Characterization of Binding Affinity and Epitope Dynamics of Anti-HIV-1 Antibodies

Abstract

A preventive vaccine is potentially the most effective way to control the HIV pandemic. Such a vaccine needs to successfully harness humoral immunity and produce cross-reactive anti-envelope antibodies that mediate direct virus neutralization and/or Fc receptor-dependent killing. For these antibodies to carry out their functions in clearing HIV infection, they must bind the virus and prevent it from infecting target CD4+ cells. The capacity of an antibody to do this is dependent on the timing, duration and extent of cognate epitope exposure before and during the attachment and entry processes. The goal of this study was (i) to quantify antibody binding to HIV, and (ii) to characterize when and for how long antibody epitopes are exposed before and during virus-cell fusion. We studied the binding properties and epitope dynamics of antibodies against HIV envelope gp120 [b12 (CD4 binding site), 2G12 (carbohydrate clusters), A32 (C1, C4, & C5 domains)], CD4-induced epitope of gp120 (17b & 19e), and the membrane-proximal external region (MPER) of gp41 (4E10). To directly quantify antibody binding to virus in solution, we developed a fluorescence correlation spectroscopy (FCS) methodology that uses fluctuations in fluorescent signals to measure diffusion and reaction kinetics of fluorescently-labeled anti-envelope Mabs as they attach to HIV-1-JRFL, HIV-1-Bal, and HIV-1-NL4-3 pseudoviruses and infectious molecular clones. We have also developed methods to visualize the temporal appearance and disappearance of cognate epitopes during virus-cell fusion using immunofluorescence and live-cell imaging techniques. In this case, viral particles were labeled with a novel SNAP-tag technology that permits tracking of particles during different stages of fusion with CD4+ target cells, and concurrent imaging of epitopes that become exposed on the HIV envelope.