| Summary: | Generalized modules for membrane antigens (GMMA) represent a technology particularly attractive for designing affordable vaccines against Gram-negative bacteria. We explored such technology for the development of O-antigen-based vaccines against <i>Shigella</i> and nontyphoidal <i>Salmonella</i>. Adsorption of GMMA on Alhydrogel was required for abrogation of pyrogenicity in rabbits, and <i>Shigella sonnei</i> GMMA on Alhydrogel was well tolerated and immunogenic in humans. Quantification of key antigens in formulated vaccines was fundamental for release and to check stability overtime. Traditionally, the direct quantification of antigens adsorbed on aluminum salts has been challenging, and the quantification of each active ingredient in multicomponent formulated vaccines has been even more complicated. To directly quantify each active ingredient and unbound drug substances in formulated vaccines, we developed the Formulated Alhydrogel competitive ELISA (FAcE) and the competitive ELISA method, respectively. The methods were both fully characterized, assessing specificity, repeatability, intermediate precision, and accuracy, for <i>S. sonnei</i> OAg quantification, both in a single component or multicomponent GMMA formulation also containing <i>S. flexneri</i> GMMA. The developed immunological methods allowed us to fully characterize <i>Shigella</i> GMMA drug products, supporting their preclinical and clinical development. The same methods, already extended to GMMA from nontyphoidal <i>Salmonella</i> and <i>Neisseria meningitidis</i>, could be potentially extended to any antigen formulated on Alhydrogel.
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