Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro
Introduction
The ability of aluminum-containing adjuvants to increase the immunogenicity of antigens was discovered in 1926 when Glenny et al. [1] found that injection of guinea pigs with diphtheria toxoid precipitated with potassium alum provided greater protection from the toxin than injection of toxoid alone. They also observed that when the precipitate was filtered, the filtrate contained none of the toxoid. This led to the belief that adsorption of the antigen to aluminum-containing adjuvants prior to administration is essential for enhancement of immunogenicity. Regulatory agencies require evidence that the antigen is adsorbed by aluminum-containing adjuvants. For example, the World Health Organization recommends that at least 80% of diphtheria and tetanus toxoids be adsorbed by aluminum-containing adjuvants in the vaccine [2].
Vaccines experience two different environments during their manufacture and use: the formulation prior to administration and interstitial fluid following intramuscular or subcutaneous administration [3]. Chang et al. [4] performed a study to determine how adsorption in the formulation affects antibody production in rabbits. The surface charge of aluminum-containing adjuvant was modified to produce vaccines in which 3, 35 or 85% of the 4.8 mg dose of lysozyme was adsorbed. The three vaccines induced much higher antibody titers than a 4.8 mg lysozyme solution but all three vaccines induced the same antibody titer. When the three vaccines were mixed with sheep lymph fluid to simulate interstitial fluid, the degree of lysozyme adsorption in each vaccine changed to 40% within 1 h. Because all of the vaccines had the same degree of lysozyme adsorption in interstitial fluid and induced the same degree of immunopotentiation, it was concluded that immunopotentiation by aluminum-containing adjuvants correlates with the degree of antigen adsorption in interstitial fluid following administration rather than the degree of antigen adsorption in the vaccine.
A recent study [5] investigated whether it was necessary for the antigen to elute or remain adsorbed to aluminum hydroxide adjuvant (AH) in order to produce immunopotentiation. The model antigens used were alpha casein (AC), which completely adsorbed to AH through the ligand exchange mechanism, and dephosphorylated alpha casein (DPAC), which completely adsorbed to AH by electrostatic attraction [6]. After exposure to interstitial fluid for 1 h, 100% of the AC remained adsorbed to AH while only 10% of the DPAC remained adsorbed. All of the AC remained adsorbed to AH even after 24 h of exposure to interstitial fluid. In comparison, the DPAC was completely eluted after 6 h of exposure to interstitial fluid. It was concluded that the strong ligand exchange adsorption mechanism, whereby a phosphate in AC displaced a surface hydroxyl to form an inner sphere complex with AH [7], maintained complete adsorption of AC in interstitial fluid [8]. However, since DPAC adsorbed to AH by forming an outer sphere complex by electrostatic attraction [7], it completely eluted from AH during exposure to interstitial fluid. It was found that AH potentiated the immune response in mice for both AC and DPAC. The results indicated that aluminum-containing adjuvants potentiate the immune response when the antigen elutes from the adjuvant following administration as well as when the antigen remains adsorbed following administration.
An important step in the induction of the immune response is the internalization of the antigen by antigen presenting cells, such as dendritic cells (DCs). Macropinocytosis and phagocytosis are two mechanisms of antigen internalization by DCs [9], [10], [11], [12], [13], [14]. Antigens that elute from the adjuvant surface are internalized by DCs by macropinocytosis while those that remain adsorbed are internalized by phagocytosis.
While the study with AC and DPAC [5] demonstrated that antigen internalization by antigen presenting cells occurs by both macropinocytosis and phagocytosis, the relative efficiency of each route was not studied. Thus, this study was undertaken to compare antigen internalization by macropinocytosis and phagocytosis. AC labeled with a green fluorescent dye was chosen as the model antigen. AC contains eight phosphate groups, is adsorbed to AH by ligand exchange and does not elute from AH when exposed to interstitial fluid [5]. Dendritic cells were incubated with a solution of fluorescent-labeled AC to model antigens that elute from the adjuvant surface following administration. Dendritic cells were also incubated with fluorescent labeled AC adsorbed to AH to model antigens that do not elute following administration. In addition, the effect of the aggregate size of the adjuvant on the internalization of AC by phagocytosis was studied. The efficiency of antigen internalization was monitored by confocal microscopy and flow cytometry.
Section snippets
Adjuvants
Aluminum hydroxide adjuvant (Rehydragel HPA, Reheis, Berkeley Heights, NJ) and aluminum phosphate adjuvant (AP) (Adju-Phos, Brenntag Biosector, Elsenbakken, Denmark) were obtained commercially and diluted to 1 mg Al/ml with doubly distilled water (ddH2O). AH was modified by pretreatment with phosphate anion at P/Al molar ratios of 1:1 or 2:1. For the 1:1 phosphate-treated AH (PTAH-1), 769 μl of commercial AH containing 13 mg Al/ml was diluted to 10 ml with 3.7 ml of 0.1 M Na3PO4 adjusted to pH 7.4
Surface charge of adjuvants
Evidence for the adsorption of phosphate by AH in PTAH-1 and PTAH-2 is seen in Table 1 as the zeta potential of AH, which is normally positive [17], became increasingly negative as the concentration of phosphate used to pretreat AH increased. The negative surface charge of PTAH-2 approached that of AP [17].
Adsorption isotherms
Adsorption isotherms of AC by the four adjuvants were determined because AC has an iep of 4.6 [18] and is negatively charged at pH 7.4. Therefore, an electrostatic attractive force will exist
Summary
The results of the in vitro cell culture experiment indicate that internalization of antigen by DCs occurs rapidly by both macropinocytosis of eluted antigen and phagocytosis of adsorbed antigen. The internalization of adsorbed antigen was more efficient as the relative fluorescence intensity within the DCs was greater when adsorbed antigen was internalized in comparison to antigen in solution. In addition, the relationship between the size of the DCs and the size of the BODIPY FL labeled
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