Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles

Profile of the antibodies tested.

a<p> <i>12 mAbs DI/II; 14 mAbs DIII; 1 unknown.</i></p>b<p><i>The percentage of neutralization was determined by single endpoint plaque reduction neutralization assay on BHK21 cells</i> (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029957#pone.0029957-SukupolviPetty1" target="_blank">[35]</a>).</p>c<p> <i>Positivity indicates at least two times background signal.</i></p>d<p> <i>n.d. denotes not determined.</i></p

Effect of immune sera on the infectious properties of immature WNV particles.

<p>Infectivity and mice experiments were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029957#pone-0029957-g005" target="_blank">Fig. 5</a>. (<b>A</b>, <b>B</b>) immune sera from mice prior vaccinated with E ectodomain. (<b>D</b>, <b>E</b>) Immune serum derived from mice prior infected with a sublethal dose of st WNV. (<b>A</b>, <b>D</b>) Values depicted on the x axis represent dilution factors. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”, (PMS) denotes polyclonal mouse serum. Student's t-tests were used to determine significance; *, <i>P</i><0.01. (<b>C</b>) Western blot analysis of immune serum from mice prior infected with a sublethal dose of WNV. On the SDS-PAGE gel, purified immature WNV was loaded.</p

Effect of anti-E mAb 4G2 on the infectious properties of immature WNV particles <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) P388D1 cells were infected with immature WNV opsonized with increasing concentrations of 4G2 at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in duplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, <i>P</i><0.01. (<b>B</b>) Immature WNV was incubated with different concentrations of anti-E 4G2 for 1 hr at 37°C, and injected in mice. A total of 3.4×10⁷ GCPs were given per mouse. Five mice were used for each experimental condition.</p

Effect of anti-E mAbs on the infectious properties of fully immature DENV particles.

<p>P388D1 cells were infected with immature (prM-containing) DENV-2 strain 16681 at MOG 1000 in the presence or absence of: (<b>A</b>) anti-prM antibody and st DENV preparation without antibody, which were used as controls and (<b>B–P</b>) different anti-E mAbs. Prior to infection, immature DENV particles were incubated with varying antibody concentrations for 1 h at 37°C. At 43 hpi supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in triplicate. Immature virus in the absence of antibodies was used as a control in all experiments (n = 20), for clarity the average of these results are only plotted in panel 1A. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, <i>P</i><0.01.</p

Effect of furin inhibitor on the infectivity of standard virus preparations.

<p>P388D1 cells were infected with antibody-opsonized st DENV particles in the presence or absence of furin inhibitor. Standard DENV particles without antibody or opsonized with anti-prM antibody 70.21 were used as controls. The error bars represent standard deviations (SD) derived from at least two separate experiments performed in duplicate. Two-tailed Student's t-tests were used to determine significance; *, <i>P</i><0.01.</p