A novel assay for detecting virus-specific antibodies triggering activation of Feγ receptors

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fc gamma receptors (Fc gamma Rs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fc gamma receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric Fc gamma R-CD3 zeta chain molecules consisting of the extracellular domain of human Fc gamma RIIIA, Fc gamma RIIIA or Fc gamma RI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3 zeta chain. Triggering of the chimeric Fc gamma R receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in Fc gamma R activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fc gamma receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs. (C) 2012 Elsevier B.V. All rights reserved.