New
anthrax vaccines currently under development are based on recombinant protective
antigen (rPA) and formulated with
aluminum adjuvant. Because long-term stability is a desired characteristic of these
vaccines, an understanding of the effects of adsorption to
aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-
Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on
aluminum adjuvant. Intrinsic fluorescence emission spectra of
tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on
Alhydrogel. Using anti-PA
monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two
monoclonal antibodies that recognize
epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-
Alhydrogel formulations in mice was assessed by measuring the induction of toxin-
neutralizing antibodies, as well as
antibodies reactive to 12-mer
peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-
neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer
peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-
Alhydrogel formulations can lead to structural alteration of the
protein and loss of the ability to elicit toxin-
neutralizing antibodies.