Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.antibody | cryoelectron microscopy | flavivirus W est Nile virus (WNV) is a human pathogen that causes a febrile illness, which can progress to encephalitis, paralysis, and death. The virus is endemic in parts of Africa, Asia, and Europe, and in the past decade has spread throughout North America and into Central and South America (1). WNV is closely related to other arthropod-transmitted, medically relevant flaviviruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These lipid-enveloped viruses enter host cells by receptor-mediated endocytosis. The singlestranded, positive-sense RNA genome is released into the cytoplasm after low pH induces the fusion of the viral lipid envelope with the endosomal membrane.Mature WNV virions are roughly spherical with a diameter of about 500 Å. The outer viral surface is composed of an icosahedral scaffold of 180 closely packed copies of the membrane-anchored envelope (E) glycoprotein. Sets of three, nearly parallel E homodimers are associated into rafts that form a herringbone pattern on the surface of mature virions. The ectodomain of E has three structural domains, DI, DII, and DIII (2-5), with domain DI positioned structurally between DII and DIII. DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2, 6-12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6, 9, 13).The humoral immune response is essential for protection against flavivirus infection and disease (14,15). The E glycoprotein is the principal antigen that elicits neutralizing antibodies agai...

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

The human antibody response to flavivirus infection is dominantly directed against a cross-reactive epitope on the fusion loop of domain II (DII-FL) of the envelope (E) protein. Although antibodies against this epitope fail to recognize fully mature West Nile virus (WNV) virions and accordingly neutralize infection poorly in vitro,their functional properties in vivo remain less well understood. Here, we show that while passive transfer of poorly neutralizing monoclonal antibodies (MAb) and polyclonal antibodies against the DII-FL epitope protect against lethal WNV infection in wild-type mice, they fail to protect mice lacking activating Fc␥ receptors (Fc␥R) and the complement opsonin C1q. Consistent with this, an aglycosyl chimeric mouse-human DII-FL MAb (E28) variant that lacks the ability to engage Fc␥R and C1q also did not protect against WNV infection in wild-type mice. Using a series of immunodeficient mice and antibody depletions of individual immune cell populations, we demonstrate that the nonneutralizing DII-FL MAb E28 does not require T, B, or NK cells, inflammatory monocytes, or neutrophils for protection. Rather, E28 treatment decreased viral load in the serum early in the course of infection, which resulted in blunted dissemination to the brain, an effect that required phagocytic cells, C1q, and Fc␥RIII (CD16). Overall, these studies enhance our understanding of the functional significance of immunodominant, poorly neutralizing antibodies in the polyclonal human antiflavivirus response and highlight the limitations of current in vitro surrogate markers of protection, such as cell-based neutralization assays, which cannot account for the beneficial effects conferred by these antibodies. West Nile virus (WNV) is a zoonotic mosquito-transmittedFlavivirus that can infect and cause disease in humans and many other vertebrate animals. The Flavivirus genus also contains other human pathogens of global relevance, including dengue virus (DENV), yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Most human WNV infections are asymptomatic, but about 20% of infected individuals experience a mild fever, and less than 1% develop severe neuroinvasive disease (67). Risk factors for symptomatic disease include an age of greater than 55 years, a compromised immune status, genetic variation in the OAS1 gene, and a CC5⌬32 genotype (17,26,40,41). Although WNV first appeared in the Western Hemisphere in 1999 in New York and spread rapidly through North America, surprisingly few human clinical infections have been reported in Central and South America, despite the migration of avian hosts and appropriate vectors for transmission (35,56).WNV infection requires attachment to cell surface receptors, which remain poorly defined, endocytosis, and acid-catalyzed fusion of the virus within the late endosome. After translation of input-strand RNA and viral replication, progeny virion assembly occurs within the endoplasmic reticulum (ER), with the capsid protein and genomic RNA associating with pre...

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-␣␤R

Acute flaccid myelitis (AFM) is a disabling, polio-like illness mainly affecting children. Outbreaks of AFM have occurred across multiple global regions since 2012, and the disease appears to be caused by non-polio enterovirus infection, posing a major public health challenge. The clinical presentation of flaccid and often profound muscle weakness (which can invoke respiratory failure and other critical complications) can mimic several other acute neurological illnesses. There is no single sensitive and specific test for AFM, and the diagnosis relies on identification of several important clinical, neuroimaging, and cerebrospinal fluid characteristics. Following the acute phase of AFM, patients typically have substantial residual disability and unique long-term rehabilitation needs. In this Review we describe the epidemiology, clinical features, course, and outcomes of AFM to help to guide diagnosis, management, and rehabilitation. Future research directions include further studies evaluating host and pathogen factors, including investigations into genetic, viral, and immunological features of affected patients, host-virus interactions, and investigations of targeted therapeutic approaches to improve the long-term outcomes in this population.

Enterovirus D68 (EV-D68) causes outbreaks of respiratory illness, and there is increasing evidence that it causes outbreaks of acute flaccid myelitis (AFM). There are no licensed therapies to prevent or treat EV-D68 infection or AFM disease. We isolated a panel of EV-D68–reactive human monoclonal antibodies that recognize diverse antigenic variants from participants with prior infection. One potently neutralizing cross-reactive antibody, EV68-228, protected mice from respiratory and neurologic disease when given either before or after infection. Cryo–electron microscopy studies revealed that EV68-228 and another potently neutralizing antibody (EV68-159) bound around the fivefold or threefold axes of symmetry on virion particles, respectively. The structures suggest diverse mechanisms of action by these antibodies. The high potency and effectiveness observed in vivo suggest that antibodies are a mechanistic correlate of protection against AFM disease and are candidates for clinical use in humans with EV-D68 infection.

Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus (WNV) in animals. Here, we assess the potential for WNV strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, as several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.

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