Mechanisms of aluminum-containing vaccine adjuvants: Implications of the relationship between the movement of antigen in vivo and immunopotentiation

The mechanism(s) of immunopotentiation by aluminum-containing adjuvants have not been clearly defined. It has been widely believed that 100% antigen adsorption is ideal and that prolonged release of antigen from a depot formed at the injection site is a major mechanism, but the movement of antigen from the injection site has not been investigated. By directly studying the movement of antigen in vivo, important information can be obtained concerning the mechanism(s) of immunopotentiation of aluminum-containing adjuvants that could lead to more effective use of these adjuvants. Four different vaccine formulations were evaluated: antigen in solution, antigen adsorbed onto aluminum hydroxide adjuvant (AH), antigen adsorbed onto aluminum phosphate adjuvant (AP), and antigen mixed with phosphate-treated aluminum phosphate adjuvant (PT-AP), which did not adsorb the antigen. The in vitro diffusion of antigen from the four vaccines was compared. Adsorption isotherms were conducted to assess the strength and capacity of antigen adsorption, and the relative strength of adsorption was assessed in vitro by antigen desorption in plasma. The persistence of antigen at the injection site in naïve rats was measured for both naïve and previously immunized rats. The accumulation of antigen in the draining lymph nodes and the antibody response were measured. In vitro, the antigen diffused at a similar rate from the solution and non-adsorbed vaccines for the first 12 hours and somewhat slower from the non-adsorbed vaccine from 18 to 72 hours. No diffusion was observed for the AH- and AP-adsorbed vaccines. Adsorption isotherms showed that AH adsorbed the antigen more strongly and to a larger extent than AP. Antigen desorbed more rapidly from AP than from AH and did not readsorb onto PT-AP when mixed with plasma. In vivo, antigen migrated from the injection site in naïve rats more slowly for the adsorbed vaccines, reflecting depot formation at the injection site. The non-adsorbed vaccine did not form a depot, indicating antigen adsorption is required for depot formation. Immunized and naïve rats produced identical results. The non-adsorbed and solution vaccines resulted in similar amounts of antigen in the lymph nodes that were higher than that of the AH- and AP-adsorbed vaccines, which were also similar. The non-adsorbed, AH-adsorbed, and AP-adsorbed vaccines all produced antibody titers significantly higher than the solution vaccine. No direct correlations were found between the persistence of antigen at the injection site, accumulation of antigen in the draining lymph nodes, and the antibody response, indicating that the major mechanism(s) of immunopotentiation are not affected by antigen adsorption. Aluminum-containing adjuvants produce inflammation at the site of injection, and several inflammation based mechanisms have been recently demonstrated. The results of this investigation indicate that these mechanisms are more important than the depot mechanism