A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein

10.1371/journal.pone.0033451PLoS ONE74

The Early Clinical Features of Dengue in Adults: Challenges for Early Clinical Diagnosis

Dengue infection in adults has become increasingly common throughout the world. As most of the clinical features of dengue have been described in children, we undertook a prospective study to determine the early symptoms and signs of dengue in adults. We show here that, overall, dengue cases presented with high rates of symptoms listed in the WHO 1997 or 2009 classification schemes for probable dengue fever thus resulting in high sensitivities of these schemes when applied for early diagnosis. However, symptoms such as myalgia, arthralgia, retro-orbital pain and mucosal bleeding were less frequently reported in older adults. This trend resulted in reduced sensitivity of the WHO classification schemes in older adults even though they showed increased risks of hospitalization and severe dengue. Instead, we suggest that older adults who present with fever and leukopenia should be tested for dengue, even in the absence of other symptoms. This could be useful for early clinical diagnosis in older adults so that they can be monitored and treated for severe dengue, which is especially important when an antiviral drug becomes available

Adaptive confidence and adaptive curiosity

SIGLEAvailable from TIB Hannover: RO 9403(149) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Selection workflow of dengue patients with warning signs that required hospitalizations and patients with no warning signs and no hospitalization required for both discovery and validation cohort.

<p>Selection workflow of dengue patients with warning signs that required hospitalizations and patients with no warning signs and no hospitalization required for both discovery and validation cohort.</p

Early prognostic performance of the top selected models with an independent validation cohort across a range of probability cutoff.

<p>Early prognostic performance of the top selected models with an independent validation cohort across a range of probability cutoff.</p

Demographic descriptions of dengue RT-PCR positive patients classified according to designated clinical outcomes.

<p>Demographic descriptions of dengue RT-PCR positive patients classified according to designated clinical outcomes.</p

Early prognostic models of warning signs and hospitalization from the discovery cohort.

<p>Early prognostic models of warning signs and hospitalization from the discovery cohort.</p

Site-directed mutagenesis of predicted epitope on prM-E protein.

<p>(A) To confirm the binding epitope of D29 Fab-IgG, residues within the P3 and P9 predicted sequences were mutated to generate mutants 1–6 (M1-6); M3/4, M3/5, M4/5 and M5/6 contain combinational-mutations as stated. (B) Reactivity of antibodies with the mutants was tested by Western blot analysis. Cleared lysate of DENV2-infected Vero cells (DV2), pCMV-prM-E- (prM-E) or mutants-transfected HEK 293 T cells (M1-5/6) were separated on 12% SDS-PAGE in non-reducing condition, followed by detection with h4G2, m2H2 and D29 Fab-IgG.</p

Localization of D29 Fab-IgG predicted epitopes in 3D crystal structures of DENV2 prM-E heterodimer (PDB 3C6E) and the binding specificity of peptide-phages.

<p>(A) Clusters of conformational epitopes predicted by Pepitope server are displayed on the prM-E heterodimer crystal structure at neutral pH with their solvent-accessible surfaces highlighted. The prM is pink, EDI is red, EDII is yellow, EDIII is blue, FP is cyan. (B) The binding specificity of peptide-phages (P1-P9) to D29 Fab-IgG was tested in a direct ELISA format with 10 µg/ml of D29 Fab-IgG, h3H5, m2H2 and non-DENV specific human antibody (Hu) immobilized on a Maxisorb plate. Ability of peptide-phages to inhibit D29 Fab-IgG binding to DENV2 was investigated by (C) ELISA and (D) Western blot analysis. For ELISA, peptide-phages (10¹² pfu/ml) were incubated with D29 Fab-IgG for 1 hr at RT before application to immobilized DENV2 for 5 min at RT. Bound D29 Fab-IgG was detected with HRP-conjugated anti-Human IgG-Fc. The percentage of inhibition of D29 Fab-IgG binding by the peptide-phages shown is the average of three experiments. Error bars represent the standard errors of the mean (***p-value <0.005). For Western blot analysis, 0.5 µg/ml of D29 Fab-IgG was incubated with 8×10⁶ pfu of purified DENV2, 5% SM or 4×10¹¹ pfu of peptide-phage clones for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT.</p