Fusion loop- and A strand-specific modified MAbs can compete for binding with fusion loop-specific MAbs of lesser neutralizing potency.

<p><b>A.</b> Fusion loop-specific mouse MAb 4G2 was incubated at 1 µg/mL with MAbs E60, 87.1 or E87 at 10, 1 or 0.1 µg/mL human MAb prior to addition to DENV2-virion coated plates (for each MAb concentration, data is represented as mean +/− SEM). Anti-mouse, Fc-specific secondary MAb was then added, followed by PNPP substrate. Optical density (OD) values are shown on the y-axis and were calculated after subtracting the average background (binding of mouse Fcγ-chain specific secondary antibody in the absence of 4G2) from the raw OD. Statistically significant differences in 4G2 binding across the different human MAb concentrations were calculated using a Kruskal-Wallis analysis from triplicate values within each experiment. This data shown is representative of seven independent experiments. <b>B–D.</b> MAb 4G2 was pre-mixed with MAb E60 N297Q, MAb 87.1 LALA or MAb E87 N297Q in ratios of 95% 4G2/5% modified MAb (<b>B</b>), 85% 4G2/15% modified MAb (<b>C</b>), or 75% 4G2/25% modified MAb (<b>D</b>). For each 4G2/modified MAb mixture, a Gaussian distribution was used to fit the enhancement curve (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003157#ppat.1003157.s002" target="_blank">Figure S2</a></b>). The area under the curve (AUC) was calculated for each curve, and relative infection was expressed by dividing the AUC in the presence of modified MAbs by the AUC measured with 4G2 (no modified MAb) only. The data displayed are the average of three to seven independent experiments +/− SEM. Comparisons between the MAb combinations E87 N297Q/4G2 and E60 N297Q/4G2 or 87.1 LALA/4G2 were performed using a Kruskal-Wallis test. <b>E.</b> AG129 mice (n = 3–6 per group from one or two experiments) were administered an enhancing quantity (20 µg) of 4G2 MAb, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAb. A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.</p

<i>In vitro</i> characteristics of intact and modified MAbs.

a<p>The data presented is the average of two to five replicates of duplicate measures.</p>b<p>The data presented is the average of two to four replicates of duplicate measures.</p>c<p>“<i>In vivo</i> therapeutic outcome” refers to lethal, virus-only DENV2 D2S10 challenge or mouse anti-DENV1-enhanced DENV2 D2S10 infection (ADE).</p>d<p>“NN” indicates that the MAb did not neutralize DENV2 D2S10.</p>e<p>“NB” indicates that the MAb did not bind to DENV2 D2S10 by direct capture ELISA.</p>f<p>“ND” indicates that the avidity for MAb 4G2 was not tested via direct capture ELISA.</p>g<p>“ND” indicates that the therapeutic efficacy for these MAbs was not assessed under the conditions specified in footnote “c”.</p

Anti-DENV MAbs are therapeutic following a virus-only or antibody-enhanced lethal infection.

<p><b>A.</b> Ribbon diagram of the DENV2 E protein homodimer (PDB ID code 1OAN) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003157#ppat.1003157-Modis1" target="_blank">[23]</a>. EDI is red, EDII is yellow and EDIII is blue. The epitopes targeted by MAbs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003157#ppat-1003157-t001" target="_blank">Table 1</a> include the fusion loop (green), dimer interface (white), C-C′ loop (orange) and A strand (magenta). <b>B.</b> AG129 mice were administered a lethal dose of DENV2 D2S10 and 24 hours later were treated with 20 µg of modified MAbs (n = 5 per group from 2 independent experiments). <b>C.</b> AG129 mice were administered an enhancing dose of polyvalent DENV1-immune mouse serum, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAbs. (n = 3–19 per group from at least 2 independent experiments for each modified MAb). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003157#ppat.1003157.s004" target="_blank">Table S1</a> for numbers of mice in each group. A Kaplan-Meier survival curve is shown (<b>B–C</b>), and log-rank analysis was used for statistical comparison.</p

A high ratio of modified to non-modified MAb is necessary to prevent enhancement <i>in vitro</i> and <i>in vivo</i>.

<p><b>A–B.</b> Non-modified and modified MAbs E60/E60 N297Q (<b>A</b>) and 87.1/87.1 LALA (<b>B</b>) were pre-mixed at ratios of 100% intact MAb, 75% intact:25% modified MAb, 50% intact:50% modified MAb, 25% intact:75% modified MAb and 10% intact:90% modified MAb. The data is plotted as the average of duplicate values where the absolute percent infection of K562 cells is shown on the y-axis. This data is representative of two or three independent experiments. <b>C.</b> AG129 mice (n = 3 per experimental group and n = 5 for non-treated control group) were administered a polyvalent DENV1-immune enhancing mouse serum, infected with DENV2 D2S10, and 24 hours later treated with a total of 20 µg of E60/E60 N297Q MAbs in the same combinations tested <i>in vitro</i> in (<b>A</b>). A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.</p

<i>In vitro</i> suppression-of-enhancement assay correlates with therapeutic efficacy of MAbs <i>in vivo</i> with enhancing polyvalent DENV-immune serum from humans.

<p><b>A.</b> The PENT (1∶540) for DENV4-immune human serum was determined in K562 cells. <b>B.</b> DENV4-immune human serum diluted 1∶540 was incubated with modified MAbs at 1,000 ng/mL. Relative infection was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003157#ppat-1003157-g005" target="_blank"><b>Figure 5</b></a>. The data displayed are combined from five independent experiments, and the mean +/− SEM is displayed for each MAb. A sign rank test was used to determine whether relative infection with each modified MAb was significantly lower than relative infection of 0.5 (50% infection), * <i>P</i><0.05, ** <i>P</i><0.08. <b>C.</b> AG129 mice (n = 3 per experimental group and n = 6 for non-treated control group) were administered an enhancing dose of DENV4-immune human serum, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAbs. A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.</p

<i>In vitro</i> suppression-of-enhancement assay predicts therapeutic efficacy of MAbs <i>in vivo</i> with enhancing polyvalent DENV-immune serum from mice.

<p><b>A.</b> The peak enhancing titer (PENT = 1∶180) for DENV1-immune mouse serum was determined in K562 cells. <b>B.</b> DENV1-immune mouse serum was diluted 1∶180 (PENT) and incubated with modified MAbs at six 2-fold dilutions beginning at 2,000 ng/mL. Relative infection was calculated by dividing the percent infection in the presence of modified MAbs by the percent infection measured with mouse DENV1-immune serum alone. The data displayed are the average of duplicate values and are representative of four independent experiments. A † indicates modified MAbs that are statistically therapeutic <i>in vivo</i> following mouse DENV1-enhanced, lethal DENV2 infection. <b>C.</b> The average infection across four experiments at 1,000 ng/mL of modified MAb (mean +/− SEM shown for each MAb). <i>P</i><0.04 was obtained when comparing the average relative infection values for therapeutic to non-therapeutic MAbs using a Wilcoxon rank-sum analysis. The solid line indicates relative infection of 0.5 (50% infection).</p

Class averages of radial averaged particle images of the WNV/E16 Fab complex at pH 6.0.

<p>The classification suggests the presence of at least four different radial expansion intermediates that represent advancing stages of the E protein layer expansion (IM-1 through 4). The number of images grouped in each class is indicated in percent below the respective class average. Most of the particles clustered in expansion stage IM-4, in which the outer protein layer, presumably composed of E and Fab molecules, reaches its maximum radius separated by an about 60 Å-wide gap from the outer lipid leaflet. A representative class average of the complex at pH 8 is shown on the left. The red lines indicate the radial limits of the nucleocapsid core (NC), the lipid bilayer (LB), the E protein layer (E) and the Fab molecules (Fab) in the pH 8 structure. Areas of high density are depicted with black pixels, low density areas are shown in white.</p

CryoEM image reconstruction of WNV in complex with E16 antibody fragments at pH 8.

<p>(A) Stereoscopic view of a surface rendering of WNV (green) complexed with E16 Fab (blue) at 23 Å resolution <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000672#ppat.1000672-Kaufmann1" target="_blank">[14]</a>, viewed down an icosahedral twofold axis. The black triangular outline identifies an icosahedral asymmetric unit. (B) Same as A, but depicting the complex of WNV and E16 scFv. E16 scFv binds WNV in a similar fashion as E16 Fab. Only two of three epitopes in the asymmetric unit are utilized. The fivefold proximal epitope is occluded because of steric hindrance.</p

CryoEM images of WNV, alone or in complex with E16 Fab, in different pH environments.

<p>(A) WNV/Fab complex at pH 8. (B) WNV/Fab complex at pH 6. Acidification triggered an expansion of the virus particles that resulted in a halo-effect around the dense nucleocapsid core. Inset: Examples of back-neutralized particles showing the irreversibility of the low-pH induced changes. (C) WNV at pH 8. (D) WNV at pH 6. Strong particle aggregation was detected when WNV was exposed to low pH in the absence of E16. Acid-induced structural changes similar to (B) were observed for some virions at the edge of the aggregates, as indicated by the arrow. The scale bars represent 500 Å.</p

Equatorial slices of cryoEM image reconstructions of WNV in complex with E16 antibody fragments in different pH environments.

<p>(A) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 8, rendered at 23 Å resolution. Red arcs (1 through 5) specify the outer radii of the nucleocapsid core (154 Å), lipid bilayer (205 Å), E glycoprotein shell (247.5 Å), scFv molecules (278 Å), and Fab fragments (318.5 Å) from the viral center. The positions of the icosahedral two-, three- and fivefold axes are indicated with black arrows and numbers. (B) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 6, rendered to 25 Å resolution. The red arcs (6 and 7) specify the outer radius of the expanded E/scFv (317.5 Å) and E/Fab (347 Å) protein layer, respectively. The low pH triggered radial expansion of the E/scFv or E/Fab protein shell resulted in a ∼60 Å wide shell of low density between the lipid bilayer and the expanded outer protein layer, as indicated by the green arrow. The scale bars represent 100 Å.</p