Figure 1.
Cycles of escape from autologous Nab in 185F and 205F.
Each panel represents a longitudinal plasma sample where Nab activity was evaluated for (A) subject 185F (5, 11, 17, 23, and 28 months) or (B) subject 205F (2, 8, 14, 20, and 26 months). Each data point represents the Nab IC50 titer (shown on the vertical axis on a log10 scale) for a single plasma-Env combination calculated from the virus infectivity curve using the Excel growth function. The Nab IC50 titers against the 0-month Envs and the contemporaneous Envs are shown for each longitudinal plasma sample (labeled along the vertical axis), and the horizontal bars indicate the median for each group of Envs. A Mann-whitney analysis was used to test for statistically significant differences between the Nab sensitivity of the 0-month and contemporaneous Envs, and p-values less than 0.05 were considered significant. Red dots indicate the Nab IC50 titer for 0-month Env that was used as a background for the chimeras. Green dots indicate the Nab resistant Envs from each time point that were selected and used to generate the chimeras.
Table 1.
Seroconvertors from ZEHRP that were evaluated for Nab escape.
Figure 2.
Moderate neutralization breadth of plasma from 185F and 205F against heterologous 0-month Envs.
A single plasma sample from 185F (23-months) and 205F (20-months) was evaluated for neutralizing activity against heterologous Envs acquired during acute/early subtype C infection of six subjects. The Nab IC50 titer for each plasma-Env combination is shown on the vertical axis on a log10 scale. Each data point represents a single plasma-Env combination. Arrows indicate the Nab IC50 titer for the autologous plasma-Env combination; all other data points represent heterologous Envs. The plasma sample is indicated below each point plot.
Figure 3.
Early escape from neutralization in 185F and 205F requires different pathways.
Neutralization of escape variants 185F 5-month PB1.1 (A and B), and 205F 2-month PB2.3 (D) and chimeras or mutants from each of these Envs in the corresponding 0-month Env was evaluated using contemporaneous plasma. Pseudoviruses were created by expressing each Env with an HIV-1 env-deficient backbone, and their infectivity for JC53-BL13 (Tzm-bl) cells was evaluated in the absence or presence of serially-diluted patient plasma with luciferase as a quantitative measure. Percent virus infectivity relative to no test plasma is plotted on the vertical axis; the reciprocal of the plasma dilution is plotted along the horizontal axis on a log10 scale. Each curve represents one Env against serial plasma dilutions, and error bars represent the standard deviation of at least two independent experiments using duplicate wells. The legends list the parental Nab resistant Env followed by the chimeric and mutant Envs created in the 0-month Env background. Amino acid alignments are shown to indicate the sequence differences between the different Envs for 185F (C) and 205F (E). The color of the text corresponds to the curves on the graph. Only regions that contained differences are shown.
Figure 4.
185F Nab resistant variants at 28-months utilize distinct escape pathways.
Neutralization of 28-month EnvPL5.1 (A) and 28-month EnvPL3.1 (B) and the chimeric Env pseudoviruses generated from each of these Envs in the 0-month Env background was evaluated using the 28-month plasma sample in JC53-BL13 cells with luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. The panels below each graph indicate the region that was transferred from the 28-month Nab resistant Env (PL5.1 in A and PL3.1 in B; gray boxes) into 0-month EnvPL3.1 (white boxes). (C) Amino acid alignment of the V5 region for the 0-month and 28-month Envs.
Figure 5.
Neutralization profile of 205F Envs by autologous monoclonal antibody-containing supernatants from B cell hybridomas 6.4C and 13.6A.
Neutralization activity of 6.4C (A) and 13.6A (B) B cell hybridoma supernatant was evaluated against pseudoviruses expressing autologous 205F Envs. Three different 0-months Envs are shown along with the longitudinal panel of Envs. Percent virus infectivity is plotted against the log10 of the reciprocal dilution of hybridoma culture supernatant in each graph. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. 0-month EnvPB1.1 was used to screen the hybridomas for neutralization activity. B cell hybridomas from 205F were generated at 49-months post-infection.
Figure 6.
Potential N-linked glycosylation sites in V1 and V2 modulate sensitivity to Mabs.
(A) Schematic diagram showing the position of potential N-linked glycans on the V1V2 sequence of the ‘founder’ 0-month Env sequence (inferred from Fig. S6), the 13.6A-resistant 0-month EnvPL6.3, and the 6.4C/13.6A-resistant 8-month EnvPB2.3. The absence of the V1 glycan shown in blue was associated with resistance against 13.6A, while addition of the V2 glycan shown in red was associated with resistance against 13.6A and 6.4C. (B) Potential N-linked glycosylation sites (NXS or NXT where X is any residue except proline) in V1V2 that were introduced singly or in combination into EnvPL6.3 are shown in blue (V1) or red (V2). The text color corresponds to the graphs in (C and D). 0-month EnvPL6.3 naturally lacks both sites. Neutralization of 0-month EnvPL6.3 and the site-directed mutants shown in panel B was evaluated for 13.6A (C) and 6.4C (D) using reduction of luciferase production in JC53-BL13 cells. Percent virus infectivity is plotted against the log10 reciprocal dilution of hybridoma supernatant. Error bars represent the standard deviation of at least two independent experiments using duplicate wells.
Table 2.
Summary of Mab neutralization data for 205F Envs.
Figure 7.
Nab escape in 185F involves multiple pathways.
(A) A 3-dimensional representation of the gp120 amino acid sequence for a different longitudinal Nab escape variant Env is shown in each panel. The time point and clone is indicated in each panel. 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). (B) Schematic representation of the Env domains contributing to Nab escape at each time point. Yellow indicates that a single domain had a major effect on Nab resistance; gray indicates that a particular domain contributed partially to Nab resistance. For more detail, see Fig. S4.
Figure 8.
Nab escape in 205F is V1V2-dependent.
(A) A 3-dimensional representation of the gp120 amino acid sequence for a different longitudinal Nab escape variant Env is shown in each panel. The time point and clone is indicated in each panel. 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). (B) Schematic representation of the Env domains contributing to Nab escape at each time point. Yellow indicates that the domain that had a major effect on Nab resistance; gray indicates the domain that made a minor contribution to Nab resistance. For more detail, see Fig. S5.
Figure 9.
Sequence variation occurs in similar regions of Env in 185F and 205F.
A 3-dimensional representation of the sequence variation in gp120 over time in 185F and 205F is shown in panels (A and B), respectively. The gp120 backbones were generated by homology modeling of the 0-month Env sequences of 185F and 205F onto the CD4-liganded HIV-1 YU-2 gp120 structure [54], with modeled V1V2 and V3 loops as described previously [25]. Blue to green to red indicates degree of sequence conservation (high to low) within the alignment.