Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.West Nile encephalitis virus (WNV) is a positive-polarity, single-stranded RNA virus of the genus Flavivirus within the family Flaviviridae. Other members of this genus that cause significant human disease include dengue virus (DENV), St. Louis encephalitis virus, Japanese encephalitis virus (JEV), yellow fever virus, and tick-borne encephalitis virus (TBEV). Flaviviruses are translated as a single polypeptide, which is then cleaved by host and viral proteases into three structural (capsid C], premembrane prM], and envelope E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (reviewed in references 42 and 43).WNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as dead-end hosts (25, 62). Infection causes syndromes ranging from a mild febrile illness to severe encephalitis and death (13, 72). WNV has spread globally and causes outbreaks with thousands of severe human cases annually in the United States. An age of greater than 55 years, a compromised immune status, and a CC5Δ32 genotype have been associated with more-severe disease (15, 20). There is currently no approved vaccine or therapy for WNV infection.The mature WNV virion has a ∼500-Å diameter and consists of a single RNA genome surrounded by the capsid protein, a lipid bilayer, and a shell of the prM/M and E proteins (31, 55). X-ray crystallography studies have elucidated the three-domain structure of the flavivirus E protein (30, 48, 50, 58, 67). Domain I (DI) is a central, eight-stranded β-barrel, which contains the only N-linked glycosylation site in WNV E. Domain II (DII) is a long, finger-like protrusion from DI and contains the highly conserved fusion peptide at its distal end. Domain III (DIII) adopts an immunoglobulin-like fold at the opposite end of DI and is believed to contain a site for receptor attachment (6, 8, 40).Within an infected cell, progeny WNV are assembled initially as immature particles. In immature virions, three pairs of E and prM interact as trimers and form 60 spiked projections with icosahedral symmetry (85, 86). Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of the E proteins and cleavage of prM to M by a furin-like protease (41, 83). Mature WNV virions are covered by 90 antiparallel E protein homodimers, which are arranged flat along the surface in a herringbone pattern with quasi-icosahedral symmetry (55).Upon binding to poorly characterized cell surface receptors, internalization of WNV is believed to occur through receptor-mediated, clathrin-dependent endocytosis (1, 79, 80). After trafficking to Rab5- and/or Rab7-positive endosomes (38, 79), the mildly acidic pH within the lumen of the endosome induces structural alterations in the flavivirus E protein (7, 49), which includes changes in its oligomeric state (7, 49, 77). During this process, also known as type II fusion, the hydrophobic peptide on the fusion loop of DII of the E protein inserts into the endosomal membrane, thus physically joining the host and viral membranes, which allows the infectious RNA genome to enter the cytoplasm (32, 33).Humoral immunity is an essential component of the protective host response against flaviviruses including WNV (reviewed in references 64 and 68). Studies by several groups have shown that the neutralization of WNV can occur after antibodies bind to a series of discrete epitopes on all three domains of the E protein (3, 12, 22, 59, 61, 71). To date, the most potently neutralizing monoclonal antibodies (MAbs) localize to an epitope on the lateral ridge of DIII (DIII-lr). One well-characterized strongly neutralizing mouse MAb, E16, blocks infection primarily at a postattachment step (57) and requires the engagement of only a fraction of its epitopes on the surface of the virion (66). Studies of the human antibody response to WNV infection reveal that, in contrast to mice, antibodies that bind the DIII-lr epitope comprise a minor component of the neutralizing humoral response in most individuals (60).In this study, we characterized two strongly neutralizing novel human MAbs (CR4348 and CR4354) that were selected from an antibody phage display library constructed from B cells of subjects that survived WNV infection (78). We demonstrate that both MAbs are WNV specific, bind weakly to recombinant or yeast surface-displayed E proteins, exhibit pH-sensitive binding to viral particles, and protect against lethal infection in mice. Our experiments suggest that these human MAbs map to distinct epitopes and neutralize infection at a postattachment stage, likely by inhibiting the acid-catalyzed viral fusion step.