FIG 11.

Virion phagocytosis is not dependent on spinoculation and requires specific Fc-FcR interaction. (A) To assess whether virion phagocytosis was dependent on the spinoculation procedure in the experimental setup, parallel conditions were set up with and without the spinoculation step (see Materials and Methods), as indicated in the legend on the figure. Spinoculation was performed at 1,200 × g for 1 h at 4°C. Antibodies were added at 12.5 μg/ml unless otherwise specified. Three pairs of antibody and virus were chosen based on their known properties for virion phagocytosis in the presence of spinoculation (top row). All three pairs retained virion phagocytosis signal even when spinoculation was omitted though overall specific and nonspecific virion phagocytosis was decreased relative to values for the experiments with spinoculation. This remained true despite decreasing the concentration of antibody (bottom left), virus (bottom-middle), or both antibody and virus (bottom right). (B and C) To further assess whether virion phagocytosis was dependent on Fc-FcR interactions, the CD4 binding site broadly neutralizing antibody CH31 was recombinantly expressed in human mIgA1, IgG1, IgG3, and IgG4 backbones and examined for virus capture of HIV-1BaL virions or phagocytosis of HIV-1BaL-Tomato virions. For virus capture, the median and range of three replicates from a single experiment are reported, and for phagocytosis, the median and range of data from four different human primary monocyte donors representing two independent experiments are reported. A positivity cutoff of 20% for infectious virus capture and 1.77 for virion phagocytosis was determined based on the mean +3 standard deviations of the values for historical negative controls. CH31 mIgA1, IgG1, and IgG3 mediated virion phagocytosis, as reported, while CH31 IgG4 did not, despite its ability to capture virions, showing that virion phagocytosis is dependent on Fc-FcR interactions.