Monoclonal antibodies reactive with immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.

FIG. 1.

Reactivity of MAbs F10F5 and F12D2 with serotype A (MU-1), B (409), C (24066), and D (9375B) GXM or de-O-acetylated serotype A (MU-1) GXM by ELISA. Serial dilutions were made from a stock 2-mg/ml antibody solution.

FIG. 2.

Reactivity of MAbs F10F5 (left panel) and F12D2 (right panel) with GXM by immunodiffusion. The assay was done with each MAb in the center well (2 mg/ml) and GXM in the outer wells (2 mg/ml). (A) serotype A (MU-1); (B) serotype B (409); (C) serotype C (24066); (D) serotype D (9375B); (E) de-O-acetylated serotype A (MU-1).

FIG. 3.

Capsule reactions of MAbs 3C2, 471, F10F5, and F12D2 with yeast cells of serotypes A (MU-1), B (409), C (24066), and D (34875).

FIG. 4.

Capsule reactions of MAbs 3C2, 471, and F12D2 with serotype A cells of C. neoformans (MU-1) and cells that have been chemically de-O-acetylated.