Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein

Abstract

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

“…Arg9 and Glu13 are also epitopes of other antibodies 35 . This particular study also showed that the N8R substitution DNA vaccine had a more neutralizing and protective effect than wild-type immunized sera, both in vitro and in vivo 35 . Arg9 is part of a salt bridge with Glu368 which maintains the structure of the E-protein in the pre-fusion state 10, 64 .…”

“…Tyr299 is part of the epitope for the cross-reactive neutralizing MAb DENV1-E102 66 . Arg188 is essential for infectivity, and is neutralized by DC4 Fab 40 , while the monoclonal antibody DD18-5 recognized residue Glu26 in DENV4 35 , a residue predicted by the Pepitope server 34 . Thus, all spatially perturbed residues identified by MEPP are known epitopes.…”

“…Firstly, a quantitative analysis of spatial and electrostatic perturbation in the pre 37 and post-fusion 12 DENV-2 E proteins was done using MEPP 38 . This revealed that highly perturbed residues are overwhelmingly conserved, and also epitopes of known neutralizing antibodies 23, 35, 39– 43 . Characterization of α -helices in E-proteins using techniques (PAGAL 44, 45 ) previously applied to the Ebola virus 46 , revealed that α 1 in ZIKV-E and DENV-E proteins is not conserved in the sequence space.…”

Computational analysis of perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

“…Apart from vaccines, other anti-viral strategies include developing peptide vaccines 28 , using peptide-inhibitors derived from the viral proteins 29 , inhibiting the fusion process 30 and anionic peptides that target cationic ‘hotspots’ 31 , 32 . Computational epitope predictors like the sequence based RANKpep 33 or the structure based Pepitope 34 have been used to validate antibody binding 35 , 36 . A detailed structural analysis of proteins of these flaviviruses will provide deeper insight into conservation than a sequential analysis does.…”

Padronização de uma nova técnica para detecção de anticorpos neutralizantes anti-dengue baseada na RT-PCR em tempo real

“…Geralmente, um sorotipo de dengue é dominante por um período de dois a seis anos, após o qual ele sofre um declínio na prevalência quando um sorotipo diferente emerge para substituí-lo (Adams et al 2006;Rabaa et al 2010) (Morens et al1985;Thomas et al 2009;Timiryasova et al 2013;Sirivichayakul et al 2014;Salje, et al 2014;Tang et al 2015) que, as reações cruzadas observadas refletem nos períodos das epidemias (Adams et al 2006).…”