Phagocytic Assay for Antibody-Dependent

ADP was performed by measuring the internalization of opsonized HA-coated beads by a phagocytic cells line (Fig 1A). The methods were similar to those previously described for HIV [19 (link), 20 (link)] with minor modifications. Briefly, FITC-labeled NeutrAvidin^® FluoSpheres^® (beads 1μm, Invitrogen, Carlsband, CA) were labeled both with internalization probe tagged with Cy5 (FIP_Cy5) [21 (link)] and 0.75μg biotinylated HA or HIV-1 gp140 then opsonized with 10μg/ml purified IgG (Protein G HP Multitrap, GE Healthcare, UK). 1x10⁵ THP-1 (ATCC TIB-202) cells were incubated with the beads for 16 hr. A 16hr incubation provided a reasonable balance between ADP and non-specific bead uptake (not shown). Cell surface FIP_Cy5 was quenched with a complimentary probe so that internalized beads (FITC+Cy5+—i.e. truly phagocytosed) can be measured. Cells were fixed and 5x10⁴ cells were analyzed by flow cytometry. Background levels of ADP activity were assessed against HIV-1 gp140 since all donors were HIV-negative and calculated as the mean plus 2 SD. Background ADP levels were reproducible across multiple experiments (11–14%) as illustrated in the dotted lines Figs 1B and 3A–3C.