Human Rhinovirus
Human rhinovirus is one of the major causes of the common cold. There are over 100 serotypes of this virus, making it unlikely that there will ever be a traditional vaccine for the common cold using conventional vaccine methods. We have examined the neutralization and dynamics of this virus to better understand how antibodies block infection and how to make more efficacious and novel vaccines.
HRV Organization
About each icosahedral 5-fold axis lies a canyon-like structure. From the atomic structures, the sites of natural escape mutations were found to lie at the top of these canyons. From this result, the dimensions of the canyon, and the conservation of some of the residues lying at the canyon floor, it was thought that the receptor might bind to the bottom of the canyon thus hiding important residues from the immune system. In this way, the virus might be allowed to change its surface residues, thwart antibody binding, but retain crucial residues necessary for viral infectivity.
Drugs for the common cold
A series of compounds (WIN) were developed by the Sterling-Winthrop company. They bind to a cavity within VP1 of HRV and prevent uncoating. We have continued analyzing the mode of action of these compounds and have shown that they stabilize or prevent the conformational changes associated with receptor interactions thereby preventing the release of its RNA genome.
HRV14 capsid 'breathing'
In a series of studies, we have used MALDI and limited proteolysis to show that portions of the HRV capsid, buried in the crystal structure, are transiently expressed in a 'breathing' process. The first sites of the capsid clipped by matrix bound trypsin are actually buried next to the RNA (yellow spheres). This 'breathing' is shut down by the the addition of WIN compounds and is affected by mutations that fill that drug binding cavity with hydrophobic compounds.
Mechanisms of antibody neutralization
When these studies were initiated, there were several proposed mechanisms for antibody mediated neutralization of viral infections. They could block several stages of viral infections; attachment, endocytosis, and uncoating. The attachment could be blocked by simply getting in the way with receptor attachment, they could rigidify the capsids, or they could induce conformational changes in the capsid. This latter was the dominant idea at the time. The other popular idea was that antibodies could aggregate the virions and decrease the number of independent particles. Again, the most dominant hypothesis was that antibodies (as few as one) could bind and distort the capsid, rendering it non-infectious.
Antibody-mediated neutralization
In order to better understand which of the above proposed mechanisms is most likely to be relevant in-vivo, our collaborators in the Reuckert lab analyzed the neutralization properties of several monoclonal antibodies. The strongest neutralizing antibodes (12 and 17) were the most straightforward - the more you add the better the neutralization. It was thought that if any antibodies were to cause conformational changes, it would be these since there is no apparent aggregation during the neutralization process. In contrast antibodies like 1 were weakly neutralizing and strongly aggregated the virions. Then there were antibodies with mix properties such as 23 and 14 that strongly aggregated leading to either strong or weak neutralization. Nevertheless, 12 and 17 clearly demonstrated that aggregation is not essential to in-vitro neutralization of HRV14.
Cryo-electron microscopy
The next step was to direct test the idea that antibodies induce large conformational changes in the virion. In collaboration with the Baker group, we determined the structures of strongly (12 and 17) and weakly (1) neutralizing antibodies. In all cases there was no evidence of large structural changes in the virion. However, the orientations of the bound Fabs of 12 and 17 suggested that they might be binding bivalently - confirmed by the structure of the IgG of mAb17 bound to HRV14. Even so, large structural changes were not observed. On the other hand, Fab1 binds to the same epitope as 12 and 17 and yet is a strongly aggregating and weakly neutralizing antibody. From the cryo-TEM structure of the complex, this is likely due to binding orientation differences that prevents bivalent attachment and promotes inter-particle cross-linking.
Atomic structure of the Fab17/HRV14 complex.
The above cryo-TEM image reconstructions clearly suggested that the antibodies do not cause conformational changes in the virion upon binding. However, the resolution of these structures were limited and finer changes might have been missed. To this end, we crystallized the Fab17/HRV14 complex and determined its crystal structure. We found, again, that the virus is neutralized without associated conformational changes. Even more important, we found that the antibody penetrates the receptor binding site. This was important in that the popular idea at the time was that the receptor binding region was canyon shaped to prevent contact with the immune system. This is clearly not the case here. In the end, these studies demonstrated that the immune system is more plastic than previously thought and that vaccine development need only to focus on production of immunogenic pathogen mimics rather than what might happen to the virus after the antibody binds.
Our relevant publications: