Antigenic variation in Cryptococcus neoformans: In vitro and in vivo selection

C. neoformans has a polysaccharide capsule that is a virulence factor. Capsulebinding antibodies can mediate protection against infection. The murine monoclonal antibody (mAb 2H1) is representative of a class of antibodies elicited by a polysaccharide-protein conjugate vaccine which are being developed as potential therapeutic agents. C. neoformans infections are chronic and often incurable in patients with impaired immunity. This raises concern about the possibility of antigenic changes during infection and the potential of antibody-based therapies to select for new antigenic variants. To investigate this question we studied the stability of the epitope defined by the protective mAb 2H1 in vivo and in vitro. In vivo experiments were done by serially passaging a colony of C. neoformans in mice given mAb 2H1. In vitro studies were done by selecting for yeast cells which did not agglutinate in the presence of mAb 2H1. The initial and passaged isolates were studied by karyotype analysis, indirect immunofluorescence, FACS and agglutination endpoint. The results indicate: 1) the epitope defined by mAb 2H1 is found in C neoformans after 5 serial passages in mice treated with mAb, suggesting stability in vivo despite antibody selection; 2) variants with markedly altered mAb 2H1 epitope localization can be selected in vitro by recovering yeast that fail to agglutinate; 3) mAb 2H1 is not opsonic for some of these variants; 4) there is considerable variation in mAb 2H1 epitope content and localization within a given C. neoformans strain. The results indicate that, under certain circumstances, mAb 2H1 can select for C. neoformans variants with altered capsular epitope phenotype and this is an important consideration in the design of antibody-based strategies for the prevention and therapy of cryptococcal infection.