Aluminum adjuvants, commonly referred to as “alum,” are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject® alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject® alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject® alum, Alhydrogel®, and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel® and precipitated alum was significantly greater than that induced by Imject® alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject® exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel® and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.

Aluminum salts, commonly referred to as “alum,” are the most common adjuvants in human vaccines and serve as a benchmark in the development of novel adjuvants. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to induce inflammatory signals that, in turn, promote antibody production. It has been noted that some formulations of alum used in animal studies, notably Imject alum®, are not the chemical equivalents of the aluminum used in human vaccines, raising concerns where Imject serves as the comparator for novel adjuvants. Here, we compared the capacity of Imject alum, Alhydrogel®, and a traditional alum-antigen (Alum-Ag) precipitate to induce humoral responses in mice to the hapten-carrier NP-CGG. Primary humoral responses elicited by Alhydrogel and the Alum-Antigen precipitate were significantly greater than those induced by Imject alum. The three alum formulations elicited comparable responses in terms of acute eosinophil and monocyte infiltration at the immunization site, but neutrophil recruitment by Imject alum was signficantly reduced compared to Alum-Ag precipitate or Alhydrogel. These findings reveal substantial differences in the adjuvant properties and inflammatory effects of distinct alum preparations, an important consideration in the analysis of new adjuvants and mechanistic studies of alum’s adjuvanticity.