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There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding "pseudoenergies", we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 microM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery.

Background: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection., Results: Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection., Conclusions: HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.

Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 microM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 microM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 microM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.