ ) ₄ -NH-C(O)-C _15-20 alkyl. In some embodiments, R ^11b is -(CH ₂ ) ₄ -NH-C(O)-C ₁₇ alkyl.

[00109] In some embodiments, the TLR7/8 agonist is a compound with any of the following structures, or pharmaceutically acceptable salts of said compounds:

[00110] In some embodiments, the TLR7/8 agonist is a compound of the following structure, or pharmaceutically acceptable salts of said compound:

[0111] In some exemplary embodiments, the TLR7/8 agonist that is used in the compositions herein comprises N-(4-{(4-amino-2-butyl-1H-imidazo(4,5-c)quinoline-1 -yl)oxy}butyl)octadecanamide) - 3M-052, described in US patent number 9242980.

TLR4 agonists

[0112] This application provides TLR4 agonists that can be used in the compositions described in this application. In some embodiments, the TLR4 agonist used in the compositions herein comprises a glucopyranosyl lipid adjuvant (GLA) such as that described in US Patent Publications US 2007/021017, US 2009/045033, US 2010/037466 and US 2010 /0310602, the contents of which are fully incorporated into this application by reference.

[0113] For example, in some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure of formula (V):

or a pharmaceutically acceptable salt of said compound, wherein:

L ₁ , L ₂ , L ₃ , L ₄ , L ₅ and L ₆ are the same or different and independently represent -O-, -NH- or -(CH ₂ )-;

L ₇ , L ₈ , L ₉ and L ₁₀ are the same or different and are independently absent or are -C(=O)-;

Y ₁ represents an acid functional group;

Y ₂ and Y ₃ are the same or different and are independently —OH, —SH, or an acid functional group;

Y ₄ is -OH or -SH;

R ₁ , R ₃ , R ₅ and R ₆ are the same or different and are independently C _8-13 alkyl; And

R ₂ and R ₄ are the same or different and are independently C _6-11 alkyl.

[0114] In some embodiments of the GLA synthetic structure, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₀ alkyl; and R ² and R ⁴ are C ₈ alkyl. In some embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0115] For example, in some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure of formula (VI):

[0116] In a particular embodiment, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₁₂ -C ₂₀ alkyl.

[0117] In another specific embodiment, the implementation of GLA corresponds to the formula described above, in which R ¹ , R ³ , R ⁵ and R ⁶ represent C ₁₁ alkyl; and R ² and R ⁴ are C ₁₃ alkyl.

[0118] In another specific embodiment, the implementation of GLA corresponds to the formula described above, in which R ¹ , R ³ , R ⁵ and R ⁶ represent C ₁₀ alkyl; and R ² and R ⁴ are C ₈ alkyl.

[0119] In another specific embodiment, the implementation of GLA corresponds to the formula described above, in which R ¹ , R ³ , R ⁵ and R ⁶ represent C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In some embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0120] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure of formula (VII):

[0121] In some embodiments of the GLA structure described above, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In some embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0122] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure of formula (VIII):

[0123] In some embodiments of the GLA structure described above, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In some embodiments, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0124] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure of formula (IX):

[0125] In some embodiments of the GLA structure described above, R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₉ -C ₂₀ alkyl. In some embodiments, R ¹ , R, R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₉ alkyl.

[0126] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure:

[0127] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure:

[0128] In some embodiments, the TLR4 agonist is a synthetic GLA adjuvant with the following structure:

[0129] In another embodiment, an attenuated lipid A derivative (ALD) is included in the compositions described herein. ALDs are lipid A-like molecules that have been altered or engineered such that the resulting molecule causes less or different adverse events than lipid A. These adverse events include pyrogenicity, local Schwartzman reactions, and toxicity, which is assessed in the chick embryo 50% lethal dose (CELD ₅₀ ) assay. ALDs useful herein include monophosphoryl lipid A (MPL) and 3-deacylated monophosphoryl lipid A (3D-MPL). MPL and 3D-MPL are known and do not need to be described in detail in this application. See, for example, US Pat. No. 4,436,727 which describes monophosphoryl lipid A and its manufacture. US Patent No. 4,912,094 and Patent Reexamination Certificate B1 of US Patent No. 4,912,094 describe 3-deacylated monophosphoryl lipid A and a process for its production. See also, for example, GB 2220211 and WO 92/116556. 3-de-O-acylated monophosphoryl lipid A is known from GB 2220211 (Ribi). Chemically it is a mixture of 3-de-O-acylated monophosphoryl lipid A with 4, 5 or 6 agitated chains and is manufactured by Ribi Immunochem Montana. One form of 3-de-O-acylated monophosphoryl lipid A is described in International Patent Application No. WO 92/116556. The descriptions of each of these patents in relation to MPL and 3D-MPL are incorporated into this application by reference.

[0130] In the TLR4 agonist compounds described above, the overall charge can be determined from the functional groups in the molecule. For example, a phosphate group may be negatively charged or neutral depending on the ionized state of the phosphate group.

Exemplary Embodiments with a TLR Agonist

[0131] In some embodiments, a TLR agonist binds to a lipid bilayer of a pegylated liposome.

[0132] In some embodiments, the TLR agonist is enclosed in a pegylated liposome.

[0133] In some embodiments, the TLR agonist is an agonist that will disrupt the structure of the non-pegylated liposome.

[0134] In some embodiments, the TLR agonist contains a hydrophobic tail. In some embodiments, the hydrophobic tail of the TLR agonist binds to the lipid bilayer of the pegylated liposome.

[0135] In some embodiments, the TLR agonist includes TLR4, SLA, GLA, MPL, 3D-MPL, R848, R837, or combinations thereof.

[0136] In some embodiments, a TLR agonist includes a TLR4 agonist.

[0137] In some embodiments, the TLR agonist includes a TLR7/8 agonist.

[0138] In some embodiments, a TLR agonist includes a TLR7 agonist.

[0139] In some embodiments, the TLR agonist includes a TLR8 agonist.

[0140] In some embodiments, the TLR agonist includes imidazoquinoline.

[0141] In some embodiments, the TLR agonist includes 3M-052.

[0142] In some embodiments, the TLR agony includes a combination of a TLR4 agonist and a TLR 7/8 agonist. In some embodiments, the PEGylated liposome contains 3M-052 and GLA.

E. Agents

[0143] The PEGylated liposomes provided herein may further comprise one or more agents, which agent may be a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent, organism, genome, or virus. In some embodiments, the pegylated liposome contains two or more agents.

[0144] In some embodiments, the agent is associated with a pegylated liposome. In some embodiments, the agent is associated with the PEGylated liposome by ligand exchange and/or by electrostatic (charge based) interaction.

[0145] In some embodiments, the agent may comprise from about 0.01 to 1% by weight of the pegylated liposome.

Polypeptides

[0146] In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide is a full length protein or a fragment thereof. In some embodiments, the polypeptide is a peptide. In some embodiments, the polypeptide is a fusion protein. In some specific embodiments, the fusion protein is capable of eliciting an immune response upon administration to an individual. In some embodiments, the polypeptide is an antigen, further described below.

Antigens

[0147] In one embodiment, the agent includes an antigen.

[0148] In some embodiments, the polypeptide antigen is involved in or derived from an allergy, cancer, or infectious disease.

[0149] In some embodiments, the compositions described herein are suitable for vaccination purposes and are provided as vaccine formulations (vaccine compositions).

[0150] An antigen can be any target epitope, molecule (including a biomolecule), molecular complex (including molecular complexes that contain biomolecules), subcellular complex, cell, or tissue against which immunoreactivity is to be induced or enhanced in a subject. Often, the term antigen will refer to the polypeptide antigen of interest. However, an antigen in this application may also refer to a recombinant construct that encodes a polypeptide antigen of interest (eg, an expression construct). In some embodiments, an antigen may be, or be prepared for, or immunologically cross-react with an infectious pathogen and/or an epitope, biomolecule, cell, or tissue that is associated with an infection, cancer, autoimmune disease, allergy, asthma, or any other condition in which it would be desirable or useful to stimulate an antigen-specific immune response.

[0151] In some embodiments, an antigen is provided that is derived from at least one infectious pathogen, such as a bacterium, virus, or fungus, including an actinobacterium, such as M. tuberculosis or M. leprae, or another mycobacterium; a bacterium such as a member of the genus Salmonella, Neisseria, Borrelia, Chlamydia or Bordetella; virus 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; a fungus such as aspergillus, blastomycetes, coccidioides and pneumocystis, or yeasts, including Candida species such as C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis; a parasite such as a protozoan, for example, Plasmodium species including P. falciparum, P. vivax, P. malariae and P. ovale; or another parasite such as one or more of Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, W uchereria bancrofti, Giardia and leishmania. In specific embodiments, the antigen may be derived from or linked to antigens involved in tuberculosis, influenza, amoebiasis, HIV, hepatitis, or leishmaniasis.

[0152] In some embodiments, the antigen is an amoebiasis-associated antigen. In some embodiments, the antigen is an amoebiasis-causing antigen. In some embodiments, the antigen is derived from an amoebiasis-causing organism. In some embodiments, the antigen is derived from Entamoeba histolytica. In one embodiment, the antigen comprises LecA. In one embodiment, the antigen is LecA.

[0153] In some embodiments, the antigen is an influenza-associated antigen. In some embodiments, the antigen is an influenza-causing antigen. In some embodiments, the antigen is derived from an influenza-causing virus. In one embodiment, the antigen comprises H5N1. In one embodiment, the antigen comprises H5N1.

[0154] For example, in some embodiments, the antigens are derived from Borrelia sp., said antigens may include a nucleic acid, a pathogen-derived antigen or antigenic preparations, recombinantly produced protein or peptides, and chimeric fusion proteins. One such antigen is OspA. OspA may be the fully mature protein in lipidated form due to its biosynthesis in the host cell (Lipo-OspA) or alternatively may be a non-lipidated derivative. Such non-lipidated derivatives include the non-lipidated NS1-OspA fusion protein, which contains the first 81 N-terminal amino acid of the non-structural protein (NS1) of the influenza virus and the full-length OspA protein, and the other MDP-OspA protein is a non-lipidated form of OspA bearing 3 additional N-terminal amino acids. .

[0155] In some embodiments, the antigen is derived from a virus such as HIV-1 (such as tat, nef, gp120 or gp160), human herpes viruses such as gD or derivatives thereof, or an immediate early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus (especially human) (such as gB or its derivatives), rotavirus (including live attenuated viruses), Epstein-Barr virus (such as gp350 or its derivatives), varicella-zoster virus (such as gpI, II and IE63 ), or from a hepatitis virus such as hepatitis B virus (for example, hepatitis B surface antigen or a derivative thereof), hepatitis A virus, hepatitis C virus, and hepatitis E virus, or from other viral pathogens such as paramyxoviruses: respiratory syncytial virus (such as F- and G-proteins or their derivatives), parainfluenza virus, measles virus, mumps virus, human papillomaviruses (for example, HPV6, 11, 16, 18, etc.), flaviviruses (for example, yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus) or influenza virus (whole live or inactivated virus, split influenza virus grown in eggs or MDCK cells, or whole influenza virus virosomes (described in Gluck and Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof such as GA, NP, NA or M proteins, or combinations thereof).

[0156] In some other embodiments, the antigen is derived from one or more bacterial pathogens such as Neisseria spp, including N. gonorrhea and N. meningitidis (e.g., capsular polysaccharides and their conjugates, transferrin binding proteins, lactoferrin binding proteins, RIS, adhesins ); S. pyogenes (eg M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans: H. ducreyi; Moraxella spp including M. catarrhalis also known as Branhamella catarrhalis (eg high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (eg, pertactin, pertussis toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, pili), B. parapertussis, and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (eg, ESAT6, antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (eg, colonization factors, heat-labile toxin or derivatives, heat-stable toxin or derivatives), enterohemorrhagic E. coli, enteropathogenic E. coli (eg, Shiga toxin-like toxin or derivatives thereof) ; Vibrio spp, including V. cholera (eg, cholera toxin or its derivatives); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (eg Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (eg toxins, adhesins and invasins) and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp, including H. pylori (eg, urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani (for example, tetanus toxin and its derivative), C. botulinum (for example, botulinum toxin and its derivative), C. difficile (for example, Clostridial toxins A or B and their derivatives); Bacillus spp., including B. anthracis (eg botulinum toxin and its derivatives); Corynebacterium spp., including C. diphtheriae (eg diphtheria toxin and its derivatives); Borrelia spp., including B. burgdorferi (e.g. OspA, OspC, DbpA, DbpB), B. garinii (e.g. OspA, OspC, DbpA, DbpB), B. afzelii (e.g. OspA, OspC, DbpA, DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and human granulocytic ehrlichiosis agent; Rickettsia spp, including R. rickettsii; chlamydia spp. including C. trachomatis (eg, MOMPs, heparin-binding proteins), C. pneumoniae (eg, MOMPs, heparin-binding proteins), C. psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum (eg rare outer membrane proteins), T. denticola, T. hyodysenteriae; or other bacterial pathogens.

[0157] In some other embodiments, the antigen is derived from one or more parasites (see, for example, John, DT and Petri, WA, Markell and Voge's Medical Parasitology- ^9th Ed., 2006, WB Saunders, Philadelphia; Bowman, DD , Georgis' Parasitology for Veterinarians, ^8th ed., 2002, WB Saunders, Philadelphia), such as Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (eg SAG2, SAG3, Tg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T. cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocysts spp., including P. carinii; Trichomonas spp., including T. vaginalis; or from a helminth capable of infecting a mammal, for example by infection with: (i) nematodes (including but not limited to: Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis and Strongyloides stercoralis); (ii) trematodes (including but not limited to: Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica); and (iii) cestodes (including but not limited to: Taenia saginata and Taenia solium). In some embodiments, the antigen is derived from Schistosoma spp., Schistosoma mansonii, Schistosoma haematobium, and/or Schistosoma japonicum, or derived from yeast such as Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans.

[0158] Other specific antigens are derived from M. tuberculosis, for example, Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO 99/51748). Proteins from M. tuberculosis also include fusion proteins and variants thereof in which at least two, three, or four or more M. tuberculosis polypeptides have been fused into a larger protein. Some fusion proteins include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV- MTI (WO 99151748). Other antigens that can be used include the antigens, combination of antigens, and fusion proteins described in US 2010/0129391 and WO 2008/124647. In one exemplary embodiment, said fusion protein is ID93. In one exemplary embodiment, the fusion protein is ID91.

[0159] Other specific antigens are derived from Chlamydia and include, for example, HWMP high molecular weight protein (WO 99/17741), ORF3 (EP 366412), and putative membrane proteins (Pmps). Other chlamydia antigens can be selected from the group described in WO 99128475. Some antigens can be obtained from Streptococcus spp, including S. pneumoniae (e.g., capsular polysaccharides and their conjugates, PsaA, PspA, streptolysin, choline-binding proteins, and pneumolysin protein antigen (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342), and its detoxified mutant derivatives (WO 90/06951; WO 99/03884) Other bacterial vaccines contain antigens derived from Haemophilus spp. ., including H. influenzae type B (e.g., PRP and its conjugates), non-typable H. influenzae, e.g., OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin-derived peptides (U.S. Patent No. 5843464) or their multicopy variants or fusion proteins.

[0160] Other specific antigens are derived from hepatitis B. Derivatives of the hepatitis B surface antigen are well known in the art and include, among others, the PreS1, Pars2 S antigens described in European patent applications EP-A414 374; EP-A-0304 578 and EP 198474. In one aspect, the antigen is gp120 from HIV-1, especially when expressed in CHO cells. In a further embodiment, the antigen is gD2t.

[0161] In other embodiments, the antigen is derived from human papillomavirus (HPV), which is believed to cause genital warts (HPV 6 or HPV 11 and others), and HPV viruses that cause cervical cancer (HPV16, HPV18 and others). Specific antigens include L1 particles or capsomeres and fusion proteins containing one or more antigens selected from E6, E7, L1 and L2 proteins of HPV 6 and HPV 11. Some forms of fusion protein include L2E7 described in WO 96/26277 and protein D (1/3)-E7 described in GB 9717953.5 (PCT/EP 98/05285). Additional possible antigens include antigens from HPV 16 or 18. For example, L1 or L2 antigen monomers, or L1 or L2 antigens presented together as a virus-like particle (VLP), or a single L1 protein presented separately in a VLP or capsomere structure. Such antigens, virus-like particles and capsomeres are known per se. See, for example, WO 94/00152, WO 94/20137, WO 94/05792 and WO 93/02184.

[0162] In other embodiments, the antigen is a fusion protein. Fusion proteins can be included in the VLP alone or as fusion proteins such as E7, E2 or F5, for example; in specific embodiments, a VLP containing L1E7 fusion proteins is provided (WO 96/11272). Specific HPV 16 antigens include early E6 or F7 proteins fused to carrier protein D to form a D-E6 or E7 fusion protein from HPV 16, or combinations thereof; or combinations of E6 or E7 with L2 (WO 96/26277). Alternatively, the early E6 and E7 proteins of HPV 16 or 18 may be present in the same molecule, such as the D-E6/E7 fusion protein. The compositions may optionally contain either or both E6 and E7 proteins from HPV 18, for example as a D-E6 or D-E7 fusion protein or a D-E6/E7 fusion protein. The compositions may additionally contain antigens from other HPV strains, for example from HPV 31 or 33 strains.

[0163] Antigens can also be obtained from parasites that cause malaria. For example, antigens from Plasmodia falciparum include RTS,S and TRAP. RTS is a fusion protein containing the substantially full-length C-terminal portion of the P. falciparum circumsporozoite (CS) protein linked via four amino acids of the preS2 portion of the hepatitis B surface antigen to the hepatitis B surface (S) antigen. International Patent No. PCT/EP 92/02591 published as WO 93/10152 claiming priority from UK Patent Application No. 9124390.7. When RTS is expressed in yeast, it is produced as a lipoprotein particle, and when it is co-expressed with HBV S antigen, a mixed particle is obtained, known as RTS.S.

[0164] TRAP antigens are described in International Patent Application No. PCT/GB 89/00895 published as WO 90/01496. In some embodiment, the present invention provides a malaria vaccine wherein the antigenic composition comprises a combination of RTS,S and TRAP antigens. Other Plasmodium antigens that are likely candidates for multi-stage malaria vaccine components are MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28 , PFS27125, Pfs16, Pfs48/45, Pfs230 from P. faciparum and their counterparts in Plasmodium spp.

[0165] In one embodiment, the antigen is derived from a cancer cell, which may be useful for immunotherapeutic treatment of cancer. For example, the antigen may be a tumor rejection antigen such as prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, or melanoma. Examples of cancer or cancer cell derived antigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens such as those described in WO 99/40188, 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 and 1998); Correale et al. (1997) , Journal of the National Cancer Institute 89, p. 293). These are just some of the examples of cancer antigens expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, for example, US Pat. No. 6,544,518.

[0166] Other tumor-specific antigens include, but are not limited to, tumor-specific or tumor-associated gangliosides such as GM ₂ and GM ₃ or their carrier protein conjugates; or an autologous peptide hormone such as full-length gonadotropin-releasing hormone (GnRH, WO 95/20600), a short 10 amino acid peptide useful in the treatment of many types of cancer. In another embodiment, prostate antigens are used, such as prostate specific antigen (PSA), prostatic acid phosphatase (PCP), prostate stem cell antigen (PSA) (e.g., Proc. Nat. Acad. Sci. USA 95(4) 1735 -1740 1998), prostate specific membrane antigen (PSMA) or, in one embodiment, the antigen known as prostasis (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 are known from WO 98/137418 and WO/004149.Another antigen is prostate epithelial antigen with six transmembrane domains (STEAP) (PNAS 96 14523 14528 7-12 1999).

[0167] Other tumor-associated antigens useful in the context of the present invention include: Plu-1 (J Biol. Chem 274 (22) 15633-15645, 1999), HASH-1, HasH-2, crypto (cripto) (Salomon et al. Bioessays 199, 21: 61-70, US patent number 5654140) and cryptin (criptin) (US patent No. 5981215). In addition, antigens particularly suitable for cancer therapy vaccines also include tyrosinase and survivin.

[0168] In other embodiments, the agents used in the compositions of the present invention include antigens associated with respiratory diseases, such as those caused or exacerbated by a bacterial infection (e.g., pneumococcal), for the prevention and treatment of conditions such as chronic obstructive disease lungs (COPD). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and/or emphysema (Am J Respir Crit Care Med. 1995, Nov; 152(5 Pt 2): S77-121). Exacerbations of COPD are often caused by a bacterial (eg, pneumococcal) infection (Clin Microbiol Rev. 2001, Apr; 14(2): 336-63).

Polynucleotides.

[0169] In some embodiments, the agent is a polynucleotide. A polynucleotide includes, but is not limited to, DNA, RNA, an aptamer, and an oligonucleotide. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the DNA or RNA is single stranded or double stranded. In some embodiments, the polynucleotide is a non-coding RNA. In some embodiments, the polynucleotide is a coding RNA. In some embodiments, the RNA is selected from the group consisting of replicon RNA, mRNA, tRNA, siRNA, shRNA, and microRNA.

[0170] In some embodiments, the polynucleotide encodes a polypeptide. In some embodiments, the polynucleotide encodes a polypeptide that is or contains an antigen. In some embodiments, the polypeptide encoded by said polynucleotide is a fusion protein. In some embodiments, the polypeptide encoded by said polynucleotide is LecA. In some embodiments, the polypeptide encoded by said polynucleotide is H5N1. In some embodiments, the polypeptide encoded by said polynucleotide is ID93.

[0171] In some embodiments, the polynucleotide is a replicon. In some embodiments, the replicon is a plasmid, cosmid, bacmid, phage, or virus that is capable of replicating, for the most part, under its own control. In some embodiments, the replicon is RNA or DNA. In some embodiments, the replicon is single stranded or double stranded. In some embodiments, the replicon is derived from an RNA virus.

Adjuvants

[0172] In some embodiments, the pegylated liposomes provided herein further comprise an adjuvant or can be administered with a coadjuvant. In some embodiments, the adjuvant is selected from the group consisting of retinoic acid (RA), AS-2, monophosphoryl lipid A, 3-de-O-acylated monophosphoryl lipid A, IFA, QS21, CWS, TOM, AGP containing CpG oligonucleotides, Toll-like receptor (TLR) agonists, Leif, saponins, saponin mimetics, biological and synthetic lipid A, imichimod, gardichimod, resiquimod, poly(I:C), flagellin, GLA, SLA, stingin (stingin) and combinations of these adjuvants.

organisms

[0173] In some embodiments, the pegylated liposomes provided herein contain an organism. For example, Entamoeba histolytica, the virus that causes influenza, or the bacterium Mycobacterium tuberculosis, which causes tuberculosis (TB). To date, vaccination with live bacteria is the most effective way to induce protective immunity against tuberculosis. The most common mycobacterium used for this purpose is Bacillus Calmette-Guérin (BCG), an avirulent strain of Mycobacterium bovis. Thus, in some embodiments, the composition comprises a pegylated liposome and a mycobacterium.

[0174] In some embodiments, the agent is a virus or a virus genome. Thus, in these embodiments, the PEGylated liposomes contain the virus or the genome of the virus.

F. Exemplary pegylated liposomes.

[0175] In some embodiments, the non-pegylated neutral lipid:cholesterol:pegylated lipid molar ratio in the pegylated liposome of the present invention is approximately 9.8:5.7:0.8.

[0176] In some embodiments, the non-pegylated neutral lipid:cholesterol:pegylated lipid molar ratio in the pegylated liposome of the present invention is approximately 18:5.5:3.

[0177] In some embodiments, the non-pegylated neutral lipid OPPC:cholesterol:pegylated DPPE-PEG-750 lipid molar ratio in the pegylated liposome of the present invention is approximately 9.8:5.7:0.8.

[0178] In some embodiments, the non-pegylated neutral lipid OPPC:cholesterol:pegylated DPPE-PEG-2000 lipid molar ratio in the pegylated liposome of the present invention is approximately 9.8:5.7:0.8.

[0179] In some embodiments, the non-pegylated neutral lipid OPPC:cholesterol:pegylated DPPE-PEG-750 lipid molar ratio in the pegylated liposome of the present invention is approximately 18:5.5:3.

[0180] In some embodiments, the pegylated liposome contains GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0181] In some embodiments, the pegylated liposome contains GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0182] In some embodiments, the pegylated liposome contains LecA antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0183] In some embodiments, the pegylated liposome contains LecA antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0184] In some embodiments, the pegylated liposome contains the H5N1 antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0185] In some embodiments, the pegylated liposome contains the H5N1 antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0186] In some embodiments, the pegylated liposome contains an ID93 antigen, GLA, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEGylated component is PEG-2000.

[0187] In some embodiments, the pegylated liposome contains the ID93 antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0188] In some embodiments, the pegylated liposome contains an ID91 antigen, GLA, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEGylated component is PEG-2000.

[0189] In some embodiments, the pegylated liposome contains the ID91 antigen, GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0190] In some embodiments, the PEGylated liposome contains 3M-052, cholesterol, a non-PEGylated neutral lipid, and a PEGylated lipid, wherein the PEGylated component is PEG-2000.

[0191] In some embodiments, the pegylated liposome contains 3M-052, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEGylated component is PEG 750.

[0192] In some embodiments, the pegylated liposome contains 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0193] In some embodiments, the pegylated liposome contains 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0194] In some embodiments, the pegylated liposome contains LecA antigen, 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0195] In some embodiments, the pegylated liposome contains LecA antigen, 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0196] In some embodiments, the pegylated liposome contains the H5N1 antigen, 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-2000.

[0197] In some embodiments, the pegylated liposome contains the H5N1 antigen, 3M-052 and GLA, cholesterol, non-pegylated neutral lipid, and pegylated lipid, wherein the PEGylated component is PEG-750.

[0198] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen or a hepatitis-associated antigen, a TLR agonist, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid. , wherein the PEG component is PEG-2000.

[0199] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen or a hepatitis-associated antigen, a TLR agonist, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid. , wherein the PEG component is PEG-750.

[0200] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen, or a hepatitis-associated antigen, GLA, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEG component is PEG-2000.

[0201] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen, or a hepatitis-associated antigen, GLA, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEG component is PEG-750.

[0202] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen, or a hepatitis-associated antigen, 3M-052, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEG component is PEG-2000.

[0203] In some embodiments, the pegylated liposome comprises a tuberculosis-associated antigen, an HIV-associated antigen, a cancer-associated antigen, an amebiasis-associated antigen, an influenza-associated antigen, or a hepatitis-associated antigen, 3M-052, cholesterol, a non-pegylated neutral lipid, and a pegylated lipid, wherein the PEG component is PEG-750.

III. Physicochemical properties of pegylated liposomes.

A. Size

[0204] In this application, the size of the pegylated liposome is in the range from about 1 nm to 450 nm, and it can be considered a pegylated nanoliposome. Such nanoliposomes can be produced and sterilized by filtration. Moreover, when delivered in vivo, such nanoliposomes containing an agent (eg, antigen and/or adjuvant) usually do not or less often show a depot effect. Moreover, in vivo delivery of such nanoliposomes containing an agent (eg, antigen and/or adjuvant) to draining lymph nodes is possible, allowing presentation to antigen-presenting cells and elicitation of an effective immune response by Th1 cells.

[0205] In some embodiments, the size of a pegylated liposome can be estimated using techniques known in the art, including, but not limited to, X-ray and laser diffraction, dynamic light scattering (DLS), CryoEM, or Malvern Zetasize. In some embodiments, the size of the pegylated liposome refers to the Z-mean diameter.

[0206] In some embodiments, the size of the pegylated liposome is in the range of about 50 nm to 75 nm. In some embodiments, the size of the pegylated liposome is in the range of about 50 nm to 100 nm. In some embodiments, the size of the pegylated liposome is in the range of about 50 nm to 150 nm. In some embodiments, the size of the pegylated liposome is in the range of about 50 nm to 200 nm. In some embodiments, the size of the pegylated liposome is in the range of about 50 nm to 300 nm. In some embodiments, the size of the pegylated liposome is in the range of about 20 nm to 100 nm. In some embodiments, the size of the pegylated liposome is in the range of about 20 nm to 50 nm. In some embodiments, the size of the pegylated liposome is in the range of about 10 nm to 200 nm. In some embodiments, the size of the pegylated liposome is in the range of about 10 nm to 100 nm. In some embodiments, the size of the pegylated liposome is in the range of about 10 nm to 50 nm. In some embodiments, the size of the pegylated liposome is about 1 nm, is about 5 nm, is about 10 nm, is about 15 nm, is about 20 nm, is about 25 nm, is about 30 nm, is about 35 nm, is about 40 is approximately 45 nm is approximately 50 nm is approximately 55 nm is approximately 60 nm is approximately 65 nm is approximately 70 nm is approximately 75 nm is approximately 80 nm is approximately 85 nm is approximately 90 nm is approximately 95 nm is approximately 100 nm is approximately 105 nm is approximately 110 nm is approximately 115 nm is approximately 120 nm is approximately 125 nm is approximately 130 nm is approximately 135 nm is approximately 140 nm is approximately 145 nm is approximately 150 nm is approximately 155 nm is approximately 160 nm is approximately 165 nm is approximately 170 nm is approximately 175 nm is approximately 180 nm is approximately 185 nm is approximately 190 nm, is about 195 nm, or is about 200 nm. In some embodiments, the size of the pegylated liposome is not greater than about 1 nm, not greater than about 5 nm, not greater than about 10 nm, not greater than about 15 nm, not greater than about 20 nm, not greater than about 25 nm, not greater than about 30 nm, not greater than about 35 nm, not greater than about 40 nm, not greater than about 45 nm, not greater than about 50 nm, not greater than about 55 nm, not greater than about 60 nm, not more less than about 65 nm, not more than about 70 nm, not more than about 75 nm, not more than about 80 nm, not more than about 85 nm, not more than about 90 nm, not more than about 95 nm, not more than about 100 nm, not more than about 105 nm, not more than about 110 nm, not more than about 115 nm, not more than about 120 nm, not more than about 125 nm, not more than about 130 nm, not more than about 135 nm, not more than about 140 nm, not more than about 145 nm, not more than about 150 nm, not more than about 155 nm, not more than about 160 nm, not more than about 165 nm, not more than about 170 nm, not greater than about 175 nm, not greater than about 180 nm, not greater than about 185 nm, not greater than about 190 nm, not greater than about 195 nm, or not greater than about 199 nm.

[0207] In preferred embodiments, this application proposes that the PEGylated liposome can be filtered through a filter with a pore diameter of at least 0.45 microns. In some embodiments, the PEGylated liposome can be filtered through a filter with a pore diameter of less than 0.45 microns. In a typical implementation, the PEGylated liposome can be filtered through a filter with a pore diameter of 0.20 or 0.22 microns.

B. Stability.

[0208] The PEGylated liposomes provided herein are stable, allowing for ease of use, manufacturing capability, portability, and storage. The present inventors have found that PEGylation of the liposome contributes to the stability of the liposome. The physico-chemical properties of the PEGylated liposome, including but not limited to its size, are maintained over time at various temperatures and under various conditions.

[0209] In some embodiments, a pegylated liposome exhibits reduced or no aggregation compared to a liposome in the absence of a pegylated lipid. In some embodiments, the pegylated liposome or composition of pegylated liposomes does not aggregate, shows little or no aggregation, shows reduced aggregation, or does not show an overall increase in average size over time from baseline size.

[0210] The stability of a pegylated liposome can be measured using techniques well known to those skilled in the art. In some embodiments, stability is observed visually. Visual observation may include observation of particles, flocculation, or aggregates. In some embodiments, stability is measured by the size of the pegylated liposome, and is optionally expressed as a change in size over time, or at different temperatures, or under certain conditions. In some embodiments, stability is determined by assessing the % aggregation of pegylated liposomes in the composition. In some embodiments, stability is measured by the ability of the PEGylated liposome to pass through a filter of a certain size, for example, through a filter with a pore diameter of 0.20, 0, 22, or 0.45 microns. In some embodiments, stability is determined by pH. In some embodiments, stability is determined by measuring the polydispersity index (PdI), for example, using dynamic light scattering (DLS) techniques.

[0211] In some embodiments, the Z-mean diameter of the PEGylated liposome increases by less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 3%, less than 1% over the analyzed period of time.

[0212] In some embodiments, the PEGylated liposome is stable at 0-8°C. In some embodiments, the PEGylated liposome is stable at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C for at least 1 minute , for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes, for at least 30 minutes, in for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes, for at least 55 minutes, for at least 1 hour, for at least 2 hours, for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least 24 hours, for at least 48 hours, for at least 72 hours, for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 1 month, for at least 2 months, for at least 3 months , for at least 4 months, for at least 5 months, for at least 6 months, for at least 7 months, for at least 8 months, for at least 9 months, in for at least 10 months, for at least 11 months, for at least 1 year, for at least 2 years, or for at least 5 years. In one exemplary embodiment, the PEGylated liposome is stable for at least 1 month at about 2°C to about 8°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 1 month at about 4°C to about 8°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 6 months at about 4°C to about 8°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 1 year at about 4°C to about 8°C.

[0213] In some embodiments, the PEGylated liposome is stable at 8-20°C. In some embodiments, the PEGylated liposome is stable at 8-20°C for at least 1 minute, for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes, for at least 30 minutes, for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes , for at least 55 minutes, for at least 1 hour, for at least 2 hours, for at least 6 hours, for at least 12 hours, for at least 18 hours, in for at least 24 hours, for at least 48 hours, for at least 72 hours, for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 7 months, for at least 8 months, for at least 9 months, for at least 10 months, for at least 11 months, for at least 1 year, for at least 2 years or for at least 5 years. In one exemplary embodiment, the PEGylated liposome is stable for at least 1 month at about 8°C to about 20°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 1 month at about 8°C to about 20°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 6 months at about 8°C to about 20°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 1 year at about 8°C to about 20°C.

[0214] In some embodiments, the PEGylated liposome is stable at 20-30°C. In some embodiments, the PEGylated liposome is stable at 25°C for at least 1 minute, for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes , for at least 25 minutes, for at least 30 minutes, for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes, in for at least 55 minutes, for at least 1 hour, for at least 2 hours, for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least at least 24 hours, for at least 48 hours, for at least 72 hours, for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 7 months for at least 8 months, for at least 9 months, for at least 10 months, for at least 11 months, for at least 1 year, for at least 2 years, or for at least 5 years. In one exemplary embodiment, the PEGylated liposome is stable for at least 1 month at a temperature of about 25°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 6 months at about 25°C. In another exemplary embodiment, the PEGylated liposome is stable for at least 1 year at about 25°C.

[0215] In some embodiments, the PEGylated liposome is stable at 30-40°C. In some embodiments, the PEGylated liposome is stable at 30°C, 31°C, 32°C, 33°C, 34°C 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C for at least 1 minute, for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes, for for at least 30 minutes, for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes, for at least 55 minutes, for at least at least 1 hour, for at least 2 hours, for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least 24 hours, for at least 48 hours, for at least 72 hours, for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 1 month, for at least 2 months, for at least 3 months for at least 4 months for at least 5 months for at least 6 months for at least 7 months for at least 8 months for for at least 9 months, for at least 10 months, for at least 11 months, for at least 1 year, for at least 2 years, or for at least 5 years. In one exemplary embodiment, the PEGylated liposome is stable for at least 1 month at a temperature of about 37°C.

[0216] In some embodiments, the PEGylated liposome is stable at 40-62°C. In some embodiments, the PEGylated liposome is stable at 40-62°C for at least 1 minute, for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes, for at least 30 minutes, for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes , for at least 55 minutes, for at least 1 hour, for at least 2 hours, for at least 6 hours, for at least 12 hours, for at least 18 hours, in for at least 24 hours, for at least 48 hours, for at least 72 hours, for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 1 month.

[0217] In one exemplary embodiment, the PEGylated liposome is stable at 4-8°C for at least one year. In another exemplary embodiment, the PEGylated liposome is stable at 25° C. for at least one year.

[0218] In some embodiments, the PEGylated liposome is stable after 1-5 freeze/thaw cycles. In some embodiments, the PEGylated liposome is stable after 1, after 2, after 3, after 4, or after 5 freeze/thaw cycles.

[0219] In some embodiments, the polydispersity index of the pegylated liposome is maintained at about 0.3 or less. In some embodiments, the polydispersity index of the pegylated liposome is maintained at about 0.25 or less. In some embodiments, the polydispersity index of the pegylated liposome is maintained at about 0.2 or less.

IV. Methods for obtaining PEGylated liposomes

[0220] Proposed in this application, the method of obtaining pegylated liposome includes (a) mixing non-pegylated neutral lipid, pegylated lipid and cholesterol in an organic solvent; (b) evaporating said organic solvent, whereby a lipid film is obtained; (c) rehydration of the lipid film in buffer; and (d) exposing the rehydrated product from step (c) to a high energy input source (eg, sonication, microfluidization, extrusion). In some embodiments, exposure to a high energy source includes microfluidization. In some embodiments, exposure to a high energy source includes sonication. In some embodiments, exposure to a high energy source includes extrusion. In some embodiments, the organic solvent is chloroform or a chloroform/methanol/water mixture. In some embodiments, step (a) further comprises mixing the TLR agonist with other components.

[0221] In an exemplary embodiment, DPPC, cholesterol, and DPPE-PEG-750 are combined with varying amounts of 3M-052 in an organic solvent (chloroform or chloroform/methanol/water). The organic solvent is then evaporated. The resulting lipid film is rehydrated in buffer and sonicated until the formulation is translucent with no large visible particles. Larger batches (100 ml) of PEGylated liposomes can be produced as described above, but with shorter sonication followed by high shear homogenization.

[0222] In some embodiment, the resulting pegylated liposome is mixed with an agent (eg, an antigen).

V. Compositions containing PEGylated liposomes

[0223] This application provides formulations, compositions and pharmaceutical compositions containing pegylated liposomes described in this application.

[0224] In some embodiments, the composition comprising the PEGylated liposome further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0225] The compositions described in this application can be administered to a subject for any of the purposes of vaccination, therapy or diagnosis.

[0226] Pharmaceutical compositions typically include the compositions described in this application, and may additionally contain one or more components described in this application, which are selected from an antigen, additional agonists, or a recombinant expression construct, in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0227] In the embodiments of this application, the pharmaceutical composition can be filtered through a filter with a pore diameter of 0.45 microns. In some embodiments, the pharmaceutical composition can be filtered through a 0.20 micron filter. In some embodiments, the pharmaceutical composition may be filtered through a 0.22 micron filter.

[0228] In one embodiment, the present invention provides a pharmaceutical composition comprising a pegylated liposome that contains a TLR7/8 agonist or a TLR4 agonist. Such a composition can be used for "monotherapy", in which the TLR7/8 agonist or TLR 4 agonist described in this application is included in the composition, and the composition is essentially free of other antigens, and it is administered to the subject in order to stimulate the immune a response, such as a non-specific immune response or an antigen-specific immune response, for the purpose of diagnosing, treating, or preventing a disease or other condition, such as infection by some organism.

[0229] In other embodiments, the pharmaceutical composition is a vaccine composition that contains both the compositions described in this application and the antigen, and may additionally contain one or more of the components proposed in this application, in combination with a pharmaceutically acceptable carrier, excipient or diluent. Exemplary carriers are generally non-toxic to recipients at the dosages and concentrations employed.

[0230] In therapeutic embodiments provided herein, a dosage of about 1 μg/kg to about 1 mg/kg of the therapeutic pharmaceutical composition is administered. Those skilled in the art will appreciate that the amount and frequency of administration will depend on the subject's response.

[0231] In embodiments based on the vaccines proposed in this application, approximately 1 μg - 25 μg of an agent (eg, adjuvant or antigen) will be administered per administration. Those skilled in the art will appreciate that the amount and frequency of administration will depend on the subject's response.

[0232] "Pharmaceutical acceptable carriers" for use in therapy are well known in the pharmaceutical art and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). For example, sterile saline and phosphate buffered saline at physiological pH can be used. The composition of the pharmaceutical composition may include preservatives, stabilizers, colorants and even flavoring agents. For example, sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters can be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents can be used. Id.

[0233] A "pharmaceutically acceptable salt" refers to salts of compounds of the present invention obtained by combining such compounds with an organic or inorganic acid (acid addition salt) or an organic or inorganic base (base addition salt). The compositions of the present invention may be used either in free base form or in salt form, both forms being considered to be within the scope of the present invention.

[0234] Pharmaceutical compositions can be in any form that allows the introduction of the specified composition to the patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Common routes of administration include, without limitation, oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal, or subcutaneous routes. The term parenteral in this application includes iontophoretic (e.g. US Pat. No. 7,033,598; 7,018,345; 6,970,739), sonophoretic (e.g., US Pat. 2; 6018678), thermal (e.g. US Pat. No. 5885211; 6685699), passive transdermal administration (for example, US patents No. 3598122; 3598123; 4286592; 4314557; 4379454; 4568343; 5464387; application for US patent No. 2232892; US patents No. 6871477; 6974588; 6676961), insertion with microneedles (for example , US Pat. No. 6,908,453; US Pat. No. 5,457,041; US Pat. In some embodiment, the composition described in this application (including vaccine and pharmaceutical compositions) is administered intradermally using a technique selected from iontophoresis, microcavitation, sonophoresis, or microneedling.

[0235] The pharmaceutical composition can be formulated in such a way as to ensure the bioavailability of its constituent active ingredients after the introduction of the specified composition to the subject. Compositions to be administered to a subject are in the form of one or more dose units, where, for example, a tablet may be a single dose unit, and an aerosol container of one or more compounds of the present invention may contain multiple dose units.

[0236] For oral administration, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethylcellulose. Coloring and/or flavoring agents may be present. An overlay can be used.

[0237] The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. If the compositions are intended for oral administration, they may contain one or more sweetening agents, preservatives, coloring/coloring agents and flavor enhancers. One or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents may be included in the composition for administration by injection.

[0238] The liquid pharmaceutical composition used in this application, either in the form of a solution, or in the form of a suspension, or in another similar form, may contain one or more of the following carriers or excipients: sterile diluents such as water for injection, saline , preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, mannide monooleate, cholesterol and/or synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as sodium chloride or dextrose.

[0239] In another embodiment, the composition according to the present invention is formulated in such a way that it can be transferred to the aerosol state.

[0240] It may also be desirable to include other components in the pharmaceutical composition, such as delivery vehicles, including, but not limited to: aluminum salts, water-in-oil emulsions, biodegradable oil carriers, oil-in-water emulsions, biodegradable microcapsules, and liposomes. . Examples of additional immunostimulatory substances (coadjuvants) for use in such vehicles are also described above and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, interferon gamma, and IL-12.

[0241] While any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier will vary depending on the route of administration and the need for sustained release. For parenteral administration, such as subcutaneous injection, the carrier may include water, saline, alcohol, fat, wax, or a buffer. For oral administration, any of the carriers described above or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate may be used. Biodegradable microspheres (eg, polylactic galactide) can also be used as carriers for pharmaceutical compositions according to the present invention. Suitable biodegradable microspheres are described in, for example, US Pat.

[0242] Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. . Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents. For example, the product may be in the form of a lyophilisate, suitable solutions of excipients (eg sucrose) are used as diluents.

[0243] The pharmaceutical composition may be intended for topical administration, in this case, the carrier may, respectively, include a solution, emulsion, ointment or gel base. The base, for example, may contain one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. If the composition is for transdermal administration, it may include a transdermal patch or iontophoresis device. Topical formulations may contain antigen (eg, vaccine formulation with GLA antigen) or GLA (eg, formulation with immunological adjuvant; GLA available from Avanti Polar Lipids, Inc., Alabaster, Alabama; eg, product number 699800) at a concentration of from about 0 .1 to about 10% w/v (mass per unit volume).

[0244] The composition may be for rectal administration, for example in the form of a suppository, which can melt in the rectum and release the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol. In the methods of the present invention, the pharmaceutical compositions/adjuvants can be administered by use of insert(s), granule(s), time-controlled formulation(s), patch(s), or rapid release formulation(s).

VI. Methods of using PEGylated liposomes

[0245] Provided herein is a method for stimulating an immune response in a subject, comprising administering any of the pegylated liposomes or compositions containing pegylated liposomes provided herein. PEGylated liposomes and compositions can be used for vaccination, therapy and diagnostics.

[0246] In one embodiment, a composition containing pegylated liposomes is used to treat or prevent cancer.

[0247] In some embodiments, a pharmaceutical composition comprising a pegylated liposome provided herein is a vaccine composition and is used as a vaccine. In some embodiments, the compositions described herein are used to stimulate an immune response in a subject (including a non-specific response and an antigen-specific response). In some embodiments, the immune response includes a systemic immune response. In some embodiments, the immune response includes a mucosal immune response.

[0248] In one embodiment, a composition containing pegylated liposomes is used to enhance protective immunity against an influenza-causing virus.

[0249] In one embodiment, a composition containing pegylated liposomes is used to enhance protective immunity against an amoebiasis-causing organism.

[0250] In one embodiment, a composition containing pegylated liposomes is used to enhance protective immunity against Entamoeba histolytica.

[0251] In one embodiment, a composition containing pegylated liposomes is used to enhance protective immunity against influenza.

[0252] In one embodiment, a composition containing pegylated liposomes is used to enhance protective immunity against amebiasis.

[0253] In one embodiment, a composition containing pegylated liposomes is used to stimulate both a systemic immune response (characterized by an IgG immune response) and a mucosal immune response (characterized by an IgA immune response) in a subject by intranasally administering any of the compositions containing pegylated liposomes proposed in this application. In one embodiment, a composition containing pegylated liposomes is used to stimulate both a systemic immune response and a mucosal immune response in a subject by intranasal administration of a pegylated liposome containing a TLR4 agonist and a TLR7/8 agonist. In a similar embodiment, a composition containing pegylated liposomes is used to stimulate both a systemic immune response and a mucosal immune response in a subject by intranasal administration of a pegylated liposome containing GLA and 3M-052. Surprisingly, the present inventors found that intranasal administration of a composition containing a pegylated liposome can induce a local mucosal response (in the nasal cavity), induce a systemic immune response, and induce a long-term mucosal response (e.g., a mucosal response in the feces, intestines, and/or vagina) .

[0254] In the embodiments provided herein, the subject is a mammal (e.g., an animal, including farm animals (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.). ) and rodents (rats, mice, etc.), or humans). In one embodiment, the subject is a human. In another embodiment, the subject is a non-human mammal. In another embodiment, said non-human mammal is a dog, cow, or horse.

VII. Kits and industrial products

[0255] In some embodiments, kits are also provided containing the pegylated liposomes described herein and compositions, which may be provided in one or more containers. In one embodiment, all components of these compositions are together in one container, but it is not intended that the embodiments of the present invention be limited to this, and they also contemplate the presence of two or more containers in which, for example, the immunological adjuvant composition is separated and does not come into contact with the component antigen. As a non-limiting theory, it is believed that in some instances it may be beneficial to administer a composition containing only PEGylated liposomes as an immunological adjuvant formulation, while in other instances such administration may be usefully separated in time and/or space (e.g., administration to different anatomical regions). ) from the introduction of the antigen, while in other additional cases it is useful to administer to the specified subject the vaccine composition described in this application and containing both the antigen and the adjuvant composition, and optionally other components described in this application.

[0256] In some embodiments, one vial of said kit contains a composition containing pegylated liposomes, and the second vial of said kit contains an agent. In some embodiments, the kit contains a third vial containing an optional agent.

[0257] Kits according to the present invention may additionally contain instructions for use described in this application, or instructions for mixing the materials contained in the vials. In some embodiments, the material in the vial is dry or lyophilized. In some embodiments, the material in the vial is liquid.

[0258] The container of such kit embodiments may be any suitable container, vial, vial, ampoule, tube, cup, box, bottle, vial, jar, bowl, well of a single-well or multi-well device, container, tank, or similar containers, or another device in which the compositions described in this application can be placed, stored and / or transported, and accessed to remove the contents. Typically, such a container may be made from a material that is suitable for the intended use and from which the contents contained therein can be easily removed. Just a few examples of such containers include glass and/or plastic stoppered or resealable tubes and ampoules, including those containing a rubber septum or other closures that are suitable for retrieving the contents with a needle and syringe. Such containers, for example, may be made of glass or a chemically compatible plastic or resin, which may be made of or coated with a material that allows efficient removal of the material from the container and/or protects the material from, for example, damaging conditions such as ultraviolet light. or temperature fluctuations, or from the inclusion of undesirable impurities, including microbial impurities. Said containers are preferably sterile or sterilizable and made from materials that will be compatible with any carrier, adjuvant, diluent, vehicle, or the like that can be used to suspend or dissolve the vaccine compositions described herein and/or immunological adjuvant compositions. , and/or antigens, and/or constructs for recombinant expression, etc.

VIII. Typical Implementations

[0259] In a first embodiment, the present invention provides, inter alia, a cholesterol-containing liposome; a non-pegylated neutral lipid; and a pegylated lipid, wherein the average molecular weight of the PEG in the pegylated lipid is about 5,000 daltons or less.

[0260] According to the present invention, in the second embodiment, any of the liposomes according to the ^1st embodiment is also provided, characterized in that the average molecular weight of PEG in the pegylated lipid is in the range from about 750 daltons to about 5000 daltons; liposomes according to the first implementation variant, characterized in that the average molecular weight of PEG in pegylated lipid is approximately 2000 daltons or less; or liposomes according to the first embodiment, characterized in that the average molecular weight of the PEG in the pegylated lipid is approximately 750 daltons.

[0261] According to the present invention, in a third embodiment, any of the liposomes according to the ^1st or ^2nd embodiment is provided, wherein the lipid component of said pegylated lipid comprises a neutral lipid; characterized in that the lipid component of said pegylated lipid contains a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain; characterized in that the lipid component of said pegylated lipid is DSPE, DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE or DMPE; characterized in that the lipid component of said pegylated lipid is DSPE; or characterized in that the lipid component of said pegylated lipid is DPPE.

[0262] According to the present invention, in a fourth embodiment, any of the 1 ^ohm , 2 ^ohm , or 3 ^ohm liposomes is provided, wherein the non-pegylated neutral lipid comprises a C14 alkyl chain, a C16 alkyl chain, or a C18 alkyl chain; characterized in that the non-pegylated neutral lipid is DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE or DMPE; or wherein the non-pegylated neutral lipid is DPPC.

[0263] According to the present invention, in the fifth embodiment, any of the liposomes according to the ^1st , ^{2nd, 3rd} , or ^4th embodiments is provided, wherein said liposome is stable; characterized in that the liposome is stable for at least 1 month at a temperature of from about 2°C to about 8°C; characterized in that the liposome is stable for at least 1 month at a temperature of approximately 25°C; or characterized in that the liposome is stable for at least 1 month at a temperature of approximately 37°C.

[0264] According to the present invention, in the sixth embodiment, any of the liposomes according to the 1 ^th , 2 ^th 3 ^th , 4 ^th , or 5 ^th embodiments are provided, characterized in that the polydispersity coefficient of said liposome is maintained at a level of about 0.3 or less.

[0265] According to the present invention, in a seventh embodiment, any of the liposomes according to 1 ^th , 2 ^th 3 ^th , 4 ^{th 5th} or 6 ^{th th} embodiments is provided, characterized in that the size of said liposome is less than or approximately equal to 450 nm; characterized in that the size of the liposome is maintained at less than or approximately equal to 450 nm; or characterized in that the size of the liposome is in the range from about 50 nm to about 300 nm.

[0266] According to the present invention, in an eighth embodiment, any of the liposomes according to 1 ^th , 2 ^th 3 ^th , 4 ^th 5 ^th , 6 ^th or 7 ^th embodiments is provided, characterized in that the molar percentage (mol.%) of pegylated lipid in the specified liposome is in the range from about 1 mol. % to about 25 mol. %; characterized in that the % pegylated lipid in the liposome ranges from about 1 mol. % to about 10 mol. %; or differing in that they say. % pegylated lipid in the liposome is approximately 5 mol. %.

[0267] According to the present invention, in the ninth embodiment, any of the liposomes according to the ^1st , ^2nd , ^3rd , ^{4th, 5th} , ^6th , ^7th, or ^8th embodiments is provided, characterized in that they say % cholesterol in the specified liposome is in the range from about 1 mol. % to about 50 mol. %; or differing in that they say. % cholesterol in the liposome is approximately 50 mol. %.

[0268] According to the present invention, in the tenth embodiment, any of the liposomes according to the ^1st , ^2nd , ^3rd , ^{4th, 5th} , ^6th , ^7th, or ^8th or ^9th embodiments are provided, characterized in that they say % non-pegylated lipid in the specified liposome is in the range from approximately 45 mol. % to about 98 mol. % or characterized in that they say. % non-pegylated lipid in the liposome is approximately 45 mol. %.

[0269] According to the present invention, in the eleventh embodiment, any of the liposomes according to the ^1st , 2nd, ^3rd , ^4th , ^5th , ^6th , ^{7th ,} or ^8th , ^9th , or ^10th embodiments are provided, characterized in that the molar the ratio between lipids non-pegylated neutral lipid:cholesterol:pegylated lipid is approximately 9.8:5.7:0.8 or characterized in that the molar ratio between lipids non-pegylated neutral lipid:cholesterol:pegylated lipid is approximately 18:5.5:3 .

[0270] According to the present invention ^, in the twelfth embodiment, any of the liposomes according to the ^1st , ^2nd , ^3rd , 4th, ^5th , ^6th , ^7th , ^8th , ^9th , ^10th , or ^11th embodiments, characterized in that said liposome further comprises at least one TLR agonist; characterized in that the liposome contains a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7/8 agonist, or a TLR9 agonist; characterized in that the liposome contains a TLR4, SLA, GLA, 3D-MPL, R837 or R848 agonist; characterized in that the liposome contains a TLR agonist with a hydrophobic tail; characterized in that the liposome contains a TLR7/8 agonist; characterized in that the liposome contains a TLR7 agonist, characterized in that the liposome contains a TLR8 agonist; characterized in that the liposome contains a TLR7/8 agonist comprising imidazoquinoline or an imidazoquinoline-containing compound; characterized in that the liposome contains 3M-052; characterized in that the liposome contains R848; characterized in that the liposome contains a TLR4 agonist; characterized in that the liposome contains 3D-MPL; characterized in that the liposome contains GLA; characterized in that the liposome contains a synthetic GLA of formula (V) proposed in this application, or a pharmaceutically acceptable salt thereof and any of the corresponding embodiments of formula (V) or a pharmaceutically acceptable salt thereof; characterized in that the liposome contains a synthetic GLA of formula (VI) proposed in this application, or a pharmaceutically acceptable salt thereof and any of the corresponding embodiments of formula (VI) or a pharmaceutically acceptable salt thereof; or characterized in that the liposome contains a synthetic GLA formula:

or a pharmaceutically acceptable salt of said compound; characterized in that the liposome contains a TLR4 agonist and a TLR7/8 agonist; characterized in that the liposome contains any of the TLR4 or TLR7/8 agonists described in this application; or characterized in that the liposome contains GLA and 3M-052.

[0271] According to the present invention, the thirteenth embodiment provides any of the liposomes according to 1 ^ohm , 2 ^ohm , 3 ^ohm , 4 ^ohm , 5 ohm, 6 ^ohm , 7 ^ohm , 8 ^ohm , 9 ^ohm , 10 ^ohm , 11 ^ohm , or ^{12 ohm} embodiments, characterized in that said liposome contains at least one agent; characterized in that the liposome contains at least one agent that includes a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent or organism; characterized in that the liposome contains at least one agent that includes the antigen; characterized in that the liposome contains at least one agent that includes an antigen, and characterized in that said antigen includes an amebiasis-associated antigen, LecA, an influenza-associated antigen, H5N1, a tuberculosis-associated antigen, ID91, ID93, an antigen from BCG, hepatitis virus-related antigen, hepatitis B antigen, hepatitis C antigen, HIV-associated antigen or cancer-associated antigen.

[0272] According to the present invention, in a fourteenth embodiment, a composition is provided comprising any of liposomes according to 1 ^ohm , 2 ^ohm , 3 ^ohm , 4 ohm, 5 ^ohm , 6 ^ohm , 7 ^ohm , 8 ^ohm , 9 ^ohm , 10 ^ohm , ^{11 ohm} , ^12th or ^13th embodiments, or a composition comprising any of the liposomes ^according to ^1st , ^2nd , 3rd, ^4th , ^5th , ^6th , ^7th , ^8th , ^9th , ^10th , ^11th , 12 ^ohm or 13 ^ohm embodiments, and a pharmaceutically acceptable carrier, excipient, or diluent. The composition may be, for example, a vaccine, therapeutic or diagnostic composition.

[0273] According to the present invention, in a fifteenth embodiment, there is provided a method for stimulating an immune response in a subject, comprising administering to said subject a liposome or composition according to any of ^1st , ^2nd , 3rd, ^4th , ^5th , ^{6th , 7th} , 8 ^th , ^9th , ^10th , ^11th , ^12th , ^13th, or ^14th embodiments, whereby an immune response is stimulated in the subject. The immune response may be, for example, a non-specific immune response; antigen-specific immune response; systemic immune response; mucosal immune response; or an immune response from the intestinal mucosa, stool, or vagina. These compositions can be used, for example, for the treatment or prevention of cancer; as a vaccine; to increase protective immunity against the influenza-causing virus; to increase protective immunity against the organism causing amoebiasis; to increase protective immunity against Entamoeba histolytica; to increase protective immunity against influenza; or to enhance protective immunity against amoebiasis. The route of administration of said composition may be oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal or subcutaneous. The liposome or composition can be administered together with a retinoic acid coadjuvant. Said subject may be, for example, a human or a non-human mammal.

[0274] According to the present invention, in a sixteenth embodiment, there is provided a method for inducing a Th1 cell response in a subject, comprising administering to said subject a liposome or composition according to any of ^1st , ^2nd , 3rd, ^4th , ^{5th , 6th} , ^7th , ^8th , ^9th , ^10th , 11th, ^{12th , 13th,} or ^14th embodiments, whereby a Th1 cell response is elicited in the subject. The immune response may be, for example, a non-specific immune response, an antigen-specific immune response, a systemic immune response, a mucosal immune response, or an intestinal, fecal or vaginal mucosal immune response. These compositions can be used, for example, for the treatment or prevention of cancer, as a vaccine; to increase protective immunity against the influenza-causing virus; to increase protective immunity against the organism causing amoebiasis; to increase protective immunity against Entamoeba histolytica; to increase protective immunity against influenza; or to enhance protective immunity against amoebiasis. The route of administration of said composition may be oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal or subcutaneous. The liposome or composition can be administered together with a retinoic acid coadjuvant. Said subject may be, for example, a human or a non-human mammal.

[0275] According to the present invention, in a seventeenth embodiment, there is provided a method for stimulating a systemic immune response and a mucosal immune response in a subject, comprising intranasally administering to the subject a liposome or a composition according to any of ^1st , ^2nd , ^3rd , ^4th , ^5th , ^6th , ^7th , ^8th , ^9th , ^10th , ^11th , ^12th , ^13th or ^14th implementation options. A mucosal immune response may include, for example, an immune response from the intestinal, fecal, or vaginal mucosa. The mucosal immune response may, for example, be distant from the nasal cavity. The liposome or composition can be administered together with a retinoic acid coadjuvant. Said subject may be, for example, a human or a non-human mammal.

[0276] According to the present invention in the eighteenth embodiment, a method for producing any of the pegylated liposomes described in this application, including: a) mixing non-pegylated neutral lipid, pegylated lipid and cholesterol in an organic solvent; b) evaporating said organic solvent, whereby a lipid film is obtained; c) rehydration of the lipid film in buffer; and sonicating, microfluidizing, or extruding the rehydrated product from step c). Step a) may further include the step of mixing with the TLR agonist. The organic solvent may be, for example, chloroform. The rehydrated product from step (c) can, for example, be sonicated and then subjected to microfluidization. Said method may further include mixing the agent with the pegylated liposome. The agent may be any of the agents described in this application.

[0277] According to the present invention, in a nineteenth embodiment, a squalene-based oil-in-water emulsion containing a TLR agonist, squalene, and unsaturated phosphatidylcholine is provided. In such embodiments, emulsions that use unsaturated phosphatidylcholine as compared to a saturated phospholipid, such as DMPC, result in a higher yield of TLR agonist, i.e., a higher TLR agonist concentration in the final formulation at the same starting agonist concentration initially. TLR.

[0278] According to the present invention, in a twentieth embodiment, there is provided a squalene-based oil-in-water emulsion comprising a TLR agonist, squalene, and unsaturated phosphatidylcholine, wherein squalene is present at a concentration of about 30 to about 40 mg/mL and unsaturated phosphatidylcholine is present. at a concentration of from about 5 to about 10 mg/ml.

[0279] According to the present invention, in a twenty-first embodiment, there is provided a squalene-based oil-in-water emulsion optimized for formulation with a TLR agonist, typically a TLR agonist that is insoluble in water (negligible water solubility), containing the TLR agonist, squalene and unsaturated phosphatidylcholine (eg, phosphatidylcholine from egg), wherein squalene is present at a concentration of about 34 mg/ml and unsaturated phosphatidylcholine is present at a concentration of from about 7-8 mg/ml or about 7.6 mg/ml.

[0280] According to the present invention, the twenty-second embodiment provides any of the emulsions of the ^19th , ^20th , or ^21st embodiments, optionally further containing a coemulsifying agent, a tonicity regulator, and a buffering agent. In any of the embodiments described herein, the co-emulsifying agent may be, for example, a non-ionic linear triblock copolymer such as a poloxamer (eg, poloxamer 188). In any of the embodiments described herein, the tonicity regulator may be, for example, a polyol such as mannitol or glycerol. In any of the embodiments described in this application, the buffering agent may be, for example, a phosphate buffering agent, such as, for example, ammonium phosphate buffer. The co-emulsifying agent, when present in said emulsion, is preferably present in the emulsion at a concentration of 0.1 to about 2 mg/mL, more preferably at a concentration of about 0.5 mg/mL, most preferably at a concentration of about 0.36 mg/mL . The tonicity regulator, when present in said emulsion, is present in an amount sufficient to cause said composition to have an osmolarity of about 250-350 mosm/kg, preferably 270-315 mosm/kg.

[0281] According to the present invention, the twenty-third embodiment provides any of the emulsions of the ^19th , ^20th , ^21st , or ^22nd embodiments, wherein the TLR agonist is (a) synthetic, (b) water-insoluble, (c ) is an imidazoquinoline or imidazoquinoline-containing compound, (d) contains a hydrophobic tail, (e) is 3M-052, or any combination of the above is valid. These emulsions may further contain at least one agent; wherein said at least one agent may be a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent, or organism. Examples of agents include, for example, amoebiasis-associated antigen, LecA, influenza-associated antigen, H5N1, tuberculosis-associated antigen, ID91, ID93, antigen from BCG, hepatitis virus-related antigen, hepatitis B antigen, hepatitis C antigen, HIV-associated antigen or a cancer-associated antigen.

[0282] According to the present invention, in a twenty-fourth embodiment, there is provided a method for preparing 19 ^ohm , 20 ^ohm , 21 ^ohm , 22 ^ohm , and 23 ^ohm emulsions, comprising combining a TLR agonist with unsaturated phosphatidylcholine in an organic solvent. Any organic solvent capable of dissolving both the TLR agonist and the unsaturated phosphatidylcholine is suitable for use, including, for example, ethanol, DMF, and chloroform. Said method may further comprise the step of (a) removing chloroform by any suitable method, including evaporation, to form a lipid film, (b) adding squalene to the dried lipid film, and (c) and mixing the resulting mixture, e.g., by sonication (e.g. , in a water bath at 60°C), or microfluidization, or simple mixing.

[0283] According to the present invention, in a twenty-fifth embodiment, pharmaceutical compositions are provided comprising any of the emulsions of 19 ^ohm , 20 ^ohm , 21 ^ohm , 22 ^ohm , and 23 ^ohm embodiments; and a pharmaceutically acceptable carrier.

[0284] According to the present invention, in a twenty-sixth embodiment, there is provided a method for stimulating an immune response in a subject, comprising administering to the subject any of the emulsions of 19 ^ohm , 20 ^ohm , 21 ^ohm , 22 ^ohm , and 23 ^ohm embodiments, or the pharmaceutical compositions of embodiment ²⁵ . The immune response may be, for example, a Th1 immune response; non-specific immune response, antigen-specific immune response, systemic immune response, mucosal immune response, or intestinal, fecal or vaginal mucosal immune response. These emulsions or compositions can be used, for example, for the treatment or prevention of cancer, as a vaccine; to increase protective immunity against the influenza-causing virus; to increase protective immunity against the organism causing amoebiasis; to increase protective immunity against Entamoeba histolytica; to increase protective immunity against influenza; or to enhance protective immunity against amoebiasis. The route of administration of said composition may be oral, topical, parenteral, sublingual, buccal, rectal, vaginal, intravenous, intradermal, transdermal, intranasal, intramucosal or subcutaneous. The liposome or composition can be administered together with a retinoic acid coadjuvant. Said subject may be, for example, a human or a non-human mammal.

[0285] The following examples are offered by way of illustration, but not by way of limitation.

EXAMPLES

Example 1 Formulation and Testing of TLR Ligand-Containing Liposomes for Amoebiasis Vaccines

Materials and methods

Adjuvanted formulations, LecA antigen

[0286] All adjuvants were obtained from the Infectious Disease Research Institute (IDRI, Seattle, Washington) and offered as 2X or 5X concentrated formulations for mixing with antigen just before injection.

[0287] The LecA antigen was produced at TECHLAB (Blacksburg, Virginia) as described (Barroso L, Abhyankar M, Noor Z, Read K, Pedersen K, White R, et al. Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine Vaccine 2014;32(10): 1218-). Glucopyranosyl lipid adjuvant (GLA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-750) ( DSPE-PEG-750) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) (DSPE-PEG-2000) were obtained from Corden Pharma (Listal, Switzerland) or Avanti Polar Lipids (Alabaster, Alabama). 3M-052 was kindly provided by 3M Drug Delivery Systems (Saint Paul, MN). Cholesterol and buffer salts were purchased from JT Baker (San Francisco, CA). The GLA used in the examples corresponds to the structure of formula (VI) where R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₁₃ alkyl.

[0288] PEGylated liposome formulations were prepared by combining DPPC, cholesterol, OBRE-PEG-750 or OPPE-PEG-2000, and 3M-052 and/or GLA in chloroform. The molar ratio between lipids was 9.8:5.7:0.8 (DPPC:cholesterol:pegylated lipid). The organic solvent was then evaporated from these formulations for at least 12 hours using a rotary evaporator. The thin lipid film was rehydrated in 25 mM ammonium phosphate buffer (pH ~5.7) and sonicated in a Crest Powersonic CP230D water bath (Trenton, NJ) at ~60°C for up to 30 min. The formulation was then subjected to microfluidization at 10,000-30,000 psi. inch with 5-6 passes at 10°C setting on the recirculating water chiller. Liposomes were filtered through a 0.8/0.2 µm double membrane polyethersulfone filter and stored at 5°C, ambient temperature, 37°C or 60°C. An alternative to microfluidization is sonication.

[0289] Squalene type emulsions in water were prepared by microfluidization (M110P, Microfluidics Corp) at 30,000 psi essentially as described (Fox et al. Vaccine 2013, 31:5848). Aluminum adsorbed formulations were prepared by mixing the aqueous lipid component of said pegylated lipid based GLA suspension or CpG 1826 aqueous solution with Alhydrogel® (Brenntag Biosector) as previously described (Fox et al. J Pharm Sci 2012, 101:4357 and Fox et al. .) Physico-chemical stability measurements

[0290] The particle sizes of the emulsion and liposome were measured using dynamic light scattering after a 1:100 dilution in water. Short-term (≤24 h) physico-chemical compatibility of the antigen-adjuvant mixture was assessed by monitoring the particle size, appearance and primary structure of the antigen immediately after mixing and 4 and 24 h after mixing, while the mixtures were stored at 5°C and ambient temperature . The antigen was first diluted in saline to 0.1 mg/ml and then mixed 1:1 by volume with the liposomal adjuvant formulation. Particle size was measured as described above, except that one cuvette was prepared instead of three. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (EPS in PAAG/SDS) was performed by mixing 50 µl of sample with 50 µl of 4x sample recovery buffer and 100 µl of 20% SDS. The sample was heated at 90°C for 5 min and stored at -20°C, after which it was reheated for 1 min at 90°C and loaded onto a polyacrylamide gel with Tris-glycine running buffer for 65 m at 180 V, and then stained with Coomassie brilliant blue.

[0291] Using a high performance liquid chromatography (HPLC) method, the concentration of 3M-052 was determined by detecting UV absorption at 320 nm, and the concentration of GLA was determined by detecting charged aerosol. The HPLC method has been described previously (Misquith A, Fung M, Dowling QM, Guderian JA, Vedvick TS, Fox CB. In vitro evaluation of TLR4 agonist activity: formulation effects. Coll Surf B: Biointerfaces. 2014; 113:312-9). Adjuvant concentrations were considered within normal limits if the measured values were within +/- 20% of the target concentration. Liposomal particle size and size polydispersity were assessed using Zetasizer Nano-S or -ZS from Malvern Instruments (Worcestershire, UK). The composition was diluted 100 times in highly purified water (18.2 mΩ) in a 1.5 ml polystyrene disposable cuvette. Three separate cuvettes were prepared for each composition. All size measurements were then performed three times for each cuvette. In rare cases, dust particles led to obvious measurement deviations: in such cases, the measurement in the questionable cuvette was discarded from the series of measurements. TLR ligand concentration, particle size and appearance were regularly monitored as indicated.

Immunizations

[0292] Four to six week old male CBA/J mice were purchased from Jackson Labs. The unlabeled LecA antigen was purified at TechLab Inc. (Blacksburg, Virginia) and applied 5 μg of antigen for each immunization of each mouse. All studies in mice were carried out strictly in accordance with the regulations of the Institutional Committee for the Care and Use of Laboratory Animals (IACUC). For subcutaneous immunization in the neck, LecA was mixed with the appropriate adjuvant (Table 1) and the volume was adjusted to 100 μl with saline for injection. Intranasal immunizations were performed under anesthesia and typically used 10 µl of the antigen-adjuvant mixture per nostril. Observed a two-week interval between successful immunizations for all regimens. For experiments involving a weekly dose of all-trans retinoic acid (RA), each mouse received ip 150 μg of all-trans retinoic acid (Sigma) dissolved in a final volume of 50 μl of DMSO. The stock solution of RA was stored at -80°C protected from light. All adjuvants were stored at 4° C. and formulations were prepared aseptically just prior to immunization.

Measurement of immunogenicity

[0293] Antibody titers were measured by ELISA using 96-well plates coated with 0.5 μg of lectin per well. Plasma samples were diluted appropriately and titers of antigen-specific IgG subtypes were measured using 1:10,000 diluted horseradish peroxidase-conjugated goat anti-mouse IgG1 and IgG2a detection antibodies (Southern Biotechnology). Fecal supernatants were prepared as described (Guo X, Barroso L, Becker SM, Lyerly DM, Vedvick TS, Reed SG and others). Protection against intestinal amoebiasis by the recombinant vaccine was T-cell mediated and mediated by interferon gamma (Infect Immun. 2009, Sep; 77(9):3909-18). Briefly, freshly collected stool samples were resuspended (5 μl diluent per mg stool) in PBS containing a protease inhibitor cocktail (Roche) and mixed vigorously for 5 minutes. Insoluble material was removed by two successive centrifugation steps at 3000 rpm and 12000 rpm and the supernatant was stored at -20°C. Appropriately diluted stool supernatants were used for ELISA and horseradish peroxidase-conjugated goat anti-mouse IgA at a dilution of 1:5000 was used as secondary antibody. Antibody units were determined using standard curves.

[0294] To measure extracellular cytokines, splenocytes were restimulated with 50 μg/ml LecA for 72 h and supernatants were analyzed with a suspension chip multiplex system using Luminex beads (BioRad) (Guo X, Barroso L, Becker SM, Lyerly DM, Vedvick TS, Reed SG, et al. Protection against intestinal amebiasis by a recombinant vaccine is transferable by T cells and mediated by gamma interferon (Infect Immun. 2009, September; 77(9):3909-18).Samples were analyzed undiluted according to manufacturer's instructions and measured in picograms per milliliter of supernatant.

Culture Conditions and Provocation Experiments

[0295] Trophozoites originally obtained from HM1: IMSS (American Type Culture Collection (ATCC)), sequentially passed through the cecum of mice, were used for provocation experiments. Trophozoites were maintained in trypsin-yeast extract-iron medium (TYI-S-33) supplemented with 2% Diamond vitamins, 13% heat-inactivated bovine serum (Gemini Labs), and 100 U/mL penicillin plus 100 μg/mL streptomycin (Invitrogen) (Diamond LS, Harlow DR, Cunnick CC A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba Trans R Soc Trop Med Hyg 1978;72(4):431-2). Mice from the immunized and control groups were challenged into the caecum four weeks after the final immunization with two million trophozoites in 150 µl of post-laparotomy medium (Houpt E, Barroso L, Lockhart L, Wright R, Cramer C, Lyerly D, et al. Prevention of intestinal amebiasis by vaccination with the Entamoeba histolytica Gal/GalNaclectin Vaccine 2004 Jan 26;22(5-6):611-7). Mice were sacrificed one week after challenge. The cecums were washed with 1 ml PBS, 300 μl of the caecal wash was cultured in TYI-S-33 broth for up to five days, and 200 μl was used for antigen load ELISA analysis. Vaccine efficacy was calculated as 100×(1-(% vaccinated mice with infection)/(% sham-immunized mice with infection) (Guo X, Barroso L, Becker SM, Lyerly DM, Vedvick TS, Reed SG, et al. Protection against intestinal amebiasis by a recombinant vaccine is transferable by T cells and mediated by gamma interferon Infect Immun 2009 Sep 77(9):3909-18 Soong CJ, Kain KS, Abd-Alla M, Jackson TF, Ravdin JI J Infect Dis. 1995, March; 171(3):645-51).

Fecal antigen detection

[0296] Fecal antigen in caecal contents was detected using the E. his II ELISA kit (TechLab Inc., Blacksburg, Virginia). An optical density at 450 nm ≥0.05 higher than the negative control was considered a positive result. A standard curve was generated using purified LecA.

Sticking analysis

[0297] Chinese hamster ovary (CHO) cells were grown in α-MEM medium and E. histolytica trophozoites were grown as described above. E. histolytica trophozoites were pre-incubated with a tenfold dilution of fecal supernatants from the control or immunized groups on ice for 1 h. Trophozoites and CHO cells were then mixed at a ratio of 1:20 and incubated for 90 min on ice in round bottom polystyrene tubes. Immediately before counting with a microscope, the tubes were quickly shaken on a vortex and the cells were counted on a hemocytometer. Adherence was measured as the number of trophozoites to which at least 3 CHO cells adhered and recorded as % rosette formation. Each sample was analyzed in triplicate and a minimum of 100 amoebae were counted (Barroso L, Abhyankar M, Noor Z, Read K, Pedersen K, White R, et al. Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine Ravdin JI, Guerrant RL Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica Study with mammalian tissue culture cells and human erythrocytes J Clin Invest 1981 November; 68(5): 1305-13).

Statistical analysis

[0298] All analyzes were performed using Graph Pad Prism software. The ratios of infected and uninfected mice from the challenge studies were analyzed using Fisher's exact test. Differences in antigenic loads, antibody titers and adhesion inhibition were analyzed using the Mann-Whitney test.

results

Physical and chemical stability.

[0299] Liposomal stability was monitored by dynamic light scattering (particle size and size polydispersity), appearance, and HPLC (GLA concentration and 3M-052). Only little or no change in particle size and size polydispersity values was detected after 12 months of storage at 5°C, although the polydispersity values for liposomes containing DSPE-PEG-2000 were significantly higher than the polydispersity values for liposomes containing DSPE-PEG -750 (Fig. 1A). The appearance of the liposomes was equally translucent and homogeneous and did not change over time. During storage at 37° C., DSPE-PEG-2000 containing liposomes exhibited greater changes in particle size and size polydispersity over time compared to DSPE-PEG-750 containing liposomes (FIGS. 1C-1D). The concentrations of GLA and 3M-052 did not change over 12 months in samples stored at 5°C. At 37° C., the rate of GLA loss was higher than that of 3M-052, with GLA loss >40% after 6 months of storage, with no detectable loss of 3M-052.

[0300] In order to assess the short-term (≤24 h) compatibility of the adjuvant and the LecA antigen after mixing, the antigen stock solution was diluted in saline and then mixed 1:1 v/v with the adjuvant to mimic the planned mixing procedure for immunization studies in vivo, described below. The formulations exhibited a translucent, homogeneous appearance before and after mixing with the antigen. In general, little or no change in particle size was observed (<15%) within 24 h after mixing with the antigen when stored at 5°C or at ambient temperature, although liposomes containing DSPE-PEG-2000 showed a slightly greater tendency to increase in size compared to liposomes containing shorter PEG (DSPE-PEG-750). Overall, the presence of GLA, 3M-052, or both agonists together did not appear to affect particle size compatibility results. Similarly, size polydispersity values did not change significantly, although polydispersity was higher for liposomes containing DSPE-PEG-2000 compared to liposomes containing DSPE-PEG-750. SDS-PAGE analysis of antigen-adjuvant mixtures by ESP showed no change in the primary structure of the antigen at any time point or under any storage condition for 24 hours, regardless of the composition of the liposomes, showing a uniform single band near the expected molecular weight. Taken together, these results indicate that the antigen-adjuvant mixture exhibits acceptable short-term compatibility of at least up to 24 hours at 5° C. or ambient temperature.

Evaluation of immunogenicity and selection of liposome composition

[0301] Using recombinant E. histolytica LecA adhesion immunogen, adjuvants were screened for that could elicit a balanced immune response. Received adjuvants containing synthetic TLR agonists, as shown in table 2, and evaluated their ability to induce an antigen-specific humoral response.

[0302] Each of these adjuvants consists of pharmaceutically acceptable components and is suitable for clinical research. The emulsion and liposome formulations showed an average particle size of 65-130 nm depending on the specific formulation and processing method, while the aluminum containing formulations contained microparticles. Received nine formulations by mixing adjuvants with purified unlabeled protein LecA and immunized mice subcutaneously. Five mice per group were immunized three times subcutaneously at 2-week intervals with LecA antigen mixed with the appropriate adjuvant. Plasma samples collected one week after the final immunization were diluted 256,000-fold and analyzed for IgG1 and IgG2a production by ELISA. The *IgG2a level elicited by the LecA formulation with GLA 3M-052-liposome adjuvant was statistically significant compared to all other groups except for EM014 (GLA-CpG-ES) and 3M-052-emulsion. Plasma IgG subclass titers were determined by ELISA (FIG. 2). In FIG. Figure 2 shows that a liposome formulation containing a mixture of GLA (TLR-4 agonist) and 3M-052 (TLR-7/8 agonist) (designated GLA-3M-052-LS) was found to have a balanced IgG response and selected for further study. .

[0303] Next, the ability of GLA-3M-052-LS to induce the production of an antigen-specific cytokine indicative of a cell-mediated immune response was examined. Splenocytes were restimulated with LecA in vitro and culture supernatants were analyzed. LecA adjuvanted with GLA-3M-052-liposome elicited strong IFN-γ and IL-17 responses, which are markers of protection in a mouse model (FIGS. 3A-3D). In particular, mice were sacrificed one week after the third immunization and LecA splenocytes were restimulated for 72 h. ml. *=p<0.05; **=p<0.001.

[0304] In addition, in peripheral blood mononuclear cells (PBMCs) from the LecA group alone, adjuvanted with GLA-3M-052-liposome, moderate but statistically significant intracellular IFN-γ staining was detected.

[0305] The possibility of co-using all-trans retinoic acid (RA) as a coadjuvant was also investigated. Mice in the respective groups received a weekly injection of RA. The PK-assisted regimen further increased IFN-γ and IL-17 levels (also see FIGS. 3A-3D). In both (adjuvant + LecA) and (adjuvant + LecA + RK) groups, an equivalent IL-2 response was formed; while the PK-assisted regimen resulted in a slightly stronger IL-4 response.

[0306] Since the liposome-based vaccine is also suitable for mucosal immunization, a mixed mucosal/parenteral immunization schedule was used for subsequent experiments to test its ability to induce an antigen-specific IgA response in the gut.

Mucosal IgA response and its ability to inhibit adhesion

[0307] Mice were primed by intranasal immunization followed by subcutaneous and intranasal booster immunizations. Mice in the RA-assisted groups received a weekly injection of RA. In FIG. 4a shows that adjuvanted LecA elicited a strong mucosal IgA response that was antigen specific. The addition of RA helped increase the IgA titer. Adherence of E. histolytica trophozoites to target cells is the primary and key step in the onset of infection.

[0308] In order to assess whether IgA in the mucosa is protective in nature, its ability to block parasite adherence to CHO cells was examined in vitro. Pre-incubation of trophozoites with fecal supernatants from immunized mice significantly reduced their ability to adhere (FIG. 4b). Stool suspensions in the (adjuvant + LecA) or (adjuvant + LecA + RK) dosing schedules showed comparable ability to inhibit adhesion. Thus, adjuvanted LecA elicited a high titer gut IgA response in vitro that was protective. The composition of nanoliposomes containing synergistic TLR agonists has the ability to protect against intestinal parasitic provocation.

[0309] The ability of the GLA-3M-052-liposome adjuvant to protect against E. histolytica challenge was investigated using a murine intestinal amebiasis model. Mice from the control and experimental immunized groups were challenged intracaecally with a virulent strain of E. histolytica and the cecum was harvested one week after challenge to assess antigenic load (Fig. 5a) as well as the presence of live parasites (Fig. 5b). Adjuvanted LecA significantly reduced antigenic load compared to control mice, and this reduction was even more pronounced when PK was used as a coadjuvant. The indicated adjuvant with this regimen showed a moderate efficacy of 34%. However, the addition of RA to the regimen essentially increased the efficacy to 69.2% (Table 3). The formula used to calculate the effectiveness of the vaccine was as follows:

[0310] Efficacy = 100×(1-(% vaccinated mice with infection)/(% sham-immunized mice with information).

[0311] Efficacy for adjuvant + LecA=100×(1-46/69.2)=100×(1-0.66)=34%

[0312] The results confirm that the nanoliposome formulation containing a mixture of synthetic synergistic TLR agonists has the ability to elicit an antigen-specific protective response and is compatible with micronutrient-assisted immunization.

Effect of PEG Length

In FIG. 6-7 show that stronger responses were observed for PEG-2000 formulations compared to PEG-750 formulations. In FIG. 6 shows balanced IgG2a and IgG1 titers for the GLA-3M-052 PEG-2000+LecA group; higher titers were also observed for this group compared to the GLA-3M-052 PEG-750+LecA group. In FIG. 7 examined the effect of PEG length on the IgA response in fecal mucosa. In FIG. 7 shows that the IgA response in feces for the GLA-3M-052 PEG-2000+LecA group was greater than for the GLA-3M-052 PEG-750+LecA group. Thus, increasing the length of the PEG in the liposome increases the IgA response in the mucosa. Effect of shipping way

[0313] Investigated whether the route of delivery during immunization on immune responses. In FIG. 8A-B depict the effect of the delivery route on immunization, with the highest titers of IgG2a and IgA obtained with the I/N (nasal) only regimen. The liposome + adjuvant elicited a strong mucosal and systemic immune response of gut Th1 IgA against LecA (16B).

[0314] FIG. 9A-9B additionally show delivery routes. The intranasal-only regimen produced equivalent or greater amounts of IFN-γ and IL-17 compared to other regimens. The regimen with the introduction of LecA + liposome + adjuvant only in the mucosa caused a strong mucosal and systemic immune response Th1 IFN-γ (9A - B, table 4). The intranasal delivery was not expected to elicit an IgA response (systemic response). This showed that mucosal delivery of GLA and 3M-052 within the LecA liposome elicited a systemic immune response. The results obtained demonstrate that non-coagulant administration of said composition elicits a strong systemic immune response, as evidenced by IgG2a/IgG1 ratios. It was not expected that the administration of said composition intranasally to the mucosal surface would cause not only a local mucosal response in the nasal passages, but also a long-term intestinal mucosal (fecal) response.

The discussion of the results

[0315] An important result of this study was the identification of a nanoliposome adjuvant containing synthetic TLR agonists that were capable of inducing a systemic as well as mucosal immune response. A liposome-based adjuvant system containing TLR4 (GLA) and TLR7/8 (3M-052) agonists elicited a balanced humoral and strong cytokine response. GLA is a synthetic TLR4 ligand that is included in lipid-based platforms used in various Phase 1 and 2 clinical trials. Modern Vaccines, ^2nd ed, in press; Smirnov et al. Vaccine 2011, 29:5434; Zhao et al. J Immunother Cancer 2014, 2:12; Singh et al. J Immunol 2014, 193:4722). The liposomal formulation with GLA and 3M-052 was suitable for a mixed mucosal/parenteral immunization regimen and also elicited a strong mucosal IgA response. Immunized mice challenged with E. histolytica showed a significant reduction in antigenic load. The composition of nanoliposomes with GLA 3M-052 was compatible with the use of all-trans retinoic acid as a coadjuvant, and this regimen further increased the effectiveness of protection and IgA levels in the mucosa.

Example 2 Preparation of 3M-052 Adjuvant Formulations and Their Testing for Influenza Vaccines

Materials and methods

Obtaining compositions of materials and production

[0316] 3M-052 was synthesized by the 3M Company. R848 was provided by 3M or purchased from Axxora Life Sciences Inc. (San Diego, California). Synthetic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC ), 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-dipalmitoyl-5p -glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-750) (DPPE-PEG-750) and egg phosphatidylcholine (PC) were purchased from Avanti polar lipids Inc (Alabaster, AL) or Lipoid LLC (Newark, NY). Jersey). Squalene was obtained from Sigma (St. Louis, MO). Cholesterol, ammonium phosphate monobasic and ammonium phosphate dibasic were purchased from J.T. Baker (San Francisco, California). Poloxamer 188 and glycerin were purchased from Spectrum Chemical (Gardina, CA). Phosphate buffered saline (1×PBS) at pH 7.2 was purchased from Invitrogen (Grand Island, NY).

[0317] Small batches (≤20 ml) of pegylated liposome formulations were prepared by combining DPPC, cholesterol and DPPE-PEG-750 with varying amounts of 3M-052 in an organic solvent (chloroform or chloroform/methanol/water). The organic solvent was then evaporated using Genevac EZ-2. The lipid film was rehydrated in PBS (pH 7.2) or 25 mM ammonium phosphate buffer (pH 5.7) and sonicated in a Crest powersonic CP230D sonication water bath (Trenton, NJ) at ~60°C for ~2-3 hours or until the formulations become translucent with no large visible particles. The same procedure was followed for neutral liposomes (DOPC, cholesterol), anionic liposomes (DPPC, cholesterol, DPPG) and cationic liposomes (DPPC, cholesterol, DPTAP). The mass ratios of the components of the liposomes are described as follows: pegylated (18:5.5:3, DPPC:chol:DPPE-PEG-750), neutral (20:5, DOPC:chol), anionic (18:5:2, DPPC:chol :DPPG), cationic (18:5:2, DPPC:hol:DPTAP). Larger batches (100 ml) of PEGylated liposomes were produced as described above, but with shorter sonication followed by high shear homogenization (Microfluidizer M110P) with 12 continuous passes at 30,000 psi.

[0318] Liposomes containing R848 were prepared by manufacturing a concentrated composition of pegylated liposomes as described above, except that the liposomes were first hydrated with a 75 mM ammonium sulfate solution. After sonication at 60°C ~ 1 hour, a PD-10 column (GE Healthcare) was used to replace the external liposome buffer with 0.9% saline. Liposomes, now with saline in external buffer and ammonium sulfate inside, were mixed with saline containing R848 and incubated for 1 h at 60°C. Finally, the liposomes were passed through another PD-10 column to remove non-liposome R848.

[0319] Stable oil-in-water (SE) emulsions were prepared by dissolving 3M-052 in chloroform with DMPC or PC from egg. The chloroform was then removed using a rotary evaporator. Squalene was then added to the dried lipid film and the glass container was placed in a sonication water bath at 60° C. for ~1 hour. This mixture was called the oil phase. Alternatively, an oil phase was prepared by dispersing 3M-052 directly in squalene and DMPC (no PC from egg or chloroform). The aqueous phase was then added to the oil phase to give a final concentration of 25 mM ammonium phosphate buffer, 0.037% (w/w) poloxamer 188, and 1.8% (v/v) glycerol (isotonic agent), 4% v/v squalene, 7 .6 mg/ml DMPC or PC from egg, and various amounts of 3M-052. Some emulsions also contained 0.02% v/v α-tocopherol. The crude emulsion was obtained by sonicating the specified mixture in a water bath at 60°C for an additional 10-15 minutes. The final emulsion was obtained by passing the crude emulsion through a high shear homogenizer (Microfluidizer M110P) 12 times without interruption at 30,000 psi.

[0320] R848 emulsions were prepared by producing the concentrated emulsion described above and mixing it with R848 dry powder or with a solution of R848 in ammonium phosphate buffer. Emulsions and liposomes were filtered through a 0.2 μm membrane prior to the in vivo experiments described below.

Determination of the characteristics of the composition

[0321] The concentrations of 3M-052 and R848 were evaluated after diluting each formulation 20-fold in a 98% ethanol/2% HCl solution in a cuvette. The absorption of light by the samples at ~322 nm was analyzed on a Hitachi U-3900H spectrophotometer (Tokyo, Japan). Particle size was assessed using Zetasizer Nano-S, -ZS or -APS from Malvern Instruments (Worcestershire, UK). The formulations were diluted 100-fold in highly purified water in a 1.5 ml disposable polystyrene cuvette. Three separate cuvettes were prepared for each composition. All size measurements were then performed three times for each cuvette. Zeta potentials were measured using Malvern Zetasizer Nano-ZS. 50 μl of each composition was combined with 950 μl of ultra pure water in a disposable capillary cuvette (Malvern Instruments, DTS1070). For each sample obtained, nine measurements were made (one sample per composition).

Virus and vaccine stock solutions

[0322] All vaccine antigens used in this study were derived from influenza A/Vietnam/1203/04 (VN1203). Recombinant HA protein (rGA) used in immunogenicity and challenge studies in mice was obtained from Protein Sciences Corp. For ferret immunization and challenge studies, a clinical grade VN1203 split vaccine was obtained from Sanofi Pasteur.

[0323] Virus stock solutions for mice and ferret provocation studies were prepared by inoculating 10 day old fertilized hen eggs with H5N1 stock solutions as described. The clarified allantoin liquid was filtered and stored at -80° C. until use. Virus titers were determined by the MDCK Plaque Formation Assay (ATCC CCL-34) using standard assays as described.

Ferret Whole Blood Stimulation Assay

[0324] Whole blood was collected from male Fitch ferrets and incubated with TLR agonist compounds including imiquimod (TLR7), TLR7/8 agonist CL057 (Invivogen) and the synthetic TLR4 agonist GLA. After incubation, RNA was collected from both stimulated samples and saline controls using a whole blood RNA isolation kit (Qiagen). TLR7, TLR8, IL-1β and IL-8 RNA levels were determined by real-time PCR. Changes in expression levels were plotted as fold change in RNA levels compared to blood samples stimulated with saline.

Mice provocation studies

[0325] For mice provocation studies, female C57B1/6 mice aged 6-8 weeks were immunized with recombinant H5N1 HA protein (strain VN1203, Protein Sciences Corp.), in combination with an adjuvant as indicated. All immunizations were administered intramuscularly in a total volume of 100 μl divided equally between both legs. Twenty-one days after immunization, mice were challenged with 1×10 ⁶ PFU of A/VN/1203/05 (H5N1 clade 1). Starting from the first day after the challenge, the weight loss of all animals was monitored and signs of severe infection were observed. Animals found to have lost >25% of body weight on day 0 were euthanized.

Research on ferret provocation

[0326] Male Fitch ferrets (Mustela putoris fero, Triple F Farms, Sayre, PA) were used for all immunization and challenge studies. Ferrets were immunized by injecting 250 µl into the quadriceps muscle. All animals received 0.5 μg of A/Vietnam/1203/04 H5N1 split vaccine (Sanofi Pasteur) obtained from government pandemic stocks. Serum samples were collected 21 days after immunization to determine antibody titers.

[0327] Provocation was started by instillation of 1×10 ⁵ - 5×10 ⁵ PFU of virus into the lung in a volume of 50 μl (25 μl/nostril). After infection, the animals were observed and weighed daily to track virus-induced morbidity. Any animal that had lost 25% of its body weight prior to challenge was euthanized.

Plaque formation analysis

[0328] Virus titers were obtained by plaque formation assay on Madin-Darby canine kidney (MDCK) cells. Briefly, cells were seeded 24 hours prior to the assay in 6-well plates at a concentration of 1 x 10 ⁶ cells/well in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. At the start of the assay, confluent MDCK monolayers were washed three times with DMEM to remove FBS. Serial dilutions of nasal wash samples were prepared, added to monolayers in a volume of 100 μl and incubated at 37°C for 60 minutes. After incubation, 2 ml of 0.8% agarose (SeaKem) in 1×L-15 medium (Lonza) was applied to the monolayers. After 48-72 hours of incubation, plaques were quantified after staining with crystal violet (BD).

Haemagglutination Inhibition Assay (AIH)

[0329] Hemagglutinin inhibition assays were performed following the WHO protocol (52). Formalin-inactivated H5N1 antigens were obtained from the National Institute for Biological Standards and Control (NIBSC). All cells were analyzed using washed 1% red blood cells (erythrocytes, RBC) horse (Lampire).

Microneutralization analysis

[0330] Neutralizing antibody titers were determined using pseudotyped lentiviral particles. Briefly, pseudotyped particles were generated by simultaneous transfection of 293T cells with plasmids expressing H5N1 and GA and 3 lentivirus packaging plasmids: pDR8.74 (55), pRSV-Rev and HR'-CMV-Luc, which is packaged into viral particles and encodes the luciferase transgene. under the control of the CMV immediate-early promoter. Virus stocks were titrated in black 96-well flat-bottom plates (Corning) that had been seeded 24 hours before with 5×10 ³ MDCK cells/well in Dulbecco's Modified Eagle's Medium (DMEM) + 10% fetal bovine serum ( FBS). Before virus titration, the cells were washed three times with 200 μl of DMEM to remove FBS, and prepared serial dilutions of virus stock solutions were applied to them. After incubation for 72 hours to allow expression of the transgene, luciferase levels in the transduced cells were determined using the BrightGlo luciferase assay system (Promega) following the manufacturer's instructions.

[0331] For the neutralization assay, prepared serial dilutions of serum after immunization were mixed at a ratio of 1:1 with diluted virus stock solutions containing 1×10 ⁴ relative units of luciferase (RLU). Virus and serum mixtures were incubated at 37° C. for 1 hour and added to plates containing washed MDCK cells as described above. Neutralizing titers were determined after assessing the level of luciferase expression in all transduced cells. The _IC90 antibody titer was defined as the highest serum dilution for which a 10-fold decrease in luciferase levels was observed compared to vector-transduced control cells.

Chips GA

[0332] HA chips containing influenza virus HA proteins have been described previously (56). In this study, second generation chips were used that contained 278 GA proteins from various influenza strains (Sinobiological). For post-vaccination immunoassay, chips were first blocked with PBS + 1% fetal bovine serum + 0.1% Tween-20, washed three times with Protein Array Wash Buffer (ArrayIt), and incubated with 300 µl of a 1:100 dilution of post-immunization mouse sera for 1 hour with shaking. After the primary incubation, the chips were washed 5 times with wash buffer and incubated with fluorophore-conjugated secondary antibodies to IgG2c (Jackson Immonoresearch, P/N: 115-495-208) and IgG1 (Life Technologies, P/N: A21123) at a dilution of 1:2000. After incubation for 30 minutes at room temperature, the chips were washed with Rinse Buffer (Arrayit) and analyzed using a 400 V chip scanner from Molecular Dynamics. After scanning, analysis was performed using Tableau analysis software.

ELISA

[0333] Final titers of GA-specific IgG, IgG1 and IgG2c were determined seven days and twenty-one days after immunization. High binding polystyrene 384-well plates were coated with recombinant HA VN1203 (Protein Sciences Corp.) (2 μg/ml) in 0.1 M bicarbonate coating buffer for 2.5 hours at room temperature. The plates were washed three times with 0.1% PBS-Tween 20 before and after blocking incubation for two hours with 0.05% PBS-Tween 20 + 1% BSA at room temperature. Prepare serial dilutions of mouse sera in 0.05% PBS-Tween 20 + 0.1% BSA using Nanonscreen NSX-1536, incubated overnight at 4°C and washed five times. The plates were incubated for 1 hour on a shaker with HRP-conjugated anti-mouse IgGT, IgG1 or IgG2c antibodies (Southern Biotechnologies). After five washes, the plates were developed on a Nanoscreen robotic device using tetramethylbenzidine SureBlue substrate (Kirkegaard & Perry Laboratories). The enzymatic reaction was stopped with 1 NH ₂ SO ₄ using a Multipette Sagian robotic device. Plates were read at 450-570 nm using a Synergy ELISA plate reader (Biotek) and Gen5 software.

Staining for intracellular cytokines

[0334] In order to quantify specific T cell responses to the vaccine, splenocytes were isolated from five mice per group after vaccination. Erythrocytes were lysed using Red Blood Cell Lysis Buffer (eBioscience) and resuspended in cRPMI 1640 (10% FBS, 1% penicillin/streptomycin; 0.1% 2-mercaptoethanol). Cells were seeded at a concentration of 10 ⁷ cells/well in 96-well plates and stimulated for 2 hours with medium or rHA antigen (10 μg/ml) at 37°C. Added GolgiPlug (BD Biosciences) 1:50 and incubated cells for an additional 8 hours at 37°C. Cells were washed and their surface stained with fluorochrome-labeled antibodies at a 1:100 dilution in 1% BSA-PBS against CD4 (clone RM4-5), CD8 (clone 53-6.7), CD44 (clone IM7) and B220 (RA3-6B2) ( BioLegend and eBioscience) in the presence of anti-CD 16/32 vehicle (clone 93) for 15 minutes in the dark at room temperature. Cells were fixed and permeabilized (permeated) with Cytofix/Cytoperm (BD Biosciences) for 30 minutes at room temperature in the dark. Cells were washed with Perm/Wash (BD Biosciences) and stained with fluorochrome labeled antibodies to detect intracellular cytokines as follows: IFN-γ (clone XMG-1.2), IL-2 (JES6-5H4), TNF (MP6-XT22), IL-5 (TRFK5 clone) and IL-10 (JES5-16E3 clone) (BioLegend and eBioscience). Staining was carried out for 15 minutes at room temperature in the dark. Cells were washed, resuspended in 1% BSA-PBS, and filtered using a 96-well PP/PE filter plate with 30-40 µm filter pore diameter (Pall Corp). Accumulated up to 10 ⁶ events on a flow cytometer with four lasers LSR Fortessa (BD Biosciences). The results were analyzed using Flow Jo software (Treestar).

results

[0335] DPPC-based liposomes modified with anionic, cationic, or pegylated phospholipids and neutral DOPC-based liposomes were prepared using a thin film technique followed by sonication. Liposomes were manufactured at a high concentration of 1 mg/ml 3M-052 to detect any incompatibility with various liposome formulations. After receiving two separate batches for each liposome, the PEGylated liposome and DOPC liposome showed a translucent appearance and the smallest particle size and polydispersity after sonication (Table 5).

[0336] The PEGylated liposome was chosen for further stability and in vivo evaluation. These PEGylated liposomes were negatively charged (Table 6), most likely due to the anionic phosphate group of DPPE-PEG-750.

[0337] For these liposomes showed little or no change in particle size for at least 6 months at 5°C (Fig. 10A-10B). In general, no obvious loss of 3M-052 was observed during production.

[0338] 3M-052 squalene (ES) oil-in-water emulsion formulations were produced by microfluidization, producing <100 nm droplets with low polydispersity and long-term particle size stability (FIGS. 10A-10B). The values of the zeta potentials of the emulsions were slightly negative (table 6). Surprisingly, emulsions made with egg PC instead of DPMC resulted in higher yields of 3M-052 after treatment. Moreover, the addition of an initial mixing step combining 3M-052 and egg-PC in chloroform further increased the yield of 3M-052 compared to sonicating the dry powder in squalene/PC from egg without chloroform. Thus, changes in the emulsion composition and processing procedure had a significant impact on the inclusion of 3M-052 in the final composition. See table 7 below.

3M-052 formulation combined with H5N1 GA protected mice from lethal H5N1 challenge after a single immunization

[0339] After demonstrating the stability of 3M-052 adjuvant formulations (FIGS. 10A and B), the ability of a given TLR agonist to enhance GA-specific vaccine responses was examined. 3M-052 in either pegylated liposomes or an oil-in-water (ES) squalene emulsion was mixed with recombinant H5N1 influenza virus HA protein (A/Vietnam/1203/strain 04 (VN1203), Protein Sciences Corp., Meriden, Conn.) and was used to immunize groups of C57Bl/6 mice (N=20/group) intramuscularly. Twenty-one days after a single immunization, all mice were challenged intranasally with 1000 LD50 VN1203. Ten animals were followed for 14 days to determine virus-induced morbidity as measured by weight loss, as well as mortality. In a liposomal formulation, adjuvant 3M-052 prevented viral-induced morbidity: 100% of animals given rG5 + 3M-052 liposomes survived compared to 50% of animals given rG5 or rG5 in combination with liposomes alone (FIG. 11A). Mice immunized with rG5 + 3M-052 liposomes showed no weight loss, while animals receiving rG5 with or without liposomes lost weight within 11 days of challenge (FIG. 11B). Animals treated with emulsion formulations showed consistent survival with and without 3M-052 (FIG. 11A), consistent with the known efficacy of emulsions as adjuvants for influenza vaccines. However, animals treated with emulsions in combination with TLR 7/8 agonists showed less body weight loss during the acute infection period compared to animals treated with rG5 + ES (FIG. 11C).

[0340] In addition to the animals monitored for survival, 5 animals/group were sacrificed on days 4 and 7 post-challenge to measure/observe influenza-induced lung pathology and determine lung virus titers. At 4 days post-challenge, animals immunized with rG5 + adjuvant 3M-052 formulations exhibited significantly reduced virus titers in the lung compared to non-immunized controls. Animals immunized with the rG5 + 3M-052-liposome formulation could not detect any virus titer at this time point (FIG. 11D). On day 7, animals treated with 3M-052 adjuvanted formulations showed minimal virus titers compared to rG5 alone. For both formulations based on liposomes and ES, the addition of 3M-052 resulted in a significant reduction in titer compared to the same formulation without TLR (FIG. 11E). In addition to reduced viral titers, animals immunized with rG5 + adjuvant 3M-052 formulations showed significantly lower lung weights (FIG. 11F) and less lung pathology (FIG. 11G). Taken together, these results demonstrate the ability of 3M-052 to protect mice even from the clinical complications of lethal avian influenza challenge. In FIG. 11H shows the virus titer in the lung.

3M-052 Adjuvanted Formulations Induce Th1 CD4 T Cell Response in Mice

[0341] In order to investigate the correlates of protection for formulations containing 3M-052 with adjuvants, CD4 T cell responses elicited after immunization with rG5 in combination with adjuvants were examined. After a single immunization, cytokine production by CD4+ T cells was examined. Low but detectable levels of Th1 cytokine (IFNγ/TNFα/IL-2) positive cells could be demonstrated in animals immunized with 3M-052 adjuvanted formulations (FIGS. 12A-C). Canonical Th1 CD+ T cells could be observed for all formulations with adjuvants that proved to be protective in animals. Consistent with previous work, these results demonstrate the ability of 3M-052 to induce a Th1-biased cellular response in mice following vaccination with low levels of antigen.

Formulations with adjuvant 3M-052 induce an extended cross-subtypic humoral response to H5N1 GA

[0342] Induction of Th1 CD4 T cells (IFNγ ⁺ TNFα ⁺ IL-2 ⁺ ) was observed after immunization with 3M-052 adjuvant formulations (FIG. 12). Using a second-generation chip with a high density of HA proteins, the ability of 3M-052 to enhance the humoral response induced by the clade 1 rG5 antigen was examined. -16. The ability of both IgG1 and IgG2c antibodies to bind to GA proteins from different subtypes was investigated. The addition of adjuvants increased the level of antibody induced after the boost. For adjuvants in ES and liposome, increased binding to H5 subtype proteins and predominant IgG1 responses were found. The addition of 3M-052 to any of the formulations resulted in a significant broadening of the humoral response, with binding to a wide range of GA subtypes, as well as the induction of cross-reactive IgG2c antibodies (results not shown).

[0343] When examining H5 strains on the chip, a clade cross-linking response was clearly demonstrated after immunization with formulations with 3M-052. Animals immunized with both 3M-052-liposomes and 3M-052-ES showed elevated levels of IgG2c antibody that binds to proteins from multiple clades of the H5N1 virus. In contrast, IgG1 levels were independent of the presence of 3M-052. In order to confirm the results obtained on the HA chips, the final titer of antibodies in the serum of mice after immunization was determined by ELISA using HA VN1203 as the coating antigen. The results observed in the serum ELISA assay mirrored those observed on the HA chip (results not shown).

TLR7/8 agonists increase receptor and cytokine mRNA levels in ferret whole blood

[0344] In order to confirm that imidazoquinolines are able to stimulate congenital TLR receptors in ferrets, a whole blood stimulation assay was performed. Whole blood from male Fitch ferrets was collected and stimulated with either imidazoquinolines or the synthetic TLR4 agonist glucopyranosyl lipid A (GLA). The ability of agonist molecules to stimulate mRNA of both cytokines and the TLR receptor was examined using real-time PCR. Whole blood stimulation with both 100 µM R848 (a known TLR7/8 agonist in other species, 3M) and 100 µM CL075 (a known TLR8 agonist in other species, Invivogen) resulted in a significant increase in IL-1B, IL-8, TLR7 and TLR8 mRNA levels. . This result demonstrates the ability of imidazoquinolines to stimulate ferret receptors and suggests that both TLR7 and TLR8 are active in ferrets, which may play a role in this influenza model.

3M-052 adjuvanted formulations increase (in combination with H5N1 antigens) protection against H5N1 challenge in ferrets after a single immunization

[0345] Given the promising results in mice and the evidence that imidazoquinolines can stimulate TLR in ferrets, the ability of adjuvants containing 3M-052 to protect ferrets from lethal H5N1 challenge after a single immunization was investigated. For these studies, adjuvants neutral liposomes and ES with and without 3M-052 were combined with a degraded H5N1 virus vaccine (Sanofi) derived from VN1203 (SP-H5). This antigen was chosen because it is now included in the United States government pandemic stockpile and has been clinically tested in previous trials. Briefly, after a dose matching study with H5N1 antigen alone (results not shown), groups of 4-6 ferrets were immunized intramuscularly with 0.5 μg of SP-H5 mixed with adjuvants as indicated (FIG. 13). Twenty-one days after immunization, ferrets were challenged intranasally with 10 ⁶ PFU A/VN/1203/04 and monitored for 14 days for weight loss, clinical performance, and survival. In addition, nasal swabs were collected every other day, starting on day one after provocation. In one study, 3M-052/ES completely protected animals from challenge compared to animals immunized with SP-H5 + ES (FIG. 13A). 3M-052/ES also reduced virus-induced morbidity compared with ES alone: animals immunized with SP-H5 + 3M-052/ES did not lose body weight after challenge (Fig. 13B) and had only minimal clinical scores at throughout the infection (Fig. 13C). Similar results were observed in the second experiment after immunization of animals with liposomal adjuvant formulations. 3M-052 liposomes protected 100% of the animals from challenge (Fig. 13E). Similar to the ES-based formulations, animals immunized with SP-H5 + 3M-052-liposomes did not lose body weight (FIG. 13F) and had negligible clinical scores (FIG. 13G). Perhaps most importantly, a study of virus titers in the lungs showed that 3M-052, both in ES and liposomal formulations, was able to reduce viral shedding in nasal washes: in animals immunized with 3M-052/ES (Fig. 13D) or 3M -052/liposomes (FIG. 13H) showed a significant reduction in virus titers as early as 3 days after challenge compared to other adjuvants or control animals. In both cases, the virus was eliminated to undetectable levels by day 5 in all animals treated with 3M-052 adjuvants.

3M-052 adjuvanted formulations elicit a neutralizing humoral clade cross-linking response in ferrets.

[0346] Based on the extensive HA-specific humoral response observed in mice and the observed strong protection against homologous virus in ferrets, neutralizing antibody titers induced after immunization with various adjuvant formulations were directly examined using a pseudotyped H5N1 HA lentiviral vector containing the transgene luciferase. Briefly, prepared serial dilutions of serum samples were incubated with lentiviral particles containing GA and NA H5N1 proteins on the surface. Serum-virus mixtures were incubated on confluent monolayers of MDCK cells. After incubation to allow expression of the virus-packaged luciferase transgene, the neutralizing capacity of the serum samples was determined by quantitative analysis of luciferase gene expression. Consistent with the increased survival observed in these studies, adjuvants 3M-052/ES and 3M-052/liposomes increased neutralizing serum IC90 titers 21 days after a single injection (Fig. 14A,D). In addition, both formulations significantly increased neutralizing titers against clade 2 viruses: elevated IC ₉₀ titers were observed against A/Indonesia/5/05 (clade 2.1) and A/whooper swan/Mongolia/244/05 (clade 2.2) (Fig. 14B-F). This finding is consistent with the ability of formulations with 3M-052 to induce broad spectrum neutralizing antibody titers against drifting strains of influenza.

3M-052-ES elicits a protective crossover response to the clade

[0347] Given the neutralizing titers that were observed against A/Whooper Cygnus/Mongolia/244/05 following immunization with VN1203 antigens, the ability of 3M-052 formulations to reduce virus replication in the lung following vaccination with VN1203 antigen was investigated. As in previous studies, ferrets were immunized once with SP-H5 in combination with 3M-052 formulations and challenged 21 days later with 5×10 ⁵ PFU of virus. Animals that received 3M-052/liposomes showed a modest decrease in virus shedding over time; animals that received 3M-052/liposomes did not detect virus 5 days after challenge, while other animals that received liposomes or antigen alone showed virus at this point in time (FIG. 15B). In contrast, animals that received 3M-052-ES cleared the virus rapidly with no plaque detected 3 days post challenge, while animals that received SP-H5 + ES showed detectable virus titers up to day 5 (Fig. .15A).

The discussion of the results

[0348] The results presented here demonstrate protection in mice and ferrets after challenge with a high dose (1000 LD ₅₀ ) of H5N1 viruses when animals were immunized with TLR7/8 agonist formulations 3M-052. 3M-052 was included in any of these formulations, and these formulations were stable for extended periods. The novel formulation of pegylated liposomes with 3M-052 was found to increase survival and reduce virus titer in the lung when combined with a low dose (100 ng) of the recombinant HA-based antigen. In addition, a formulation that contained 3M-052 and DMPC or PC from egg in an oil-in-water emulsion of squalene was investigated and showed increased survival and reduced virus titer in the lung when this formulation was used in combination with rHA. Observed the induction of Th1 CD4+ T cells in animals immunized with 3M-052. Consistent with the induction of Th1 CD4+ T cells, an increase in IgG2c antibodies was observed in 3M-052 immunized animals. The ability of 3M-052 adjuvanted formulations to induce protection against both homologous and heterologous virus strains, as well as the longer-term stability of 3M-052 adjuvanted formulations, suggests that these formulations are suitable for stockpiling. Moreover, we have demonstrated the compatibility of these adjuvants with the pre-pandemic vaccine antigens currently in national stocks, and showed that the combination vaccine is able to protect ferrets from both the respective virus strain and drifting isolates.

Example 3 Versatility of PEGylated Liposomes

[0349] In order to test the versatility of the composition of PEGylated liposomes, additional lipids were added to the basic composition of cholesterol, non-PEGylated neutral lipid and PEGylated lipid. The addition of one or more additional neutral lipids and one or more additional cationic lipids did not adversely affect the stability or potency of the resulting liposome formulation (results not shown).

Claims (34)

1. A liposome for stimulating an immune response or inducing a Th1 cell response in a subject, comprising: (a) cholesterol; (b) non-pegylated neutral lipid; (c) a pegylated lipid, wherein the average molecular weight of the PEG in the pegylated lipid is approximately 2000 daltons; And (d) a TLR4 agonist and a TLR7/8 agonist, wherein said TLR4 agonist is GLA (glucopyranosyl lipid adjuvant) and the TLR7/8 agonist is 3M-052. 2. A liposome according to claim 1, wherein the lipid component of said pegylated lipid comprises a neutral lipid. 3. Liposome according to any one of paragraphs. 1, 2, characterized in that the lipid component of said pegylated lipid is DSPE, DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE or DMPE. 4. Liposome according to any one of paragraphs. 1-3, characterized in that the non-pegylated neutral lipid is DPPC, DOPC, DLPC, DMPC, DSPC, POPC, DPPE or DMPE. 5. Liposome according to any one of paragraphs. 1-4, characterized in that (a) the specified liposome is stable for at least 1 month at a temperature of from about 2°C to about 8°C; and/or (b) the polydispersity index of the liposome is maintained at about 0.3 or less; and/or (c) the size of the liposome is less than or approximately equal to 450 nm. 6. Liposome according to any one of paragraphs. 1-5, characterized in that the molar percentage (mol.%) pegylated lipid in the liposome is in the range from about 1 mol. % to about 25 mol. %, mol. % cholesterol in the liposome is in the range from about 1 mol. % to about 50 mol. % and mol. % non-pegylated lipid in the liposome ranges from about 45 mol. % to about 98 mol. %. 7. Liposome according to any one of paragraphs. 1-6, characterized in that the molar ratio between non-pegylated neutral lipid:cholesterol:pegylated lipid is approximately 9.8:5.7:0.8 or approximately 18:5.5:3. 8. Liposome according to any one of paragraphs. 1-7, characterized in that said at least one TLR4 agonist includes GLA. 9. Liposome according to claim 8, characterized in that GLA is synthetic GLA formulas

or a pharmaceutically acceptable salt of said compound; or synthetic GLA formula (VI)

(VI) or a pharmaceutically acceptable salt of said compound, where R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ -C ₂₀ alkyl; and R ² and R ⁴ are C ₁₂ -C ₂₀ alkyl, preferably R ¹ , R ³ , R ⁵ and R ⁶ are C ₁₁ alkyl; and R ² and R ⁴ are C ₁₃ alkyl. 10. Liposome according to any one of paragraphs. 1-9, characterized in that the GLA content is at least about twice that of 3M-052 by weight. 11. Composition containing a liposome according to any one of paragraphs. 1-10 and antigen. 12. Composition according to claim 11, characterized in that the antigen comprises H5N1. 13. Composition according to claim 11, characterized in that the antigen comprises LecA. 14. A method of stimulating an immune response in a subject or inducing a Th1 cell response in a subject, comprising administering to said subject a liposome according to any one of paragraphs. 1-10, whereby an immune response is stimulated or a Th1 cell response is induced in the subject. 15. The method of claim. 14, characterized in that the immune response is a non-specific immune response or that the immune response is an antigen-specific immune response. 16. The method of obtaining pegylated liposome according to any one of paragraphs. 1-10, including: (a) mixing non-pegylated neutral lipid, pegylated lipid and cholesterol in an organic solvent; (b) evaporating said organic solvent, whereby a lipid film is obtained; (c) rehydration of the lipid film in buffer; And (d) sonicating, microfluidizing, or extruding the rehydrated product from step (c).

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