Stanley L. Hem

Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.

In vivo absorption of aluminium-containing vaccine adjuvants using 26Al

Aluminium hydroxide (AH) and aluminium phosphate (AP) adjuvants, labelled with 26Al, were injected intramuscularly (i.m.) in New Zealand White rabbits. Blood and urine samples were collected for 28 days and analysed for 26Al using accelerator mass spectrometry to determine the absorption and elimination of AH and AP adjuvants. 26Al was present in the first blood sample (1 h) for both adjuvants. The area under the blood level curve for 28 days indicates that three times more aluminium was absorbed from AP adjuvant than AH adjuvant. The distribution profile of aluminium to tissues was the same for both adjuvants (kidney > spleen > liver > heart > lymph node > brain). This study has demonstrated that in vivo mechanisms are available to eliminate aluminium-containing adjuvants after i.m. administration. In addition, the pharmacokinetic profiles of AH and AP adjuvants are different.

ALUMINUM COMPOUNDS USED AS ADJUVANTS IN VACCINES

The structure of nine commercially manufactured aluminum-containing adjuvants was investigated by X-ray diffraction, infrared spectroscopy, transmission electron micrography, and energy dispersive spectrometry. Seven samples which were labeled as aluminum hydroxide were identified as boehmite, a crystalline aluminum oxyhydroxide [AIO(OH)]. However, the degree of crystallinity varied between the samples. Two samples which were labeled as aluminum phosphate were found to be amorphous aluminum hydroxyphosphate. Buffer anions and sulfate anions substitute for hydroxyls in the amorphous aluminum hydroxide formed by the in situ alum precipitation method. Finally, the aluminum-containing adjuvant in diphtheria and tetanus toxoid, U.S.P., produced by three manufacturers was characterized.

Predicting the adsorption of proteins by aluminium-containing adjuvants.

The adsorption of two model proteins, albumin and lysozyme, by boehmite or amorphous aluminium hydroxyphosphate adjuvants was studied. Electrostatic, attraction has a major role in adsorption. At physiological pH, boehmite, which has a point of zero charge above 7.35, extensively adsorbed albumin, which has an isoelectric point of 4.8, but was not effective in adsorbing lysozyme (isoelectric point, 11.0). Conversely, amorphous aluminium hydroxyphosphate (point of zero charge, 4.0) was effective in adsorbing lysozyme but adsorbed relatively little albumin. The results suggest that the selection of either boehmite or amorphous aluminium hydroxyphosphate as an adjuvant should be based in part on the isoelectric point of the antigen.

A preliminary study of the dermal absorption of aluminium from antiperspirants using aluminium-26

Aluminium chlorohydrate (ACH), the active ingredient in many antiperspirants, was labeled with the radioisotope 26Al. The labeled ACH was then fractionated into about 100 samples using gel filtration chromatography. Each fraction was analyzed for 26Al and total aluminium content. Aluminium-26 was only detected in the fractions that also contained aluminium, which verified that the ACH was uniformly labeled. 84 mg of the labeled ACH was then applied to a single underarm of two adult subjects with blood and urine samples being collected over 7 weeks. Tape-stripping and mild washings of the skin were also collected for the first 6 days. Results indicate that only 0.012% of the applied aluminium was absorbed through the skin. At this rate, about 4 microg of aluminium is absorbed from a single use of ACH on both underarms. This is about 2.5% of the aluminium typically absorbed by the gut from food over the same time period. Therefore, a one-time use of ACH applied to the skin is not a significant contribution to the body burden of aluminium.

Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminium-containing adjuvants.

The effect of ionic strength and ethylene glycol on the adsorption of bovine serum albumin (BSA) or lysozyme by a commercial aluminium hydroxide or aluminium phosphate adjuvant was studied at pH 7.4 and 25 degrees C. The adsorption of BSA by aluminium hydroxide adjuvant and lysozyme by aluminium phosphate adjuvant was found to be inversely related to ionic strength. This indicates that electrostatic attractive forces contribute to adsorption. The adsorption of lysozyme by aluminium phosphate adjuvant was reduced by the addition of ethylene glycol. However, no change in the adsorption of BSA by aluminium hydroxide adjuvant was noted when up to 40% ethylene glycol was present. This behaviour indicates that hydrophobic forces contribute to the adsorption of lysozyme but not of BSA. However, virtually no adsorption was observed when the protein and the adjuvant had the same surface charge. Thus, attractive forces may not be sufficient to produce adsorption of an antigen by an aluminium-containing adjuvant if electrostatic repulsive forces are present.

Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Structure and Properties of Aluminum-Containing Adjuvants

This chapter is concerned with the identification, characterization, and behavior of aluminum-containing adjuvants with proteins and anions similar to those occurring in vaccines and interstitial fluid. Aluminum-containing adjuvants referred to commercially as aluminum hydroxide have been identified as poorly crystalline aluminum oxyhydroxide with the structure of the mineral boehmite. Relevant properties of this material include its high surface area and its high pI, which provide the adjuvant with a high adsorptive capacity for positively charged proteins. Aluminum phosphate and alum-precipitated adjuvants may be classified as amorphous aluminum hydroxyphosphate with little or no specifically adsorbed sulfate. Variations in the molar PO4/A1 ratio of amorphous aluminum hydroxyphosphates result in PI values that range from 5 up to 7; the materials are negatively charged at a physiological pH of 7.4. The amorphous nature of these compounds gives them high surface area and high protein adsorptive capacity for positively charged proteins. Observations on the interactions of anions and charged proteins with charged adjuvant surfaces have provided a framework for predicting behavior of complex systems of vaccines and for designing specific combinations of adjuvants and antigens to optimize the stability and efficacy of vaccines.

Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response.

Hepatitis B surface antigen (HBsAg) is known to adsorb to aluminum hydroxide adjuvant (AH) by ligand exchange between its accessible phosphate groups and surface hydroxyl groups of the adjuvant. To study the effect of the binding strength, five vaccines were prepared with AH or four samples of AH that were modified by pretreatment with different concentrations of potassium dihydrogen phosphate. The adsorptive coefficients ranged from 3660 to 250mL/mg based on the Langmuir adsorption isotherm and degrees of elution ranged from 1 to 31% when the vaccines were exposed to interstitial fluid in vitro. When tested in mice the four vaccines containing phosphate-treated AH (PTAH) induced significantly greater antibody responses than the vaccine containing AH, which had the highest adsorptive coefficient and the smallest degree of elution of HBsAg. The results indicated that antibody production is reduced when the antigen is adsorbed too strongly. Thus, the strength of adsorption of the antigen to an aluminum-containing adjuvant can affect the immunogenicity of the vaccine and should be optimized during vaccine formulation.

Effect of the Degree of Phosphate Substitution in Aluminum Hydroxide Adjuvant on the Adsorption of Phosphorylated Proteins

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.