Relationship of adsorption mechanism of antigens by aluminum-containing adjuvants to in vitro elution in interstitial fluid
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.
References (20)
Aluminium adjuvants in retrospect and prospect
Vaccine
(2004)- et al.
Degree of antigen adsorption in the vaccine or interstitial fluid and its effect in the antibody response in rabbits
Vaccine
(2001) - et al.
Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production
Vaccine
(2003) - et al.
Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant
Vaccine
(2004) - et al.
Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid
Vaccine
(2005) - et al.
Effect of anions on model aluminum-adjuvant-containing vaccines
J Colloid Interface Sci
(1995) - et al.
The in-vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins
Vaccine
(1999) - et al.
Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro
Vaccine
(2005) - et al.
Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminum-containing adjuvants
Vaccine
(1995) - et al.
Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants
Vaccine
(2003)
Cited by (26)
Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen
2022, Saudi Pharmaceutical JournalAdsorption of protein antigen to the cationic liposome adjuvant CAF®01 is required for induction of Th1 and Th17 responses but not for antibody induction
2021, European Journal of Pharmaceutics and BiopharmaceuticsUse of a quartz crystal microbalance platform to study protein adsorption on aluminum hydroxide vaccine adjuvants: Focus on phosphate-hydroxide ligand exchanges
2020, International Journal of PharmaceuticsEffect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens
2020, Journal of Pharmaceutical SciencesCitation Excerpt :On the other hand, Hem et al. have shown that antigens formulated as unbound to aluminum adjuvant can elicit equal or better immune response compared with adjuvant-bound antigens.28-30 Hem et al. have shown that several model proteins that bind AH because of electrostatic attractions elute readily on contact with interstitial fluid,31 a potentially better parameter to predict immune responses.32 Finally, the strength of antigen adsorption to AH is another important parameter for immune responses.
The kinetics of soluble and particulate antigen trafficking in the afferent lymph, and its modulation by aluminum-based adjuvant
2010, VaccineCitation Excerpt :No further increase was noted in the few samples collected after 72 h (not shown). The depot effect of aluminium-based adjuvant has recently been questioned, as most antigen is rapidly desorbed from alum in vitro after the addition of lymph fluid [4,5]. However, the real in vivo kinetics of antigen release from aluminium adjuvant into the lymphatics after injection has not been examined.