PGT145 treatment selects for amino acid changes in Env that confer resistance to ADCC and to neutralization.

(A) Viral RNA was isolated from plasma at weeks 4 and 8 post-infection and sequenced. The predicted amino acid sequences in Env from PGT145-treated animals (top, bold) and from DEN3-treated animals (bottom, plain text) were aligned to SIV_mac239 K180S Env. Regions of Env with substitutions in multiple animals are shown. The K180S substitution is indicated in magenta and residues N171 and N202 are underlined in the SIV_mac239 K180S Env reference sequence. Positions of identity are indicated by periods and amino acid differences are identified by their single letter code. Amino acid ambiguities are indicated with non-standard letters as follows: U = M/L, J = S/K, O = A/I (week 4) and O = I/K (week 8). The frequencies of each substitution within the virus population are indicated by color. (B) Antibody binding to Env was assessed by staining CEM._NKR-CCR5-sLTR-Luc cells infected with each of the indicated SIV_mac239 K180S mutants with PGT145 (top row) and with SIV+ plasma (bottom row) followed by AF647-conjugated goat anti-human IgG F(ab′)₂ after gating on virus-infected (CD4_low, p27⁺) cells. The shaded histograms represent non-specific staining with DEN3. (B & C) SIV_mac239 K180S mutants with substitutions in Env selected only in PGT145-treated animals (specific) or in both PGT145- and DEN3-treated animals (non-specific) were tested for susceptibility to ADCC (B) and neutralization (C) mediated by PGT145. The dotted lines indicate half-maximal ADCC or neutralization and the error bars indicate standard deviation of the mean for triplicate wells at each antibody concentration.