Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage

<div><p>Broadly neutralizing antibodies (bnAbs) to HIV-1 can evolve after years of an iterative process of virus escape and antibody adaptation that HIV-1 vaccine design seeks to mimic. To enable this, properties that render HIV-1 envelopes (Env) capable of eliciting bnAb responses need to be defined. Here, we followed the evolution of the V2 apex directed bnAb lineage VRC26 in the HIV-1 subtype C superinfected donor CAP256 to investigate the phenotypic changes of the virus populations circulating before and during the early phases of bnAb induction. Longitudinal viruses that evolved from the VRC26-resistant primary infecting (PI) virus, the VRC26-sensitive superinfecting (SU) virus and ensuing PI-SU recombinants revealed substantial phenotypic changes in Env, with a switch in Env properties coinciding with early resistance to VRC26. Decreased sensitivity of SU-like viruses to VRC26 was linked with reduced infectivity, altered entry kinetics and lower sensitivity to neutralization after CD4 attachment. VRC26 maintained neutralization activity against cell-associated CAP256 virus, indicating that escape through the cell-cell transmission route is not a dominant escape pathway. Reduced fitness of the early escape variants and sustained sensitivity in cell-cell transmission are both features that limit virus replication, thereby impeding rapid escape. This supports a scenario where VRC26 allowed only partial viral escape for a prolonged period, possibly increasing the time window for bnAb maturation. Collectively, our data highlight the phenotypic plasticity of the HIV-1 Env in evading bnAb pressure and the need to consider phenotypic traits when selecting and designing Env immunogens. Combinations of Env variants with differential phenotypic patterns and bnAb sensitivity, as we describe here for CAP256, may maximize the potential for inducing bnAb responses by vaccination.</p></div

Early VRC26 escape results in virus variants with reduced entry fitness.

<p><b>A</b> and <b>B:</b> Replicative capacity for CAP256 Env reporter pseudoviruses was tested by infecting A3.01-CCR5 cells either with free virus preparations (<b>A</b>) or transfected 293-T cells to probe cell-cell transmission (<b>B</b>). Infectivity for free virus (<b>A</b>, black) and cell-cell transmission (<b>B</b>, red) is displayed in relative light units (RLU). Means of two (free virus) to three (cell-cell) independent experiments in duplicates with SD are shown. <i>SU-like VRC26 sensitive</i> viruses were compared to <i>SU-like VRC26 early escape</i> viruses using the Mann-Whitney test. P values are indicated. <b>C:</b> Correlation between free virus and cell-cell infectivity were determined by Spearman correlation on untransformed data sets. R and p values are indicated. PI-like, <i>SU-like VRC26 sensitive</i>, <i>SU-like VRC26 early escape</i> and PI/SU recombinant viruses are marked in red, light blue, dark blue and black respectively. <b>D:</b> Results of the free virus (black) and cell-cell (red) infectivity assay were normalized to the PI-virus, 6-wk^PI, and are indicated as relative infectivity. Relative means are shown. <b>E:</b> The ratios of relative infectivity values for cell-cell and free virus transmission are depicted. Early viruses (wk 6 to wk 34) were compared to late viruses (wk 42 to wk 176) using the Mann-Whitney test. The P value is indicated. PI-like, <i>SU-like VRC26 sensitive</i>, <i>SU-like VRC26 early escape</i> and PI/SU recombinant viruses are marked in red, light blue, dark blue and black respectively.</p

Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage - Fig 3

<p><b>Heterologous bnAbs exhibit different neutralization efficiencies in CAP256 Env free virus and cell-cell transmission A</b> and <b>B:</b> IC50 (in μg/ml) of heterologous bnAbs targeting the V2 apex (red), the CD4 binding site (grey), the V3 glycan supersite (black) or the MPER region (blue) against free virus (<b>A</b>) and cell-cell transmission (<b>B</b>) are shown. Data are means of 2 to 4 independent experiments. <b>C</b>: The change in neutralization activity for cell-cell compared to free virus transmission is displayed as the log of the ratio of IC50^cell-cell/IC50^free-virus (log fold change). Zero denotes equal activity in both transmission pathways. 10-fold higher (log₁₀ = 1) and lower (log₁₀ = -1) IC50 in cell-cell over free virus transmission are indicated by dotted lines.</p

CAP256-VRC26 bnAbs display high activities at the pre- and post-CD4 attachment step.

<p><b>A</b> and <b>B:</b> Pre- and post- CD4 attachment activities of the VRC26 bnAbs are displayed relative to the total activity of each bnAb-virus combination. Data were derived from two independent experiments <b>A:</b> Data displayed are sorted by bnAbs, PI-like, <i>SU-like VRC26 sensitive</i> and <i>SU-like VRC26 early escape</i> viruses are marked in red, light blue and dark blue respectively. <b>B:</b> Same data as in A, sorted by virus strains. Pre- and post-attachment activity are shown in black or blue respectively.</p

Virus evolution alters the entry kinetics of autologous CAP256 viruses.

<p><b>A:</b> Schematic presentation of the free virus entry kinetic assay on TZM-bl target cells testing the half-maximal time to CD4-attachment and fusion. <b>B:</b> Mean half-maximal times of infection (t_1/2) to CD4 attachment (orange) and virus fusion (black) of 2 to 4 independent experiments are displayed. <b>C:</b> Interrelations of IC50s (in μg/ml) for VRC26 bnAb neutralization, viral infectivity and mean half-maximal time (t_1/2) to CD4-attachment, fusion and from CD4 attachment to fusion were determined for viruses analyzed in Fig 6B and displayed for all SU-like, <i>SU-like VRC26 sensitive</i> and <i>SU-like VRC26 early escape</i> viruses for both free virus and cell-cell transmission. Spearman correlation on untransformed data sets are shown with R and p values. Green fields denote positive, orange fields negative correlations. n.s. indicates no significance.</p

Co-evolution of CAP256 virus strains and the VRC26 bnAb lineage.

<p>Time line of the evolution of the CAP256 virus strains (PI-like, SU-like, PI/SU recombinants) and the VRC26 lineage bnAbs analyzed in the current study. Codes of bnAb and virus Env clones are indicated at the approximate time points of isolation. Classification of SU-like Env into <i>SU-like VRC26 sensitive</i>, <i>SU-like VRC26 early escape and SU-like VRC26 complete escape</i> is indicated.</p

Activity of autologous plasma against cell-cell transmission of CAP256 viruses is strongly driven by VRC26 bnAb activity.

<p><b>A:</b> 50% neutralizing titers (in plasma dilution) of autologous CAP256 plasma from week 145 p.i. against free virus (black) and cell-cell transmission (red) of CAP256 virus strains is depicted. Data are means of 2 to 3 independent experiments. Inhibition of <i>SU-like VRC26 sensitive viruses</i> and <i>SU-like VRC26 early escape</i> viruses in free virus and cell-cell transmission were compared using the Mann-Whitney test. P values are indicated in black and red respectively. <b>B:</b> The change in neutralization activity for cell-cell compared to free virus transmission is shown as the ratio of NT50^free-virus/NT50^cell-cell (Fold change Neutralizing titer). Zero denotes equal activity in both transmission pathways. A 10-fold higher activity in free virus over cell-cell transmission reflects a log₁₀ = 1, a 10-fold lower activity a log₁₀ = -1 as indicated by dotted lines. Viruses that were not neutralized to 50% at the half lowest plasma concentration were denoted as resistant (res.) and their fold changes were estimated. <b>C:</b> Interrelations of neutralizing titers for plasma and IC50s for bnAb neutralization were determined separately for PI-like and SU-like viruses during free virus and cell-cell transmission. <b>D:</b> Interrelations of virus infectivity in free virus and cell-cell transmission, IC50s and neutralizing titers (NT50) were determined for SU-like viruses. <b>C+D:</b> Spearman correlations on untransformed data sets were used, R and p values are indicated. Orange fields negative correlations. N.s. denotes no significance.</p

The first wave of broadly neutralizing antibodies targets residues in the V2 region.

<p><b>A</b>) Longitudinal neutralization of ConC V2 mutants. ConC wild-type (wt) is shown in red. V2 mutants F159A, N160A, R166A, K168A, K169E, K171A, and I181A that abrogated wave 1 neutralization are shown in purple. The D167N mutation that enhanced wave 1 neutralization is shown in orange, while the L165A mutation that resulted in universal neutralization sensitivity is shown in grey. The timing of wave 1 (red), wave 2 (green), and wave 3 (brown) neutralization is summarized above as horizontal lines, while the peak titers at each wave are indicated with dotted lines. ID₅₀ titers (y-axis) are shown versus weeks p.i. (x-axis). <b>B</b>) The dependence of CAP257 wave 1 neutralizing antibodies (at 67 weeks p.i.) on V2 residues in ConC, compared to monoclonal antibodies PGT145, CH01-04, and PG9/16. Complete abrogation of neutralization is colored red, 2–10 fold reductions in IC₅₀ are colored yellow, and >10 fold reductions in IC₅₀ are colored orange.</p

<i>SU-like sensitive</i> and <i>SU-like VRC26 early escape</i> viruses are differently neutralized post CD4 attachment.

<p><b>A</b>: Interdependencies of pre- or post-attachment activity and IC50 for free virus (left) or cell-cell neutralization (right). Spearman correlations on untransformed data sets with R and p values are shown. Neutralization data are derived from data sets depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006825#ppat.1006825.g002" target="_blank">Fig 2</a>, pre- and post- attachment data are derived from data sets depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006825#ppat.1006825.g007" target="_blank">Fig 7</a>. PI-like, <i>SU-like VRC26 sensitive</i> and <i>SU-like VRC26 early escape</i> viruses are marked in red, light blue and dark blue respectively. <b>B:</b> Pre- and post-attachment inhibition and loss in activity after CD4 attachment based on data depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006825#ppat.1006825.g007" target="_blank">Fig 7</a> were compared for <i>SU-like VRC26 sensitive</i> and <i>SU-like early escape</i> viruses using the Mann-Whitney test. P values are indicated.</p

CAP256-VRC26 bnAbs maintain neutralization activity against cell-cell transmission of autologous viruses.

<p><b>A</b> and <b>B:</b> 50% inhibitory concentrations (IC50 in μg/ml) of CAP256-VRC26 bnAbs against free virus (<b>A</b>) and cell-cell transmission (<b>B</b>) of indicated CAP256 virus strains are shown. Data are means of 2 to 4 independent experiments. Black lines depict the median IC50 of all sensitive antibody-virus combinations. <i>SU-like VRC26 sensitive</i> viruses were compared to <i>SU-like VRC26 early escape</i> viruses using the Mann-Whitney test. P values are indicated. <b>C:</b> The change in neutralization activity for cell-cell compared to free virus transmission is displayed as the log of ratio of IC50^cell-cell/IC50^free-virus (log fold change). Zero denotes equal activity in both transmission pathways. 10-fold higher (log₁₀ = 1) and lower (log₁₀ = -1) IC50 in cell-cell over free virus transmission are indicated by dotted lines. Black lines show median fold change IC50^cell-cell/IC50^free-virus for all sensitive VRC26/virus combinations. Different colors depict different bnAbs. <b>D+E:</b> Spearman correlation analyses of VRC26 bnAb neutralization in free virus and cell-cell transmission. R and p values are indicated. <b>D:</b> Neutralization activity in free virus and cell-cell spread (IC50 in μg/ml) of VRC26 bnAbs against autologous virus is tightly correlated (Spearman correlation, R: 0.9210, p<0.0001). <b>E:</b> Loss in cell-cell neutralization activity is associated with higher resistance of viruses to VRC26 bnAbs in the free virus pathway (Spearman correlation, R: -0.5369, p<0.0001).</p