Interactions of the Cryptococcus neoformans polysaccharide capsule with monoclonal antibody: Epitope location, variation, and stability

Abstract

Cryptococcus neoformans is a unique pathogenic yeast which can be classified into four major serotypes (A, B C and D) on the basis of its polysaccharide capsule. C. neoformans var. neoformans comprise serotypes A and D, and represent most of the isolates recovered from immunocompromised patients. C. neoformans var. gattii comprise serotypes B and C which, although pathogenic, is rarely recovered from immunocompromised patients. The persistence of C. neoformans infection is a major clinical problem in immunosuppressed patients. Capsule binding murine monoclonal antibodies (mAbs) can mediate protection against murine infection, however, the mechanism(s) by which C. neoformans persist and escape antibody-mediated immune clearance is (are) not understood. To address this question, we studied capsule-mAb interactions for the protective mAbs 2H1 (IgG1), 12A1 (IgM) and the non-protective (for serotype D strains) mAb 13F1 (IgM). MAbs 2H1, 12A1, and 13F1 are representative of a class of antibodies elicited by a polysaccharide-protein conjugate vaccine being developed as potential therapeutic agents.;The structure of the C. neoformans capsule in the presence and absence of mAbs was examined by Scanning Electron Microscopy (SEM). My results demonstrate that cells, in absence of mAb were covered with a loose fibrillar network that was identified as capsular material. In the presence of protective mAbs, a structural change occurred as capsule borders became more distinct. I addressed whether the 2H1 epitope, which elicits protective mAbs, is present in diverse C. neoformans strains and found that it is prevalent in over 21 different laboratory and clinical strains. The IgGI mAb 2H1 prolongs survival of lethally infected mice, however, the infection is seldom eradicated. To investigate whether epitope variants arise during the course of infection, I first determined whether variants of C. neoformans that differ in 2H1 epitope expression exist by selecting for cells that did not agglutinate in the presence of mAb 2H1. I also evaluated whether antigenic variation occurred in vivo, by examining isolates derived from the serial passage of three strains of C. neoformans in mice given mAb 2H1.;My studies of polysaccharide-mAb interactions have shown that: (1) C. neoformans capsular polysaccharide can undergo structural changes in the presence of protective mAbs or serum proteins; (2) An epitope that elicits protective mAbs is prevalent in diverse C. neoformans strains; (3) mAbs can be used to discriminate between serotype A and D strains; (4) Epitope location determines antibody efficacy in functional assays; (5) Variants with reduced binding to protective mAbs exist that have differences in capsular structure; (6) Variation in capsular structure affects biological and serological assays, (7) Persistence of infection in the presence of specific antibody does not result from selection of escape variants that no longer bind antibody, and (8) Changes in mAb epitope expression can occur in vivo. (Abstract shortened by UMI.).