Relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response
Introduction
Aluminum-containing adjuvants are used in vaccine formulations to enhance the antibody response. In 1926, Glenny et al.[1] laid the groundwork for the traditional view that adsorption of antigen to aluminum-containing adjuvants prior to administration is essential for the enhancement of immunogenicity. They observed that injecting alum-precipitated diphtheria toxoid led to a significant increase in the immune response. Furthermore, when the precipitate was filtered, the filtrate was devoid of the toxoid. This observation led to the conclusion that the antigen must be adsorbed to the aluminum-containing adjuvant. Thus, a goal of vaccine formulation is to maximize the adsorption of the antigen. Antigen adsorption to aluminum-containing adjuvants occurs by two principle mechanisms. Electrostatic attraction is the most prevalent and occurs when the adjuvant and antigen have opposite charges [2]. Ligand exchange occurs when an antigen contains a phosphate group (e.g. DNA, phosphorylated antigens, phospholipid-bound antigens, and PRP-containing antigens) that can displace a hydroxyl group on the adjuvant surface to form an inner-sphere surface complex with aluminum that is the inorganic equivalent of a covalent bond [3]. Ligand exchange is the strongest adsorption force and can occur even when an electrostatic repulsive force is present [4].
Two parameters are important when considering adsorption: the maximum amount that can be adsorbed as a monolayer, which is characterized by the adsorptive capacity and the strength of the adsorption force, which is characterized by the adsorptive coefficient [5]. The effect of adsorptive capacity on the immune response has been studied. These studies suggest that the percentage of the antigen dose that is adsorbed in the vaccine is not related to immunogenicity. For example, equivalent immunopotentiation was observed for three lysozyme vaccines in which the degree of adsorption was 3, 35 or 85% [6]. The degree of adsorption of each vaccine changed to 40% when the vaccines were mixed with the sheep interstitial fluid in vitro. The study concluded that immunopotentiation was not related to the degree of adsorption in the vaccine formulation but was correlated to the degree of adsorption following administration. Another study [7] showed that aluminum phosphate adjuvant potentiated the immune response to alpha casein, ovalbumin or lysozyme when the antigen was not adsorbed in the vaccine formulation nor when mixed in vitro with interstitial fluid. The authors hypothesized that the antigens, even though not adsorbed, were trapped in void spaces within the adjuvant aggregates, resulting in uptake of antigen by dendritic cells. The adsorption of Bacillus anthracis recombinant protective antigen by aluminum phosphate adjuvant was not required for immunopotentiation [8].
The strength of adsorption (adsorptive coefficient) by ligand exchange is related to both the number of phosphate groups on the antigen [9], and the number of surface hydroxyl groups on the aluminum-containing adjuvant [10]. No reports were found in the literature of the relationship between the adsorptive coefficient of the antigen to an aluminum-containing adjuvant and the immune response. Thus, a study was undertaken using alpha casein (CAS), which contains eight phosphate groups and dephosphorylated alpha casein (DP-CAS) with two phosphate groups [11]. The two adjuvants used in the study were aluminum hydroxide adjuvant (AH) in which all of the surface groups were hydroxyls, and phosphate-treated aluminum hydroxide adjuvant (PT-AH) which contained a mixture of hydroxyl and phosphate groups on the surface. Four vaccines were prepared by combining these two antigens and two adjuvants. The vaccine composed of CAS and AH had the greatest potential for adsorption by ligand exchange and the vaccine composed of DP-CAS and PT-AH had the least potential for adsorption by ligand exchange. The immunogenicity of the four vaccines was tested in mice.
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Materials
Alpha casein, DP-CAS, Incomplete Freund's adjuvant (IFA), MOPS [3-(N-morpholino) propanesulfonic acid], MOPS sodium salt, were ACS grade or better, and were used as supplied (Sigma, St. Louis, MO). The bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) was used to measure the concentration of antigen in vitro. The aluminum hydroxide adjuvant (Rehydragel HPA, Reheis, Berkeley Heights, NJ) was an aqueous suspension containing 2.2% (w/w) equivalent Al2O3. Endotoxin was removed from CAS
Results
Two phosphorylated proteins were selected for this study: CAS (eight phosphates, 26,000 Daltons, iep ∼4.6) and DP-CAS (two phosphates, 25,500 Daltons, iep ∼4.6) [11]. All the surface sites in AH contain a hydroxyl that is available for ligand exchange. Phosphate treatment of AH reduces the number of surface sites that contain a hydroxyl and thereby reduces the potential for ligand exchange [12]. The four vaccines studied were designed to have different adsorptive coefficients. Adsorption forces
Discussion
The activation of naïve vaccine-specific B and T-cells occurs primarily in lymph nodes draining the site of vaccination. Antigens reach the lymph node via the afferent lymphatics either as free antigens or intracellularly in dendritic cells [15]. Protein antigens taken up by dendritic cells are partially degraded by proteolytic enzymes into peptides in acidic endosomal compartments. The peptides bind to MHC II molecules and the MHC II/peptide complexes are presented to antigen-specific T-cells
Acknowledgements
This research was supported in part by a Purdue University Ross Fellowship and Fonner Fellowship to B.H. and Merck Research Laboratories.
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