Summary Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcγ receptors (FcγR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII- or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcγR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibody-based therapy to control severe dengue.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

West Nile virus (WNV), a positive-sense RNA virus and a member of the Flaviviridae family, recently became endemic in North America, with annual outbreaks of severe encephalitis occurring mostly in immunocompromised or elderly individuals. There is currently no vaccine approved for human use, and treatment is primarily supportive. The WNV genome encodes three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). During the course of WNV infection, antibodies are raised against prM/M and E as well as NS1, NS3, and NS5, with a majority of the protective antibody response against the E protein (12, 63).The crystal structure of the ectodomain of the E protein has been determined for dengue virus (DENV), tick-borne encephalitis virus (TBEV), and WNV (43,45,48,56,65). Flavivirus E proteins have three separate domains and form headto-tail homodimers on the surface of the virion. Domain I (DI) is the central structural domain and consists of a 10-stranded ␤-barrel. DII is formed from two extended loops that project from DI. At the end of DII is a highly conserved loop, amino acid residues 98 to 110, that has been implicated in the acidcatalyzed type II fusion event (1,7,44). In the E dimer, the fusion loop lies in a pocket at the DI-DIII interface of the adjacent E protein. DIII, located on the opposite side of DI, forms a seven-stranded immunoglobulin-like fold and has been implicated in receptor binding (5, 10, 14). Short, flexible linker regions connect the domains and allow for the conformational changes associated with virus maturation and fusion (65).The structure of the WNV virion has been defined by cryoelectron microscopy (36, 47). The mature WNV is ϳ500 Å in diameter and has a relatively smooth surface with no apparent spikes or large projections. The 180 E monomers lay flat along the virion surface as sets of three parallel dimers. The arrangement of the 180 E monomers has quasi-icosahedral symmetry such that there are three E monomers in the asymmetric unit and three distinct chemical environments available for antibody or ligand binding (47). The reduced pH in the endosome causes the E protein to convert from a homodimer to a homotrimer and exposes the fusion loop (44).Antibodies are critical for the control of flavivirus infection in vivo (4,17,18,20,23,50,59), and this protection has been correlated with neutralizing activity in vitro (32,53,58). However, there have been reports of strong and weak in vivo protection with nonneutralizing (6,11,29,31,34,58) and neutralizing (30, 32, 41) monoclonal antibodies (MAbs), respectively. Several recent studies suggest that specific epitopes elicit flavivirus-reactive MAbs with particular functional activities (3,37,38,50,57,60). Most type-specific neutralizing antibodies map to DIII of the E protein. Cross-reactive, neutralizing MAbs bind to regions outside DIII and have been mapped to

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals.Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.Dengue fever (DF), the most prevalent arthropod-borne viral illness in humans, is caused by dengue virus (DENV). The four serotypes of DENV are transmitted to humans primarily by the mosquitoes Aedes aegypti and Aedes albopictus. DENV is a member of the Flaviviridae family and is related to the viruses that cause yellow fever and the Japanese, St. Louis, and West Nile encephalitides (8). Infection by DENV causes a spectrum of clinical disease, ranging from an acute, debilitating, selflimited febrile illness (DF) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever/dengue shock syndrome). At present, no approved antiviral treatment or vaccine is available, and therapy is supportive in nature. DENV causes an estimated 25 to 100 million cases of DF and 250,000 cases of dengue hemorrhagic fever per year worldwide, with 2.5 billion people at risk for infection (27,48).DENV is an enveloped virus with a single-stranded, positivesense RNA genome (11). The 10.7-kilobase genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus-and host-encoded proteases. The 500-Å DENV mature virion has a well-organized outer protein she...

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.