We next investigated the adaptations that were responsible for escape from contemporaneous Nab in 185F and 205F. The 0month Envs were potently neutralized by plasma from all subsequent time points ( Fig. 1A and B) and were used to provide a neutralization sensitive background, which remained more than 95% conserved at the amino acid level with subsequent variants and could be used to investigate the molecular determinants of escape for each Nab resistant variant. To do this, two approaches were used: (i) where sequence changes were limited in the Nab resistant Env, site-directed mutagenesis was used to introduce potential escape mutations into the 0-month Env and (ii) where multiple sequence changes were present in the Nab resistant Env, larger Env subregions (i.e. V1 to V5, V3 to V5, V1V2, etc.) were transferred from the Nab resistant Env into the 0-month Env. The neutralization sensitivity of the chimeric and parental Envs was then evaluated using plasma contemporaneous with the Nab resistant Env.