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

Abstract

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.

The antiviral activity of antibodies in vitro and in vivo

“…Rappaport (1970) determined that an average of four antibody molecules bound per neutralized MS2 bacteriophage represented a neutralizing dose. Studies by Collono and Smith and their colleagues have indicated that HRV is neutralized by inhibition of attachment, and that the binding of 6-7 antibody molecules per HRV virion is required to reduce HRV attachment by 63% (Colonno et al, 1989;Smith et al, 1993). Icenogle et al (1983) determined that neutralization of poliovirus requires the attachment of 4-5 antibody molecules, as discussed above.…”

“…However, in this case it is particularly clear that the induced conformational changes do not play a major role in picornavirus neutralization (reviewed in Hewat and Blaas, 2001;Smith, 2001). The most convincing data come from cryo-electron microscopy (cryo-EM) and X-ray crystallography of virus-antibody complexes which show that rhinovirus can be neutralized without the induction of large conformational changes in the virion capsid (Hewat and Blaas, 1996;Smith, 2001;Smith et al, 1993Smith et al, , 1996. Furthermore, as the change in isoelectric point has been observed following binding of a large number of different antibodies against a range of epitopes on rhinovirus, it appears unlikely that all these antibodies would induce a common conformational change required for neutralization.…”

“…The strongly neutralizing mAb 17-IA binds to an epitope located on a loop between the B and C strands of the capsid protein VP1 ␤-barrel and situated close to the canyon which harbors the binding site for the cellular HRV receptor ICAM-1 (reviewed in Smith, 2001). mAb 17-IA neutralizes by inhibition of attachment, and it has been demonstrated that attachment of 6-7 antibody molecules per HRV particle is required to reduce attachment by 63% (Smith et al, 1993). Significantly, Colonno et al (1989) have shown that Fab fragments of antibodies against all four neutralizing epitopes on HRV inhibit attachment, some of which are located more-distal from the HRV receptor binding site.…”

“…In particular, the binding of about 6-7 IgG molecules to HRV prevents its attachment to the target cell thereby neutralizing the virus. (Colonno et al, 1989;Icenogle et al, 1983;Smith et al, 1993).…”

Clinical Virology of Rhinoviruses

“…Neutralization of virus infectivity by antibody occurs when IgG binds to the viral surface so that access of the host cell receptor to the canyon is blocked (1). The receptor binding site in the base of the canyon is inaccessible to antibody and is highly conserved across immunotypes (2).…”