Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A

Abstract

Spleen cells from mice immunized with a Bordetefla pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0.22 and 2.2 pg mAb ml-l (0.1 and 1 pg per 450 p1 assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.

Tn5-induced lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane function

“…Several studies have characterized the ability of Gramnegative endotoxin to activate complement and form the membrane attack complex which in turn leads to cell lysis (Archambault et al, 1991;Byrd et al, 1991;Fernandez & Weiss, 1994). Thus, assays examining the ability of the variant strains to fix complement in the presence of the LPSA and LPSB mAbs served as initial attempts to characterize the consequences of LPS alterations on an important function of the outer membrane.…”

“…Escherichia cofi strain HBlOl containing the TnS plasmid, pUW964, was maintained on L-agar containing 25 pg kanamycin ml-'. LPSA-and LPSB-specific mAbs, BL-2 and BL-8, respectively, were characterized previously by Archambault et al (1991) and Martin et af. (1992 (Weiss et af., 1983).…”

“…These experiments were made possible by the existence of a less frequently isolated clinical strain of B. pertussis whose only LPS component is LPSB. We utilized a mAb recently developed against LPSB to screen Tn5 mutants of the wildtype LPSAB strain for LPS alterations (Archambault et al, 1991;Martin et al, 1992) Our laboratory has produced by Tn5 insertion ten LPS mutants, which contain varying quantities of LPSA and LPSB as well as two clinically undocumented intermediate LPS types which we have designated IntA and IntB (respective to the band to which they are closest in molecular mass). This study deals with the phenotypic and genetic characterization of these mutants as well as the initial analysis of the effect of these mutations on outer membrane function.…”

Structural characterization of the lipid A of Bordetella pertussis 1414 endotoxin

“…The serological properties of the rough-type endotoxin of Bordetella pertussis, the agent of whooping cough, have been well documented, and the determination of its complex glycosidic structure is nearing completion (2,5,12,14,20,22,27).…”

Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine

“…Several articles indicate that serum IgG anti-OS antibodies can confer complement mediated killing effect upon B. pertussis (10,11). B. pertussis LPS is comprised of Lipid A and a branched dodecasaccharide core, composed of unusual sugars with free amino and carboxylic acid groups but with no O-specific polysaccharide (O-SP; ref.…”