Project 1: To enhance the affinity, avidity, and stability of ICAM-1 D1 for higher potency in human rhinovirus inhibition The major group of human rhinoviruses (HRVs), consisting of ~90 serotypes, utilizes the Intercellular adhesion molecule 1(ICAM-1) (2) as their receptor. A minor group of ~10 HRV serotype uses members of the low-density lipoprotein receptor family to attach to cells. It was suggested that N-terminal domain (D1) of the receptor binds to the rhinovirus. ‘Canyon’ or depression in the capsid that surrounds the virus 5-fold axis (2). ICAM-1 D1 is the domain free of glycan (1), which make it feasible to produce in bacterial. Thus ICAM-1 is attractive agent against human rhinovirus, especially N-terminal domain of the receptor. However, ICAM1-1 D1 expressed in bacterial is very unstable according to our experimental result (unpublished), and show a little binding to rhinoviruses HRV14 and HRV 16. So we can use our yeast surface display system to produce ICAM-1 D1 mutagenesis libraries to select antibodies with high stability, avidity and higher binding affinity to rhinovirus, which make it potent in inhibiting rhinovirus inhibition.

Project 2: To redesign of antibodies against human rhinovirus that have introduced neutralization escape mutations

It have been tested the different mouse monoclonal antibodies against human rhinovirus in a neutralization assay, the mutants of antibodies fell into four antigenic group, which called neutralization immnogens: NIm-IA, -IB, -II, and -III. According to sequencing results reveals that the amino acid substitutions was correlate exactly with the immunogenic groups (3). Such as NIm-III mutants at VP1 amino acid residue 91 or 95. Engineering of antibodies to regain potency to re-neutralize escape mutants will be important in quick developing antibodies for passive immunotherapy to treat rapidly evolving rhinoviruses and find the potential immogenic sites. This can pave the way for further studying the interaction between different antibodies and escape mutant viruses and wild type virus. We already displayed mAb17, which can neutralize HRV14 (Figure 1) and showed good binding to rhinovirus HRV14. We will producing different escape mutant viruses using mAb17 and other important antibodies, such as mAb12, and then screening mAb mutagenesis library with escape mutant viruses to produce antibodies, which can re-neutralize the virus using our yeast surface display system.

Figure 1, Shaded, surface representations of the cryo-TEM image reconstructions of HRV14-Fab17, HRV14-MAb17. Fab17, mAb17, are blue, brown, respectively, with the surface of HRV14 shown in gray. (3)

References: 1. David Jimenez, Pedro Roda-Navarro, Timothy A. Springer, and Jose M. Casasnovas, Contribution of N-linked Glycans to the Conformation and Function of Intercellular Adhesion Moleculars (ICAMs), The Journal of Biological Chemistry, 2005, 280(7): 5854-5861. 2. Jordi Bella, Parsanna R. Kolatkar, Christopher W. Marlor, Jeffrey M. Greve, and Michael G. Rossmann. The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an IFA-1 integrin ligand. Proc. Natl. Acad. Sci. USA. Biochemistry 95: 4140-4145. 3. ZhiWei Che, Norman H. Olson, Donna Leippe, Wai-Ming Lee, Anne G. Mosser, Roland R.Rueckert, Timothy S. Baker, and Thomas J. Smith. Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscope and X-ray crystallography of virus-Fab complexes. Journal of Virology, 1998, 72(6): 4610