BackgroundThe envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.Methodology/Principal FindingsWe developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.Conclusions/SignificanceOur analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.

Viral envelope proteins are required for productive viral entry and initiation of infection. Although the humoral immune system provides ample evidence for targeting envelope proteins as an antiviral strategy, there are few pharmacological interventions that have this mode of action. In contrast to classical antiviral targets such as viral proteases and polymerases, viral envelope proteins as a class do not have a well-conserved active site that can be rationally targeted with small molecules. We previously identified compounds that inhibit dengue virus by binding to its envelope protein, E. Here, we show that these small molecules inhibit dengue virus fusion and map the binding site of these compounds to a specific pocket on E. We further demonstrate inhibition of Zika, West Nile, and Japanese encephalitis viruses by these compounds, providing pharmacological evidence for the pocket as a target for developing broad-spectrum antivirals against multiple, mosquito-borne flavivirus pathogens.

It is known that solid tumors recruit new blood vessels to support tumor growth, but the molecular diversity of receptors in tumor angiogenic vessels might also be used clinically to develop better targeted therapy. In vivo phage display was used to identify peptides that specifically target tumor blood vessels. Several novel peptides were identified as being able to recognize tumor vasculature but not normal blood vessels in severe combined immunodeficiency (SCID) mice bearing human tumors. These tumor-homing peptides also bound to blood vessels in surgical specimens of various human cancers. The peptidelinked liposomes containing fluorescent substance were capable of translocating across the plasma membrane through endocytosis. With the conjugation of peptides and liposomal doxorubicin, the targeted drug delivery systems enhanced the therapeutic efficacy of the chemotherapeutic agent against human cancer xenografts by decreasing tumor angiogenesis and increasing cancer cell apoptosis. Furthermore, the peptide-mediated targeting liposomes improved the pharmacokinetics and pharmacodynamics of the drug they delivered compared with nontargeting liposomes or free drugs. Our results indicate that the tumor-homing peptides can be used specifically target tumor vasculature and have the potential to improve the systemic treatment of patients with solid tumors.

BackgroundDengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.Methodology/Principal FindingsWe generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.Conclusions/SignificanceWe used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.