Figure 5.

Antibody size is not a limit to neutralisation of cell-cell spread by J3. (A) IC50 values (μM) generated from duplicate titrations of the J3 VHH and full length heavy chain only J3 (J3-Fc) incubated for 1 h with HIV-1 (NL4.3) infected Jurkat cells and then mixed with target 1G5 luciferase-encoding cells for 24 h as for Figure 1. Student’s t-test was performed using data from 3 independent experiments. (B) The percentage cell-cell spread when J3 was added during (t = 0 h) or after (t = 1 h and t = 3 h) mixing HIV-1 infected Jurkat cells and uninfected 1G5 target cells. Statistical analysis was performed using a student’s t- test. (C) Immunofluorescence staining showing J3 (top panel) and J3-Fc (bottom panel) can access the virological synapse formed during cell-cell contact between a Gag + HIV-1 infected T cell and an uninfected target T cell (asterisk). Gag (red), J3 (green) and DAPI (blue). Scale bar is 5 microns. (D) Inhibition of virological synapse formation by J3 and J3-Fc. HIV-1 (NL4.3) infected T cells were mixed with uninfected T cells in the presence of J3, J3-Fc or control (Lab5) and incubated for 1 h at 37°C. Cells were then fixed and permeabilised, stained for HIV-1 Gag and imaged by immunofluorescence microscopy. Random fields were selected and the number of synapses formed between HIV-1 infected T cells and uninfected target T cells (defined by polarisation of Gag to the cell-cell interface) were scored. Data are the mean and SEM from 3 independent experiments. Statistical analysis was performed by comparing to control Lab5 using an Anova with Tukey’s post-test.