Effect of virion maturation state on neutralization sensitivity of WNV E T198F RVPs.

<p><b>(A)</b> We assessed the level of V5-tagged prM in WNV RVPs prepared using standard (Std) conditions, or those increasing the efficiency of prM cleavage (Furin) by SDS-PAGE and Western blotting of pelleted virions using a mouse monoclonal antibody against V5 (top panel). The level of E protein (bottom panel), as detected by mouse monoclonal antibody, 4G2, was used as a loading control for each RVP preparation. Data are representative of three independent experiments performed with independent RVP preparations. <b>(B)</b> We concurrently tested Std and Furin preparations of WT and T198F RVPs for sensitivity to neutralization by mAb E60. Representative dose-response neutralization curves are shown, with the y- and x-axes representing percent infectivity and mAb concentration, respectively. Infectivity was normalized to levels observed in the absence of antibody. Error bars indicate the range of infectivity from duplicate infections. <b>(C)</b> Mean EC50 values for E60 against Std or Furin preparations of WT and T198F RVPs. Error bars represent the SEM. The indicated p-values were obtained from paired t-tests. <b>(D)</b> Mean percentages of Std or Furin WT and T198F RVPs resistant to neutralization at the highest concentration of E60 tested (10 μg/ml). Error bars represent the SEM. The indicated p-values were obtained from paired t-tests. For <b>(C)</b> and <b>(D)</b>, mean values were obtained from three independent experiments performed in duplicate using independent RVP preparations.</p

Neutralizing activity of WNV-immune sera.

<p>Sensitivity of WNV WT and T198F RVPs to neutralization by 2-mercaptoethanol (2-ME)-treated or untreated (unt) sera pooled from WT-immune (n = 5) <b>(A and C)</b> or T198F-immune (n = 5) <b>(B and D)</b> five-week old WT C57BL/6J mice. Sera were obtained at 6 <b>(A and B)</b> and 9 <b>(C and D)</b> days post-infection. IgG purified from pooled <b>(E)</b> WT-immune (n = 5) or <b>(F)</b> T198F-immune (n = 5) sera obtained from 9 days post-infection were tested for the ability to neutralize WNV WT and T198F RVPs. Error bars indicate the range of infection from duplicate wells. Data in <b>(A-F)</b> are representative of four independent experiments performed using independent RVP stocks.</p

Effect of amino acid chemistry on neutralization sensitivity of WNV E T198 RVP variants.

<p>We tested WT WNV RVPs containing threonine at E residue 198 (black curves) for sensitivity to neutralization by mAbs <b>(A)</b> E16 and <b>(B)</b> E60 concurrently with RVPs incorporating amino acid variants representing distinct chemical groups at residue 198, including aromatic (F; grey), small (A; red), nucleophilic (C, S; cyan), hydrophobic (L, M; magenta), acidic (D; orange), basic (K; blue), and amide (N; green). Error bars indicate the range of infectivity from duplicate infections. Percent infectivity versus mAb concentration is shown in each graph. Infectivity was normalized to levels observed in the absence of antibody. We compared the mean EC50 values for <b>(C)</b> E16 and <b>(D)</b> E60 against each variant to those of WT RVPs using a one-way ANOVA followed by Dunnett’s multiple comparisons test. Data were obtained from three independent experiments performed in duplicate. Error bars indicate the SEM. The color scheme for distinct amino acid chemical groups corresponds to that in <b>(A)</b> and <b>(B)</b>. *, p<0.05.</p

Stability of WNV E T198F RVPs.

<p><b>(A)</b> Intrinsic decay of the infectivity of WT and T198F RVPs. Standard or Furin RVP preparations were equilibrated at 37°C for 1 h and further incubated for additional lengths of time as indicated on the x-axis, after which aliquots were harvested and frozen. Samples from each time point were thawed concurrently and used to infect Raji-DC-SIGN-R cells. Data were normalized to the infectivity of RVPs incubated at 37°C for 1 h and fitted to a one-phase exponential decay curve. Representative decay curves for WT and T198F RVPs are shown. Error bars indicate the SEM from triplicate infections. <b>(B)</b> Paired t-tests were used to compare the half-life of infectivity of WT and T198F RVPs. Shown are mean half-life values obtained from three independent experiments performed in triplicate. Error bars represent the SEM.</p

Kinetic aspects of neutralization of WNV E T198F RVPs.

<p>Furin WT and Furin T198F WNV RVPs were incubated with mAb E16 (top panel) or E60 (bottom panel) for 1 h at room temperature and were either immediately used to infect Raji-DC-SIGN-R cells or were further incubated at 37°C for additional lengths of time as indicated above the graphs prior to the addition of Raji-DC-SIGN-R cells. Percent infectivity versus mAb concentration is shown in each graph. Infectivity was normalized to levels observed in the absence of antibody. Error bars indicate the range of infectivity from duplicate infections. Neutralization curves shown are representative of five independent experiments performed in duplicate.</p

<i>In vivo</i> effects of WNV E T198F.

<p>Five-week old WT C57BL/6J mice were inoculated subcutaneously with 10² FFU of WT (n = 15) or T198F (n = 15) WNV and monitored for <b>(A)</b> survival and <b>(B)</b> weight loss. Data are pooled from three independent experiments. Error bars in <b>(B)</b> represent the standard deviation. <b>(C)</b> Nine- to ten-week old WT C57BL/6J mice were injected via an intraperitoneal route with 0.5 mg each of blocking antibody against mouse IFN-α/β receptor (MAR1-5A3, n = 10) or an isotype control antibody against human IFN-γ receptor 1 (GIR-208, n = 10) one day prior to subcutaneous inoculation with 10² FFU of WT or T198F WNV. Mice were monitored for survival up to 21 days post-infection. Data are pooled from two independent experiments. <b>(D)</b> Eight-week old μMT mice were inoculated subcutaneously with 10² FFU of WT (n = 8) or T198F (n = 8) WNV and monitored for survival. Data are pooled from two independent experiments. <b>(E)</b> Five-week old WT C57BL/6J mice were infected with WT (n = 6) or T198F WNV (n = 6) as in <b>(A)</b>. Serum samples were collected on days 2 and 4 post-infection and viral burden was quantified by qRT-PCR. Horizontal lines across data points indicate the median viral burden. <b>(F)</b> Determination of infectious virus titer in the serum and spleen of five-week old WT C57BL/6J mice infected with WT (n = 8) or T198F (n = 8) virus at day 4 post-infection by plaque assay on Vero or BHK21 cells, respectively. Horizontal lines across data points indicate the median viral titer. <b>(G)</b> Five-week old WT C57BL/6J mice were infected with WT (n = 8) or T198F WNV (n = 8) as in <b>(A)</b>. Infectious titer in the brain at 6 and 8 days post-infection was determined by plaque assay on BHK21 cells. Horizontal lines across data points indicate the median viral titer. Data in <b>(E-G)</b> were pooled from two independent experiments. The p-values shown in <b>(A)</b> and <b>(D)</b> were obtained from a log-rank test; those in <b>(E-G)</b> were obtained by a Mann-Whitney test.</p

Effect of natural antibodies on WNV intrinsic decay.

<p>Representative intrinsic decay curves of fully infectious WT and T198F viruses in serum obtained from <b>(A)</b> naïve WT C57BL/6J mice, <b>(B)</b> naïve μMT C57BL/6J mice, and <b>(C)</b> media. Experiments were performed as described in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006178#ppat.1006178.g004" target="_blank">Fig 4A</a></b>. Error bars indicate the SEM from triplicate infections. <b>(D)</b> Average half-life values of WT and T198F viruses following prolonged incubation in serum obtained from naïve WT mice, naïve μMT mice, or media obtained from two independent experiments performed using independent serum samples and virus preparations. Error bars indicate the SEM. Fold-differences in half-life between WT and T198F WNV in each incubation condition are indicated.</p

Characteristics of DENV1 E F193T and ZIKV E F198T RVPs.

<p><b>(A)</b> We assessed the efficiency of prM cleavage of standard (Std) preparations of WT and F193T DENV1 RVPs by SDS-PAGE and Western blotting of pelleted virions using a mouse prM-reactive mAb (top panel). The level of E protein, as detected using mouse mAb 4G2, was used as a loading control (bottom panel). Data are representative of three independent experiments performed using independent RVP preparations. <b>(B)</b> Representative dose-response neutralization curves for Std DENV1 WT and F193T RVPs against mAb E60. Percent infectivity versus mAb concentration is shown. Error bars indicate the range of infectivity from duplicate infections. <b>(C)</b> Comparison of mean EC50 values of E60 against Std DENV1 WT and F193T RVPs using a paired t-test. Values were obtained from five independent experiments performed in duplicate. Error bars indicate the SEM. <b>(D)</b> Representative intrinsic decay curves of Std DENV1 WT and F193T RVPs. Experiments were performed as described in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006178#ppat.1006178.g004" target="_blank">Fig 4A</a></b>. Percent infectivity versus hours of incubation is shown. Error bars indicate the SEM obtained from triplicate infections of Raji-DC-SIGN-R cells. <b>(E)</b> Comparison of the mean half-life of infectivity of Std DENV1 WT and F193T RVPs using a paired t-test. Values were obtained from five independent experiments performed in triplicate. Error bars indicate the SEM. <b>(F)</b> Intrinsic decay of Std ZIKV WT and F198T RVPs. Experiments were performed as described in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006178#ppat.1006178.g004" target="_blank">Fig 4A</a></b>. Percent infectivity versus hours of incubation is shown. Decay curves shown are representative of three independent experiments. Error bars indicate the SEM obtained from triplicate infections of Raji-DC-SIGN-R cells. <b>(G)</b> Neutralization of Std ZIKV WT and F198T RVPs by mAb E60. Percent infectivity, normalized to levels observed in the absence of antibody, versus mAb concentration is shown. Curves shown are representative of four independent experiments. Error bars indicate the range of infectivity from duplicate infections.</p

Neutralization sensitivity of WNV E T198F RVPs to mAbs.

<p><b>(A)</b> Top (left panel) and side (right panel) views of the crystal structure of the WNV E protein monomer (PDB 2HG0) are shown, with domains I, II, and III (DI, DII, and DIII), and the fusion loop of DII (DII-FL) indicated below the structure. The side view was obtained by rotating the structure in the left panel 90 degrees towards the page. On the structure, amino acid residues important for recognition by mAb E60 in the DII-FL [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006178#ppat.1006178.ref058" target="_blank">58</a>] and by mAb E16 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006178#ppat.1006178.ref059" target="_blank">59</a>] in DIII are indicated by the green and purple spheres, respectively. Gray spheres indicate residues within DI-DII hinge that were mutated for epitope mapping studies. The blue spheres and black arrow indicate the location of threonine at E protein residue 198 described in this study <b>(B)</b> Representative dose-response neutralization curves for WT and T198F WNV RVPs tested concurrently against mAbs E60 and <b>(C)</b> E16. The y- and x-axes indicate percent infectivity and mAb concentration, respectively. Infectivity was normalized to levels observed in the absence of antibody. Error bars indicate the range of infectivity from duplicate wells. We performed paired t-tests to compare the EC50 values of <b>(D)</b> E60 and <b>(E)</b> E16 against WT and T198F RVPs obtained from six independent experiments performed in duplicate. Error bars indicate the standard error of the mean (SEM).</p

The Type-Specific Neutralizing Antibody Response Elicited by a Dengue Vaccine Candidate Is Focused on Two Amino Acids of the Envelope Protein

<div><p>Dengue viruses are mosquito-borne flaviviruses that circulate in nature as four distinct serotypes (DENV1-4). These emerging pathogens are responsible for more than 100 million human infections annually. Severe clinical manifestations of disease are predominantly associated with a secondary infection by a heterotypic DENV serotype. The increased risk of severe disease in DENV-sensitized populations significantly complicates vaccine development, as a vaccine must simultaneously confer protection against all four DENV serotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of ongoing vaccine development efforts. However, a recent large clinical trial of a candidate live-attenuated DENV vaccine revealed low protective efficacy despite eliciting a neutralizing antibody response, highlighting the need for a better understanding of the humoral immune response against dengue infection. In this study, we sought to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. We constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination.</p></div