Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated mAbs against domain III of the WNV E protein. MAbs that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from human patients who had recovered from WNV infection also detected this epitope. One mAb, E16, neutralized 10 different strains in vitro, and demonstrated therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity, and neutralizing activity. In post-exposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and thus, may be a viable treatment option against WNV infection in humans.WNV is a single-stranded, positive-polarity RNA Flavivirus that is related to dengue fever, yellow fever, and Saint Louis, tick-borne, and Japanese encephalitis viruses. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis, and the elderly and immunocompromised are at greatest risk for severe disease 1 . At present, treatment is supportive and no vaccine exists for human use.The innate and adaptive immune responses prevent dissemination to and within the central nervous system (CNS) 2,3 . Recently, two groups demonstrated therapeutic efficacy of immune human γ-globulin in mice infected with WNV 4,5 . Even after virus had spread to the CNS, passive administration of immune heterologous γ-globulin improved survival 5 . In theory, a potently neutralizing mAb could have the same or better benefit with a lower dose and improved safety profile.Most neutralizing antibodies against flaviviruses recognize the envelope (E) protein. In general, virus-specific rather than cross-reactive antibodies have the strongest neutralizing activity in NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript vitro and greatest protection in vivo 6 . Crystallographic analysis of the soluble ectodomain of flavivirus E proteins has revealed three domains 7,8 . Domain I is an 8-stranded β-barrel 7-9 that participates in the conformational changes associated with the acidification in the endosome 10 . Domain II contains 12 β-strands and has roles in dimerization, trimerization, and fusion 7,8,10 . Domain III (DIII) adopts an immunoglobulin-like fold, and contains the loops that are most distal from the surface in the mature virion 11,12 and the site for putative receptor attachment 6,8,13,14 . Based on the sequencing of in vitro neutralization escape variants, many neutralizing antibodies against flaviviruses localize to DIII 15-22 .Here, we define further the molecular basis of antibody-mediated neutralization of WNV using a large panel of newly generated mAbs against WNV E protein. Humanized versions o...

Antibody binding to the icosahedral arrangement of envelope proteins on the surface of flaviviruses can result in neutralization or enhancement of infection. We evaluated how many antibodies must bind to a given epitope on West Nile virus (WNV) to achieve neutralization. The most potent monoclonal antibodies (mAbs) block infection at concentrations that result in low occupancy of accessible sites on the virion, with neutralization occurring when as few as 30 of 180 envelope proteins are bound. In contrast, weakly neutralizing mAbs recognize fewer sites on the virion and require almost complete occupancy to inhibit WNV infection. For all mAbs studied, enhancement of infection is possible in cells bearing activating Fc-gamma receptors when the number of mAbs docked to the virion is not sufficient for neutralization. Thus, neutralization is best described by a model requiring "multiple hits" with the cumulative functional outcome determined by interplay between antibody affinity and epitope accessibility.

West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.

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...