Resistance analysis of an antibody that selectively inhibits dengue virus serotype-1.

The four serotypes of dengue virus (DENV) are the causative agents of the most prevalent mosquito-borne viral disease in human. No clinically approved antiviral therapy is currently available. Therapeutic antibodies represent a viable approach for potential treatment of DENV infection. We recently isolated a human monoclonal antibody (HM14c10) that selectively neutralizes DENV serotype 1 (DENV-1), but not serotypes 2, 3, and 4. Here we report the resistance profile of DENV-1 against HM14c10 in cell culture. Escape mutant viruses readily emerged by culturing wild-type DENV-1 in the presence of the HM14c10 antibody. Sequencing of resistant viruses revealed a single T51K substitution in the domain I/II hinge region of the viral envelope protein. Residue T51 is located within the HM14c10 epitope and is highly conserved among various DENV-1 isolates. Recombinant DENV-1 containing the T51K mutation could not be neutralized by HM14c10 in vitro or in vivo. Biochemical assay revealed that the T51K mutation completely abolished the antibody binding to the DENV-1 virion. Collectively, the results demonstrate that a single amino acid change in DENV envelope protein can confer resistance to a potent antibody through abolishing the antibody-virus interaction.

The structural basis for serotype-specific neutralization of dengue virus by a human antibody.

Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.

A single amino acid in nonstructural protein NS4B confers virulence to dengue virus in AG129 mice through enhancement of viral RNA synthesis.

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.

A non mouse-adapted dengue virus strain as a new model of severe dengue infection in AG129 mice.

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-alpha/beta and -gamma receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.

Pathogenesis and prevention of dengue virus infection: state-of-the-art.

PURPOSE OF REVIEW: The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes has made this mosquito-borne virus of great global public health importance. This article reviews the latest findings in the field of DEN pathogenesis, vector control and vaccine development. RECENT FINDINGS: Atypical clinical manifestations as well as a shift in the age of DEN-infected patients have forced a revisit of the current definitions and classifications of the DEN-associated diseases. The antibody-mediated internalization of DEN virus triggers intracellular pathways that further enhance viral replication and output. A correlation between the human leukocyte antigen system and disease severity was found. The development of tetravalent DEN vaccine candidates is reaching its final stages, though the antibody levels required to protect against the four DEN serotypes are still unknown. SUMMARY: Efforts have been devoted to identify the host target cells that are permissive or resistant to DEN virus replication and to decipher the downstream events that would result in either disease enhancement or protection. Strong emphasis has recently been made on genetic polymorphisms that confer DEN protection versus susceptibility to severe DEN. The increasing knowledge gained from basic science should help to better design effective DEN vaccines.

Induction of neutralizing antibodies against dengue virus type 2 upon mucosal administration of a recombinant Lactococcus lactis strain expressing envelope domain III antigen.

Mucosal vaccines present several advantages over conventional parenteral vaccines including their ease of administration and low cost, both criteria being priorities for developing countries plagued by infectious diseases. A recombinant Lactococcus lactis strain producing the envelope domain III (EDIII) antigen from dengue virus serotype 2 was engineered, and the ability of the live recombinant bacteria to trigger a systemic anti-EDIII IgG antibody response upon nasal or oral administration to BALB/c and C57BL/6 mice was investigated. Results showed that the antibody response depended on the route of administration and on the mouse strain inoculated. Out of six, two and three C57BL/6 mice orally and nasally inoculated with the recombinant bacteria, respectively, displayed anti-EDIII antibody responses higher than that obtained in the mouse group intraperitoneally (i.p.) immunized with heat-inactivated dengue 2 virus. The protective potential of the immune sera was measured using the plaque reduction neutralizing test (PRNT) and results indicated that high anti-EDIII antibody levels did not correlate directly with high neutralizing activities. Immune sera from orally inoculated mice were found the most potent to neutralize in vitro dengue infection with neutralizing antibody activities in some cases higher than that obtained with the immune sera from mice i.p. injected with heat-inactivated virus.