Synthetic glucuronoxylomannans enable molecular insights into antibody structure-function and fungal capsule architecture

Antibodies play critical roles in protection against infectious diseases and can be passively administered to protect patients. In the case of antibodies against glucuronoxylomannan (GXM), the major constituent of the Cryptococcus neoformans capsule, this results in protective, non-protective and disease enhancing outcomes. We sought to improve our basic understanding of these functionally different antibodies. Here we report a library of twenty-six synthetic GXM oligosaccharides, consisting of M2 (serotype A), M4 (serotype C), and M1 motifs (serotype D). These GXM glycans range in size from 1-mers to an 18-mer and were used to construct a microarray containing both O-acetylated and non-acetylated glycans. This allowed mapping of the binding preferences of sixteen functionally different monoclonal antibodies to the Cryptococcus capsule. This data is complemented by sequence comparison analysis of fragment antigen-binding (Fab) region which revealed how small changes (<13) in Fab sequence can affect antibody antigen specificity and function. While, changes in the constant region (isotype switching) altered glycan specificity. Furthermore, by combining immunofluorescence imaging of fungal cells and knowledge of antibody binding specificities, we gained direct evidence for the molecular complexity of cryptococcal capsules. Our results highlight the complex relationship between antibody epitope, affinity and isotype class, all of which act synergistically to contribute to a protective or non-protective immune response, meaning that binding specificity alone is not sufficient to predict an antibodys function. These results are relevant to the design of vaccines against Cryptococcosis.