WO2025216920A1

WO2025216920A1 - Freezable aqueous aluminum salt adjuvants

Info

Publication number: WO2025216920A1
Application number: PCT/US2025/022472
Authority: WO
Inventors: Carl Richard ALVING; Mangala Rao; Gary R. Matyas; Erwin G. ABUCAYON
Original assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc; United States Department of the Army
Current assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc; United States Department of the Army
Priority date: 2024-04-08
Filing date: 2025-04-01
Publication date: 2025-10-16
Anticipated expiration: 2026-10-08

Abstract

Disclosed herein are aqueous aluminum salt adjuvants and compositions thereof which may be frozen without loss of their adjuvanticity.

Description

FREEZABLE AQUEOUS ALUMINUM SALT ADJUVANTS

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 63/631,026, filed April 8, 2024, which is herein incorporated by reference in its entirety.

[0003] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with Government support under W81XWH- 18-2-0040 awarded by the United States Army Medical Research and Development Command. The Government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] 1. FIELD OF THE INVENTION

[0007] The field of the invention generally relates to adjuvants and pharmaceutical compositions comprising adjuvants.

[0008] 2. DESCRIPTION OF THE RELATED ART

[0009] In 1926 Glenny and colleagues initiated the field of aluminum salts as vaccine adjuvants when they demonstrated that precipitation of diphtheria toxoid with potassium alum (A1K(SO4)2*12H2O) resulted in an insoluble particle, which upon injection into guinea pigs, resulted in an enhanced immune response to the toxoid. This marked the beginning of aluminum salt adjuvants in which aluminum-based particles comprise antigen adsorbed thereon. Such aluminum-based particles include preformed gels of particles of aluminum hydroxide (AH) or aluminum phosphate (AP). It should be noted that all prior art aluminum adjuvants are in particulate form, which can be understood from their preparation. Particularly, aluminum particulates are precipitated out of solution by mixing with an alkali hydroxide, or by conjugating to a solid particle.

[0010] Unfortunately, vaccine compositions comprising aluminum particulate adjuvants must not be frozen because freezing irreversibly destroys their adj uvanti city. Freezing is believed to irreversibly damage the structure of the aluminum particle and thereby reduce or eliminate the adsorption of antigen thereon and/or detrimentally alter the conformation of the adsorbed antigen such that the immune system fails to learn to recognize the antigen when properly folded and having the correct conformation. Thus, regulatory agencies mandate that vaccine compositions comprising aluminum particulate adjuvants that suffer a freeze event must be discarded. Consequently, in a literature review “analyzing the prevalence of vaccine exposure [of “freeze-sensitive vaccines] to temperatures below recommendations throughout various segments of the cold chain”, Hansen et al. (2017) report that “Among [45] reviewed articles, the percentage of vaccine exposure to temperatures below recommended ranges during storage was 33% in wealthier countries and 37.1% in lower income countries. Vaccine exposure to temperatures below recommended ranges occurred during shipments in 38% of studies from higher income countries and 19.3% in lower income countries. Worse yet accidental freeze events may go unnoticed such that the damaged vaccines are administered such that the recipients falsely believe they are vaccinated and protected against infection.

[0011] Additionally, aluminum particulate adjuvants cannot be sterile-filtered, autoclaved, and stored at room temperature. Further, absorption of antigens on aluminum particulate adjuvants is dependent on and thereby limited by electrostatic interactions and the tertiary conformation of the antigens can be detrimentally altered as a result of being adsorbed on aluminum particulates.

[0012] SUMMARY OF THE INVENTION

[0013] Provided herein are aqueous adjuvant compositions comprising or consisting essentially of an aqueous aluminum salt, a buffer, and a pH of about 5.0-7.5, preferably about 6.0-7.5 or 6.0-7.0. In some embodiments, the aqueous aluminum salt is aqueous aluminum chloride, aqueous aluminum sulfate, or aqueous aluminum triacetate. In some embodiments, the buffer is a sodium acetate buffer, a tromethamine buffer, or a sodium citrate dihydrate buffer. In some embodiments, the buffer is a sodium acetate buffer. In some embodiments, the buffer is a tromethamine buffer (e.g., TRIS), or a sodium citrate dihydrate buffer. In some embodiments, the aqueous adjuvant compositions comprise 0.4-220.6 mg/mL sodium acetate, 0.2-1.0 of a tromethamine, or 0.6-442.1 mg/mL sodium citrate dihydrate. In some embodiments, the concentration of the aqueous aluminum salt is about 0.5-1.5 mg/mL. In some embodiments, the aqueous adjuvant compositions exclude hydroxide salts, alkaline salts, and carbonate salts.

[0014] Also provided herein are compositions comprising or consisting essentially of the aqueous adjuvant compositions and a given antigen of interest. In some embodiments, the compositions lack insoluble particles containing aluminum. In some embodiments, the compositions exclude hydroxide salts, alkaline salts, and carbonate salts.

[0015] Methods of making the aqueous adjuvant compositions and compositions thereof are also provided. In some embodiments, the methods comprise titrating the aqueous aluminum salt with the buffer to obtain a solution having a pH of about 5.0-7.5, preferably about 6.0-7.5 or 6.0-7.0. In some embodiments, the methods comprise adding a given antigen of interest to an aqueous adjuvant composition as described herein. In some embodiments, the methods further comprise maintaining the pH in the range of about 5.0-7.5, preferably about 6.0-7.5 or 6.0-7.0.

[0016] Methods of immunizing subjects are also provided and said methods comprise injecting a subject with an aqueous adjuvant composition comprising a given antigen of interest. In some embodiments, the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum.

[0017] Also included are methods of enhancing an immune response in subjects, which comprise injecting a subject with an aqueous adjuvant composition comprising an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum, and wherein the immune response is greater than that obtained with a control composition containing the given antigen of interest without any aluminum. In some embodiments, the methods comprise (a) freezing the composition and thawing the composition, and/or (b) storing the composition at a temperature of -70- 10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc., before injecting the subject with the composition.

[0018] Methods of storing the aqueous adjuvant compositions are also provided. In some embodiments, the storage methods comprise maintaining the aqueous adjuvant composition at a temperature of -70-10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc.

[0019] Also provided are medicaments comprising the aqueous adjuvant compositions disclosed herein. In some embodiments, the medicaments further a given antigen of interest. In some embodiments, the given antigen of interest is present in an immunogenic amount.

[0020] Kits comprising one or more aqueous adjuvant compositions packaged together devices, e.g., pipettes, containers, pH measurement devices, etc., for mixing with another ingredient, e.g., a given antigen of interest, are also contemplated herein.

[0021] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention. [0022] DESCRIPTION OF THE DRAWINGS

[0023] This invention is further understood by reference to the drawings wherein:

[0024] FIG. 1 : Titration of aqueous aluminum chloride (40 mg/mL) in water to pH 6.0-

7.0 with 5 M sodium acetate buffer (pH=9.0). Each point represents the mean ± SD of the resulting pH (n=3). The SDs are too small to be seen in the FIG..

[0025] FIG. 2: Physical appearance of aqueous aluminum chloride adjuvant in sodium acetate buffer under different conditions. Alhydrogel® was used as a reference. Photographs were taken immediately (0 h) or after 1 h, 2 h, 4 h, 24 h and 48 h at 22 °C (A) Freshly prepared adjuvant at 22 °C, (B) Freshly prepared adjuvant stored at 4 °C, and (C) Frozen adjuvant at -20 °C and thawed at 22 °C.

[0026] FIG. 3: A244 gpl20-specific IgG antibody responses in mouse serum induced with different concentrations of aqueous aluminum chloride in PBS (30, 60, 90 pg Al; pH ~2.9 to —3.5), AH in PBS (30 pg Al), and aqueous aluminum sulfate in PBS (30 pg Al). Each bar represents the mean ± SEM of individual serum samples (n=5), analyzed in triplicate. *p<0.05; **p<0.01.

[0027] FIG. 4: A244 gpl20-specific IgG antibody titers at week 12 in mouse serum induced with different concentrations of aqueous aluminum chloride in PBS (30, 60, 90 pg Al), AH in PBS (30 pg Al), and AH in sodium acetate (60 pg Al). Each bar represents the mean ± SEM of individual serum samples (n=5) analyzed in triplicate, ns = not significant.

[0028] FIG. 5: A244 gpl20-specific IgG antibody responses in mouse serum induced with AH in sodium acetate (30 pg Al, first bars in each group) and AH in PBS (30 pg Al, second bars in each group). Each bar represents the mean ± SEM of individual serum samples (n=10 for AH in sodium acetate through week 10; n=5 for all others), analyzed in triplicate.

[0029] FIG. 6: A244 gpl20-specific IgG antibody responses in mouse serum with aqueous aluminum chloride in sodium acetate (60 pg Al, first bars in each group), aqueous aluminum triacetate in sodium acetate (60 pg Al, second bars in each group), and AH in sodium acetate (30 pg Al, third bars in each group). Each bar represents the mean ± SEM of individual serum samples (n=10 through week 10; n=5 for weeks 12- 23), analyzed in triplicate. *p<0.05.

[0030] FIG. 7: A244 gpl20-specific IgG2a antibody responses in mouse serum with aluminum chloride/sodium acetate (60 pg Al, first bars in each group), aluminum triacetate/sodium acetate (60 pg Al, second bars in each group), and AH/sodium acetate (30 pg Al, third bars in each group). Each bar represents the mean ± SEM of individual serum samples (n=10 through week 10; n=5 for weeks 12-23), analyzed in triplicate. *p<0.05.

[0031] FIG. 8: CRM197-specific IgG antibody responses in mouse serum induced with aqueous aluminum chloride in sodium acetate (30 pg Al) and AH in sodium acetate (60 pg Al). Each bar represents the mean ± SEM of individual serum samples (n=5).

[0032] FIG. 9: A table providing the pH ranges and buffer concentrations in which the indicated amounts aluminum chloride, aluminum sulfate, and aluminum triacetate remain in aqueous form.

[0033] FIG. 10, FIG. 11, FIG. 12: UV-visible spectra of particulate Alhydrogel suspension and soluble aluminum chloride and aluminum triacetate. (FIG. 10) Absorbance scans of freshly prepared AH (top curve), aluminum chloride and aluminum triacetate (bottom line, shown in two overlapping traces) in sodium acetate; (FIG. 11) absorbance scans of frozen/thawed AH (top curve), aluminum chloride and aluminum triacetate (bottom line, shown in two overlapping traces) in 5 M sodium acetate; and (FIG. 12) absorbance scans of freshly prepared AH (top curve), aluminum chloride and aluminum triacetate (bottom line, shown in two overlapping traces) in DPBS.

[0034] FIG. 13: Comparison of binding antibody titers in BALB/c mice following immunization with adjuvanted immunogens subjected to freeze-thaw cycles three times. Recombinant hepatitis B surface antigen (rHBsAg) was adsorbed to Alhydrogel (AH) in 100 mM sodium acetate buffer, pH 6.5 or rHBsAg was mixed with aluminum chloride (AlCh) in 100 mM sodium acetate buffer, pH 6.5. Each of the vaccine preparations was freeze-thawed three times and then injected separately into female BALB/c mice (N=6/group). Each mouse received a total dose of 5 pg of the antigen and 150 pg of Al³⁺, which was equally distributed in each hind leg (50 pl/hind leg containing 2.5 pg Ag and 75 pg Al³⁺). Blood was collected at week 4, serum was separated from the clotted blood following centrifugation, and the serum was stored in aliquots at -20 °C until use. Individual serum samples were tested in triplicate for binding antibody titers against rHSBsAg by ELISA. The results are expressed as endpoint titers, defined as the reciprocal dilution that gives an absorbance value that is greater than or equal to twice the background value (antigen-coated wells that did not contain the test sera, but had all other components added). The end point titers are represented as geometric mean + geometric SD. Each dot represents the average value of the triplicate measurements from each animal at the time point indicated. Statistical comparisons between different adjuvant groups at the time point shown were performed using Mann-Whitney test. [0035] FIG. 14: Light microscopy images. Panel A: Recombinant hepatitis B surface antigen (rHBsAg) was adsorbed to Alhydrogel (AH) in 100 mM sodium acetate buffer, pH 6.5 (unfrozen) and then examined under brightfield light Olympus BH2-RFCA microscope equipped with an Olympus DP71 camera with 75X magnification. The picture shows non-aggregated AH. Panel B: Recombinant hepatitis B surface antigen (rHBsAg) was adsorbed to Alhydrogel (AH) in 100 mM sodium acetate buffer, pH 6.5 and freeze-thawed 3X and then examined under brightfield light microscope as described above. The picture shows aggregation of AH because of freeze-thawing. Panels C, D: Recombinant hepatitis B surface antigen (rHBsAg) was mixed with AlCh in 100 mM sodium acetate buffer, pH 6.5 (Panel C), or the mixture was freeze-thawed 3X (Panel D) and then examined under brightfield light microscope as described above. Both pictures show no aggregates.

[0036] FIG. 15: Comparison of rHBsAg-specific IgG antibodies in BALB/c mice following immunization with adjuvanted immunogens subjected to freeze-thaw cycles three times. Recombinant hepatitis B surface antigen (rHBsAg) was adsorbed to Alhydrogel (AH) in 100 mM sodium acetate buffer, pH 6.5 or rHBsAg was mixed with aluminum chloride (AlCh) in 100 mM sodium acetate buffer, pH 6.5. Each of the vaccine preparations was freeze-thawed three times and then injected separately into female BALB/c mice (N=6/group). Each mouse received a total dose of 5 pg of the antigen and 150 pg of Al³⁺, which was equally distributed in each hind leg (50 pl/hind leg containing 2.5 pg Ag and 75 pg Al³⁺). Blood was collected at week 4, serum was separated from the clotted blood following centrifugation, and the serum was stored in aliquots at -20 °C until use. Individual serum samples were tested in triplicate for rHBsAg-specific IgG antibodies by ELISA according to the method of Clapp et al. The results are expressed as rHBsAg-specific IgG antibodies (pg/mL) based on a standard curve. Mouse monoclonal HBsAg IgGl (MyBioSource, San Diego, CA) was used as the positive control. The antigen-specific IgG antibody concentrations are represented as geometric mean + geometric SD. Each dot represents the average value of the triplicate measurements from each animal at the time point indicated. Statistical comparisons between different adjuvant groups at the time point shown were performed using Mann- Whitney test.

[0037] FIG. 16: IgGl Standard curve. Goat anti-rHBsAg coated plates were blocked followed by the addition of rHBsAg and varying amounts of mouse monoclonal HBsAg IgGl antibody (0 to 20 ng/mL). Then goat anti-mouse IgG-HRP was added, color was developed by the addition of HRP substrate. The color development was stopped after 20 min by the addition of 2M sulfuric acid and the optical density was measured at 450 nm. A standard graph was generated in prism. The graph shows a sigmoidal curve. Each dot represents the average of triplicate concentrations + SEM. The 3 sigmoidal lines on the graph represent 3 independent measurements showing the reproducibility of the experiment.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] Disclosed herein are non-particulate aluminum adjuvants and compositions thereof. The non-particulate aluminum adjuvants are solutions of aqueous forms of aluminum salts at a pH of 5.0 to 7.5. Aqueous forms of aluminum chloride, aqueous aluminum sulfate, and aqueous aluminum triacetate are exemplified herein. These “aqueous aluminum adjuvants” elicit immune responses that are at least equivalent to that obtained with Alhydrogel® (AH), a commercially available aluminum particulate adjuvant (hereafter “particulate adjuvant”), which comprises insoluble aluminum hydroxide particles in suspension. AH was chosen as an exemplary particulate adjuvant because of its widespread commercial use. In some embodiments, the aqueous aluminum adjuvants and compositions thereof contain not more than 1% of their total aluminum content as water insoluble particles.

[0040] The results herein indicate that solutions of aluminum salts in aqueous form exhibit substantially similar levels of adjuvanticity compared to particulate adjuvants, the aluminum salts remain in aqueous form over a pH range of 6.0-7.5 when buffered, and the aqueous aluminum adjuvants may be frozen without loss of adjuvanticity. As disclosed herein, the aluminum salts remain in aqueous form at pH values suitable for administration via injection in the presence of a buffer. The aqueous aluminum adjuvants produced adjuvanticity comparable to AH when tested with two proteins as model antigens: HIV-1 A244 gpl20 or CRM197. Aqueous aluminum chloride buffered with sodium acetate remained soluble after being frozen at -20 °C and thawed in a water bath at room temperature (22 °C). That is, no visible aluminum chloride solute was observed thereby indicating the salt was in aqueous form. Unlike particulate adjuvants, after freezing, the thawed aqueous aluminum adjuvants retained their adjuvanticity.

[0041] Solubility of Aqueous Aluminum Adjuvants

[0042] The solubilities of the aqueous aluminum adjuvants were readily characterized by visual inspection, using Alhydrogel® as a positive control. As shown in FIG. 2, 1.5 mg Al/mL aluminum chloride in 5 M sodium acetate buffer remained visually soluble as a function of time at various ranges of different storage temperatures. Aluminum chloride in sodium acetate maintained its solubility for 4 h at room temperature (RT). At 4 °C, there were no visible particulates in solution for >10 weeks (shown in FIG. 1 are photographs up to 48 h). That is, the aluminum salts remained in aqueous form. Further, freezing the solution for 24 h at -20 °C did not result in the formation of particulates during re-solubilization, thereby suggesting that freezing had no effect on the solubility of aluminum chloride in 5 M sodium acetate.

[0043] The solubility of aluminum chloride or aluminum triacetate in sodium acetate buffer was further characterized using UV-visible scattering experiments. Alhydrogel in sodium acetate exhibited absorbance/scattering from 300 nm to 700 nm, while there was no absorbance/scattering observed in the freshly prepared and freeze-thawed aluminum chloride and aluminum triacetate (FIG. 10, FIG. 11). Since aluminum phosphate in aqueous systems is particulate in nature at pH ~3.0 to ~7.5, this observation further confirmed the solubility and the absence of particulate aluminum chloride and aluminum triacetate in sodium acetate buffer. A similar observation was found for aluminum chloride in DPBS (FIG. 12), where there was no absorbance/scattering observed from 300 nm to 700 nm, suggesting the absence of particulate aluminum phosphate salt in DPBS.

[0044] Immune Responses Induced with Aqueous Aluminum Adjuvants

[0045] The adjuvant activities of aqueous aluminum chloride (30, 60, and 90 pg Al), and aqueous aluminum sulfate (60 pg Al), in comparison to AH (30 pg Al) in PBS, with A244 gpl20 as an antigen were investigated in mice. As shown in FIG. 3, immunization with A244 gpl20 adjuvanted with aqueous aluminum chloride, aqueous aluminum sulfate, or AH resulted in variable induction of immune responses to A244 gpl20. Formulation with 60 pg Al in aqueous aluminum chloride mounted higher anti-A244 gpl20 endpoint titers compared to those with 30 pg or 90 pg Al, particularly at weeks 10 and 12. Starting at week 8 (2 weeks after the third immunization), anti-A244 gpl20 endpoint titers elicited with both aqueous aluminum chloride in PBS and AH in PBS were essentially equivalent. The immune responses induced with the aqueous aluminum sulfate were consistently inferior at weeks 5 to 12, with endpoint titers significantly lower compared to those of aqueous aluminum chloride (60 pg Al) and AH (FIG. 3).

[0046] As depicted in FIG. 4, at week 12, the anti-A244 gpl20 endpoint titers induced with AH (30 pg Al and 60 pg Al) and aqueous aluminum chloride (60 pg Al) were statistically equivalent. Endpoint titers elicited by aqueous aluminum chloride (60 pg Al) were equivalent to those induced by AH. [0047] Immune Responses Induced with Aqueous Aluminum Chloride and AH in Sodium Acetate Buffer

[0048] In water, aluminum chloride (40 mg/mL) is strongly acidic with a pH of about 2.9. As shown in FIG. 1, the acidity of aqueous aluminum chloride was neutralized by 5 M sodium acetate, raising the pH from about 2.9 to about 7.0, without any visual precipitate formation. In the subsequent experiments using aqueous aluminum salts buffered with sodium acetate, a pH range of 6.0-7.0 was maintained. As shown in FIG. 5, the anti-A244 gpl20 endpoint titers induced with AH in sodium acetate are statistically similar to those in PBS, throughout the duration of the study. That is, sodium acetate buffer as compared to PBS does not significantly impact the elicited antibody response over time.

[0049] In sodium acetate buffer, aqueous aluminum chloride can potentially react with acetate ions forming aluminum triacetate via a chloride ligand exchange reaction. Because of this, aqueous aluminum triacetate as an adjuvant was investigated and compared to the adjuvant activity of aqueous aluminum chloride and AH in sodium acetate buffer. Aqueous aluminum triacetate was also soluble in sodium acetate buffer at pH ~6.5. As shown in FIG. 6, the antibody endpoint titers to A244 gpl20 induced with aqueous aluminum triacetate were statistically similar to those of aqueous aluminum chloride, except for weeks 14, 22, and 23, and were also statistically comparable to those of AH, except for weeks 4, 10, and 20. Particularly, the formulations adjuvanted with soluble aluminum chloride and aluminum acetate exhibited IgG2a endpoint titers that were not statistically different than those obtained with particulate Alhydrogel (FIG. 7).

[0050] The adjuvant activity of aqueous aluminum chloride in sodium acetate was also tested with CRM197 as an antigen in mice. As shown in FIG. 8, immunization with CRM197 adjuvanted with aqueous aluminum chloride in sodium acetate resulted in the induction of anti-CRM197 antibodies with endpoint titers that are statistically equivalent to those of AH.

[0051] ADVANTAGEOUS EFFECTS

[0052] The fact that aqueous aluminum salts are capable of enhancing an immune response against a given antigen to about the same degree as particulate adjuvants is surprising because it is believed that the aluminum particulates enhance immune responses by way of acting as “carriers” and presenting the given antigens to immune cells. This mechanism of action is supported by the fact that freezing aluminum particulate adjuvants irreversibly destroys their adj uvanti city. Freezing causes significant structural and chemical damage to the particulates and thereby detrimentally impacts the adsorption of antigens thereon as evidenced by the results shown in FIG. 13 and FIG. 15. Specifically, the data of FIG. 13 and FIG. 15 show that the antibody titers induced by aluminum particulate adjuvants that were frozen are significantly lower than those induced by aluminum particulate formulations that were not frozen and both frozen and non-frozen aqueous aluminum adjuvant formulations. Particularly, the data of FIG. 13 and FIG. 15 show that: there is no significant difference between the immunogenicity of the non-frozen particulate adjuvant formulation (Ag + AH) and the non-frozen aqueous adjuvant formulation (Ag + AlCh); there is a significant loss of immunogenicity when the particulate adjuvant formulation is frozen; and there is no significant difference in immunogenicity between the frozen and non-frozen aqueous adjuvant formulations. That is, vaccines formulated with aqueous aluminum adjuvants as described herein may be frozen and thawed multiple times without any significant loss of immunogenicity.

[0053] The aqueous aluminum adjuvants described herein are advantageous over particulate adjuvants because:

• The aqueous aluminum adjuvants may be frozen;

• The aqueous aluminum adjuvants may be autoclaved;

• The aqueous aluminum adjuvants may be stored at room temperature;

• The aqueous aluminum adjuvants may be sterile-filtered;

• The aqueous aluminum adjuvants may be used with any antigen because there is no need for adsorption on particulates, and therefore there are no surface charge limitations and the antigens may be provided in their native soluble form without adverse conformational changes resulting from adsorption on a particle; and

• The concentration/dose of antigen can be accurately provided and determined.

[0054] Compositions

[0055] Generally, (unbuffered) solutions of aluminum salts such as aluminum chloride, aluminum triacetate, and aluminum sulfate are acidic and precipitate out of solution at pH values of about 6.0 and higher, in hydroxide bases. However, as exemplified in the detailed examples, buffers such as sodium acetate maintain aluminum salts in aqueous form over a pH range of about 6.0-7.5. Thus, aqueous aluminum adjuvants according to the present invention comprise, consist essentially of, or consist of an aluminum salt in aqueous form and a buffer. Suitable buffers include sodium acetate and tromethamine base. [0056] FIG. 9 is a table showing the concentrations of various buffers that result in solutions having the indicated pH values wherein the given concentrations of aluminum chloride, aluminum triacetate, and aluminum sulfate each remain in aqueous form. The given concentrations of the aluminum salts are the maximum concentrations that remain in aqueous form at the indicated pH and indicated concentration of the given buffer at room temperature. For example, at a pH of 7.0 and about 139.4 mg/mL sodium acetate, up to 1.5 mg/mL remains in aqueous form at room temperature. Thus, aqueous aluminum chloride adjuvants comprising about 139.4 mg/mL of sodium acetate and a pH of about 7.0 may contain up to about 1.5 mg/mL of aqueous aluminum chloride at room temperature (z.e., about 20-25 °C). The aqueous aluminum adjuvants and compositions thereof may contain water soluble aluminum salt solute in addition to the aqueous form of the aluminum salt. In some embodiments, the aqueous aluminum adjuvants and compositions thereof contain not more than 1% of their total aluminum content as water soluble aluminum salt solute when at room temperature.

[0057] Therefore, in some embodiments, aqueous aluminum adjuvants and compositions thereof comprise, consist essentially of, or consist of:

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 1.0-25.0 mg/mL sodium acetate at a pH of about 5.0-6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 1.0-4.0 mg/mL sodium acetate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 12.9-25.0 mg/mL sodium acetate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 1.0-220.6 mg/mL sodium acetate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 1.0-139.4 mg/mL sodium acetate at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 12.9-139.4 mg/mL sodium acetate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 76.0-139.4 mg/mL sodium acetate at a pH of about 7.0. • A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 158.0-220.6 mg/mL sodium acetate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 1.0-220.6 mg/mL sodium acetate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 12.9-

139.4 mg/mL sodium acetate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 1.0-4.0 mg/mL sodium acetate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 12.9-25.0 mg/mL sodium acetate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 76.0-

139.4 mg/mL sodium acetate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 158.0— 220.6 mg/mL sodium acetate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 1.0-23.2 mg/mL sodium acetate at a pH of about 5.0-6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 1.0-4.0 mg/mL sodium acetate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 9.0-23.2 mg/mL sodium acetate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.4-

214.4 mg/mL sodium acetate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 8.4- 116.0 mg/mL sodium acetate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.4-2.5 mg/mL sodium acetate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 8.4-18.0 mg/mL sodium acetate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 115.0—

214.4 mg/mL sodium acetate at a pH of about 7.0.

214.4 mg/mL sodium acetate at a pH of about 7.5. • A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.3-0.9 mg/mL tromethamine at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.3-0.9 mg/mL tromethamine at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.3-0.8 mg/mL tromethamine at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.3-0.9 mg/mL tromethamine at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.4-0.9 mg/mL tromethamine at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.9 mg/mL tromethamine at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.9 mg/mL tromethamine at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.8 mg/mL tromethamine at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.9 mg/mL tromethamine at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.4-0.9 mg/mL tromethamine at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.2-1.0 mg/mL tromethamine at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.2-0.9 mg/mL tromethamine at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.2-0.8 mg/mL tromethamine at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.2-0.8 mg/mL tromethamine at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.3-0.9 mg/mL tromethamine at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.4-1.0 mg/mL tromethamine at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 0.9-442.1 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.5. • A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 0.9-177.9 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 2.3-177.9 mg/mL sodium citrate dihydrate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 0.9 - 2.9 mg/mL sodium citrate dihydrate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 2.3 - 7.3 mg/mL sodium citrate dihydrate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 50.5 - 177.9 mg/mL sodium citrate dihydrate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 166.3 - 442.1 mg/mL sodium citrate dihydrate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 0.9-442.1 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 2.3-177.9 mg/mL sodium citrate dihydrate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 0.9-2.9 mg/mL sodium citrate dihydrate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 2.3-7.3 mg/mL sodium citrate dihydrate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 50.5- 177.9 mg/mL sodium citrate dihydrate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 166.3— 442.1 mg/mL sodium citrate dihydrate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.9-284.0 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 1.5-101.5 mg/mL sodium citrate dihydrate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.9-3.5 mg/mL sodium citrate dihydrate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 1.5-5.0 mg/mL sodium citrate dihydrate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 29.3-101.5 mg/mL sodium citrate dihydrate at a pH of about 7.0. • A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 85.5-284.0 mg/mL sodium citrate dihydrate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.6- 273.3 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.9-62.1 mg/mL sodium citrate dihydrate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.6-2.1 mg/mL sodium citrate dihydrate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 0.9-4.7 mg/mL sodium citrate dihydrate at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 28.8- 62.1 mg/mL sodium citrate dihydrate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 77.4- 273.3 mg/mL sodium citrate dihydrate at a pH of about 7.5.

[0058] The aqueous aluminum adjuvants and compositions thereof exclude amounts of compounds that will react with the given aluminum salt and form insoluble precipitate. Those skilled in the art of chemistry can readily determine which compounds and amounts will react with the aluminum ions and form insoluble precipitates. In some embodiments, solutions of the aqueous aluminum adjuvants and compositions thereof exclude hydroxide salts, alkaline salts, and carbonate salts.

[0059] Compositions comprising aqueous aluminum adjuvants may include an active agent, e.g., a given antigen of interest. The compositions may further include additional ingredients such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, for pharmaceutical administration, so long as the additional ingredients do not cause the aluminum salts to form insoluble precipitate. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th ed (2000) Lippincott Williams & Wilkins, Baltimore, MD.

[0060] The total amount of aluminum in the compositions may be selected using methods in the art, e.g., using the standardized aluminum adjuvant dose guidelines promulgated by Vecchi et al. (2012) “Aluminum adjuvant dose guidelines in vaccine formulation for preclinical evaluations” J Pharm Sci 101(1): 17-20) and applicable regulatory agency, e.g., U.S. Food and Drug Administration guidelines.

[0061] Methods of Making and Using

[0062] Contemplated herein are methods of making aqueous aluminum adjuvants and compositions thereof, e.g., vaccines.

[0063] As an example, 700 pl of a vaccine having a pH of 6.0 to 7.5 and 6 mg of Al per 1 mg/mL of a given antigen in 5 M sodium acetate may be prepared by adding 140 pl of the given antigen (e.g., 200 pg to 1 mg per mL) to 560 pl of an aqueous aluminum salt in 5M sodium acetate (1.5 mg Ah⁺/mL) and mixing. The vaccine may be frozen or stored at 4 °C. These amounts and volumes may be scaled up or down as desired. The methods exclude the addition of amounts of compounds that will react with the given aluminum salt and form insoluble precipitate. Again, those skilled in the art of chemistry can readily determine which compounds and amounts will react with the aluminum ions and form insoluble precipitates. In some embodiments, the methods exclude the addition of hydroxide salts, alkaline salts, and carbonate salts.

[0064] The aqueous aluminum adjuvants may be stored at temperatures ranging from - 70-10 °C for any desired time period, frozen, sterile-filtered, and/or autoclaved before being compounded with a given antigen of interest without any significant loss of their adj uvanti city. Vaccine compositions comprising the aqueous aluminum adjuvants and a given antigen of interest may be stored at temperatures ranging from -70-10 °C for any desired time period and/or frozen (and then thawed) before immunizing a subject therewith without any significant loss of immunogenicity as compared to freshly prepared vaccine compositions.

[0065] The following examples are intended to illustrate but not to limit the invention.

[0066] EXAMPLES

[0067] Materials and Reagents

[0068] Aluminum chloride hexahydrate (A1C13*6H2O, catalog number A0718), aluminum sulfate octahydrate (A12(SO4)3*8H2O), catalog number 1017364) and sodium acetate (NaCOOCHi, catalog number S5636) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Aluminum triacetate, also known as aluminum acetate (A1(COOCH3)3, catalog number NCZ-AE- 104/21) was purchased from NanoChemazone (Leduc, AB, Canada).

[0069] Antigens A244 gpl20 and CRM 197 were purchased from Immune Technology, Inc. (New York, NY, USA) and Fina Biosolutions, LLC (Rockville, MD, USA), respectively. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Origene (Rockville, MD, USA). HRP-conjugated goat anti-mouse IgG2a and mouse IgG2a antibody were purchased from Southern Biotech. The KPL ABTS Peroxidase 2-component substrate system was purchased from SeraCare (Gaithersburg, MD, USA). Immulon® 96-well U-bottom plates and NUNC Maxisorp™ 96-well flat-bottom plates were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium, 10% sodium dodecyl sulfate (SDS) and 10X phosphate-buffered saline were purchased from Quality Biological, Inc. (Gaithersburg, MD, USA). Tween® 20 was purchased from Millipore Sigma (St. Louis, MO, USA). Powdered nonfat milk was purchased from a local grocery store. Alhydrogel® (aluminum hydroxide gel) was purchased from Brenntag, Inc. (Reading, PA, USA) and Croda, Inc. (Princeton, NJ, USA). Sterile filtration was conducted with a polyvinylidene difluoride (PVDF) syringe (VWR, #76478-992, Bridgeport, NJ, USA). Stock solutions were prepared in ultrapure water purchased from KD Medical (Columbia, MD, USA). pH measurements were performed using a Thermo Scientific Orion VersaStar Pro pH meter equipped with an Orion Ross Microprobe (Waltham, MA, USA).

[0070] Preparation of Adjuvants

[0071] Aqueous Aluminum Chloride Adjuvant

[0072] A fresh stock solution of 40 mg/mL aqueous aluminum chloride (4.47 mg Al/mL) was prepared in ultrapure water or DPBS. From this stock solution, 1.125 mL was added to 2.235 mL 5 M sodium acetate buffer or DPBS to obtain two aqueous aluminum chloride adjuvants: 1.5 mg/mL aqueous aluminum chloride buffered with sodium acetate at pH ~6.5 to ~7.0, and 1.5 mg/mL aqueous aluminum chloride buffered with DPBS at pH ~2.9 to ~3.5. The aqueous adjuvant solutions were sterile-filtered through a 0.22 pm PVDF syringe filter in a biosafety cabinet.

[0073] Aqueous Aluminum Triacetate Adjuvant

[0074] A fresh stock solution of 33.9 mg/mL aqueous aluminum triacetate (4.48 mg Al/mL) was prepared by dissolving aluminum triacetate powder in ultrapure water. From this stock solution, 1.125 mL was added to 2.235 mL 5 M sodium acetate buffer to obtain a final concentration of 1.5 mg/mL aqueous aluminum triacetate buffered with sodium acetate at pH ~6.5 to ~7.0. Similarly, 0.56 mL of the aluminum triacetate stock solution and 0.56 mL of ultrapure water were added to 2.235 mL of 5 M sodium acetate buffer to obtain a 0.75 mg/mL final concentration of aqueous aluminum triacetate. The aqueous adjuvant solutions were sterile-filtered through 0.22 pm PVDF syringe filters in a biosafety cabinet.

[0075] Particulate Adjuvant - Alhydrogel® (Aluminum Hydroxide Gel; AH)

[0076] An aliquot of 0.182 mL of stock solution (10.3 mg Al/mL) of AH was added to 2.318 mL of 0.1 M sodium acetate buffer (pH —6.5) or DPBS (pH —7.4) to obtain a concentration of 0.75 mg/mL Al (resulting in pHs of ~6.6 and ~7.5, respectively. The preparation was made in a biosafety cabinet. Because AH is a particulate adjuvant, preparations comprising AH cannot be sterile-filtered.

[0077] Adjustment of pH of Aqueous Aluminum Chloride Adjuvant

[0078] A solution of 40 mg/mL aqueous aluminum chloride (4.47 mg Al/mL) in ultrapure water with a starting pH of ~2.9 was titrated against 5 M sodium acetate (pH —9.0). An incremental volume of 50, 100, 200, or 500 pL of 5 M sodium acetate was added to the aqueous aluminum chloride solution and stirred for about 1 minute, and the pH was recorded using a calibrated pH meter. A pH range of 6.0-7.0 was obtained after the addition of a total volume of 2-8 mL of 5 M sodium acetate. The titration curve for the adjustment of the pH of the aqueous aluminum chloride solution with the 5 M sodium acetate is presented in FIG. 1.

[0079] Physical Characterization of Adjuvant Compositions

[0080] The changes in the physical appearance of the aqueous aluminum chloride in 5 M sodium acetate (“aluminum chloride/sodium acetate”) after different storage conditions were investigated as a function of time. Aluminum chloride/sodium acetate stored at 22 °C was observed at 0 h, 1 h, and 4 h; aluminum chloride/sodium acetate stored at 4 °C was observed at 0 h, 24 h, and 48 h; aluminum chloride/sodium acetate frozen at -20 °C was slowly thawed at 22 °C and observed at 0 h, 2 h, 4 h, and 24 h. Photographs were taken at every time point using a Canon PowerShot G1 X Mark II camera (Ota, Tokyo, Japan). To minimize the ambient reflective light from the outside surfaces of the tubes in FIG. 2, the brightnesses of all the composite images were adjusted together to improve the transparent image clarity. [0081] UV-Visible Scattering Measurement

[0082] An amount of 1.125 mL of 40 mg/mL aluminum chloride (4.47 mg Al/mL) or 33.9 mg/mL aluminum triacetate (4.48 mg Al/mL) was added to 2.235 mL of 5 M sodium acetate buffer to obtain a final concentration of 1.5 mg/mL Al for each aluminum solution. A separate suspension of 0.75 mg Al/mL Alhydrogel was prepared by adding 0.15 mL of 10.3 mg Al/mL Alhydrogel to 1.85 mL of 5 M sodium acetate. The 1 mL aliquots of each final solution (1.5 mg/mL Al) were stored at -20 °C overnight. An amount of 200 pL of each remaining solution or suspension was transferred to a 96-well Corning UV-transparent flat-bottom plate. A negative control consisted of 5 M sodium acetate. The absorbance scan from 300 nm to 700 nm was then collected on a BioTek Epoch2 Microplate Reader (Agilent, Santa Clara, CA, USA). On the following day, the frozen solutions were slowly thawed and equilibrated to RT, and their absorbances were measured in a similar manner as described above. The absorbance profiles of each freshly prepared 1.5 mg/mL Al in aluminum chloride and Alhydrogel in DPBS were also measured as described above.

[0083] Adsorption Capacity Measurement

[0084] The antigen adsorption capacities of the Alhydrogel in PBS and sodium acetate were indirectly determined from the protein antigen measured in the supernatant by BCA assay after centrifugation. The sample was prepared by adding 140 pL of A244 gpl20 (1 mg/mL) to 560 pL AH (0.75 mg Al/mL) in 100 mM sodium acetate or DPBS in sterile 1.5 mL microcentrifuge tubes. The formulations were mixed in a roller for 15 min, followed by incubation at 4 °C for 1 h. The samples were then centrifuged at 21,130 *g for 10 min, and the supernatants were recovered carefully.

[0085] The amount of protein antigen in the supernatant was determined by BCA assay as per the manufacturer’s instruction (PierceTM, Rockford, IL, USA, Thermo Scientific). Briefly, a series of BSA calibration standards in the range of 25-1000 mg/mL in DPBS was prepared. A working reagent (WR) was then prepared by adding 50 parts of Pierce BCA Protein Assay Reagent A to 1 part of Pierce BCA Protein Assay Reagent B. 25 pL of each calibration standard was used in triplicate, and the samples were pipetted into the 96-well plate. An amount of 200 pL of WR was then added to each well, and the plate was placed in the shaker for 30 sec. The plate was then covered with optical adhesive film and incubated at 37 °C for 30 min. The absorbance was measured at 562 nm. The protein antigen concentrations in the samples were interpolated from the regression analysis of the calibration standards. The percent (%) adsorption was determined from the equation below. The A244 gpl20 adsorption capacities for Alhydrogel in DPBS and in sodium acetate were 96.26 ± 0.64% and 100%, respectively.

% Adsorption

[0086] Vaccine Formulation

[0087] Vaccines were prepared by mixing appropriate amounts of antigen and adjuvant to generate formulations containing 10 pg of HIV-1 A244 gpl20 or CRM197 and 30, 60, or 90 pg of Al in 50 pL immunizations. Briefly, 140 pL of A244 gpl20 (1 mg/mL) or CRM197 (1 mg/mL) was mixed with 560 pL aqueous aluminum chloride (1.5 mg/mL of Al) or AH (1.5 mg/mL of Al) in 5 M sodium acetate or DPBS, in a sterile microfuge tube. The vaccine formulations were further mixed by shaking on a roller for 15 min, followed by incubation at 4 °C for 1 h prior to immunization. The final pH values of all vaccine formulations were in the range of 6.0 to 7.4.

[0088] Immunization Studies

[0089] Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Walter Reed Army Institute of Research [Assurance number DI 6-00596 (A4117-01)]. Blood collection was performed as per WRAIR/NMRC VSP Guidelines on blood collection. Mice were euthanized using CO2 administered in a CO2 chamber, via regulated flow valve (in accordance with the most current 2020 AVMA Guidelines on Euthanasia), followed by cervical dislocation at the various time points mentioned in the study to collect spleen, lymph nodes, and bone marrow. Following euthanasia, mice were exsanguinated by cardiac puncture. Sample collection and processing are described below.

[0090] Female BALB/c mice (5-6 weeks of age) were obtained from The Jackson Laboratory. Animals were housed in groups and fed standard chow diets. Mice (N = 5 or 10) were immunized intramuscularly in the hind leg quadriceps. All immunizations contained 10 pg of HIV- 1 A244 gpl20 or CRM 197, with the given adjuvant (z.e., aqueous aluminum chloride in sodium acetate buffer, aqueous aluminum chloride in DPBS, aqueous aluminum triacetate in 5 M sodium acetate buffer, aqueous aluminum sulfate in DPBS, AH in 5 M sodium acetate buffer, or AH in PBS, with the given amount of aluminum (z.e., 30, 60, or 90 pg Al) in a total volume of 50 pL). Mice were immunized at weeks 0, 3, and 6. At week 10, half of each group was euthanized, and relevant tissues were harvested from these mice for further analysis. Mice were bled at 2-week intervals for 20 or 23 weeks. Blood samples were centrifuged at 14,000 x g for 5 min at 4 °C. Serum was collected and stored at -25 °C until use. Blood was also collected before each immunization.

[0091] ELISA assays were performed using methods in the art. Briefly, 96-well Immulon® high-binding U-bottom plates were coated with 100 pL of either HIV-1 A244 gpl20 protein (1 pg/mL) or CRM197 (1 pg/mL) in DPBS (pH 7.4) and stored overnight at 4 °C. The plates were then blocked with 250 pL of blocking buffer, PBS containing 0.5% milk and 0.1% Tween® 20 at room temperature (RT) for 2 h. Individual serum samples were diluted in blocking buffer to an appropriate starting dilution and added to the first row of the 96-well plates in triplicates, where they were serially diluted two-fold in the blocking buffer in each row of the plates. The plates were then incubated at RT for 1 h, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse secondary IgG (1 :40,000 dilution in blocking buffer) and incubation at RT for 1 h. Color was developed by addition of peroxidase substrate components A and B and incubation in reduced lighting at RT for 1 h. The reaction was stopped by addition of 100 pL per well of 1% sodium dodecyl sulfate, and the absorbance was measured within 30 min at 405 nm in a Spectramax M2 microplate reader (Molecular Devices, San Jose, CA). The results are expressed as endpoint titers defined as the reciprocal dilution that gives an absorbance value that is greater than or equal to twice the background value (in an antigen-coated well that did not contain the serum but had all the other components added). Antibody-positive (anti-A244 gpl20 or anti-CRM197 seropositive samples from other studies) and antibody-negative controls were included on each plate.

[0092] Subclass IgG2a ELISAs were performed in a similar manner as described above for the antigen-specific IgG ELISA, with few modifications. This was performed on NUNC Maxisorp™ 96-well flat-bottom plates. An HRP-conjugated goat anti-mouse IgG2a secondary IgG (1 : 1000 dilution in blocking buffer) was used. Mouse IgG2a standard was utilized as a positive control.

[0093] Statistical comparison between multiple groups, was performed using an ordinary one-way ANOVA with Tukey’s multiple comparison test. Statistical comparison between 2 groups, was performed using unpaired t-test with Welch’s correction. Differences among values are statistically significant if p < 0.05. [0094] Freeze-Thaw Activity Assays

[0095] The following assays were performed to determine the effect freezing has on the immunogenicity of vaccines adjuvanted with aluminum particulate adjuvants compared to aqueous aluminum adjuvants.

[0096] Materials: Aqueous Al adjuvant = aluminum chloride diluted in 5M sodium acetate; Stock preparation = 3.0 Ah⁺ mg/mL: 5.37 mL aluminum chloride Hexahydrate in water (40 mg AlCh/mL = 4.47 mg Al/mL) + 2.63 mL 5 M sodium acetate in water; filtered with 0.22 um PVDF syringe filter. Particulate Al adjuvant = Alhydrogel; Stock preparation = Alhydrogel (Croda, 10.0 mg/mL Al) diluted to a final formulation concentration of 1.5 mg/mL Al in 100 mM sodium acetate. Antigen: rHBsAg (Saccharomyces cerevisiae-derived recombinant hepatitis B surface antigen (rHBsAg), subtype adw (purity >98%) purchased from Advanced Immunochemical Services, Inc. (Long Beach, CA). Buffers: 100 mM sodium acetate, pH 6.5 (10 mL 100 mM sodium acetate (pH 7.88) + 20 pL 0.5 M Acetic Acid (pH 2.37) filtered with 0.22 pm PVDF syringe filter); and 5 M Sodium Acetate, pH 9.0. All reagents were equilibrated to room temperature before use.

[0097] Female BALB/c mice (5-6 weeks of age) were obtained from The Jackson Laboratory and divided into 4 formulation treatment groups:

- Ag+AH (positive control for Al particulate adjuvant): AH in 100 mM sodium acetate mixed with rHBsAg for 15 min and then incubated at 4 °C for 1 h;

- Ag+AH Freeze-thawed 3x: AH in 100 mM sodium acetate mixed with rHBsAg for 15 min then subjected to 3 freeze-thaw cycles, each cycle comprising freezing at -20 °C for 24 h then thawing for about 4 h at 4 °C;

- Ag+AlCh: Aqueous AlCh in 5 M sodium acetate mixed with rHBsAg for 15 min and then incubated at 4 °C for 1 h; and

- Ag+AlCh Freeze-thawed 3x: Aqueous AlCh in 5 M sodium acetate mixed with rHBsAg for 15 min then subjected to 3 freeze-thaw cycles, each cycle comprising freezing at -20 °C for 24 h then thawing for about 4 h at 4 °C.

[0098] 100 pl of the given formulation was administered by i.m. injection, i.e., 50 pL in each hind leg to result in 2.5 pg antigen and 75 pg Al per leg. Animals were housed in groups and fed standard chow diets.

[0099] The ELISA procedure for the determination of antigen-specific IgG antibodies as described by Clapp et al. was used. Each well of a 96-well Immulon-IB high-binding polystyrene plate was coated with 100 pL of 5 pg/mL goat anti-rHBsAg in sodium bicarbonate/carbonate buffer (100 mM and pH 9.3) overnight at 4 °C. Plates were washed three times with 8 mM Na2HPO4, 150 mM NaCl, 2 mM KH2PO4, 3 mM KC1, 0.05% Tween® 20, pH 7.4 (PBS-T) using a plate washer. The plate was then blocked with a solution of 50 mM Tris, 100 mM NaCl, and 1% BSA (TNN) for 30 min, rinsed 3x with PBS-T, followed by the addition of 100 pL of 1.67 pg/mL rHBsAg in TNN. The plate was rinsed with PBS-T and either serial 2-fold dilutions of test sera or varying amounts of mouse monoclonal HBsAg IgGl (MyBioSource, San Diego, CA) antibody in TNN were added to generate the standard curve. The mouse monoclonal antibody also served as the positive control. Plates were washed with PBS- T, and then goat antimouse IgG (gamma chain specific) conjugated to horseradish peroxidase (HRP) at a dilution of 1 : 1000 in 20 mM Tris, 150 mM NaCl and 1% BSA (TBS-T) was added to each well. After 1 h incubation at room temperature, the plate was washed 5X with PBS-T, and 1 : 1 mix of HRP substrate TMB (3,3,5,5-tetramethylbenzidine) (1-step Ultra TMB- Substrate) was added to all wells. After 20 min of color development, the reaction was stopped by the addition of 2 M H2SO4, and the optical density was measured at 450 nm. A standard graph was generated (FIG. 16) and the amount of rHBsAg-specific IgG antibodies in the serum was determined from the standard graph.

[0100] REFERENCES

[0101] The following references are herein incorporated by reference in their entirety with the exception that, should the scope and meaning of a term conflict with a definition explicitly set forth herein, the definition explicitly set forth herein controls:

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16. Beck, et al. Immune response to antigen adsorbed to aluminum hydroxide particles: Effects of co-adsorption of ALF or ALFQ adjuvant to the aluminum-antigen complex. J Control Release 2018, 275, 12-19.

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[0102] ADDITIONAL EMBODIMENTS

[0103] Embodiment Al : An adjuvant composition comprising or consisting essentially of an aqueous aluminum salt, a sodium acetate buffer, and a pH of about 5.0-7.5, preferably about 6.0-7.5 or about 6.0-7.0.

[0104] Embodiment A2: The adjuvant composition according to Embodiment Al, wherein the aqueous aluminum salt is aqueous aluminum chloride, aqueous aluminum sulfate, or aqueous aluminum triacetate. [0105] Embodiment A3 : The adjuvant composition according to Embodiment Al or Embodiment A2, wherein the adjuvant composition comprises 0.4-220.6 mg/mL sodium acetate.

[0106] Embodiment A4: The adjuvant composition according to any one of Embodiments Al to A4, wherein the concentration of the aqueous aluminum salt is about 0.5-1.5 mg/mL.

[0107] Embodiment A5: The adjuvant composition according to any one of Embodiments Al to A5, wherein the adjuvant composition excludes hydroxide salts, alkaline salts, and carbonate salts.

[0108] Embodiment A6: A composition comprising or consisting essentially of the adjuvant composition according to any one of Embodiments Al to A5 and a given antigen of interest.

[0109] Embodiment A7: The composition according to Embodiment A6, wherein the composition lacks insoluble particles containing aluminum.

[0110] Embodiment A8: The composition according to Embodiment A6 or Embodiment A7, wherein the composition excludes hydroxide salts, alkaline salts, and carbonate salts.

[0111] Embodiment A9: A method of making the adjuvant composition according to any one of Embodiments Al to A5, which comprises titrating the aqueous aluminum salt with the sodium acetate buffer to obtain a solution having a pH of about 5.0-7.5, preferably about 6.0-7.5 or about 6.0-7.0.

[0112] Embodiment A10: A method of making the composition according to any one of Embodiments A6 to A 8, which comprises adding the given antigen of interest to the adjuvant composition according to any one of Embodiments Al to A5.

[0113] Embodiment Al 1 : The method according to Embodiment A9 or Embodiment A10, which further comprises maintaining the pH in the range of about 5.0-7.5, preferably about 6.0-7.5 or about 6.0-7.0.

[0114] Embodiment A12: A method of immunizing a subject, which comprises injecting the subject with the composition according to any one of Embodiments A6 to A8, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum.

[0115] Embodiment A13 : A method of enhancing an immune response in a subject, which comprises injecting the subject with the composition according to any one of Embodiments A6 to A8, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum, and wherein the immune response is greater than that obtained with a control composition containing the given antigen of interest without any aluminum.

[0116] Embodiment A14: The method according to Embodiment A12 or Embodiment A13, which comprises (a) freezing the composition and thawing the composition, and/or (b) storing the composition at a temperature of -70-10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc., before injecting the subject with the composition.

[0117] Embodiment A15: A method of storing the composition according to any one of Embodiments A6 to A8, which comprises maintaining the composition at a temperature of -70-10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc.

[0118] In Embodiments Al to Al 5, the adjuvant compositions and compositions thereof may comprise or consist essentially of:

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 76.0-139.4 mg/mL sodium acetate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride or aqueous aluminum triacetate and about 158.0-220.6 mg/mL sodium acetate at a pH of about 7.5. • A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 1.0-220.6 mg/mL sodium acetate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 12.9-

139.4 mg/mL sodium acetate at a pH of about 6.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 76.0-

139.4 mg/mL sodium acetate at a pH of about 7.0.

214.4 mg/mL sodium acetate at a pH of about 5.0-7.5.

214.4 mg/mL sodium acetate at a pH of about 7.0.

214.4 mg/mL sodium acetate at a pH of about 7.5.

[0119] Embodiment Bl : An adjuvant composition comprising or consisting essentially of an aqueous aluminum salt, a buffer, and a pH of about 5.0-7.5, preferably about 6.0- 7.5 or about 6.0-7.0, wherein said buffer is a tromethamine buffer (e.g., TRIS base) or sodium citrate dihydrate. [0120] Embodiment B2: The adjuvant composition according to Embodiment Bl, wherein the aqueous aluminum salt is aqueous aluminum chloride, aqueous aluminum sulfate, or aqueous aluminum triacetate.

[0121] Embodiment B3: The adjuvant composition according to Embodiment Bl or Embodiment B2, wherein the adjuvant composition comprises 0.2-1.0 of tromethamine or 0.6-442.1 mg/mL sodium citrate dihydrate.

[0122] Embodiment B4: The adjuvant composition according to any one of Embodiments B 1 to B7, wherein the concentration of the aqueous aluminum salt is about 0.5-1.5 mg/mL.

[0123] Embodiment B5: The adjuvant composition according to any one of Embodiments B 1 to B8, wherein the adjuvant composition excludes hydroxide salts, alkaline salts, and carbonate salts.

[0124] Embodiment B6: A composition comprising or consisting essentially of the adjuvant composition according to any one of Embodiments Bl to B8 and a given antigen of interest.

[0125] Embodiment B7: The composition according to Embodiment B9, wherein the composition lacks insoluble particles containing aluminum.

[0126] Embodiment B8: The composition according to Embodiment B9 or Embodiment B10, wherein the composition excludes hydroxide salts, alkaline salts, and carbonate salts.

[0127] Embodiment B9: A method of making the adjuvant composition according to any one of Embodiments Bl to B8, which comprises titrating the aqueous aluminum salt with the buffer to obtain a solution having a pH of about 5.0-7.5, preferably about 6.0- 7.5 or about 6.0-7.0.

[0128] Embodiment B10: A method of making the composition according to any one of Embodiments B9 to B 11, which comprises adding the given antigen of interest to the adjuvant composition according to any one of Embodiments Bl to B8.

[0129] Embodiment Bl 1 : The method according to Embodiment B12 or Embodiment B13, which further comprises maintaining the pH in the range of about 5.0-7.5, preferably about 6.0-7.5 or about 6.0-7.0.

[0130] Embodiment B 12: A method of immunizing a subject, which comprises injecting the subject with the composition according to any one of Embodiments B9 to Bl 1, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum. [0131] Embodiment B 13: A method of enhancing an immune response in a subject, which comprises injecting the subject with the composition according to any one of Embodiments B9 to Bl 1, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum, and wherein the immune response is greater than that obtained with a control composition containing the given antigen of interest without any aluminum.

[0132] Embodiment B 14: The method according to Embodiment B15 or Embodiment Bl 6, which comprises (a) freezing the composition and thawing the composition, and/or (b) storing the composition at a temperature of -70-10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc., before injecting the subject with the composition.

[0133] Embodiment B15: A method of storing the composition according to any one of Embodiments B9 to Bl 1, which comprises maintaining the composition at a temperature of -70-10 °C, preferably -25-5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc.

[0134] In Embodiments Bl to B15, the adjuvant compositions and compositions thereof may comprise:

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate or aqueous aluminum triacetate and about 0.3-0.9 mg/mL tromethamine at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.8 mg/mL tromethamine at a pH of about 5.0. • A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 0.3-0.9 mg/mL tromethamine at a pH of about 6.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 0.9-442.1 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum salt and about 0.9-177.9 mg/mL sodium citrate dihydrate at a pH of about 5.0-7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 2.3-177.9 mg/mL sodium citrate dihydrate at a pH of about 6.0-7.0. • A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 0.9-2.9 mg/mL sodium citrate dihydrate at a pH of about 5.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum chloride and about 166.3—

442.1 mg/mL sodium citrate dihydrate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 29.3-101.5 mg/mL sodium citrate dihydrate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum sulfate and about 85.5-284.0 mg/mL sodium citrate dihydrate at a pH of about 7.5.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 28.8-

62.1 mg/mL sodium citrate dihydrate at a pH of about 7.0.

• A solution of about 0.5-1.5 mg/mL aqueous aluminum triacetate and about 77.4- 273.3 mg/mL sodium citrate dihydrate at a pH of about 7.5. [0135] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0136] As provided herein, a “given antigen of interest” may be any desired compound, e.g, protein, polysaccharide, lipid, etc. that is intended to provoke an immune response in a subject when administered thereto.

[0137] As used herein, the terms “subject”, “patient”, and “individual” are used interchangeably to refer to humans and non-human animals. The terms “non-human animal” and “animal” refer to all non-human vertebrates, e.g, non-human mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0138] As used herein, “and/or” means “and” or “or”. For example, “A and/or B” means “A, B, or both A and B” and “A, B, C, and/or D” means “A, B, C, D, or a combination thereof’ and said “A, B, C, D, or a combination thereof’ means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

[0139] As used herein, the phrase “one or more of’, e.g., “one or more of A, B, and/or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more of A and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.

[0140] As used herein, the phrase “consists essentially of’ in the context of a given ingredient in a composition, means that the composition may include additional ingredients so long as the additional ingredients do not adversely impact the activity, e.g, biological or pharmaceutical function, of the given ingredient. In the context of the compositions described herein, “consists essentially of’ means that the compositions comprising an aqueous aluminum adjuvant may comprise additional ingredients so long as the additional ingredients do result in the formation of insoluble aluminum precipitates.

[0141] The phrase “comprises, consists essentially of, or consists of A” is used as a tool to avoid excess page and translation fees and means that in some embodiments the given thing at issue: comprises A, consists essentially of A, or consists of A. For example, the sentence “In some embodiments, the composition comprises, consists essentially of, or consists of A” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition consists essentially of A. In some embodiments, the composition consists of A.”

[0142] Similarly, a sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C ” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C ”

[0143] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0144] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

What is claimed is:

1. An adjuvant composition comprising or consisting essentially of an aqueous aluminum salt, a buffer, and a pH of about 5.0-7.5.

2. The adjuvant composition according to claim 1, wherein the aqueous aluminum salt is aqueous aluminum chloride, aqueous aluminum sulfate, or aqueous aluminum triacetate.

3. The adjuvant composition according to claim 1 or claim 2, wherein the buffer is a sodium acetate buffer, a tromethamine buffer, or a sodium citrate dihydrate buffer.

4. The adjuvant composition according to claim 1 or claim 2, wherein the buffer is a sodium acetate buffer.

5. The adjuvant composition according to claim 1 or claim 2, wherein the buffer is a tromethamine buffer or a sodium citrate dihydrate buffer.

6. The adjuvant composition according to claim 1 or claim 2, wherein the adjuvant composition comprises 0.4-220.6 mg/mL sodium acetate, 0.2-1.0 of tromethamine, or 0.6-442.1 mg/mL sodium citrate dihydrate.

7. The adjuvant composition according to any one of claims 1 - 6, wherein the concentration of the aqueous aluminum salt is about 0.5-1.5 mg/mL.

8. The adjuvant composition according to any one of claims 1 - 7, wherein the adjuvant composition excludes hydroxide salts, alkaline salts, and carbonate salts.

9. A composition comprising or consisting essentially of the adjuvant composition according to any one of claims 1 - 8 and a given antigen of interest.

10. The composition according to claim 9, wherein the composition lacks insoluble particles containing aluminum.

11. The composition according to claim 9 or claim 10, wherein the composition excludes hydroxide salts, alkaline salts, and carbonate salts.

12. A method of making the adjuvant composition according to any one of claims 1 - 8, which comprises titrating the aqueous aluminum salt with the buffer to obtain a solution having a pH of about 5.0-7.5.

13. A method of making the composition according to any one of claims 9 - 11, which comprises adding the given antigen of interest to the adjuvant composition according to any one of claims 1 - 8.

14. The method according to claim 12 or claim 13, which further comprises maintaining the pH in the range of about 5.0-7.5.

15. A method of immunizing a subject, which comprises injecting the subject with the composition according to any one of claims 9 - 11, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum.

16. A method of enhancing an immune response in a subject, which comprises injecting the subject with the composition according to any one of claims 9 - 11, wherein the composition comprises an immunogenic amount of the given antigen of interest and lacks insoluble particles containing aluminum, and wherein the immune response is greater than that obtained with a control composition containing the given antigen of interest without any aluminum.

17. The method according to claim 15 or claim 16, which comprises (a) freezing the composition and thawing the composition, and/or (b) storing the composition at a temperature of -70 °C to 10 °C, preferably -25 °C to 5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc., before injecting the subject with the composition.

18. A method of storing the composition according to any one of claims 9 - 11, which comprises maintaining the composition at a temperature of -70 °C to 10 °C, preferably -25 °C to 5 °C, for a given period of time, e.g., 30 minutes or more, 1 hour or more, 1 month or more, 6 months or more, 1 year or more, etc.

19. The adjuvant composition according to any one of claims 1 - 8, the composition according to any one of claims 9 - 11, and the method according to any one of claims 12 - 19, wherein the adjuvant composition or the composition thereof contains not more than 1% of the total aluminum content as water insoluble particles.