The V2-based mutation that conferred resistance against 6.4C and 13.6A appeared during the first eight months of infection; however, 13.6A and 6.4C were recovered from memory B cells circulating 41 months later. As such, it was not possible to determine whether related B cells were circulating during early infection in 205F. The V1-based change that conferred resistance against 13.6A was present at 0-months (at which time the subject was seropositive), but only transiently. If this mutation occurred in response to immune pressure from 13.6A, then this antibody must have been present within ,48 days from the calculated time of infection. Indeed we have observed very low level neutralizing activity (IC50 = ,1:40) in the 0-month plasma of 205F against 0-month Envs [15] and (data not shown), consistent with this concept. While others have observed that the very early antibody response (within the first ,40 days after infection) lacks neutralizing activity and is directed predominantly against gp41 [40] , our findings raise the possibility that viral neutralization and escape could occur earlier than previously thought. Individual Mabs, derived from B cells early in infection The blue gp120 backbones were generated by homology modeling of the 0-month Env sequence onto the CD4-liganded HIV-1 YU-2 gp120 structure [54] , with modeled V1V2 and V3 loops as described previously [25] . Red indicates amino acid changes relative to the 0-month Env sequence. Spheres indicate changes in potential N-linked glycosylation sites (green = loss, white = gain and tested against founder virus Envs, may be required to detect this initial Nab activity. It will therefore be important to determine whether Nab activity and viral escape is present in other subtype C infected subjects at very early time points, as well as whether early escape mutations are associated with decreased viral fitness. The blue gp120 backbones were generated by homology modeling of the 0-month Env sequence onto the CD4-liganded HIV-1 YU-2 gp120 structure [54] , with modeled V1V2 and V3 loops as described previously [25] . Red indicates amino acid changes relative to the 0-month Env sequence. Spheres indicate changes in a potential N-linked glycosylation sites (green = loss, white = gain Spatial properties of escape pathways Importantly in this study, not all sequence changes were linked directly with Nab escape. For example, one of the first sequence changes that was present in the 185F 5-month Env was a substitution in the first position of the a2 helix (E335A); however, this change did not alter Nab sensitivity to contemporaneous plasma when introduced into the 0-month Env. Reversal of this mutation (A335E) in the 5month escape variant also did not increase its sensitivity to autologous Nab (Murphy et al., in preparation) . The 205F Nab escape variants also exhibited variation in the a2 helix beginning at 14-months, but again this region did not appear to contribute independently to Nab escape. These findings support that a2 was not targeted directly by Nab in these instances, despite ongoing sequence evolution. The a2 helix has been linked to autologous and heterologous Nab sensitivity of subtype C Envs from Zambia, South Africa, and India by our group and others [23, 25, 41] . However, its exact role(s) in Nab sensitivity or escape remains undetermined [23, 25] . We have speculated that the a2 helix plays an ancillary role in Nab escape, and perhaps participates in maintenance of the tertiary structure of the gp120 outer domain or the quaternary structure of the trimer [25, 42] . The findings presented here support our earlier findings, but do not rule out the possibility that the a2 helix is targeted by Nab in some instances, or by low titer Nab specificities. Studies are ongoing in our laboratory to more precisely define the role of changes in the a2 helix in the context of autologous Nab. In addition to the a2 helix, sequence variation was observed in V1V2, V5, and other regions of the outer domain in both 185F and 205F Envs ( Fig. 9A and B, respectively) . However, in 185F, the V3V5 region had the strongest effect on Nab resistance, while in 205F V1V2 was the major determinant. A companion study of four subtype C infected seroconvertors and our own previous study of subtype C chronically infected subjects reported consistent findings, in that V1V2 was commonly involved in Nab escape, but to varying degrees [24] and (Moore et al., in press). Importantly, the combined biological results and spatial analysis of these two subjects demonstrate how the perpetual flexibility of the V1V2 and V5 domains provides a formidable defense against Nab. This could be due in part to their ability to simultaneously mask multiple epitopes through limited changes, but may also involve direct escape.