Determination of % anti-FL Abs in serum of a dengue patient by VLP-capture ELISA.

<p>(A) Serial dilutions of the serum were subjected to a capture ELISA using DENV1 WT and mutant VLPs containing mutations in the FL epitope (W101A+F108A). The bar graph displaying results from an anti-E ELISA shows that comparable amounts of WT and mutant VLPs were added based on recognition of E by pooled human dengue-immune sera. % anti-FL Abs = [1 – endpoint titer to mutant VLPs/endpoint titer to WT VLPs]×100%. (B) The same serum was subjected to a capture ELISA using DENV2 WT and mutant VLPs (W101A+F108A). Data are presented as in (A). Data are means with standard deviation of duplicates from one representative experiment of two. For endpoint titers, only means are shown.</p

Relationship between [anti-FL Abs] and NT₅₀ against current infecting and “non-exposed” serotypes in secondary DENV infection.

<p>The current infecting serotype, previous infecting serotype(s) and “non-exposed serotypes” were determined as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#s2" target="_blank">Methods</a>. [anti-FL Abs] and NT₅₀ against the current infecting serotype (A, E) and “non-exposed” serotypes (B to D, F to H) in patients with secondary DENV3 (A to D) and secondary DENV2 (E to H) infections.</p

Measurement of [anti-E Abs] in human sera by capture ELISA.

<p>(A) Virion-capture ELISA was performed using serial dilutions of a human mAb 82.11 with known <b>c</b>oncentrations to generate a standard curve. (B) Sequential human serum samples from a dengue patient were tested simultaneously with the standard and the OD values were interpolated to determine [anti-E Abs]. Data are means with standard deviation of duplicates from one representative experiment of two. (C) The binding specificity of mAb 82.11 was determined by Western blot analysis using cell lysates collected from Vero cells infected with mock, DENV1 (Hawaii strain), DENV2 (NGC strain), DENV3 (H87 strain), DENV4 (H241 strain) or WNV (NY99 strain) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#pntd.0002451-Lin1" target="_blank">[16]</a>. The NT₅₀<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#pntd.0002451-Beltramello1" target="_blank">[25]</a> and dissociation constant (Kd) of mAb 82.11 are summarized on the right. The viron-capture ELISA of DENV1, DENV2 and DENV3 was performed using serial dilutions of mAb 82.11; the Kd was determined using the program GraphPad Prism 5.0. Data are means of duplicates from one representative experiment of two. ND, not done.</p

Kinetics of [anti-E Abs] and [anti-FL Abs] in sera of dengue patients.

<p>(A) [anti-E Abs] and (B) [anti-FL Abs] in samples collected 3, 6, 12 and 18 months post-infection. Green closed symbols, patients with primary DENV infection; red open symbols, patients with secondary DENV infection.</p

Anti-DENV2 does not enhance D2S10 infection <i>in vitro</i>.

<p>Neither purified anti-DENV2 IgY nor control purified IgY enhanced DENV2 D2S10, whereas control MAb E60 generated ~15% enhancement at the peak enhancing titer. Percent infection is shown on the y-axis and log reciprocal antibody concentration on the x-axis. These data are representative of two independent experiments.</p

Concentration of anti-E Abs and anti-FL Abs and proportion of anti-FL Abs in sequential serum samples from 10 dengue cases.

a<p>Primary or secondary DENV infection was determined as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#s2" target="_blank">Methods</a>.</p>b<p>The current infecting serotype was determined as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#s2" target="_blank">Methods</a>. With the exception of two DHF/DSS cases (274, 233), all others were DF cases. D = DENV.</p>c<p>Sampling time was determined relative to onset of fever.</p>d<p>[anti-E Abs], [anti-FL Abs] and % anti-FL Abs were determined as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#s2" target="_blank">Methods</a>.</p>e<p>Below the limit of detection (BD). The limit of detection of % anti-FL Abs is 4% as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002451#s2" target="_blank">Methods</a>.</p

Anti-DENV2 IgY antibody characterization.

<p><b>A)</b> Comparison of rabbit-derived IgG and goose-derived IgY isolated using mercapto-ethyl pyridine (MEP) HyperCel Hydrophobic Charged Induced Chromatography (HCIC) sorbent. Rabbit IgG was isolated from rabbits that were immunized with goose IgY. Anti-DENV2 goose IgY was isolated from egg yolks of geese immunized with DENV2 killed antigen. Separation of full length IgY and alternatively spliced IgY occurred prior to HCIC using differential ammonium sulfate precipitation (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005721#sec002" target="_blank">methods</a>). Lane 1, molecular weight marker, Lane 3, rabbit IgG anti IgY; lane 7. IgY full length; lane 8, IgY alternatively spliced and lane 9, IgY full length and alternatively spliced (2 bands). <b>B)</b> Egg yolks were collected from geese immunized with killed DENV2 virus. Arrows indicate boost immunizations at 2 and 4 weeks post-first immunization. Anti-dengue IgY antibody titer did not differ among weeks (ANOVA on ranks, p = 0.157). Data are presented as mean ± SE. N = 4, 6, 22, 24, 21, and 27 at 2, 3, 4, 5, 6, and 7 weeks post vaccination respectively.</p

Identification of DENV2 epitopes recognized by IgY.

<p>The amino acid sequences of four DENV proteins (E, NS1, NS3, and prM) were used to generate 15-mer overlapping peptides that were spotted onto microarray slides. Slides were incubated with alternatively spliced anti-DENV2 full-length anti-DENV2 IgY, or naïve IgY. Reactivity is measured based on a graded color scale ranging from no reactivity in black to high reactivity (based on highest value) in red. Values represent MFI minus background. Selected peptide amino acid sequences are listed in the order that they would appear in the full-length protein. Not all peptides are included for each protein.</p

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