Molecular characterization of the humoral responses to Cryptococcus neoformans infection and glucuronoxylomannan-tetanus toxoid conjugate immunization.

Abstract

Suml'naryThe molecular characteristics of the humoral immune response to a serotype A Cr~tococcus neoformans infection were compared with the response elicited by a cryptococcal ghicuronoxylomannan-tetanus toxoid (GXM-TT) conjugate. Anticryptococcal monoclonal antibodies (mAbs) isolated from both responses have previously been shown to recognize the same antigenic determinant of cryptococcal GXM. Southern blot and sequence analyses indicate that the hybridomas isolated from each response arose from only a few precursor B cells. All the mAbs generated from the infected and GXM-TT conjugate-immunized mice utilize the same VH7183 family member: J.2/J.4, v~5.1, and J~l; mAbs generated by different B cells had complementarity-determining region Ys (CDR3s) composed of seven amino acids with a common sequence motif. Thus, the molecular analysis of these anticryptococcal mAb-producing hybridomas indicated that the response to both cryptococcal infection and conjugate immunization was oligoclonal and highly restricted with regard to immunoglobulin gene utilization. The GXM-TT conjugate primarily stimulated isotype switching and clonal proliferation, and did not result in hybridomas expressing additional immunoglobulin repertoires. The mAbs from both responses had a number of replacement mutations at the 5' end of CDR2 that appear to be the result of antigen-driven selection. Somatic mutation also resulted in altered epitope specificity for one mAb, 13F1. Passive administration of representative mAbs from different clones generated in response to the GXM-TT conjugate prolonged survival of lethally infected mice.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides

“…mAb 18B7 Sequencing-Since the 18B7 mAb to GXM was initially isolated and characterized, several studies over the intervening years have found nucleotide and amino acid sequences for the V region containing discrepancies at a number of positions in both H and L chains (24,37,38). These earlier sequences were obtained by either direct mRNA sequencing of the hybridoma cell line or by sequencing cDNA generated by the hybridoma through RT-PCR.…”

“…Previous studies in our laboratory have led to the development of a murine monoclonal antibody (mAb) library against glucuronoxylomannan (GXM), the major component of the polysaccharide capsule in the fungal pathogen Cryptococcus neoformans (23)(24)(25)(26)(27). Earlier studies suggested that two of these mAbs, 3E5 and 18B7, possess hydrolytic activity against a peptide antigen mimetic known as P1 (28,29).…”

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

“…44 -46 This may allow C. neoformans to evade the host defense mechanisms, thus explaining the poor and delayed inflammation usually associated with cryptococcal meningitis 18,47 and the apparent inefficacy of the spontaneous antibody response that we know is based on a restricted repertoire. 48,49 In any case, the ability of C. neoformans to undergo active and rapid antigenic variability should be kept in mind for the future development of immunotherapy.…”

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

“…The 18B7 V H and V L nucleotide sequences have been previously reported based on direct mRNA sequencing (12). The 18B7 V H and V L region cDNA was cloned from 18B7 hybridoma cells by RT-PCR and inserted into human Ig expression vectors as described (8).…”