Figure 5. Inhibition of MuHV-4 infection by gp70-Fc and gHL-Fc blocking mAbs.

A. Wild-type virions (200 p.f.u.) were incubated with mAbs as shown (2h, 37°C) then plaque-assayed on BHK-21 cells. We compared pairs of treatment arms by scoring (+ or -) for each antibody dilution whether the titer of a given treatment arm was more or less than the mean of both. We then performed an exact Chi-squared test on the comparisons. By this measure, mAb 230-4A2 but not mAb LT-6E8 gave significant neutralization, and 230-4A2 plus LT-6E8 was significantly better than 230-4A2 alone (p<0.0001). Equivalent data were obtained in 2 further experiments. B. Wild-type virions were incubated with mAbs (2 h, 37°C, 1 µg/1000 p.f.u.) then bound to either BHK-21 or NMuMG cells (2 h, 37°C, 3 p.f.u./cell). Bound virions were detected by washing, fixing and permeabilizing the cells, then staining them for gN with mAb 3F7. Secondary detection was with Alexa488-conjugated goat anti-mouse IgG pAb. This would have detected mAbs LT-6E8 and 230-4A2 even if the gN epitope were masked. The cells were scored simply as positive or negative on staining. Each bar shows the % positive of 10,000 cells, based on a gate where unexposed cells were 0% positive. The differences in binding between no mAb block/single mAb block, and between single mAb block/dual mAb block were highly significant (p<0.0001 by Student's t test). Equivalent data were obtained in 1 further experiment. C. Wild-type, gp70-deficient or gL-deficient virions (200 p.f.u.) were each incubated with mAbs (2h, 37°C, 1 µg/1000 p.f.u.), then with BHK-21 cells (2h, 37°C, 3 p.f.u./cell) as in B. The cells were then washed x3 in PBS, fixed and permeabilized, stained for gN with mAb 3F7 (green), counterstained with DAPI (blue) and examined microscopically for virion uptake. As in B, the anti-mouse secondary antibody would also have detected bound 230-4A2 or LT-6E8, so we could be sure no virions were missed because mAb binding had masked gN. D. Wild-type, gp70-deficient or gL-deficient virions (200 p.f.u.) were each incubated with mAbs as shown (2h, 37°C) then plaque assayed on BHK-21 cells. 230-4A2 and 230-5B2 both block gHL-Fc binding; LT-6E8 blocks gp70-Fc binding. Titers are plotted as % of p.f.u. without mAb for each virus. We compared treatment arms by an exact Chi-squared test of the pairwise comparisons at each antibody dilution, as in A. By this measure, mAb LT-6E8 neutralized the gL knockout but not the wild-type or gp70 knockout, and mAbs 230-4A2 and 230-5B2 neutralized the wild-type and gp70 knockout-the gp70 knockout significantly better than the wild-type-but not the gL knockout (p<0.004). The data are from 1 of 5 equivalent experiments.