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

Abstract

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis inWNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as deadend 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 CC5⌬32 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 ϳ500-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 ␤-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 receptormediated, clathrin-dependent endocytosis (1,79,80). After trafficking to 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

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

“…Little to no conservation of the identified epitope residues was observed among other flaviviruses (Fig. S4), consistent with the strict type-specificity of CR4354 for WNV (21).…”

“…2 and 3). This is consistent with the inability of CR4354 to bind to recombinant, soluble E ectodomain or recognize virus particles at low pH, which have undergone a conformational change (21). Although the epitope spreads over two molecules, weak binding of a single E molecule by CR4354 should be considered, at the least for the largest contact region on a single E molecule (DI and DI-II interface).…”

“…2). Lys136, identified previously by neutralization escape mutant analysis (21), was verified as a central part of the CR4354 epitope. Hydrophobic interactions account for much of the binding energy (Table 1 and Table S4).…”

“…Although the most potent inhibitory mouse antibodies against WNV bind to DIII of E (17)(18)(19), these appear to play a less dominant role in the human immune response against WNV infection (20). CR4354 is a strongly neutralizing human monoclonal antibody (MAb) against WNV that was isolated from a patient infected with WNV and inhibits infection subsequent to cell attachment (21). This antibody blocks virus fusion with liposomes in vitro and recognizes virus particles, but not the recombinant E ectodomain, suggesting that its epitope requires an oligomeric E arrangement present only in virions or subviral particles (SVP).…”

In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain

“…The amino acid differences between the vaccine strains and the GI strains did not exceed 2.4 %, indicating that the difference may not influence the efficacy of the vaccines. Previous studies have identified individual residues within the E protein that are related to the recognition of JEV by neutralizing antibodies (Crill & Chang, 2004;Goncalvez et al, 2008;Kobayashi et al, 1985;Morita et al, 2001;Vogt et al, 2009;Wu et al, 2003). Although the sequence variation between the vaccine strains and GV strains do not correspond to epitope residues, only a limited number of residues could be responsible for the decreased neutralization ability of the vaccines against GV strains.…”