Nanoparticle formulations

Abstract

The present disclosure relates to nanoparticle vaccine adjuvants and vaccine compositions containing nanoparticle vaccine adjuvants, methods of preparing such adjuvants and compositions, and methods of using such compositions and adjuvants for vaccination. The vaccine adjuvants disclosed herein are effective in enhancing the immune response to vaccination.

Description

[Technical field]

[0001] The present disclosure relates to nanoparticle vaccine adjuvants and vaccine compositions containing nanoparticle vaccine adjuvants, methods of preparing such adjuvants and compositions, and methods of using such compositions and adjuvants for vaccination. The vaccine adjuvants disclosed herein are effective in enhancing the immune response to vaccination. [Background technology]

[0002] Vaccination is an important public health tool, as highlighted by the recent SARS-CoV-2 pandemic. Effective vaccines against infectious diseases, when administered broadly throughout a population, can both slow the transmission of the disease and reduce the severity of symptoms experienced by the vaccinated population. Vaccines can also be effective in treating certain types of diseases, including proliferative disorders, such as cancer.

[0003] Vaccines work by inducing an immune response that operates to protect the vaccinated individual. The immune system is stimulated by exposure to a suitable immunogen, such as a pathogen antigen or a cancer-associated antigen, which directly or indirectly stimulates a protective adaptive immune response. The adaptive immune response can be mediated by B cells and / or T cells. The goal can be to provide long-term immunity against the antigen and / or the pathogen, cell or entity that carries the antigen.

[0004] Several different types of vaccines have been developed. Vaccines developed for protection against infectious diseases include inactivated and attenuated live vaccines; toxoid vaccines; viral vector vaccines; subunit, recombinant, polysaccharide and conjugate vaccines; and mRNA vaccines. Vaccines developed for protection against cancer include autologous immune cell vaccines derived from patients, recombinant virus vaccines expressing tumor antigens, peptide vaccines, DNA vaccines, and heterologous whole cell vaccines derived from established human tumor cell lines. In each case, the vaccine contains an antigen capable of inducing an immune response, or a polynucleotide that codes for an antigen capable of inducing an immune response.

[0005] Many vaccines also contain adjuvant components. These are substances that can improve the immune response induced by the vaccine. This can be important, especially when the vaccine antigen has low immunogenicity, or when the vaccine is administered to immunologically suppressed, immunologically depleted, or immunologically immature patients, such as infants or the elderly. The enhancing effect of adjuvants can also allow the dose of vaccine per patient to be reduced, which is important for vaccine sparing in vaccine-deficient situations. Vaccine adjuvants approved for human use include aluminum-based inorganic salts (Alum), MF59, monophosphoryl lipid A (MPL), and CpG oligodeoxynucleotide (CpG 1018).

[0006] Another recognized vaccine adjuvant is imiquimod (R-837), also known as 1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine (CAS number: 99011-02-6), R-837 and S-26308, and has the structural formula:

[0007] [ka] is a small molecule imidazoquinoline drug having the formula:

[0008] A variety of active structural analogs of imiquimod (R-837) have been synthesized and characterized. These include the imidazoquinolines resiquimod (R-848), gardiquimod, CL097, S28690, 852-A and 854A; the thiazoloquinolone CL075, and others, including those shown in Table 1 below:

[0009] [Table 1-1]

[0010] [Table 1-2]

[0011] [Table 1-3]

[0012] Another exemplary structural analog of imiquimod (R-837) is the small molecule TLR7 agonist S28690, described in Hicks et al, Blood (2004) 104(11):3481.

[0013] These structural analogs of imiquimod (R-837) are active TLR7 / 8 ligands with similar properties and activities as imiquimod (R-837), optionally including pH-dependent solubility. In this disclosure, the term "imiquimod" hereafter refers to imiquimod (R-837), but also encompasses and refers to structural analogs of imiquimod (R-837), which are TLR7 / 8 agonists or TLR7 / 8 ligands, including but not limited to those listed above. Suitably, structural analogs of imiquimod (R-837) may exhibit pH-dependent solubility with increased solubility at lower pH. The term "imiquimod" as used herein is therefore an active TLR7 / 8 ligand, and has the basic molecular structure:

[0014] [ka] (In the formula, R 1 is typically N and R 2 is typically H or C, and the imidazoquinoline is optionally substituted at one or more of the indicated attachment points with one or more substituents, which may be branched, linear or cyclic alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl groups, particularly C 1~10 Alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl group, or hydroxyl group, or amine group, or N-(C 1~10 The term "imiquimod" as used herein also includes similar structural derivatives of these imidazoquinolines, including thiazoloquinoline derivatives that are active TLR7 / 8 ligands.

[0015] Imiquimod stimulates the innate and adaptive immune systems by activating Toll-like receptors 7 and / or 8 (TLR7 / 8). It is FDA-approved as the active ingredient in two topical cream formulations, Aldara® and Zyclara®.

[0016] Various studies have shown that topical imiquimod can improve vaccine-induced immune responses. In particular, topical imiquimod improves both antibody and cellular responses to subcutaneous immunization with ovalbumin, shifting the immune response to a Th1 phenotype with significant improvements in IgG2a, IgG2b and CD8+ T cell responses (Johnston et al, Vaccine 2006 Mar 10;24(11):1958-65). Pretreatment with topical imiquimod also significantly improves the immunogenicity of influenza vaccination in both young and elderly individuals (Hung et al, Lancet Infect Dis. 2016 Feb;16(2):209-18). Similar results were reported by Adams et al, J. Clin. Oncol. 25(18) suppl. 8545, which evaluated the safety and adjuvant activity of imiquimod when administered with the NY-ESO-1 protein vaccine.

[0017] These studies have demonstrated that imiquimod is effective as a vaccine adjuvant. However, topical application of imiquimod in the form of a cream is inconvenient as reported in these studies, making its use in routine vaccination unfeasible. Topical application presents compliance issues, as the cream must remain on the skin for several hours to be effective. This may result in skin irritation, or may be poorly tolerated by patients for other reasons. Consistency of imiquimod delivery is also an issue. For practical purposes, it is preferable to formulate the adjuvant with other vaccine ingredients for administration as a single vaccine formulation. It is preferable to formulate the adjuvant as an injectable composition.

[0018] However, formulating imiquimod for injection is not a simple undertaking. Imiquimod exhibits extremely low solubility in aqueous solutions at physiological pH. Figure 1 shows the aqueous solubility of imiquimod as a function of pH using Britton-Robinson buffer, indicating that imiquimod has maximum solubility in aqueous solutions at pH 2 or less (Chollett et al Pharm. Dev. Technol. 1999 Jan; 4(1):35-43). The aqueous solubility of imiquimod decreases rapidly with increasing pH above pH 2, and it is only sparingly soluble above pH 6. For this reason, attempts to develop aqueous or water-based formulations of imiquimod have typically involved the use of acidic solvents below pH 4, which are unacceptable for injection. Hayashi et al. (Int. J. Urol. 2010 May; 17(5):483-90) described the formulation of imiquimod using 0.1 M lactic acid, poloxamer and HP-β-CD at slightly acidic pH. Guedes et al. (J. Braz. Chem. Soc., Vol. 31, No. 8, 1732-1745, 2020) similarly described the solubilization of imiquimod in β-cyclodextrin in the presence of citric acid at pH 3. Ramineni et al. (J. Pharm. Sci. 2013 Feb;102(2):593-603) developed a mucoadhesive film containing imiquimod and HP-β-CD using a mixture of acetate buffer and methanol at pH 4. Meanwhile, Fox et al. (Journal of Nanobiotechnology 2014, 12:17) disclose an anionic liposomal formulation containing imiquimod solubilized in a lactic acid core at pH 2.5-3.5.

[0019] Imiquimod can be formulated in low pH aqueous formulations as described in these and similar references, but this approach does not readily allow for co-formulation of imiquimod with vaccine antigens and polynucleotides that denature or change morphology at low pH, and low pH formulations are not suitable for parenteral administration.

[0020] In an alternative approach, polylactide (PLA)-based micelles have been core-loaded with imiquimod and surface-functionalized with antigen protein (HIV-1 Gag p24) for the purpose of antigen delivery (Jimenez-Sanchez, et al Pharm. Res. (2015) 32:311-320). Although imiquimod is encapsulated in the hydrophobic PLA core, and the p24 antigen is covalently attached via lysine and N-terminal amine groups to N-succinimidyl pendant groups in the micelle corona, release of imiquimod from the particles was found to be extremely rapid (50% in 1 h and approximately 75% in 4 to 5 h). Nanomedicine formats that slow the release of adjuvants are believed to be more likely to support stronger immune responses.

[0021] Against this background, the present inventors have successfully developed and described herein a nanoparticle vaccine adjuvant that includes imiquimod, is suitable for injection, and is capable of obtaining sustained release of imiquimod over an extended period of time, thereby improving the performance of imiquimod as a vaccine adjuvant. The disclosed nanoparticle vaccine adjuvant also provides a vaccine composition comprising the imiquimod vaccine adjuvant, which can be formulated with existing vaccines or co-formulated with vaccine antigens and / or polynucleotides, and additional vaccine components, if required, thereby being administered parenterally, including by injection. Summary of the Invention

[0022] According to one embodiment, the present disclosure provides a vaccine adjuvant comprising a plurality of nanoparticles comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell, the inner aqueous core comprising imiquimod and a host molecule capable of reversibly forming a complex with imiquimod. The vaccine adjuvant may be or comprise an aqueous solution, dispersion or suspension of the disclosed nanoparticles, or may be or comprise a dried or lyophilized preparation that can be hydrated to produce an aqueous solution, dispersion or suspension of the disclosed nanoparticles. The inner aqueous core may have a pH of about 6.5 or greater and / or may comprise a hydrogel. The imiquimod and the host molecule may be dispersed within the hydrogel.

[0023] In a further aspect, the present disclosure provides a vaccine composition comprising (a) an antigen capable of inducing an immune response and / or a polynucleotide encoding the antigen capable of inducing an immune response; and (b) a vaccine adjuvant comprising a nanoparticle according to the present disclosure. The vaccine composition may be or may comprise an aqueous solution, dispersion or suspension of the disclosed components (a) and (b), or may be or may comprise a dried or lyophilized preparation that can be hydrated to produce an aqueous solution, dispersion or suspension of the disclosed components (a) and (b). In some embodiments, some or all of the antigen and / or polynucleotide of component (a) is releasably attached, bound and / or encapsulated within the outer lipid shell of the nanoparticle of component (b). Component (a) may further or alternatively comprise a delivery vehicle, e.g., a nanoparticle delivery vehicle, such as a plurality of nanoparticles comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell, in which case the antigen and / or polynucleotide is loaded into or on the delivery vehicle.

[0024] The vaccine composition may include additional ingredients and excipients, including further adjuvants. The vaccine composition may, in some embodiments, include some or all of the active ingredients and / or excipient components of a vaccine formulation developed for prophylactic or therapeutic use, such as an approved vaccine formulation. Advantageously, the approved vaccine formulation may include an antigen capable of inducing an immune response and / or a polynucleotide encoding the antigen capable of inducing an immune response. The vaccine composition of the present disclosure may include an approved vaccine formulation that includes an antigen capable of inducing an immune response and / or a polynucleotide encoding the antigen capable of inducing an immune response supplemented with a vaccine adjuvant comprising a nanoparticle according to the present disclosure.

[0025] In a further aspect, the present disclosure provides the disclosed vaccine adjuvants for use in a method of improving an immune response to a vaccine in a subject, e.g., a human subject. The present disclosure further provides a method of improving an immune response to a vaccine in a subject, e.g., a human subject, comprising administering to the subject a vaccine adjuvant disclosed herein, the vaccine adjuvant being administered to the subject before, simultaneously with, and / or after administering the vaccine. The vaccine can be any vaccine capable of inducing an immune response in a subject. The immune response can be, for example, a protective immune response capable of protecting the subject from a disease, disorder, or pathogen, including an infectious or proliferative disease or disorder, or a viral, bacterial, or fungal pathogen. The immune response can be a therapeutic immune response capable of alleviating or reducing the symptoms or manifestations of a disease or disorder, including an infectious disease or disorder.

[0026] In a further aspect, the present disclosure provides the disclosed vaccine composition for use in a method of inducing an immune response in a subject, e.g., a human subject. The present disclosure further provides a method of inducing an immune response in a subject, e.g., a human subject, comprising administering to the subject a vaccine composition disclosed herein. The immune response can be a protective immune response that can protect the subject from, e.g., an infectious or proliferative disease or disorder, or a disease, disorder or pathogen, including a viral, bacterial or fungal pathogen. The immune response can be a therapeutic immune response that can alleviate or reduce the symptoms or manifestations of a disease or disorder, including an infectious disease or disorder.

[0027] The present disclosure further provides a method for producing the disclosed vaccine adjuvants, comprising the following sequential steps: (a) solubilizing imiquimod with a host molecule in an aqueous solution having a pH buffered to about pH 6 or less; preferably about pH 4-6, or about pH 4.5-6, or about pH 4-5.5, or about pH 5-6; (b) combining the resulting aqueous solution with lipids to form lipid shell nanoparticles that encapsulate imiquimod; and (c) raising the buffered pH of the formulation to about pH 6.5 or greater, or to pH 7 or greater, or to about pH 6.5-9, or to about pH 6.5-8.5, or to about pH 6.5-8, or to about pH 7-9, or to about pH 7-8.5, or to about pH 7-8, or to about pH 7.5-9.

[0028] The present disclosure provides a method for producing the disclosed vaccine composition, comprising combining the disclosed vaccine adjuvant with an antigen capable of inducing an immune response and / or with a polynucleotide encoding an antigen capable of inducing an immune response. The method may comprise combining the disclosed vaccine adjuvant with an approved vaccine formulation comprising an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response. The present disclosure provides a method for producing the disclosed vaccine composition, comprising producing the vaccine adjuvant according to the methods disclosed herein, and adding during or after step (a) an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response according to the methods disclosed herein.

[0029] The disclosed vaccine adjuvants and vaccine compositions, buffered at physiological pH, can be administered to a subject parenterally, optionally by injection, and can be used in the course of immunization against or against a wide range of diseases, disorders and pathogens, including infectious diseases and pathogens and cancer.

[0030] The compositions of the present disclosure are designed to facilitate controlled, sustained and optionally localized delivery and / or release of imiquimod for the purpose of improving immune responses to vaccines. The disclosed adjuvants can result in sustained delivery and / or release of imiquimod over a period of time that can improve immune response enhancement.

[0031] The disclosed vaccine adjuvant comprises the small molecule imiquimod complexed with a host molecule and encapsulated in liposomes. Imiquimod (R-837) is contained in approved and marketed pharmaceuticals (Aldara® and Zyclara®) and is well characterized and well understood by oncologists. As disclosed herein, although it has not been previously formulated for convenient parenteral administration and for controlled delivery and release, research and clinical trials have demonstrated the efficacy of imiquimod as a vaccine adjuvant. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows a graph illustrating the effect of pH on the solubility of imiquimod in aqueous solution. [Diagram 2] FIG. 1 shows the structure of an exemplary vaccine adjuvant nanoparticle according to the present disclosure. [Diagram 3] FIG. 1 shows a Cryo TEM image of vaccine adjuvant nanoparticles according to the present disclosure. [Figure 4] FIG. 1 shows a flow chart depicting steps of an exemplary method of producing a vaccine adjuvant according to the present disclosure. [Diagram 5] FIG. 1 shows the results of a release assay illustrating the release profile of imiquimod from a vaccine adjuvant according to the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] definition The terms used in this disclosure should be understood to have their usual meaning in the art unless otherwise indicated. In particular, "antigen" is intended to include any molecule or entity capable of inducing an immune response in vivo in humans and / or animals. "Polynucleotide" is intended to include any nucleic acid containing more than one type of nucleotide. "Immune response" is intended to include any protective or defensive reaction of the immune system to an antigen, including innate, adaptive and reactive immune responses, type 1 and type 2 responses, cell-mediated and humoral and inflammatory immune responses. Within the context of the present invention, "immune response" may be generally understood to mean a protective or therapeutic immune response, and / or an immune response effective to protect against or treat a disease, disorder or medical condition. "Immunization" is intended to include the process of inducing an immune response, particularly a protective immune response, against an antigen in a subject, and a subject becomes "immune" after effective immunization. "Vaccine" is a substance or composition that can be used to immunize a subject. A "vaccine adjuvant" is a substance that may be effective in expanding, enhancing, amplifying, modulating, augmenting, or in any way improving the immune response induced by a vaccine. A "hydrogel" is a matrix of optionally crosslinked water-swellable hydrophilic polymers, and "hydrogel polymer" is to be interpreted accordingly. The term "imiquimod" refers to imiquimod (R-837), but also encompasses and refers to structural analogs of imiquimod (R-837) that are TLR7 / 8 agonists or TLR7 / 8 ligands as defined herein above and that exhibit pH-dependent solubility, including, but not limited to, imidazoquinolines and thiazoloquinolones, as shown in Table 1.

[0034] Detailed Description The present disclosure provides a vaccine adjuvant comprising a plurality of nanoparticles, each nanoparticle comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell, the inner aqueous core comprising imiquimod and a host molecule capable of reversibly forming a complex with imiquimod. The aqueous core may comprise imiquimod complexed with the host molecule. The aqueous core of the nanoparticle may comprise uncomplexed imiquimod and / or the host molecule.

[0035] The inner aqueous core may optionally comprise a hydrogel. The imiquimod and host molecule may optionally be dispersed, dissolved or suspended in the hydrogel.

[0036] The inner aqueous core may have a pH of about 6.5 or greater. Preferably, the inner aqueous core has a pH of at least 7, or at least 7.5. The inner aqueous core may have a pH of about pH 9 or less, or about pH 8.5 or less, or preferably a pH of about pH 6.5-9, or about pH 6.5-8.5, or about pH 6.5-8, or about pH 7-9, or about pH 7-8.5, or about pH 7-8, or about pH 7.5-9.

[0037] The outer lipid shell of the nanoparticles comprises one or more lipid layers or bilayers, which surround the central core. The lipids forming the shell can be neutral, zwitterionic, anionic or cationic lipids at physiological pH. The lipids within and / or between each lipid layer or bilayer can be optionally cross-linked. The outer lipid shell can thus be composed of one or more concentric lipid layers, which are optionally cross-linked, and the lipids can be neutral, anionic or cationic lipids at physiological pH. The composition of the lipid shell and the extent of cross-linking within or between the lipid layers can be varied to modify and optimize the release profile of imiquimod from the nanoparticles.

[0038] In some preferred embodiments, one or more lipid layers or bilayers may comprise lipids selected from the group consisting of cholesterol, phospholipids, lysolipids, lysophospholipids and sphingolipids, and derivatives thereof.Suitable lipids include phosphatidylcholine (PC) including 1,2-diacyl-glycero-3-phosphocholine (e.g. egg PC, soybean PC); phosphatidylserine (PS); phosphatidylglycerol; phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin; sphingoglycolipids (also known as 1-ceramidylglucosides), such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids; sterols containing a carboxylic acid group, such as cholesterol or a derivative thereof; and 1,2-diacyl-sn-glycero-3-phosphoethanolamines, including, but not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-dioleoylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dimyristoylphosphatidylcholine (DMPC). Suitable lipids also include naturally occurring lipids, such as L-α-phosphatidylcholine derived from tissues: egg yolk, heart, brain, liver, soybean) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholine, 1-acyl-2-acyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine) derivatives of these lipids.

[0039] The outer lipid shell may also be or may include cationic lipids, including but not limited to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts, also referred to as TAP lipids, such as methyl sulfate salts. Suitable TAP lipids include but are not limited to DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-) and DSTAP (distearoyl-). Other suitable cationic lipids include dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, N"[1-(2,3-dioleyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dialkyloxy-3-dimethylammonium propane, dioctadecylamidoglycylspermine (DOGS), 3-[N-(N' 5N'-Dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); β-alanylcholesterol; cetyltrimethylammonium bromide (CTAB); diC 14-amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonioacetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N,N,N',N'-tetramethyl-,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER). 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives such as 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM) and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). and 2,3-dialkyloxypropyl quaternary ammonium derivatives containing a hydroxyalkyl moiety on the quaternized amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP ... These include 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethylammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethylammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

[0040] In some embodiments, the outer lipid shell may comprise neutral, zwitterionic, cationic or anionic lipids, including DSPE PEG (2000 MW) and DSPE PEG (5000 MW), such as PEGylated derivatives of mPEG-DSPE. Surface presentation of PEG or other suitable hydrophilic polyalkylene oxides on the outer shell of the nanoparticles may function to reduce the uptake of the nanoparticles by the reticuloendothelial system ("RES") when the nanoparticles are present in vivo, thereby increasing in vivo residence time and systemic circulation time, and / or allowing the nanoparticles to produce sustained and extended immunostimulatory effects. Further examples of suitable PEGylated lipids include dipalmitoyl-glycero-succinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol and cholesteryl-polyethylene glycol.

[0041] In some embodiments, the outer lipid shell may comprise a mixture of phospholipids and cholesterol, such as N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero 3-phosphoethanolamine sodium salt (mPEG-DSPE), phosphatidylcholine, such as fully hydrogenated soy phosphatidylcholine (HSPC), and cholesterol. These lipids are well known, well characterized, and used in approved commercial products, such as Doxil®. ​​Alternative suitable phospholipids known to those skilled in the art may be used in place of DSPE-PEG and / or HSPC. The lipids may be mixed and used in any desired molar ratio. For example, the molar ratio of phospholipid to cholesterol may range from about 1:1 to about 6:1, more preferably from about 1:1 to about 3:1, and most preferably about 2:1. When the phospholipid comprises DSPE-PEG and HSPC, these components may be present in a molar ratio of DSPE-PEG:HSPC of about 1:1 to 1:200 or 1:10 to 1:200, preferably 1:1 to 1:100 or 1:1 to 1:50 or 1:1 to 1:30 or 1:10 to 1:30, advantageously 1:15 to 1:25. In some embodiments, the molar ratio of HSPC:DSPE-PEG:cholesterol may be about 2:0.1:1 or about 2:0.01:1 or about 2:0.2:1.

[0042] The outer lipid shell of the nanoparticle surrounds an inner aqueous core that may include one or more hydrogel polymers that may function to further stabilize and / or control the release of imiquimod from the nanoparticle, and any other active agents that may be contained within the inner core of the nanoparticle, such as antigens or polynucleotides, as described in more detail below. The hydrogel polymers may be covalently and / or non-covalently crosslinked, or may be capable of being covalently and / or non-covalently crosslinked, or may have no crosslinks. Hydrogel polymers include, for example, poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), polyhydroxyalkanoates, such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); poly(glycolide-co-caprolactone); polycarbonates; polyamides, polypeptides, and poly(amino acids); polyesteramides; other biocompatible polyesters; poly(dioxanones); poly(alkylene alkylates; hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polysiloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), polyvinyl alcohol, polyvinylpyrrolidone; poly(alkylene oxide); cellulose, polyacrylic acid, albumin, collagen, gelatin, prolamines and / or polysaccharides. In particular, the hydrogel polymer may comprise a block copolymer or a copolymer comprising a blend of any of the aforementioned hydrogel polymers. In some embodiments, the inner aqueous core of the nanoparticles may comprise a polyethylene glycol polymer, such as polyethylene glycol 4000. PEG 4000 is widely used in pharmaceutical formulations, including parenteral formulations, such as INVEGA SUSTENNA®.The inner aqueous core may additionally or alternatively comprise a block copolymer containing one or more poly(alkylene oxide) segments, such as polyethylene glycol, and one or more aliphatic polyester segments, such as polylactic acid.

[0043] The nanoparticles may have a diameter of about 300 nm or less, as measured by standard art-recognized dynamic light scattering (DLS) techniques. In some embodiments, the diameter of the nanoparticles (measured by DLS) may be about 200 nm or less, or about 150 nm or less, or about 130 nm or less, or about 120 nm or less. The diameter of the nanoparticles (measured by DLS) may be at least 15 nm, or at least 20 nm, or at least 30 nm, or at least 50 nm. Suitably, the diameter of the nanoparticles (measured by DLS) may be about 20-300 nm, or about 20-150 nm, or about 20-100 nm, or about 20-50 nm, or about 30-300 nm, or about 50-150 nm, or about 80-125 nm, or about 90-110 nm. DLS may be performed according to ISO 22412:2017 or similar techniques.

[0044] Advantageously, the nanoparticles can be spherical or spheroidal and / or unilamellar. Exemplary nanoparticles according to the present disclosure viewed under cryo-transmission electron microscopy (cryo-TEM) can be seen in Figure 3, where it is seen that the majority of the nanoparticles are spherical and unilamellar, and that the interior of the nanoparticles is denser than the surrounding buffer, consistent with the loading of imiquimod, HP-β-CD and PEG4000 polymer within the nanoparticles.

[0045] The inner aqueous core of the nanoparticles contains imiquimod. Some or all of the imiquimod is complexed with the host molecule. As mentioned above, the term "imiquimod" as used herein refers to 1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine (CAS number: 99011-02-6), R-837 and S-26308, as well as the following:

[0046] [ka] These include the imidazoquinolines known as

[0047] The term further encompasses structural analogs of imiquimod (R-837) as defined herein, which are active TLR7 / 8 ligands including, but not limited to, resiquimod, gardiquimod, CL097, S28690, 852-A, 854A, CL075, etc., known in the art and defined above. Imiquimod, as defined herein, is a small synthetic guanosine analog with recognized immune stimulatory capabilities, and is known to be effective in activating TLR7 and / or TLR8 in particular. In some preferred embodiments and aspects of the present disclosure, the imiquimod is imiquimod (R-837).

[0048] Imiquimod (R-837) is approved for therapeutic administration as a skin cream and is commercially available as a drug substance manufactured and tested in accordance with current Good Manufacturing Practices (cGMP) under a valid drug master file. Alternatively, imiquimod can be readily synthesized as a small molecule based on available raw materials and using methods well known in the art.

[0049] The inner aqueous core of the nanoparticle further comprises a host molecule capable of reversibly forming a complex, for example an inclusion complex, with imiquimod. An inclusion complex can be formed when an imiquimod molecule, or a portion of an imiquimod molecule, inserts into a cavity of a host molecule or a group of host molecules. The host molecule can assist in solubilizing imiquimod in the aqueous core of the nanoparticle and / or controlling the release of imiquimod from the nanoparticle. Imiquimod can thus be present in the form of an inclusion complex with the host molecule.

[0050] The host molecule may be, for example, a cyclodextrin; preferably α-cyclodextrin; β-cyclodextrin; γ-cyclodextrin; methyl α-cyclodextrin; methyl β-cyclodextrin; methyl γ-cyclodextrin; ethyl β-cyclodextrin; butyl α-cyclodextrin; butyl β-cyclodextrin; butyl γ-cyclodextrin; pentyl γ-cyclodextrin; hydroxyethyl β-cyclodextrin; hydroxyethyl γ-cyclodextrin; 2-hydroxypropyl α-cyclodextrin; Cyclodextrin;2-Hydroxypropyl β-cyclodextrin;2-Hydroxypropyl γ-cyclodextrin;2-Hydroxybutyl β-cyclodextrin;Acetyl α-cyclodextrin;Acetyl β-cyclodextrin;Acetyl γ-cyclodextrin;Propionyl β-cyclodextrin;Butyryl β-cyclodextrin;Succinyl α-cyclodextrin;Succinyl β-cyclodextrin;Succinyl γ-cyclodextrin;Benzoyl β-cyclodextrin;Palmityl β-cyclodextrin cyclodextrin;toluenesulfonyl β-cyclodextrin;acetyl methyl β-cyclodextrin;acetyl butyl β-cyclodextrin;glucosyl α-cyclodextrin;glucosyl β-cyclodextrin;glucosyl γ-cyclodextrin;maltosyl α-cyclodextrin;maltosyl β-cyclodextrin;maltosyl γ-cyclodextrin;α-cyclodextrin carboxymethyl ether;β-cyclodextrin carboxymethyl ether;γ-cyclodextrin carboxymethyl ether The cyclodextrin may include a cyclodextrin selected from: butyl ether; carboxymethyl ethyl β-cyclodextrin; phosphate ester α-cyclodextrin; phosphate ester β-cyclodextrin; phosphate ester γ-cyclodextrin; 3-trimethylammonium-2-hydroxypropyl β-cyclodextrin; sulfobutyl ether β-cyclodextrin; carboxymethyl α-cyclodextrin; carboxymethyl β-cyclodextrin; carboxymethyl γ-cyclodextrin, and combinations thereof.However, many other host molecules are known in the art and can be used in accordance with the present disclosure, such as polysaccharides, cryptands, cryptophanes, cavitands, crown ethers, dendrimers, ion exchange resins, calixarenes, valinomycin, nigericin, catenanes, polycatenanes, carcerands, cucurbiturils, and spherands, which are well known to those of skill in the art.

[0051] In a preferred embodiment, the host molecule is or includes 2-hydroxypropyl-β-cyclodextrin. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) (CAS number 128446-35-5) is a partially substituted poly(hydroxypropyl) ether of beta-cyclodextrin (0.59-0.73 molar substitution per anhydroglucose unit). It can reversibly complex with imiquimod, for example to improve solubilization of imiquimod in the aqueous inner core of the nanoparticles while providing controlled release of imiquimod from the nanoparticles. HP-β-CD is currently used in several marketed products, including Mitozytrex™, a formulation of HP-β-CD and mitomycin approved in the United States to treat gastric or pancreatic adenocarcinoma.

[0052] Preferred embodiments of the present disclosure include a host molecule complexed with imiquimod. Some embodiments may also include uncomplexed imiquimod and / or host molecule in the inner aqueous core of the nanoparticle. In particular, the inner aqueous core of the nanoparticle may also include imiquimod dispersed, dissolved or suspended in the aqueous core, and / or imiquimod present in the form of a precipitate. Optionally, the nanoparticle may not include IL-2 and / or may not include a protein cytokine that is releasably attached, bound and / or encapsulated in the outer lipid shell.

[0053] The vaccine adjuvants of the present disclosure may be or may comprise an aqueous solution, dispersion or suspension of the disclosed nanoparticles, or may comprise a dried or lyophilized preparation that can be hydrated to produce an aqueous solution, dispersion or suspension of the disclosed nanoparticles. The adjuvants are typically used in hydrated form, but can be conveniently prepared in lyophilized form, optionally for storage prior to use.

[0054] The vaccine adjuvant of the present disclosure may further comprise one or more additional adjuvant components, such as some or all of the ingredients of approved vaccine adjuvants. This may further improve the efficacy of the vaccine adjuvant to enhance immune response. The vaccine adjuvant of the present disclosure may comprise one or more additional adjuvant components, such as TLR agonists, including, for example, TLR4 agonists, such as monophosphoryl lipid A (MPL), and / or TLR9 agonists, such as CpG 1018. The vaccine adjuvant may further comprise, for example, MPL and commercially available saponin QS-21. The additional adjuvant components may be present in the vaccine adjuvant in free form and / or in combination with a delivery vehicle, such as liposomes. Additionally or alternatively, the additional adjuvant components may be loaded into or onto the vaccine adjuvant nanoparticles.

[0055] Also provided in accordance with the present disclosure is a vaccine composition comprising: (a) an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response; and (b) a vaccine adjuvant comprising a plurality of nanoparticles according to the present disclosure.

[0056] In some embodiments, some or all of the antigens and / or polynucleotides of component (a) are releasably attached, bound, and / or encapsulated in the outer lipid shell of the nanoparticles of component (b). The antigens and / or polynucleotides may be encapsulated in the lipid shell of the nanoparticles of component (b) and / or dispersed in the aqueous core of the nanoparticles of component (b) and / or releasably attached to or bound to the lipid shell of the nanoparticles of component (b). Some or all of the antigens and / or polynucleotides may optionally be reversibly associated with host molecules in the aqueous core of the nanoparticles of component (b). In some embodiments, the antigens and / or polynucleotides may be non-covalently attached to the lipid shell of the nanoparticles of component (b), for example, by ionic interactions, hydrogen bonds, or van der Waals interactions. In some other embodiments, the antigen and / or polynucleotide may be covalently attached to the lipid shell of the nanoparticle of component (b) by a cleavable linking group, which can be cleaved under suitable conditions, e.g., at a certain ambient pH or in the presence of a certain cleaving agent(s), to release the antigen and / or polynucleotide.

[0057] Component (a) may also or alternatively comprise a delivery vehicle that is loaded with the antigen and / or polynucleotide and is capable of releasing the antigen and / or polynucleotide in vivo. The delivery vehicle may be, for example, a nanoparticle vehicle, such as a polymeric nanoparticle, liposome or nanoparticle, that comprises an outer lipid shell as described herein and an inner aqueous core encapsulated within the outer lipid shell. The delivery vehicle may be, for example, a nanogel or PLGA nanoparticle as described in Look et al, Biomaterials 35(2014) 1089-1095. Some or all of the antigen and / or polynucleotide may be releasably attached, bound and / or encapsulated within the nanoparticle delivery vehicle. When the nanoparticle delivery vehicle comprises a core / shell nanoparticle of the type described herein, some or all of the antigen and / or polynucleotide may be releasably attached, bound and / or encapsulated within the outer lipid shell of the nanoparticle. The outer lipid shell of the nanoparticle may comprise one or more lipid layers or bilayers surrounding a central core as described herein. The inner aqueous core of the nanoparticle may comprise a hydrogel as described herein. The inner aqueous core of the nanoparticle may further comprise a host molecule as described herein.

[0058] In some embodiments of the vaccine composition, component (a) may comprise a polynucleotide that is a DNA or RNA molecule, such as an mRNA molecule, or an siRNA molecule. DNA and RNA vaccines are known in the art. These known vaccines contain DNA or RNA polynucleotides that can be expressed in vivo to obtain an antigen that can stimulate a protective or therapeutic immune response. Recent examples include mRNA vaccines developed to protect against SARS-CoV-2 infection and COVID-19 disease. DNA vaccines for protecting against and treating cancer are also described in the art. Component (a) of the vaccine composition may thus comprise a DNA or RNA molecule, such as an mRNA molecule, that can express an antigen that can stimulate a protective or therapeutic immune response in vivo.

[0059] In some embodiments, component (a) of the vaccine composition may comprise a viral antigen, and / or a bacterial antigen, and / or a fungal antigen, and / or a disease-associated and / or cancer-associated antigen; and / or a polynucleotide encoding a viral antigen, and / or a bacterial antigen, and / or a fungal antigen, and / or a disease-associated and / or cancer-associated antigen. Component (a) may comprise an antigen that is a peptide, protein, carbohydrate, nucleic acid and / or lipid molecule or structure. Component (a) may comprise a polynucleotide encoding a peptide antigen or a protein antigen.

[0060] In some embodiments, component (a) of the vaccine composition is a coronavirus or coronavirus-related antigen, such as a SARS-CoV, MERS-CoV or SARS-CoV-2 antigen; or an influenza or influenza-related antigen, such as an influenza A, influenza B, influenza C or influenza D antigen; or a herpes simplex (HSV-1 or HSV-2) or HSV-related antigen; or a cytomegalovirus (CMV) or a CMV-related antigen; or a Lyme disease (Borrelia) or a Lyme disease-related antigen; or a respiratory syncytial virus (RSV) or a RSV-related antigen; or an Epstein-Barr virus (EBV) or a RSV-related antigen; or EBV or an EBV-related antigen; or Zika virus or a Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles or a measles-related antigen; or mumps or a mumps-related antigen; or rubella or a rubella-related antigen; or chickenpox (varicella) or a chickenpox-related antigen; or herpes zoster (shingles) or a shingles-related antigen; or diphtheria or a diphtheria-related antigen; or tetanus or a tetanus-related antigen; or poliomyelitis or a poliomyelitis-related antigen; or dengue virus or a dengue virus-related antigen; or Haemophilus influenzae influenzae (Hib) or a Hib related antigen; or rotavirus or a rotavirus related antigen; or Streptococcus pneumoniae (Streptococcus) or a Streptococcus related antigen; or human papillomavirus (HPV) or an HPV related antigen; or pertussis or a pertussis related antigen; or hepatitis or a hepatitis related antigen; or tuberculosis or a tuberculosis related antigen; or human immunodeficiency virus (HIV) or an HIV related antigen; or adenovirus or an adenovirus related antigen; or anthrax or anthrax related antigen; or cholera or a cholera related antigen; or Japanese encephalitis (JE) or a JE related antigen; or rabies or a rabies related antigen; or smallpox or a smallpox related antigen; or typhoid fever (typhoid fever) or a typhoid related antigen; or yellow fever or a yellow fever related antigen;or Ebola or an Ebola-related antigen; or a cancer or a cancer-related antigen.

[0061] In some embodiments, component (a) of the vaccine composition is a coronavirus or coronavirus-related antigen, such as a SARS-CoV, MERS-CoV or SARS-CoV-2 antigen; or an influenza or influenza-related antigen, such as an influenza A, influenza B, influenza C or influenza D antigen; or a herpes simplex (HSV-1 or HSV-2) or HSV-related antigen; or a cytomegalovirus (CMV) or a CMV-related antigen; or a Lyme disease (Borrelia) or a Lyme disease-related antigen; or a respiratory syncytial virus (RSV) or a RSV-related antigen; or an Epstein-Barr virus (EBV) or a RSV-related antigen; or EBV or an EBV-related antigen; or Zika virus or a Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles or a measles-related antigen; or mumps or a mumps-related antigen; or rubella or a rubella-related antigen; or chickenpox (varicella) or a chickenpox-related antigen; or herpes zoster (shingles) or a shingles-related antigen; or diphtheria or a diphtheria-related antigen; or tetanus or a tetanus-related antigen; or poliomyelitis or a poliomyelitis-related antigen; or dengue virus or a dengue virus-related antigen; or Haemophilus influenzae influenzae (Hib) or a Hib related antigen; or rotavirus or a rotavirus related antigen; or Streptococcus pneumoniae (Streptococcus) or a Streptococcus related antigen; or human papillomavirus (HPV) or an HPV related antigen; or pertussis or a pertussis related antigen; or hepatitis or a hepatitis related antigen; or tuberculosis or a tuberculosis related antigen; or human immunodeficiency virus (HIV) or an HIV related antigen; or adenovirus or an adenovirus related antigen; or anthrax or anthrax related antigen; or cholera or a cholera related antigen; or Japanese encephalitis (JE) or a JE related antigen; or rabies or a rabies related antigen; or smallpox or a smallpox related antigen; or typhoid fever (typhoid fever) or a typhoid related antigen; or yellow fever or a yellow fever related antigen;or an Ebola or Ebola-associated antigen; or a polynucleotide encoding a cancer or cancer-associated antigen;

[0062] The vaccine composition may be or include an aqueous solution, dispersion or suspension. Alternatively, the vaccine composition may be provided in a dry or lyophilized form. The vaccine composition may optionally include one or more additional excipients and / or active ingredients, including, but not limited to, aluminum or aluminum salts, MF59 (squalene oil), thiomersal, gelatin, sorbitol, lipids (including ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol), potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate dihydrate, sucrose, tromethamine and tromethamine hydrochloride; for example, stabilizers, preservatives, emulsifiers, buffers and / or additional adjuvants. Some or all of the antigen, and / or polynucleotide, and / or vaccine adjuvant, and / or additional ingredients may be mixed, dissolved, dispersed or suspended in the vaccine composition.

[0063] The vaccine composition may, in some embodiments, comprise some or all of the active ingredients and / or excipient components of a vaccine formulation developed for prophylactic or therapeutic use, e.g., an approved vaccine formulation. Advantageously, the approved vaccine formulation may comprise an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response. In these embodiments, the vaccine composition of the present disclosure may comprise ingredients of the approved vaccine formulation mixed or separately formulated with a vaccine adjuvant comprising nanoparticles according to the present disclosure. An approved vaccine formulation may be, for example, a vaccine approved against a coronavirus, e.g., SARS-CoV, MERS-CoV or SARS-CoV-2, and / or a vaccine approved against influenza, and / or a vaccine approved against herpes simplex (HSV-1 or HSV-2), and / or a vaccine approved against cytomegalovirus (CMV), and / or a vaccine approved against Lyme disease (Borrelia), and / or a vaccine approved against respiratory syncytial virus (RSV), and / or a vaccine approved against Epstein-Barr virus (EBV), and / or a vaccine approved against Zika virus. approved vaccines, and / or approved vaccines against meningitis, and / or approved vaccines against measles, and / or approved vaccines against mumps, and / or approved vaccines against rubella, and / or approved vaccines against chickenpox (varicella), and / or approved vaccines against herpes zoster (shingles), and / or approved vaccines against diphtheria, and / or approved vaccines against tetanus, and / or approved vaccines against poliomyelitis, and / or approved vaccines against dengue virus, and / or approved vaccines against Haemophilus influenzae (Hib), and / or approved vaccines against rotavirus, and / or approved vaccines against Streptococcus pneumoniaeThe vaccine may be an approved vaccine against rabies, typhoid fever, and / or an approved vaccine against human pneumonia, and / or an approved vaccine against human papillomavirus (HPV), and / or an approved vaccine against whooping cough, and / or an approved vaccine against hepatitis, and / or an approved vaccine against tuberculosis, and / or an approved vaccine against human immunodeficiency virus (HIV), and / or an approved vaccine against adenovirus, and / or an approved vaccine against anthrax, and / or an approved vaccine against cholera, and / or an approved vaccine against Japanese encephalitis (JE), and / or an approved vaccine against rabies, and / or an approved vaccine against smallpox, and / or an approved vaccine against typhoid fever, and / or an approved vaccine against yellow fever, and / or an approved vaccine against Ebola, and / or an approved vaccine against cancer.

[0064] An exemplary nanoparticle according to the present disclosure is illustrated in FIG. 2. As shown in this figure, the exemplary nanoparticle has an external liposomal shell formed from a lipid bilayer containing mPEG-DSPE, where mPEG chains are presented on the exterior and interior surfaces of the liposomal shell. The internal aqueous core of the nanoparticle comprises a PEG 4000 hydrogel and a hydroxypropyl-β-cyclodextrin host molecule. The host molecule reversibly associates with an imiquimod molecule in the internal aqueous core of the nanoparticle (not shown). In some embodiments, as disclosed herein, an antigen or polynucleotide according to the present disclosure may be loaded into the nanoparticle, reversibly attached to or within the external liposomal shell of the nanoparticle, and / or disposed entirely or partially within the internal core of the nanoparticle (not shown).

[0065] The vaccine adjuvant and / or vaccine composition of the present disclosure is preferably suitable for administration to humans or animals for prophylactic or therapeutic purposes.The adjuvant and / or composition may be suitable for parenteral administration, particularly by infusion, injection or deposition.The adjuvant and / or composition is preferably sterile.The nanoparticles contained in the vaccine adjuvant and vaccine composition are preferably biodegradable.

[0066] The adjuvant and / or composition may be buffered to a pH of at least about 6.5, or at least about 7, and preferably below pH 9, or below pH 8.5. Suitably, the adjuvant and / or composition may be buffered to a pH that is preferably about pH 6.5-9, or about pH 6.5-8.5, or about pH 6.5-8, or about pH 7-9, or about pH 7-8.5, or about pH 7-8, or about pH 7.5-9. This is beneficial and desirable in terms of therapeutic applications, and the adjuvant or composition should ideally be buffered to a pH that is about physiological pH (pH 7.4). Buffering of the adjuvant or composition may be accomplished using any suitable and acceptable buffering agent, for example citric acid / sodium citrate.

[0067] The vaccine adjuvants and / or vaccine compositions of the present disclosure may suitably be substantially free of unencapsulated imiquimod, which helps to avoid undesirable precipitation of imiquimod in or from the adjuvant or composition.

[0068] The vaccine adjuvant and / or vaccine composition may suitably comprise about 0.01-50 μg / ml of imiquimod. In some embodiments, the adjuvant or composition may comprise about 1-30 μg / ml of imiquimod, or about 5-25 μg / ml of imiquimod, or about 5-15 μg / ml of imiquimod. The adjuvant or composition may comprise at least about 1 μg / ml of imiquimod, or at least about 2 μg / ml of imiquimod, or at least about 3 μg / ml of imiquimod, or at least about 5 μg / ml of imiquimod. The adjuvant or composition may comprise about 20 μg / ml or less of imiquimod, or about 15 μg / ml or less of imiquimod, or about 12 μg / ml or less of imiquimod. The adjuvant or composition may contain about 5 μg / ml, or about 10 μg / ml, or about 15 μg / ml of imiquimod.

[0069] The amount or concentration of antigen or polynucleotide in a vaccine composition can be determined by one of skill in the art, taking into account the usual considerations, including the strength of the response required, the immunogenicity of the antigen, toxicity issues and other criteria well known to those of skill in the art.

[0070] The vaccine adjuvant and / or vaccine composition may suitably comprise between about 1 and 100 mg / ml lipid. In some embodiments, the adjuvant or composition may comprise between about 5 and 50 mg / ml lipid, or between about 10 and 40 mg / ml lipid, or between about 20 and 30 mg / ml lipid. The adjuvant or composition may comprise at least about 5 mg / ml lipid, or at least about 10 mg / ml lipid, or at least about 15 mg / ml lipid, or at least about 20 mg / ml lipid. The adjuvant or composition may comprise up to about 50 mg / ml lipid, or up to about 40 mg / ml lipid, or up to about 30 mg / ml lipid, or up to about 25 mg / ml lipid.

[0071] The average diameter of the nanoparticles in the adjuvant or composition, as measured by standard art-recognized dynamic light scattering (DLS) techniques, preferably according to ISO 22412:2017, may suitably be about 300 nm or less, or about 200 nm or less, or about 150 nm or less, or about 130 nm or less, or about 120 nm or less. Herein, the average size of the nanoparticles in the adjuvant or composition may refer to the average diameter of the nanoparticles in the adjuvant or composition, or may refer to the median particle size D50 of the nanoparticles in the adjuvant or composition. In some embodiments, the average diameter of the nanoparticles in the adjuvant or composition may be at least about 15 nm, or at least about 20 nm, or at least about 30 nm, or at least about 50 nm. In some embodiments, the average diameter of the nanoparticles in the adjuvant or composition and / or the range of sizes of the nanoparticles in the adjuvant or composition may be about 20-300 nm, or about 20-150 nm, or about 20-100 nm, or about 20-50 nm, or about 30-300 nm, or about 50-150 nm, or about 80-125 nm, or about 90-110 nm.

[0072] As described in more detail below, the vaccine adjuvants and compositions of the present disclosure can provide sustained release and / or delivery of imiquimod to a subject over an extended period of time, thereby enabling effective enhancement of vaccine-induced immune responses. Imiquimod is known in the art as a vaccine adjuvant that can enhance immune responses induced by vaccine antigens, including infectious disease or pathogen antigens and cancer-related antigens. The present disclosure provides an approach and platform that allows imiquimod to be co-administered by injection with existing approved adjuvants and / or vaccine ingredients for combined adjuvant and / or immunogenic effects, and the modes and techniques of administration and delivery can improve vaccine efficacy, reduce systemic exposure and associated toxicity, improve pharmacokinetics, and / or provide preventative and therapeutic benefits at doses significantly lower than the approved therapeutic doses of imiquimod.

[0073] The vaccine adjuvants of the present disclosure may be suitable for enhancing vaccine-induced protective or therapeutic immune responses against a variety of different diseases and medical conditions, including viral, bacterial or fungal diseases, colonization or infections, and proliferative disorders, including cancer. Another aspect of the present disclosure thus provides a method of enhancing an immune response to a vaccine in a subject, such as a human subject, comprising administering to the subject a vaccine adjuvant as disclosed herein, where the vaccine adjuvant is administered to the subject prior to, simultaneously with, and / or after administration of the vaccine.

[0074] The vaccine compositions of the present disclosure may be suitable for inducing an immune response in a subject that is effective to prevent or treat a variety of different diseases or medical conditions, including viral, bacterial or fungal diseases, colonization or infections, and proliferative disorders, including cancer. Another aspect of the present disclosure thus provides a method for inducing a protective or therapeutic immune response in a subject, e.g., a human subject, and / or for immunizing a subject, e.g., a human subject, against a viral, bacterial or fungal disease, or colonization or infection, or against a proliferative disorder, e.g., cancer, comprising administering to the subject a vaccine composition as disclosed herein.

[0075] The vaccine adjuvant and / or vaccine composition may be administered to a subject parenterally, for example, by intravenous, intramuscular or subcutaneous injection or injection or deposition. The vaccine adjuvant or vaccine composition may be administered orally, intranasally, intramuscularly, intradermally, transdermally, intravenously, intraperitoneally, intrathecally, intravesically, cutaneously, subcutaneously, or ocularly, including subconjunctivally, retrobulbarly, intracamerally, and intravitreally. The vaccine adjuvant or vaccine composition may be administered systemically, for example, by intravenous infusion or injection, or locally, for example, by injection at or in the immediate locality of a lesion, such as a tumor. Local administration may be particularly relevant for immunization against or treatment of cancer. In such cases, localized administration of the vaccine composition or vaccine adjuvant may have certain advantages over systemic administration. Among other things, localized administration means that systemic exposure to the vaccine or adjuvant is minimized, the RES and tumor vasculature barriers are bypassed and local / regional spread of cancer can be addressed more efficiently.

[0076] The vaccine adjuvant or vaccine composition may be administered as a single dose or as multiple doses. Each dose may preferably contain about 1 ng to 100 μg of imiquimod per dose; preferably at least about 1 ng, or at least about 5 ng, or at least about 10 ng, or at least about 50 ng, or at least about 100 ng, or at least about 500 ng, or at least about 1 μg, or at least about 5 μg, or at least about 10 μg of imiquimod. Additionally or alternatively, each dose may preferably contain about 100 μg or less, or about 75 μg or less, or about 50 μg or less, or about 25 μg or less, or about 20 μg or less, or about 10 μg or less, or about 5 μg or less, or about 1 μg or less of imiquimod. Each dose may suitably contain about 1-100 ng, or about 100 ng-1 μg, or about 1-10 μg, or about 10-100 μg of imiquimod.

[0077] Further encompassed within the scope of the present disclosure are vaccine adjuvants suitable for and / or provided for use in enhancing an immune response to a vaccine in a subject, and vaccine compositions suitable for and / or provided for use in inducing an immune response in a subject.Also provided are vaccine adjuvants according to the present disclosure for use in the manufacture of a composition for use in enhancing an immune response to a vaccine in a subject.Also provided are vaccine compositions according to the present disclosure for use in the manufacture of a composition for use in inducing an immune response in a subject.

[0078] The present disclosure further provides a method for producing the vaccine adjuvants disclosed herein, comprising the following sequential steps: (a) solubilizing imiquimod with a host molecule in an aqueous solution having a buffered pH of about pH 6 or less, preferably about pH 4-6, or about pH 4.5-6, or about pH 4-5.5, or about pH 5-6; (b) combining the resulting aqueous solution with lipids to form lipid shell nanoparticles encapsulating imiquimod; and (c) increasing the buffered pH of the formulation to about pH 6.5 or above, or to pH 7 or above, or to about pH 6.5-9, or to about pH 6.5-8.5, or to about pH 6.5-8, or to about pH 7-9, or to about pH 7-8.5, or to about pH 7-8, or to about pH 7.5-9.

[0079] This method allows for adjustment of the pH during the process to obtain a formulation at neutral (physiological) pH while minimizing or avoiding precipitation of unencapsulated imiquimod from the formulation. The method is compatible with the addition of antigens or polynucleotides as described herein.

[0080] Suitably, step (a) of the process may involve solubilising imiquimod at a buffered pH of about pH 4-6, or about pH 4.5-6, or about pH 4-5.5, or about pH 5-6. Step (a) may involve, for example, solubilising imiquimod in an aqueous solution in the presence of a hydroxy acid, such as citric acid, tartaric acid, lactic acid, glycolic acid or malic acid. Step (a) may involve solubilising imiquimod in an aqueous solution in the presence of a host molecule as disclosed herein, such as a cyclodextrin, in particular HP-β-CD. In particular, step (a) may involve combining imiquimod with a host molecule, such as a cyclodextrin, in a solution buffered to about pH 6 or less, preferably about pH 4-6, or about pH 4.5-6, or about pH 4-5.5, or about pH 3-5.5, or about pH 5-6, or about pH 5.

[0081] Step (b) may involve mixing the solution of (a) with lipids to form a solution or suspension of multilamellar structures, as disclosed herein, and processing these multilamellar structures to form lipid-shelled nanoparticles that encapsulate imiquimod. The lipids may optionally be solubilized in an alcohol solution. Processing the structures may include extruding the solution or suspension of multilamellar structures through a membrane to form lipid-shelled nanoparticles that encapsulate imiquimod, or drying the solution or suspension of multilamellar structures to form a film, solubilizing the film, and shaking or sonicating the resulting solution to form lipid-shelled nanoparticles that encapsulate imiquimod; or using microfluidic mixing techniques to form lipid-shelled nanoparticles that encapsulate imiquimod.

[0082] In other embodiments, step (b) may involve mixing the solution of (a) with empty liposomes to form lipid shell nanoparticles that encapsulate imiquimod. In this case, the empty liposomes may be formed from lipids as disclosed herein.

[0083] Step (c) of the process may involve raising the buffered pH of the formulation to about pH 6.5 or above, or to pH 7 or above, or to about pH 6.5-9, or to about pH 6.5-8.5, or to about pH 6.5-8, or to about pH 7-9, or to about pH 7-8.5, or to about pH 7-8, or to about pH 7.5-9. In some embodiments, the buffered pH of the formulation may be raised in step (c) by known techniques, such as diafiltration or buffer exchange.

[0084] The method may optionally further comprise the step of adding a hydrogel polymer, such as a PEG 4000 polymer, after step (a), as disclosed herein. The addition of the hydrogel polymer thus allows the hydrogel polymer to be incorporated into the aqueous core of the nanoparticle. Optionally, the method may not comprise the step of adding IL-2 or a protein cytokine, such as IL-2, to the formulation of (c) to load the nanoparticle with a protein cytokine.

[0085] In some embodiments, the method may further comprise, after step (b) and before step (c), depleting the nanoparticle formulation of (b) of unencapsulated imiquimod, which may optionally be done by ultracentrifugation or by diafiltration using a membrane sized to retain the nanoparticles but allow free imiquimod to pass through, or by other techniques known in the art.

[0086] The method may further include standard processing steps, including concentration adjustment, addition of suitable excipients, and sterilization. In particular, the method may include the step of adding one or more additional adjuvant components disclosed herein. The one or more additional adjuvant components may be added during or after step (a). In some embodiments, one or more, or all, additional ingredients may be added after step (c), after the buffered pH of the formulation is increased.

[0087] Once produced, the vaccine adjuvant may optionally be dried or lyophilized and / or subdivided into containers for storage or administration. Alternatively, the vaccine adjuvant may be further processed to obtain a vaccine composition according to the present disclosure, as further described below.

[0088] The present disclosure further provides a method for producing a vaccine composition as disclosed herein, comprising obtaining a vaccine adjuvant as disclosed herein and adding an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response. The step of adding the antigen or polynucleotide may, for example, include adding one or more ingredients of an approved vaccine, and in particular may include adding an approved vaccine formulation.

[0089] In some embodiments, the method may include producing a vaccine adjuvant according to the methods disclosed herein and adding an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response. The antigen and / or polynucleotide may be added during or after step (a). In particular, the antigen and / or polynucleotide may be added during step (a), or between step (a) and step (b), or during step (b), or between step (b) and step (c), or during step (c), or after step (c). In some embodiments, the method may include adding a polynucleotide during step (a), between step (a) and step (b), or during step (b). If the antigen is a small molecule or a substance or material that is not harmed by acidic pH, the method may include adding the antigen during step (a), between step (a) and step (b), or during step (b). If the antigen is or comprises a large molecule and / or is a substance or material that is impaired by acidic pH, the method may include adding the antigen after step (c). "Impaired" in this context includes any change that significantly affects the immunogenicity of the antigen.

[0090] In other embodiments, the method may include obtaining a vaccine adjuvant as disclosed herein and combining the vaccine adjuvant with an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response. The step of obtaining the vaccine adjuvant may optionally include manufacturing the vaccine adjuvant according to the method disclosed herein. Alternatively, the step of obtaining the vaccine adjuvant may include obtaining a previously manufactured vaccine adjuvant. The step of combining the vaccine adjuvant with the antigen and / or polynucleotide may include mixing the vaccine adjuvant with the antigen and / or polynucleotide. It is recognized that this may involve adding the vaccine adjuvant to the antigen and / or polynucleotide, or adding the antigen and / or polynucleotide to the vaccine adjuvant.

[0091] An example of a manufacturing process is illustrated in FIG. 4 and described in Examples 1-3. The illustrated and described process utilizes an acidic pH buffer and an optimized HP-β-CD concentration for incorporation into imiquimod:cyclodextrin nanoparticles during the extrusion process. The inventors have found that since acidic pH conditions are not compatible with lipid and antigen / polynucleotide stability and are not suitable for parenterally administered drugs, the product raw material should not be exposed to a pH below pH 4 during the manufacturing process, and the pH must be raised to a near neutral pH in the final product. This may or may not be before the addition of any antigen or polynucleotide. Since imiquimod solubility is significantly reduced at near neutral pH, the concentration of unencapsulated "free" imiquimod in the in-process solution must be reduced prior to any increase in pH to prevent precipitation of unencapsulated "free" imiquimod, which would block filters used for diafiltration and sterile filtration, preventing removal from the bulk product and preventing correct sterile filtration. The reduction in the concentration of the non-encapsulated "free" imiquimod can be achieved by methods such as ultracentrifugation or diafiltration using a membrane of suitable size that retains the nanoparticles but allows the passage of free imiquimod and cyclodextrin into the permeate.Diafiltration is a process that is well adapted to clinical and commercial scale drug manufacturing and may be preferred for this reason.The concentration of the non-encapsulated "free" imiquimod in the solution during the process can be reduced sufficiently to avoid precipitation at approximately neutral pH, and then the pH can be increased, and diafiltration can be continued to remove the outer cyclodextrin and imiquimod, and the product can be formulated into the final buffer of the formulation.

[0092] Vaccine adjuvants can be mixed with antigen or polynucleotide for the production of vaccine composition. This step can be carried out during or after the production of vaccine adjuvant. If polynucleotide is to be added, it may be effective to add polynucleotide in the early stage of adjuvant production. If large protein antigen or cellular antigen is to be added, it may be effective to add large protein antigen or cellular antigen in the later stage of adjuvant production or after, once pH is adjusted to approximately neutral as disclosed herein. This may be before or after all the required diafiltration and purification steps are completed, as described in Example 2.

[0093] A batch process performed according to cGMP standards can produce approximately 10 liters of vaccine adjuvant in the form of a sterile suspension of liposomes containing imiquimod at a concentration of approximately 10 μg / ml. This composition can be used as an adjuvant in a vaccine in combination with other vaccine ingredients, including antigens or polynucleotides encoding antigens.

[0094] Below are specific examples according to the present disclosure.

[0095] [Example 1] Nanoparticle Components Several raw materials and excipients important to the structure and quality of the formulation were used to produce the nanoparticles. With the exception of cholesterol (see below), all of the raw materials and excipients were synthetic or derived from plants.

[0096] The liposome shell was composed of three components. ●N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero 3-phosphoethanolamine sodium salt (MPEG-DSPE) ●Fully hydrogenated soy phosphatidylcholine (HSPC), and Cholesterol The aqueous core consisted of two components. ●2-Hydroxypropyl-β-cyclodextrin ●Polyethylene glycol 4000 hydrogel All of these ingredients are commercially available, known and well-characterized excipients.

[0097] [Example 2] Preparation of nanoparticle vaccine adjuvants containing imiquimod The steps of the process to generate imiquimod-loaded nanoparticles are illustrated in Figure 4. Imiquimod was solubilized in aqueous solution at pH 5 in the presence of HP-β-CD. A lipid solution was generated by dissolving fully hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero 3-phosphoethanolamine sodium salt (MPEG-DSPE) and cholesterol in ethanol at a molar ratio of approximately 1.9:0.1:1. This solution was mixed with the imiquimod solution to form multilamellar structures. These structures were then extruded through membranes of appropriate pore size (80 to 100 nm) to generate nanoparticles or "nanolipogels", which were core-shell structures with a lipid shell encapsulating imiquimod and HP-β-CD.

[0098] The nanolipogels were incubated with a solution of PEG-4000 in a pH 5 buffer to load the PEG-4000 inside the nanoparticles.

[0099] Diafiltration was performed to remove external imiquimod and cyclodextrin. A further diafiltration step was then performed to increase the pH of the formulation to pH 7.4. The resulting solution is suitable for use as a vaccine adjuvant.

[0100] [Example 3] Preparation of vaccine compositions To prepare a vaccine composition, the nanoparticles of Example 2 are mixed with a suitable vaccine antigen, such as a vaccine material containing the SARS-CoV-2 spike protein, in a solution at pH 7.4. The SARS-CoV-2 spike protein is utilized as an antigen in various approved vaccine formulations, including the NUVAXOVID® SARS-CoV-2 vaccine, and is commercially available.

[0101] The nanoparticles were further subjected to diafiltration using hollow fiber membranes (500Kd) and 1% trehalose PBS pH 7.4 buffer at 25° C. for 4 diafiltration volumes to further reduce the external cyclodextrin concentration, remove external imiquimod, and add 1% trehalose. This step may alternatively be performed during the production of the nanoparticles according to Example 2.

[0102] The concentration of the formulation is then adjusted and the formulation is sterilized to the standard required for therapeutic use. Sterilization is carried out by filtration using a 0.2 μm filter.

[0103] The formulation is supplied in a single dose concentration containing 10 μg / mL of imiquimod. It is a sterile, white, opaque liquid. The container closure system consists of a 5 mL clear borosilicate glass vial, a 13 mm synthetic chlorobutyl rubber stopper, and a flip-off crimp seal. The composition of IMP in a 5 ml vial is listed in Table 2 below.

[0104] [Table 2]

[0105] [Example 4] Properties of the formulation Cryo-TEM images of imiquimod-loaded nanoparticles shown in Figure 3. Cryo transmission electron microscopy (cryo TEM) demonstrates that the majority of these nanoparticles are spheres with a single lamellar outer shell. The cryo TEM images also provide information related to the state of the encapsulated drug and the internal environment of the nanoparticles. The nanoparticles have a denser interior than the surrounding buffer, which is consistent with the loading of the liposomes with imiquimod, HP-β-CD and PEG 4000 polymer in the interior of the liposomes.

[0106] [Example 5] Nonclinical toxicity and adjuvant efficacy studies The baseline study in this example indicates that imiquimod utilized in the presently disclosed nanoparticles is well tolerated in animals at doses exceeding the clinical doses disclosed herein, and studies demonstrating the adjuvant activity of imiquimod at doses similar to the amounts of imiquimod delivered by the nanoparticles disclosed herein.

[0107] Various studies have shown that topical imiquimod can improve vaccine-induced immune responses. Pretreatment with topical imiquimod also significantly improves the immunogenicity of influenza vaccination in both young and elderly individuals (Hung et al, Lancet Infect. Dis. 2016 Feb; 16(2):209-18). Similar results were also reported by Adams et al, J. Clin. Oncol. 25(18) suppl 8545, which evaluated the safety and adjuvant activity of imiquimod when administered with NY-ESO-1 protein vaccine. The topical administration of imiquimod in these studies allows for accurate assessment of the imiquimod dose delivered to the target immune cells.

[0108] In vitro treatment of macrophages with micelle-encapsulated imiquimod resulted in significant activation of NF-κB and MAPK pathways at a concentration of 0.2 μg / ml (Jimenez-Sanchez, et al 2014), indicating that local imiquimod concentrations of several hundred ng / ml activate the immune system. In vivo studies by Zhang et al. (Clinical and Vaccine Immunology 2014. 21:4 pp570 -579) found that 50 μg imiquimod injected intraperitoneally in mice in combination with influenza vaccine significantly accelerated and enhanced the humoral immune response against the virus and significantly protected the mice against early lethal viral challenge.

[0109] The FDA Pharmacology / Toxicology review of Zyclara (imiquimod) cream 3.75% (registration number 201153) described several animal studies in different species of the toxicology, pharmacokinetics, and metabolism of imiquimod, and findings from these studies include the following: - The lethal dose of a single subcutaneous dose in rats was 20 mg / kg. Intravenous doses of 0.5 to 5.0 mg / kg imiquimod produced some evidence of cardiac stimulation, central nervous system stimulation, and autonomic nervous system inhibition in dogs. Intravenous doses of 0.5, 1, and 2 mg / kg / day of imiquimod were administered to pregnant female rabbits during the period of organogenesis (days 6-18 of gestation). No treatment-related effects on embryofetal toxicity or teratogenicity were observed at 2 mg / kg / day.

[0110] The EMA's toxicology review of Aldara (Imiquimod) 5% cream (https: / / www.ema.europa.eu / en / documents / scientific-discussion / aldara-epar-scientific-discussion_en.pdf) describes several animal studies of the toxicology, pharmacokinetics and metabolism of imiquimod. Overall, it reports that "the toxicology program indicates a high degree of safety, with no target organ toxicity other than that attributable to excessive pharmacological activity. Imiquimod did not affect fertility and was not teratogenic or genotoxic. Carcinogenicity studies in mice showed no increase in the incidence of tumors or non-neoplastic lesions as a result of dermal exposure to imiquimod." Specific findings from these studies include: Single-dose toxicity of imiquimod studied in mice, rats, and monkeys indicates a high degree of safety. Adverse effects limited to the central nervous system produced several signs before death, usually convulsions. - Two dermal toxicity studies in rabbits using doses of 2000 and 5000 mg / kg under occlusion showed no mortality and no signs of toxicity other than mild, transient erythema at the application site. Repeated-dose toxicity of imiquimod after oral administration for up to 6 months in rats and monkeys showed only minor adverse effects, i.e. hyperplasia of B- and T-cell lymphoid tissues, increased plasma cell counts, spleen and lymph node enlargement, Kupffer cell hyperplasia, mononuclear / macrophage cell accumulation or proliferation, apart from minor effects on body weight and food consumption that could be the result of excessive pharmacological activity. In both species, there were no other target organs and a no observed adverse effects level (NOAEL) of 3 mg / kg was established.

[0111] The maximum imiquimod dose of the nanoparticles disclosed herein is several thousand times lower than the current dose step or MRHD of the approved drug based on multiple animal studies, or a well-tolerated 30 mg subcutaneous dose in healthy human volunteers (Soria, Myhre, et al., 2000). See also Table 3, which compares the amount of imiquimod in a proposed 100 μl dose of the disclosed nanoparticles (NPs) with the approved safe dose of Aldara® (imiquimod).

[0112] [Table 3] Lipid nanoparticles with hydrogel cores utilized in embodiments of the present disclosure have been shown to be preferentially taken up by antigen presenting cells (APCs), providing advantages in immunomodulation. Look et al, Biomaterials 35(2014) 1089-1095 showed that lipid nanoparticles with hydrogel cores were subject to more efficient uptake by dendritic cells compared to PLGA nanoparticles, demonstrating a >100x fold increase in uptake by flow cytometry analysis and confocal imaging.

[0113] [Example 6] In vitro release assay An in vitro release assay (IVRA) was developed for imiquimod. The results of the IVRA are illustrated in Figure 5, where nanoparticles produced according to Examples 1-3 were diluted with an equal volume of PBS buffer and incubated at 37°C with gentle shaking. Samples were removed at 0, 4, 6 and 24 hours and immediately processed using a 300 kd filter to obtain the released free (unencapsulated) imiquimod in the filtrate. A reverse phase HPLC assay was used to determine the concentration of imiquimod in the filtrate.

[0114] The results showed that imiquimod was released from the nanolipogels at a linear rate over time, with approximately 25% of the drug released after 24 hours and expected to be fully released over several days. The in vitro results demonstrate the unexpected ability of the nanolipogels to provide sustained release and delivery of effective amounts of imiquimod over an extended period of several days, and compare highly favorably with prior art imiquimod formulations, where imiquimod was found to be released much more rapidly. Studies have shown that the immune stimulatory effects of TLR7 / 8 agonists, such as imiquimod, are improved by sustained delivery (Auderset et al Front Immunol. 2020 Nov 11;11:580974). The delayed release achieved by the formulations disclosed herein therefore improves the adjuvant effect of the formulation when administered as part of a vaccine.

[0115] [Example 7] Use in community vaccination settings A dose of the vaccine composition of Example 3 is administered by parenteral intramuscular injection into the upper arm of a patient at risk of contracting COVID-19 disease. The adjuvant component functions to enhance the protective immune response induced by the vaccine.

Claims

1. 1. A vaccine adjuvant comprising a plurality of nanoparticles comprising an outer lipid shell and an inner aqueous core encapsulated within said outer lipid shell, said inner aqueous core comprising an imidazoquinoline and a host molecule capable of reversibly forming a complex with the imidazoquinoline, The imidazoquinoline has the following structure: 【Chemistry 2】 wherein R 1 is N, R 2 is H or C, and said imidazoquinoline is unsubstituted or substituted at one or more of the indicated points of attachment with one or more substituents independently selected from branched, straight chain or cyclic alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl groups, in particular C 1-10 alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl groups, or hydroxyl groups, or amine groups, or N—(C 1-10 alkyl)methanesulfonamido groups. is an active TLR7 / 8 ligand having the formula The host molecule is a cyclodextrin. Vaccine adjuvant.

2. The vaccine adjuvant described in claim 1, wherein the imidazoquinoline is imiquimod R-837, resiquimod, gardiquimod, S28690, 852-A, 854A, CL075, or CL097.

3. The inner aqueous core of the nanoparticles comprises a hydrogel, which hydrogel is optionally selected from the group consisting of poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); poly(glycolide-co-caprolactone); polycarbonates; polyamides, polypeptides, and poly(amino acids); polyesteramides; other biocompatible polyesters; poly(dioxanone); poly(amino acids). poly(alkylene alkylates); hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polysiloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), polyvinyl alcohol, polyvinylpyrrolidone; poly(alkylene oxides); cellulose, polyacrylic acid, albumin, collagen, gelatin, prolamines, polysaccharides, derivatives, copolymers and blends thereof; and / or The host molecule may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl α-cyclodextrin, methyl β-cyclodextrin, methyl γ-cyclodextrin, ethyl β-cyclodextrin, butyl α-cyclodextrin, butyl β-cyclodextrin, butyl γ-cyclodextrin, pentyl γ-cyclodextrin, hydroxyethyl β-cyclodextrin, hydroxyethyl γ-cyclodextrin, 2-hydroxypropyl α- Cyclodextrin; 2-hydroxypropyl β-cyclodextrin; 2-hydroxypropyl γ-cyclodextrin; 2-hydroxybutyl β-cyclodextrin; acetyl α-cyclodextrin; acetyl β-cyclodextrin; acetyl γ-cyclodextrin; propionyl β-cyclodextrin; butyryl β-cyclodextrin; succinyl α-cyclodextrin; succinyl β-cyclodextrin; succinyl γ-cyclodextrin; benzoyl β-cyclodextrin Cyclodextrin; Palmityl β-cyclodextrin; Toluenesulfonyl β-cyclodextrin; Acetyl methyl β-cyclodextrin; Acetyl butyl β-cyclodextrin; Glucosyl α-cyclodextrin; Glucosyl β-cyclodextrin; Glucosyl γ-cyclodextrin; Maltosyl α-cyclodextrin; Maltosyl β-cyclodextrin; Maltosyl γ-cyclodextrin; α-Cyclodextrin carboxymethyl ether; β-Cyclodextrin carboxymethyl ether; γ-Cyclodextrin carboxymethyl ether; Carboxymethylethyl β-cyclodextrin; Phosphate ester α-cyclodextrin; Phosphate ester β-cyclodextrin; Phosphate ester γ-cyclodextrin; 3-Trimethylammonium-2-hydroxypropyl β-cyclodextrin; Sulfobutyl ether β-cyclodextrin; Carboxymethyl α-cyclodextrin; Carboxymethyl β-cyclodextrin;3. The vaccine adjuvant according to claim 1 or 2, comprising a cyclodextrin selected from the group consisting of carboxymethyl γ-cyclodextrin, carboxymethyl γ-cyclodextrin, and combinations thereof, advantageously wherein said cyclodextrin is or comprises 2-hydroxypropyl-β-cyclodextrin; 4. A vaccine adjuvant as described in claim 1 or 2, comprising an imidazoquinoline complexed with the host molecule, optionally wherein the imidazoquinoline is present in the form of an inclusion complex with the host molecule.

5. an aqueous solution, dispersion or suspension of nanoparticles, optionally the solution, suspension or dispersion is buffered to a pH of at least about 6.5, preferably at least pH 7, suitably about pH 6.5-9, or about pH 6.5-8.5, or about pH 6.5-8, or about pH 7-9, or about pH 7-8.5, or about pH 7-8, or about pH 7.5-9; and / or the solution, suspension or dispersion is free of unencapsulated imidazoquinoline and / or free of unencapsulated, non-solubilized imidazoquinoline; and / or the solution, suspension or dispersion comprises one or more additional adjuvant components, the one or more additional adjuvant components optionally comprising liposomes; 3. The vaccine adjuvant according to claim 1 or 2.

6. (a) an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response; and (b) comprising nanoparticles; A vaccine composition comprising the vaccine adjuvant of claim 1.

7. Some or all of the antigen and / or polynucleotide are releasably attached, bound, and / or encapsulated within the outer lipid shell of the nanoparticle; and / or the polynucleotide is a DNA molecule or an RNA molecule, such as an mRNA molecule; and / or the antigen is a viral, bacterial, fungal, or disease- or cancer-associated antigen and / or the polynucleotide encodes a viral, bacterial, fungal, or disease- or cancer-associated antigen; and / or the antigen is a peptide, protein, carbohydrate, nucleic acid and / or lipid molecule or structure, and / or the polynucleotide encodes a peptide or protein antigen; and / or the antigen is a coronavirus or coronavirus-related antigen, such as a SARS-CoV, MERS-CoV or SARS-CoV-2 antigen; or an influenza or influenza-related antigen, for example an influenza A, influenza B, influenza C or influenza D antigen; or a herpes simplex (HSV-1 or HSV-2) or HSV-related antigen; or a cytomegalovirus (CMV) or CMV-related antigen; or a Lyme disease (Borrelia) or Lyme disease-related antigen; or respiratory syncytial virus (RSV) or an RSV-related antigen; or Epstein-Barr virus (EBV) or an EBV-related antigen; or Zika virus or a Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles or a measles-related antigen; or mumps or a mumps-related antigen; or rubella or a rubella-related antigen; or chickenpox (varicella) or a chickenpox-related antigen; or herpes zoster (shingles) or a shingles-related antigen; or diphtheria or a diphtheria-related antigen; or tetanus or a tetanus-related antigen; or acute poliomyelitis or an acute poliomyelitis-related antigen; or dengue virus or a dengue virus-related antigen; or Haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus or a rotavirus-related antigen; or Streptococcus pneumoniae (Streptococcus pneumoniae) (Streptococcus) or Streptococcus-related antigens; or human papillomavirus (HPV) or HPV-related antigens; or whooping cough or whooping cough-related antigens; or hepatitis or hepatitis-related antigens; or tuberculosis or tuberculosis-related antigens; or human immunodeficiency virus (HIV) or HIV-related antigens; or adenovirus or adenovirus-related antigens; or anthrax or anthrax-related antigens; or cholera or cholera-related antigens; or Japanese encephalitis (JE) or JE-related antigens; or rabies or rabies-related antigens; or smallpox or smallpox-related antigens; or or typhoid fever (typhoid fever) or typhoid fever-related antigen; or yellow fever or yellow fever-related antigen; or Ebola or Ebola-related antigen; or cancer or cancer-related antigen, and / or said polynucleotide is a coronavirus or coronavirus-related antigen, such as a SARS-CoV, MERS-CoV or SARS-CoV-2 antigen; or influenza or influenza-related antigen, for example an influenza A, influenza B, influenza C or influenza D antigen; or herpes simplex (HSV-1 or HSV-2) or HSV-related antigen; or cytomegalovirus (CMV) or CMV-related antigen;or Lyme disease (Borrelia) or Lyme disease-related antigens; or respiratory syncytial virus (RSV) or an RSV-related antigen; or Epstein-Barr virus (EBV) or an EBV-related antigen; or Zika virus or a Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles or a measles-related antigen; or mumps or a mumps-related antigen; or rubella or a rubella-related antigen; or chickenpox (varicella) or a chickenpox-related antigen; or herpes zoster (shingles) or a shingles-related antigen; or diphtheria or a diphtheria-related antigen; or tetanus or a tetanus-related antigen; or acute poliomyelitis or an acute poliomyelitis-related antigen; or dengue virus or a dengue virus-related antigen; or Haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus or a rotavirus-related antigen; or Streptococcus pneumoniae (Streptococcus pneumoniae (Streptococcus) or a Streptococcus-related antigen; or human papillomavirus (HPV) or an HPV-related antigen; or pertussis or a pertussis-related antigen; or hepatitis or a hepatitis-related antigen; or tuberculosis or a tuberculosis-related antigen; or human immunodeficiency virus (HIV) or an HIV-related antigen; or adenovirus or an adenovirus-related antigen; or anthrax or anthrax-related antigen; or cholera or a cholera-related antigen; or Japanese encephalitis (JE) or a JE-related antigen; or rabies or a rabies-related antigen; or smallpox or a smallpox-related antigen; or typhoid fever (typhoid) or a typhoid-related antigen; or yellow fever or a yellow fever-related antigen; or Ebola or an Ebola-related antigen; or cancer or a cancer-related antigen.

8. 7. A vaccine composition according to claim 6 for use in a method for inducing an immune response in a subject, comprising: The method comprises administering to the subject the vaccine composition, and optionally the vaccine composition is administered to the subject by intravenous, intramuscular or subcutaneous injection, or orally, intranasally, intradermally, intraperitoneally or transdermally; Vaccine compositions.

9. The vaccine adjuvant of claim 1 for use in a method for inducing an immune response in a subject, comprising: The method comprises administering to the subject the vaccine adjuvant together with a vaccine, and optionally the vaccine adjuvant is administered to the subject by intravenous, intramuscular, or subcutaneous injection, or orally, intranasally, intradermally, intraperitoneally, or transdermally; Vaccine adjuvant.

10. 10. A vaccine composition according to claim 8 or a vaccine adjuvant according to claim 9 for immunising said subject against a viral, bacterial or fungal infection, colonisation or disease, or against a proliferative disorder, such as cancer.

11. The following sequential steps: (a) solubilizing the imidazoquinoline with said host molecule in an aqueous solution at a pH buffered to about pH 6 or less; preferably about pH 4-6, or about pH 4.5-6, or about pH 4-5.5, or about pH 5-6; (b) combining the resulting aqueous solution with lipids to form lipid-shelled nanoparticles encapsulating the imidazoquinoline; and (c) increasing the buffered pH of the formulation to about pH 6.5 or greater, or to pH 7 or greater, or to about pH 6.5-9, or to about pH 6.5-8.5, or to about pH 6.5-8, or to about pH 7-9, or to about pH 7-8.5, or to about pH 7-8, or to about pH 7.5-9 2. A method for producing the vaccine adjuvant of claim 1, comprising:

12. step (b) comprising mixing said solution of (a) with lipids, optionally solubilized in an alcohol solution, to form a solution or suspension of multilamellar structures, and processing these structures to form lipid-shelled nanoparticles encapsulating the imidazoquinoline; Optionally, the step of fabricating the structure further comprises: (i) extruding said solution or suspension of multilamellar structures through a membrane to form lipid-shelled nanoparticles encapsulating the imidazoquinoline; or (ii) drying the solution or suspension of multilamellar structures to form a film, solubilizing the film, and shaking or sonicating the resulting solution to form lipid-shelled nanoparticles encapsulating the imidazoquinoline; or (iii) using microfluidic mixing techniques to form lipid shell nanoparticles encapsulating imidazoquinolines; 12. The method of claim 11, comprising any one of:

13. step (b) comprising mixing said solution of (a) with empty liposomes to form lipid-shelled nanoparticles encapsulating the imidazoquinoline; and / or increasing the buffered pH of the formulation in step (c) by diafiltration or buffer exchange; and / or step (a) comprises solubilising the imidazoquinoline in aqueous solution in the presence of a hydroxy acid, such as citric acid, tartaric acid, lactic acid, glycolic acid or malic acid, optionally in the presence of said host molecule; and / or step (a) comprises combining the imidazoquinoline with the host molecule in a solution at a pH of about 4 to 5.5, preferably at a pH of about 5; and / or and / or further comprising the step of adding a hydrogel polymer, such as a PEG polymer, during or after step (a); and / or further comprising, after step (b) and before step (c), optionally depleting the nanoparticle formulation of (b) of unencapsulated imidazoquinoline by ultracentrifugation or diafiltration using a membrane sized to retain the nanoparticles but allow free imidazoquinoline to pass through; and / or The method does not include the step of adding a protein cytokine, such as IL-2, to the formulation of (c) to load the nanoparticles with the protein cytokine; 13. The method of claim 11 or 12.

14. 7. A method for producing a vaccine composition according to claim 6, comprising providing a vaccine adjuvant according to claim 1 and adding an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response, optionally comprising: A method wherein said step of adding an antigen or polynucleotide comprises adding one or more or all components of an approved vaccine.

15. 15. The method of claim 14, comprising producing an adjuvant according to the method of claim 11 and adding, during or after step (a), an antigen capable of inducing an immune response and / or a polynucleotide encoding an antigen capable of inducing an immune response.

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