CN118119375A

CN118119375A - Nanoparticle formulations

Info

Publication number: CN118119375A
Application number: CN202280068770.2A
Authority: CN
Inventors: B·霍斯伯勒; M·D·穆迪
Original assignee: New Immunology Second Co ltd
Current assignee: New Immunology Second Co ltd
Priority date: 2021-10-11
Filing date: 2022-10-11
Publication date: 2024-05-31

Abstract

The present disclosure relates to nanoparticle vaccine adjuvants and vaccine compositions containing the nanoparticle vaccine adjuvants; methods of making the adjuvants and compositions; and methods of vaccinating using the compositions and adjuvants. The vaccine adjuvants disclosed herein are effective in enhancing the immune response to vaccination.

Description

Nanoparticle formulations

Technical Field

Background

Vaccination is an important public health measure, as highlighted by the recent pandemic of SARS-CoV-2. Effective vaccines against infectious diseases, if widely vaccinated in humans, can both slow down the spread of the disease and reduce the severity of symptoms experienced by vaccinated humans. Vaccines are also effective in the treatment of certain types of diseases, including proliferative diseases such as cancer.

Vaccines function by inducing an immune response that serves to protect the vaccinated individual. The immune system is stimulated by exposure to a suitable immunogen (e.g., pathogen antigen or cancer-associated antigen), which directly or indirectly elicits a protective adaptive immune response. The adaptive immune response may be mediated by B cells and/or T cells. The aim may be to provide long-term immunity against the antigen and/or the pathogen, cell or entity carrying the antigen.

Several different types of vaccines have been developed. Vaccines developed for the prevention of infectious diseases include inactivated vaccines and attenuated live vaccines; toxoid vaccine; a viral vector vaccine; subunits, recombinations, polysaccharides and conjugate vaccines; and mRNA vaccines. Vaccines developed for the prevention of cancer include autologous patient derived immune cell vaccines, recombinant viral 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 encoding an antigen capable of inducing an immune response.

Many vaccines also contain an adjuvant component. These agents are capable of enhancing vaccine-induced immune responses. This may be important, especially in cases where the vaccine antigen has low immunogenicity, or where the vaccine is administered to immunocompromised, immunocompromised or immunocompromised patients (e.g. infants or elderly). The boosting effect of the adjuvant may also allow for a reduction in vaccine dose per patient, which is important to save vaccine in case of insufficient vaccine. Vaccine adjuvants approved for use in humans include aluminum-based mineral salts (Alum), MF59, monophosphoryl lipid A (MPL), and CpG oligodeoxynucleotides (CpG 1018).

Another well-known vaccine adjuvant is imiquimod (R-837). 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-is a small molecule imidazoquinoline drug having the structural formula:

Various active structural analogs of imiquimod (R-837) have been synthesized and characterized. These include the imidazoquinolines resiquimod (R-848), galdammod (gardiquimod), CL097, S28690, 852-A and 854A; thiazoloquinolone CL075,075; and others, including those shown in table 1 below:

TABLE 1

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

These structural analogs of imiquimod (R-837) are active TLR7/8 ligands that have properties and activity similar to imiquimod (R-837), optionally including pH dependent solubility. In the present disclosure, the term "imiquimod" hereinafter means imiquimod (R-837); but also include and refer 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, the structural analogue of imiquimod (R-837) may exhibit pH dependent solubility, with increased solubility at lower pH. Thus, the term "imiquimod" as used herein includes imidazoquinolines (imidazoquinolines), which are active TLR7/8 ligands and have a basic molecular structure:

Wherein R ¹ is typically N and R ² is typically H or C and wherein the imidazoquinoline is optionally substituted at the addition point indicated by one or more arrows with one or more substituents which may be independently selected from branched, straight or cyclic alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl, in particular C _1-10 alkyl, alkenyl, alcohol, alkylamine, alkoxy or alkoxyalkyl, or hydroxy, or amine, or N- (C _1-10 alkyl) methanesulfonamide. The term "imiquimod" as used herein also includes compact structural derivatives of these imidazoquinolines, including thiazoloquinoline (thiazoloquionoline) derivatives, which are active TLR7/8 ligands.

Imiquimod stimulates the innate and adaptive immune system by activating Toll-like receptors 7 and/or 8 (TLR 7/8). It is approved by the FDA as two topical cream formulationsAnd/>Is an active ingredient of (a).

Various studies have shown that topical imiquimod can enhance vaccine-induced immune responses. In particular, topical imiquimod enhances antibody and cellular responses to subcutaneous immunity to ovalbumin; the immune response shifted to the Th1 phenotype, with significantly enhanced IgG2a, igG2b and CD8+ T cell responses (Johnston et al, vaccine 2006Mar 10;24 (11): 1958-65). Pretreatment with topical imiquimod also significantly increased the immunogenicity of influenza vaccinations in young and old humans (Hung et al LANCET INFECT Dis.2016Feb;16 (2): 209-18). Similar results were also reported by Adams et al j.clin.oncol.25 (18) suppl.8545, assessing safety and adjuvant activity of imiquimod when administered with an NY-ESO-1 protein vaccine.

These studies demonstrate that imiquimod is effective as a vaccine adjuvant. However, as reported in these studies, topical application of imiquimod in the form of a cream is inconvenient and not feasible for conventional vaccination. Topical application causes compliance problems because the cream must remain on the skin for several hours to function. It may cause skin irritation or be intolerable to the patient for other reasons. Consistency of imiquimod delivery is also a problem. For practical purposes, it is preferred that the adjuvant is formulated with other vaccine components for administration as a single vaccine formulation. The adjuvant is preferably formulated as an injectable composition.

However, the formulation of imiquimod for injection is not a simple task. At physiological pH, the solubility of imiquimod in aqueous solutions is very low. FIG. 1 illustrates the water solubility of imiquimod as a function of pH using Britton-Robinson buffer; imiquimod was shown to have maximum solubility in aqueous solutions at pH 2 or below (Chollett et al Pharm. Dev. Technology 1999Jan;4 (1): 35-43). As the pH rises above 2, the water solubility of imiquimod drops dramatically and only slightly at pH above 6. Thus, efforts to develop aqueous or water-based formulations of imiquimod have generally involved the use of acidic solvents below pH 4, which is unacceptable for injection. Hayashi et al (int.J.urol.2010, 5; 17 (5): 483-90) describe an imiquimod formulation using 0.1M lactic acid, poloxamer and HP-beta-CD at low acidic pH. Guedes et al (J. Braz. Chem. Soc., vol.31, no.8,1732-1745,2020) similarly describe the solubilization of imiquimod in beta-cyclodextrin at pH 3 in the presence of citric acid (solubilisation). Ramineni et al (J.Pharm. Sci.2013, month 2; 102 (2): 593-603) developed an adhesive film containing imiquimod and HP-beta-CD using a mixture of acetate buffer at pH 4 and methanol. Fox et al (Journal ofNanobiotechnology 2014, 12:17) also disclose an anionic liposome formulation comprising imiquimod dissolved in the lactic acid core at pH 2.5-3.5.

Although imiquimod may be formulated as a water-based formulation at low pH as described in these and similar references, this approach does not readily allow imiquimod to be co-formulated with vaccine antigens and polynucleotides that denature or alter their form at low pH. Low pH formulations are also unsuitable for parenteral administration.

In another approach, a polylactic acid (PLA) -based micelle core is loaded with imiquimod and surface functionalized with an antigen protein (HIV-1 gag p 24) for antigen delivery purposes (Jimenez-S nchez et al Pharm.Res. (2015) 32:311-320). Imiquimod is encapsulated in a hydrophobic PLA core, while the p24 antigen is covalently linked to the N-succinimidyl pendant group of the micelle crown through lysine and an N-terminal amine group. However, the release of imiquimod from the particles was found to be very fast (50% in 1 hour, about 75% in 4 to 5 hours). The slower rate of adjuvant release of the nanoparticulate form is believed to potentially support a more effective immune response.

Against this background, the present inventors have successfully developed and described herein a nanoparticulate vaccine adjuvant comprising imiquimod which is suitable for injection and which is capable of providing sustained release of imiquimod over an extended period of time, which may improve the performance of imiquimod as a vaccine adjuvant. The disclosed nanoparticle vaccine adjuvants may be formulated with existing vaccines or with vaccine antigens and/or polynucleotides, if desired with additional vaccine components; thus also provided are vaccine compositions comprising imiquimod vaccine adjuvants, which can be administered parenterally, including by injection.

Disclosure of Invention

According to one aspect, 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; wherein the internal aqueous core comprises imiquimod and a host molecule capable of reversibly forming a complex with imiquimod. The vaccine adjuvant may be or may comprise an aqueous solution, aqueous dispersion or aqueous suspension of the disclosed nanoparticles, or may be or may comprise a dry or lyophilized formulation that can be hydrated to produce an aqueous solution, aqueous dispersion or aqueous suspension of the disclosed nanoparticles. The inner aqueous core may have a pH of about 6.5 or higher, and/or may comprise a hydrogel. Imiquimod and host molecules may be dispersed within the hydrogel.

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 an antigen capable of inducing an immune response; and (b) a vaccine adjuvant comprising nanoparticles according to the present disclosure. The vaccine composition may be or may comprise an aqueous solution, aqueous dispersion or aqueous suspension of the disclosed components (a) and (b), or may be or may comprise a dry or lyophilized formulation that may 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) may be releasably linked to, associated with, and/or encapsulated within the outer lipid shell of the nanoparticle of component (b). In addition or alternatively, component (a) may comprise a delivery vehicle, such as a nanoparticle delivery vehicle, e.g. a plurality of nanoparticles comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell, wherein the antigen and/or polynucleotide is loaded into or onto the delivery vehicle.

The vaccine composition may comprise further ingredients and excipients, including further adjuvants. In some embodiments, the vaccine composition may comprise some or all of the active ingredient and/or excipient ingredients of a vaccine formulation that has been developed for prophylactic or therapeutic use; such as approved vaccine formulations. Conveniently, 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. The vaccine compositions of the present disclosure may comprise 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, supplemented with a vaccine adjuvant comprising nanoparticles according to the present disclosure.

In a further aspect, the present disclosure provides a vaccine adjuvant as disclosed for use in a method of enhancing an immune response to a vaccine in a subject (e.g., a human subject). The present disclosure further provides methods of enhancing an immune response to a vaccine in a subject (e.g., a human subject), comprising the step of administering a vaccine adjuvant disclosed herein to the subject, wherein the vaccine adjuvant is administered to the subject prior to, concurrently with, and/or after the vaccine administration. The vaccine may be any vaccine capable of inducing an immune response in a subject. The immune response may, for example, be a protective immune response capable of protecting a 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 may be a therapeutic immune response that is capable of alleviating or reducing the symptoms or manifestations of a disease or disorder, including an infectious disease or disorder.

In a further aspect, the present disclosure provides a vaccine composition as disclosed for use in a method of inducing an immune response in a subject (e.g., a human subject). The present disclosure also provides methods of inducing an immune response in a subject, e.g., a human subject, comprising the step of administering to the subject a vaccine composition disclosed herein. The immune response may, for example, be a protective immune response capable of protecting a 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 may be a therapeutic immune response that is capable of alleviating or reducing the symptoms or manifestations of a disease or disorder, including an infectious disease or disorder.

The present invention also provides a method for manufacturing the disclosed vaccine adjuvant comprising the following sequential steps:

(a) Dissolving imiquimod with a host molecule in an aqueous solution buffered to a pH of about pH 6 or less; preferably to about pH 4-6, or to about pH 4.5-6, or to about pH 4-5.5, or to about pH 5-6;

(b) Combining the resulting aqueous solution with a lipid to form lipid shell nanoparticles encapsulating imiquimod; and then

(C) Increasing the buffered pH of the formulation to about pH 6.5 or higher, or to pH 7 or higher, 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.

The present disclosure provides a method for manufacturing the disclosed vaccine composition, comprising the steps of: the disclosed vaccine adjuvants are combined 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 adjuvants 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 manufacturing the disclosed vaccine composition, comprising the steps of: manufacturing 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 during or after step (a).

The disclosed vaccine adjuvants and vaccine compositions buffered to physiological pH can be administered parenterally to a subject, optionally by injection; and may be used in or in the course of immunization against a variety of diseases, disorders and pathogens, including infectious diseases and pathogens and cancer.

The compositions of the present disclosure are designed to facilitate controlled, sustained, and optionally local delivery and/or release of imiquimod to enhance an immune response to a vaccine. The disclosed adjuvants are capable of providing sustained delivery and/or release of imiquimod over a period of time, which allows for improved enhancement of immune responses.

The disclosed vaccine adjuvants comprise a small molecule imiquimod complexed with a host molecule and encapsulated in a liposome. Imiquimod (R-837) is contained in approved and marketed drugsAnd/>) And are well characterized and understood by medical oncologists. Research and clinical trials have shown efficacy of imiquimod as a vaccine adjuvant, although it has not previously been formulated for convenient parenteral administration as well as for controlled delivery and release as disclosed herein.

Drawings

Fig. 1 shows a graph illustrating the effect of pH on the solubility of imiquimod in an aqueous solution.

Fig. 2 shows the structure of an exemplary vaccine adjuvant nanoparticle according to the present disclosure.

Figure 3 shows a frozen TEM image of vaccine adjuvant nanoparticles according to the present disclosure.

Fig. 4 shows a flow chart illustrating the steps of an exemplary method for producing a vaccine adjuvant according to the present disclosure.

Fig. 5 shows the results of a release test illustrating the release profile of imiquimod from a vaccine adjuvant according to the present disclosure.

Detailed Description

Definition of the definition

Unless otherwise indicated, terms used in this disclosure should be understood to have their normal meaning in the art. In particular, "antigen" shall include any molecule or entity capable of inducing an immune response in a human and/or animal. "Polynucleotide" shall include any nucleic acid comprising more than one nucleotide. An "immune response" shall include any protective or defensive response of the immune system to an antigen, including innate, adaptive and responsive immune responses, type 1 and type 2 responses, cell-mediated, and humoral and inflammatory immune responses. In the context of the present disclosure, an "immune response" is generally understood to mean a protective or therapeutic immune response, and/or an immune response effective to prevent or treat a disease, disorder, or medical condition. "immunization" shall include the process of inducing an immune response, in particular a protective immune response, against an antigen in a subject; after effective immunization, the subject is "immunized". A "vaccine" is a substance or composition that can be used to immunize a subject. A "vaccine adjuvant" is a substance that can effectively expand, enhance, amplify, modulate, augment, or in any way improve the immune response induced by a vaccine. A "hydrogel" is a matrix of a water-swellable, hydrophilic polymer, optionally crosslinked; "hydrogel polymer" is to be construed accordingly. The term "imiquimod" means imiquimod (R-837); but also encompasses and refers to structural analogs of imiquimod (R-837) as defined above, which are TLR7/8 agonists or TLR7/8 ligands and may exhibit pH dependent solubility, including but not limited to imidazoquinolines and thiazoloquinolones as shown in table 1.

Detailed Description

The present disclosure provides vaccine adjuvants comprising a plurality of nanoparticles, each nanoparticle comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell; wherein the internal aqueous core comprises imiquimod and a host molecule capable of reversibly forming a complex with imiquimod. The aqueous core may comprise imiquimod complexed with a host molecule. The aqueous core of the nanoparticle may comprise uncomplexed imiquimod and/or a host molecule.

The inner aqueous core may optionally comprise a hydrogel. Imiquimod and host molecules may optionally be dispersed, dissolved or suspended in the hydrogel.

The internal aqueous core may have a pH of about 6.5 or higher. Suitably, the internal aqueous core may have a pH of at least 7, or a pH of at least 7.5. The internal aqueous core may have a pH of no more than about pH 9 or no more than about pH 8.5; or may suitably have a pH of about pH 6.5-9 or about 6.5-8.5 or about 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.

The outer lipid shell of the nanoparticle comprises one or more lipid layers or bilayers, which surround the central core. The shell-forming lipids may be neutral, zwitterionic, anionic or cationic lipids at physiological pH. The lipids within and/or between each lipid layer or bilayer may optionally be cross-linked. Thus, the outer lipid shell may consist of one or more concentric lipid layers, optionally crosslinked, wherein the lipid may be a neutral, anionic or cationic lipid at physiological pH. The composition of the lipid shell and the degree of cross-linking within or between the lipid layers can be varied to alter and optimize the release profile of imiquimod from the nanoparticle.

In some advantageous embodiments, one or more lipid layers or bilayers may comprise a lipid selected from cholesterol, phospholipids, lysolipids, lysophospholipids, and sphingolipids, and derivatives thereof. Suitable lipids include, but are not limited to, phosphatidylcholine (PC) (e.g., egg PC, soy PC), including 1, 2-diacyl-propanetriyl-3-phosphorylcholine; phosphatidylserine (PS); phosphatidylglycerol; phosphatidylinositol (PI); a glycolipid; sphingomyelins (sphingophospholipids), such as sphingomyelin; glycosphingolipids (also known as 1-ceramide-based glucosides), such as ceramide galactopyranoside, ganglioside, and cerebroside; a fatty acid; sterols containing carboxylic acid groups, such as cholesterol or derivatives thereof; and 1, 2-diacyl-sn-propan-3-phosphate ethanolamines (l, 2-diacyl-sn-glycero-3-phosphoethanolamines), including 1, 2-dioleoyl-sn-propan-3-phosphate ethanolamine or 1, 2-dioleoyl-propan-3-phosphate ethanolamine (DOPE), 1, 2-bis-hexadecyl phosphate ethanolamine (DHPE), 1, 2-distearoyl phosphatidylcholine (DSPC), 1, 2-dipalmitoyl phosphatidylcholine (DPPC), and 1, 2-dimyristoyl phosphatidylcholine (DMPC). Suitable lipids also include natural lipids, such as L- α -phosphatidyl of the following tissue origin: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1, 2-diacyl-SN-propanetriyl-3-phosphorylcholine, l-acyl-2-acyl-SN-propanetriyl-3-phosphorylcholine, l, 2-diheptanoyl-SN-propanetriyl-3-phosphorylcholine) derivatives of these lipids.

The external lipid shell may also or alternatively comprise a cationic lipid including, but not limited to, N- [1- (2, 3-dioleoyloxy) propyl ] -N, N-trimethylammonium salt, also known as TAP lipid, e.g. methylsulfate. Suitable TAP lipids include, but are not limited to DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Other suitable cationic lipids include Dioctadecyl Dimethyl Ammonium Bromide (DDAB), 1, 2-diacyloxy-3-trimethylpropane ammonium, N "[1- (2, 3-diacyloxy) propyl ] -N, N-dimethylamine (dotap), 1, 2-diacyloxy-3-dimethylpropane ammonium, N- [1- (2, 3-dioleyloxy) propyl ] -N, N-trimethylammonium chloride (DOTMA), 1, 2-dialkoxy-3-dimethylpropane ammonium, dioctadecyl aminoglycinamide (DOGS), 3- [ N- (N '5N' -dimethylamino-ethane) carbamoyl ] cholesterol (DC-Chol); 2, 3-Dioleoyloxy-N- (2- (spermioylamino) -ethyl) -N, N-dimethyl-1-propylamine trifluoroacetic acid (DOSPA), beta-alanylcholesterol, cetyltrimethylammonium bromide (CTAB), di-C ₁₄ -amidine, N-tert-butyl-N '-tetradecyl-3-tetradecylaminopropionamidine (N-tert-butyl-N' -tetradecyl-3-

Tetradecylamino-propionamidine), N- (alpha-trimethylammonioacetyl) didodecyl-D-glutamate chloride (N- (alpha-trimethylammonioacetyl) didodecyl-D-glutamate

Chloride (TMAG)), ditetradecanoyl-N- (trimethylammonium-acetyl) diethanol chloride (ditetradecanoyl-N- (trimethylammonio-acetyl) diethanolaminechloride), 1, 3-dioleoyloxy-2- (6-carboxy-arginyl) -propionamide (l, 3-dioleoyloxy-2- (6-carboxy-spermyl) -propylamide (DOSPER)) and N, N, N ' N ' -tetramethyl-, N ' -bis (2-hydroxyethyl) -2, 3-dioleoyloxy-1, 4-butanediammonium iodide, 1- [2- (acyloxy) ethyl ] -2-alkyl (alkenyl) -3- (2-hydroxyethyl) -imidazoline derivatives, such as 1- [2- (9 (Z) -octadecenoyloxy) ethyl ] -2- (8 (Z) -heptadecenyl-3- (2-hydroxyethyl) imidazoline chloride (DOTIM) and 1- [2- (hexadecyloxy) ethyl ] -2-pentadecyl-3- (2-hydroxyethyl) imidazoline chloride (DPTIM), and amine-containing 2, 3-dialkyloxy-2-alkyl (alkenyl) -3- (2-hydroxyethyl) imidazoline.g. quaternary ammonium bromide derivatives, such as quaternary ammonium bromide derivatives 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1, 2-dioleoyloxy-propyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1, 2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (dmriie), 1, 2-dipalmitoxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), and 1, 2-distearoxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE).

In some embodiments, the outer lipid shell may comprise a pegylated derivative of a neutral, zwitterionic, cationic or anionic lipid, such as mPEG-DSPE, including DSPE PEG (2000 MW) and DSPE PEG (5000 MW). When the nanoparticle is present in vivo, surface display of PEG or other suitable hydrophilic polyalkylene oxide on the shell of the nanoparticle can be used to reduce uptake of the nanoparticle by the reticuloendothelial system ("RES"); thereby extending residence and systemic circulation time in the body and/or allowing the nanoparticles to provide a sustained and prolonged immunostimulatory effect. Other examples of suitable pegylated lipids include dipalmitoyl-propanetriyl-succinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol, and cholesterol polyethylene glycol.

In some embodiments, the outer lipid shell may comprise a mixture of phospholipids and cholesterol, such as a mixture of N- (carbonyl-) methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-propanetriyl 3-phosphoethanolamine sodium salt (mPEG-DSPE), phosphatidylcholine, such as fully Hydrogenated Soybean Phosphatidylcholine (HSPC), and cholesterol. These lipids are well known and have good properties for use in approved commercial products, such asAlternative 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 be 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 to HSPC of about 1:1-1:200 or 1:10-1:200; suitably 1:1 to 1:100 or 1:1 to 1:50 or 1:1 to 1:30 or 1:10 to 1:30; advantageously from 1:15 to 1:25. In some embodiments, the molar ratio of HSPC to DSPE-PEG to cholesterol may be about 2:0.1:1 or about 2:0.01:1 or about 2:0.2:1.

The outer lipid shell of the nanoparticle surrounds an inner aqueous core, which may comprise one or more hydrogel polymers, which may be used to further stabilize and/or control the release of imiquimod and any other active agent (e.g., an antigen or polynucleotide as described in more detail below) that may be contained within the inner core of the nanoparticle from the nanoparticle. The hydrogel polymer may be covalently and/or non-covalently crosslinked, or may be capable of covalent and/or non-covalent crosslinking, or may not have crosslinking. The hydrogel polymer may be or comprise, for example, poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), polyhydroxyalkanoates such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate; polycaprolactone; poly (orthoesters); polyanhydrides; poly (phosphazene); poly (lactide-co-caprolactone); poly (glycolide-co-caprolactone); a polycarbonate; polyamides, polypeptides, and poly (amino acids); a polyester amide; other biocompatible polyesters; poly (dioxanone); poly (alkylene alkylate); hydrophilic polyethers; polyurethane; a polyether ester; polyacetal; polycyanoacrylates; a polysiloxane; poly (oxyethylene)/poly (oxypropylene) copolymers; polyketal; polyphosphates; polyhydroxyvalerate; polyalkylene oxalates; polyalkylene succinates; poly (maleic acid), polyvinyl alcohol, polyvinylpyrrolidone; poly (alkylene oxide); cellulose, polyacrylic acid, albumin, collagen, gelatin, prolamine and/or polysaccharide. In particular, the hydrogel polymer may comprise a copolymer, including a block copolymer, or a blend of any of the foregoing hydrogel polymers. In some embodiments, the inner aqueous core of the nanoparticle may comprise a polyethylene glycol polymer, such as polyethylene glycol 4000.PEG 4000 is widely used in pharmaceutical formulations, including parenteral formulations, such as INVEGAThe internal 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.

The nanoparticles may have a diameter of no more than about 300nm as measured by standard, art-recognized Dynamic Light Scattering (DLS) techniques. In some embodiments, the diameter of the nanoparticle (as measured by DLS) may be no more than about 200nm, or no more than about 150nm, or no more than about 130nm, or no more than about 120nm. The diameter of the nanoparticle (measured by DLS) may be at least 15nm or at least 20nm or at least 30nm or at least 50nm. Suitably, the diameter of the nanoparticle (as measured by DLS) may be from about 20 to 300nm or from about 20 to 150nm or from about 20 to 100nm or from about 20 to 50nm or from about 30 to 300nm or from about 50 to 150nm or from about 80 to 125nm or from about 90 to 110nm. DLS may be performed according to ISO 22412:2017 or similar techniques.

Advantageously, the nanoparticles may be spherical or spheroid, and/or may be monolayer. Exemplary nanoparticles according to the present disclosure, as viewed under a frozen transmission electron microscope (frozen TEM), can be seen in fig. 3. Here, it can be seen that most nanoparticles are spherical and monolayer; and the interior of the nanoparticle is denser than the surrounding buffer, consistent with the loading of imiquimod, HP-beta-CD, and PEG 4000 polymers inside the nanoparticle.

The inner aqueous core of the nanoparticle comprises imiquimod. Some or all of the imiquimod is complexed with a host molecule. As described above, the term "imiquimod" as used herein includes imidazoquinolines known as 1- (2-methylpropyl) imidazo [4,5-c ] quinolin-4-amine (CAS No. 99011-02-6), R-837 and S-26308 are as follows:

The term also includes structural analogs of imiquimod (R-837) as defined herein, which are active TLR7/8 ligands, including but not limited to, remiquinimod, gardimmod, CL097, S28690, 852-a, 854A, CL075 and other ligands known in the art and as defined above. Imiquimod, as defined herein, is a small synthetic guanosine analogue which is recognised by its immunostimulatory capacity and is known to be effective in activating TLR7 and/or TLR8, amongst others. In some preferred embodiments and aspects of the present disclosure, the imiquimod is imiquimod (R-837).

Imiquimod (R-837) is approved for therapeutic administration as a skin cream and is commercially available as a pharmaceutical substance manufactured and tested according to the current good manufacturing practice (Good Manufacturing Practices, cGMP) under the active pharmaceutical main file (Drug MASTER FILE). Or imiquimod can be readily synthesized as a small molecule based on available starting materials and using methods well known in the art.

The inner aqueous core of the nanoparticle further comprises a host molecule capable of reversibly forming a complex (e.g., an inclusion complex) with imiquimod. An inclusion complex may be formed when an imiquimod molecule or a portion of an imiquimod molecule is inserted into a cavity of a host molecule or group of host molecules. The host molecule may aid in dissolving imiquimod in the aqueous core of the nanoparticle and/or control the release of imiquimod from the nanoparticle. Thus imiquimod may exist as an inclusion complex with the host molecule.

The host molecule may, for example, comprise cyclodextrin; preferably, the cyclodextrin is selected from alpha-cyclodextrin; beta-cyclodextrin; gamma-cyclodextrin; methyl alpha-cyclodextrin; methyl beta-cyclodextrin; methyl gamma-cyclodextrin; ethyl beta-cyclodextrin; butyl alpha-cyclodextrin; butyl beta-cyclodextrin; butyl gamma-cyclodextrin; amyl γ -cyclodextrin; hydroxyethyl beta-cyclodextrin; hydroxyethyl gamma-cyclodextrin; 2-hydroxypropyl α -cyclodextrin; 2-hydroxypropyl beta-cyclodextrin; 2-hydroxypropyl gamma-cyclodextrin; 2-hydroxybutyl beta-cyclodextrin; acetyl α -cyclodextrin; acetyl beta-cyclodextrin; acetyl gamma-cyclodextrin; propionyl beta-cyclodextrin; butyryl beta-cyclodextrin; succinyl alpha-cyclodextrin; succinyl beta-cyclodextrin; succinyl gamma-cyclodextrin; benzoyl beta-cyclodextrin; palmityl beta-cyclodextrin; tosyl beta-cyclodextrin; acetylmethyl beta-cyclodextrin; acetyl butyl beta-cyclodextrin; glucosyl α -cyclodextrin; glucosyl beta-cyclodextrin; glucosyl gamma-cyclodextrin; maltosyl alpha-cyclodextrin; maltosyl beta-cyclodextrin; maltosyl gamma-cyclodextrin; alpha-cyclodextrin carboxymethyl ether; beta-cyclodextrin carboxymethyl ether; gamma-cyclodextrin carboxymethyl ether; carboxymethyl ethyl beta-cyclodextrin; phosphate alpha-cyclodextrin; phosphate beta-cyclodextrin; phosphate gamma-cyclodextrin; 3-trimethylammonium-2-hydroxypropyl beta-cyclodextrin; sulfobutyl ether beta-cyclodextrin; carboxymethyl alpha-cyclodextrin; carboxymethyl beta-cyclodextrin; carboxymethyl gamma-cyclodextrin; and combinations thereof. However, many other host molecules are known in the art and may be used in accordance with the present disclosure, such as polysaccharides, cryptands, cage molecules (cryptophane), cryptands (cavitands), crown ethers, dendrimers, ion exchange resins, calixarenes, valinomycin, nigericin (nigericin), chordons, polysofar, air prison molecules (carcerand), cucurbiturils (cucurbituril), and sphere ligands, among other compounds familiar to those skilled in the art.

In certain advantageous embodiments, the host molecule is or includes 2-hydroxypropyl- β -cyclodextrin. 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) (CAS number 128446-35-5) is a partially substituted poly (hydroxypropyl) ether of beta-cyclodextrin (the molar substitution per anhydroglucose unit is 0.59-0.73). It is capable of reversibly complexing with imiquimod, for example improving the solubility of imiquimod in the aqueous core of the nanoparticle, while allowing a controlled release of imiquimod from the nanoparticle. HP-beta-CD is currently used in many marketed products, including Mitozytrex ^TM, a formulation of HP-beta-CD and mitomycin, approved for use in the treatment of the stomach or pancreas in the United states.

Preferred embodiments of the present disclosure include imiquimod complexed with a host molecule. Some embodiments may also include uncomplexed imiquimod and/or host molecules within the inner aqueous core of the nanoparticle. In particular, the inner aqueous core of the nanoparticle may further comprise imiquimod dispersed, dissolved or suspended in the aqueous core, and/or imiquimod in the form of a precipitate. Optionally, the nanoparticle may not include IL-2 and/or may not include a protein cytokine releasably attached to, associated with, and/or encapsulated within the outer lipid shell.

The vaccine adjuvants of the present disclosure may be or may comprise an aqueous solution, aqueous dispersion or aqueous suspension of the disclosed nanoparticles, or may comprise a dry or lyophilized formulation that may be hydrated to produce an aqueous solution, dispersion or suspension of the disclosed nanoparticles. The adjuvant will generally be used in hydrated form but may conveniently be prepared in lyophilized form, optionally stored prior to use.

The vaccine adjuvants of the present disclosure may also comprise one or more additional adjuvant ingredients, such as some or all of the ingredients of approved vaccine adjuvants. This may further improve the efficacy of the vaccine adjuvant in enhancing the immune response. The vaccine adjuvants of the present disclosure may, for example, comprise one or more additional adjuvant ingredients that are TLR agonists, including TLR4 agonists such as monophosphoryl lipid a (MPL) and/or TLR9 agonists such as CpG 1018. Vaccine adjuvants may, for example, also comprise MPL and QS-21 (commercially available saponins). The additional adjuvant component may be present in the vaccine adjuvant in free form and/or may be present in combination with a delivery vehicle such as a liposome. Additionally or alternatively, additional adjuvant ingredients may be loaded into or onto the vaccine adjuvant nanoparticles.

There is also provided according to the present disclosure 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.

In some embodiments, some or all of the antigen and/or polynucleotide of component (a) may be releasably linked to, associated with, and/or encapsulated within the outer lipid shell of the nanoparticle of component (b). The antigen and/or polynucleotide may be encapsulated within the lipid shell of the nanoparticle of component (b), and/or may be dispersed within the aqueous core of the nanoparticle of component (b), and/or may be releasably attached to or associated with the lipid shell of the nanoparticle of component (b). Some or all of the antigen and/or polynucleotide may optionally be reversibly associated with a host molecule within the aqueous core of the nanoparticle of component (b). In some embodiments, the antigen and/or polynucleotide may be non-covalently attached to the lipid shell of the nanoparticle of component (b), e.g., by ionic interactions, by hydrogen bonding, or by van der waals interactions. In some other embodiments, the antigen and/or polynucleotide may be covalently linked to the lipid shell of the nanoparticle of component (b) through a cleavable linking group that can cleave under suitable conditions (e.g., at an environmental pH or in the presence of certain cleavage agents), e.g., to release the antigen and/or polynucleotide.

Additionally or alternatively, component (a) may comprise a delivery vehicle loaded with antigen and/or polynucleotide and capable of releasing antigen and/or polynucleotide in vivo. The delivery vehicle may be, for example, a nanoparticle vehicle, such as a polymeric nanoparticle, a liposome, or a nanoparticle comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell as described herein. The delivery vehicle may be, for example, a nanogel or PLGA nanoparticles, as described in Look et al, biomaterials35 (2014) 1089-1095. Some or all of the antigen and/or polynucleotide may be releasably attached to, associated with, and/or encapsulated within the nanoparticle delivery vehicle. When the nanoparticle delivery vehicle comprises core/shell nanoparticles of the type described herein, some or all of the antigen and/or polynucleotide may be releasably attached to, associated with, and/or encapsulated within the outer lipid shell of the nanoparticle. As described herein, the outer lipid shell of the nanoparticle may comprise one or more lipid layers or bilayers surrounding a central core. The inner aqueous core of the nanoparticle may comprise a hydrogel, as described herein. The inner aqueous core of the nanoparticle may also comprise a host molecule, as described herein.

In some embodiments of the vaccine composition, component (a) may comprise a polynucleotide, which is a DNA molecule or an 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 produce antigens that can stimulate a protective or therapeutic immune response. Recent examples include mRNA vaccines developed to prevent SARS-CoV-2 infection and COVID-19 disease. DNA vaccines for the prevention and treatment of cancer are also described in the art. Thus, component (a) of the vaccine composition may comprise a DNA or RNA molecule, such as an mRNA molecule, which is capable of expressing an antigen capable of stimulating a protective or therapeutic immune response in vivo.

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 may comprise a polynucleotide encoding a viral antigen, and/or a bacterial antigen, and/or a fungal antigen, and/or a disease-related and/or cancer-related antigen. Component (a) may comprise an antigen, which 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.

In some embodiments, component (a) of the vaccine composition may comprise 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, b, c or d antigen; or herpes simplex virus (HSV-1 or HSV-2) or an HSV-related antigen; or Cytomegalovirus (CMV) or CMV-related antigen; or lyme disease (borrelia burgdorferi (borrelia)) or a lyme disease-associated antigen; or Respiratory Syncytial Virus (RSV) or RSV-associated antigen; or Epstein-Barr virus (EBV) or EBV-associated antigen; or a Zika virus or Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles associated antigens; or mumps-associated antigens; or rubella or a rubella-related antigen; or varicella (varicella, chickenpox) or varicella-associated antigen; or Herpes zoster (Herpes zoter, shingles) or Herpes zoster-related antigens; or diphtheria-associated antigen; or tetanus-related antigens; or polio or a polio-related antigen; or dengue virus-related antigen; or haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus-related antigen; or streptococcus pneumoniae (streptococcus) or streptococcus-related antigens; or Human Papilloma Virus (HPV) or HPV-associated antigen; or pertussis-related antigens; or hepatitis-related antigens; or tuberculosis or a tuberculosis-related antigen; or Human Immunodeficiency Virus (HIV) or HIV-associated antigen; or adenovirus or an adenovirus-associated antigen; or anthrax-related antigen; or cholera-related antigens; or Japanese Encephalitis (JE) or a JE-related antigen; or rabies-associated antigens; or smallpox-related antigen; or typhoid fever (typhoid) or typhoid associated antigens; or yellow fever or an antigen associated with yellow fever; or ebola related antigens; or cancer or a cancer-related antigen.

In some embodiments, component (a) of the vaccine composition may comprise a polynucleotide encoding a coronavirus or a 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, b, c or d antigen; or herpes simplex virus (HSV-1 or HSV-2) or an HSV-related antigen; or Cytomegalovirus (CMV) or CMV-related antigen; or lyme disease (borrelia burgdorferi) or lyme disease-associated antigens; or Respiratory Syncytial Virus (RSV) or RSV-associated antigen; or Epstein-Barr virus (EBV) or EBV-associated antigen; or a Zika virus or Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles associated antigens; or mumps-associated antigens; or rubella or a rubella-related antigen; or varicella (varicella, chickenpox) or varicella-associated antigen; or Herpes zoster (Herpes zoter, shingles) or Herpes zoster-related antigens; or diphtheria-associated antigen; or tetanus-related antigens; or polio or a polio-related antigen; or dengue virus-related antigen; or haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus-related antigen; or streptococcus pneumoniae (streptococcus) or streptococcus-related antigens; or Human Papilloma Virus (HPV) or HPV-associated antigen; or pertussis-related antigens; or hepatitis-related antigens; or tuberculosis or a tuberculosis-related antigen; or Human Immunodeficiency Virus (HIV) or HIV-associated antigen; or adenovirus or an adenovirus-associated antigen; or anthrax-related antigen; or cholera-related antigens; or Japanese Encephalitis (JE) or a JE-related antigen; or rabies-associated antigens; or smallpox-related antigen; or typhoid fever (typhoid) or typhoid associated antigens; or yellow fever or an antigen associated with yellow fever; or ebola related antigens; or cancer or a cancer-related antigen.

The vaccine composition may be or may comprise an aqueous solution, an aqueous dispersion or an aqueous suspension. Or the vaccine composition may be provided in dry or lyophilized form. The vaccine composition may optionally comprise one or more additional excipients and/or active ingredients; such as stabilizers, preservatives, emulsifiers, buffers and/or further adjuvants; including but not limited to aluminum or aluminum salts, MF59 (squalene oil), thimerosal (thiomersal), gelatin (gelatine), sorbitol, lipid (including 4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 2[ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, 1, 2-distearoyl-sn-propan-3-phosphorylcholine, cholesterol), potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate dihydrate, sucrose, tromethamine, and tromethamine hydrochloride. 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.

In some embodiments, the vaccine composition may comprise some or all of the active ingredient and/or excipient ingredients of a vaccine formulation that has been developed for prophylactic or therapeutic use; such as an approved vaccine formulation. Conveniently, 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 compositions of the present disclosure may comprise ingredients of an approved vaccine formulation mixed or otherwise formulated with a vaccine adjuvant comprising nanoparticles according to the present disclosure. The approved vaccine formulation may for example be an approved anti-coronavirus (e.g. SARS-CoV, MERS-CoV or SARS-CoV-2) vaccine, and/or an approved anti-influenza vaccine, and/or an approved anti-Herpes simplex (HSV-1 or HSV-2) vaccine, and/or an approved anti-Cytomegalovirus (CMV) vaccine, and/or an approved anti-lyme disease (borrelia burgdorferi) vaccine, and/or an approved anti-Respiratory Syncytial Virus (RSV) vaccine, and/or an approved anti-Epstein-Barr virus (EBV) vaccine, and/or an approved anti-Zika virus vaccine, and/or an approved anti-meningitis vaccine, and/or an approved anti-measles vaccine, and/or an approved anti-adenitis vaccine, and/or an approved anti-rubella vaccine, and/or an approved pox (varicella), chickenpox) a vaccine, and/or an approved anti-Herpes zoster (hepes zoter, shinles) vaccine, and/or an approved anti-diphtheria vaccine, and/or an approved anti-tetanus vaccine, and/or an approved anti-polio vaccine, and/or an approved anti-dengue virus vaccine, and/or an approved anti-haemophilus influenzae (Hib) vaccine, and/or an approved anti-rotavirus vaccine, and/or an approved anti-streptococcus pneumoniae vaccine, and/or an approved anti-Human Papillomavirus (HPV) vaccine, and/or an approved anti-pertussis vaccine, and/or an approved anti-hepatitis vaccine, and/or an approved anti-tuberculosis vaccine, and/or an approved anti-human immunodeficiency virus vaccine (HIV), and/or an approved anti-adenovirus vaccine, and/or an approved anti-anthrax vaccine, and/or an approved anti-cholera vaccine, and/or an approved anti-Japanese Encephalitis (JE) vaccine, and/or an approved anti-rabies vaccine, and/or an approved anti-smallpox vaccine, and/or an approved anti-typhoid vaccine, and/or an approved anti-yellow fever vaccine, and/or an approved anti-ebola vaccine, and/or an approved anti-cancer vaccine.

Fig. 2 illustrates an exemplary nanoparticle according to the present disclosure. As shown, exemplary nanoparticles have an outer lipid shell formed from a lipid bilayer comprising mPEG-DSPE, wherein mPEG chains are displayed on the outer and inner surfaces of the lipid shell. The inner aqueous core of the nanoparticle comprises a PEG 4000 hydrogel and a hydroxypropyl- β -cyclodextrin host molecule. The host molecule binds reversibly to imiquimod molecules in the aqueous core inside the nanoparticle (not shown). In some embodiments, an antigen or polynucleotide according to the present disclosure may be loaded into a nanoparticle as disclosed herein; and may be reversibly attached to or within the liposome shell of the nanoparticle and/or disposed entirely or partially within the inner core of the nanoparticle (not shown).

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

The adjuvant and/or composition may be buffered to a pH of at least about 6.5 or at least about 7; and preferably does not exceed pH 9 or does not exceed pH 8.5. Suitably, the adjuvants and/or compositions may be buffered to a suitable pH, suitably between about pH 6.5-9 or between about pH 6.5-8.5 or between about pH 6.5-8 or between about pH 7-9 or between about pH 7-8.5 or between about pH 7-8 or between about pH 7.5-9. This is beneficial and desirable when referring to therapeutic uses, where the adjuvant or composition should ideally be buffered to a pH close to physiological pH (pH 7.4). Buffering of the adjuvant or composition may be accomplished using any suitable and acceptable buffer, such as citric acid/sodium citrate.

The vaccine adjuvants and/or vaccine compositions of the present disclosure may suitably be substantially free of unencapsulated imiquimod. This will help to avoid unwanted precipitation of imiquimod in or from the adjuvant or composition.

The vaccine adjuvant and/or vaccine composition may suitably comprise imiquimod about 0.01-50 μg/ml. In some embodiments, the adjuvant or composition may comprise about 1-30 μg/ml imiquimod or about 5-25 μg/ml imiquimod or about 5-15 μg/ml imiquimod. The adjuvant or composition may comprise at least about 1 μg/ml imiquimod, or at least about 2 μg/ml imiquimod, or at least about 3 μg/ml imiquimod, or at least about 5 μg/ml imiquimod. The adjuvant or composition may comprise no more than about 20 μg/ml imiquimod or no more than about 15 μg/ml imiquimod or no more than about 12 μg/ml imiquimod. The adjuvant or composition may comprise imiquimod about 5 μg/ml, or about 10 μg/ml, or about 15 μg/ml.

The amount or concentration of antigen or polynucleotide in the vaccine composition can be determined by the skilled artisan, taking into account general considerations including the strength of the desired reaction, immunogenicity of the antigen, toxicity issues and other criteria familiar to the skilled artisan.

The vaccine adjuvant and/or vaccine composition may suitably comprise about 1-100mg/ml lipid. In some embodiments, the adjuvant or composition may comprise about 5-50mg/ml lipid or about 10-40mg/ml lipid or about 20-30mg/ml lipid. The adjuvant or composition may comprise at least about 5mg/ml lipid or at least about 10mg/ml lipid or at least about 15mg/ml lipid or at least about 20mg/ml lipid. The adjuvant or composition may comprise no more than about 50mg/ml of lipid or no more than about 40mg/ml of lipid or no more than about 30mg/ml of lipid or no more than about 25mg/ml of lipid.

The average diameter of the nanoparticles in the adjuvant or composition may suitably be no more than about 300nm, or no more than about 200nm, or no more than about 150nm, or no more than about 130nm, or no more than about 120nm, as measured by standard, art-recognized Dynamic Light Scattering (DLS) techniques, preferably according to ISO 22412:2017. 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 diameter 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 15nm or at least about 20nm or at least about 30nm or at least about 50nm. In some embodiments, the average diameter of the nanoparticles in the adjuvant or composition and/or the size of the nanoparticles in the adjuvant or composition may range from about 20 to 300nm or from about 20 to 150nm or from about 20 to 100nm or from about 20 to 50nm or from about 30 to 300nm or from about 50 to 150nm or from about 80 to 125nm or from about 90 to 110nm.

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, which allows for effective enhancement of vaccine-induced immune responses. Imiquimod is known in the art as a vaccine adjuvant that is capable of enhancing the immune response induced by vaccine antigens, including infectious or pathogen antigens and cancer-associated antigens. The present disclosure provides a method and platform that allows for co-administration of imiquimod by injection with existing approved adjuvants and/or vaccine components to achieve combined adjuvant and/or immunogenic effects, wherein the mode and manner of administration and delivery can enhance vaccine activity, reduce systemic exposure and associated toxicity, improve pharmacokinetics, and/or provide prophylactic and therapeutic benefits at doses well below imiquimod approved therapeutic doses.

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 infection, and proliferative diseases including cancer. Accordingly, another aspect of the present disclosure provides a method of enhancing an immune response to a vaccine in a subject (e.g., a human subject), comprising the step of administering a vaccine adjuvant disclosed herein to the subject, wherein the vaccine adjuvant is administered to the subject prior to, concurrently with, and/or after administration of the vaccine.

The vaccine compositions of the present disclosure may be suitable for inducing an immune response in a subject that is effective in preventing or treating a variety of different diseases or medical conditions, including viral, bacterial or fungal diseases, colonization or infection, and proliferative diseases including cancer. Thus, another aspect of the present disclosure provides a method for inducing a protective or therapeutic immune response in a subject (e.g., a human subject), and/or a method for immunizing a subject (e.g., a human subject) against a viral, bacterial, or fungal disease or colonization or infection, or against a proliferative disease, such as cancer, comprising the step of administering to the subject a vaccine composition disclosed herein.

The vaccine adjuvant and/or vaccine composition may be administered parenterally to the subject, for example by intravenous, intramuscular or subcutaneous injection or infusion or deposition. The vaccine adjuvant or vaccine composition may be administered orally, intranasally, intramuscularly, intradermally, transdermally, intravenously, intraperitoneally, intrathecally, intravesically, dermally, subcutaneously, or ocularly, including subconjunctival, retrobulbar (retrobulbarly), intracameral (intramedia), and intravitreally. The vaccine adjuvant or vaccine composition may be administered systemically, for example by intravenous injection or infusion; or may be administered locally, for example by injection into the lesion or the immediate vicinity of the lesion, for example a tumor. Topical administration may be particularly relevant to immunization or treatment of cancer. In this case, local administration of the vaccine composition or vaccine adjuvant may have certain advantages over systemic administration. Notably, topical administration means minimizing systemic exposure of vaccine or adjuvant, bypassing RES and tumor vasculature barriers, and may more effectively address local/regional spread of cancer.

The vaccine adjuvant or vaccine composition may be administered as a single dose or as multiple doses. Each dose may suitably comprise about 1ng to 100 μg of imiquimod; suitably, at least about 1ng or at least about 5ng or at least about 10ng or at least about 50ng or at least about 100ng or at least about 500ng or at least about 1 μg or at least about 5 μg or at least about 10 μg imiquimod per dose. Additionally or alternatively, each dose may suitably comprise no more than about 100 μg or no more than about 75 μg or no more than about 50 μg or no more than about 25 μg or no more than about 20 μg or no more than about 10 μg or no more than about 5 μg or no more than about 1 μg of imiquimod. Each dose may suitably comprise about 1-100ng or about 100ng-1 μg or about 1-10 μg or about 10-100 μg imiquimod.

Also included within the scope of the present disclosure are vaccine adjuvants suitable and/or provided for enhancing an immune response to a vaccine in a subject; and vaccine compositions suitable for and/or provided for 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 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 inducing an immune response in a subject.

The present disclosure also provides a method for manufacturing the vaccine adjuvant disclosed herein, comprising the following consecutive steps:

(b) Combining the resulting aqueous solution with a lipid to form lipid shell nanoparticles encapsulating imiquimod; and

The method allows the pH to be adjusted in the process in such a way as to achieve a neutral (physiological) pH formulation while minimizing or avoiding precipitation of unencapsulated imiquimod from the formulation. The method is compatible with the addition of an antigen or polynucleotide, as described herein.

Suitably, step (a) of the method may comprise dissolving imiquimod at a pH buffered to about pH 4-6, or to about pH 4.5-6, or to about pH 4-5.5, or to about pH 5-6. Step (a) may for example comprise dissolving 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 comprise dissolving imiquimod in an aqueous solution in the presence of a host molecule disclosed herein, such as cyclodextrin, particularly HP- β -CD. In particular, step (a) may comprise combining imiquimod with a host molecule, such as cyclodextrin, in a solution that is pH buffered to about pH 6 or less, preferably to about pH 4-6, or to about pH 4.5-6, or to about pH 4-5.5, or to about pH 3-5.5, or to about pH 5-6, or to about pH 5.

Step (b) may comprise mixing the solution of (a) with a lipid disclosed herein to form a solution or suspension of a multilayer structure; and processing these multilayer structures to form lipid shell nanoparticles encapsulating imiquimod. The lipid may optionally be dissolved in an alcohol solution. The step of processing these structures may include extruding a solution or suspension of the multilayer structure through a membrane to form lipid shell nanoparticles encapsulating imiquimod; or may include drying a solution or suspension of the multilayer structure to form a film, dissolving the film, and shaking or sonicating the resulting solution to form lipid shell nanoparticles encapsulating imiquimod; or may include the use of microfluidic mixing techniques to form lipid shell nanoparticles encapsulating imiquimod.

In other embodiments, step (b) may comprise mixing the solution of (a) with empty liposomes to form lipid shell nanoparticles encapsulating imiquimod. In this case, empty liposomes can be formed from the lipids disclosed herein.

Step (c) of the method may comprise increasing the buffered pH of the formulation to about pH 6.5 or higher, or to pH 7 or higher, 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 increased in step (c) by known techniques such as diafiltration or buffer exchange.

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

In some embodiments, the method may further comprise reducing unencapsulated imiquimod from the nanoparticle formulation of (b) after step (b) and before step (c). This may optionally be accomplished 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.

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

Once produced, the vaccine adjuvant may optionally be dried or lyophilized, and/or may be dispensed into a container for storage or administration. Or the vaccine adjuvant may be further processed to provide a vaccine composition according to the present disclosure, as further described below.

The present disclosure further provides methods for producing the vaccine compositions disclosed herein, comprising providing the vaccine adjuvants 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 an antigen or polynucleotide may, for example, comprise adding one or more components of an approved vaccine; and may include, inter alia, the addition of approved vaccine formulations.

In some embodiments, the methods can include making 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 comprise adding a polynucleotide during step (a), between step (a) and step (b), or during step (b). When the antigen is a small molecule or a substance or material that is not compromised by an acidic pH, the method may comprise adding the antigen during step (a), between step (a) and step (b), or during step (b). When the antigen is or comprises a macromolecule and/or is a substance or material that is damaged by an acidic pH, the method may comprise adding the antigen after step (c). "compromised" as used herein includes any change that substantially affects the immunogenic properties of an antigen.

In other embodiments, the method may comprise providing a vaccine adjuvant 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 providing a vaccine adjuvant may optionally comprise manufacturing the vaccine adjuvant according to the methods disclosed herein. Or the step of providing a vaccine adjuvant may comprise obtaining a previously manufactured vaccine adjuvant. The step of combining the vaccine adjuvant with the antigen and/or polynucleotide may comprise mixing the vaccine adjuvant with the antigen and/or polynucleotide. It will be appreciated that this may involve the addition of a vaccine adjuvant to the antigen and/or polynucleotide, or the addition of an antigen and/or polynucleotide to a vaccine adjuvant.

An example of a manufacturing process is shown in fig. 4 and described in examples 1-3. The process shown and described utilizes an acidic pH buffer and HP-beta-CD concentration that is optimized for incorporating imiquimod-cyclodextrin into nanoparticles during extrusion. The present inventors have recognized that since acidic pH conditions are incompatible with lipid and antigen/polynucleotide stability and are not suitable for parenterally administered drugs, the product ingredients should not be exposed to a pH below pH 4 during manufacture and the pH must be raised to a pH near neutral for the final product. This may or may not be prior to the addition of any antigen or polynucleotide. Since the solubility of imiquimod decreases substantially near neutral pH, the concentration of "free" unencapsulated imiquimod in the process solution must be reduced before the pH rises to prevent precipitation of "free" unencapsulated imiquimod, thereby blocking the filter used for diafiltration and sterile filtration, preventing its removal from the bulk product and preventing successful sterile filtration. The reduction in the concentration of "free" unencapsulated imiquimod can be achieved by methods such as ultracentrifugation or diafiltration using a suitably sized membrane that retains the nanoparticles but allows free imiquimod and cyclodextrin to pass into the permeate. Diafiltration is a process well suited for clinical and commercial scale pharmaceutical production and therefore may be the preferred process. After the concentration of "free" unencapsulated imiquimod in the process solution is sufficiently reduced to not precipitate at near neutral pH, the pH can be raised and diafiltration continued to remove the external cyclodextrin and imiquimod and the product formulated into a pharmaceutical final buffer.

Vaccine adjuvants may be mixed with antigens or polynucleotides to produce vaccine compositions. This step may be performed during or after the production of the vaccine adjuvant. When a polynucleotide is to be added, it may be considered advantageous to add the polynucleotide at an early stage of adjuvant production. When larger protein antigens or cellular antigens are to be added, once the pH is adjusted to near neutral, it may be considered advantageous to add the larger protein antigens or cellular antigens during or after a later stage of adjuvant production, as disclosed herein. This may be before or after all necessary diafiltration and purification steps are completed, as described in example 2.

A batch process performed according to cGMP standards can produce about 10 liters of vaccine adjuvant in the form of a liposome sterile suspension containing imiquimod at a concentration of about 10 μg/ml. The composition may be used as an adjuvant in a vaccine by combining with other vaccine components including an antigen or a polynucleotide encoding an antigen.

The following are specific embodiments according to the present disclosure.

Example 1: nanoparticle composition

The production of nanoparticles uses a number of raw materials and excipients that are important to the structure and quality of the drug. All raw materials and excipients are synthetic or derived from plants except cholesterol (see below).

The liposome shell consists of three parts:

n- (carbonyl-) methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-propanetriyl 3-phosphato ethanolamine sodium salt (MPEG-DSPE)

Perhydrogenated soybean phosphatidylcholine (HSPC), and

Cholesterol

The aqueous core consists of two components:

2-hydroxypropyl-beta-cyclodextrin

Polyethylene glycol 4000 hydrogel

These ingredients are all commercially available and are known and well characterized excipients.

Example 2: preparation of nanoparticle vaccine adjuvant comprising imiquimod

The process steps for producing imiquimod-loaded nanoparticles are shown in fig. 4. Imiquimod was dissolved in an aqueous solution at pH 5 in the presence of HP- β -CD. The lipid solution was produced by dissolving fully Hydrogenated Soybean Phosphatidylcholine (HSPC), N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-glycerol 3-phosphate ethanolamine sodium salt (MPEG-DSPE) and cholesterol in ethanol (at a molar ratio of about 1.9:0.1:1). The solution was mixed with imiquimod solution to form a multilayer structure. These structures are then extruded through a membrane with appropriate pore size (80 to 100 nm) to produce nanoparticles or "nanolipid gels", which are core-shell structures with lipid shells encapsulating imiquimod and HP- β -CD.

The nanolipid gel was incubated with a solution of PEG-4000 in pH 5 buffer, allowing PEG-4000 to be loaded into the interior of the nanoparticle.

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

Example 3: preparation of vaccine compositions

To prepare a vaccine composition, the nanoparticle of example 2 is mixed with a vaccine component comprising a suitable vaccine antigen, such as SARS-CoV-2 spike protein, in a solution at pH 7.4. SARS-CoV-2 spike protein is used as an antigen in a variety of approved vaccine formulations, includingSARS-CoV-2 vaccine and has been marketed.

The nanoparticles were further diafiltered 4-fold by volume with 1% trehalose PBS pH 7.4 buffer at 25 ℃ using a hollow fiber membrane (500 Kd) to further reduce the external cyclodextrin concentration and remove external imiquimod, and 1% trehalose was added. Or this step may be performed during the production of the nanoparticle according to example 2.

The concentration of the formulation is then adjusted and the formulation sterilized to the standard required for therapeutic use. Sterilization was performed by filtration using a 0.2 μm filter.

The formulation is provided in a single dose concentration containing 10 μg/mL imiquimod. It is a sterile white opaque liquid. The container closure system consisted of a 5mL clear borosilicate glass vial, a 13mm synthetic butyl rubber stopper, and a flip-off CRIMP SEAL bead seal. The composition of IMP in a 5ml vial is listed in Table 2 below.

TABLE 2

* The concentrations of PEG 4000 and 2-hydroxypropyl-beta-cyclodextrin were the maximum possible concentrations based on the batch formulation.

Example 4: drug properties

Figure 3 shows a frozen TEM image of imiquimod-loaded nanoparticles. Frozen transmission electron microscopy (frozen TEM) showed that the majority of these nanoparticles were spherical with a single shell. The frozen TEM images also provide information about the encapsulated drug state and the nanoparticle internal environment. The interior of the nanoparticle is denser than the surrounding buffer, consistent with the loading of imiquimod, HP-beta-CD and PEG 4000 polymer inside the liposome.

Example 5: non-clinical toxicity and adjuvant efficacy studies

This example references studies showing that imiquimod used in the nanoparticles of the present disclosure is well tolerated in animals at doses greater than the clinical doses disclosed herein; and studies demonstrating the adjuvant activity of imiquimod at doses similar to the amount of imiquimod delivered by the nanoparticles of the present disclosure.

Various studies have shown that topical imiquimod is able to enhance vaccine-induced immune responses. Pretreatment with topical imiquimod also significantly increased the immunogenicity of influenza vaccinations in young and old (Hung et al, lancet select. Dis.2016Feb;16 (2): 209-18). Similar results were also reported by Adams et al, j.clin.oncocol.25 (18) suppl 8545, assessing the safety and adjuvant activity of imiquimod when administered with an NY-ESO-1 protein vaccine. The local administration of imiquimod in these studies made it impossible to accurately evaluate the dose of imiquimod delivered to the target immune cells.

In vitro treatment of macrophages with micelle-encapsulated imiquimod at a concentration of 0.2 μg/ml significantly activated NF- κb and MAPK pathways (jimenez-S nchez et al, 2014), indicating that imiquimod at a local concentration of hundreds of ng/ml activates the immune system. In vivo studies by Zhang et al (CLINICAL AND VACCINE Immunology 2014.21:4pp 570-579) found that intraperitoneal injection of 50 μg imiquimod into mice, in combination with influenza vaccine, significantly accelerated and enhanced humoral immune responses against the virus and conferred significant protection to mice from early deadly viral challenges.

The FDA pharmacological/toxicological examination of Zyclara (imiquimod) cream 3.75% (application No. 201153) describes a number of animal studies of imiquimod toxicology, pharmacokinetics, and metabolism in different species, the results of which include:

subcutaneous lethal dose of single subcutaneous injection in rats was 20mg/kg.

Intravenous injection of 0.5-5.0mg/kg imiquimod produces some evidence of cardiac stimulation, central nervous system stimulation, and autonomic nervous system inhibition in dogs.

Intravenous injection of imiquimod 0.5, 1 and 2 mg/kg/day into pregnant female rabbits during the organogenesis phase (day 6-18 of gestation). No treatment-related effects on embryo fetal toxicity or teratogenicity were found at 2 mg/kg/day.

Toxicology examination (https://www.ema.europa.eu/en/documents/scientific-discussion/aldara-epar-scientific-discussion_en.pdf) of Aldara (imiquimod) 5% cream by EMA a number of animal studies of the toxicology, pharmacokinetics and metabolism of imiquimod are described. Overall, toxicology programs have been reported to demonstrate high safety, with no target organ toxicity other than excessive pharmacological activity. Imiquimod does not affect fertility, neither teratogenesis nor genotoxicity. In the carcinogenicity study performed in mice, skin contact with imiquimod did not lead to an increased incidence of tumor or non-tumor lesions. Specific results of these studies included:

The toxicity of the imiquidone Mo Teshan dose studied in mice, rats and monkeys suggests a high safety. Adverse reactions are limited to the central nervous system, leading to many clinical symptoms, usually tics, before death.

In two skin toxicity studies of 2000mg/kg and 5000mg/kg performed on rabbits under closed conditions, there was no death and no signs of toxicity other than the slight transient erythema at the site of use.

Repeated dose toxicity up to 6 months after oral imiquimod in rats and monkeys suggests that, in addition to a slight effect on body weight and food consumption, the only adverse effect is considered to be the result of excessive pharmacological activity, i.e. B and T cell lymphoproliferation, increased plasma cell numbers, splenomegaly and lymphadenectasis, kupfer cell proliferation, mononuclear/macrophage aggregation or proliferation. No other target organs were found for both species and no adverse effect level (noobserved ADVERSE EFFECT LEVEL, NOAEL) build up of 3mg/kg was observed.

The maximum imiquimod dose of the nanoparticles disclosed herein was thousands of times lower than the current dose level of approved drug or the well-tolerated 30mg subcutaneous dose in MRHD or healthy human volunteers based on a large number of animal studies (Soria, myhre et al, 2000). See also Table 3, which compares the amount of imiquimod in the proposed 100 μl dose of the disclosed Nanoparticles (NPs) toThe approved safe doses of (imiquimod) are compared.

TABLE 3 Table 3

MRHD = human maximum recommended dose

Lipid nanoparticles having a hydrogel core as used in embodiments of the present disclosure have been shown to be preferentially absorbed by Antigen Presenting Cells (APCs), which may provide advantages in immunomodulation. Look et al, biomaterials 35 (2014) 1089-1095 showed that lipid nanoparticles with hydrogel cores were more easily taken up by dendritic cells than PLGA nanoparticles, with >100 fold increase in uptake demonstrated by flow cytometry analysis and confocal imaging.

Example 6: in vitro release assay

An in vitro release assay for imiquimod has been developed (IVRA). IVRA are shown in figure 5. Here, the nanoparticles produced according to examples 1-3 were diluted with an equal volume of PBS buffer and incubated with gentle shaking at 37 ℃. Samples were taken at 0,4, 6 and 24 hours and immediately treated with a 300kd filter to obtain free (unencapsulated) imiquimod released in the filtrate. The concentration of imiquimod in the filtrate was determined using a reverse phase HPLC assay.

The results show that imiquimod is released from the nanolipid gel at a linear rate over time, about 25% of the drug being released after 24 hours, and is expected to be completely released within a few days. This in vitro result demonstrates that the nanolipid gel is capable of providing surprisingly sustained release and delivering effective amounts of imiquimod over an extended period of days, which is very advantageous compared to prior art imiquimod formulations, wherein imiquimod release is found to be much faster in prior art imiquimod formulations. Studies have shown that sustained delivery enhances the immunostimulatory effect of a TLR7/8 agonist (e.g., imiquimod) (Auderset et al Front immunol.2020, 11/11; 11: 580974). Thus, the delayed release achieved by the formulations of the present disclosure enhances the adjuvant effect of the formulation when administered as part of a vaccine.

Example 7: use in community vaccination environments

A dose of the vaccine composition of example 3 was administered by parenteral intramuscular injection into the upper arm of a patient at risk of infection COVID-19. The adjuvant component is used to enhance the vaccine-induced protective immune response.

Claims

1. A vaccine adjuvant comprising a plurality of nanoparticles, the nanoparticles comprising an outer lipid shell and an inner aqueous core encapsulated within the outer lipid shell; wherein the internal aqueous core comprises imiquimod and a host molecule capable of reversibly forming a complex with the imiquimod.

2. A vaccine adjuvant according to claim 1, wherein the imiquimod is imiquimod (R-837) or an active structural analogue of imiquimod (R-837), such as resiquimod, gardimmod (gardiquimod), S28690, 852-a, 854A, CL075 or CL097.

3. A vaccine adjuvant according to claim 1 or 2 comprising imiquimod complexed with the host molecule.

4. A vaccine adjuvant according to any one of claims 1-3, wherein the inner aqueous core of the nanoparticle comprises a hydrogel, optionally selected from poly (lactic acid), poly (glycolic acid), poly (lactic-co-glycolic acid), polyhydroxyalkanoates such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate; polycaprolactone; poly (orthoesters); polyanhydrides; poly (phosphazene); poly (lactide-co-caprolactone); poly (glycolide-co-caprolactone); a polycarbonate; polyamides, polypeptides, and poly (amino acids); a polyester amide; other biocompatible polyesters; poly (dioxanone); poly (alkylene alkylate); hydrophilic polyethers; polyurethane; a polyether ester; polyacetal; polycyanoacrylates; a polysiloxane; poly (oxyethylene)/poly (oxypropylene) copolymers; polyketal; polyphosphates; polyhydroxyvalerate; polyalkylene oxalates; polyalkylene succinates; poly (maleic acid), polyvinyl alcohol, polyvinylpyrrolidone; poly (alkylene oxide); cellulose, polyacrylic acid, albumin, collagen, gelatin, prolamine, polysaccharides, derivatives, copolymers and blends thereof.

5. The vaccine adjuvant of any one of claims 1-4, wherein the host molecule is or comprises cyclodextrin; preferably, the cyclodextrin is selected from alpha-cyclodextrin; beta-cyclodextrin; gamma-cyclodextrin; methyl alpha-cyclodextrin; methyl beta-cyclodextrin; methyl gamma-cyclodextrin; ethyl beta-cyclodextrin; butyl alpha-cyclodextrin; butyl beta-cyclodextrin; butyl gamma-cyclodextrin; amyl γ -cyclodextrin; hydroxyethyl beta-cyclodextrin; hydroxyethyl gamma-cyclodextrin; 2-hydroxypropyl α -cyclodextrin; 2-hydroxypropyl beta-cyclodextrin; 2-hydroxypropyl gamma-cyclodextrin; 2-hydroxybutyl beta-cyclodextrin; acetyl α -cyclodextrin; acetyl beta-cyclodextrin; acetyl gamma-cyclodextrin; propionyl beta-cyclodextrin; butyryl beta-cyclodextrin; succinyl alpha-cyclodextrin; succinyl beta-cyclodextrin; succinyl gamma-cyclodextrin; benzoyl beta-cyclodextrin; palmityl beta-cyclodextrin; tosyl beta-cyclodextrin; acetylmethyl beta-cyclodextrin; acetyl butyl beta-cyclodextrin; glucosyl α -cyclodextrin; glucosyl beta-cyclodextrin; glucosyl gamma-cyclodextrin; maltosyl alpha-cyclodextrin; maltosyl beta-cyclodextrin; maltosyl gamma-cyclodextrin; alpha-cyclodextrin carboxymethyl ether; beta-cyclodextrin carboxymethyl ether; gamma-cyclodextrin carboxymethyl ether; carboxymethyl ethyl beta-cyclodextrin; phosphate alpha-cyclodextrin; phosphate beta-cyclodextrin; phosphate gamma-cyclodextrin; 3-trimethylammonium-2-hydroxypropyl beta-cyclodextrin; sulfobutyl ether beta-cyclodextrin; carboxymethyl alpha-cyclodextrin; carboxymethyl beta-cyclodextrin; carboxymethyl gamma-cyclodextrin; and combinations thereof; advantageously, wherein the cyclodextrin is or comprises 2-hydroxypropyl- β -cyclodextrin.

6. A vaccine adjuvant according to any one of the preceding claims, wherein the imiquimod is present as an inclusion complex with the host molecule.

7. A vaccine adjuvant according to any one of claims 1-6 comprising an aqueous solution, aqueous dispersion or aqueous suspension of nanoparticles.

8. A vaccine adjuvant according to claim 7, wherein the solution, suspension or dispersion is buffered to a pH of at least about 6.5, preferably at least pH 7; suitably between about pH 6.5-9 or between about pH 6.5-8.5 or between about pH 6.5-8 or between about pH 7-9 or between about pH 7-8.5 or between about pH 7-8 or between about pH 7.5-9.

9. A vaccine adjuvant according to claim 7 or 8 wherein the solution, suspension or dispersion is substantially free of unencapsulated imiquimod and/or substantially free of unencapsulated non-dissolved imiquimod.

10. A vaccine adjuvant according to any one of the preceding claims, further comprising one or more additional adjuvant ingredients, wherein the one or more additional adjuvant ingredients optionally comprise liposomes.

11. 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) The nanoparticle-containing vaccine adjuvant of any one of claims 1-10.

12. The vaccine composition of claim 11, comprising some or all of the components of an approved vaccine formulation.

13. The vaccine composition of claim 11 or 12, wherein some or all of the antigen and/or polynucleotide is releasably attached to, associated with and/or encapsulated within an outer lipid shell of a nanoparticle.

14. The vaccine composition according to any one of claims 11-13, wherein the polynucleotide is a DNA molecule or an RNA molecule, such as an mRNA molecule.

15. The vaccine composition according to any one of claims 11-14, wherein the antigen is a viral, bacterial, fungal or disease or cancer associated antigen; and/or wherein the polynucleotide encodes a viral, bacterial, fungal or disease or cancer associated antigen.

16. The vaccine composition according to any one of claims 11-15, wherein the antigen is a peptide, protein, carbohydrate, nucleic acid and/or lipid molecule or structure; and/or wherein the polynucleotide encodes a peptide antigen or a protein antigen.

17. Vaccine composition according to any one of claims 11-16, wherein 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, such as an influenza a, b, c or d antigen; or herpes simplex virus (HSV-1 or HSV-2) or an HSV-related antigen; or Cytomegalovirus (CMV) or CMV-related antigen; or lyme disease (borrelia burgdorferi) or lyme disease-associated antigens; or Respiratory Syncytial Virus (RSV) or RSV-associated antigen; or Epstein-Barr virus (EBV) or EBV-associated antigen; or a Zika virus or Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles associated antigens; or mumps-associated antigens; or rubella or a rubella-related antigen; or varicella (varicella, chickenpox) or varicella-associated antigen; or Herpes zoster (Herpes zoter, shingles) or Herpes zoster-related antigens; or diphtheria-associated antigen; or tetanus-related antigens; or polio or a polio-related antigen; or dengue virus-related antigen; or haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus-related antigen; or Streptococcus pneumoniae (Streptococcus) or Streptococcus-related antigens; or Human Papilloma Virus (HPV) or HPV-associated antigen; or pertussis-related antigens; or hepatitis-related antigens; or tuberculosis or a tuberculosis-related antigen; or Human Immunodeficiency Virus (HIV) or HIV-associated antigen; or adenovirus or an adenovirus-associated antigen; or anthrax-related antigen; or cholera-related antigens; or Japanese Encephalitis (JE) or a JE-related antigen; or rabies-associated antigens; or smallpox-related antigen; or typhoid fever (typhoid) or typhoid associated antigens; or yellow fever or an antigen associated with yellow fever; or ebola related antigens; or cancer or a cancer-related antigen; and/or wherein the antigen encoded by the polynucleotide 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, b, c or d antigen; or herpes simplex virus (HSV-1 or HSV-2) or an HSV-related antigen; or Cytomegalovirus (CMV) or CMV-related antigen; or lyme disease (borrelia burgdorferi) or lyme disease-associated antigens; or Respiratory Syncytial Virus (RSV) or RSV-associated antigen; or Epstein-Barr virus (EBV) or EBV-associated antigen; or a Zika virus or Zika virus-related antigen; or meningitis or a meningitis-related antigen; or measles associated antigens; or mumps-associated antigens; or rubella or a rubella-related antigen; or varicella (varicella, chickenpox) or varicella-associated antigen; or Herpes zoster (Herpes zoter, shingles) or Herpes zoster-related antigens; or diphtheria-associated antigen; or tetanus-related antigens; or polio or a polio-related antigen; or dengue virus-related antigen; or haemophilus influenzae (Hib) or a Hib-related antigen; or rotavirus-related antigen; or Streptococcus pneumoniae (Streptococcus) or Streptococcus-related antigens; or Human Papilloma Virus (HPV) or HPV-associated antigen; or pertussis-related antigens; or hepatitis-related antigens; or tuberculosis or a tuberculosis-related antigen; or Human Immunodeficiency Virus (HIV) or HIV-associated antigen; or adenovirus or an adenovirus-associated antigen; or anthrax-related antigen; or cholera-related antigens; or Japanese Encephalitis (JE) or a JE-related antigen; or rabies-associated antigens; or smallpox-related antigen; or typhoid fever (typhoid) or typhoid associated antigens; or yellow fever or an antigen associated with yellow fever; or ebola related antigens; or cancer or a cancer-related antigen.

18. The vaccine composition according to any one of claims 11-17, further comprising one or more additional excipients, adjuvants and/or active ingredients.

19. A method for inducing an immune response in a subject comprising the step of administering the vaccine composition of any one of claims 11-18 to a subject; or comprising the step of administering the vaccine adjuvant of any one of claims 1-10 in combination with a vaccine to a subject.

20. The method of claim 19, wherein the vaccine composition or vaccine adjuvant is administered to the subject by intravenous, intramuscular, or subcutaneous injection, or wherein the vaccine composition or vaccine adjuvant is administered orally, intranasally, intradermally, or transdermally.

21. The method of claim 19 or 20, for immunizing the subject against a viral, bacterial or fungal infection, colonization or disease, or a proliferative disease, such as cancer.

22. A method of preparing a vaccine adjuvant according to any one of claims 1-10, comprising the sequential steps of:

(a) Dissolving imiquimod with a host molecule in an aqueous solution, the pH of the aqueous solution buffered to about pH 6 or less; preferably to about pH 4-6, or to about pH 4.5-6, or to about pH 4-5.5, or to about pH 5-6;

23. The method of claim 22, wherein step (b) comprises mixing the solution of (a) with a lipid, optionally dissolved in an alcoholic solution, to form a solution or suspension of multilayer structures, and treating the structures to form lipid shell nanoparticles encapsulating imiquimod.

24. The method of claim 23, wherein the step of treating the structure comprises extruding a solution or suspension of the multilayer structure through a membrane to form lipid shell nanoparticles encapsulating imiquimod; or by drying the solution or suspension of the multilayer structure to form a film, dissolving the film, and shaking or sonicating the resulting solution to form lipid shell nanoparticles encapsulating imiquimod; or by using microfluidic mixing techniques to form lipid shell nanoparticles encapsulating imiquimod.

25. The method of any one of claims 22-24, wherein step (b) comprises mixing the solution of (a) with empty liposomes to form lipid shell nanoparticles encapsulating imiquimod.

26. The method of any one of claims 22-25, wherein the buffered pH of the formulation is increased in step (c) by diafiltration or buffer exchange.

27. The method of any one of claims 22-26, wherein step (a) comprises dissolving 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, optionally in the presence of a host molecule, such as cyclodextrin.

28. The method of any one of claims 22-27, wherein step (a) comprises mixing imiquimod with a host molecule, such as cyclodextrin, at a pH between about 4-5.5; preferably in a solution having a pH of about 5.

29. The method of any one of claims 22-28, further comprising the step of adding a hydrogel polymer, such as a PEG polymer, during or after step (a).

30. The method of any one of claims 22-28, further comprising reducing unencapsulated imiquimod from the nanoparticle formulation of (b) after step (b) and before step (c), optionally by ultracentrifugation or by diafiltration using a membrane sized to retain nanoparticles but allow free imiquimod to pass through.

31. The method according to any one of claims 22-30, which does not comprise the step of adding a protein cytokine, such as IL-2, to the formulation of (c) such that the nanoparticle is loaded with the protein cytokine.

32. A method of producing the vaccine composition of any one of claims 11-18, comprising providing the vaccine adjuvant of any one of claims 1-10 and adding an antigen capable of inducing an immune response and/or a polynucleotide encoding an antigen capable of inducing an immune response.

33. The method of claim 32, wherein the step of adding an antigen or polynucleotide comprises adding one or more or all components of an approved vaccine.

34. A method according to claim 32 or 33, comprising manufacturing an adjuvant according to the method of any one of claims 22 to 31, and adding an antigen capable of inducing an immune response and/or a polynucleotide encoding an antigen capable of inducing an immune response during or after step (a).

35. A method according to claim 32 or 33, comprising providing an adjuvant according to any one of claims 1 to 10 and combining the adjuvant with an antigen capable of inducing an immune response and/or a polynucleotide encoding an antigen capable of inducing an immune response.