281 A Quantitative Assay to Measure Cell-to-Cell Fusion Mediated by HIV-1 Env Glycoprotein Y. Ataman-Önal*1, P-Y. Durand1, F. Reynard1, F. Bedin1, R. Altmeyer2, M. Girard3, B. Verrier1 1UMR2142 CNRS-bioMerieux, Lyon, France; 2Inst Pasteur, Paris, France; and 3Fndn Merieux, Lyon, France
Background: HIV1-target cell fusion requires the Env protein to bind CD4 and a chemokine coreceptor on the cell surface. Infected cells expressing Env can initiate syncytium formation through cell-to-cell fusion, a process that contributes significantly to the spread of HIV-1 infection and AIDS pathogenesis. Methods to quantify cell-to-cell fusion include counting syncytia by light microscopy, resonance energy transfer based assays, and indirect reporter gene using techniques. These methods are time consuming or prone to subjectivity.
Methods: We developed a direct assay with a fluorescent readout to measure cell-to-cell fusion mediated by HIV1 Env protein. The protein was expressed in vitro by infecting BHK21 cells with a recombinant Semliki Forest virus, and the cells were labeled using a rhodamine-derived dye, CMTMR. Target CD4+ X4R5+ cells were labeled with a fluorescein-derived dye, CMFDA. The 2 cell populations were co-cultured during 5 hrs and analyzed by FACS to determine the percentage of double labeled population, which corresponds to fused cells.
Results: We have analyzed cell fusion capacity of Env proteins of 9 primary isolates (clades A-E, G) and prototype HXB2 strain. After 5 hrs of co-culture, fusion percentage was between 1.5%-6%, with background values below 0.5%. Two (2) of the primary Env proteins (MBA, G365) and the HXB2Env were highly fusogenic, whereas the remainder were less. We tested different receptor expressing cells: GHOSTX4R5 cells yielded large syncitia, not suitable for accurate FACS analysis; donor PBMC showed increased sensitivity, but reproducibility was an issue; and HeLaP4P cells were an interesting compromise. We also used the assay to measure inhibition of cell-to-cell fusion. The ID90 for the anti-CD4 RPAT4 antibody was 1 µg/ml, for RANTES it was 400 ng/ml. Fusion mediated by HXB2Env was inhibited 50% by 10/10 HIV-1 neutralizing sera, whereas fusion mediated by primary MBA-Env was inhibited only by 4/10. No correlation was observed between neutralization titers of sera and their ability to inhibit cell-to-cell fusion. This suggests that anti-HIV antibodies may be more potent to neutralize virus infectivity than to block cell-to-cell fusion.
Conclusions: Our assay provides a numerically accurate, quick, objective manner to estimate the fusogenic competence of HIV-1 Env proteins, and the effects of potential inhibitors or enhancers of fusion. It may be used to test if immune sera generated by vaccination can inhibit cell-to-cell fusion.
