FIG 9.
Substitutions at V4 loop P421 site lead to a loss of recognition of V4 loop-specific MAbs, and P421Q confers neutralization resistance to V4 loop-specific MAbs in neutralization-sensitive background. (A) We tested the effect of SIVmac239 adaptations at position 421 on antibody recognition by a panel of Env-specific MAbs with specificity to the V1, V3, and V4 loops. Fifty nanograms of recombinant SIVmac239 gp140 WT or mutant protein were added to 96-well plates and incubated overnight at 4°C. Binding was measured over seven serial 6-fold dilutions of MAb in triplicate at a starting concentration of 10 μg/ml against soluble gp140 WT or mutant proteins. An HRP-conjugated anti-IgG antibody was used with TMB substrate for detection of binding. Absorbance values were read at 450 nm and experiments were performed in triplicate. (B) The level of virus neutralization of Env-specific MAbs was measured over 10 serial 2-fold dilutions of MAb against SIVmac316 WT or with SIVmac316 P421Q in triplicate. Ten nanograms of p27 of WT and mutant virus stock was used to infect 5 × 103 C8166-SEAP cells/well in a 96-well plate. MAb 3.11H targets a V3 linear epitope and was used as a control. Infectivity is reported as the level of SEAP activity above background from uninfected C8166-SEAP cells.