Comparison of bnAb escape mutant occurrence probabilities via cell-cell versus free virus transmission.

<p>Graphs display how much more likely it is that an escape variant arose via the cell-cell pathway in comparison to free virus spread. The analysis was performed for all tested bnAb-virus pairs. Light grey areas indicate the concentrations for which an escape variant is more likely to be formed during cell-cell spread. The dark grey areas correspond to the concentration range that favours formation during free virus spread.</p

Interplay of bnAb features that shape neutralization potency.

<p>Differential features shape the neutralization activity of bnAbs. Two main reactivity patterns can be observed: (i) BnAbs with high potency against free virus spread and high pre-attachment activity. (ii) BnAbs with lower efficacy against free virus spread, decreased pre-attachment but high post-CD4 attachment activity and retained activity during cell-cell transmission.</p

T-20 potently inhibits free virus and cell-cell transmission across diverse virus strains.

<p>Inhibition of free virus (black circles) and cell-cell (red circles) transmission of subtype A, B and C viruses by T-20 is shown. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope).</p

The capacity to neutralize free viruses prior to completion of CD4 attachment varies across bnAb classes.

<p><b>A:</b> Schematic of free virus infection of A3.01-CCR5 target cells to measure total and pre-attachment activity of bnAbs. Total activity of each bnAb-virus combination (where bnAbs were present throughout the infection process) was set to 100% and pre-attachment inhibition displayed relative to it. To assess pre-attachment activity, bnAbs were removed following attachment by spinoculation. <b>B: Pre-attachment neutralization activity is strain- and epitope-dependent.</b> Pre-attachment activity (relative, %) of the bnAb classes CD4bs, V3 glycans, V1V2 loop, MPER was assessed against the indicated virus strains. Data are means of two to three independent experiments. <b>C: Pre-attachment activity correlates with high potency against free virus, decreased post- attachment inhibition capacity and loss in activity during cell-cell transmission.</b> Summary of the interdependencies of pre-attachment, post-attachment inhibition, IC₅₀ for free virus inhibition, IC₅₀ for cell-cell inhibition and loss in activity during cell-cell inhibition. Correlations were determined by Spearman correlation based on the untransformed data sets and are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s013" target="_blank">S8 Fig</a>. R and p values are depicted. n.s. indicates a non-significant correlation. Direct correlation is denoted in blue, inverse correlation in orange.</p

Evaluating A3.01-CCR5 T cell-based free virus and cell-cell transmission.

<p><b>A to F:</b> As indicated, the Gaussia Luciferase reporter virus NLinGluc and the firefly luciferase reporter virus system NLlucAM were used to study free virus and cell-cell transmission. Infectivity was measured by assessing Gaussia and firefly luciferase reporter activity in the supernatant and from the lysed cells, respectively. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates. Panels C-F depict curve fits to sigmoid dose response curves (variable slope). <b>A: DEAE dependency of the NLinGluc reporter virus.</b> JR-FL NLinGluc free virus was titrated on TZM-bl cells in 96-well plates in presence (black circles) or absence (green squares) of 10 µg/ml diethylaminoethyl (DEAE). <b>B: Absence of Gaussia luciferase activity in 293-T donor cells but high signal in 293-T-A3.01-CCR5 co-cultures allows for specific detection of cell-cell transmission.</b> 293-T cells transfected with JR-FL <i>env</i> and NLinGluc reporter virus were titrated and incubated in presence of 1.5*10⁴ A3.01-CCR5 target cells per 96 plate culture well (+A3.01, black circles) or in medium alone (-A3.01, green diamonds) in the absence of DEAE. <b>C: Assessing the sensitivity to antiretrovirals to validate free virus and cell-cell assays.</b> Testing the sensitivity of A3.01-CCR5 infection by free virus (JR-FL NLlucAM firefly luciferase reporter virus; left) and cell-cell (JR-FL NLinGluc transfected 293-T; right) transmission to the RT inhibitor Zidovudine (black circles) or the protease inhibitor Atazanavir (open circles) to verify that both pathways are sensitive to RT inhibition but only cell-cell transmission is affected by protease inhibition. <b>D: NLinGluc (Gaussia) and NLlucAM (firefly) luciferase reporter viruses yield comparable results in free virus inhibition assays.</b> Free virus inhibition of JR-FL NLinGluc (yellow circles) and JR-FL NLlucAM (black circles) reporter viruses by bnAbs VRC01 and 2F5 was compared. For both viruses, 100% infectivity was determined in cultures without inhibitor. <b>E: Equal neutralization sensitivity of free virus infection on A3.01-CCR5 and PBMC target cells.</b> Inhibition of free virus infection of PBMC (blue squares) or A3.01-CCR5 (black circles) by bnAbs VRC01 and 2F5 was studied using the firefly reporter virus JR-FL NLlucAM. For both cell types 100% infectivity was determined in cultures without inhibitor. <b>F: Equal sensitivity to neutralization in 293-T-A3.01-CCR5 and PBMC-PBMC cell-cell transmission.</b> The capacity of bnAbs VRC01 and 2F5 to block cell-cell transmission was assessed in co-cultures of JR-FL NLinGluc-transfected 293-T with A3.01-CCR5 (black circles) and JR-FL infected PBMC with rhTRIM5α-transduced PBMC (blue squares). Infectivity was assessed via determination of Gaussia luciferase activity in the 293-T-A3.01-CCR5 co-cultures or via intracellular p24 staining and flow cytometry analysis for the PBMC co-cultures. For both co-cultures, 100% infectivity was determined in cultures without inhibitor.</p

Unique properties of bnAbs CAP256-VRC26.08 and CAP256-VRC26.09.

<p><b>A: Comparable potency of bnAbs CAP256-VRC26.08 and CAP256-VRC26.09 during free virus and cell-cell neutralization.</b> Inhibition of free virus (black circles) and cell-cell (red circles) transmission of the indicated virus strains by bnAbs CAP256-VRC26.08 and CAP256-VRC26.09 is shown. The graphs depict means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope). <b>B-C: Comparison of inhibitory activity of CAP256-VRC26.08 and CAP256-VRC26.09</b>. <b>B:</b> Comparison of IC₅₀ values of free virus and cell-cell inhibition determined in <b>(A)</b> Subtype A and C viruses are denoted in blue and orange, respectively. <b>C:</b> Comparison of the change in inhibitory activity in cell-cell transmission relative to free virus inhibition (fold change IC₅₀). <b>D: Comparison of the capacity to neutralize post-CD4 receptor engagement.</b> Activity of bnAbs added after attachment of virions to A3.01-CCR5 target cells was assessed and compared to samples, where bnAbs were present before and after attachment (total activity, set to 100% inhibition). Post- attachment inhibition is displayed relative to the total inhibition activity. Subtype A and C virus are denoted in blue and orange, respectively. One representative of two independent experiments is shown. <b>E: Comparison of the capacity to neutralize pre-CD4 receptor engagement.</b> The relative activity of bnAbs in the pre-attachment phase compared to total activity (bnAbs present before and after attachment) was assessed. Data are means of two to three independent experiments.</p

The capacity to neutralize post-CD4 receptor engagement differs between bnAbs and virus strains.

<p><b>A, B:</b> Schematic of free virus infection of A3.01-CCR5 target cells to measure total and post-attachment activity of bnAbs. <b>C:</b> Total activity of each bnAb-virus combination (where bnAbs were present throughout the infection process) was set to 100% and post-attachment inhibition displayed relative to it. Subtype A, B and C viruses are denoted in blue, black and orange, respectively. Mean values of two to three independent experiments are shown.</p

Decrease of bnAb activity during cell-cell transmission is variable and strain-dependent.

<p><b>A: Diverse pattern of bnAb activity loss within and across viral strains.</b> Inhibition of free virus (black circles) and cell-cell (red circles) transmission of subtype B virus strains PVO.4 and THRO by the indicated bnAbs was studied. The graphs show means and standard error of means (SEM, error bars) of two to three independent experiments performed in duplicates and curve fits to sigmoid dose response curves (variable slope). <b>B, C: Inhibitory activity of bnAbs in free virus and cell-cell transmission of divergent virus subtypes.</b> 50% inhibitory concentrations (IC₅₀ in µg/ml) of bnAbs against a panel of subtype A (blue), B (black) and C (orange) virus strains in free virus <b>(B)</b> and cell-cell <b>(C)</b> transmission. Graphs depict IC₅₀ values of all bnAb-virus pairs for which the virus proved sensitive. Non-sensitive combinations are recorded in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s003" target="_blank">S3 Table</a>. Grey bars depict median IC₅₀ values for all virus strains sensitive to a given bnAb. <b>D: bnAbs predominantly lose activity in cell-cell transmission.</b> The change in inhibitory activity in cell-cell transmission compared to free virus inhibition is expressed as the ratio of IC₅₀ cell-cell/IC₅₀ free virus (fold change IC₅₀). Subtype A, B and C viruses are denoted in blue, black and orange, respectively. A fold change IC₅₀ of 0 indicates an equal activity during free virus and cell-cell transmission (black line). Median fold change IC₅₀ (grey bars) for all virus strains sensitive to a given bnAb are shown.</p

Differential loss in cell-cell neutralization across bnAb classes.

<p>Interdependencies of inhibitory activity (IC₅₀) against free and cell-cell transmitted virus <b>(A)</b> and the fold change in IC₅₀ between free and cell-cell transmission <b>(B)</b> was determined for individual bnAb classes and all classes combined by Spearman correlation based on the untransformed data sets. R and p values are depicted. n.s. indicates a non-significant correlation. Data are derived from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.g002" target="_blank">Fig 2</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s005" target="_blank">S5A–S5P Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004966#ppat.1004966.s003" target="_blank">S3 Table</a>. Subtype A, B and C viruses are denoted in blue, black and orange, respectively.</p

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