Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.Picornaviruses are among the largest of animal virus families and include the well-known poliovirus, rhinovirus, foot-and-mouth disease virus (FMDV), coxsackievirus, and hepatitis A virus. The rhinoviruses, of which there are more than 100 serotypes subdivided into two groups, are major causative agents of the common cold in humans (42). The viruses are nonenveloped and have an ∼300-Å-diameter protein shell that encapsidates a single-stranded, plus-sense RNA genome of about 7,200 bases. The human rhinovirus 14 (HRV14) capsid exhibits a pseudo-T=3 (P=3) icosahedral symmetry and consists of 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, with VP4 at the RNA-capsid interface (40). An ∼20-Å deep canyon lies roughly at the junction of VP1 (forming the north rim) with VP2 and VP3 (forming the south rim) and surrounds each of the 12 icosahedral fivefold vertices. The canyon regions of HRV14 and HRV16, both major receptor group rhinoviruses, were shown to contain the binding site of the cellular receptor, intercellular adhesion molecule 1 (ICAM-1) (8, 24a, 37). Four major neutralizing immunogenic (NIm) sites, NIm-IA, NIm-IB, NIm-II, and NIm-III, were identified by studies of neutralization escape mutants with monoclonal antibodies (MAbs) (46, 47) and then mapped to four protruding regions on the viral surface (40).Several mechanisms of antibody-mediated neutralization have been proposed. Perhaps the simplest is based on aggregation of virions (5, 53, 54), which generally occurs over a narrow range of antibody/virus ratios. This limited range has raised questions about the role of aggregation in vivo. Alternative suggestions are that antibodies may neutralize virions by inducing extensive conformational changes in the capsid (15, 29), abrogate virus attachment to the host cell (8, 14), or prevent uncoating (57). There is no universal acceptance of a single neutralization mechanism, and the various MAbs may neutralize with different combinations of these mechanisms.Neutralizing MAbs against HRV14 have been divided into three groups: strong, intermediate, and weak neutralizers (26, 34). All strongly neutralizing antibodies bind to the NIm-IA site, which was defined by natural escape mutations at residues D1091 and E1095 of VP1 on the loop between the β-B and β-C strands of the VP1 β-barrel (the letter designates the amino acid, the first digit identifies the viral protein, and the remaining three digits specify the sequence number). Because strongly neutralizing antibodies form stable, monomeric virus-antibody complexes with a maximum stoichiometry of 30 antibodies per virion, it was concluded that they bind bivalently to the virions (26, 34). Weakly neutralizing antibodies form unstable, monomeric complexes with HRV14 and bind with a stoichiometry of ∼60 antibodies per virion (26, 52). The remaining antibodies, all of which precipitate the virions, are classified as intermediate neutralizers (26, 34).The structures of two complexes, the strongly neutralizing antibody MAb17-IA and its Fab fragment, Fab17, bound to HRV14, were determined by means of cryo-transmission electron microscopy (cryo-TEM) and three-dimensional image reconstruction (51, 52) and interpreted on the basis of model-building studies that used the atomic structures of HRV14 (40) and Fab17 (28). These studies showed that no observable conformational changes were induced in the viral capsid upon Fab or MAb binding. Modeling and site-directed mutagenesis studies demonstrated that electrostatic interactions play a key role in the binding of Fab17 to HRV14 (52). In the complex, the loop of the NIm-IA site on HRV14 sits clamped in the cleft between the heavy- and light-chain hypervariable regions and forms complementary electrostatic interactions with Lys58^H (on the heavy chain) and Arg91^L (on the light chain) of Fab17. In addition, a cluster of lysines on HRV14 (K1236, K1097, and K1085) interact with two acidic residues, Asp45^H and Asp54^H, in the CDR2 (CDR stands for complementarity-determining region) of the Fab heavy chain (49). Earlier modeling studies also suggested that bidentate binding of MAb17-IA to HRV14 is facilitated by rotation of the Fab constant domains about the elbow axes towards the viral twofold axes (51). This suggested that the flexibility of the elbow region (the junction between the variable and constant domains) plays a role in the bivalent binding process, which in turn increases antibody avidity. Finally, the 4-Å-resolution crystal structure of the Fab17-HRV14 complex clearly showed that the virion does not undergo conformational changes upon Fab binding (49). This crystal structure determination also revealed that the earlier docking of the HRV14 and Fab17 atomic structures into the 22-Å cryo-TEM density map (50) yielded a pseudo-atomic model that was very close to the real structure of the complex.We have expanded our complementary X-ray crystallography and cryo-TEM microscopy studies to examine the structures of two more Fab-virus complexes, using Fab fragments from two other NIm-IA antibodies, MAb1-IA (MAb1) and MAb12-IA (MAb12), bound to HRV14. MAb1 and MAb12 are weak and strong neutralizing antibodies, respectively. Image reconstructions of these two complexes are interpreted on the basis of pseudo-atomic models, which substantiate the previous hypothesis that neutralizing efficacy and binding valency are interrelated (34). Electrostatic interactions at the epitope-paratope interface are highly conserved and apparently important for the antibody binding to the virion surface. Like Fab17, Fab1 and Fab12 penetrate the canyon. There are, however, differences between the orientations of the strongly and weakly neutralizing antibodies and in the contacts made with the receptor binding region of the canyon. Finally, data suggesting that antibody binding to HRV14 is alone sufficient for neutralization and that other possible mechanisms are not required are presented.     

Structure of a neutralizing antibody bound bivalently to human rhinovirus 2.

The structure of a complex between human rhinovirus serotype 2 (HRV2) and the weakly neutralizing monoclonal antibody 8F5 has been determined to 25 A resolution by cryo-electron microscopy and 3-D reconstruction techniques. THe antibody is seen to be bound bivalently across the icosahedral 2-fold axis, despite the very short distance of 60 A between the symmetry-related epitopes. The canyon around the 5-fold axis is not obstructed. Due to extreme flexibility of the hinge region the Fc domains occupy random orientations and are not visible in the reconstruction. The atomic coordinates of Fab-8F5 complexes with a synthetic peptide derived from the viral protein 2 (VP2) epitope were fitted to the structure obtained by cryo-electron microscope techniques. The X-ray structure of HRV2 is not unknown, so that of the closely related HRV1A was placed in the electron microscopic density map. The footprint of 8F5 on the viral surface is largely on VP2, but also covers the VP3 loop centred on residue 3060. C alpha atoms of VP1 and 8F5 come no closer than 10 A. Based on the fit of the X-ray coordinates to the electron microscope data, the synthetic 15mer peptide starts and ends in close proximity to the corresponding amino acids of VP2 on HRV1A. However, the respective loops diverge considerably in their overall spatial disposition. It appears from this study that bivalent binding of an antibody directed against a picornavirus exists for a smaller spanning distance than was previously thought possible. Also bivalent binding does not ensure strong neutralization.     

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.     

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.     

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.     

Docking of a human rhinovirus neutralizing antibody onto the viral capsid

The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods.¹ The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known.² The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 Å from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone³ indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding,⁴ might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating. © 1995 Wiley-Liss, Inc.     

Structure of human rhinovirus serotype 2 (HRV2)

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

The recognition of parvovirus capsids by antibodies

The parvoviruses are small, non-enveloped icosahedral viruses which infect many animals, including vertebrates and arthropods. Vertebrate parvoviruses can be classified into the autonomous and the adeno-associated viruses — the autonomous parvoviruses have been examined in detail for antigenic structure. The protective immunity against parvoviruses in animals appears to be primarily antibody-mediated. The capsid of the autonomous parvoviruses is assembled from two proteins, VP1 and VP2, which overlap in sequence, with VP1 having additional N-terminal residues. Empty capsids can be assembled from VP2 alone.The structures of canine parvovirus (CPV) and feline panleukopenia virus (FPV) have been solved to better than 3·5 Å resolution, and the structure of human parvovirus, B19, has been solved to 8 Å resolution. In each case the T = 1 icosahedron is made up to 60 copies of a structural motif common to VP1 and VP2, consisting of an eight-stranded anti-parallel β-barrel. The surface of the capsid is made up primarily of large elaborate loops which connect the β-strands that make up the barrel. Antigenic epitopes have been mapped utilizing escape mutants, natural variants, peptide analysis and by expression of viral proteins. In CPV two major antigenic determinants were defined by escape mutant analysis, while peptide analysis revealed antigenic determinants in many different regions of the capsid protein, including the amino terminus of VP2. Neutralizing epitopes of B19 were found by peptide analysis in the VP1-unique region and in sequences common to VP1 and VP2. Other antigenic, but non-neutralizing, epitopes were found in the VP1–VP2 junction, as well as various other parts of the VP2 protein.The binding of a Fab derived from one neutralizing anti-CPV Mab has been examined by cryo-electron microscopy image reconstruction, which showed that 60 copies of the Fab were bound per virion. The Fab footprint covered approximately 796 Ųof the capsid surface, in a region where escape mutations to that Mab had been previously shown to cluster. The mechanism of neutralization was not clear, but could involve interference with cell attachment, cell entry or uncoating during the process of cell infection.     

Nonneutralizing human rhinovirus serotype 2-specific monoclonal antibody 2G2 attaches to the region that undergoes the most dramatic changes upon release of the viral RNA

The monoclonal antibody 2G2 has been used extensively for detection and quantification of structural changes of human rhinovirus serotype 2 during infection. It recognizes exclusively A and B subviral particles, not native virus. We have elucidated the basis of this selectivity by determining the footprint of 2G2. Since viral escape mutants obviously cannot be obtained, the structures of complexes between Fab fragments of 2G2 and 80S subviral B particles were determined by cryoelectron microscopy. The footprint of the antibody corresponds to the capsid region that we predicted would undergo the most dramatic changes upon RNA release.     

Cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating

Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-A-diameter channel on the fivefold axis to allow exit of the RNA genome. The beta-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.     

A Novel Universal Neutralizing Monoclonal Antibody against Enterovirus 71 That Targets the Highly Conserved “Knob” Region of VP3 Protein

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the “knob” region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.     

Humanized monoclonal antibodies derived from chimpanzee Fabs protect against Japanese encephalitis virus in vitro and in vivo

Japanese encephalitis virus (JEV)-specific Fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated JE-VAX and then boosted with attenuated JEV SA14-14-2. From a panel of 11 Fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed Fabs A3, B2, and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys(179) (domain I), that for Fab B2 was Ile(126) (domain II), and that for Fab E3 was Gly(302) (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized monoclonal antibodies (MAbs) exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED(50)) of 0.84 microg, followed by MAb A3 (ED(50) of 5.8 microg) and then MAb E3 (ED(50) of 24.7 microg) for a 4-week-old mouse. Administration of 200 microg/mouse of MAb B2 1 day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to that of mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.     

A cellular receptor of human rhinovirus type 2, the very-low-density lipoprotein receptor,binds to two neighboring proteins of the viral capsid

The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-A-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor "staples" the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.     

Structural studies on the mechanisms of antibody-mediated neutralization of human rhinovirus

Antibodies represent a major component of the mammalian immunological defense against picornavirus infection. The work reviewed here examines structural details of antibody-mediated neutralization of human rhinovirus 14 (HRV14) using a combination of crystallography, molecular biology and electron microscopy. The atomic structures of the Fab fragment from a neutralizing monoclonal antibody (Fab17-IA) and HRV14 were used to interpret the ∼25Å resolution cryo-electron microscopy structure of the Fab17-IA/HRV14 complex. While there were not any observable antibody-induced conformational changes in the HRV14 upon antibody binding, there was evidence that charge interactions dominate the paratope-epitope interface and that the intact antibody might bind bivalently across icosahedral two-fold axes. Site-directed mutagenesis results confirmed that charge interactions dominate antibody binding and electron microscopy studies on the mAb17-IA/HRV14 complex confirmed that this neutralizing antibody binds bivalently across icosahedral two-fold axes.     

Chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis

Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Comparison of the three-dimensional structure of two human rhinoviruses (HRV2 and HRV14)

An attempt has been made to build a model of human rhinovirus 2 (HRV2) based on the known human rhinovirus 14 (HRV14) structure. HRV2 was selected because its amino acid sequence is known and because it belongs to the minor rhinovirus receptor class as compared to HRV14, which belongs to the major class. Initial alignment of HRV2 with HRV14 based on the primary sequence and the knowledge of the three-dimensional structure of HRV14 showed that the most probable position of the majority of insertions and deletions occurred in the vicinity of the neutralizing immunogenic sites (NIm). Out of a total of 855 amino acids present in one copy of each of the capsid proteins VP1 through VP4 of HRV14, 411 are different between the two viruses. There are also 6 amino acid residues inserted and 14 residues deleted in HRV2 relative to HRV14. Examination of amino acid interactions showed several cases of conservation of function, e.g., salt bridges or the filling of restricted space. The largest variation amongst the residues lining the canyon, the putative receptor binding site, was in the carboxy-terminal residues of VP1.