The NS1 Glycoprotein Can Generate Dramatic Antibody-Enhanced Dengue Viral Replication in Normal Out-Bred Mice Resulting in Lethal Multi-Organ Disease

Andrew K I Falconar,Fernando Martinez,Bradley S Schneider

doi:10.1371/journal.pone.0021024

Abstract

Antibody-enhanced replication (AER) of dengue type-2 virus (DENV-2) strains and production of antibody-enhanced disease (AED) was tested in out-bred mice. Polyclonal antibodies (PAbs) generated against the nonstructural-1 (NS1) glycoprotein candidate vaccine of the New Guinea-C (NG-C) or NSx strains reacted strongly and weakly with these antigens, respectively. These PAbs contained the IgG2a subclass, which cross-reacted with the virion-associated envelope (E) glycoprotein of the DENV-2 NSx strain, suggesting that they could generate its AER via all mouse Fcγ-receptor classes. Indeed, when these mice were challenged with a low dose (<0.5 LD50) of the DENV-2 NSx strain, but not the NG-C strain, they all generated dramatic and lethal DENV-2 AER/AED. These AER/AED mice developed life-threatening acute respiratory distress syndrome (ARDS), displayed by diffuse alveolar damage (DAD) resulting from i) dramatic interstitial alveolar septa-thickening with mononuclear cells, ii) some hyperplasia of alveolar type-II pneumocytes, iii) copious intra-alveolar protein secretion, iv) some hyaline membrane-covered alveolar walls, and v) DENV-2 antigen-positive alveolar macrophages. These mice also developed meningo-encephalitis, with greater than 90,000-fold DENV-2 AER titers in microglial cells located throughout their brain parenchyma, some of which formed nodules around dead neurons. Their spleens contained infiltrated megakaryocytes with DENV-2 antigen-positive red-pulp macrophages, while their livers displayed extensive necrosis, apoptosis and macro- and micro-steatosis, with DENV-2 antigen-positive Kuppfer cells and hepatocytes. Their infections were confirmed by DENV-2 isolations from their lungs, spleens and livers. These findings accord with those reported in fatal human “severe dengue” cases. This DENV-2 AER/AED was blocked by high concentrations of only the NG-C NS1 glycoprotein. These results imply a potential hazard of DENV NS1 glycoprotein-based vaccines, particularly against DENV strains that contain multiple mutations or genetic recombination within or between their DENV E and NS1 glycoprotein-encoding genes. The model provides potential for assessing DENV strain pathogenicity and anti-DENV therapies in normal mice.

Highlights

Dengue viruses (DENVs), which occur as four discrete serotypes, are the most important vector-borne human viruses [1]
No contaminating E or prM glycoproteins were detected in immunoblot assays using high (200 ng) concentrations of the purified DENV-2 NS1 glycoproteins of the DENV-2 16681, NGC or NSx strains with monoclonal antibodies (MAbs) specific for each of these DENV glycoproteins (Figure S1)
Whilst polyclonal antibodies (PAbs) generated in mice to live DENV-2 infections showed higher titers against the DENV-2 E glycoprotein, they more weakly reacted with the NS1 glycoprotein of the DENV-2 NSx strain than the 16681 or New Guinea-C (NG-C) strains

Summary

Introduction

Dengue viruses (DENVs), which occur as four discrete serotypes, are the most important vector-borne human viruses [1]. The greatest DENV AER was, obtained in vitro using undiluted polyclonal antibodies (PAbs) obtained from children during the acute-phase of DENV infections that subsequently developed DHF/DSS [10], or at the age when most DHF/DSS cases occurred [11]. Despite these findings and their importance for understanding of DENV pathogenesis, the ability of undiluted PAbs raised against DENV to subsequently generate AER of a heterologous DENV serotype in vivo was assessed in only one study [12]. The ability of DENV E glycoprotein-specific antibodies to either neutralize or generate AER was dependent on their ability to fix complement and, their IgG subclasses [19]

Methods

Results

Conclusion