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

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

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

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

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

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

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

Mode of virus transmission differentially steers susceptibility to entry inhibition.

<p><b>(A) DEAE-Dextran is not required for effective cell-cell transmission of HIV-1_JR-FL to TZM-bl cells.</b> Serial dilutions of JR-FL infected PBMC were incubated with TZM-bl cells in presence (black circles) or absence (red circles) of 10 µg/ml DEAE-Dextran. Infectivity was measured by enzymatic activity of the luciferase reporter (relative light units (RLU)). Each infected cell input was probed in triplicate. Error bars represent SD (standard deviation). One of four independent experiments is shown. <b>(B) Omission of DEAE-Dextran as media supplement abolishes cell-free JR-FL infection of TZM-bl cells.</b> Serial dilutions of cell-free JR-FL virus were used to infect the luciferase reporter cell line TZM-bl in presence (black squares) or absence (red squares) of 10 µg/ml DEAE-Dextran. Infectivity was measured by induction of the luciferase reporter (relative light units (RLU)). Each virus dilution was probed in quadruplicates. Bars represent SD . One of four independent experiments is shown. <b>(C) Cell-cell transmission but not enforced contact between virus and target cell overcomes entry restriction.</b> The infectivity of cell-free virus without enforced attachment to TZM-bl target cells (gravity sedimentation), or upon spinoculation, magnetic bead virus adsorption and during cell-cell transmission was assessed in presence (solid lines) or absence (dotted lines) of 10 µg/ml DEAE-Dextran. Infection was determined by measuring luciferase production after 48 h (recorded as RLU). Each virus dilution was probed in duplicates. Bars represent SD. One of three independent experiments is shown. <b>(D) Inhibitory profiles of CD4-IgG2 and T-20 during cell-cell and cell-free transmission.</b> TZM-bl target cells were either cocultivated with JR-FL infected PBMC (red circles, no DEAE) or cell-free virus (black squares, with 10 µg/ml DEAE) in the presence of increasing doses of CD4-IgG2 (left panel) or T-20 (right panel). Infection was determined by measuring luciferase production after 48 h and recorded as RLU. Red and black values denote IC50 (nM) of during cell-cell and cell-free transmission, respectively. Data points represent means of duplicates from three independent inhibition experiments. Bars represent SEM. Lines depict fitted dose response curves. <b>(E) Decreased CD4-IgG2 sensitivity during cell-cell transmission is due to an inherent feature of cell-cell transmission.</b> TZM-bl target cells were mixed with replication competent infected JR-FL^rc PBMC in the presence of CD4-IgG2 or T-20 (red bars) in medium lacking DEAE Dextran. Cell-free JR-FL^rc was either spinoculated (hatched bars), adsorbed by magnetic beads (checkered bars) or added without enforced adsorption (grey bars) onto TZM-bl target cells in medium containing DEAE Dextran in the presence of the inhibitor. Fold increases in IC50 of cell-cell compared to cell-free infection are indicated on top of the respective bars. Bars depict means of three independent experiments in duplicates. Lines denote SD. Inhibition of cell-cell transmission by CD4-IgG2 and T-20 (red bars) was significantly less efficient than blocking of cell-free virus (grey bars) infection (Student t-test, p<0.0001 in both cases). <b>(F) Single round infection is highly resistant to CD4-IgG2 inhibition during cell-cell transmission.</b> TZM-bl target cells (no DEAE) were co-cultivated with JR-FL pseudovirus transfected 293-T cells in the presence of CD4-IgG2 or T-20. Cell-free JR-FL^pp-lucAM was added to the TZM-bl (with 10 µg/ml DEAE) in the presence of both inhibitors. Fold increases in IC50 of cell-cell compared to cell-free infection are indicated on top of the respective bars. Bars depict means of three independent experiments performed in duplicates. Lines denote SD. Inhibition of cell-cell transmission by CD4-IgG2 and T-20 (red bars) was significantly less efficient than blocking of cell-free virus (grey bars) infection (Student t-test, p<0.0001 in both cases).</p