Molecular Basis for Immunoglobulin M Specificity to Epitopes inCryptococcus neoformansPolysaccharide That Elicit Protective and Nonprotective Antibodies

Abstract

The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V H ) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V H binding cavity to the corresponding residues in the 13F1 V H altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an antiidiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.The protective efficacy of antibodies (Abs) to the humanpathogenic fungus Cryptococcus neoformans depends on the Ab isotype and specificity (reviewed in references 3 and 46). The evidence that Ab specificity is critical for protective efficacy comes from studies of two clonally related immunoglobulin M (IgM) monoclonal Abs (MAbs) known as 12A1 and 13F1 (3, 31, 39, 46). Although these MAbs originated from the same B-cell precursor and use the same variable (V)-region genes, they differ in specificity as a result of V-region somatic mutations that translate into 12 amino acid differences (31, 39). The differences in specificity are manifested by differences in the indirect immunofluorescence (IF) binding pattern such that MAbs 12A1 and 13F1 produce annular and punctate patterns, respectively, after binding to serotype D C. neoformans cells (11,31,39). The annular binding pattern is correlated with opsonic efficacy, capsular reaction patterns, and complement activation kinetics (27) and Ab protection against serotype D organisms (31, 39). Since the MAb pair 12A1 and 13F1 have markedly different biological properties yet differ in sequence by only a few amino acids, they provide a unique opportunity for the study of Ab specificity.MAbs to C. neoformans capsular glucuronoxylomannan (GXM) have been grouped into five classes based on V-region usage and idiotype and serotype specificity (5). Class II MAbs include a large set of MAbs that bind to an immunodominant epitope found in all cryptococcal serotypes and are characterized by the use of V H 7183, J H 2, V 5.1, and J 1 gene eleme...

Isotype Can Affect the Fine Specificity of an Antibody for a Polysaccharide Antigen

“…Before this study, the punctate IF pattern had been observed only with two murine IgMs known as 13F1 and 21D2 (15). Recently, we used site-directed mutagenesis to establish that changing two amino acids in the in V region of an IgM mAb could change the IF pattern from annular to punctate (10). In this study, we demonstrate that the same V can produce annular or punctate patterns depending on which human or mouse C region it is associated with.…”

“…Prior studies had established that the IF pattern is a property of the V H (10). The 18B7 V H -was expressed with the V L of mAbs 12A1 and 13F1, which produce annular and punctate IF patterns, respectively, as a function of their V H sequences (10). Binding of 18B7 murine IgM to C. neoformans produced a different IF pattern than that observed with the parent IgG1 mAb in combination with both the 12A1 and 13F1 V L (Fig.…”

“…DNA containing the V H was amplified using the primers 5Ј-ATAAAGCTTGCGGGGATATCCACCATGGAC and 3Ј-ATAGCTA GCTGAGGAGACTGTGA. The amplified DNA was cloned into the PCR 2.1 vector (Invitrogen, San Diego, CA) for sequencing and subcloned into a murine IgM expression vector, as described (10). The plasmid was then transfected into H chain nonproducing 18B7, 12A1, and 13F1 hybridoma cells by electroporation.…”

A Peptide Mimotope of Type 8 Pneumococcal Capsular Polysaccharide Induces a Protective Immune Response in Mice

“…Our data showing that PUB1 inhibited the type 8 PS binding of human IgAs, but not other isotypes, suggest that PUB1 could be a surrogate for an epitope that elicits naturally occurring type 8 PS-reactive IgA. In view of evidence that isotype influences polysaccharide and peptide specificity (30,32,35), our data suggest that the specificity of type 8 PS-specific IgA could differ from that of IgM or IgG. Alternatively, our findings could reflect differences in affinity or avidity or unique characteristics of the primary or secondary reagents that were used.…”

Unexpected Diversity in the Fine Specificity of Monoclonal Antibodies That Use the Same V Region Gene to Glucuronoxylomannan of Cryptococcus neoformans

“…The significance of confirming the contribution of the variable domain recognition of GXM is important in light of new data showing that the isotype of human chimeric Abs to GXM, which contained identical variable domains, can affect the fine specificity of the Ab (43). Site-directed mutagenesis studies of mAbs 12A1 and 13F1 have elucidated two amino acids responsible for their immunofluorescent staining differences (44). Closer examination of the amino acid differences among the Abs to GXM evaluated with the EDC mutants may similarly reveal residues important in binding the polysaccharide.…”