Elsevier

Vaccine

Volume 24, Issue 10, 6 March 2006, Pages 1665-1669
Vaccine

Relationship of adsorption mechanism of antigens by aluminum-containing adjuvants to in vitro elution in interstitial fluid

https://doi.org/10.1016/j.vaccine.2005.09.048Get rights and content

Abstract

The objective of this research was to determine how the mechanism by which antigens adsorb to aluminum-containing adjuvants affects the elution upon exposure to interstitial fluid. Antigens (alpha lactalbumin, bovine serum albumin, lysozyme and myoglobin) that adsorb to aluminum-containing adjuvants principally by electrostatic attraction were found to elute readily in vitro when exposed to interstitial fluid. Phosphorylated antigens (alpha casein, hepatitis B surface antigen and phosphorylated bovine serum albumin) that adsorb to aluminum-containing adjuvants principally by ligand exchange exhibit little if any elution during 12–24 h in vitro exposure to interstitial fluid. Dephosphorylated alpha casein, which contains less than two phosphate groups, was less strongly adsorbed by ligand exchange in comparison to alpha casein, which contains eight phosphate groups. Dephosphorylated alpha casein was completely eluted when exposed to interstitial fluid. The results of this study lead to the generalization that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange.

Introduction

The goal in the formulation of vaccines using aluminum-containing adjuvants is adsorption of the antigen by the adjuvant [1]. However, it is now realized that the degree of adsorption may change following intramuscular or subcutaneous administration of the vaccine [2], [3], [4], [5], [6]. This is because the composition of interstitial fluid is different than the composition of the vaccine formulation. Components of interstitial fluid such as phosphate anion [7], citrate anion [8], or fibrinogen [9] have been found to cause elution of the antigen.

Shi et al. [3] reported that lysozyme was completely eluted from aluminum phosphate (AP) adjuvant after in vitro exposure to interstitial fluid for 15 min. In contrast, ovalbumin was only partially eluted from aluminum hydroxide (AH) adjuvant during a 50 h in vitro exposure period [3]. A recent study [6] in which the phosphate content of ovalbumin was varied from 3.2 to 0.14 mol phosphate/mol ovalbumin showed that elution from AH upon in vitro exposure to interstitial fluid was inversely related to the degree of phosphorylation of the ovalbumin.

Iyer et al. [4] studied the effect of the degree of adsorption of the antigen after in vitro exposure to interstitial fluid on the potentiation of the immune response by AH. They found that AH potentiated the immune response to dephosphorylated alpha casein, which eluted completely when exposed to interstitial fluid for 4 h, as well as to alpha casein, which did not elute when exposed to interstitial fluid for 24 h. Iyer et al. [5] also found that hepatitis B surface antigen did not elute from aluminum hydroxide adjuvant or from a commercial hepatitis B vaccine containing aluminum hydroxyphosphosulfate during in vitro exposure to interstitial fluid. As an aluminum-containing adjuvant potentiated the immune response to alpha casein, dephosphorylated alpha casein and hepatitis B surface antigen, it was concluded that antigen-presenting cells take up eluted as well as adsorbed antigen. However, in vitro studies of antigen uptake by dendritic cells indicate that antigen uptake is enhanced when the antigen remains adsorbed to aluminum-containing adjuvants [10].

The two most important mechanisms of antigen adsorption by aluminum-containing adjuvants are electrostatic attraction and ligand exchange whereby the phosphate group of a phosphorylated antigen exchanges with a surface hydroxyl of the adjuvant [11]. Ligand exchange is a stronger adsorption force than electrostatic attraction. Since the adsorption state in vivo affects how dendritic cells interact with the antigen, it is important to understand how the antigen adsorption state may change upon vaccine administration. In an effort to determine if elution of an adsorbed antigen upon exposure to interstitial fluid can be related to the adsorption mechanism, a survey of the adsorption mechanism of eight antigens by aluminum-containing adjuvants and the degree of antigen adsorption when exposed in vitro to interstitial fluid was undertaken.

Section snippets

Materials

Aluminum hydroxide adjuvant, which contained 2.0% (w/w) equivalent Al2O3 (10.6 mg Al/mL) (Rehydragel HPA, Reheis, Berkeley Heights, NJ) was obtained commercially. Alpha casein, alpha lactalbumin, bovine serum albumin, dephosphorylated alpha casein, myoglobin (equine skeletal muscle), and phosphorylated bovine serum albumin were obtained from Sigma (St. Louis, MO). Myoglobin (human cardiac muscle) used for the interstitial fluid experiment was obtained from BioTrend (Köln, Germany).

Adsorption isotherms

Adsorption

Results and discussion

Eight antigens were selected for this study. Four, alpha lactalbumin, BSA, lysozyme and myoglobin do not contain any phosphate groups and, therefore, cannot adsorb to aluminum-containing adjuvants by ligand exchange. Four antigens, alpha casein, dephosphorylated alpha casein, PBSA and HBsAg contain phosphate groups and have the potential to adsorb by ligand exchange. Ovalbumin is heterogeneous in phosphate content [16] and was not included in this study. A detailed study of the adsorption and

Significance

The results of this study lead to the generalization that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange. Antigens that elute rapidly may reach the lymph node to be taken up by residential dendritic cells [19] or may be internalized by local antigen-presenting cells by macropinocytosis and transported to the lymph node. In contrast, antigens

Acknowledgment

This work was supported in part by a gift from Merck Research Laboratories.

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