The specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. To study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (DEN) virus E-glycoprotein-specific monoclonal antibodies (MAbs) to block virus adsorption to Vero cells. In contrast to previous studies, the binding sites of these MAbs were localized to one of three structural domains (I, II, and III) in the E glycoprotein. The results indicate that most MAbs that neutralize virus infectivity do so, at least in part, by the blocking of virus adsorption. However, MAbs specific for domain III were the strongest blockers of virus adsorption. These results extend our understanding of the structure-function relationships in the E glycoprotein of DEN virus and provide the first direct evidence that domain III encodes the primary flavivirus receptor-binding motif.The flavivirus E glycoprotein is the primary antigen inducing protective immunity, is essential for membrane fusion, and mediates binding to cellular receptors. Therefore, this protein directly affects host range, cellular tropism, and, in part, the virulence of these viruses (17, 18). The crystal structure of the ectodomain of the tick-borne encephalitis (TBE) virus E-glycoprotein homodimer was recently solved at high resolution (16). Multiple lines of evidence indicate that this E-glycoprotein structure is strongly conserved across the Flaviviridae (16). This protein contains three structural domains. The central domain, domain I (DI), contains predominately type-specific nonneutralizing epitopes and is theorized to be the molecular hinge region involved in low-pH-triggered conformational changes (19). The dimerization domain, domain II (DII), makes important contacts with itself in the homodimer, is involved in virus-mediated membrane fusion, and contains many cross-reactive epitopes eliciting neutralizing and nonneutralizing monoclonal antibodies (MAbs) (16,19). Domain III (DIII) is characterized by an immunoglobulin-like structure containing the most distal projecting loops from the virion surface. It contains multiple type-and subtype-specific epitopes eliciting only virus-neutralizing MAbs and has been hypothesized to contain the host cell-binding antireceptor (16,18,19). As part of our ongoing research to elucidate the structure-function relationships of the dengue (DEN) virus E glycoprotein, we have assessed the ability of a well-characterized panel of E-glycoprotein-specific MAbs to block virus adsorption to Vero cells. These results provide the first direct evidence that E glycoprotein DIII encodes a receptor-binding motif.

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus speciesspecific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus crossreactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.

Dengue virus (DENV) is a serious mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation dengue hemorrhagic fever (DHF). Nearly half of the world's population is at risk of dengue disease and there are estimated to be millions of infections annually; a situation which will continue to worsen with increasing expansion of the mosquito vectors and epidemic DF/DHF. Currently there are no available licensed vaccines or antivirals for dengue, although significant effort has been directed toward the development of safe and efficacious dengue vaccines for over 30 years. Promising vaccine candidates are in development and testing phases, but a better understanding of immune responses to DENV infection and vaccination is needed. Humoral immune responses to DENV infection are complex and may exacerbate pathogenicity, yet are essential for immune protection. In this report, we develop DENV-2 envelope (E) protein epitope-specific antigens and measure immunoglobulin responses to three distinct epitopes in DENV-2 infected human serum samples. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary DENV-2 infected sera. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. This report begins to tease apart complex humoral immune responses to DENV infection and is thus important for improving our understanding of dengue disease and immunological correlates of protection, relevant to DENV vaccine development and testing.