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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\u00b0C 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.<\/jats:p>","DOI":"10.1128\/cvi.00174-12","type":"journal-article","created":{"date-parts":[[2012,7,19]],"date-time":"2012-07-19T11:58:23Z","timestamp":1342699103000},"page":"1465-1473","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":44,"title":["Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant"],"prefix":"10.1128","volume":"19","author":[{"given":"Leslie","family":"Wagner","sequence":"first","affiliation":[{"name":"Center for Biologics Evaluation and Research, Food and Drug Administration Bethesda, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anita","family":"Verma","sequence":"additional","affiliation":[{"name":"Center for Biologics Evaluation and Research, Food and Drug Administration Bethesda, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce D.","family":"Meade","sequence":"additional","affiliation":[{"name":"Meade Biologics, Hillsborough, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karine","family":"Reiter","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David L.","family":"Narum","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rebecca A.","family":"Brady","sequence":"additional","affiliation":[{"name":"Center for Biologics Evaluation and Research, Food and Drug Administration Bethesda, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephen F.","family":"Little","sequence":"additional","affiliation":[{"name":"U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Drusilla L.","family":"Burns","sequence":"additional","affiliation":[{"name":"Center for Biologics Evaluation and Research, Food and Drug Administration Bethesda, Maryland, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.bbapap.2006.12.007","article-title":"Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy","volume":"1774","author":"Agopian A","year":"2007","unstructured":"AgopianA. 2007. 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