Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans.
Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.
Department of Medicine, Division of Infectious Disease, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Diane C
DC
McFadden
Arturo
A
Casadevall
D000908:Antibodies, Fungal / Q000096:biosynthesis / Q000145:classification / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000145:classification / Q000378:metabolism*; D000916:Antibody Diversity / Q000235:genetics*; D000918:Antibody Specificity / Q000235:genetics; D000946:Antigens, Fungal / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics*; D002240:Carbohydrate Sequence; D003455:Cryptococcus neoformans / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D005816:Genetic Complementation Test; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018448:Models, Immunological; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D007135:Immunoglobulin Variable Region; D011134:Polysaccharides; C027478:glucuronoxylomannan