Nanocochleates containing N-Octylglicoside extracted Vibrio cholerae antigens elicited high vibriocidal antibodies titers after intragastric immunization in a mice model

Abstract

Biological detergents are used in research laboratories, to extract or solubilize proteins from cell membranes. In order to evaluate the ability to extract antigens from the bacterial cell surface of the wild Vibrio cholerae strain C7258 and study their immunogenic potential by forming proteoliposomes and cochleate and preserving their immunogenicity, the non-ionic detergent, n-Octylglucoside (n-OG), and the Zwitterionic detergent (3-cholamidopropyl dimethylammonio 1-propanesulfonate; CHAPS) were tested in concentrations between 5 and 15%. The anionic detergent sodium deoxycholate (DOC) was used as a reference. Electrophoretic, immunochemical and electron microscopy techniques have characterized the extracts and their chromatographic fractions. With CHAPS and n-OG detergents in concentrations between 5 and 15%, a higher yield was obtained in the extraction of proteins and lipopolysaccharides (LPS) and other components from the bacterial surface compared to 10% DOC. When using 10% DOC, 15% CHAPS and n-OG between 5 and 15%, stable proteoliposomes were formed, of average size between 82 and 93 nm in diameter, with known proportions of proteins, LPS and other components. In some of the concentrations, liposomes were formed with almost pure proteins. Some cholera outer membrane proteins like the 17 kDa protein, which corresponds to the mannose-sensitive hemagglutinin (MSHA), which mediates the adhesion to the brush border of the small intestine and the outer membrane protein U (OMPU) were identified with monoclonal antibodies (mAbs) and purified. The fundamental components of liposomes, proteins and LPS, retained their molecular weights, when compared with known standards and by processing programs of electrophoretic profiles and their antigenicity, without alterations due to the extraction procedure, as could be verified by immune identification techniques with monoclonal antibodies in the case of LPS, significant antigens in this pathogen. The main purpose of the present work was to show that a new anticholera vaccine formulation based on cochleates, containing selected protein and LPS fraction extracted by detergents, is able to elicit protective high titers of bactericidal antibodies after intragastric immunization in the mice model. The objective was achieved.

Keywords: Bactericidal antibodies; Cochleate; Detergents; Intragastric immunization; Lipopolysaccharide; Outer membrane proteins; Proteoliposomes; SEM; TEM; Vibrio cholerae; n-Octylglucoside.