Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model.

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and ß-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to ß-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic.