Nanometer alum particles containing sizing agent

Abstract

The name of the invention is nano alum particles containing a sizing agent. Provided herein are nano alum particles comprising an aluminum salt and a sizing agent, wherein the particle has a size in the range of about 1 nm to 450 nm. Such nano alum particles are stable and are suitable for terminal sterilization steps prior to loading into vials. Compositions comprising the nano alum particles as well as the preparation and use of the nano alum particles are also provided.

Description

[0001] This application is a divisional application of the patent application with the filing date of May 31, 2017, the application number of 2017800336635, and the title of "Nanoscale Alum Particles Containing an Adhesive Agent".

[0002] Cross - reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 344,347, filed on Jun. 1, 2016, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0004] The present invention relates to the field of pharmaceutical and vaccine formulations. More specifically, the embodiments described herein relate to nanoscale alum particles, compositions comprising the nanoscale alum particles, and methods of making and using the nanoscale alum particles. BACKGROUND ART

[0005] Aluminum salts (collectively referred to as alum) have been used in vaccines for over eighty years due to their favorable safety profile and their ability to induce enhanced immune responses against adsorbed vaccine antigens [1, 2]. As one of the few classes of adjuvants approved by the US FDA, aluminum salts have an established regulatory pathway as opposed to many newer adjuvant formulations [1]. When dispersed in an aqueous solution, aluminum salts form heterogeneous polymeric microparticles with sizes on the order of about 0.5 micrometers (μm) to 10 micrometers, which may make it difficult to characterize quality control compared to formulations with monodisperse size populations such as oil-in-water emulsions. This complexity is exacerbated by the fact that there are multiple types of aluminum salts available with different properties, including aluminum phosphate, aluminum hydroxyphosphate sulfate, and aluminum hydroxide.

[0006] Several studies have suggested that the average particle size of an adjuvant formulation is a key factor that can affect the biological activity of a vaccine (1). Recently, novel synthetic methods have been employed to remanufacture new synthetic formulations containing alum nanoparticles using aluminum salts. These synthetic nanoparticles have been described as generating stronger immune responses while reducing inflammation at the injection site compared to microparticles [1, 4, 5]. However, in each of these studies, a bottom-up synthetic method was used to fabricate the aluminum particles, and no comparison was made with clinical aluminum salt adjuvants such as etc., making it difficult to interpret the value of the novel formulations relative to clinically approved materials.

[0007] In addition, from a regulatory perspective, clinically relevant aluminum-based microparticles cannot be terminally sterilized by filtration through 0.45 micron or 0.20 micron filters and can only be sterilized by radiation or autoclaving, rendering their manufacture unsuitable for the terminal sterilization step when combined with antigens or adjuvants. There is a need to provide aluminum-based nanoparticles that exhibit little to no aggregation or reduced aggregation and can be terminally sterilized prior to filling into vials. SUMMARY OF THE INVENTION

[0008] The present disclosure provides nanoalum particles, compositions comprising the nanoalum particles, and methods of making and using the nanoalum particles. The nanoalum particles can be used in the field of pharmaceutical and / or vaccine formulations. Provided herein are compositions (including formulations) comprising a plurality of nanoalum particles comprising an aluminum salt and a sizing agent, wherein the particles in the composition have a size of less than 1 μm. The term nanoalum particle is used herein to refer to particles that comprise aluminum and have a size measured in nanometers, typically from 1 nm to about 450 nm. In some embodiments, the composition is suitable for terminal sterilization by filtration of the product (e.g., using a ≤0.45 micron filter) according to FDA regulations. In some embodiments, the particles present in the composition have a size in the range of about 1 nm to about 450 nm. In some embodiments, the average size of the particles in the composition is in the range of about 1 nm to about 450 nm. In some embodiments, the average size of the particles in the composition is in the range of about 1 nm to about 200 nm. The nanoalum particles described herein can be produced by treating or milling aluminum hydroxide in the presence of a sizing agent via standard techniques known in the art, including but not limited to microfluidization, sonication, and high shear mixing. High shear mixing can be performed using a high shear mixer. Silverson is a company that produces high shear mixers that can be used in the methods of the present invention.

[0009] The nanoalum particles in the composition are stable and exhibit little to no aggregation or reduced aggregation and are suitable for the terminal sterilization step prior to filling into vials. The nanoalum particles provided herein can be used to deliver agents to an individual, such as polypeptides or polynucleotides. By way of example only, the nanoalum particles provided herein can be used to deliver an antigen and / or an adjuvant to a host to generate an immune response.

[0010] The present disclosure provides a nanoalum particle comprising: (a) an aluminum salt; and (b) a sizing agent; wherein the particle has a size in the range of about 1 nm to about 450 nm.

[0011] In certain embodiments, the average size of the particles is the Z-average as determined by dynamic light scattering.

[0012] In certain embodiments, the aluminum salt is selected from the group consisting of aluminum hydroxide, aluminum hydroxide gel, AlPO4, AlO(OH), Al(OH)(PO4), and KAl(SO4)2.

[0013] In certain embodiments, the sizing agent is selected from the sizing agents presented in Table 1. The sizing agent can be selected from the group consisting of PAA, PEG, and PEG linked to a lipid. The sizing agent can be selected from the group consisting of chitosan, dextran (e.g., dextran sulfate), or poly(allylamine). The sizing agent can be selected from the group consisting of PAA, PEG, PEG linked to a lipid, chitosan, dextran sulfate, or poly(allylamine).

[0014] In certain embodiments, the sizing agent is PEG linked to a phospholipid. In certain embodiments, the sizing agent is PEG and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons. In certain embodiments, the sizing agent is PEG linked to a lipid (optionally a phospholipid) and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons. In certain embodiments, the lipid is selected from the group consisting of DSPE, DPPE, and DMPE. In certain embodiments, the sizing agent is PAA and the average molecular weight of the PAA is in the range of about 750 daltons to about 7000 daltons.

[0015] When the sizing agent is chitosan, it can be low molecular weight chitosan (e.g., having a molecular weight of about 15 kDa to about 190 kDa), medium molecular weight chitosan (e.g., having a molecular weight of about 190 kDa to about 700 kDa), or high molecular weight chitosan (e.g., having a molecular weight of about 700 kDa to about 1000 kDa). The degree of deacetylation (DDA) of chitosan will vary depending on the purification method and reaction conditions. The degree of deacetylation of chitosan is generally in the range of about 40% to about 90%, where commercial chitosan typically has a DDA of about 70% to about 90%. However, chitosan with a DDA greater than 90% or less than 40% can be used in the methods of the present invention, as can chitosan with a DDA of about 40% to about 90%, preferably about 70% to about 90%. In some embodiments, at least one primary amino group on the C2 carbon of chitosan can be used as a covalent binding site. Thus, as used herein, the term chitosan encompasses chitosan conjugates, including but not limited to mannosylated chitosan or fluorescently labeled chitosan. Chitosan for use in the methods of the present invention is commercially available from many sources, including SIGMA-ALDRICH TM 。

[0016] When the sizing agent is dextran, it can be any one of Class 1, Class 2, or Class 3 dextran with a molecular weight equal to or greater than 1000 daltons. A particularly preferred dextran used as a sizing agent is dextran sulfate. Dextran sulfate is typically sold in its sodium salt form - thus, as used herein, the term dextran sulfate also encompasses its salt forms, including its sodium salt form. Like chitosan, when the sizing agent is dextran sulfate, it can be of low molecular weight (e.g., 5000 daltons to 100 kDa), medium molecular weight (e.g., 100 kDa to 500 kDa), or high molecular weight (e.g., 500 kDa to 1000 kDa or even 2000 kDa). The preferred molecular weight of dextran sulfate is from 20 kDa to about 80 kDa.

[0017] Poly(allylamine) is a water-soluble cationic polymer having free primary amino groups that can be used as a sizing agent as described herein. The molecular weight of poly(allylamine) is preferably from about 5 kDa to about 100 kDa, most preferably from about 5 kDa to about 50 kDa, and most preferably from about 5 kDa to about 25 kDa. The free base form of poly(allylamine) or any of its salt forms (e.g., hydrochloride) can be used. Those skilled in the art will understand that poly(allylamine) polymers with a molecular weight greater than 100 kDa can be used in the methods described herein, and additionally, when using a salt form of poly(allylamine), its molecular weight will increase.

[0018] In certain embodiments, the nanoalum particles are in a filter-sterilized liquid formulation. In certain embodiments, the nanoalum particles are stable in the liquid formulation for at least about 1 month, at least about 6 months, or at least about 1 year at about 0 °C to about 8 °C. In certain embodiments, the nanoalum particles are stable in the liquid formulation for at least about 1 month at about 37 °C. In certain embodiments, the sizing agent is associated with the aluminum salt.

[0019] The present disclosure provides a method for making nanoalum particles, the method comprising subjecting an aluminum salt to a high energy source in the presence of a sizing agent, thereby producing nanoalum particles, and wherein the size of the nanoalum particles is in the range of about 1 nm to about 450 nm.

[0020] As those skilled in the art will appreciate, the nanoalum particles of the present invention can be made from larger particles of micron size. Accordingly, the present disclosure provides a method for making the described nanoalum particles from precursor aluminum salt particles having a size of 0.5 μm to 20 μm or a size of 0.5 μm to 10 μm.

[0021] The present disclosure provides a method for making nanoalum particles, the method comprising (a) subjecting an aluminum salt to a high energy source to produce nanoalum particles having a size in the range of about 1 nm to about 450 nm and (b) mixing a sizing agent with the nanoalum particles within about 30 minutes after step (a).

[0022] In certain embodiments, the high energy source is generated from a microfluidizer, an extruder, an ultrasonic generator, a high shear mixer (e.g., a silverson mixer), or a homogenizer. Two or more high energy sources may be used. For example, the high energy source may be generated from a microfluidizer and a high shear mixer, and a mixture comprising the aluminum salt and the sizing agent may pass through the microfluidizer one or more times (e.g., from one time to about 30 times or more). In certain embodiments, the high energy source is generated from a microfluidizer, and the mixture comprising the aluminum salt and the sizing agent passes through the microfluidizer from one time to about 15 times. In certain embodiments, the aluminum salt is selected from the group consisting of aluminum hydroxide, aluminum hydroxide gel, AlPO4, AlO(OH), Al(OH)(PO4), and KAl(SO4)2. In certain embodiments, the sizing agent is selected from the group consisting of PAA, PEG, and lipid-linked PEG. Alternatively, the sizing agent may be selected from the sizing agents listed in Table 1 or from chitosan, dextran, or poly(allylamine). In certain embodiments, the sizing agent is PEG and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons. In certain embodiments, the sizing agent is lipid (optionally phospholipid)-linked PEG and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons. In certain embodiments, the lipid is selected from the group consisting of DSPE, DPPE, and DMPE. In certain embodiments, the sizing agent is PAA and the average molecular weight of the PAA is in the range of about 750 daltons to about 7000 daltons. In certain embodiments, the method further comprises filter-sterilizing the nanoalum particles. In certain embodiments, the ratio of aluminum salt to PEG is between about 2:1 and about 7.5:1. In embodiments where the sizing agent is chitosan or poly(allylamine), the aluminum salt will undergo surface modification via phosphate ligand exchange.

[0023] The present disclosure provides a nanoalum particle capable of being obtained or produced by the method disclosed herein, wherein the nanoalum particle has a size in the range of about 1 nm to about 450 nm.

[0024] The present disclosure provides a composition comprising the nanoalum particles disclosed herein.

[0025] In certain embodiments, the composition further comprises a bioactive agent. In certain embodiments, the active agent is associated with the nanoalum particles in the composition. In certain embodiments, as determined by gel electrophoresis, greater than about 75% of the bioactive agent is associated with the nanoalum particles in the composition. In certain embodiments, the bioactive agent is a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent, or organism. In certain embodiments, the bioactive agent is a polypeptide. In certain embodiments, the polypeptide is an antigen, fusion protein, full-length protein, peptide, or peptidomimetic. In certain embodiments, the antigen is a Rig I agonist. In certain embodiments, the bioactive agent is a polynucleotide. In certain embodiments, the polynucleotide is DNA. In certain embodiments, the DNA comprises a sequence encoding a polypeptide. In certain embodiments, the DNA is an oligonucleotide. In certain embodiments, the polynucleotide is RNA. In certain embodiments, the RNA is selected from the group consisting of replicon RNA, mRNA, tRNA, siRNA, shRNA, and microRNA. In certain embodiments, the RNA comprises a sequence encoding a polypeptide. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the adjuvant is selected from the group consisting of: AS-2, monophosphoryl lipid A, 3-de-O-acylated monophosphoryl lipid A, IFA, QS21, CWS, TOM, AGP, CpG oligonucleotide-containing, Toll-like receptor (TLR) agonist, Leif, saponin, saponin mimetic, biological and synthetic lipid A, imiquimod, gardiquimod, resiquimod, poly I:C, flagellin, GLA, SLA, STING, and combinations thereof.

[0026] In certain embodiments, the composition is a liquid formulation. In certain embodiments, the composition is capable of being filtered through a 0.20 micron filter or a 0.45 micron filter. In certain embodiments, the composition is capable of terminal sterilization prior to filling into vials. In certain embodiments, the composition is stable at about 0 °C to about 8 °C for at least about 1 month, at least about 6 months, or at least about 1 year. In certain embodiments, the composition is stable at about 37 °C for at least about 1 month. In certain embodiments, the composition further comprises liposomes. In certain embodiments, the average size of the particles in the composition ranges from about 1 nm to about 450 nm.

[0027] The present disclosure provides a kit that includes a first vial containing the composition disclosed herein. In certain embodiments, the kit further includes a second vial containing another medicament.

[0028] The present disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject a composition disclosed herein, thereby stimulating the immune response of the subject.

[0029] In certain embodiments, the immune response is a non-specific immune response. In certain embodiments, the immune response is an antigen-specific immune response. In certain embodiments, the immune response involves activation of B cells, activation of T cells, production of antibodies, or release of cytokines. In certain embodiments, the composition is for use in monotherapy. In certain embodiments, the composition is for treating allergies, addictions, cancer, or autoimmune diseases. In certain embodiments, the route of administration of the composition is oral, intravenous, intradermal, transdermal, nasal, subcutaneous, or rectal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the non-human mammal is a dog, a cat, a cow, or a horse.

[0030] The present disclosure provides a method of delivering a bioactive agent to cells of a subject, the method comprising administering to the subject a composition comprising (a) nanoalum particles and (b) a bioactive agent, thereby delivering the bioactive agent to the cells of the subject, the nanoalum particles comprising an aluminum salt and a sizing agent, wherein the particles have a size in the range of about 1 nm to about 450 nm.

[0031] In certain embodiments, the bioactive agent is delivered into the cells. In certain embodiments, the bioactive agent is an RNA comprising a sequence encoding a polypeptide, and the polypeptide is expressed by the cells. In certain embodiments, the composition generates an immune response in the subject.

[0032] The present disclosure provides a method of making a composition, the method comprising mixing a nanoalum particle disclosed herein with a bioactive agent.

[0033] The present disclosure provides a method of making a composition, the method comprising the steps of: (a) subjecting an aluminum salt to a high energy source in the presence of a sizing agent, thereby producing nanoalum particles, and wherein the nanoalum particles have a size in the range of about 1 nm to about 450 nm; and (b) mixing the nanoalum particles produced in step (a) with a bioactive agent.

[0034] The present disclosure provides a method of making a composition, the method comprising the steps of: (a) subjecting an aluminum salt to a high energy source to produce nanoalum particles having a size in the range of about 1 nm to about 450 nm; (b) mixing a sizing agent with the nanoalum particles within about 30 minutes after step (a); and (c) mixing the nanoalum particles with a bioactive agent during or after step (b).

[0035] These and other aspects of the present invention will be apparent from the following detailed description and the accompanying drawings. Additionally, various references that more particularly describe certain aspects of the present invention are listed herein and are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figures 1A to 1F : Nano-alum formulations with PAA and PEG sizing agents and stability analysis of nano-alum formulations. Figure 1A Demonstrated that the average particle size of nano-alum formulations processed or milled in the presence of PAA2000 when the sizing agent was subjected to three to six passes at 30K PSI was approximately 100 nm, with a polydispersity of approximately 0.25 to 0.3. Increasing the milling to 10 to 15 passes resulted in nano-alum formulations of approximately 78 nm to 87 nm, and the polydispersity did not increase. Figures 1B to 1C Depicts the particle diameter of nano-alum formulations over time with PEG phospholipid (PEG 5000-DSPE to alum ratio of 2:1) or PAA as the sizing agent. Formulations with an initial particle size of approximately 78 nm were stored at 4°C for up to one year and their particle size and polydispersity were tested at the indicated time points. Samples were taken in triplicate. Figures 1D to 1F : Thermal stability of nano-alum formulations. The thermal stability of nano-alum formulations with a particle size less than 100 nm formulated with polyethylene glycolated lipids with different PEG lengths (5000, 2000, and 750) and / or different acyl chain lengths (18, 16, or 14 carbons) was evaluated at 25°C, 37°C, or 60°C at 0 weeks, 2 weeks, or 4 weeks. QG194 is PEG5000-DSPE; QG195 is PEG2000-DMPE; QG196 is PEG2000-DPPE; QG197 is PEG750-DSPE; QG198 is PEG2000-DSPE.

[0037] Figure 2 : Nano-alum formulations with various sizing agents contain the predicted alum content. Nano-alum formulations prepared with different lengths of PEG sizing agents linked to phospholipids with different acyl chain lengths have the predicted alum content. Nano-alum formulations including sizing agents with phospholipid 18C (DSPE) and different PEG lengths 5000 (sample 1), 2000 (sample 4), and 750 (sample 5) contain approximately equal amounts of the predicted alum starting value of 4 mg / ml, with a range measured by ICP-OES from 3.9 mg / ml for PEG750-DSPE (sample 5) to 4.5 mg / ml for (PEG2000-DPPE sample 3).

[0038] Figures 3A to 3D: Mice were immunized with 2.5 μg of ID97 alone or adjuvanted with alum, PAA, nano-alum PAA, nano-alum PEG, or the TLR4 agonist GLA-SE. One week after immunization, splenocytes were isolated and either left unstimulated or stimulated with ID97 protein at 37 °C for 8 hours in the presence of brefeldin A. Cells were then stained for surface expression of CD4, CD8, or CD44 and intracellular expression of CD154, IFN-γ, TNF-α, IL-2, GM-CSF, IL-5, and IL-17A. Antigen-specific responses were calculated as the frequency of CD4 + T cells responding in the ID97-stimulated samples minus the unstimulated samples. Figure 3A shows the frequency of CD4 + T cells making each response to ID97. Serum was collected from immunized animals one week after immunization and IgG isotypes ( Figure 3B ), IgG1 subclass ( Figure 3C ), and IgG2 subclass ( Figure 3D ) ID97-binding antibody titers were estimated by ELISA. Data demonstrate that nano-alum PAA enhanced the Th1 response. Figure 3B The legend for Figure 3A is the same as Figure 3C and the figure for Figure 3D is the same as

[0039] Figures 4A to 4C : Female mice were immunized with saline, alum, nano-alum PAA, or nano-alum PEG. One day later, draining lymph nodes were removed and secreted cytokines and chemokines were analyzed by Luminex assay. Data demonstrate that nano-alum PAA enhanced Th1-biased cytokines in the draining lymph nodes of mice.

[0040] Figure 5 : Wild-type mice and IL-18R− / − mice were immunized with nano-alum PAA adjuvanted with 2.5 μg ID97 and 1 μg of a PE recombinant antigen. One week after immunization, splenocytes were isolated and either left unstimulated or stimulated with ID97 protein at 37 °C for 8 hours in the presence of brefeldin A. Cells were then stained for surface expression of CD4, CD8, and CD44 and intracellular expression of CD154, IFN-γ, TNF, IL-2, GM-CSF, IL-5, and IL-17A. Antigen-specific responses were calculated as the frequency of CD4 T cells responding in the ID97-stimulated samples minus the unstimulated samples. Data demonstrate that nano-alum PAA enhanced the Th1 response via an IL-18R-dependent mechanism.

[0041] Figures 6A to 6C: Immunize mice with RNA replicon expression vectors formulated with nanoalum. Data demonstrate that at 24 hours post-injection, RNA replicons admixed with cationic emulsion and at doses 30-fold and 300-fold lower than unformulated RNA (30 μg) (1 μg or 0.1 μg) exhibited expression equal to that of unformulated RNA, while PAA nanoalum exhibited lower expression( Figure 6A ). However, at day 4 and day 7 post-injection, RNA admixed with either control cationic emulsion or PAA nanoalum exhibited roughly equal expression at doses of 1 μg (mcg) and 0.1 μg (mcg)( Figure 6B and Figure 6C ).

[0042] Figures 7A to 7D : Expression of RNA replicon vectors formulated with nanoalum RNA is not due to the sizing agent in the nanoalum formulation. In Figures 7A to 7D , data grouped according to the dose (mcg, unformulated replicons were described as +, 1 μg (mcg), and 0.1 μg (mcg) respectively) of replicon vector delivered within 24 hours and the formulation demonstrated that at doses of 0.1 μg or 1.0 μg, PAA alone (upper right panel) did not deliver and / or induce expressible levels of RNA replicons, while the same dose RNA replicons formulated or admixed with control cationic emulsion or PAA nanoalum exhibited detectable luciferase expression.

[0043] Figures 8A to 8C : Mice immunized with mRNA formulated with nanoalum express the RNA-encoded gene product in vivo. Data demonstrate that nanoalum formulations are capable of delivering and expressing from mRNA and are dose-sparing compared to unformulated mRNA. Figure 8A Depicts the relative luminescence observed in animals injected with mRNA encoding luciferase and imaged for expression of the luciferase gene. When estimated at 24 hours post-injection, unformulated mRNA exhibited detectable expression at both 10 μg and 1 μg but not at 0.1 μg (left panel). However, both the control cationic formulation and the PAA nanoalum formulation (middle panel and right panel) not only expressed equal levels of mRNA-encoded gene compared to each other at all doses (10 μg, 1 μg, and 0.1 μg), but also demonstrated increased expression levels (>30-fold) at a dose of 1 μg and detectable expression levels at a dose of 0.1 μg compared to unformulated mRNA, thus demonstrating the dose-sparing nature of the nanoalum formulation. Figure 8BDepicts the relative immunofluorescence of animals imaged for mRNA expression 5 days after injection. When estimated 5 days after injection, unformulated mRNA showed detectable expression of the mRNA-encoded gene at 10 μg but not at lower doses (1 μg and 0.1 μg), and mRNA formulated in a control cationic formulation showed no detectable expression at any dose delivered (10 μg, 1 μg, and 0.1 μg) (left and middle panels). The expression of the PAA nanoalum formulation (rightmost panel) was >10-fold higher than that of the mRNA-encoded luciferase at 10 μg and showed detectable expression at 1 μg that was approximately equal to the level observed with 10 μg of unformulated mRNA, thus demonstrating the dose-sparing nature of the nanoalum formulation even 5 days after mRNA delivery. Figure 8C Depicts the relative in vivo expression of the mRNA-encoded luciferase gene 6 hours, 24 hours, and 5 days after admixture with unformulated, control cationic emulsion formulation, or PAA nanoalum and in vivo injection. The data demonstrate that animals immunized with mRNA formulated with nanoalum had increased and relatively stable levels of mRNA-encoded luciferase gene expression (□) over five days compared to unformulated mRNA (·) or mRNA formulated in a control cationic emulsion (Δ) whose expression rapidly declined.

[0044] Figures 9A to 9E : Nanoalum formulations stabilize RNA. This figure demonstrates that when a nanoalum formulation (middle panel, PAA nanoalum) is admixed with RNA, stored as a single vial formulation at 4 °C for 1 hour, 4 hours, or 24 hours, and subsequently used to immunize mice, the admixed formulation is capable of delivering the replicon RNA construct such that the luciferase expression level of the replicated RNA is equal to or greater than that of the formulation admixed and administered immediately at time zero, when analyzed on day 1 ( Figure 9A middle panel) or day 5 ( Figure 9B middle panel). Unformulated RNA replicons showed no detectable expression after storage for 4 hours or 24 hours when gene expression was measured 24 hours or 5 days after administration and showed detectable expression when administered immediately or 1 hour after admixture. Similarly, the control cationic formulation showed protection of the RNA replicon when admixed and stored at 4 °C for 1 hour, 4 hours, or 24 hours, when gene expression was measured 1 day or 5 days after injection. Figures 9C to 9E Is a scatter plot of data directly comparing the control cationic formulation, PAA nanoalum, and unformulated replicons. RNA was administered immediately after admixture with replicon RNA (T = 0, left Figure 9C ), after admixture and storage at 4 °C for 4 hours (T = 1 hour, middle Figure 9D) or blended and stored at 4°C for 24 hours (T = 24 hours, right Figure 9E ). Gene expression was measured on day 5 after administration to the animals in relative light units. Luciferase expression levels demonstrated that RNA formulated with nanoalum was stable when blended at 4°C for up to 24 hours as a single vial formulation compared to unformulated RNA.

[0045] Figures 10A to 10E : Mice immunized with an RNA replicon expression vector encoding a Leishmania fusion protein formulated with nanoalum expressed RNA in vivo and elicited a dose-sparing antigen-specific immune response. This figure demonstrates that mice immunized with an RNA replicon vector encoding the Leishmania fusion polynucleotide EMCH generated an antigen-specific response. Figures 10A to 10D Demonstrated that immunization with EMCH RNA formulated with a control cationic emulsion or PAA nanoalum at a 100-fold lower dose generated approximately equal percentages of CD4+CD44high CD154, IFNγ, IL-2, or TNFα cytokines producing T cells as 10 μg of unformulated RNA, compared to little or no cytokine induction after using 0.1 μg of unformulated RNA replicon. Figure 10E : The characteristics of a protective Leishmania immune response include the presence of multifunctional antigen-specific T cells that secrete multiple cytokines. Further analysis of the multifunctional T cell response of CD4+CD44high T cells was performed. The data demonstrated that mice immunized with 100 ng of EMCH RNA replicon formulated with PAA nanoalum (hatched bars) or with a control cationic emulsion (diagonal cross-hatched bars) had an equal number of triple-positive CD44high IFN-γ+IL-2+TNFα+CD4+ T cells as animals immunized with 10 μg of unformulated RNA (black solid bars). There were also double-positive cells expressing IFN-γ and IL-2 or IL-2 and TNFα. The data demonstrated that the PAA nanoalum formulation was able to deliver RNA expressed at levels sufficient to generate a relevant antigen-specific immune response.

[0046] Figures 11A to 11B Demonstrated that mice immunized with nanoalum formulations with nanoalum particle sizes of 400 nm, 130 nm, or 75 nm adsorbed to the TB fusion peptide ID93 elicited an antigen-specific immune response. Figure 11A Demonstrated that the nanoalum formulation elicited antigen-specific IgG1 antibody titers indicative of a Th2 bias. Figure 11B Demonstrated that the nanoalum formulation plus the TLR4 agonist SLA elicited antigen-specific IgG2 antibody titers indicative of a Th1 bias.

[0047] Figures 12A to 12CIt was demonstrated that immunization of mice with PEGylated nanoalum particles elicited an antigen-specific immune response, the PEGylated nanoalum particles comprising a PEGylated phospholipid adjuvant conjugated to phospholipids with different acyl chain lengths and having different PEG lengths or the same PEG length and admixed with the TB fusion peptide ID93 plus the TLR4 agonist SLA.

[0048] Figures 13A to 13B . Figure 13A It was demonstrated the effect of the number of microfluidization passes at 30,000 psi on the hydrodynamic size of nanoalum synthesized using a precursor and 120 kDa chitosan with a DD of 75% to 85%. Figure 13B It was demonstrated the use of an adjuvant as a precursor and the hydrodynamic diameter and PDI of nanoalum fabricated using different amounts of 120 kDa chitosan with a DD of 75% to 85%. The samples were microfluidized discontinuously 22 times at 30,000 psi.

[0049] Figures 14A to 14B . Figure 14A The particle size of nanoalum (0.2% w / v Al) stabilized by 40 kDa dextran sulfate and derived was provided. Microfluidization was completed at 30,000 psi. Figure 14B The particle size stability data of nanoalum-dextran lot number QG774 (0.2% w / v aluminum + 0.22% dextran sulfate - 40 kDa) stored at 5 °C, 25 °C, and 37 °C were shown.

[0050] Figures 15A to 15B : Figure 15A The ζ-potential of native adjuvant before and after treatment with PBS buffer containing 67 mM phosphate was provided. Figure 15B It was demonstrated the particle diameter (Z-average) and size distribution of nanoalum (0.2% w / v Al or 2 mg Al / ml) stabilized by different amounts of chitosan and derived. Size data were collected immediately after microfluidization and before sterile filtration.

[0051] Figure 16 It was demonstrated the effect of poly(allylamine) (PAH) fractions on the particle size and size distribution of nanoalum synthesized by . DETAILED DESCRIPTION

[0052] The present disclosure described herein provides nanoalum particles, compositions comprising the nanoalum particles, and methods of making and using the nanoalum particles.

[0053] I. DEFINITIONS

[0054] Unless otherwise indicated, the following terms have the following meanings. Any terms that are not defined have their recognized meanings in the art.

[0055] In this specification, the terms “about” and “consisting essentially of” mean ±20% of the indicated range, value, or structure, unless otherwise indicated. In some embodiments, the terms “about” and “consisting essentially of” mean ±15%; ±10%; or ±5% of the indicated range, value, or structure, unless otherwise indicated.

[0056] The use of alternatives (e.g., “or”) should be understood to mean one, both, or any combination of the alternatives.

[0057] As used herein, the terms “comprising,” “having,” and “including” are used synonymously, and the terms and variations thereof are intended to be understood as non-limiting.

[0058] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include their plural referents unless the context clearly dictates otherwise.

[0059] As used herein, the term “bioactive agent” refers to any material to be delivered by the nano-alum formulations of the present disclosure and includes, but is not limited to: macromolecules, peptides, proteins, peptidomimetics, nucleic acids, oligonucleotides, deoxynucleotides, ribonucleotides, mRNA, RNAi, RigI, replicon RNA, adjuvants comprising TLR agonists (e.g., TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 agonists), saponins, whole virus particles, virus fragments, cell fragments. Also included within the term bioactive agent are, for example, aptamers, carbohydrates, carbohydrate-binding, and virus-like particles.

[0060] As used herein, the term “macromolecule” refers to macromolecules exemplified, but not limited to, by peptides, proteins, oligonucleotides, polynucleotides of biological or synthetic origin. Also included within the term macromolecule are, for example, carbohydrates.

[0061] The terms “polypeptide”, “peptide” and “protein” as used interchangeably herein refer to amino acid polymers of any length. The polymers can be linear or branched, it can include modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeled component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art, and peptidomimetic compounds derived from peptides and proteins by structural modification using non-natural amino acids.

[0062] The term “isolated” means that the molecule has been removed from its natural environment.

[0063] “Purified” means that the molecular purity has been increased such that it exists in a form with a higher purity than that present in its natural environment and / or when first synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity. In some embodiments, purification can mean a purity that is 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% higher than that present in its natural environment and / or when first synthesized and / or amplified under laboratory conditions.

[0064] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If there are modifications to the nucleotide structure, they are made either before or after the assembly of the polymer. The polynucleotides of the present disclosure include ribonucleotides (e.g., the terms RNA, RNAi, tRNA and mRNA as well known in the art) and deoxyribonucleotides (DNA) known in the art, and can be single-stranded molecules or double-stranded molecules.

[0065] As used herein, “oligonucleotide” generally refers to a short, usually single-stranded, usually synthetic polynucleotide with a length usually but not necessarily less than about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description of polynucleotides above applies equally and fully to oligonucleotides. Examples include Rig I agonists.

[0066] As used herein, "replicon" includes any genetic element capable of replicating extensively under its own control, such as plasmids, cosmids, phagemids, bacteriophages or viruses. A replicon can be RNA or DNA and can be single-stranded or double-stranded.

[0067] "Individual" or "subject" is any mammal. Mammals include but are not limited to humans, primates, farm animals, sport animals, pets (such as cats, dogs, horses) and rodents.

[0068] "Alkyl" is a straight-chain or branched-chain saturated hydrocarbon. For example, an alkyl group can have from 1 to 30 carbon atoms (i.e., (C1-C 30 ) alkyl), or from 1 to 20 carbon atoms (i.e., (C1-C 20 alkyl) or from 1 to 10 carbon atoms (i.e., (C1-C 10 ) alkyl), or from 1 to 8 carbon atoms (i.e., (C1-C8) alkyl), or from 1 to 6 carbon atoms (i.e., (C1-C6) alkyl), or from 1 to 4 carbon atoms (i.e., (C1-C4) alkyl). By way of example, this term includes straight-chain and branched-chain hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), neopentyl ((CH3)3CCH2-), and n-hexyl (CH3(CH2)5-).

[0069] "Halogen" or "halide" refers to fluorine, chlorine, bromine and iodine.

[0070] "Hydroxyl" or "hydroxy" refers to the group -OH.

[0071] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. By way of example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentyloxy, etc.

[0072] "Carboxyl ester" (Carboxyl ester or carboxy ester) refers to the following groups: -C(O)O-alkyl and -C(O)O-substituted alkyl, where alkyl and substituted alkyl are as defined herein.

[0073] II. General Technology

[0074] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of molecular biology, recombinant DNA technology, biochemistry, and chemistry, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al., U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames and S.J. Higgins eds., 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); treatise: Methods In Enzymology (Academic Press, Inc., New York); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[0075] III. Nano-alum particles

[0076] The nano-alum particles provided herein include an aluminum salt (alternatively referred to as alum) and a sizing agent, wherein the particles are sized in the range of about 1 nm to 450 nm. Discussions of the aluminum salt and the sizing agent are provided below.

[0077] A. Aluminum salt

[0078] The compositions described herein may include aluminum salts, which may be referred to herein as alum. Suitable aluminum salts include aluminum hydroxide, aluminum trihydrate, hydroxy aluminum oxide, aluminum phosphate, hydroxy aluminum phosphate, hydroxy aluminum phosphate sulfate, and potassium alum. Aluminum salts may also be referred to by the following formulas: Al(OH)3, AlH3O3, AlH6O3, AlO(OH), Al(OH)(PO4), and KAl(SO4)2. Those skilled in the art will understand that hydroxy aluminum phosphate is non-stoichiometric and, although it is represented herein as Al(OH)(PO4), the ratio of surface hydroxyl groups to phosphate varies depending on the manufacturing conditions and is thus more accurately represented by the formula: Al(OH) x (PO4) y .

[0079] Aluminum salts used as adjuvants are advantageous because they have a good safety record, increase antibody responses, stabilize antigens, and are relatively simple to produce on a large scale. (Edelman 2002 Mol. Biotechnol. 21:129 - 148; Edelman, R. 1980 Rev. Infect. Dis. 2:370 - 383.)

[0080] In certain embodiments, the aluminum salt is aluminum hydroxide or hydroxy aluminum oxide. Has a total positive charge and can readily absorb negatively charged moieties. May also be referred to as Amphojel; aluminum hydroxide gel; hydrated aluminum oxide; aluminum trihydroxide; or Alugelibye.

[0081] In certain embodiments, the aluminum salt is aluminum phosphate. Has a total negative charge and can readily absorb positively charged moieties.

[0082] Those skilled in the art will understand that in embodiments where the aluminum salt to be used and the sizing agent have the same surface charge, it is desirable to subject the aluminum salt to surface modification such that its charge can be reversed, thereby allowing attraction between the sizing agent and the aluminum salt. As an example, when the aluminum salt has a cationic surface charge (e.g., ALO(OH)) and the sizing agent has a cationic surface charge (e.g., chitosan, poly(allylamine)), ligand exchange (e.g., phosphate ligand exchange) is used to change the surface charge of the aluminum salt to anionic, thereby allowing interaction between the sizing agent and the aluminum salt.

[0083] B. Sizing Agent

[0084] In some embodiments, when compared to nanoalum without a sizing agent but including an aluminum salt, the size of the nanoalum particles is maintained because the sizing agent reduces, blocks, or delays the polymerization of the treated or milled aluminum salt.

[0085] In some embodiments, the sizing agent is added during the process of achieving the desired nanoalum particle size by high energy input such as sonication or microfluidization of the aluminum salt. In some embodiments, the sizing agent is added after achieving the desired nanoalum particle size by high energy input such as sonication or microfluidization of the aluminum salt. In some embodiments, when the sizing agent is added after achieving the desired nanoalum particle size by high energy input such as sonication or microfluidization of the aluminum salt, the sizing agent is added immediately after the treatment or at about 0.5 minute, 0.5 minute to 1.0 minute, 1.0 minute to 1.5 minute, 1.5 minute to 2.0 minute, 2.0 minute to 2.5 minute, 2.5 minute to 3.0 minute, 3.0 minute to 3.5 minute, 3.5 minute to 4.0 minute, 4.0 minute to 4.5 minute, 4.5 minute to 5.0 minute, 5.05 minute to 5.5 minute, 5.5 minute to 6.0 minute, 6.0 minute to 6.5 minute, 6.5 minute to 7.0 minute, 7.0 minute to 7.5 minute, 7.5 minute to 8.0 minute, 8.0 minute to 8.5 minute, 8.5 minute to 9.0 minute, about 10 minutes, about 12 minutes, about 14 minutes, about 16 minutes, about 18 minutes, about 20 minutes, about 22 minutes, about 24 minutes, about 26 minutes, about 28 minutes, about 30 minutes after treating the aluminum salt to achieve the desired nanoalum particle size.

[0086] In some embodiments, the sizing agent is a substance that modifies the surface properties of the nanoalum. In some embodiments, the sizing agent is a substance that stabilizes the size of the nanoalum.

[0087] In some embodiments, the sizing agent is a substance that stabilizes or protects a bioactive agent. Examples of bioactive agents include, but are not limited to: antigens, adjuvants, TLR agonists, peptidomimetics, peptides, polypeptides, proteins, nucleotides, polynucleotides, RNA, DNA, entire viral genomes, and entire viruses. The bioactive agent can be delivered by the nanoalum formulations of the present disclosure. In some embodiments, the sizing agent protects or shields the bioactive agent from oxidation. In some embodiments, the sizing agent protects or shields the bioactive agent from heat stress, which can include heating temperature and time factors. In some embodiments, the sizing agent protects or shields the bioactive agent from cold stress, which can include low temperature and time factors. In some embodiments, the sizing agent protects or shields the bioactive agent from degradation. In some embodiments, the sizing agent is a substance that protects or shields the bioactive agent delivered by the nanoalum formulations of the present disclosure from degradation or inactivation upon exposure to serum or blood components. In some embodiments, the sizing agent protects or shields the bioactive agent such that the agent can be formulated with nanoparticles as a stable single vial formulation.

[0088] In some embodiments, the presence of the sizing agent reduces, blocks, or delays the polymerization or repolymerization of the aluminum salt by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90% compared to the nanoalum particles formed in the absence of the sizing agent, or even blocks nearly 100% repolymerization of the aluminum salt.

[0089] In the nanoalum particles provided herein, the sizing agent associates with the aluminum salt. In some embodiments, the sizing agent binds directly to the aluminum salt. In some embodiments, the sizing agent is adsorbed onto the nanoalum particles. In some embodiments, the sizing agent associates with the aluminum salt by ligand exchange. In some embodiments, the sizing agent associates with the aluminum salt by charge / electrostatic interactions. In some embodiments, the sizing agent associates with the aluminum salt through phosphate head groups found in the sizing agent. In some embodiments, the sizing agent is further linked to a lipid. In some embodiments, the sizing agent is further linked to a phospholipid.

[0090] Table 1 provides a non-limiting list of sizing agents included in the nanoalum particles provided herein.

[0091] Table 1: Sizing Agent

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] In some embodiments, the sizing agent is polyacrylic acid (PAA). In some embodiments, the average molecular weight of the PAA ranges from about 500 to 7000; 1000 to 7000; 1500 to 7000; 2000 to 7000; 2500 to 7000; 3000 to 7000; 3500 to 7000; 4000 to 7000; 4500 to 7000; 5000 to 7000; 5500 to 7000; 6000 to 7000; or 6500 to 7000. In some embodiments, the average molecular weight of the PAA ranges from about 500 to 1000; 500 to 1500; 500 to 2000; 500 to 2500; 500 to 3000; 500 to 3500; 500 to 4000; 500 to 4500; 500 to 5000; 500 to 5500; 500 to 6000; 500 to 6500; or 500 to 7000. In some embodiments, the average molecular weight of the PAA is from about 1000 to 3000 or 1500 to 2500. In some embodiments, the average molecular weight of the PAA is about 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1250, 1200, 1100, 1000, or 500. In some embodiments, the average molecular weight of the PAA is about 5000, 2000, 1250, 1200, or 1000. In some embodiments, the average molecular weight of the PAA is about 2000.

[0098] In some embodiments, the sizing agent is polyethylene glycol (PEG). In some specific embodiments, the average molecular weight range or PEG length range of PEG is from about 500 Daltons to about 6000 Daltons. In some specific embodiments, the average molecular weight range or PEG length range of PEG is from about 750 Daltons to about 5000 Daltons. In some embodiments, the average molecular weight range or PEG length range of PEG is from about 750 Daltons to 1000 Daltons; 750 Daltons to 1500 Daltons; 750 Daltons to 2000 Daltons; 750 Daltons to 2500 Daltons; 750 Daltons to 3000 Daltons; 750 Daltons to 3500 Daltons; 750 Daltons to 4000 Daltons; 750 Daltons to 4500 Daltons; or 750 Daltons to 5000 Daltons. In some embodiments, the average molecular weight range or PEG length range of PEG is from about 4500 Daltons to 5000 Daltons; 4000 Daltons to 5000 Daltons; 3500 Daltons to 5000 Daltons; 3000 Daltons to 5000 Daltons; 2500 Daltons to 5000 Daltons; 2000 Daltons to 5000 Daltons; 1500 Daltons to 5000 Daltons; 1000 Daltons to 5000 Daltons; or 750 Daltons to 5000 Daltons. In some embodiments, the average molecular weight range or PEG length range of PEG is from about 500 Daltons to 1000 Daltons; 500 Daltons to 750 Daltons; or 750 Daltons to 1000 Daltons. In some embodiments, the average molecular weight range or PEG length range of PEG is from about 1500 Daltons to 2500 Daltons; 1500 Daltons to 2000 Daltons; or 2000 Daltons to 2500 Daltons. In some embodiments, the average molecular weight range or PEG length range of PEG is from about 4500 Daltons to 5500 Daltons; 4500 Daltons to 5000 Daltons; or 5000 Daltons to 5500 Daltons. In one exemplary embodiment, the sizing agent is PEG750. In one exemplary embodiment, the sizing agent is PEG2000. In one exemplary embodiment, the sizing agent is PEG5000.

[0099] C. Lipids Linked to the Sizing Agent

[0100] In some embodiments, the sizing agent is further linked to a lipid or phospholipid. Table 2 provides a non-limiting list of lipids that can be linked to the sizing agent. In one exemplary embodiment, the sizing agent is PEG, and PEG is linked to DSPE. In certain embodiments, the sizing agent is PEG, and PEG is linked to DPPE. In certain embodiments, the sizing agent is PEG, and PEG is linked to DMPE.

[0101] In certain embodiments, the lipid is a phospholipid or a quaternary ammonium lipid. In certain embodiments, the lipid is a phospholipid, and the phospholipid is phosphatidylcholine or phosphoglyceride. In certain embodiments, the lipid comprises any one of the following moieties:

[0102]

[0103] where X - is an alkali metal counterion, and Y + is a halide counterion.

[0104] In certain embodiments, the surfactant is poloxamer:

[0105] where a is from 2 to 130 and b is from 15 to 67.

[0106] In certain embodiments, the lipid comprises a C 10-20 alkyl chain. In certain embodiments, the lipid comprises a C 12-18 alkyl chain.

[0107] In certain embodiments, the lipid is anionic. In certain embodiments, the lipid is cationic. In certain embodiments, the lipid is overall uncharged. In certain embodiments, the lipid is zwitterionic.

[0108] In certain embodiments, suitable lipids are shown in Table 2.

[0109] Table 2 - Lipids

[0110]

[0111]

[0112]

[0113] In certain embodiments, the lipid is poloxamer 188.

[0114] In certain embodiments, the lipid is selected from DLPG, DMPG, DPPG, DSPG, DOPG, DSTAP, and DPTAP. In certain embodiments, the lipid is selected from DLPG, DMPG, DPPG, DSPG, and DOPG. In certain embodiments, the lipid is selected from DSTAP and DPTAP.

[0115] In certain embodiments, the lipid is DSPG. In certain embodiments, the lipid is DSTAP. In certain embodiments, the lipid is DPTAP.

[0116] In certain embodiments, the lipid is selected from DSPG, DSTAP, and poloxamer 188.

[0117] In certain embodiments, the lipid is selected from DLPC, DMPC, DPPC, DSPC, DOPC, and POPC. In certain embodiments, the lipid is selected from DLPC, DSPC, and DOPC.

[0118] In certain embodiments, the lipid is DSPE. In an exemplary embodiment, the sizing agent PEG is linked to the DSPE in the nanoalum particles.

[0119] In certain embodiments, the lipid is DPPE. In an exemplary embodiment, the sizing agent PEG is linked to the DPPE in the nanoalum particles.

[0120] In certain embodiments, the lipid is DMPE. In an exemplary embodiment, the sizing agent PEG is linked to the DMPE in the nanoalum particles.

[0121] In certain embodiments, the lipid is DLPE. In an exemplary embodiment, the sizing agent PEG is linked to the DLPE in the nanoalum particles.

[0122] D. Method for Making Nanoalum Particles

[0123] Provided herein are nanoalum particles comprising an aluminum salt and a sizing agent, wherein the nanoalum particles are in the range of about 1 nm to 450 nm in size. The present disclosure provides a method for preparing such nanoalum particles.

[0124] A method for making nanoalum particles includes subjecting an aluminum salt to a high energy source or high energy shear force in the presence of a sizing agent, thereby reducing the size of the aluminum salt and producing nanoalum particles, and wherein the nanoalum particles are in the range of about 1 nm to about 450 nm in size.

[0125] In certain embodiments, alum is treated or milled in the presence of a sizing agent or the sizing agent is added to the milled alum at least seconds, minutes, or hours after treatment. In some embodiments, alum is treated and immediately lyophilized or dried and the sizing agent is added at the time of reconstitution or within seconds, minutes, hours of reconstitution. Treatment or milling is performed using standard techniques known in the art including sonication, high shear mixing (e.g., silverson mixing), or microfluidization. Another standard technique known in the art that can be used in the methods of the present disclosure is high pressure homogenization.

[0126] In some embodiments, the high energy source provides at least 5000 PSI, at least 10,000 PSI, at least 15,000 PSI, at least 20,000 PSI, at least 25,000 PSI, at least 30,000 PSI, at least 35,000 PSI, at least 40,000 PSI, at least 45,000 PSI, or at least 50,000 PSI. In some embodiments, the high energy source provides from about 5000 PSI to 50000 PSI; 5000 PSI to 10000 PSI; 5000 PSI to 15000 PSI; 5000 PSI to 20000 PSI; 5000 PSI to 25000 PSI; 5000 PSI to 30000 PSI; 5000 PSI to 35000 PSI; 5000 PSI to 40000 PSI; 5000 PSI to 45000 PSI; or 5000 PSI to 50000 PSI. In some embodiments, the high energy source provides from about 45000 PSI to 50000 PSI; 40000 PSI to 50000 PSI; 35000 PSI to 50000 PSI; 30000 PSI to 50000 PSI; 25000 PSI to 50000 PSI; 20000 PSI to 50000 PSI; 15000 PSI to 50000 PSI; 10000 PSI to 50000 PSI; or 5000 PSI to 50000 PSI. In some embodiments, the high energy source provides from about 25000 PSI to 35000 PSI; 25000 PSI to 30000 PSI; or 30000 PSI to 35000 PSI. In some embodiments, the high energy source provides about 30000 PSI.

[0127] In some embodiments, the high energy source is a high shear source.

[0128] In some embodiments, the high energy source is a microfluidizer. Microfluidization is used to describe the process by which a composition is exposed to high shear forces. In some embodiments of the present disclosure, the composition is processed by an instrument or device referred to as .

[0129] In some embodiments, the high energy source is an extruder.

[0130] In some embodiments, the high energy source is an ultrasonic generator.

[0131] In some embodiments, the high energy source is a homogenizer.

[0132] In some embodiments, the aluminum salt and the sizing agent are subjected to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, or 100 high shear forces. In some embodiments, the aluminum salt and the sizing agent are subjected to 1 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, or 91 to 100 high shear forces. In some embodiments, the aluminum salt and the sizing agent are subjected to 3, 6, or 10 high shear forces.

[0133] In some embodiments, the method for making the nano-alum particles of the present disclosure is carried out at 0 °C, 4 °C, 25 °C, 30 °C, 50 °C, or 60 °C. In some embodiments, the method for making the nano-alum particles of the present disclosure is carried out at 0 °C to 4 °C, 5 °C to 10 °C, 11 °C to 15 °C, 16 °C to 20 °C, 21 °C to 25 °C, 26 °C to 30 °C, 31 °C to 35 °C, 36 °C to 40 °C, 41 °C to 45 °C, 46 °C to 50 °C, 51 °C to 55 °C, or 56 °C to 60 °C. In some embodiments, the method for making the nano-alum particles of the present disclosure is carried out at 4 °C.

[0134] In some embodiments, the starting concentration of the aluminum salt is 10 mg / ml. In some embodiments, the starting concentration of the aluminum salt is 4 mg / ml. In some embodiments, the starting concentration of the aluminum salt is 2 mg / ml. In some embodiments, the starting concentration of the aluminum salt is 0.5 mg / ml to 10 mg / ml, 1 mg / ml to 10 mg / ml, 0.5 mg / ml to 5 mg / ml; 1 mg / ml to 5 mg / ml; 0.5 mg / ml to 4 mg / ml; 0.5 mg / ml to 3 mg / ml; or 0.5 mg / ml to 3 mg / ml.

[0135] In some embodiments, the starting size of the aluminum salt is 1 μm. In some embodiments, the starting size of the aluminum salt is 0.5 μm to 5 μm; 0.5 μm to 4 μm; 0.5 μm to 3 μm; 0.5 μm to 2 μm; or 0.5 μm to 1 μm.

[0136] In some embodiments, the nanoalum particles are produced by grinding or treating according to the methods described herein in the presence of a sizing agent and have an average particle size of 1 nm to 450 nm. In certain embodiments, the synthetic nanoalum may comprise a synthetic alum as described in the art, which is resynthesized to produce an appropriate alum particle size, and the sizing agent of the present disclosure has been added to the alum to produce a stable aqueous nanoalum formulation. The nanoalum particles of the formulation may be mixed with pharmaceutically acceptable excipients known in the art to produce a nanoalum composition or formulation.

[0137] As used herein, the terms "grinding", "sizing" or "treating" refer to methods of treating an alum solution to achieve nano-sized particles. The methods include processing an alum composition (including a formulation) by a high energy source or input to reduce the aggregation of alum particles having a reduced average particle size as measured to be less than 0.5 μm to 10 μm. Suitable examples of energy input for achieving a nanoalum composition include, but are not limited to: high shear mixing (such as sonication or high shear mixing with a Silverson high shear mixer), extrusion, homogenization, and microfluidization. In some embodiments, the high shear mixing is carried out at 1000 rpm, 2000 rpm, 5000 rpm, or 10,000 rpm for 1 minute, 2 minutes, 5 minutes, or 10 minutes. In some embodiments, the microfluidizer is a Microfluidics M110P (Newton, Massachusetts), which is equipped with a diamond F12Y interaction chamber, followed by a ceramic H30Z auxiliary processing module. In some embodiments, the alum composition is microfluidized at a pressure of 3,000 PSI, 5,000 PSI, 10,000 PSI, 15,000 PSI, or 30,000 PSI. In some embodiments, the alum solution is processed through the microfluidizer at a recycle water temperature of 60 °C, 40 °C, 20 °C, or 4 °C to achieve a nanoalum composition. In some embodiments, the alum solution is ground or treated at least about 1 time, 3 times, 6 times, 10 times, 15 times, 20 times, or 30 times to repeatedly achieve nano-sized particles with an average particle size of 1 nm to 450 nm as disclosed herein. In some embodiments, the alum solution is microfluidized at 30,000 PSI up to 10 times at a recycle water temperature of 4 °C to prevent temperature rise during the treatment. In some embodiments, the alum solution is treated in the presence of a sizing agent. In some embodiments, the ratio of the sizing agent to alum is 30:1, 20:1, 15:1, 10:1, 7.5:1, 4:1, 3:1, 2:1, 1.5:1, 0.5:1, or 0.25:1. In some embodiments, the ratio of the sizing agent to alum is 7.5:1, 4:1, 3:1, 2:1, or 1:1.

[0138] It should be understood that certain variables may be controlled by the method of preparing the nanoscale alum particles of the preparation examples. Certain variables include but are not limited to: sizing agent, type of high energy source, pressure applied by the high energy source, number of times the mixture passes through the high energy source, temperature at which the process occurs, sizing agent concentration, method point at which the sizing agent is added to the aluminum, and ratio of aluminum salt to sizing agent by weight.

[0139] Table 3. Effect of PEG length or molecular weight and grinding process on the sizing agent

[0140]

[0141]

[0142] * Except for PAA with alum at 1.6 mg / ml, alum is a 4 mg / ml solution.

[0143] The bolded sizing agent mg / ml, alum:sizing agent ratio, and Z-Ave (d.nm) ± error value in Table 3 represent the conditions for producing the nanoscale alum of the present disclosure.

[0144] It should be understood that certain variables and their combinations may be involved in the method of preparing the nanoscale alum particles of the preparation examples, as shown in Table 3.

[0145] In certain embodiments, for the method of preparing nanoscale alum particles, where the sizing agent is PEG5000, the method may have one or more of the following characteristics:

[0146] a) The type of high energy source is a microfluidizer;

[0147] b) The pressure applied by the high energy source is about 30 k psi;

[0148] c) The number of times the mixture passes through the high energy source is from 1 to 10 times, such as 3 times, 6 times, or 10 times;

[0149] d) The temperature at which the process occurs is about 4 °C;

[0150] e) The alum concentration is about 4 mg / ml;

[0151] f) The sizing agent concentration is about 8 mg / ml; and

[0152] g) The ratio of aluminum salt to sizing agent is about 1:2.

[0153] In one variant, the method complies with at least one of features (a) to (g). In another variant, the method complies with two or more of features (a) to (g) (and in some variants, complies with all features). In a specific variant, the method complies with feature (a). In another variant, the method complies with features (a), (b), and (c). In another variant, the method complies with features (a), (b), (c), and (d). In another variant, the method complies with features (a), (b), (c), (d), and (e). In another variant, the method complies with features (a), (b), (c), (d), and (f). In another variant, the method complies with features (a), (b), (c), (d), and (g).

[0154] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PEG2000, the method may have one or more of the following features:

[0155] a) The type of high-energy source is a microfluidizer;

[0156] b) The pressure applied by the high-energy source is about 30 k psi;

[0157] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times, or 10 times;

[0158] d) The temperature at which the process occurs is about 4 °C;

[0159] e) The alum concentration is 4 mg / ml;

[0160] f) The sizing agent concentration is about 10 mg / ml; and

[0161] g) The ratio of the aluminum salt to the sizing agent is about 1:2.5.

[0162] In one variant, the method complies with at least one of features (a) to (g). In another variant, the method complies with two or more of features (a) to (g) (and in some variants, complies with all features). In a specific variant, the method complies with feature (a). In another variant, the method complies with features (a), (b), and (c). In another variant, the method complies with features (a), (b), (c), and (d). In another variant, the method complies with features (a), (b), (c), (d), and (e). In another variant, the method complies with features (a), (b), (c), (d), and (f). In another variant, the method complies with features (a), (b), (c), (d), and (g).

[0163] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PEG750, the method may have one or more of the following characteristics:

[0164] a) The type of high-energy source is a microfluidizer;

[0165] b) The pressure applied by the high-energy source is about 30 k psi;

[0166] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times or 10 times;

[0167] d) The temperature at which the process occurs is about 4 °C;

[0168] e) The alum concentration is 4 mg / ml;

[0169] f) The sizing agent concentration is about 30 mg / ml; and

[0170] g) The ratio of aluminum salt to sizing agent is about 1:7.5.

[0171] In one variant, the method conforms to at least one of the characteristics (a) to (g). In another variant, the method conforms to two or more of the characteristics (a) to (g) (and in some variants, to all of the characteristics). In a specific variant, the method conforms to characteristic (a). In another variant, the method conforms to characteristics (a), (b) and (c). In another variant, the method conforms to characteristics (a), (b), (c) and (d). In another variant, the method conforms to characteristics (a), (b), (c), (d) and (e). In another variant, the method conforms to characteristics (a), (b), (c), (d) and (f). In another variant, the method conforms to characteristics (a), (b), (c), (d) and (g).

[0172] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PEG750, the method may have one or more of the following characteristics:

[0173] a) The type of high-energy source is a microfluidizer;

[0174] b) The pressure applied by the high-energy source is about 30 k psi;

[0175] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times or 10 times;

[0176] d) The temperature at which the process occurs is about 4 °C;

[0177] e) The alum concentration is 4 mg / ml;

[0178] f) The sizing agent concentration is about 20 mg / ml; and

[0179] g) The ratio of aluminum salt to sizing agent is about 1:5.

[0180] In one variant, the method meets at least one of the features (a) to (f). In another variant, the method meets two or more of the features (a) to (f) (and in some variants, all the features. In a specific variant, the method meets feature (a). In another variant, the method meets features (a), (b), and (c). In another variant, the method meets features (a), (b), (c), and (d). In another variant, the method meets features (a), (b), (c), (d), and (e). In another variant, the method meets features (a), (b), (c), (d), and (f). In another variant, the method meets features (a), (b), (c), (d), and (g).

[0181] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PAA, the method may have one or more of the following features:

[0182] a) The type of high-energy source is a microfluidizer;

[0183] b) The pressure applied by the high-energy source is about 30 k psi;

[0184] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times, or 10 times;

[0185] d) The temperature at which the process occurs is about 4°C;

[0186] e) The alum concentration is 1.6 mg / ml;

[0187] f) The sizing agent concentration is about 4.8 mg / ml; and

[0188] g) The ratio of aluminum salt to sizing agent is about 1:3.

[0189] In one variant, the method meets at least one of features (a) to (f). In another variant, the method meets two or more of features (a) to (f) (and in some variants, all features. In a specific variant, the method meets feature (a). In another variant, the method meets features (a), (b), and (c). In another variant, the method meets features (a), (b), (c), and (d). In another variant, the method meets features (a), (b), (c), (d), and (e). In another variant, the method meets features (a), (b), (c), (d), and (f). In another variant, the method meets features (a), (b), (c), (d), and (g).

[0190] Table 4. Effect of acyl chain length on the lipid coating agent

[0191]

[0192]

[0193] * Alum is a 4 mg / ml solution. The coating agent mg / ml, alum:coating agent ratio, and Z-Ave (d.nm) ± error value in bold in Table 4 represent the conditions for producing the nano-alum of the present disclosure.

[0194] It should be understood that certain variables and their combinations may be involved in the method for preparing the nano-alum particles of the preparation examples, as shown in Table 4.

[0195] In certain embodiments, for the method of preparing nano-alum particles, where the coating agent is PEG5000 with DSPE-18C, the method may have one or more of the following features:

[0196] a) The type of high-energy source is a microfluidizer;

[0197] b) The pressure applied by the high-energy source is about 30 k psi;

[0198] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times, or 10 times;

[0199] d) The coating agent concentration is about 8 mg / ml; and

[0200] e) The ratio of aluminum salt to coating agent is about 1:2.

[0201] In one variant, the method conforms to at least one of features (a) to (e). In another variant, the method conforms to two or more of features (a) to (e) (and in some variants, conforms to all features). In a specific variant, the method conforms to feature (a). In another variant, the method conforms to features (a), (b) and (c). In another variant, the method conforms to features (a), (b), (c) and (d). In another variant, the method conforms to features (a), (b), (c) and (e).

[0202] In certain embodiments, for a method of preparing nanoalum particles, where the sizing agent is PEG2000 with DSPE-18C, the method may have one or more of the following features:

[0203] a) The type of high-energy source is a microfluidizer;

[0204] b) The pressure applied by the high-energy source is about 30 k psi;

[0205] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times or 10 times;

[0206] d) The sizing agent concentration is about 10 mg / ml; and

[0207] e) The ratio of aluminum salt to sizing agent is about 1:2.5.

[0208] In one variant, the method conforms to at least one of features (a) to (e). In another variant, the method conforms to two or more of features (a) to (e) (and in some variants, conforms to all features). In a specific variant, the method conforms to feature (a). In another variant, the method conforms to features (a), (b) and (c). In another variant, the method conforms to features (a), (b), (c) and (d). In another variant, the method conforms to features (a), (b), (c) and (e).

[0209] In certain embodiments, for a method of preparing nanoalum particles, where the sizing agent is PEG5000 with DPPE-16C, the method may have one or more of the following features:

[0210] a) The type of high-energy source is a microfluidizer;

[0211] b) The pressure applied by the high-energy source is about 30 k psi;

[0212] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times or 10 times;

[0213] d) The concentration of the sizing agent is from about 1 mg / ml to about 8 mg / ml, such as 4 mg / ml or 8 mg / ml; and

[0214] e) The ratio of the aluminum salt to the sizing agent is about 1:1 or 1:2.

[0215] In one variant, the method conforms to at least one of features (a) to (e). In another variant, the method conforms to two or more of features (a) to (e) (and in some variants, conforms to all features). In a specific variant, the method conforms to feature (a). In another variant, the method conforms to features (a), (b), and (c). In another variant, the method conforms to features (a), (b), (c), and (d). In another variant, the method conforms to features (a), (b), (c), and (e).

[0216] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PEG2000 with DPPE-16C, the method may have one or more of the following features:

[0217] a) The type of the high-energy source is a microfluidizer;

[0218] b) The pressure applied by the high-energy source is about 30 k psi;

[0219] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times, or 10 times;

[0220] d) The concentration of the sizing agent is about 10 mg / ml; and

[0221] e) The ratio of the aluminum salt to the sizing agent is about 1:2.5.

[0222] In one variant, the method conforms to at least one of features (a) to (e). In another variant, the method conforms to two or more of features (a) to (e) (and in some variants, conforms to all features). In a specific variant, the method conforms to feature (a). In another variant, the method conforms to features (a), (b), and (c). In another variant, the method conforms to features (a), (b), (c), and (d). In another variant, the method conforms to features (a), (b), (c), and (e).

[0223] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is PEG2000 with DMPE-14C, the method may have one or more of the following features:

[0224] a) The type of the high-energy source is a microfluidizer;

[0225] b) The pressure applied by the high-energy source is about 30 k psi;

[0226] c) The number of times the mixture passes through the high-energy source is from 1 to 10 times, such as 3 times, 6 times or 10 times;

[0227] d) The sizing agent concentration is about 10 mg / ml; and

[0228] e) The ratio of aluminum salt to sizing agent is about 1:25.

[0229] In one variant, the method meets at least one of the features (a) to (e). In another variant, the method meets two or more of the features (a) to (e) (and in some variants, all the features. In a specific variant, the method meets feature (a). In another variant, the method meets features (a), (b) and (c). In another variant, the method meets features (a), (b), (c) and (d). In another variant, the method meets features (a), (b), (c) and (e).

[0230] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is chitosan and the aluminum salt is Al(OH)(PO4) (for example, ), the method can have any combination of the following features:

[0231] a) The high-energy source type is a high-shear mixer followed by a microfluidizer;

[0232] b) The pressure applied by the high-energy source is about 30 k psi;

[0233] c) The number of times the mixture passes through the microfluidizer is from 1 to 30 times, preferably from 10 to 30 times;

[0234] d) The high-shear mixer mixes at about 5,000 rpm;

[0235] e) The alum concentration is about 2 mg aluminum / ml;

[0236] f) The sizing agent concentration is about 2 mg / ml; and

[0237] g) The mass ratio of aluminum salt to sizing agent is about 1:1;

[0238] h) The sizing agent is low molecular weight chitosan.

[0239] In certain embodiments, for a method of preparing nano-alum particles, where the sizing agent is dextran (for example, sodium salt of dextran sulfate) and the aluminum salt is AlO(OH) (for example, ), the method can have any combination of the following features:

[0240] a) The high - energy source type is a high - shear mixer, followed by a microfluidizer;

[0241] b) The pressure applied by the high - energy source is about 30 k psi;

[0242] c) The number of times the mixture passes through the microfluidizer is from 1 to 30 times, preferably 10 to 30 times;

[0243] d) The high - shear mixer mixes at about 5,000 rpm;

[0244] e) The alum concentration is about 2 mg of aluminum / ml;

[0245] f) The sizing agent concentration is about 0.5 mg / ml (e.g., 0.44 mg / ml); and

[0246] g) The mass ratio of the aluminum salt to the sizing agent is about 4.5:1;

[0247] h) The sizing agent is sodium salt of low - molecular - weight dextran sulfate.

[0248] In certain embodiments, for a method of preparing nano - alum particles, where the sizing agent is chitosan and the aluminum salt is AlO(OH) (e.g., ), the method can have any combination of the following characteristics:

[0249] a) The high - energy source type is a high - shear mixer, followed by a microfluidizer;

[0250] b) The pressure applied by the high - energy source is about 30 k psi;

[0251] c) The number of times the mixture passes through the microfluidizer is from 1 to 30 times, preferably 10 to 30 times;

[0252] d) The high - shear mixer mixes at about 5,000 rpm;

[0253] e) The alum concentration is about 2 mg of aluminum / ml;

[0254] f) The sizing agent concentration is about 1 mg / ml; and

[0255] g) The mass ratio of the aluminum salt to the sizing agent is about 2:1;

[0256] h) The sizing agent is low - molecular - weight chitosan.

[0257] i) Before mixing the aluminum salt and the sizing agent, the aluminum salt is subjected to ligand exchange (e.g., phosphate ligand exchange).

[0258] In certain embodiments, for a method of preparing nanoalum particles, where the sizing agent is poly(allylamine) and the aluminum salt is AlO(OH) (e.g., ), the method can have any combination of the following characteristics:

[0259] a) The high energy source type is a high shear mixer followed by a microfluidizer;

[0260] b) The pressure applied by the high energy source is about 30 k psi;

[0261] c) The number of times the mixture passes through the microfluidizer is from 1 to 30 times, preferably from 10 to 30 times;

[0262] d) The high shear mixer mixes at about 5,000 rpm;

[0263] e) The alum concentration is about 2 mg aluminum / ml;

[0264] f) The sizing agent concentration is about 0.5 mg / ml; and

[0265] g) The mass ratio of the aluminum salt to the sizing agent is about 4:1;

[0266] h) The sizing agent is about 15 kDa;

[0267] i) Before mixing the aluminum salt and the sizing agent, the aluminum salt is subjected to ligand exchange (e.g., phosphate ligand exchange).

[0268] E. Size of the nanoalum particles

[0269] As provided herein, the size of the nanoalum particles comprising the aluminum salt and the sizing agent is in the range of about 1 nm to 450 nm.

[0270] In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 75 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 100 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 150 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 200 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 300 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 400 nm. In some embodiments, the size of the nano-alum particles ranges from about 50 nm to 450 nm. In some embodiments, the size of the nano-alum particles ranges from about 20 nm to 100 nm. In some embodiments, the size of the nano-alum particles ranges from about 20 nm to 50 nm. In some embodiments, the size of the nano-alum particles ranges from about 10 nm to 200 nm. In some embodiments, the size of the nano-alum particles ranges from about 10 nm to 100 nm. In some embodiments, the size of the nano-alum particles ranges from about 10 nm to 50 nm. In some embodiments, the size of the nano-alum particles is about 1 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, about 200 nm, about 210 nm, about 220 nm, about 240 nm, about 250 nm, about 260 nm, about 280 nm, about 200 nm, about 300 nm, about 320 nm, about 340 nm, about 350 nm, about 360 nm, about 380 nm, about 400 nm, about 420 nm, about 440 nm or about 450 nm.In some embodiments, the size of the nano-alum particles is not greater than about 1 nm, not greater than about 5 nm, not greater than about 10 nm, not greater than about 15 nm, not greater than about 20 nm, not greater than about 25 nm, not greater than about 30 nm, not greater than about 35 nm, not greater than about 40 nm, not greater than about 45 nm, not greater than about 50 nm, not greater than about 55 nm, not greater than about 60 nm, not greater than about 65 nm, not greater than about 70 nm, not greater than about 75 nm, not greater than about 80 nm, not greater than about 85 nm, not greater than about 90 nm, not greater than about 95 nm, not greater than about 100 nm, not greater than about 105 nm, not greater than about 110 nm, not greater than about 115 nm, not greater than about 120 nm, not greater than about 125 nm, not greater than about 130 nm, not greater than about 135 nm, not greater than about 140 nm, not greater than about 145 nm, not greater than about 150 nm, not greater than about 155 nm, not greater than about 160 nm, not greater than about 165 nm, not greater than about 170 nm, not greater than about 175 nm, not greater than about 180 nm, not greater than about 185 nm, not greater than about 190 nm, not greater than about 195 nm, not greater than about 199 nm, not greater than about 210 nm, not greater than about 230 nm, not greater than about 250 nm, not greater than about 270 nm, not greater than about 290 nm, not greater than about 310 nm, not greater than about 330 nm, not greater than about 350 nm, not greater than about 370 nm, not greater than about 390 nm, not greater than about 410 nm, not greater than about 430 nm, not greater than about 440 nm, or not greater than about 449 nm, or not greater than about 450 nm.

[0271] In some embodiments, the nano-alum particles are capable of filtering through a filter of at least 0.45 microns. In some embodiments, the nano-alum particles are capable of filtering through a filter with a pore size of 0.45 microns or smaller. In some embodiments, the nano-alum particles are capable of filtering through a 0.45-micron filter. In some embodiments, the nano-alum particles are capable of filtering through a 0.20-micron filter. In some embodiments, the nano-alum particles are capable of filtering through a 0.22-micron filter.

[0272] F. Stability

[0273] In some embodiments provided herein, the nano-alum particles, which include an aluminum salt and a sizing agent and have a size from 1 nm to 450 nm, are stable because the size of the nano-alum particles smaller than 450 nm is maintained and because the aluminum salt has reduced polymerization or no polymerization when compared to an aluminum salt lacking the sizing agent.

[0274] In some embodiments, "stable" means that a nano-alum formulation or composition composed of nano-alum particles that do not "aggregate" shows little to no aggregation or reduced aggregation and indicates that the average particle size or polydispersity of the formulation increases little to not at all over time compared to the initial particle size.

[0275] The stability of the nano-alum particles can be measured by techniques familiar to those skilled in the art. In some embodiments, stability is visually observed. Visual inspection can include examining for particulates, flocculation, or aggregation. In some embodiments, stability is determined by the size of the nano-alum particles. For example, size can be evaluated by techniques known in the art, including but not limited to x-ray and laser diffraction, dynamic light scattering (DLS), CryoEM, or Malvern Zetasize. In some embodiments, the size of the nano-alum particles refers to the Z-average diameter. In some embodiments, stability is evaluated by the percentage of polymerization of the aluminum salt in the nano-alum particles. In some embodiments, stability is evaluated by the ability of the nano-alum particles to pass through a filter of a specific size, such as a 0.20, 0.22, or 0.45 micron filter. In some embodiments, stability is determined by pH. In some embodiments, stability is determined by measuring the polydispersity index (PdI) using, for example, dynamic light scattering (DLS) techniques.

[0276] In some embodiments, the Z-average diameter of the nanoparticles increases less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 3%, less than 1% during the measured time period. In some embodiments, the polydispersity index (PdI) of the nanoparticles increases less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 12%, less than 10%, less than 7%, less than 5%, less than 3%, less than 1% during the measured time period.

[0277] In some embodiments, the nano-alum particles are stable at 0°C to 8°C. In some embodiments, the nano-alum particles are stable at 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 5 years.

[0278] In some embodiments, the nano-alum particles are stable at 20°C to 30°C. In some embodiments, the nano-alum particles are stable at 25°C for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 5 years.

[0279] In some embodiments, the nano-alum particles are stable at 35°C to 40°C. In some embodiments, the nano-alum particles are stable at 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 5 years.

[0280] In some embodiments, the nano-alum particles are stable at 57 °C to 62 °C. In some embodiments, the nano-alum particles are stable at 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, or 62 °C for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month.

[0281] In an exemplary embodiment, the nano-alum particles are stable at 4 °C for at least 2 years. In an exemplary embodiment, the nano-alum particles are stable at 4 °C for at least 4 years. In an exemplary embodiment, the nano-alum particles are stable at 4 °C for at least 5 years. In an exemplary embodiment, the nano-alum particles are stable at 25 °C for at least one month. In an exemplary embodiment, the nano-alum particles are stable at 37 °C for at least two weeks. In an exemplary embodiment, the nano-alum particles are stable at 60 °C for at least two weeks.

[0282] In some embodiments, the nano-alum particles are stable after 1 to 4 freeze-thaw cycles. In some embodiments, the nano-alum particles are stable after 1, 2, 3, or 4 freeze-thaw cycles.

[0283] IV. Nano-alum Particle Composition

[0284] The present disclosure provides a composition comprising nano-alum particles, wherein the nano-alum particles comprise an aluminum salt and a sizing agent, and wherein the nano-alum particles have a size of from about 1 nm to 450 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 75 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 100 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 150 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 200 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 300 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 400 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 50 nm to 450 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 20 nm to 100 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 20 nm to 50 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 10 nm to 200 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 10 nm to 100 nm. In some embodiments, the average size of the nano-alum composition is in the range of from about 10 nm to 50 nm. In some embodiments, the average size of the nano-alum composition is about 1 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, about 200 nm, about 210 nm, about 220 nm, about 240 nm, about 250 nm, about 260 nm, about 280 nm, about 200 nm, about 300 nm, about 320 nm, about 340 nm, about 350 nm, about 360 nm, about 380 nm, about 400 nm, about 420 nm, about 440 nm or about 450 nm.In some embodiments, as measured by DLS, the average size of the nano-alum composition is not greater than about 1 nm, not greater than about 5 nm, not greater than about 10 nm, not greater than about 15 nm, not greater than about 20 nm, not greater than about 25 nm, not greater than about 30 nm, not greater than about 35 nm, not greater than about 40 nm, not greater than about 45 nm, not greater than about 50 nm, not greater than about 55 nm, not greater than about 60 nm, not greater than about 65 nm, not greater than about 70 nm, not greater than about 75 nm, not greater than about 80 nm, not greater than about 85 nm, not greater than about 90 nm, not greater than about 95 nm, not greater than about 100 nm, not greater than about 105 nm, not greater than about 110 nm, not greater than about 115 nm, not greater than about 120 nm, not greater than about 125 nm, not greater than about 130 nm, not greater than about 135 nm, not greater than about 140 nm, not greater than about 145 nm, not greater than about 150 nm, not greater than about 155 nm, not greater than about 160 nm, not greater than about 165 nm, not greater than about 170 nm, not greater than about 175 nm, not greater than about 180 nm, not greater than about 185 nm, not greater than about 190 nm, not greater than about 195 nm, not greater than about 199 nm, not greater than about 210 nm, not greater than about 230 nm, not greater than about 250 nm, not greater than about 270 nm, not greater than about 290 nm, not greater than about 310 nm, not greater than about 330 nm, not greater than about 350 nm, not greater than about 370 nm, not greater than about 390 nm, not greater than about 410 nm, not greater than about 430 nm, not greater than about 440 nm, or not greater than about 449 nm.

[0285] In some embodiments, the composition can be filtered and finally sterilized before being filled into vials. In some embodiments, the composition can be filtered through a 0.45 micron filter. In some embodiments, the composition can be filtered through a 0.20 micron filter. In some embodiments, the composition can be filtered through a 0.22 micron filter.

[0286] In some embodiments, the composition is maintained as an aqueous formulation. In some embodiments, the composition is maintained as a lyophilized formulation. In some embodiments, the composition is maintained as a spray-dried formulation.

[0287] In some embodiments, the composition comprises nano-alum and an emulsion. In some embodiments, the emulsion of the composition is a water-in-oil emulsion. In some embodiments, the emulsion of the composition is a Pickering emulsion. In some embodiments, the emulsion of the composition is an oil-in-water emulsion. In some embodiments, the oil of the emulsion is a biodegradable oil. In additional embodiments, the oil is squalene. In other embodiments, the oil is a synthetic biodegradable oil.

[0288] Liposomes and liposome-derived nanovesicles are known in the art [8] and can be used with the nanoalums of the present disclosure. In some embodiments, the composition comprises liposomes containing nanoalum particles. In some embodiments, the composition comprises nanoalum and liposomes, wherein the liposomes are cationic liposomes. In some embodiments, the composition comprises nanoalum and liposomes, wherein the liposomes are anionic liposomes. In some embodiments, the composition comprises nanoalum and liposomes, wherein the liposomes are neutral liposomes. In some embodiments, the composition comprises nanoalum and liposomes, wherein the liposomes are tetraether liposomes. In some embodiments, the composition comprises nanoalum and liposomes, wherein the liposomes are virosomes.

[0289] A. Bioactive agent

[0290] In some embodiments, the composition further comprises one or more bioactive agents. For example, the bioactive agent can be a polypeptide, polynucleotide, antigen, adjuvant, diagnostic agent, therapeutic agent, organism, virus, viral genome. In some embodiments, the composition comprises two or more bioactive agents. In some embodiments, the bioactive agent is associated with the nanoalum particles. In some embodiments, the bioactive agent is associated with the nanoalum particles by ligand exchange and / or by electrostatic (charge-based) interactions. In some embodiments, at least 25%, at least 40%, at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of the bioactive agent present in the composition is associated with the nanoalum particles. In some embodiments, the percentage association of the bioactive agent with the nanoalum particles is determined by gel electrophoresis or UV spectroscopy. An exemplary method for determining the percentage association is shown in the examples.

[0291] i. Macromolecules

[0292] In some embodiments, the bioactive agent is a macromolecule. Macromolecules can include, but are not limited to, polynucleotides, polypeptides, or antigens. In some embodiments, the macromolecule is naturally occurring. In some embodiments, the macromolecule is synthetic. In some embodiments, the macromolecule is labeled or tagged.

[0293] a. Polynucleotides

[0294] Proteins, protein subunits, and inactivated pathogens are effective stimulants of the antibody response (humoral immunity) and have been successfully developed into successful vaccines against many infectious diseases, where humoral immunity is strongly correlated with protection. However, for some chronic infectious diseases or cancers, in addition to humoral immunity, a classical cell or cytolytic T cell response may be required. In the context of major histocompatibility molecules, the classically generated cellular immune response occurs from endogenous or intracellular antigen presentation. This has led researchers to speculate that delivering nucleic acids encoding intracellular antigens may lead to more successful vaccination strategies against chronic infections and cancers. RNA vaccines are particularly attractive for nucleic acid delivery that is theoretically based on the ability to more effectively present proteins transcribed from RNA in the context of the host's major histocompatibility molecules. RNA vaccine delivery in the art includes, for example, delivering messenger RNA and replicating RNA constructs expressed from alphavirus constructs, both of which rely on the delivery and expression of RNA-encoded proteins in cells. While this approach has appeared promising thus far in theory, the development of RNA vaccines is limited by the cost of producing RNA, relatively inefficient RNA delivery in vivo, the instability of naked RNA, and the relative in vivo expression levels of RNA. Simply put, all of these limitations may be attributed to the lack of effective delivery of RNA in vivo. Recently, many strategies have been employed to address these limitations, including incorporating chemically modified nucleotides, modifying RNA structures containing an ARCA cap and an elongated poly(A) tail, and evaluating delivery strategies for RNA and polymeric delivery vehicles ranging from naked RNA to cationic lipids (1 to 5). Perhaps the most well-studied formulation for delivering RNA is the cationic emulsion, which is composed of the cationic lipid, DOTAP, dehydrated sorbitol trioleate, polysorbate, and squalene (5). It has been demonstrated that these cationic liposomes self-assemble into synthetic lipid nanoparticles, with RNA encapsulated in the particle core. While these cationic liposomes have demonstrated the ability to deliver RNA vaccines and induce an immune response, the manufacture of these formulations is relatively complex and expensive. What is needed in the art is a stable, inexpensive, and terminally sterilizable formulation suitable for large-scale manufacture that includes a polynucleotide for delivering RNA and DNA.

[0295] In some embodiments, the bioactive agent is a polynucleotide. Polynucleotides include, but are not limited to, DNA, RNA, aptamers, and oligonucleotides. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the DNA or RNA is single-stranded or double-stranded. In some embodiments, the polynucleotide is non-coding RNA. In some embodiments, the polynucleotide is coding RNA. In some embodiments, the RNA is selected from the group consisting of replicon RNA, mRNA, tRNA, siRNA, shRNA, Rig I, and microRNA.

[0296] In some embodiments, a polynucleotide encodes a polypeptide. In some embodiments, a polynucleotide encodes a polypeptide that is an antigen or comprises an antigen as further described herein. In some embodiments, the polypeptide encoded by the polynucleotide is a fusion protein. In some embodiments, the polypeptide encoded by the polynucleotide is ID93.

[0297] In one specific embodiment, the nanoparticle comprises a PEG coating agent, and the agent is RNA.

[0298] In one specific embodiment, the nanoparticle comprises a PAA coating agent, and the agent is RNA.

[0299] 1. Recombinant expression construct

[0300] According to certain embodiments disclosed herein, the compositions described herein can contain at least one recombinant expression construct that comprises a promoter operably linked to a nucleic acid sequence encoding a polypeptide. In certain additional embodiments, the recombinant expression construct is present in a viral vector, such as an adenovirus, adeno-associated virus, herpesvirus, lentivirus, poxvirus, or retroviral vector. Compositions and methods for making and using such expression constructs and vectors are known in the art, such as for expressing polypeptides as provided herein, e.g., according to Ausubel et al. (eds.), Current Protocols in Molecular Biology, 2006, John Wiley & Sons, New York. Non-limiting examples of recombinant expression constructs can generally be found, for example, in U.S. Patent Nos. 6,844,192; 7,037,712; 7,052,904; 7,001,770; 6,106,824; 5,693,531; 6,613,892; 6,875,610; 7,067,310; 6,218,186; 6,783,981; 7,052,904; 6,783,981; 6,734,172; 6,713,068; 5,795,577 and 6,770,445 and elsewhere, the teachings of which for expressing polypeptide antigens as provided herein can be adapted for certain presently disclosed embodiments.

[0301] 2. Alternative internucleoside linkages and nucleic acid analogs

[0302] In some embodiments, the polynucleotide includes alternative internucleoside linkages or nucleic acid analogs. For example, in one embodiment, the polynucleotide includes phosphorothioate or dithiophosphate linkages, although phosphodiester and other internucleotide linkages are within the scope of the present disclosure, including oligonucleotides having mixed internucleoside linkages. Methods for generating phosphorothioate oligonucleotides or dithiophosphates are described in U.S. Patent Nos. 5,666,153, 5,278,302, and WO95 / 26204.

[0303] 3. Replicon

[0304] In some embodiments, the polynucleotide is a replicon. In some embodiments, a replicon is a plasmid, cosmid, bacmid, phage, or virus that is capable of replicating greatly under its own control. In some embodiments, the replicon is RNA or DNA. In some embodiments, the replicon is single-stranded or double-stranded. In some embodiments, the replicon is derived from an RNA virus.

[0305] b. Polypeptide

[0306] In some embodiments, the bioactive agent is a polypeptide. Thus, in some embodiments, the described compositions include the nanoalum particles provided herein and further include a polypeptide. In some embodiments, the polypeptide is a full-length protein or a fragment thereof. In some embodiments, the polypeptide is a peptide. In some embodiments, the polypeptide is a fusion protein. In some specific embodiments, the fusion protein is capable of inducing an immune response when administered to an individual. In some embodiments, the polypeptide is an antigen, as further described below.

[0307] c. Antigen

[0308] In some embodiments, the bioactive agent is an antigen. In some embodiments, the antigen is a polypeptide encoded by a polynucleotide. In some embodiments, the antigen is a polypeptide encoded by a polynucleotide. In some embodiments, the antigen is a DNA polynucleotide encoding a polypeptide that delivers the nanoalum formulation of the present disclosure. In some embodiments, the antigen is an RNA polynucleotide encoding a polypeptide that delivers the nanoalum formulation of the present disclosure. Thus, in some embodiments, the described compositions include any of the nanoalum particles provided herein and further include an antigen, wherein the antigen of the nanoalum particle is provided as a polypeptide or a polynucleotide.

[0309] In some embodiments, the antigen is related to or derived from an allergic, cancer, or infectious disease.

[0310] In some embodiments, the compositions described herein are for vaccination purposes and are provided as a vaccine formulation (vaccine composition).

[0311] An antigen can be any target epitope, molecule (including biomolecules), molecular complex (including molecular complexes containing biomolecules), subcellular assembly, cell or tissue for which it is desired to induce or enhance the immunoreactivity of a subject. Often, the term antigen will refer to a polypeptide antigen of interest. However, as used herein, an antigen can also refer to a recombinant construct (e.g., an expression construct) encoding a polypeptide antigen of interest. In certain embodiments, the antigen can be or can be derived from or can immunologically cross-react with: infectious pathogens and / or epitopes, biomolecules, cells or tissues associated with infection, cancer, autoimmune diseases, allergies, asthma or any other condition where it would be desirable or beneficial to stimulate an antigen-specific immune response.

[0312] Accordingly, certain embodiments contemplate antigens derived from at least one infectious pathogen such as a bacterium, virus or fungus, including actinomycetes such as Mycobacterium tuberculosis or Mycobacterium leprae or another Mycobacterium species; bacteria, such as members of Salmonella, Neisseria, Borrelia, Chlamydia or Bordetella; viruses, such as herpes simplex virus, human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), cytomegalovirus, varicella zoster virus, hepatitis virus, Epstein - Barr virus (EBV), respiratory syncytial virus, human papillomavirus (HPV) and cytomegalovirus; HIV, such as HIV - 1 or HIV - 2; fungi, such as Aspergillus, Blastomyces, Coccidioides and Pneumocystis or yeasts, including Candida species, such as Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida tropicalis and Candida parapsilosis; parasites, such as protozoa, e.g., Plasmodium species, including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale; or another parasite, such as Acanthamoeba, Entamoeba histolytica, Angiostrongylus cantonensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Cryptosporidium, hookworm, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba nana, Wuchereria bancrofti, Giardia and Leishmania.

[0313] For example, in certain embodiments, where the antigen is derived from the genus Borrelia, the antigen can comprise nucleic acids, pathogen - derived antigens or antigen preparations, recombinantly produced proteins or peptides, and chimeric fusion proteins. One such antigen is OspA. OspA can be the fully mature protein in its lipidated form (Lipo - OspA) by virtue of its biosynthesis in a host cell or alternatively can be a non - lipidated derivative. Such non - lipidated derivatives include the non - lipidated NS1 - OspA fusion protein having the first 81 N - terminal amino acids (NS1) of the non - structural protein of influenza virus and the intact OspA protein, and another MDP - OspA is the non - lipidated form of OspA carrying 3 additional N - terminal amino acids.

[0314] In certain embodiments, the antigen is derived from a virus such as derived from HIV-1 (such as tat, nef, gp120 or gp160), human herpesvirus such as gD, or a derivative thereof, or an immediate early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus (especially human) (such as gB or a derivative thereof), rotavirus (including live attenuated virus), Epstein-Barr virus (such as gp350 or a derivative thereof), varicella-zoster virus (such as gpl, II and IE63), or derived from hepatitis virus such as hepatitis B virus (e.g., hepatitis B surface antigen or a derivative thereof), hepatitis A virus, hepatitis C virus and hepatitis E virus, or derived from other pathogenic viruses such as paramyxovirus: respiratory syncytial virus (such as F and G proteins or a derivative thereof), parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, 11, 16, 18, etc.), flavivirus (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, Zika virus, Powassan virus), or influenza virus (whole live virus or inactivated virus), split influenza virus, cells grown in eggs or MDCK cells, or intact influenza virus (such as described by Gluck, Vaccine, 1992, 10, 915-920), or a purified or recombinant protein thereof such as HA, NP, NA or M protein, or a combination thereof).

[0315] In certain other embodiments, the antigen is derived from one or more bacterial pathogens, such as Neisseria spp., including Neisseria gonorrhoeae and Neisseria meningitidis (e.g., capsular polysaccharides and their conjugates, transferrin-binding proteins, lactoferrin-binding proteins, PilC, adhesins); Streptococcus pyogenes (e.g., M protein or fragments thereof, C5A protease, lipoteichoic acid), Streptococcus agalactiae, Streptococcus mutans; Haemophilus ducreyi; Moraxella spp., including Moraxella catarrhalis, also known as Branhamella catarrhalis (e.g., high molecular weight and low molecular weight adhesins and invasins); Bordetella spp., including Bordetella pertussis (e.g., pertussis adhesin, pertussis toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, fimbriae), Bordetella parapertussis, and Bordetella bronchiseptica; Mycobacterium spp., including Mycobacterium tuberculosis (e.g., ESAT6, antigen 85A, -B, or -C), Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium smegmatis; Legionella spp., including Legionella pneumophila; Escherichia spp., including enterotoxigenic Escherichia coli (e.g., colonization factor, heat-labile enterotoxin or derivatives thereof, heat-stable enterotoxin or derivatives thereof), enterohemorrhagic Escherichia coli, enteropathogenic Escherichia coli (e.g., Shiga-like toxin or derivatives thereof); Vibrio spp., including Vibrio cholerae (e.g., cholera toxin or derivatives thereof); Shigella spp., including Shigella sonnei, Shigella dysenteriae, Shigella flexneri; Yersinia spp., including Yersinia enterocolitica (e.g., Yop proteins), Yersinia pestis, Yersinia pseudotuberculosis; Campylobacter spp., including Campylobacter jejuni (e.g., toxins, adhesins, and invasins) and Campylobacter coli; Salmonella spp., including Salmonella typhi, Salmonella paratyphi A, Salmonella choleraesuis, Salmonella enteritidis; Listeria spp., including Listeria monocytogenes; Helicobacter spp., including Helicobacter pylori (e.g., urease, catalase, vacuolating toxin); Pseudomonas spp., including Pseudomonas aeruginosa; Staphylococcus spp., including Staphylococcus aureus, Staphylococcus epidermidis; Enterococcus spp., including Enterococcus faecalis, Enterococcus faecium; Clostridium spp., including Clostridium tetani (e.g., tetanus toxin and derivatives thereof), Clostridium botulinum (e.g., botulinum toxin and derivatives thereof), Clostridium difficile (e.g., Clostridium toxin A or B and derivatives thereof); Bacillus spp., including Bacillus anthracis (e.g., botulinum toxin and derivatives thereof); Corynebacterium spp., including Corynebacterium diphtheriae (e.g., diphtheria toxin and derivatives thereof); Borrelia spp., including Borrelia burgdorferi (e.g., OspA, OspC, DbpA, DbpB), Borrelia garinii (e.g., OspA, OspC, DbpA, DbpB), Borrelia afzelii (e.g., OspA, OspC, DbpA, DbpB), Borrelia andersonii (e.g., OspA, OspC, DbpA, DbpB), Borrelia hermsii;Agents of the genus Ehrlichia, including Ehrlichia equi and human granulocytic ehrlichiosis; Rickettsia, including Rickettsia rickettsii; Chlamydia, including Chlamydia trachomatis (e.g., MOMP, heparin-binding protein), Chlamydia pneumoniae (e.g., MOMP, heparin-binding protein), Chlamydia psittaci; Leptospira, including Leptospira interrogans; Treponema, including Treponema pallidum (e.g., rare outer membrane protein), Treponema denticola, Treponema dysenteriae; or other bacterial pathogens.

[0316] In certain other embodiments, the antigen is derived from one or more parasites (see, e.g., John, D.T. and Petri, W.A., Markell and Voge's Medical Parasitology - 9th Edition, 2006, WB Saunders, Philadelphia; Bowman, D.D., Georgis' Parasitology for Veterinarians - 8th Edition, 2002, WB Saunders, Philadelphia), such as Plasmodium, including Plasmodium falciparum; Toxoplasma, including Toxoplasma gondii (e.g., SAG2, SAG3, Tg34); Entamoeba, including Entamoeba histolytica; Babesia, including Babesia microti; Trypanosoma, including Trypanosoma brucei gambiense; Giardia, including Giardia lamblia; Leishmania, including Leishmania major; Pneumocystis, including Pneumocystis carinii; Trichomonas, including Trichomonas vaginalis; or is derived from worms capable of infecting mammals, such as: (i) nematode infections (including but not limited to Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Dracunculus medinensis, Trichinella spiralis, and Strongyloides stercoralis); (ii) trematode infections (including but not limited to Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, Clonorchis sinensis, Paragonimus spp., Fasciola hepatica, Fasciola gigantica, Fasciolopsis buski); and (iii) cestode infections (including but not limited to Taenia saginata and Taenia solium). In certain embodiments, the antigen is derived from Schistosoma, Schistosoma mansoni, Schistosoma haematobium, and / or Schistosoma japonicum, or is derived from yeasts such as Candida, including Candida albicans; Cryptococcus, including Cryptococcus neoformans.

[0317] Other specific antigens are derived from Mycobacterium tuberculosis, such as Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd14, DPV, MTI, MSL, mTTC2, and hTCC1 (WO 99 / 51748). Proteins directed against Mycobacterium tuberculosis also include fusion proteins and variants thereof, wherein at least two, three, four, or more polypeptides of Mycobacterium tuberculosis are fused to a larger protein. Some fusions include Ra12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2, Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99151748). Other antigens that can be used include the antigens, antigen combinations, and fusion proteins described in US2010 / 0129391 and WO 2008 / 124647. In one exemplary embodiment, the fusion protein is ID93. In one exemplary embodiment, the fusion protein is ID91.

[0318] Other specific antigens are derived from Leishmania, for example, the Leishmania polypeptides and polynucleotides of the present disclosure can be prepared or isolated using any one of a variety of procedures and using any one of a variety of Leishmania species, including but not limited to Leishmania donovani, Leishmania chagasi, Leishmania infantum, Leishmania major, Leishmania amazonensis, Leishmania braziliensis, Leishmania panamensis, Leishmania mexicana, Leishmania tropica, and Leishmania subgenus guyanensis. Such species are available from the American Type Culture Collection (ATCC), Rockville, Maryland. Proteins directed against Leishmania also include fusion proteins and variants thereof, wherein at least two, three, four, or more polypeptides of Leishmania are fused to a larger protein as described in WO2009 / 012166, WO 2014 / 160987, WO 2014 / 160985. In one exemplary embodiment, the fusion protein is EMCH as described herein.

[0319] Other specific antigens are derived from Chlamydia and include, for example, high molecular weight proteins (HWMP) (WO 99 / 17741), ORF3 (EP 366 412), and putative membrane proteins (Pmp). Other Chlamydia antigens can be selected from the group described in WO 99128475. Certain antigens can be derived from the genus Streptococcus, including Streptococcus pneumoniae (e.g., capsular polysaccharides and their conjugates, PsaA, PspA, streptolysin, choline-binding proteins), and the protein antigen pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337-342) and its detoxified derivatives of mutants (WO 90 / 06951; WO 99 / 03884). Other bacterial vaccines include antigens derived from the genus Haemophilus, which includes Haemophilus influenzae type B (e.g., PRP and its conjugates), nontypeable Haemophilus influenzae such as OMP26, high molecular weight adhesins, P5, P6, D protein, and D lipoprotein, and fimbrin and fimbrin or its multi-copy variants or fusion proteins derived from peptides (U.S. Patent No. 5,843,464).

[0320] Other specific antigens are derived from hepatitis B. Derivatives of hepatitis B surface antigen are well known in the art and include, in particular, those described in European patent applications EP-A414 374; EP-A-0304 578, and EP 198474, the PreS1, Pars2S antigens. In one aspect, the antigen is HIV-1 gp120, especially when expressed in CHO cells. In additional embodiments, the antigen is gD2t.

[0321] In other embodiments, the antigen is derived from human papillomavirus (HPV), which is thought to be responsible for genital warts (HPV 6 or HPV 11 and others), and the HPV virus is responsible for cervical cancer (HPV16, HPV18 and others). Specific antigens include L1 particles or capsids, and fusion proteins comprising one or more antigens selected from the HPV 6 and HPV 11 proteins E6, E7, L1 and L2. Certain forms of the fusion protein include L2E7 as disclosed in WO 96 / 26277 and Protein D(1 / 3)-E7 as disclosed in GB 9717953.5 (PCT / EP98 / 05285). Additional possible antigens include HPV 16 or 18 antigens. For example, L1 or L2 antigen monomers, or L1 or L2 antigens presented together as virus-like particles (VLPs), or the L1 protein alone presented in a VLP or capsid structure. Such antigens, virus-like particles and capsids are known per se. See, for example, WO 94 / 00152, WO 94 / 20137, WO 94 / 05792 and WO 93 / 02184.

[0322] In other embodiments, the antigen is a fusion protein. The fusion protein can be included alone or as a fusion protein such as, for example, E7, E2 or F5; specific embodiments include VLPs comprising an L1E7 fusion protein (WO 96 / 11272). Specific HPV 16 antigens include the early proteins E6 or F7, which are fused to a Protein D carrier to form a Protein D-E6 or E7 fusion from HPV 16 or a combination thereof; or a combination of E6 or E7 with L2 (WO 96 / 26277). Alternatively, the HPV 16 or 18 early proteins E6 and E7 can be presented as a single molecule, such as a Protein D-E6 / E7 fusion. The composition can optionally include either or both of the E6 and E7 proteins from HPV 18, for example in the form of a Protein D-E6 or Protein D-E7 fusion protein or a Protein D E6 / E7 fusion protein. The composition can additionally include antigens from other HPV strains, such as from strain HPV 31 or 33.

[0323] The antigen can also be derived from the parasite that causes malaria. For example, antigens from Plasmodium falciparum include RTS,S and TRAP. RTS is a hybrid protein that includes substantially all of the C-terminal portion of the circumsporozoite (CS) protein of Plasmodium falciparum, which is linked to the surface (S) antigen of hepatitis B virus by four amino acids of the preS2 portion of the hepatitis B surface antigen. Its complete structure was disclosed in International Patent Application No. PCT / EP92 / 02591, which claims priority from UK Patent Application No. 9124390.7 and is published as WO 93 / 10152. When expressed in yeast, RTS is produced as a lipoprotein particle, and when co-expressed with the S antigen from HBV, it produces a hybrid particle called RTS,S.

[0324] The TRAP antigen was described in International Patent Application No. PCT / GB89 / 00895, published as WO 90 / 01496. An embodiment of the present disclosure is a malaria vaccine in which the antigen preparation comprises a combination of RTS,S and TRAP antigens. Other Plasmodium falciparum antigens that may be candidates for components of a multistage malaria vaccine are Plasmodium falciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, clag, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16, Pfs48 / 45, Pfs230 and their analogs in other Plasmodium species.

[0325] In one embodiment, the antigen is derived from cancer cells and can be used for immunotherapeutic treatment of cancer. For example, the antigen can be a tumor rejection antigen, such as those for prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, or melanoma. Exemplary cancer or cancer cell-derived antigens include MAGE 1, 3, and MAGE 4 or other MAGE antigens, such as those disclosed in WO99 / 40188, PRAME, BAGE, Lage (also known as NY Eos 1), SAGE, and HAGE (WO 99 / 53061), or GAGE (Robbins and Kawakami, 1996, Current Opinions in Immunology, 8th Edition, pages 628 to 636; Van den Eynde et al., International Journal of Clinical & Laboratory Research (1997 and 1998); Correale et al. (1997), Journal of the National Cancer Institute, 89th Edition, page 293. These non-limiting examples of cancer antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518.

[0326] Other tumor-specific antigens include, but are not limited to: tumor-specific or tumor-associated gangliosides for the treatment of many cancers, such as GM2 and GM3 or their conjugates to carrier proteins; or autologous peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH, WO 95 / 20600), 10-amino acid long short peptides. In another embodiment, prostate antigens are used, such as prostate-specific antigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA, 95(4) 1735-1740 1998), PSMA, or an antigen referred to as prostate in one embodiment (e.g., Nelson et al., Proc. Natl. Acad. Sci. USA (1999) 96:3114-3119; Ferguson et al., Proc. Natl. Acad. Sci. USA 1999 96, 3114-3119; WO 98 / 12302; U.S. Patent No. 5,955,306; WO 98 / 20117; U.S. Patent Nos. 5,840,871 and 5,786,148; WO 00 / 04149. Other prostate-specific antigens are known from WO 98 / 137418 and WO / 004149. Another one is STEAP (PNAS 96 14523 14528 7-12 1999).

[0327] Other tumor-associated antigens for use in the context of the present disclosure include: Plu-1 (J Biol Chem 274(22) 15633-15645, 1999), HASH-1, HasH-2, Cripto (Salomon et al., Bioessays 199, 21:61-70, U.S. Patent No. 5,654,140) and Criptin (U.S. Patent No. 5,981,215). In addition, antigens particularly relevant to cancer therapy vaccines also include tyrosinase and survivin.

[0328] In other embodiments, the agent used in the compositions of the present disclosure comprises an antigen associated with a respiratory disease, such as those respiratory diseases caused or exacerbated by a bacterial infection (e.g., pneumococcal infection), for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD). COPD is physiologically defined by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (American Journal of Respiratory and Critical Care Medicine, November 1995; 152(5 Pt 2): S77-121). Exacerbations of COPD are typically caused by a bacterial (e.g., pneumococcal) infection (Clinical Microbiology Reviews, April 2001; 14(2): 336-63).

[0329] ii. Adjuvant

[0330] In some embodiments, the agent is an adjuvant, and thus the compositions comprising any of the nanoalum particles described herein include an adjuvant in the presence or absence of an antigen.

[0331] In some embodiments, the adjuvant is selected from the group consisting of: AS-2, monophosphoryl lipid A, 3-de-O-acylated monophosphoryl lipid A, IFA, QS21, CWS, TOM, AGP, CpG oligonucleotide-containing, Toll-like receptor (TLR) agonists, Leif, saponins, saponin mimetics, biological and synthetic lipid A, imiquimod, gardiquimod, resiquimod, poly I:C, flagellin, GLA, SLA, STING, and combinations thereof.

[0332] In one exemplary embodiment, the adjuvant is GLA. In one exemplary embodiment, the adjuvant is SLA.

[0333] a. TLR agonist

[0334] As described herein, certain embodiments of the present disclosure contemplate including nanoalum particles as described herein and further comprising one or more Toll-like receptor agonists (TLR agonists). Toll-like receptors (TLRs) comprise cell surface transmembrane receptors of the innate immune system that confer early recognition capabilities to host cells of a variety of conserved microbial molecular structures as may be present in or on a large number of infectious pathogens. (See, e.g., Armant et al., 2002, Genome Biol. 3(8): commentary 3011.1-3011.6; Fearon et al., 1996, Science 272:50; Medzhitov et al., 1997, Curr. Opin. Immunol. 9:4; Luster, 2002, Curr. Opin. Immunol. 14:129; Lien et al., 2003, Nat. Immunol. 4:1162; Medzhitov, 2001, Nat. Rev. Immunol. 1:135; Takeda et al., 2003, Ann Rev Immunol. 21:335; Takeda et al., 2005, Int. Immunol. 17:1; Kaisho et al., 2004, Microbes Infect. 6:1388; Datta et al., 2003, J. Immunol. 170:4102).

[0335] Inducing TLR-mediated signal transduction to enhance immune responses initiated through the innate immune system is affected by TLR agonists on the surface of conjugate cells. For example, lipopolysaccharide (LPS) can be a TLR agonist through TLR2 or TLR4 (Tsan et al., 2004, Journal of Leukocyte Biology, 76:514; Tsan et al., 2004, American Journal of Physiology-Cell Physiology, 286:C739; Lin et al., 2005, Shock, 24:206); polyinosinic-polycytidylic acid (poly I:C) can be a TLR agonist through TLR3 (Salem et al., 2006, Vaccine, 24:5119); CpG sequences (oligodeoxynucleotides containing unmethylated cytosine-guanosine or "CpG" dinucleotide motifs, e.g., CpG 7909, Cooper et al., 2005, AIDS, 19:1473; CpG10101, Bayes et al., Methods Find Exp Clin Pharmacol, 27:193; Vollmer et al., Expert Opinion on Biological Therapy, 5:673; Vollmer et al., 2004, Antimicrob Agents Chemother, 48:2314; Deng et al., 2004, Journal of Immunology, 173:5148) can be a TLR agonist through TLR9 (Andaloussi et al., 2006, Glia, 54:526; Chen et al., 2006, Journal of Immunology, 177:2373); peptidoglycan can be a TLR2 and / or TLR6 agonist (Soboll et al., 2006, Biology of Reproduction, 75:131; Nakao et al., 2005, Journal of Immunology, 174:1566); 3M003 with a molecular weight of 318 Da from 3M Pharmaceuticals of St. Paul, Minnesota (4-amino-2-(methacryloyl)-α,α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1-ethanol hydrate, which is also the source of related components 3M001 and 3M002; Gorden et al., 2005, Journal of Immunology) can be a TLR7 agonist (Johansen, 2005, Clin. Exp. Allerg. 35:1591) and / or a TLR8 agonist (Johansen, 2005); flagellin can be a TLR5 agonist (Feuillet et al., 2006, Proc. Nat. Acad. Sci. USA 103:12487); and hepatitis C can be used as a TLR agonist via TLR7 and / or TLR9 (Lee et al., 2006, Proc. Nat. Acad. Sci. USA 103:1828; Horsmans et al., 2005, Hepatol. 42:724). Other TLR agonists are known (e.g., Schirmbeck et al., 2003, J. Immunol. 171:5198) and can be used according to certain embodiments described herein.

[0336] b. TLR7 / 8 agonist

[0337] Provided herein are TLR7 / 8 agonists that can be used in the compositions described herein. As used herein, "TLR7 / 8 agonist" refers to an agonist that affects the biological activity thereof through its interaction with TLR7, TLR8, or both. Such biological activity includes, but is not limited to, inducing TLR7- and / or TLR8-mediated signal transduction to enhance immune response inhibition through the innate immune system.

[0338] c. TLR4 agonist

[0339] Provided herein are TLR4 agonists that can be used in the compositions described herein. In certain embodiments, the TLR4 agonist for use in the compositions herein includes glucopyranosyl lipid adjuvant (GLA), such as those described in U.S. Patent Publication Nos. US2007 / 021017, US2009 / 045033, US2010 / 037466, and US 2010 / 0310602, the contents of which are incorporated herein by reference in their entirety.

[0340] For example, in certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the structure of formula (IV) below:

[0341]

[0342] or a pharmaceutically acceptable salt thereof, wherein:

[0343] L1, L2, L3, L4, L5, and L6 are the same or different and independently are -O-, -NH-, or -(CH2)-;

[0344] L7, L8, L9, and L 10 are the same or different and independently are absent or -C(=O)-;

[0345] Y1 is an acid functional group;

[0346] Y2 and Y3 are the same or different and independently are -OH, -SH, or an acid functional group;

[0347] Y4 is -OH or -SH;

[0348] R1, R3, R5, and R6 are the same or different and independently are C 8-13 alkyl; and

[0349] R2 and R4 are the same or different and independently are C 6-11 alkyl.

[0350] In some embodiments of synthesizing the GLA structure, R 1 , R 3 , R 5 , and R 6 are C 10 alkyl; and R 2 , and R 4 are C8 alkyl. In certain embodiments, R 1 , R 3 , R 5 , and R 6 are C 11 alkyl; and R 2 , and R 4 are C9 alkyl.

[0351] For example, in certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the structure of formula (V) below:

[0352]

[0353] In a specific embodiment, R1, R3, R5, and R6 are C11-C20 alkyl; and R2 and R4 are C12-C20 alkyl.

[0354] In another specific embodiment, GLA has the above formula, where R1, R3, R5, and R6 are C11 alkyl; and R2 and R4 are C13 alkyl.

[0355] In another specific embodiment, GLA has the above formula, where R1, R3, R5, and R6 are C10 alkyl; and R2 and R4 are C8 alkyl.

[0356] In another specific embodiment, GLA has the above formula, wherein R 1 , R 3 , R 5 and R 6 are C 11 -C 20 alkyl; and R 2 and R 4 are C9-C 20 alkyl. In certain embodiments, R 1 , R 3 , R 5 and R 6 are C 11 alkyl; and R 2 and R 4 are C9 alkyl.

[0357] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (V):

[0358]

[0359] In certain embodiments of the above GLA structure, R1, R3, R5, and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5, and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.

[0360] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (VI):

[0361]

[0362] In certain embodiments of the above GLA structure, R1, R3, R5, and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5, and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.

[0363] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the structure of the following formula (VII):

[0364]

[0365] In certain embodiments of the above GLA structure, R 1 , R 3 , R 5 and R 6 are C 11 -C 20 alkyl; and R 2 and R4 is a C9-C 20 alkyl group. In certain embodiments, R 1 , R 3 , R 5 and R 6 are C 11 alkyl groups; and R 2 and R 4 are C9 alkyl groups.

[0366] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure (SLA):

[0367]

[0368] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0369]

[0370] In certain embodiments, the TLR4 agonist is a synthetic GLA adjuvant having the following structure:

[0371]

[0372] In another embodiment, an attenuated lipid A derivative (ALD) is incorporated into the compositions described herein. An ALD is a lipid A-like molecule that has been altered or engineered such that the molecule exhibits fewer or different adverse reactions of lipid A. These adverse reactions include pyrogenicity, local Shwarzman reactivity, and toxicity as evaluated in the chicken embryo 50% lethal dose assay (CELD 50 ). ALDs useful according to the present disclosure include monophosphoryl lipid A (MLA) and 3-deacylated monophosphoryl lipid A (3D-MLA). MLA and 3D-MLA are known and need not be described in detail herein. See, for example, U.S. Patent No. 4,436,727, issued March 13, 1984, and assigned to Ribi ImmunoChem Research, Inc., which discloses monophosphoryl lipid A and its manufacture. U.S. Patent No. 4,912,094 and Reexamination Certificate B1 U.S. Patent No. 4,912,094 embody 3-deacylated monophosphoryl lipid A and methods for its manufacture. The disclosures of each of these patents regarding MLA and 3D-MLA are incorporated herein by reference.

[0373] In the above TLR4 agonist compounds, the total charge can be determined based on the functional groups in the molecule. For example, a phosphate group can be negatively charged or neutral, depending on the ionization state of the phosphate group.

[0374] d. CpG nucleotides

[0375] In one embodiment, the adjuvant is an immunostimulatory oligonucleotide containing unmethylated CpG dinucleotides (e.g., U.S. Patent No. 6,544,518). Immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides ("CpG") are known as adjuvants. In some embodiments, the CpG oligonucleotides of the present disclosure may contain two or more dinucleotide CpG motifs separated by at least two, at least four, at least five, or at least six, or more nucleotides.

[0376] Examples of CpG oligonucleotide sequences are disclosed in the following publications; for certain embodiments disclosed herein, the sequences may contain phosphorothioate-modified internucleotide linkages:

[0377] CPG 7909: Cooper et al., "CPG 7909 Adjuvant Improves Hepatitis B Virus Vaccine Serum Protection in HIV-Infected Adults on Antiretroviral Therapy" AIDS, September 23, 2005; 19(14):1473-9.

[0378] CpG 10101: Bayes et al., "Towards Clinical Trials" Methods Find. Exp. Clin. Pharmacol., April 2005; 27(3):193-219.

[0379] Vollmer J, "Progress in the Development of Immunostimulatory CpG Oligodeoxynucleotide Ligands for TLR9" Expert Opinion on Biological Therapy, May 2005; 5(5):673-682.

[0380] Alternative CpG oligonucleotides may include variants of the sequences described in the above publications, except that they have inconsequential nucleotide sequence substitutions, insertions, deletions, and / or additions made to them. The CpG oligonucleotides utilized in certain embodiments of the present disclosure can be synthesized by any method known in the art (e.g., EP 468520). Conveniently, such oligonucleotides can be synthesized using an automated synthesizer. Oligonucleotides are typically deoxynucleotides. In one embodiment, the internucleotide bond in the oligonucleotide is a phosphorodithioate or phosphorothioate bond, although phosphodiester is also within the scope of currently contemplated embodiments. Oligonucleotides including different internucleotide linkages are also contemplated, such as mixed phosphorodithioate phosphodiester. Other internucleotide bonds that stabilize the oligonucleotide can also be used.

[0381] iii. Organisms and Viruses

[0382] In some embodiments, the agent is an organism. Thus, in some embodiments, the described compositions comprise the nanoalum particles provided herein and further comprise an organism.

[0383] For example, the bacterium Mycobacterium tuberculosis causes tuberculosis (TB). Currently, vaccination with live bacteria is the most effective method for inducing protective immunity against TB. The most common mycobacterium used for this purpose is Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis. Thus, in some embodiments, the composition comprises nanoalum particles and mycobacteria.

[0384] In some embodiments, the agent is a virus or viral genome. Thus, in some embodiments, the described compositions comprise the nanoalum particles provided herein and further comprise viral particles, isolated viral envelopes, or viral genomes.

[0385] B. Associated with Nanoalum Particles

[0386] In the embodiments provided herein, the agent of the compositions provided herein is associated with the nanoalum particles. In some embodiments, the agent of the compositions provided herein binds to the nanoalum particles. In some embodiments, the agent of the compositions provided herein adsorbs onto the nanoalum particles. Such binding or adsorption refers to the interaction between molecules or portions thereof that generally exhibit an ability to attract or bind to each other due to specific or non-specific binding or interactions, including but not limited to biochemical, physiological, and / or chemical interactions. In certain embodiments, the binding to the nanoalum particles can be determined by ultraviolet spectroscopy or gel electrophoresis.

[0387] Adsorption onto the nanoalum particles can generally occur by the following mechanisms, but is not limited to: electrostatic interactions and ligand exchange. Electrostatic interactions utilize the presence of opposite charges on the components under certain solution conditions. Ligand exchange utilizes a phosphate group in one of the components to exchange with a hydroxyl group of another component. For ligand exchange, accessible phosphate and hydroxyl groups in the components are used. In some embodiments, in order to prepare a composition with an agent, antigen, or adjuvant, the charge on the agent and the presence of phosphate and hydroxyl groups on the agent are considered.

[0388] i. Ligand Exchange

[0389] In certain embodiments regarding the ligand exchange mechanism, there may be ligand exchange between the agent and the aluminum salt.

[0390] In certain embodiments, there may be ligand exchange between the adjuvant and the aluminum salt. Certain components in the adjuvant composition include phosphate groups, while certain other components include hydroxyl groups, and thus are capable of ligand exchange. For example, certain TLR4 agonists include phosphate groups. Comprising a phosphate group. The hydroxyl group is present in at least one of the following components: antigen, TLR agonist, lipid / surfactant, and

[0391] ii. Electrostatic interaction

[0392] In certain embodiments regarding the mechanism of electrostatic interaction, ligand exchange may occur between the agent and the aluminum salt.

[0393] In certain embodiments regarding the mechanism of electrostatic interaction, at about physiological pH, the vaccine composition is substantially uncharged. If the antigen of the vaccine composition is charged, the components of the adjuvant composition can be selected to neutralize the charge of the antigen to provide a substantially uncharged vaccine composition. If the antigen of the vaccine composition is substantially uncharged, the components of the adjuvant composition can be selected to keep the antigen substantially uncharged to provide a substantially uncharged vaccine composition.

[0394] As described above, each of the components in the composition can be characterized as negatively charged, positively charged, or uncharged.

[0395] C. Dose sparing

[0396] In some embodiments, a composition comprising the nanoalum particles provided herein and further comprising an agent has dose sparing and / or a high level of in vivo expression. In one embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95% or even at least 99% less of the agent compared to the amount of the agent that would be used to achieve the same biological and / or physiological effect. A composition comprising any of the nanoalum particles provided herein is not used for delivery. In one embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg, 10 ng or 1 ng to achieve a biological and / or physiological effect. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg or 10 ng, 1 ng of a polypeptide to achieve the desired biological and / or physiological effect. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg or 10 ng, 1 ng of a polypeptide to achieve an immune response. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg, 10 ng or 1 ng of a polynucleotide to achieve the desired biological and / or physiological effect. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg or 10 ng, 1 ng of a polynucleotide to achieve an immune response. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 100 ng, 50 ng, 30 ng, 10 ng or 1 ng of an RNA polynucleotide of an RNA agent to achieve an immune response. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg, 10 ng or 1 ng of a replicon RNA vector polynucleotide to achieve an immune response. In a specific embodiment, use of a composition comprising any of the nanoalum particles provided herein allows use of a dose of the agent of about 10 μg, 5 μg, 2 μg, 1 μg, 10 ng or 1 ng of an mRNA vector polynucleotide to achieve an immune response.

[0397] In one embodiment, delivery of an RNA polynucleotide as compared to unformulated RNA results in a dose savings of RNA as little as 300-fold less RNA to achieve polypeptide antigen expression to generate an immune response. In one embodiment, delivery of an RNA polynucleotide as compared to unformulated RNA results in a dose savings of RNA as little as 100-fold less RNA to achieve polypeptide antigen expression to generate an immune response. In one embodiment, delivery of an RNA polynucleotide as compared to unformulated RNA results in a dose savings of RNA as little as 50-fold less RNA to achieve polypeptide antigen expression to generate an immune response. In one embodiment, delivery of an RNA polynucleotide as compared to unformulated RNA results in a dose savings of RNA as little as 30-fold less RNA to achieve polypeptide antigen expression to generate an immune response. In one embodiment, delivery of an RNA polynucleotide as compared to unformulated RNA results in a dose savings of RNA as little as 10-fold less RNA to achieve polypeptide antigen expression to generate an immune response.

[0398] D. Pharmaceutical Compositions

[0399] Provided herein are pharmaceutical compositions comprising nanoalum particles and the compositions described herein. In some embodiments, the compositions comprising nanoalum particles further comprise a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is a vaccine composition. For any therapeutic or diagnostic purpose, the compositions described herein can be administered to a subject. In some embodiments, the compositions described herein are used to stimulate an immune response (including non-specific and antigen-specific responses) in a subject. In the embodiments provided herein, the subject is a mammal (e.g., an animal including farm animals (cows, pigs, goats, horses, etc.), pets (cats, dogs, etc.), and rodents (rats, mice, etc.) or a human). Specifically, the formulations and compositions of the present invention that promote a Th1 immune response can be used to stimulate such a response in a subject.

[0400] Pharmaceutical compositions generally comprise the compositions described herein and can further comprise one or more components selected from antigens, additional agonists, or recombinant expression constructs in combination with a pharmaceutically acceptable carrier, excipient, or diluent as provided herein.

[0401] In the embodiments provided herein, the pharmaceutical composition is capable of filtering through a 0.45 micron filter. In some embodiments, the pharmaceutical composition is capable of filtering through a 0.20 micron filter. In some embodiments, the pharmaceutical composition is capable of filtering through a 0.22 micron filter.

[0402] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising nanoalum particles, the nanoalum particles comprising a TLR7 / 8 agonist or a TLR4 agonist. Such a composition can be used in "monotherapy", wherein, as described herein, the TLR7 / 8 agonist or TLR4 agonist is formulated in the composition and the composition is substantially devoid of other antigens and administered to a subject to stimulate an immune response such as a non-specific immune response or an antigen-specific immune response in order to diagnose, treat or prevent a disease or other condition, such as an organism infection.

[0403] In other embodiments, the pharmaceutical composition is a vaccine composition, the vaccine composition comprising the composition described herein and an antigen, and may further comprise one or more components in combination with a pharmaceutically acceptable carrier, excipient or diluent as provided herein. Exemplary carriers will be non-toxic to the recipient at the dosages and concentrations employed.

[0404] Exemplary carriers will be non-toxic to the recipient at the dosages and concentrations employed.

[0405] In the embodiments provided herein, a pharmaceutical composition is administered at a dosage of from about 1 ng / kg to about 1 mg / kg. In the embodiments provided herein, a pharmaceutical composition is administered at a dosage of from about 1 ng to about 1 mg. In some embodiments, a pharmaceutical composition is administered at a dosage of about 500 μg, 200 μg, 100 μg, 50 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 2 μg, 1 μg, 10 ng or 1 ng. It will be apparent to those skilled in the art that the amount and frequency of administration will depend on the response of the subject. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985). For example, sterile saline and phosphate buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of o-hydroxybenzoic acid can be added as preservatives. Intradermal injection is carried out at 1449. In addition, antioxidants and suspending agents can be used for intradermal injection.

[0406] "Pharmaceutically acceptable salts" refer to salts of compounds of the present disclosure derived from combinations of such compounds with organic or inorganic acids (acid addition salts) or organic or inorganic bases (base addition salts). The compositions of the present disclosure can be used in the free base or free salt form, with both forms being considered within the scope of the present disclosure.

[0407] The pharmaceutical composition can be in any form that permits the administration of the composition to a patient. For example, the composition can be in solid, liquid, or gaseous (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral (e.g., sublingual or buccal), sublingual, rectal, vaginal, and intranasal (e.g., as a spray). As used herein, the term parenteral includes: iontophoretic injection (e.g., U.S. Pat. Nos. 7,033,598; 7,018,345; 6,970,739), sonophoresis injection (e.g., U.S. Pat. Nos. 4,780,212; 4,767,402; 4,948,587; 5,618,275; 5,656,016; 5,722,397; 6,322,532; 6,018,678), thermoinjection (e.g., U.S. Pat. Nos. 5,885,211; 6,685,699), passive transdermal injection (e.g., U.S. Pat. Nos. 3,598,122; 3,598,123; 4,286,592; 4,314,557; 4,379,454; 4,568,343; 5,464,387; British Patent Specification No. 2232892; U.S. Pat. Nos. 6,871,477; 6,974,588; 6,676,961), microneedle injection (e.g., U.S. Pat. Nos. 6,908,453; 5,457,041; 5,591,139; 6,033,928) and jet injection administration as well as subcutaneous injection, intravenous, intramuscular, intracardiac, intracavemous, intrathecal, intraductal, intraurethral injection or infusion techniques. In a specific embodiment, the compositions (including vaccines and pharmaceutical compositions) described herein are administered intradermally by a technique selected from iontophoresis, microcavitation, sonophoresis, or microneedles.

[0408] The pharmaceutical composition can be formulated to permit the active ingredient contained therein to be bioavailable when the composition is administered to a subject. The composition to be administered to a subject is in the form of one or more doses, where, for example, a tablet can be a single dose unit, and a container of one or more compounds in aerosol form according to the present disclosure can contain multiple dose units.

[0409] For oral administration, excipients and / or binders can be present. Examples are sucrose, kaolin, starch dextrin, sodium alginate, carboxymethyl cellulose, and ethyl cellulose. Colorants and / or flavorants can be present. A coating shell can be employed.

[0410] The composition can be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or for delivery by injection. When intended for oral administration, the composition can contain one or more of a sweetening agent, a preservative, a dye / colorant, and a flavoring agent. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizing agent, and an isotonic agent can be included.

[0411] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form as used herein, can contain one or more of the following carriers or excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as squalene, squalene, mineral oil, mannitol monooleate, cholesterol and / or synthetic monoglycerides or diglycerides that can be used as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate, or phosphate and agents for tonicity adjustment such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0412] In another embodiment, the composition of the present disclosure is formulated in a manner that can be atomized.

[0413] It may also be desirable to include other components in the pharmaceutical composition, such as delivery vehicles, including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules and liposomes. Examples of additional immunostimulatory substances (adjuvants) for such vehicles are also described above and can include N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), glucan, IL-12, GM-CSF, interferon-γ and IL-12.

[0414] Although any suitable carrier known to those of ordinary skill in the art can be used in the pharmaceutical compositions of the present disclosure, the type of carrier can vary depending on the mode of administration and whether sustained release is desired. For parenteral administration such as subcutaneous injection, the carrier can include water, saline, ethanol, fats, waxes, or buffering agents. For oral administration, any of the above carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate. Biodegradable microspheres (e.g., polylactide-co-glycolide) can also be used as carriers for the pharmaceutical compositions of the present disclosure. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. In this regard, it is preferred that the microspheres be greater than about 25 microns.

[0415] The pharmaceutical compositions can also contain diluents such as buffering agents, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrin, chelating agents such as EDTA, glutathione, and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are suitable exemplary diluents. For example, the product can be formulated as a lyophilizate using a suitable excipient solution (e.g., sucrose) as a diluent.

[0416] As described above, in certain embodiments, the present disclosure encompasses compositions capable of delivering nucleic acid molecules encoding a desired antigen. Such compositions include recombinant viral vectors (e.g., retroviruses (see WO 90 / 07936, WO 91 / 02805, WO 93 / 25234, WO 93 / 25698, and WO 94 / 03622), adenoviruses (see Berkner, Biotechniques 6:616 - 627, 1988; Li et al., Hum. Gene Ther. 4:403 - 409, 1993; Vincent et al., Nat. Genet. 5:130 - 134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA 91:215 - 219, 1994), poxviruses (see U.S. Patent No. 4,769,330; U.S. Patent No. 5,017,487; and WO 89 / 01973)), recombinant expression construct nucleic acid molecules complexed with polycationic molecules (see WO 93 / 03709), and nucleic acids associated with liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851, 1987). In certain embodiments, DNA can be linked to killed or inactivated adenoviruses (see Curiel et al., Hum. Gene Ther. 3:147 - 154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Other suitable compositions include DNA ligands (see Wu et al., J. Biol. Chem. 264:16985 - 16987, 1989) and lipid - DNA combinations (see Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 - 7417, 1989).

[0417] In certain embodiments, liquid compositions intended for parenteral or oral administration should contain an amount such that a suitable dose of the vaccine composition will be obtained. Generally, this amount is at least 0.01 wt% antigen in the composition. When intended for oral administration, this amount can vary between 0.1% and about 70% of the weight of the composition. Oral compositions can contain between about 4% and about 50% antigen. Compositions and formulations can be prepared such that parenteral dosage units contain between about 0.01% and 1% by weight of the active composition.

[0418] The pharmaceutical composition can be intended for topical administration, in which case the carrier can suitably include a solution, an emulsion, a cream or a gel matrix. For example, the matrix can include one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and ethanol, and emulsifiers and stabilizers. A thickening agent can be present in the pharmaceutical composition for topical administration. If it is intended for transdermal administration, the composition can contain a transdermal patch or an iontophoresis device. The topical administration can contain an antigen (e.g., GLA antigen vaccine composition) or GLA (e.g., immunoadjuvant composition; GLA is available from Avanti Polar Lipids, Inc., Alabaster, Alabama; e.g., product number 699800) at a concentration of about 0.1% w / v to about 10% w / v (weight per unit volume).

[0419] A composition in the form of, for example, a suppository can be intended for rectal administration, and the suppository will melt in the rectum and release the drug. The composition for rectal administration can contain an oleaginous matrix as a suitable non-irritating excipient. Such a matrix includes but is not limited to lanolin, cocoa butter, and polyethylene glycol. In the methods of the present disclosure, the vaccine composition / adjuvant can be administered by using one or more inserts, one or more beads, one or more sustained-release formulations, one or more patches, or one or more rapid-release formulations.

[0420] V. Uses of Nanoalum Particles and Compositions

[0421] A. Treatment

[0422] In some embodiments, the agent can be used for therapeutic purposes. Thus, in some embodiments, the described composition includes the nanoalum particles provided herein and further includes an agent for treating a disease, condition, or disorder.

[0423] In some embodiments, the agent can be used to treat or prevent allergies, cancer, infectious diseases, autoimmunity, or addiction.

[0424] In the embodiments disclosed herein, the composition includes a cancer antigen. In some embodiments, the vaccine composition includes a cancer antigen or other cancer-specific or cancer-associated antigen that will be used to combat any cancer characterized by the expression of a tumor-associated antigen such as HER-2 / neu expression.

[0425] Compositions and methods according to certain embodiments of the present disclosure can also be used for preventing or treating autoimmune diseases, which include diseases, conditions or disorders in which the immune system of a host or subject unfavorably mediates an immune response against "self" tissues, cells, biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins, lipoproteins, proteolipids, lipids, glycans, nucleic acids such as RNA and DNA, oligosaccharides, polysaccharides, proteoglycans, glycosaminoglycans, etc. and other molecular components of the subject's cells and tissues) or epitopes (e.g., specific immunologically defined recognition structures such as those recognized by antibody variable region complementarity determining regions (CDRs) or by T cell receptor CDRs).

[0426] Thus, autoimmune diseases are characterized by an abnormal immune response involving cells or antibodies directed against normal autologous tissue in any given situation. Mammalian autoimmune diseases are generally classified into one of two distinct categories: cell-mediated diseases (i.e., T cells) or antibody-mediated disorders. Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto's thyroiditis, type I diabetes (juvenile diabetes), and autoimmune uvoretinitis. Antibody-mediated autoimmune disorders include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (or SLE), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune asthma, cryoglobulinemia, thrombotic thrombocytopenic purpura, primary biliary sclerosis, and pernicious anemia. One or more antigens associated with systemic lupus erythematosus are small nuclear ribonucleoprotein (snRNP); one or more antigens associated with Graves' disease are the thyroid-stimulating hormone receptor, thyroglobulin, and other components of thyroid epithelial cells (Akamizu et al., 1996; Kellerman et al., 1995; Raju et al., 1997; and Texier et al., 1992); one or more antigens associated with pemphigus are cadherin-like pemphigus antigens such as desmoglein 3 and other adhesion molecules (et al., 1996: Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993: and one or more antigens associated with thrombotic thrombocytopenic purpura are platelet antigens (see, for example, U.S. Patent 6,929,796; Gorski et al. (eds.), Autoimmunity, 2001, Kluwer Academic Publishers, Norwell, MA; Radbruch and Lipsky, P.E. (eds.) Current Concepts in Autoimmunity and Chronic Inflammation (Curr. Top. Microbiol. and Immunol.), 2001, Springer, New York).

[0427] Autoimmunity plays a role in more than 80 different diseases, including type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, scleroderma, and thyroid diseases. There is a lack of robust quantitative assessment of the incidence of most autoimmune diseases. The most recent studies conducted in the late 1990s indicated that autoimmune diseases are the third most common serious illnesses in the United States; and the most common autoimmune diseases affect more than 8.5 million Americans. Current estimates of the prevalence of the diseases range from 5% to 8% of the US population. Most autoimmune diseases disproportionately affect women. Women with autoimmune diseases may be 2.7 times more likely than men. Women are more prone to autoimmune diseases; men exhibit higher levels of natural killer cell activity than women. (Jacobsen et al., Clinical Immunology and Immunopathology, 84:223-243, 1997.)

[0428] The compositions provided herein can be used to induce protective immunity, such as protective immunity against tuberculosis, including the use of polypeptides containing at least one immunogenic portion of one or more mycobacterial proteins, as well as DNA and RNA molecules encoding such polypeptides. Additionally, such compounds can be formulated into vaccines and / or pharmaceutical compositions for immunization against mycobacterial infections. (U.S. Patent Nos. 6,949,246 and 6,555,653).

[0429] In certain embodiments, the compositions of the present disclosure will be particularly suitable for treating the elderly and / or immunosuppressed patients, including subjects undergoing renal dialysis, subjects undergoing chemotherapy and / or radiotherapy, transplant recipients, and the like. Such individuals typically exhibit a reduced immune response to vaccines, and thus, the use of the compositions of the present disclosure can enhance the immune response achieved in these subjects.

[0430] In other embodiments, the compositions of the present disclosure include antigens associated with respiratory diseases, such as those respiratory diseases caused or exacerbated by bacterial infections (e.g., pneumococcal infections), for the prevention and treatment of conditions such as chronic obstructive pulmonary disease (COPD).

[0431] In addition to direct in vivo processes, ex vivo processes can be used in which cells are removed from a host, modified, and placed in the same or another host animal. It will be apparent that one can utilize any of the compositions noted above in an ex vivo context to introduce antigen-encoding nucleic acid molecules into tissue cells. Protocols for viral, physical, and chemical uptake methods are well known in the art.

[0432] Accordingly, the present disclosure can be used to enhance or elicit an immune response in a host, patient, or cell culture. As used herein, the term "subject" refers to any mammal. A patient may be afflicted with an infectious disease, a cancer such as breast cancer, or an autoimmune disease or may be normal (i.e., without detectable disease and / or infection). A "cell culture" is any preparation containing immunocompetent cells or isolated cells of the immune system (including but not limited to T cells, macrophages, monocytes, B cells, and dendritic cells). Such cells can be isolated by any of a variety of techniques known to those of skill in the art (e.g., Ficoll-hypaque density centrifugation). The cells may (but need not) be isolated from a patient afflicted with cancer and can be reintroduced into the patient after treatment.

[0433] B. Vaccine

[0434] The present disclosure thus provides compositions for altering (e.g., in a statistically significant manner such as increasing or decreasing relative to an appropriate control familiar to those of skill in the art) the immune response of a host capable of mounting an immune response. As will be known to those of ordinary skill in the art, an immune response can be any active alteration of the immune state of a host, which can include any alteration in the structure or function of one or more tissues, organs, cells, or molecules involved in the maintenance and / or regulation of the host immune state. Generally, an immune response can be detected by any of a variety of well-known parameters, including but not limited to the following in vivo or in vitro determinations: soluble immunoglobulins or antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors, and other soluble small peptides, carbohydrates, nucleotides, and / or lipid mediators; changes in cell activation state as determined by altered functional or structural properties of the cells of the immune system, e.g., cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cell differentiation of the cells of the immune system, including an altered surface antigen expression profile or the occurrence of apoptosis (programmed cell death); or any other criterion by which an immune response can be detected.

[0435] The determination of the immune response induced by the compositions of the present disclosure can be established by any of a number of well-known immunological assays that will be readily familiar to those of ordinary skill in the art. Such assays include, but are not limited to, the following in vivo or in vitro determinations: soluble antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors, and other soluble small peptides, carbohydrates, nucleotides, and / or lipid mediators; changes in the state of cell activation determined by altered functional or structural properties of the cells of the immune system, such as cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cell differentiation of the cells of the immune system, including changes in the surface antigen expression profile or the occurrence of apoptosis (programmed cell death). The procedures for performing these and similar assays are widely known and can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998; see also Current Protocols in Immunology; see also, for example, Weir, Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, 1979, Freeman Publishing, San Francisco, CA; Green and Reed, 1998, Science, 281:1309 and references cited therein).

[0436] The detection of the proliferation of antigen-reactive T cells can be accomplished by various known techniques. For example, T cell proliferation can be detected by measuring the rate of DNA synthesis, and antigen specificity can be determined by controlling the stimuli to which the candidate antigen-reactive T cells are exposed (such as, for example, antigen-presenting cells pulsed with a specific desired antigen or a control antigen). T cells that have been stimulated to proliferate exhibit an increased rate of DNA synthesis. A typical method for measuring the rate of DNA synthesis is, for example, by pulse-labeling a culture of T cells with tritiated thymidine, which is a nucleoside precursor incorporated into newly synthesized DNA. A liquid scintillation spectrophotometer can be used to determine the amount of incorporated tritiated thymidine. Other ways to detect T cell proliferation include measuring an increase in interleukin-2 (IL-2) production, Ca2+ flux, or dye uptake rate, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured, or the relative number of T cells that can respond to a specific antigen can be quantified.

[0437] The detection of antigen-specific antibody production can be achieved, for example, by assaying a sample (such as, for example, an immunoglobulin-containing sample such as serum, plasma, and blood) from a host treated with a vaccine according to the present disclosure using in vitro methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, or solid-phase immunoblotting methods including Western blotting. In an embodiment, the ELISA assay can further include antigen capture immobilization of the target antigen with a solid-phase monoclonal antibody specific for the antigen, for example, to enhance the sensitivity of the assay. The refinement of soluble mediators (such as cytokines, chemokines, lymphokines, prostaglandins) can also be readily determined, for example, by enzyme-linked immunosorbent assay (ELISA) using methods, devices, and reagents that are readily available from commercial sources (such as, for example, Sigma, St. Louis, MO; see also the R&D Systems 2006 Catalog, R&D Systems, Minneapolis, MN).

[0438] Any number of other immunological parameters can be monitored using conventional assays well known in the art. These conventional assays can include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) assays, secondary antibody in vitro responses, flow immunocytofluorescence analysis of various peripheral blood or lymphomonocyte subsets using established marker antigen systems, immunohistochemistry, or other related assays. These and other assays can be found, for example, in Rose et al. (editors), Manual of Clinical Laboratory Immunology, 5th Edition, 1997, American Society of Microbiology, Washington, DC.

[0439] Accordingly, it is contemplated that the compositions provided herein will be capable of eliciting or enhancing in a host at least one immune response selected from the group consisting of a Th1-type T lymphocyte response, a Th2-type T lymphocyte response, a cytotoxic T lymphocyte (CTL) response, an antibody response, a cytokine response, a lymphokine response, a chemokine response, and an inflammatory response. In certain embodiments, the immune response can include at least one of the following: production of one or more cytokines, wherein the cytokines are selected from interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α); production of one or more interleukins, wherein the interleukins are selected from IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, and IL-23; production of one or more chemokines, wherein the chemokines are selected from MIP-1α, MIP-1β, RANTES, CCL4, and CCL5; and a lymphocyte response selected from memory T cells, memory B cells, effector T cell responses, cytotoxic T cell responses, and effector B cell responses. See, e.g., WO 94 / 00153; WO 95 / 17209; WO 96 / 02555; U.S. 6,692,752; U.S. 7,084,256; U.S. 6,977,073; U.S. 6,749,856; U.S. 6,733,763; U.S. 6,797,276; U.S. 6,752,995; U.S. 6,057,427; U.S. 6,472,515; U.S. 6,309,847; U.S. 6,969,704; U.S. 6,120,769; U.S. 5,993,800; U.S. 5,595,888; Smith et al., 1987, J Biol Chem. 262:6951; Kriegler et al., 1988, Cell 53:45 53; Beutler et al., 1986, Nature 320:584; U.S. 6,991,791; U.S. 6,654,462; U.S. 6,375,944.

[0440] The nano-alum formulations of the present invention can be used to treat or prevent diseases such as whooping cough, tuberculosis, leprosy, malaria, HIV, leishmaniasis, and influenza. Specifically, the ability of the nano-alum formulations to promote Th1 immunity makes them particularly useful in this regard.

[0441] C. Diagnostic agents

[0442] In some embodiments, the agent is a diagnostic agent. Thus, in these embodiments, the described composition includes the nanoalum particles provided herein and further includes a diagnostic agent and can be used to diagnose any disease, disorder, or condition.

[0443] In some embodiments, the diagnostic agent can be used to detect cancer. Compositions and methods for identifying a subject having cancer or at risk of developing cancer are described herein. Diagnosis of cancer in a subject having cancer or at risk of developing cancer can be accomplished by any of a wide range of methods accepted in the art, which can vary depending on various factors, including clinical manifestations, degree of cancer progression, type of cancer, and other factors. Examples of cancer diagnosis include histopathological, histochemical, immunohistochemical, and immunopathohistological examination of a patient sample (e.g., blood, skin biopsy, other tissue biopsy, surgical specimen, etc.); PCR assays of defined genetic (e.g., nucleic acid) markers; serological assays of circulating cancer-associated antigens or cells bearing such antigens or of antibodies with defined specificities; or other methods familiar to those skilled in the art. See, for example, U.S. Patent Nos. 6,734,172; 6,770,445; 6,893,820; 6,979,730; 7,060,802; 7,030,232; 6,933,123; 6,682,901; 6,587,792; 6,512,102; 7,078,180; 7,070,931; JP5-328975; Waslylyk et al., 1993, Eur. J Bioch. 211(7):18. Any one or more of these diagnostic agents can be included in a composition comprising the nanoalum particles described herein.

[0444] In some embodiments, the diagnostic agent can be used to detect autoimmune diseases. Thus, the detection of autoantibodies allows for the earlier detection or identification of the presence of an autoimmune disease or the risk of developing an autoimmune disease. Based on these findings, various autoantibodies against autoantigens have been discovered and the autoantibodies against autoantigens have been measured in clinical trials (e.g., U.S. Pat. Nos. 6,919,210, 6,596,501, 7,012,134, 6,919,078), while other autoimmune diagnostics may involve the detection of related metabolites (e.g., U.S. Pat. No. 4,659,659) or immunobioreactivity (e.g., U.S. Pat. Nos. 4,614,722 and 5,147,785, 4,420,558, 5,298,396, 5,162,990, 4,420,461, 4,595,654, 5,846,758, 6,660,487). Thus, in some embodiments, a composition comprising any of the nanoalum particles described herein further comprises an autoantibody that can be used to detect an autoantigen.

[0445] In some embodiments, the diagnostic agent can be used to detect infectious diseases. Compositions and methods for identifying a subject having an infection with an infectious pathogen or suspected of having a risk of having an infection with an infectious pathogen are known in the art.

[0446] For example, the bacterium Mycobacterium tuberculosis encompasses tuberculosis (TB). Thus, in some embodiments, a composition comprising any of the nanoalum particles described herein further comprises an agent for diagnosing tuberculosis. Diagnostic kits containing such polypeptides or DNA sequences and suitable detection reagents can be used to detect mycobacterial infections in patients and biological samples. Antibodies against such polypeptides are also provided.

[0447] In some embodiments, a composition comprising any of the nanoalum particles described herein further comprises an agent for diagnosing malaria, using any of the diagnostic agents described below. In vitro diagnostic methods for diagnosing malaria in an individual are known and include contacting a tissue or biological fluid obtained from the individual with a molecular or polypeptide composition under conditions that allow an in vitro immune reaction to occur between the composition and an antibody that may be present in the tissue or biological fluid, wherein the molecular or polypeptide composition comprises one or more peptide sequences that carry all or a portion of one or more T epitopes of a protein produced by the infective activity of Plasmodium falciparum; and in vitro detection of the formed antigen - antibody complex (see, e.g., U.S. Pat. No. 7,087,231).

[0448] The expression and purification of the extracellular domain of recombinant Plasmodium falciparum (3D7) AMA-1 have been described. Previous methods have produced highly purified proteins that maintain the folded and disulfide-bridged native molecule. Recombinant AMA-1 can be used for antibody production like a diagnostic reagent and can be used as a protein for use alone or as part of a vaccine for the prevention of malaria. (U.S. Patent 7,029,685).

[0449] Polynucleotides encoding Plasmodium vivax malaria peptide antigens have been described herein, which are proteins or fragments of proteins that are secreted into the plasma of a susceptible mammalian host after infection, such as monoclonal or polyclonal antibodies against these antigens. Peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are utilized in assays for the diagnosis of malaria and for determining whether Plasmodium vivax is the species responsible for the infection. (U.S. Patent 6,706,872) Species-specific Plasmodium vivax malaria peptide antigens have also been reported, which are proteins or fragments of proteins that are secreted into the plasma of a susceptible mammalian host after infection, such as monoclonal or polyclonal antibodies against these antigens. Peptide antigens, monoclonal antibodies, and / or polyclonal antibodies are utilized in assays for the diagnosis of malaria and for determining whether Plasmodium vivax is the species responsible for the infection (see, for example, U.S. Patent 6,231,861).

[0450] The extracellular domain of recombinant Plasmodium falciparum (3D7) AMA-1 has also been expressed by a method that produces highly purified proteins that maintain the folded and disulfide-bridged native molecule. Recombinant AMA-1 can be used as a diagnostic reagent, for antibody production, and as a vaccine. (U.S. Patent 7,060,276) Similarly, the expression and purification of recombinant Plasmodium falciparum (3D7) MSP-142, which maintains the folded and disulfide-bridged native molecule, are known. Recombinant MSP-142 can be used as a diagnostic reagent, for antibody production, and as a vaccine. (U.S. Patent 6,855,322).

[0451] Thus, based on these and related disclosures, methods for detecting human malaria infections to identify subjects infected with a malaria-infected pathogen or at risk of being infected with a malaria-infected pathogen are known. Specifically, for example, a blood sample is combined with a reagent containing 3-acetylpyridine adenine dinucleotide (APAD), a substrate (e.g., lactate or lactic acid), and a buffer. The reagent is designed to detect a unique glycolytic enzyme produced by the malaria parasite. This enzyme is called parasite lactate dehydrogenase (PLDH). Using the above reagent, PLDH can be easily distinguished from host LDH. Combining the reagent with a parasitized blood sample results in a reduction of APAD. However, APAD is not reduced by host LDH. The reduced APAD can then be detected by various techniques, including spectroscopy, fluorescence, electrophoresis, or colorimetric analysis. Detecting the reduced APAD in the foregoing manner provides a positive indication of malaria infection (e.g., U.S. Patent 5,124,141). In another method for diagnosing malaria, a polypeptide comprising a characteristic amino acid sequence derived from the Plasmodium falciparum antigen GLURP is identified in a test sample by specific antibodies produced against or reactive with the polypeptide. (U.S. Patent 5,231,168).

[0452] In some embodiments, a composition comprising any of the nanoalum particles described herein further comprises an agent useful for diagnosing leishmaniasis, using any of the diagnostic agents described below. Leishmaniasis is a widespread parasitic disease that frequently spreads in the Indian subcontinent, Africa, and Latin America and is a top priority for the World Health Organization (WHO) to develop a vaccine. A series of different diseases, Leishmania parasites cause lethal infections of internal organs and severe skin diseases. One of the most devastating forms of leishmaniasis is the disfiguring infection of the nose and mouth. The number of cases of leishmaniasis is increasing, and leishmaniasis is now out of control in many regions. Due to HIV infection, leishmaniasis is also on the rise in some developed countries, especially southern Europe. Available drugs are toxic, relatively expensive, and require long-term daily injections.

[0453] Leishmania protozoa are protozoan parasites that inhabit macrophages or white blood cells of the immune system. The parasite is transmitted through the bites of small blood-sucking insects (sand flies), which are difficult to control because they inhabit many regions of the planet.

[0454] Visceral leishmaniasis is the most dangerous of the three manifestations of the disease. It is estimated that there are about 500,000 new cases of the visceral form (kala-azar or "the killing disease") each year. Currently, more than 200 million people are at risk of contracting visceral leishmaniasis. More than 90% of visceral leishmaniasis cases occur in India, Bangladesh, Sudan, Brazil, and Nepal. Most deaths occur in children. Those with the cutaneous form are often permanently disfigured.

[0455] Leishmania infections are difficult to diagnose and typically involve histopathological analysis of tissue biopsy samples. However, several serological and immunological diagnostic assays have been developed. (U.S. Patent 7,008,774; Senaldi et al., (1996), Journal of Immunological Methods 193:95; Zijlstra et al., (1997), Transactions of the Royal Society of Tropical Medicine and Hygiene 91:671-673; Badaro et al., (1996), Journal of Infectious Diseases 173:758-761; Choudhary, S. et al., (1992), Journal of Communication Disorders 24:32-36; Badaro, R. et al., (1986), American Journal of Tropical Medicine and Hygiene 35:72-78; Choudhary, A. et al., (1990), Transactions of the Royal Society of Tropical Medicine and Hygiene 84:363-366; Reed, S.G. et al., (1990), American Journal of Tropical Medicine and Hygiene 43:632-639). Promastigotes release metabolites into the culture medium to produce conditioned medium. These metabolites are immunogenic to the host. See Schnur, L.F. et al., (1972), Israel Journal of Medical Sciences 8:932-942; Sergeiev, V.P. et al., (1969), Medical Parasitology 38:208-212; El-On, J. et al., (1979), Experimental Parasitology 47:254-269; and Bray, R.S. et al., (1966), Transactions of the Royal Society of Tropical Medicine and Hygiene 60:605-609; U.S. Patent No. 6,846,648; U.S. Patent 5,912,166; U.S. Patent 5,719,263 U.S. Patent 5,411,865).

[0456] In some embodiments, a composition comprising any of the nanoalum particles described herein further comprises an agent useful for diagnosing HIV, using any of the diagnostic agents described below. Methods for diagnosing HIV infection are known and include viral culture, PCR of limited nucleic acid sequences of patient samples, and antibody tests for the presence of anti-HIV antibodies in patient sera (see, for example, U.S. Patent Nos. 6,979,535, 6,544,728, 6,316,183, 6,261,762, 4,743,540).

[0457] VI. Kits

[0458] In certain embodiments, kits comprising a composition comprising nanoalum particles as described herein are also contemplated, and the composition may be provided in one or more containers. In one embodiment, all components of the composition are present in a single container, but the embodiments are not intended to be so limited and two or more containers are also contemplated in which, for example, the immunoadjuvant composition is separated from and not in contact with the antigen component. By way of non-limiting theory, it is believed that in some cases it may be beneficial to administer only the immunoadjuvant composition, while in other cases it may be beneficial for such administration to be separated from the administration of the antigen in time and / or space (e.g., at different anatomical sites), and in still other cases it may be beneficial to administer to a subject a vaccine composition as described herein and containing both an antigen and an adjuvant composition and optionally other components as described herein.

[0459] In some embodiments, a vial of the kit comprises a composition comprising nanoalum particles.

[0460] In some embodiments, one vial of the kit comprises a composition comprising nanoalum particles and a second vial of the kit contains a bioactive agent. In some embodiments, the kit comprises a third vial containing an adjuvant.

[0461] In some embodiments, one vial of the kit comprises a composition comprising nanoalum particles and a second vial of the kit contains an adjuvant. In some embodiments, the kit comprises a third vial containing a bioactive agent.

[0462] The kits of the present disclosure may further comprise instructions for use as described herein or instructions for mixing the materials contained in the vials. In some embodiments, the materials in the vials are dry or lyophilized. In some embodiments, the materials in the vials are liquid.

[0463] The container according to an embodiment of such a kit can be any suitable container, vessel, vial, ampoule, tube, cup, case, bottle, flask, jar, dish, well of a single-well or multi-well device, reservoir, tank, etc., or other devices into which the compositions disclosed herein can be placed, stored, and / or transported and into which inclusions can be removed. Typically, such containers can be made of materials that are compatible with the intended use and that can readily effect recovery of the contained inclusions. Non-limiting examples of such containers include glass and / or plastic sealed or resealable tubes and ampoules, including those having rubber septa or other sealing means compatible with the use of a needle or syringe to withdraw the inclusions. Such containers can be made, for example, of glass or a chemically compatible plastic or resin that can be made of or coated with a material that permits efficient recovery of material from the container and / or protects the material from, for example, degrading conditions such as ultraviolet light or temperature extremes or from introduction of unwanted contaminants including microbial contamination. The container is preferably sterile or sterilizable and is made of a material that will be compatible with any carrier, excipient, solvent, vehicle, etc., such as carriers, excipients, solvents, vehicles, etc. that can be used to suspend or dissolve the vaccine compositions and / or immunoadjuvant compositions and / or antigens and / or recombinant expression constructs described herein.

[0464] By way of illustration and not limitation, the following examples are provided.

[0465] Example

[0466] Example 1. Preparation of PEG and PAA nano - alum formulations

[0467] Prepare a nanoalum formulation. Purchase aluminum hydroxide 2% or Al(OH)3, aluminum hydroxide, hydroxy aluminum oxide 2% from EM Sargeant ( 85) As a wet gel suspension. The following lipids were purchased from Corden Pharma (Liestal, Switzerland): distearoyl phosphatidylethanolamine (DSPE), N - [carbonyl - (methoxypolyethylene glycol - 750)] - 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine (mPEG750 - DSPE), N - [carbonyl - (methoxypolyethylene glycol - 2000)] - 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine (mPEG2000 - DSPE), N - [carbonyl - (methoxypolyethylene glycol - 5000)] - 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine (mPEG5000 - DSPE), 1,2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine - N - [methoxy(polyethylene glycol) - 2000] (mPEG2000 - DPPE), 1,2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine - N - [methoxy(polyethylene glycol) - 5000] (mPEG5000 - DPPE), N - [carbonyl - (methoxypolyethylene glycol - 2000)] - 1,2 - dimyristoyl - sn - glycero - 3 - phosphoethanolamine (mPEG2000 - DMPE), and N - [carbonyl - (methoxypolyethylene glycol - 5000)] - 1,2 - dimyristoyl - sn - glycero - 3 - phosphoethanolamine (mPEG5000 - DMPE). ‘85’ was purchased from EM Sergeant Pulp and Chemical Company (Clifton, New Jersey) and manufactured by Brenntag (Mulheim an der Ruhr, Germany). Poly(acrylic acid) (PAA) was purchased from Sigma Aldrich.

[0468] Prepare the nano - alum formulation. Briefly, by taking 40 ml PEG nanoalum was manufactured by diluting (10 mg / ml aluminum) into 60 ml of water and heating for 2 hours in a Crest Powersonic CP230D (Crest Ultrasonic CP230D) (Trenton, NJ) water bath at approximately 60 °C. DSPE or PEGylated DSEP phospholipids were added to the heated solution at the indicated concentrations, with phospholipid ranging from approximately 0.5 mg / ml to 30 mg / ml (Table 3). All formulations were returned to the water bath at approximately 60 °C to dissolve the visible phospholipid polymers. A Microfluidics M110P (Newton, MA) equipped with a diamond F12Y interaction chamber and subsequent ceramic H30Z assist treatment module was used to process the formulation up to 10 passes at 30,000 psi with recirculated cold water to prevent temperature increase during processing. 50-μl aliquots were removed between selected passes for particle size characterization by dynamic light scattering. The remaining formulation was collected after the 10th pass and placed according to a stability protocol to monitor particle size. Prior to in vivo bioactivity assessment, selected formulations made on the 110P microfluidizer were filtered through a 0.2-μm Supor membrane. Instead of microfluidization, a Silverson high shear mixer (East Longmeadow, MA) was used to process selected formulations at 5000 rpm for 5 minutes.

[0469] For PAA nanoalum formulations, PAA with an average molecular weight of 2000 was purchased from Sigma-Aldrich. A stock solution of 50% wt in water was diluted in water to produce a stock solution of 30% wt in water. 16 g of 30% wt PAA was combined with 160 g of 10 mg / ml stock solution, and the pH was adjusted to 6.6 with 10 M NaOH. The formulation was processed using the 110P microfluidizer at 30 k PSI, 4 °C for 1 pass, 3 passes, 6 passes, 10 passes, 15 passes, 20 passes.

[0470] Particle analysis of nano-alum formulations. The formulations were characterized by dynamic light scattering (DLS) using a Zetasizer Nano-S or Nano-ZS from Malvern Instruments (Worcestershire, UK) and by laser diffraction particle analysis using an LS230 from Beckman Coulter (Brea, California). Particle size information was also obtained by sedimentation analysis (described below) and cryoTEM (cryogenic transmission electron microscopy). For DLS analysis, the alum formulations were diluted 1:100 in water in 1.5-ml disposable polystyrene cuvettes. DLS measurements were performed in triplicate and the values were reported as the mean particle diameter Z-average based on scattering intensity. Samples run on the DLS were measured against polystyrene standards of 60 nm and 200 nm (polystyrene refractive index = 1.55 to 1.59); the refractive index of aluminum is 1.24. For laser diffraction-based measurements, the alum sample was placed directly into a water-filled sample cell to achieve a polarization intensity difference scattered (PIDS) value between 50% ± 5%. The bias (establishing an electrical noise baseline by measuring the voltage of the circuit with the laser off) option was set to 60 seconds, the background measurement was 90 seconds, the run length was 90-second intervals, and the pump speed was 50%. The LS230 was degassed three times before and during sample analysis.

[0471] Sedimentation analysis. Laser scattering spectroscopy was performed using a LUMiReader from LUM GmbH (Boulder, Colorado) equipped with three lasers with wavelengths of 470 nm, 630 nm, and 870 nm. The particle sedimentation rate was determined based on changes in the laser transmission spectrum according to the vertical cross-section of the sample cuvette. 4 mL of undiluted formulation was added directly to a cylindrical glass cuvette for analysis. The sample was measured at a maximum tilt angle of 30° at 25 °C for 2 to 4 hours, and measurement scans were collected every 60 seconds. In addition, based on the multi-wavelength analysis method (2), the particle sedimentation rate can be used to calculate the volume-based particle size distribution of particles larger than approximately 0.5 μm.

[0472] Antigen adsorption. Binding of the antigen to the nano-alum formulation was determined by silver-stained SDS-PAGE. Before centrifugation, samples were mixed in the following order: alum formulation, TLR ligand, antibody, and diluent (saline or glycerol solution). Samples were then centrifuged at 35,000 x g for 30 minutes at 4 °C in a Beckman Coulter Optima Max-XP ultracentrifuge (Brea, CA). 30 μl of the sample was mixed with 10 μl of 4X-reduced LDS sample buffer, after which 20 μl was loaded onto a 12-lane SDS-PAGE gel with 8 μl of SeeBlue2 prestained standards. Each gel was run at 190 V for 55 minutes and then placed in 50:40:10 EtOH:CH3COOH:H2O fixative for at least 2 hours or up to overnight. Gels were then stained according to the instructions provided in the ProteoSilver Plus Silver Staining Kit from Sigma-Aldrich (St. Louis, MO).

[0473] TLR ligand adsorption. Binding of the TLR-9 ligand to the nano-alum formulation was determined by silver-stained SDS-PAGE using the same centrifugation and dilution preparation, gel conditions, and staining kit. The presence of a dark brown band between 3 kDa and 6 kDa indicated the presence of TLR-9 on the gel. Binding of the TLR-4 ligand to the nano-alum formulation was determined by centrifuging the TLR-4 ligand with the nano-alum formulation and testing for the presence of unbound TLR-4 ligand in the supernatant diluted 1:5 into mobile phase A (75:15:10 [v:v:v] methanol:chloroform:water with 20 mM ammonium acetate and 1% acetic acid). Each supernatant sample was injected in a 50-μl volume onto a Waters Co. (Milford, MA) Xbridge BEH Shielf RP18 column attached to an Agilent Model 1100 HPLC (Santa Clara, CA). A gradient consisting of mobile phases A and B (1:1 [v:v] methanol:chloroform with 20 mM ammonium acetate and 1% acetic acid) was run for over 25 minutes. Detection was accomplished by an ESA Biosciences Coronoa charged aerosol detector (CAD) (Chelmsford, MA). Quantification was performed using GLA standards spiked at different volumes in mobile phase B to create a standard curve.

[0474] XRD. X-ray powder diffraction analysis was performed on four samples sent to Triclinic Labs (West Lafayette, IN) to determine the effect of different treatments on the same PEGylated lipid / Effect of the composition. The samples were ultracentrifuged and x-ray powder diffraction (XRPD) analysis was performed on the still-wet solid as well as the supernatant. The reflection Bragg-Brentano geometry was configured for a Rigaku Smart-Lab (Rigaku Intelligent Laboratory) x-ray diffraction system (Woodlands, Texas) using a line source x-ray beam. The S-ray source is a long, fine-focus copper tube operating at 40 kV and 444 mA. That source provided an incident beam spectrum at the sample that varied from a narrow line at high angles to a wide rectangle at low angles. Beam conditioning slits were used on the x-ray line source to ensure that the maximum beam size along the line and normal to the line was less than 10 mm. The Bragg-Brentano geometry is a para-focusing geometry controlled by non-divergent and receiving slits, where the sample itself acts as an optical focusing component. The inherent resolution of the Bragg-Brentano geometry is controlled in part by the diffractometer radius and the width of the receiving slit used. Typically, the Rigaku Smart-Lab is operated to give a peak width of 0.1° 2θ or less. The axial divergence of the x-ray beam is controlled by 5.0° Soller slits in both the incident beam path and the diffracted beam path. The samples were placed in a low-background silicon holder so that light manual pressure was used to keep the sample surface flat and flush with the reference surface of the holder. Each sample was analyzed from 2° 2θ to 40° 2θ using a continuous scan of 6° 2θ per minute with an affected step size of 0.02° 2θ. Digital filtering was performed on each data set to remove low-frequency responses. Examination of the resulting patterns allowed the identification of two distinct crystalline responses: Gaussian peaks and Lorentzian peaks. Gaussian peaks are generally associated with microcrystalline materials and are used to refer to materials that contain both crystalline and amorphous regions. Lorentzian peaks are generally associated with nanocrystalline materials and contain microcrystals of nanometer size.

[0475] CryoEM (cryogenic electron microscopy) analysis of nano - alum formulations

[0476] CryoEM analysis was performed by NanoImaging Services. Briefly, samples for EM analysis were preserved in vitreous ice supported by a perforated carbon film on a 400-mesh copper grid. Individual samples were prepared by applying 3 μL of the sample suspension dropwise to the cleaned grid, blotting with filter paper, and immediately proceeding with vitrification in liquid ethane. The grids were stored under liquid nitrogen until transferred to the electron microscope for imaging. Electron microscopy was performed using a FEI Tecnai T12 electron microscope equipped with a FEI Eagle 4k x 4k CCD camera and operating at 120 keV. A cryo-stage that maintained the grid at a temperature below -170 °C was used to transfer the vitreous ice grids into the electron microscope. Images of individual grids were acquired at multiple scales to estimate the overall distribution of the sample. After identifying potentially suitable target areas for imaging at lower magnifications, high-magnification images were acquired at nominal magnifications of 110,000x (0.10 nm / pixel), 52,000x (0.21 nm / pixel), and 21,000x (0.50 nm / pixel). Images were acquired at nominal defocus values of -2 μm (110,000x), -3 μm to -2 μm (52,000x), and -5 μm (21,000x) and an electron dose of approximately to the following images.

[0477] Generating nano - alum formulations - developing sizing agents

[0478] PEG nano - alum - polyethylene glycolated phospholipid sizing agent

[0479] Hydroxyaluminum oxide (alum) in solution typically polymerizes into larger, typically crystalline arrays or flakes. Untreated In untreated alum formulations, larger, typically aluminum hydroxide crystalline arrays or flakes 1 micron or greater in size are formed. In data not shown, experiments were performed to determine whether milling or treatment by individually microfluidizing aluminum hydroxide solutions under various conditions would yield nano-alum formulations; however, we were unable to determine the conditions under which individually microfluidizing stock aluminum hydroxide would yield stable nano-alum formulations.

[0480] Without wishing to be bound by theory, it is theorized that it may be necessary to add a sizing agent or stabilizer to prevent or disrupt the aggregation of aluminum hydroxide molecules. Phospholipids are commonly added as emulsifiers and stabilizers for microspheres in aqueous solutions, and phospholipids were initially selected as sizing agents. Preliminary experiments were performed to test whether the inclusion of a sizing agent such as phospholipid during the milling or processing of the alum solution could yield a nano-alum formulation. Experiments were performed with single phospholipid species DSPE, DPPE, and DMPE having acyl chain lengths of 18 carbons, 16 carbons, and 14 carbons, respectively. In data not shown, it was found that the phospholipids tested were not effective sizing agents and did not prevent alum molecule aggregation.

[0481] Additional experiments were performed to determine whether the addition of a polyethylene glycol moiety linked to the phospholipid would yield an effective sizing agent. To evaluate whether the inclusion of a sizing agent would disrupt the aggregation of alum molecules to yield a nano-alum formulation, a PEG5000-DSPE stock solution was milled by microfluidizing 10 passes at 30 k PSI and immediately mixed with an aluminum hydroxide stock solution on the bench top to yield an 8 mg / ml PEG5000-DSPE:4 mg / ml formulation. CryoEM analysis of the blended microfluidized polyethylene glycolated phospholipid:aluminum hydroxide formulation demonstrated that blending aluminum hydroxide with the microfluidized polyethylene glycolated lipid did not disrupt the formation of larger crystal aggregates of alum and did not yield a nano-alum formulation.

[0482] Subsequent experiments were performed to determine whether a nano-alum formulation could be produced by adding a sizing agent during the milling or processing of the stock alum formulation. A series of experiments were performed to evaluate DSPE, DPPE, and DMPE polyethylene glycolated phospholipids, where a series of molecular weight polyethylene glycol moieties (ranging from 750 kD Mr to 5000 kD Mr) were linked to various phospholipids having different acyl chain lengths (acyl chain lengths of DSPE, DPPE, and DMPE are 18 carbons, 16 carbons, and 14 carbons, respectively) and blended with the stock alum formulation during milling or sizing under various conditions to determine whether adding a sizing agent during the milling or processing of alum could yield a nano-alum formulation. The particle size of the formulation was analyzed by Malvern analysis and cryoEM analysis. Milling or processing alum in the presence of a polyethylene glycolated phospholipid such as yielded nano-alum formulations with particle sizes ranging from approximately 400 nm to 70 nm (Table 4 and data not shown).

[0483] Analysis of the presented data demonstrated that the methods or conditions for grinding alum in the presence of a sizing agent can produce nano-alum formulations with differently defined particle sizes. The size of untreated alum was approximately 1000 nm to 10,000 nm (Table 4). Mixing alum at 5000 rpm for 5 minutes using a Silverson mixer in the presence of PEG-DSPE with a molecular weight of 5000 kD as a sizing agent (e.g., sizing agent: alum at 8 mg:4 mg) produced a nano-alum formulation with an average particle size of approximately 400 nm (Table 3 and data not shown). Microfluidizing the alum admixed with PEG-DSPE with a molecular weight of 5000 kD (e.g., sizing agent: alum at 8 mg:4 mg) at 10 k PSI for 1 pass produced a nano-alum formulation with an average particle size of approximately 120 nm to 130 nm. Treating the admixed alum 5000 kD molecular weight PEG-DSPE solution 1 pass, 10 passes, 15 passes, or up to 20 passes produced a nano-alum formulation with an average particle size of 70 nm (Table 3 and data not shown). The data in Table 4 and the data not presented demonstrated that altering the treatment by changing the grinding or sizing equipment for alum (e.g., Silverson mixer or microfluidizer) or the grinding conditions (e.g., for the microfluidizer, by changing PSI or number of passes) in the presence of a sizing agent can produce nano-alum formulations with a range of nanoparticle sizes (400 nm, 120 nm, 70 nm). Based on the presented data, one of ordinary skill in the art can use well-known techniques and equipment such as high energy sources or high energy inputs to grind or treat alum in the presence of a sizing agent such as polyethylene glycolated lipids to achieve nano-alum formulations within a desired size range.

[0484] Further analysis of the data in Table 4 and the data not shown demonstrated that PEG moieties with a wide range of molecular weights linked to DSPE phospholipids produced effective sizing agents. As demonstrated in Figure 4 and the data not shown, different PEG lengths of 750 kD to 2000 kD to 5000 kD (Table 3 and data not shown) did not affect the particle size of the nano-alum formulations when ground under the grinding conditions determined to produce a nano-alum formulation with a 70 nm particle size. Thus, the polyethylene glycolated phospholipid sizing agents of the present disclosure can include PEG moieties with a wide range of molecular weights.

[0485] Experiments were performed to determine whether varying the ratio of alum to polyethylene glycolated phospholipid sizing agent can be used to control the particle size of the nano-alum formulation.

[0486] The data presented in Table 4 indicate that varying the ratio of sizing agent to alum can be used to affect the particle size of the nano-alum formulations. For example, to reproducibly produce nano-alum with an average particle size of approximately 300 nm to 400 nm, for the DSPE-PEG5000 sizing agent it can be produced at an alum to sizing agent ratio of 1:1, while increasing the sizing agent to 1:1.5 or 1:2 reproducibly produced nano-alum of approximately 100 nm or 70 nm to 80 nm, respectively. Comparing the DSPE-PEG2000 sizing agent, the DPPE-PEG-5000 sizing agent, or the PAA200 sizing agent demonstrated that an optimal ratio of alum to sizing agent in the range of 1:2 to 1:3 reproducibly produced nano-alum with 70 nm to 80 nm alum. Additionally, the data in Table 4 and data not shown demonstrate that the acyl chain length of the polyethylene glycolated phospholipids does not affect the ability of the sizing agent to produce nano-alum with a desired size range. When mixed with the aluminum formulation and ground by the same process, polyethylene glycolated phospholipids with different acyl chain lengths having 18 carbons (18C DSPE), 16 carbons (16C DPPE), and 14 carbons (14C DMPE) all produced nano-alum formulations with the same particle size (Table 4 and data not shown). Thus, the polyethylene glycolated phospholipid sizing agents of the present disclosure can include phospholipids having different acyl chain lengths.

[0487] Lowering the pH via concentrated HCL, HNO3, and propionic acid

[0488] Upon subjecting to grinding via sonication for five minutes, using concentrated acid to rapidly lower the pH of aluminum produced the nano-alum formulations of the present disclosure. Concentrated hydrochloric acid and nitric acid were tested to achieve a final pH of 1.0, and both produced nano-alum formulations of 324 nm with a high polydispersity (data not shown). Thus, for some aspects of the present disclosure, concentrated acid solutions may be suitable as sizing agents to produce nano-alum formulations. However, since a final pH of 1.0 of the nano-alum formulations may not be favorable for all aspects of the present disclosure, including the delivery of proteins, peptides, and nucleic acids, additional acids with a lower overall pH profile were further evaluated.

[0489] Briefly, 4 mM oleic acid was mixed with water to obtain 30 ml of emulsion. This mixture was treated with a sonication probe (40% power) for 10 minutes. Equal volumes of a 1.6% by weight volume stock solution were mixed with equal volumes of the emulsified oleic acid solution to produce a 0.8 v%:2 mM oleic acid formulation, and the The oleic acid formulation was further sonicated for 10 minutes using an ultrasonic probe at 40% power. The resulting nano-alum had a particle size of 194 nm and a final pH of 2.4. Thus, for some aspects, oleic acid is a suitable sizing agent for nano-alum formulations.

[0490] The aluminum was sonicated as described above with stirring and addition of 5% acetic acid until the pH was adjusted to 6.1, 5.1, or 4.5. Acetic acid produced an effective nano-alum formulation with an average particle size of approximately 100 nm to 130 nm as a sizing agent. Additionally, the nanoparticles of the formulation were positively charged as measured by zeta potential. Thus, for some aspects of the present disclosure, acetic acid can be an effective sizing agent for producing the nano-alum of the present disclosure.

[0491] PAA nano - alum - polyacrylic acid (PAA) as a sizing agent

[0492] For preliminary experiments, the use of PAA as a sizing agent to produce nano-alum was evaluated by mixing 40 g of 0.4 wt% aluminum with 20 wt% PAA solution under strong stirring and adjusting the pH to pH 6.0, where concentrated ammonium hydroxide produced nano-alum with a particle size of approximately 140 nm and negatively charged nanoparticles as measured by zeta potential.

[0493] For subsequent experiments, briefly, 20 wt% of PAA was mixed with the aluminum stock solution, the pH was adjusted to 6.6 with sodium hydroxide, and milling was performed using a 110P microfluidizer. Based on data from the development of PEG nano-alum formulations, the formulation was milled by recycling via microfluidization at 4 °C and evaluated at 30 k psi at 3, 6, 10, and 15 passes, respectively. Passes 3 to 6 produced nano-alum with a particle size of approximately 100 nm, and no perceivable particle size effect was observed between 3 and 6 passes. Increasing the number of passes from 10 to 15 consistently produced nano-alum with a particle size of approximately 70 nm to 85 nm and good polydispersity. For subsequent experiments, 10 passes were utilized. The data indicate that PAA is an effective sizing agent for nano-alum formulations.

[0494] Stability and characterization of nano - alum formulations

[0495] Data shows that nano-alum formulations can be generated by milling in the presence of suitable sizing agents such as polyethylene glycolylated lipids and polyacrylic acid. However, for formulations to be commercialized as drug or biopharmaceutical delivery formulations, a desirable property of the formulation is that the particle size should be stable over time. Experiments were performed to determine whether aqueous PEG nano-alum or PAA nano-alum formulations are stable and maintain the initial particle size or do not increase in size or aggregate beyond an average size of 200 nm when milled to an initial particle size of 70 nm. Briefly, as described previously, PEG nano-alum formulations and PAA nano-alum formulations were prepared and, as shown, stored at 4 °C. Triplicate samples were removed at 1 week, 2 weeks, and 1 month, 3 months, 6 months, 9 months, and 12 months after preparation, and their particle size and polydispersity were estimated as described herein. Figure 1B Data for the PAA nano-alum formulation in Figure 1B demonstrate that the PAA nano-alum formulation is incredibly stable and maintains an average particle size of approximately 75 nm as tested for 1 month, 3 months, and 6 months and up to 12 months (data not shown). Similarly, Figure 1C the PEG nano-alum formulation shown in Figure 1C is also very stable and maintains an average particle size of approximately 75 nm for a period of 1 month, 3 months, 6 months, 9 months, and up to 12 months when measured by dynamic light scattering using a Malvern ζ-sizer.

[0496] Long-term stability at 2 °C to 8 °C is an important property of vaccine formulations, but maintaining cold-chain storage can be a limiting factor in delivering vaccines for global health. We tested whether the nano-alum formulations of the present disclosure are thermally stable within a temperature range (25 °C, 37 °C, and 65 °C) over a period of 4 weeks. Briefly, samples were stored in triplicate at the desired temperature and the change in their average particle size and polydispersity was determined as measured by dynamic light scattering using a Malvern ζ-sizer. By evaluating PEG5000-DSPE (acyl chain length of 18 carbons), PEG2000-DMPE (acyl chain length of 14 carbons), PEG2000-DPPE (acyl chain length of 16 carbons), PEG750-DSPE (acyl chain length of 18 carbons), and PEG200-DSPE (acyl chain length of 18 carbons), we further analyzed the possible effect of PEG length and acyl chain length on the thermal stability of aqueous nano-alum formulations. Figures 1D to 1FData in [the reference] demonstrate that PEG2000-DSPE is extremely stable at temperatures up to 25 °C and 37 °C, with little to no aggregation or change in particle size over 0, 2, or 4 weeks, and is even stable at 60 °C for up to 2 weeks. Even at 60 °C over 4 weeks, the PEG2000-DSPE nanoalum formulation demonstrated only a slight increase in particle size and an average particle size of 114 nm, indicating that nanoalum containing the sizing agent may not require cold-chain storage. The nanoalum formulations PEG5000-DSPE (PEG length 5000 and acyl chain length 18C), PEG2000-DPPE (PEG length 2000 and acyl chain length 16C), and PEG750-DSPE (PEG length 750 and acyl chain length 18C) demonstrated thermal stability at 25 °C and 37 °C for 0, 2, and 4 weeks, but were thermally unstable at 60 °C and demonstrated aggregation of particles with an average particle size greater than 2000 nm at 2 and 4 weeks. Interestingly, the nanoalum formulation PEG2000-DMPE (PEG length 2000 and acyl chain length 14C) was stable at 25 °C for up to 4 weeks and at 37 °C for 2 weeks, but was unstable at 37 °C and 60 °C and demonstrated aggregation of particles with an average particle size greater than 2000 nm. Thus, the nanoalum formulations of the present disclosure demonstrate enhanced thermal stability. The thermal stability of the nanoalum formulations can not only allow for broader global availability without specialized cold-chain storage, but also reduce the overall cost of the formulation. In the figure legend, QG194 is PEG5000-DSPE; QG195 is PEG2000-DMPE; QG196 is PEG2000-DPPE; QG197 is PEG750-DSPE; QG198 is PEG2000-DSPE.

[0497] To further assess the stability of nanoalum, we evaluated the effect of freeze-thaw cycles on the colloidal thermal stability of aluminum and nanoalum formulations. The pre-frozen particle size of the formulations was measured using: Horiba LA-960, for aluminum formulations; and Malvern ζ-sizer, for nanoalum formulations. The formulations were frozen in a dry ice / acetone bath and then thawed in a 37 °C water bath. Particle size was measured and compared before and after the freeze-thaw cycles. The average particle size of 85 (aluminum) increased by 160% after the freeze-thaw cycles, indicating failure of colloidal stability. The average particle size of (nanoalum-poly(acrylic acid)) showed no significant change in particle size after 3 freeze-thaw cycles. The average particle size of nanoalum-PEG increased by 273% after 1 freeze-thaw cycle, indicating failure of colloidal stability. The 85 adjuvant is colloidally unstable after one freeze-thaw cycle, indicating poor resistance to the destabilizing effect of freezing. On the other hand, the nanoalum stabilized by poly(acrylic acid) showed good stability after repeated freeze-thaw cycles, which can facilitate the long-term cryopreservation of the nanoalum-PAA formulation.

[0498] Aluminum is an attractive adjuvant that has been described as binding or adsorbing protein antigens through: electrostatic interactions (involving Al3+ ions or negatively charged counterions)

[11] ; the cooperation of metal ions with water molecules and hydroxyl groups and the binding of hydrogen to water molecules and hydroxyl groups [9],

[10] , and

[12] ; and, in some cases, hydrophobic interactions

[13] . In the art, there is some debate regarding the extent (if any) of protein adsorption required for the adjuvant properties of alum. We tested whether the nanoalum of the present disclosure, which has a much smaller surface area and particle size compared to aluminum, would efficiently adsorb antigens. Briefly, before centrifugation, samples were mixed in the following order: aluminum formulation, TLR ligand (GLA or CpG 5 μg), antibody (TB fusion protein ID93 (0.5 μg)), and diluent (saline or glycerol solution). Samples were centrifuged at 35,0000 x g for 30 minutes at 4 °C, and 30 μl of the supernatant that did not precipitate was mixed with 10 μl of LDS sample buffer reduced 4X. 20 μl was loaded onto a 12-lane SDS-PAGE gel with 8 μl of SeeBlue2 prestained standard. Each gel was run at 190 V for 55 minutes and then placed in 50:40:10 EtOH:CH3COOH:H2O fixative for at least 2 hours or up to overnight. The gels were then stained with the ProteoSilver Plus silver stain kit to determine whether ID93 was present in the supernatant or precipitated due to adsorption to aluminum. Data demonstrated (data not shown) that the sizing agent present in the nanoalum formulation did not interfere with the binding or association of antigens or adjuvants and was suitable as a delivery vehicle for the bioactive agents of the present disclosure. To confirm that the sizing agent PEG-5000DSPE generally did not interfere with the adsorption of proteins to aluminum or interfere with the assay, the ID93 fusion protein was admixed with the TLR4 adjuvant, GLA, and aluminum. The absence of the 62Kd ID93 band on the gel confirmed that DSPE-PEG5000 did not block the adsorption of the antigen to aluminum particles of micron (0.5 micron to 1.0 micron) size. As demonstrated by the absence of the 62Kd ID93 band on the gel, the nanoalum formulation of the present disclosure with a particle size of less than 100 nm and containing the sizing agent PEG DSPE was also able to adsorb the fusion protein ID93 (data not shown), the sizing agent PEG DSPE having different PEG lengths of 5000, 2000, or 750 or a fixed PEG length of 2000, linked to phospholipids having different acyl chain lengths of 18 carbons (DSPE), 16 carbons (DPPE), or 14 carbons (DMPE). Thus, a decrease in the average surface area or particle size of the nanoalum formulation did not result in a decrease in the adsorption of protein antibodies, making it a particularly useful formulation for vaccines.

[0499] Next, we characterize the concentration of aluminum hydroxide present in the nano-alum particles of the present disclosure. Briefly, nano-alum formulations containing sizing agents with different PEG lengths (5000, 2000, 750) or phospholipids with different acyl chain lengths (18C-DSPE, 16C-DPPE or 14C DMPE) are processed as described herein, and the aluminum content is estimated by ICP-OES testing ( Figure 2 ). Figure 2 The data in Figure 2 demonstrate that nano-alum formulations containing different sizing agents contain the predicted alum content when prepared with PEG sizing agents of different PEG lengths linked to phospholipids of different acyl chain lengths. Nano-alum formulations including those with sizing agents of phospholipid 18C (DSPE) and different PEG lengths 5000 (Sample 1), 2000 (Samples 2 to 4) and 750 (Sample 5) generated from a 4 mg / ml stock alum formulation contain approximately equal amounts of the predicted starting value of 4 mg / ml, as measured by ICP-OES testing, ranging from 3.9 mg / ml for PEG750-DSPE (Sample 5) to 4.5 mg / ml for PEG2000-DPPE (Sample 3), and the indicated sizing agents are milled by microfluidization 10 times at 30,000 psi at 4°C. Interestingly, alum milled in the absence of a sizing agent and untreated alum both contain decreased alum content (3.2 mg / ml and 3.4 mg / ml for Samples 6 and 7, respectively).

[0500] The data demonstrate that treatment or milling of aluminum hydroxide in the presence of a suitable sizing agent can produce stable nano-alum formulations suitable for delivering the agents of the present disclosure. The sizing agents of the present disclosure include, but are not limited to, polyethylene glycolated phospholipids and PAA.

[0501] Example 2. Using PEG5000 and PAA nano - alum formulations to deliver proteins or peptides (e.g., ID97) to stimulate immune response

[0502] To estimate aluminum hydroxide To explore the possibility of modification, we generated two nano-alum adjuvants, one based on polyacrylic acid (PAA) and one based on PEG5000. The modification led to smaller alum particles with a size of 100 nm to promote a Th1-biased immune response. To test the adjuvant potential of these candidates, we immunized 8-week-old female C57Bl / 6 mice (5 per group) purchased from The Jackson Laboratory, USA, with recombinant antibody ID97 - a recombinant fusion of the following four proteins from Mycobacterium tuberculosis: Rv1886, Rv3478, Rv3619, and Rv2875. ID97 was delivered alone or adjuvanted with alum (100 μg), PAA, nano-alum PAA1:1 (100 μg alum, 70 nm particle size), nano-alum PEG (100 μg alum, 70 nm particle size), or the TLR4 agonist adjuvant GLA-SE as a positive control for Th1 induction. Mice were immunized intramuscularly once. Seven days after immunization, we estimated the ID97-specific CD4 + T cell response by stimulating splenocytes with ID97 or leaving the cells unstimulated in the presence of the Golgi inhibitor brefeldin A. Cells were then stained for surface expression of CD4, CD8, and CD44 and intracellular expression of CD154, IFN-γ, TNF, IL-2, GM-CSF, IL-5, and IL-17A. The antigen-specific response was calculated as the frequency of CD4 + T cells responding in the ID97-stimulated sample minus the unstimulated sample. Figure 3A The results of the Th1 response in each group are shown. As expected, based on the recall expression of CD154 (a marker for antigen specificity but not specific for Th1, Th2, or Th17 polarization), the adjuvant-free group, the alum-adjuvanted group, and the PAA-adjuvanted group showed a low frequency of ID97-specific CD4+ T cells. Surprisingly, PAA-based nano-alum induced a robust CD4 + T cell response characterized by the production of Th1-signature cytokines IFN-γ, TNF, and IL-2. The response level was similar to that achieved by the positive control adjuvant GLA-SE. The quality of the humoral response was also estimated 7 days after immunization. Only PAA-based nano-alum and the positive control GLA-SE increased the ID97-specific IgG2c and IgG titers ( Figures 3B to 3D)。The class switch to IgG2c is influenced by IFN-γ produced by Th1 cells, so this skew supports that PAA-based nanoalum increases the Th1 response. Surprisingly, unlike GLA-SE, PAA-based nanoalum also boosts the IgG1 antibody titer, indicating that it may have a unique mode of action. PAA nanoalum has unique and surprising adjuvant properties for programming the TH1 response. Further, these responses are not just properties of the PAA component, as it does not have Th1 adjuvant activity on its own.

[0503] To elucidate the mechanism by which PAA-based nanoalum increases Th1 immunity to vaccine antigens, we estimated the concentrations of cytokines in the draining lymph nodes of immunized mice with increased key Th1 at 1 day after intramuscular immunization ( Figures 4A to 4C )。Both IL-12p70 and IL-18 are crucial for inducing IFN-γ, and IP-10 is an early IFN-γ-inducible cytokine. Compared with saline or alum immunization, PAA-based nanoalum increased the expression of both IL-18 and IL-12p70 at 1 day after immunization. This is likely to increase the early expression of IFN-γ, as IP-10 expression also increased in animals given PAA-based nanoalum. PEG-based nanoalum also increased IL-18 expression but did not increase IL-12p70 or IP-10 expression, further indicating the unique properties of PAA-based nanoalum. To determine whether this early induction of IL-18 is important for Th1 programming, we determined the Th1 CD4 - / - mouse and IL-18R + T cell profiles of mice. Compared with wild-type mice, PAA-based nanoalum failed to induce a Th1 response to the ID97 antigen ( Figure 5 )。

[0504] Collectively, these data support the finding that PAA-based nanoalum adjuvants and potentially other nanoparticle-based alum adjuvants have unique adjuvant properties compared with alum. These properties specifically include inducing innate cytokines (including IL-18 and IL-12p70) that program Th1 immunity and IFN-γ-responsive cytokines such as IP-10. Further, compared with alum, PAA-based nanoalum and potentially other nanoalums increased CD4 with a Th1 profile (IFN-γ, TNF, and IL-2 secretion upon antigen stimulation) +Induction of T cells and increased IgG2c class switching and antigen - specific antibody titers. These processes depend on the activation of the IL - 18:IL18R signaling axis. The increased Th1 response to vaccine antigens depends mainly on the inclusion of known Toll - like receptor (TLR) antigens such as MPL, GLA, SLA, CpG, polyIC:LC, or Pam2CSK4. To our knowledge, this is the first non - TLR adjuvant that can robustly enhance Th1 immunity. This could have many possible vaccine adjuvant applications, including vaccines against diseases such as whooping cough, tuberculosis, leprosy, malaria, HIV, leishmaniasis, and influenza.

[0505] In data not shown, the PAA nano - alum formulation with the TB vaccine antibody ID93 also demonstrated increased Th1 - type adjuvant activity compared to untreated alum.

[0506] Example 3. Using PAA nano - alum formulations to deliver nucleic acid agents

[0507] Based on the improved stability, inexpensive, and ultimately sterilizable large-scale applicable manufacture of nanoalum of the present disclosure, we evaluated whether the nanoalum formulation could efficiently deliver RNA. We benchmarked the performance of the nanoalum formulation against cationic emulsions described in the art. Briefly, a cationic emulsion (5) was prepared as described in the art, and the resulting emulsion was 0.5% w / vol Span 85, 5.0% v / vol squalene, 0.4% w / vol DOTAP, and 0.5% w / vol Tween 80. The replicon RNA was derived from a modified alphavirus genome in which the structural proteins containing capsid and E glycoproteins (C-E3-E2-6K-E1) were removed and replaced with a luciferase gene. Briefly, the RNA expression vector was a replicon RNS vector expressing luciferase driven by a subgenomic promoter constructed from a modified alphavirus genome that lacked the structural proteins containing capsid and E glycoproteins (C-E3-E2-6K-E1) but contained all non-structural genes (ns1 to ns5) necessary for replication and expression of RNA in cells. To analyze luciferase in vivo delivered by the RNA replicon and PAA nanoalum, C57 / BL6 mice were anesthetized, shaved, and intramuscularly immunized (intramuscular injection (i.m.)) in the thigh with 250 ul of the indicated formulation plus or minus the indicated dose of RNA. Before injection, the RNA dose (concentration) was confirmed by measurement using a Nanodrop spectrophotometer. The immunized mice were anesthetized, shaved, and the IVIS Illumina II imager was used to estimate RNA expression for sixty seconds at 24 hours, 4 days, and 7 days after injection. The animals were imaged and relative light units on a logarithmic scale were obtained. In this article, examples using the PAA nanoalum formulation are presented, but the examples should not be construed as limiting the nanoalum disclosed herein in scope.

[0508] Mice immunized with an RNA replicon expression vector formulated with nanoalum express RNA in vivo. To evaluate the ability of nanoalum to deliver RNA, mice were injected (3 mice per group) with 250 ul of a 1:3 PAA nanoalum formulation as described or a control cationic emulsion formulation as described herein and replicon RNA at a dose of 1 ug or 0.1 μg. Controls included saline vehicle, cationic emulsion, PAA nanoalum, or naked replicon RNA at a dose of 30 μg, 1 μg, or 0.1 μg. Unformulated replicon RNA was undetectable at 24 hours, except for one animal that received the highest dose of 30 μg luciferase replicon RNA (data not shown). However, at day 4 and day 7, all animals immunized with 30 μg of the unformulated luciferase replicon RNA vector had detectable expression compared to the vehicle (saline) control (data not shown). Animals that received 1 μg or 0.1 μg of naked replicon RNA had no detectable expression. In the case of RNA (1 μg RNA replicon) at a dose 30-fold lower admixed with the control cationic emulsion formulation, these three animals had detectable luciferase expression at 24 hours, 4 days, or 7 days post-delivery (data not shown), demonstrating that the cationic emulsion enhanced delivery of the RNA replicon, resulting in a dose sparing as defined by an effect of equal or greater RNA replicon expression compared to the unformulated material. The same RNA (1 μg RNA replicon) at a dose 30-fold lower admixed with the PAA nanoalum formulation also demonstrated luciferase expression in one-third of the animals at 24 hours and in all immunized animals at 4 days and 7 days, respectively (data not shown). In the case of RNA (100 ng RNA replicon) at a dose 300-fold lower admixed with the control cationic formulation, 3 out of 3 animals expressed detectable luciferase at 24 hours and 2 out of 3 animals expressed detectable luciferase at day 4 and day 7 (data not shown). In the case of RNA (100 ng RNA replicon) at a dose 300-fold lower admixed with the PAA nanoalum formulation, 1 out of 3 animals expressed detectable luciferase at 24 hours and 2 out of 3 animals expressed detectable luciferase at day 4 and day 7 (data not shown).

[0509] The above image data were quantified via a circular ROI from Living Image software and presented graphically in Figures 6A to 6C . Relative luminescence data were expressed on a logarithmic scale, according to formulation (unformulated, control cationic emulsion, and PAA nanoalum, left, middle, and right graphs, respectively) and by time at 24 hours ( Figure 6A ), 4 days ( Figure 6B ), and 7 days ( Figure 6C) were grouped according to the replicon vector doses delivered at (0 μg (mcg), 1 μg (mcg), and 0.1 μg (mcg), respectively). The data demonstrated that at 24 hours post-injection, RNA replicons formulated with cationic emulsion at doses 30-fold and 300-fold lower than the unformulated RNA (30 μg) (1 μg or 0.1 μg) demonstrated expression equivalent to that of the unformulated RNA. Compared to the cationic emulsion, at 24 hours, the same doses of replicon RNA formulated with PAA nanoalum at 30-fold and 300-fold lower than the unformulated RNA (30 μg) (1 μg or 0.1 μg) demonstrated lower expression ( Figure 6A ), but at day 4 and day 7 post-injection, RNA formulated with the control cationic emulsion or PAA nanoalum demonstrated roughly equivalent expression at doses of 1 μg (mcg) and 0.1 μg (mcg) ( Figure 6B and Figure 6C ). The data demonstrated that the PAA nanoalum formulation was able to deliver and express replicon RNA and was dose-sparing compared to the unformulated RNA.

[0510] We next tested whether the sizing agent PAA in the nanoalum formulation affected the delivery or expression of the RNA replicon vector (Figure 7). To determine whether PAA alone was responsible for the luciferase expression of the RNA replicon vector, unformulated RNA replicon was used at doses of 30 μg of unformulated replicon or 1 μg and 100 ng of formulated RNA replicon ( Figure 7A ), PAA alone plus RNA replicon ( Figure 7B ), control cationic emulsion plus RNA replicon ( Figure 7C ), or PAA nanoalum plus RNA replicon ( Figure 7D ) to immunize mice. The IVIS Illumina II imager was used to estimate luciferase expression, and the image data were quantified via circular ROIs as described at 24 hours post-injection. The data demonstrated that at doses of 0.1 μg or 1.0 μg, PAA alone ( Figure 7B ) did not deliver and / or induce expressible levels of the RNA replicon, while the same doses of RNA replicon formulated or formulated with the control cationic emulsion or PAA nanoalum demonstrated detectable luciferase expression at levels roughly equal to those 30-fold to 300-fold higher when delivering the unformulated RNA replicon. The data demonstrated that the sizing agent PAA alone was unable to deliver and / or induce expressible levels of protein from the RNA replicon.

[0511] Previous experiments demonstrated that the nanoalum formulations of the present disclosure are capable of delivering RNA replicons that can be expressed and exhibit dose-sparing properties compared to naked RNA replicons. We next determined whether the nanoalum formulations could efficiently deliver messenger RNA (Figure 8). To test this, we purchased from Trilink Biotechnologies a capped (cap 0) and polyadenylated mRNA FLuc mRNA that mimics fully processed mature mRNA (Luc mRNA), which is optimized for the mammalian system and modified with pseudouridine and 5-methylcytidine. The mRNA expresses the luciferase protein originally isolated from the firefly Photinus pyralis. Briefly, as described, mice (3 per group) were immunized with unformulated RNA, mRNA formulated with PAA nanoalum, or mRNA formulated with a control cationic emulsion, and RNA at RNA doses of 10 μg, 1 μg, or 0.1 μg. RNA expression was estimated using an IVIS Illumina II imager at 6 hours, 24 hours ( Figure 8A ), and day 5 ( Figure 8B ), and the imaging data were quantified via circular ROIs. Figure 8A Data at 24 hours post-injection in Figure 8A demonstrate that animals receiving unformulated mRNA (left panel) had detectable luciferase expression at 10 μg and 1 μg mRNA dose levels, but not at 0.1 μg. However, both the control cationic formulation and the PAA nanoalum formulation ( Figure 8A , middle and right panels) not only expressed equal levels of mRNA compared to each other at all doses (10 μg, 1 μg, and 0.1 μg), but also demonstrated increased expression levels (>30-fold) at a dose of 1 μg compared to unformulated mRNA and had detectable expression levels at an RNA dose of 0.1 μg, thus demonstrating the dose-sparing nature of the nanoalum formulation. At day 5 post-injection ( Figure 8B), The unformulated mRNA demonstrated detectable expression of LUC at an RNA dose of 10 μg, but at a lower level, and no luciferase mRNA expression was detected at the lower doses of 1 μg and 0.1 μg. Interestingly, five days after injection, mice receiving control cationically formulated mRNA demonstrated no detectable expression of mRNA at any of the delivery doses of 10 μg, 1 μg, 0.1 μg (left and middle groups). However, mice receiving PAA nanoalum formulated mRNA (rightmost group) not only expressed levels >10-fold higher than the level of mRNA at the 10 μg dose, but also demonstrated detectable expression at the 1 μg dose, thus demonstrating that the nanoalum formulation still has dose-sparing properties even five days after delivering the mRNA. Then, we compared the expression kinetics at 6 hours, 24 hours, and 5 days after in vivo delivery in animals receiving unformulated mRNA, cationic emulsion formulated mRNA, or PAA nanoalum formulated mRNA ( Figure 8C ) at an mRNA dose of 10 μg. The data demonstrated that animals immunized with mRNA formulated with nanoalum had increased and relatively steady-state mRNA expression levels (□) over five days compared to unformulated mRNA (·) or control cationic emulsion formulated mRNA (Δ) whose expression rapidly declined.

[0512] It has been reported in the literature that mRNA expression declines on day 5 when delivered via control cationic liposomes and this decline in expression is not unexpected. However, the sustained expression of unformulated mRNA or nanoalum formulated mRNA is surprising and very interesting, with a 10-fold dose-sparing effect still observed on day 5. The relative expression level at the 10 μg dose is roughly equal to the RNA dose of 1 μg of nanoalum formulated mRNA. Without wishing to be bound by theory, we hypothesize that the nanoalum formulations of the present disclosure can stabilize the mRNA constructs.

[0513] Based on the surprising stability of in vivo expression of mRNA formulated with the nanoalum of the present disclosure, we further examined whether the nanoalum of the present disclosure stabilizes RNA in vitro. To test this, we combined 1 μg of RNA replicon with control cationic or PAA formulations and stored this admixture as a single vial formulation at 4 °C for 1 hour, 4 hours, or 24 hours. Unformulated replicon RNA stored at 4 °C for 1 hour, 4 hours, or 24 hours was used as a control. Then, these single vial formulations of the admixture were used to immunize mice (3 per group), and 1 day ( Figure 9A ) and 5 days ( Figure 9B)An imager was used to estimate RNA expression and quantify the data via circular ROIs. The data demonstrated that unformulated RNA replicons had no detectable expression when stored at 4 °C for 4 hours or 24 hours after admixture, regardless of whether the measurement was performed 24 hours or 5 days after in vivo delivery. However, when estimated 24 hours or 5 days after in vivo delivery of unformulated, cationic lipid-formulated, or PAA nanoalum-formulated RNA replicons, detectable expression was exhibited if the RNA replicons were administered immediately (time = 0) or 1 hour after storage at 4 °C. When stored at 4 °C for 4 hours or 24 hours, unformulated RNA had no detectable expression due to the relative instability of RNA as already reported in the literature. Comparing the data of unformulated RNA replicons with those of control cationic or nanoalum-formulated RNA replicons, the RNA replicons exhibited comparable expression to that measured at 1 day ( Figure 9A ) or 5 days ( Figure 9B ) when admixed and stored as a single vial at 4 °C for 1 hour, 4 hours, or 24 hours before in vivo administration. We further analyzed the data by analyzing the scatter plot ( Figures 9C to 9E ) and directly comparing the data of the control cationic formulation, PAA nanoalum, and unformulated replicon RNA individually. When administered immediately after admixture with replicon RNA (T = 0, Figure 9C ), 4 hours after admixture and storage at 4 °C (T = 1 hour, Figure 9D ), or admixture and storage at 4 °C for 24 hours (T = 24 hours, Figure 9E ), the RNA had comparable expression levels on day 5 after administration, demonstrating that nanoalum-formulated RNA was stable for up to 24 hours at 4 °C when admixed as a single vial formulation.

[0514] By administering the RNA-encoded reporter gene in the examples presented herein, we have demonstrated that the nanoalum of the present disclosure: (1) is capable of delivering an in vivo expressible form of a polynucleotide agent and specifically an RNA agent, regardless of whether the delivered RNA form is mRNA or an expression vector RNA construct; (2) the nanoalum formulation allows for dose-sparing delivery of RNA vectors, meaning that comparable RNA expression of the nanoalum formulation was achieved at an RNA dose at least 30-fold to 300-fold lower than that of unformulated RNA; and (3) the nanoalum formulation of the present disclosure enhances the stability of RNA agents in vivo and in vitro. After developing and characterizing the properties of the nanoformulations for delivering RNA, we evaluated the ability of the nanoalum formulations to deliver RNA that elicits a stimulatory host immune response.

[0515] To evaluate the ability of the nanoalum formulations of the present example to deliver RNA antigens, we analyzed the immune responses of mice immunized with RNA replicons expressing the EMCH fusion polypeptide formulated with the nanoalum of the present disclosure.

[0516] Constructing the EMCH fusion polypeptide. The fusion polypeptide designated EMCH was generated by the tandem ligation of: pairs of polynucleotides added to the methionine start codon (ATG) at the 5' end of a fragment of the carboxyl-terminus of a putative mitochondrial HSP70 (8E or 8) polypeptide, the carboxyl-terminal fragment of the open reading frame encoding the malate dehydrogenase polypeptide, the carboxyl-terminal fragment of the cysteine protease B polypeptide (CpB, CPB or C), and the open reading frame encoding the amino-terminal fragment of the histone H2BN polypeptide (H2BN, h2Bn or H). EMCH has: a 2,631 polynucleotide sequence encoding amino acids 509 to 660 of the carboxyl-terminus of the putative mitochondrial HSP70 (8E or 8) polypeptide of Leishmania infantum polynucleotide; 460 to 1425, which encodes amino acids 1 to 322 of the carboxyl-terminus of the malate dehydrogenase gene of Leishmania infantum; polynucleotides 1426 to 2295, which encodes amino acids 154 to 443 of the carboxyl-terminal fragment of the cysteine protease B polypeptide (B); and polynucleotides 2297 to 2631, which encodes amino acids 1 to 111 of the amino-terminus of the histone H2BN (H) polypeptide of Leishmania infantum. The 877 amino acid fusion polypeptide was expressed in E. coli and purified by column chromatography. The nucleic acid components and methods of making and using are described more fully in WO2014 / 160985, which is incorporated herein by reference in its entirety for all purposes.

[0517] Briefly, at time zero, mice were immunized with an alphavirus RNA replicon vector encoding Leishmania fused RNA polynucleotide EMCH at 10 μg or 0.1 μg, the alphavirus RNA replicon vector being as an unformulated naked RNA control, an RNA replicon admixed with a control cationic liposome, or an RNA replicon admixed with an RNA PAA nanoalum formulation, and all groups increased after three weeks. Four weeks after the last increase, splenocytes were harvested and analyzed for recall antigen-specific T cell responses as determined by intracellular cytokine staining after in vivo stimulation with the EMCH polypeptide. Cytokine production of immunized mouse splenocytes was analyzed for EMCH-specific CD44hi CD4+ memory T cells as measured by flow cytometry. Antigen-stimulated splenocytes were identified by intracellular cytokine staining based on CD3 and CD4 expression and further gated on CD44 high cells. CD44 high CD4+ T cells were further stained for intracellular CD154, IFN-γ, IL2, TNFα, GM-CSF, IL-17, and IL-5. EMCH-specific CD44 high CD4+ T cells exhibited a polyfunctional T cell response positive for IFN-γ, TNFα, and IL-2, typical of an antigen-specific Leishmania response. Data( Figures 10A to 10D ) demonstrated that immunization with 0.1 μg of EMCH RNA, which is 100-fold lower in dose when formulated with a control cationic liposome or PAA nanoalum, generated a percentage of CD4+CD44 high CD154, IFN-γ, IL-2, or TNFα single-positive cytokine-stained T cells roughly equivalent to that of 10 μg of unformulated RNA. The unformulated RNA replicon at a dose of 0.1 μg showed little or no detectable staining. Thus, the nanoalum formulations of the present disclosure are capable of delivering RNA encoding a pathogen antigen as a vaccine formulation to stimulate an immune response in a vaccinated host.

[0518] We further characterized the quality of the immune response to the Leishmania polypeptide expressed when the RNA vector was delivered formulated with the nanoalum of the present disclosure. A protective Leishmania immune response is characterized by the presence of polyfunctional antigen-specific T cells that secrete multiple cytokines. We analyzed the polyfunctional T cell response of CD4+CD44 high T cells. Data( Figure 10E)Proof that mice immunized with 100 ng of EMCH RNA replicon formulated with PAA nanoalum (hatched bars) or with a control cationic emulsion (diagonal cross-hatched bars) had equal numbers of triple-positive IFN-γ, IL-2, and TNFα CD4+CD44high T cells compared to animals immunized with 10 μg of unformulated RNA (black solid bars). There were also double-positive cells expressing IFN-γ and IL-2 or IL-2 and TNFα. The data demonstrate that the PAA nanoalum formulation is capable of delivering RNA expressed at a level sufficient to generate a relevant antigen-specific immune response with vaccine properties.

[0519] Example 4. Using PEG nano - alum formulations (with various lengths of PEG) to deliver proteins or peptides

[0520] (ID93) to stimulate immune response

[0521] Experiments were performed to test whether the nanoalum formulations of the present disclosure can deliver protein or polypeptide agents, either alone or in combination with other agents, specifically TLR agonists, to stimulate an immune response in a host.

[0522] Animal model. Briefly, experimental animals and 6- to 8-week-old female CB57BL / 6 mice were purchased from The Jackson Laboratory or Charles River Laboratories in the United States and maintained under specific pathogen-free conditions.

[0523] ID93 is a fusion protein incorporating four Mycobacterium tuberculosis peptides Rv1813, Rv2620, and Rv2608, and Rv3619 generated as previously described

[14] .

[0524] According to the treatment protocol, splenocytes were isolated from four or five animals. Red blood cells were lysed using Red Blood Cell Lysis Buffer (eBioscience) and resuspended in RPMI 1640, 10% FBS. The total number of viable cells was counted by ViaCount assay using a PCA system (Guava Technologies), seeded at 2×106 cells / well in 96-well plates, and stimulated with medium or ID93 (10 μg / mL) for 2 hours at 37°C. GolgiPlug (BD Biosciences) was added and the cells were incubated for an additional 8 hours at 37°C. In the presence of anti-mouse CD16 / 32, the cells were washed and surface stained with fluorescent dye-conjugated antibodies CD4 (clone GK1.5), CD44 (clone IM7), and CD8 (clone 53-6.7) (BioLegend and eBioscience) for 20 minutes at 4°C. The cells were washed and permeabilized with Cytofix / Cytoperm (BD Biosciences) for 20 minutes at room temperature. The cells were washed twice with Perm / Wash (BD Biosciences) and intracellularly stained with fluorescent dye-conjugated antibodies CD154 (clone MR1), IFN-γ (clone XMG-1.2), TNF (MP6-XT22), GM-CSF (MP1-22E9), IL-17A (clone TC11-18H10), and IL-5 (clone TRFK5) (BioLegend and eBioscience) for 20 minutes at room temperature. The cells were washed and resuspended in PBS. Up to 10 6 events were collected on a four-laser LSR Fortessa flow cytometer (BD Biosciences). Cells were gated as singlets > lymphocytes > CD4+CD8- > cytokine-positive or CD44hi > positive cytokine. The ID93-specific response frequency was determined by subtracting the frequency of the positive response in unstimulated cells from that in ID93-stimulated cells in matched samples.

[0525] Antibody response

[0526] Mouse sera were prepared by collecting retro-orbital blood into microvette serum collection tubes (VWR International, West Chester, PA) and then centrifuging at 10,000 rpm for 5 minutes. Each serum sample was then analyzed by antibody capture ELISA. Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 2 μg / ml recombinant antibody ID93 in 0.1 M bicarbonate buffer and blocked with 1% BSA-PBS. Then, serum samples were serially diluted and, after washing in PBS / Tween 20 in sequential order, anti-mouse IgG1 or IgG2c-HRP (both from SouthernBiotech, Birmingham, AL) and ABTS-H2O2 (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added to the plates. The plates were analyzed at 405 nm (ELX808, Bio-Tek Instruments Inc, Winooski, VT). Prism software (GraphPad Software, Inc.) was used to calculate the median titers in order to determine the sigmoidal dose-response curve using the least-squares fitting method.

[0527] Previously published Mycobacterium tuberculosis model data demonstrated that immunization of mice with the fusion polypeptide ID93 formulated with GLA / SE induced a greater ID-93 specific IgG2c response, indicative of a Th1-biased response (Baldwin 2012). However, the published data also indicated that alum formulations had generally induced a greater Th2 response, as evidenced by a greater IgG1 antibody response. We evaluated whether altering the average particle size of the nano-alum formulation would affect the quality of the immune response generated to the ID93 fusion polypeptide. To evaluate this, on day zero, animals were immunized intramuscularly in the quadriceps with 0.5 μg ID93 admixed with 100 μg of micron alum (e.g., commercially available untreated alum) used as a control formulation or with 0.5 μg ID93 admixed on day zero with 100 μg of a PEG nano-alum formulation (PEG 5000-DSPE, as a sizing agent ground such that the resulting formulation had an average particle size of 400 nm, 130 nm, or 75 nm) plus or minus 5 μg of the TLR4 agonist SLA. On day 21 post-immunization, the animals were bled as described, and sera were collected and analyzed for ID93-specific antibody responses. ID93-specific IgG1 ( Figure 11A ) and IgG2c ( Figure 11B)Antibody titers in sera on day 21 demonstrated that animals immunized with ID93 admixed with SLA-SE formulation generated ID93-specific IgG1 and IgG2c antibody titers as we previously described, where a slight increase in IgG2c titer indicated a Th1 response. As expected, immunization of mice with ID93 fusion polypeptide alone did not generate measurable IgG1 or IgG2c antibody titers. As predicted in the literature, immunization with an alum formulation with a particle size of 1 to 10 microns showed a significant bias towards a Th2 response, as indicated by high IgG1 antibody titers and low IgG2c titers. These controls were compared to PEG nanoalum formulations including PEG-5000DSPE as an adjuvant, which were milled or sized by varying the method as described in Example 1 (silverson mixing at 5000 rpm for 5 minutes, microfluidizing once at 10 kPSI or 10 times at 30 kPSI) to produce nanoalum with particle sizes of 400 nm, 130 nm or 75 nm respectively. Data demonstrated ( Figure 11A ), that the 400 nm PEG nanoalum formulation induced the same IgG1 endpoint titer as the untreated aluminum formulation. The 130 nm and 75 nm sized PEG nanoalum formulations also produced high IgG1 midpoint titers at day 21, but were reduced by approximately half compared to alum or the 400 nm sized PEG nanoalum. Data demonstrated that none of the PEG nanoalum formulations tested generated measurable IgG2c ID93 antibody titers in immunized mice. To determine whether addition of a TLR4 agonist to the admixed nanoalum formulations could bias the response to generate a Th1 response as measured by IgG2c, mice were also immunized with the TLR4 agonist SLA plus ID93 antigen and PEG nanoalum formulations. Data demonstrated that admixing the TLR4 agonist SLA with the alum formulation or the 400 nm sized PEG nanoalum formulation had a negligible effect on the midpoint titer of the ID93-specific IgG1 response, but admixing SLA with the 130 nm or 75 nm sized PEG nanoalum tended to increase the midpoint ID93-specific IgG1 response. Similarly, analysis of the Figure 11BThe data presented demonstrate that the addition of SLA to PEG-nanoalum formulations of particle sizes 400 nm, 130 nm, or 75 nm induced ID93 IgG2c titers, compared to undetectable titers in animals immunized with ID93 / PEG-nanoalum in the absence of SLA. The data demonstrate that PEG-nanoalum is capable of eliciting a Th2-biased immune response, but the ID93 IgG1 titers of PEG-nanoalum formulations of particle size 130 nm or less were reduced by approximately half compared to alum or 400 nm PEG-nanoalum. Interestingly, the addition of the TLR4 agonist SLA restored the magnitude of the Th2-biased response almost to that of the conventional alum formulation. Additionally, while no ID93-specific IgG2c antibody titers were detected in mice immunized with ID93 PEG-nanoalum, the addition of the TLR4 agonist SLA to the ID93 / PEG-nanoalum vaccine composition resulted in IgG2c production, indicating some bias of the response towards Th1 by SLA.

[0528] Mice immunized with PEG-nanoalum formulations elicited antigen-specific immune responses, where the PEG-nanoalum particles included a polyethyleneglycolated phospholipid adjuvant with different PEG lengths or the same PEG length linked to phospholipids with different acyl chain lengths and were admixed with the TB fusion peptide ID93 plus the TLR4 agonist SLA. Table 5 presents a table comparing experimental groups adsorbed with 0.5 μg of the fusion protein ID93 with: a 100 μg conventional alum formulation batch of particle sizes from 1 μm to 10 μm mixed with PEG-5000 DSPE (without milling or treatment) plus 5 μg of the TLR4 agonist SLA; and microfluidized nanoalum formulations adsorbed to 0.5 μg of the fusion protein ID93 plus 5 μg of SLA and nanoalum formulations with a polyethyleneglycolated phospholipid adjuvant, including adjuvants PEG-DSPE with different PEG lengths 5000, 2000, or 750, where the polyethyleneglycolated phospholipid adjuvant has a defined PEG length of 2000 and phospholipids with different acyl chain lengths of 18 carbons (DSPE), 16 carbons (DPPE), and 14 carbons (DMPE).

[0529] Table 5

[0530] Grouping Vaccine PEG length Acyl chain length Alum size 1 ID93 ------- ---------- ----------- 2 ID93 - SLA - alum / PEG 5000 DSPE 18C 1μm to 10μm 3 ID93 - SLA - PEG - nano - alum 5000 DSPE 18C Approximately 70nm 4 ID93 - SLA - PEG nano - alum 2000 DSPE 18C Approximately 70nm 5 ID93 - SLA - PEG nano - alum 750 DSPE 18C Approximately 70nm 6 ID93 - SLA - PEG nano - alum 2000 DPPE 16C Approximately 70nm 7 ID93 - SLA - nano - alum 2000 DMPE 14C Approximately 70nm 8 ID93 + SLA - alum 1μm to 10μm

[0531] Mice immunized with a dose of 5 μg of TLR agonist SLA and a dose of 0.5 μg of ID93 fusion protein adsorbed to 100 μg of a PEG-nanoalum formulation (PEG-DSPE as the coating agent) with a PEG length of 5000, 2000 or 750 and a particle size of approximately 70 nm elicited ID93 antigen-specific IgG1 antibody titers, which were measured as the geometric mean titer on day 21. Figure 12A Demonstrated that equal IgG1 titers were elicited in mice immunized with a conventional alum formulation with a particle size of 1 μm to 10 μm and a 70 nm nanoalum formulation, the 70 nm nanoalum formulation comprising a coating agent PEG-DSPE with a PEG length of 5000, 2000 or 750 or a PEG length of 2000 linked to a phospholipid with an acyl chain length of 18 carbons (DSPE) or 16 carbons (DPPE). The IgG1 titer of the nanoalum formulation with a phospholipid having an acyl chain length of 14 carbons (DMPE) and a PEG length of 2000 was reduced by approximately half compared to other nanoalum formulations. Figure 12B Demonstrated that a nanoalum formulation with a particle size of 70 nm and a dose of 100 μg elicited antigen-specific IgG2c antibody titers indicative of a Th1 bias, but the response was approximately half of that seen with an alum formulation with a particle size of 1 μm to 10 μm, the nanoalum formulation comprising a coating agent PEG-DSPE with a PEG length of 5000, 2000 or 750 or a PEG length of 2000 linked to a phospholipid with an acyl chain length of 18 carbons (DSPE) or 16 carbons (DPPE), adsorbed to 0.5 μg ID93 plus 5 μg TLR4 agonist SLA. The nanoalum formulation with a phospholipid having an acyl chain length of 14 carbons (DMPE) and a PEG length of 2000 did not show any appreciable ID93 IgG2c. Figure 12CProof that the ID93 nano-alum formulation induced antigen-specific CD4+ T cells. Cytokine production of immunized mice was analyzed for ID93-specific CD44hi CD4+ memory T cells as measured by flow cytometry. Splenocytes of vaccinated mice stimulated with ID93 for 12 hours in the presence of GolgiStop and ID93-stimulated splenocytes were identified by intracellular cytokine staining based on CD3 and CD4 expression and further gated for CD44hi cells. CD44hi CD4+ T cells were further stained for intracellular CD154, IFN-γ, TNF, GM-CSF, IL-17, and IL-5. ID93-specific CD44hi CD4+ T cells exhibited a typical multi-functional T cell response positive for TNFα and IL-5 in response to antigen-specific ID93, demonstrating that these nano-alums can be effective vehicles for the TLR4 agonist SLA in inducing Th1 immunity against the ID93 antigen.

[0532] Example 4. Generating chitosan -, dextran -, and poly(allylamine) - nano - alum formulations

[0533] Aluminum-containing adjuvants have been administered in humans and animals since the mid-1920s. The term alum is widely used to classify any aluminum-based adjuvant used in vaccines in general, but chemically, these are mainly hydroxyaluminum oxide (AlO(OH)) or aluminum phosphate (AlPO4, also known as Al(OH) x (PO4) y ). AlO(OH) is poorly crystalline as evidenced by its x-ray diffraction (XRD) pattern, with a crystal structure of boehmite, which is one of many metastable phases of the corundum (α-Al2O3) stable phase. The surface of AlO(OH) is cationic and thus most suitable for adsorbing anionic antigens. TEM imaging showed fibrous nanoparticles with a calculated average size of 4.5 nm × 2.2 nm × 10 nm, which form aggregates with a wide size distribution of 5 to 10 microns in suspension. Aluminum phosphate, contrary to its name, consists of non-stoichiometric amounts of phosphate counterions and hydroxide counterions, has a negative (anionic) net surface charge and is thus most suitable for adsorbing cationic antigens. Unlike AlO(OH), aluminum phosphate is anhydrous to x-rays and consists of approximately 50 nm discoid particles forming loose aggregates with a median diameter of about 4 μm. Examples of aluminum-based nanoparticle adjuvants (nano-alums) are described herein that are made using commercially available micron-sized seals (e.g., or ) as starting materials and microfluidizing them in the presence of stabilizers.

[0534] Using (Al(OH)x (PO4) y ) Nanoalum - chitosan of adjuvant: The following describes a general method for synthesizing nanoalum using as an alum precursor and low - molecular - weight chitosan with a degree of deacetylation (DD) of 75% to 85% (50,000 Da to 190,000 Da, with a viscosity of 20 cP to 300 cP for a 1 wt% solution in 1% acetic acid at 25 °C) as a stabilizer.

[0535] Materials

[0536]

[0537] Make The adjuvant concentration (10 ml at 5 mg Al / ml; 50 mg Al) is kept constant and stabilized with different amounts of chitosan. Before mixing, a predetermined amount of chitosan is dissolved in 40 ml of a weakly acidic 0.12 M sodium acetate / 0.02 M acetic acid buffer with pH = 5.4. After complete dissolution, the chitosan solution (40 ml) is mixed with 10 ml (50 mg of aluminum), mixed in a silverson high - shear mixer at 5,000 rpm for 5 minutes and then microfluidized 22 discontinuous passes at 30,000 psi in an LM20 high - shear microfluidizer (Microfluidics). The microfluidized material is visually turbid but translucent. Table 6 below provides the composition of various derived nanoalum stabilized with chitosan. As Figure 13A shown, the hydrodynamic diameter is reduced by the number of passes. For the same homogenization process, the hydrodynamic diameter tends to be lower when the chitosan fraction increases ( Figure 13B ). On average, the ζ - potential of the nanoalum - chitosan formulation is +20 mV.

[0538] Table 6. Composition of nanoalum manufactured using as a starting material and low - molecular - weight chitosan (approx. 120,000 Da, with a minimum DD of 85%) as a stabilizer.

[0539] Lot # if applicable QG777 QG778 Aluminum phosphate [% w / v Al] 0.10％ 0.10％ 0.10％ 0.10％ 0.10％ Chitosan (low mw) [% w / v] 0.07％ 0.30％ 0.59％ 0.033％ 0.007％ Final volume [ml] 50 50 50 50 50

[0540] Use Nanoalum - dextran of (AlO(OH)) adjuvant - The following describes a general method for synthesizing nanoalum using as an alum precursor and dextran sulfate (40,000 Da) as a stabilizer.

[0541] Materials

[0542]

[0543] Make The adjuvant concentration (10 ml at 10 mg Al / ml; 100 mg aluminum) was kept constant and stabilized with different amounts of dextran sulfate. Before mixing, a predetermined amount of dextran sulfate was dissolved in 40 ml of DI water. 10 ml (100 mg Al) was added to 40 ml of the dextran sulfate solution, mixed for 5 minutes in a Silverson high-shear mixer at 5,000 rpm and then microfluidized discontinuously 15 times at 30,000 psi in an LM20 high-shear microfluidizer (Microfluidics). The microfluidized material was clear to translucent and sterile filtered through a 200 nm PES membrane. The composition of various derived nanoalums stabilized with dextran sulfate is provided in Table 7 below. As Figure 14A shown, the hydrodynamic diameter was reduced by the number of passes. On average, the ζ-potential of the nanoalum-dextran formulations was -40 mV. Particle stability data available at the time of this report showed that up to 3 months after the manufacturing date, the size of nanoalum-dextran (lot QG774, shown as an example) did not change significantly ( Figure 14B ).

[0544] Table 7 Composition of nanoalums manufactured using aluminum hydroxide gel as the starting alum material and dextran sulfate (40 kDa) as the stabilizer

[0545]

[0546] Use (AlO(OH)) adjuvant nanoalum-chitosan. The general method for synthesizing nanoalum using as the alum precursor and chitosan (15,000 Da, minimum DD of 85%) as the stabilizer is described below.

[0547] Materials

[0548]

[0549] Natural aluminum hydroxide gel (AlO(OH)) has a cationic surface charge and thus electrostatically repels chitosan, which is also cationic. To adsorb chitosan onto the latter must undergo surface modification via phosphate ligand exchange. For phosphate exchange, (10 mg Al / ml) was mixed with 10x PBS in a 1:2 volume ratio and allowed to react on an orbital shaker at 37 °C for 24 to 48 hours. The phosphate-exchanged Centrifuge at 2500 rpm for 15 minutes and decant the clear supernatant. Then the precipitated is dispersed in DI water, and the centrifugation - decantation step is repeated 3 times to wash out the phosphate buffer. The finally washed precipitate is dispersed in DI water at a concentration of 10 mg Al / ml and stored at room temperature. ζ - potential measurements before and after phosphate exchange confirm that the surface charge has been successfully converted from cationic to anionic ( ). A 2% w / v chitosan solution in 1% v / v acetic acid is prepared as a stock solution for mixing with Figure 15A . 10 ml (100 mg Al) is mixed with different amounts of chitosan prepared by diluting the 2% chitosan stock solution with DI water. The example mixing conditions are listed in Table 8. (100 mg Al) is mixed with different amounts of chitosan prepared by diluting the 2% chitosan stock solution with DI water. The example mixing conditions are listed in Table 8.

[0550] Table 8. Examples of mixing conditions with chitosan.

[0551]

[0552] Each 50 ml of the chitosan mixture is homogenized with a silverson high - shear mixer at 5,000 rpm for 5 minutes and then microfluidized at 30,000 psi for 5 minutes in continuous mode at 110 ml / min using an M110P microfluidizer (Microfluidics). The microfluidized material is milky white and almost transparent. The composition of the synthesized example batches is provided in Table 9. Figure 15B The particle size of the pre - filtered nano - alum - chitosan material from DLS is shown. Overall, the Z - average diameter is positively correlated with the amount of chitosan used. The formulation is filtered through a 200 nm PES membrane when possible and stored at 4 °C. On average, the ζ - potential of the nano - alum - chitosan formulation is +20 mV. Table 9. Composition of nano - alum derived stabilized by chitosan (15 kDa, DD minimum 85%).

[0553] Name Batch size [ml] Aluminum [mg / ml] Chitosan - 15kDa [mg / ml] Acetic acid [mM] QG851 50 2 1 8.7 QG850 50 2 0.5 4.4 QG849 50 2 1 8.7 QG845 50 2 2 17.5

[0554] The z - average hydrodynamic diameter increases over time, but depending on the amount of chitosan used, the stabilization level is approximately 300 nm to 500 nm. Secondly, the rate of size increase is temperature - dependent - the size increases faster at higher temperatures - indicating that the size increase is endothermic and may be driven by an increase in entropy.

[0555] Using (AlO(OH)) adjuvant nano-alum - poly(allylamine) - The following describes a general method for synthesizing nano-alum using as an alum precursor and poly(allylamine) (15,000 Da) as a stabilizer.

[0556] Materials

[0557]

[0558] Natural (AlO(OH)) has a cationic surface charge and thus electrostatically repels poly(allylamine), which is also cationic. To adsorb poly(allylamine) onto the latter must undergo surface modification via phosphate ligand exchange. For phosphate exchange, (10 mg Al / ml) is mixed with 10x PBS at a volume ratio of 1:2 and allowed to react on an orbital shaker at 37 °C for 24 to 48 hours. The phosphate-exchanged is centrifuged at 2500 rpm for 15 minutes and the clear supernatant is decanted. The precipitated is then dispersed in DI water, and the centrifugation-decantation step is repeated 3 times to wash out the phosphate buffer. The finally washed PE-aluminum gel precipitate is dispersed in DI water at a concentration of 10 mg Al / ml and stored at room temperature. ζ-potential measurements of before and after phosphate exchange confirm that the surface charge has been successfully converted from cationic to anionic. To synthesize poly(allylamine)-stabilized nano-alum, 10 ml of PE-aluminum gel (100 mg Al) is mixed with different amounts of 15% w / v poly(allylamine); the example mixing ratios are summarized in Table 10. Since the free base form of poly(allylamine) is used, the pH of the mixture of PE-aluminum gel and poly(allylamine) is between 8 and 11, and thus 6M HCl is needed to adjust it to 7.

[0559] Table 10. Examples of mixing ratios for preparing poly(allylamine)-stabilized derived nano-alum.

[0560]

[0561] To produce stable nano-alum, The mixture with poly(allylamine) was mixed using a Silverson high-shear mixer at 5,000 rpm for 5 minutes and then microfluidized using an M110P microfluidizer (Microfluidics) at 110 ml / min at 30,000 psi for 5 minutes. The microfluidized material was almost transparent and was sterile filtered through a 200 nm PES membrane. Figure 16 The size of the shown nano-alum particles increases with the poly(allylamine) content. On average, the ζ-potential of the nano-alum-poly(allylamine) formulation is approximately +20 mV. The composition of the prepared example nano-alum-poly(allylamine) formulations is provided in Table 11.

[0562] Table 11. Stabilized by poly(allylamine) Examples of derived nano-alum formulations

[0563] Lot number # Batch size [ml] Aluminum [mg / ml] Poly(allylamine) [mg / ml] QG861 50 2 0.5 QG860 50 2 1 QG859 50 2 2 QG858 50 2 10 QG854 50 2 20

[0564] Nano-alum-poly(allylamine) for the preparation of RNA-based vaccines - To evaluate the compatibility of nano-alum-poly(allylamine) for complexing with RNA, we mixed 1 μg of 10 kb self-replicating RNA encoding the Zika antigen with diluted nano-alum-poly(allylamine) formulations containing 1 mg / ml (lot number QG860), 2 mg / ml (lot number QG859), or 20 mg / ml (lot number QG854) poly(allylamine). Gel retardation assays (GRA) were performed on the nano-alum complexed RNA samples and naked RNA controls to evaluate the ability of each formulation to bind RNA and the loading capacity. QG859 (2 mg / ml poly(allylamine) undiluted) bound 100% of the RNA at a dilution of 1 / 200 (0.01 mg / ml poly(allylamine)). Similarly, QG860 (1 mg / ml poly(allylamine) undiluted) bound 100% of the RNA at a dilution of 1 / 100 (0.01 mg / ml poly(allylamine)). These two formulations showed similar binding characteristics related to the amount of poly(allylamine). On the other hand, QG854 (20 mg / ml poly(allylamine) undiluted) bound nearly 100% of the RNA even at a dilution of 1 / 4000 (0.005 mg / ml poly(allylamine)).

[0565] References

[0566] 1. Shah RR, Dodd S, Schaefer M, Ugozzoli M, Singh M, Otten GR, Amiji MM, O'Hagan DT, Brito LA., The Development of Self-Emulsifying Oil-in-Water Emulsion Adjuvant and an Evaluation of the Impact of Droplet Size on Performance, Journal of Pharmaceutical Sciences, 2015

[0567] 2. Edited by Weichert R, Determination of Extinction Efficiency and Particle Size Distribution by Photosedimentation using Light of Different Wavelengths, Particle Size Analysis 1981 Proc 4th Conf, held at Loughborough Univ of Technology, September 21 - 24, 1981, edited by N G Stanley-Wood and T Allen Chichester, Wiley, 1982; 1981

[0568] 4. Xiang SD, Scholzen A, Minigo G, David C, Aspostolopoulos v, Mottram PL, Plebanski M., Pathogen Recognition and Development of Particulate Vaccines: Does Size Matter?, Methods, 2006: 1 - 9

[0569] 5. Kalkanidis M, Pietersiz GA, Ziang SD, Mottram PL, Crimeen-Irwin B, Ardipradja K, Plebanski M., Methods for Nano-Particle Based Vaccine Formulation and Evaluation of their Immunogenicity, Methods 2006:20-29.

[0570] 6. Fung HWM, Mikasa TJT, Vergara J, Sivananthan SJ, Guderian JA, Duthie MS, Vedvick TS, Fox CB., Optimizing Manufacturing and Composition of a TLR4 Nanosuspension: Physicochemical Stability and Vaccine Adjuvant Activity, Journal of Nanobiotechnology 2013:11-43.

[0571] 7. Edited by Weichert R, Determination of Extinction Efficiency and Particle Size Distribution by Photosedimentation using Light of Different Wavelengths, Particle Size Analysis 1981 Proc 4th Conf, held at Loughborough Univ of Technology, September 21 - 24, 1981, edited by N G Stanley-Wood and T Allen, Chichester, Wiley, 1982; 1981.

[0572] 8. Schwendener RA., Liposomes as vaccine delivery systems: a review of the recent advances, Ther Adv Vaccines, November 2014; 2(6): 159-182.

[0573] 9. A.L. Nail, J.L. White, S.L. Hem, Structure of aluminum hydroxide I: initial precipitate, J Pharm Sci, 65(1976), pp. 1188-1191.

[0574] 10. E.B. Lindblad., Aluminium adjuvants, in D.E.S. Stewart-Tull (ed.), The theory and practical application of adjuvants, John Wiley & Sons, Ltd, New York (1995), pp. 21-35.

[0575] 11. S.J. Seeber, J.L. White, S.L. Hem., Predicting the adsorption of proteins by aluminum-containing adjuvants, Vaccine, 9(1991), pp. 201-203.

[0576] 12. S. Iyer, R.S. Robin Robinett, H. HogenEsch, S.L. Hem., Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant, Vaccine, 22(2004), pp. 1475-1479.

[0577] 13. J.V. Rinella Jr., R.F. Workman, M.A. Hermondson, J.L. White, S.L. Hem., Elutability of proteins from aluminum-containing vaccine adjuvants by treatment with surfactants

[0578] Journal of Colloid and Interface Science, 197 (1998), pp. 48 - 56.

[0579] 14. Baldwin et al. 2009, Bertholet et al., A Defined Tuberculosis Vaccine Candidate Boosts BCG and Protects Against Multidrug-Resistant Mycobacterium tuberculosis, 2010, Sci Transl Med 2, 53ra74 (2010); Baldwin et al., The Importance of Adjuvant Formulation in the Development of a Tuberculosis Vaccine, The Journal of Immunology, 2012, 188: 000 - 000.

Claims

1. A nano-alum particle, comprising: (a) an aluminum salt; and (b) a sizing agent; wherein the size of the particles is in the range of about 1 nm to about 450 nm.

2. The nano-alum particle according to claim 1, wherein the average size of the particles is the Z-average as determined by dynamic light scattering.

3. The nano-alum particle according to claim 1, wherein the aluminum salt is selected from the group consisting of aluminum hydroxide, aluminum hydroxide gel, AlPO4, AlO(OH), Al(OH)(PO4), and KAl(SO4)2.

4. The nano-alum particle according to claim 1, wherein the sizing agent is selected from the sizing agents presented in Table 1.

5. The nano-alum particle according to claim 1, wherein the sizing agent is selected from the group consisting of PAA, PEG, lipid-linked PEG, chitosan, dextran, or poly(allylamine).

6. The nano-alum particle according to claim 1, wherein the sizing agent is selected from the group consisting of PAA, PEG, and lipid-linked PEG.

7. The nano-alum particle according to claim 1, wherein the sizing agent is PEG linked to a phospholipid.

8. The nano-alum particle according to claim 1, wherein the sizing agent is PEG and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons.

9. The nano-alum particle according to claim 1, wherein the sizing agent is PEG linked to a lipid (optionally, a phospholipid) and the average molecular weight of the PEG is in the range of about 750 daltons to about 5000 daltons.

10. The nano-alum particle according to claim 9, wherein the lipid is selected from the group consisting of DSPE, DPPE, DMPE, and DLPE.

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