# Carrie Cook, PhD 1996

## Thesis Title: Effects of neutralizing antibodies on extracellular events in human rhinovirus replication

Antibodies can be extremely effective at neutralizing virus, but a clear picture of how they accomplish this has yet to emerge. This is explored in detail for human rhinovirus 14 (HRV14) and two monoclonal antibodies, mAb12 & mAb1, which are directed to the same neutralizing immunogenic site (NIm1A) on the virus. A mathematical model was developed that calculates the equilibrium size and stoichiometric distributions of mAb:HRV complexes in solution, information which is not experimentally accessible. The average size of mAb:HRV complexes prepared at several mAb/virus ratios was measured by static and dynamic light scattering. Binding isotherms were constructed. The model was employed in conjunction with these experimental data, plus scanning electron microscopy and sucrose density gradient data from the literature, to determine the three equilibrium constants. These values on a per-site basis for mAb 12:HRV14 are: 1.2*10^8 M-1 for univalent binding of antibody to virus, 4 for bivalent binding, and 2*10^7 M-1 for antibody-mediated crosslinking of separate virions. For mAb1:HRV14 the corresponding values are 1*10^6 M-1, and 3*10^7 M-1.

The second part of this work extended the experimental system to incorporate cellular interactions. The kinetics of HRV14 attachment to the ICAM-1 receptor on HeLa cells were measured at 4 C, 20 C, and 34 C. Forward rate constants are of the form k=$(2.2\times 10\sp{12})$exp($-$7.4 kcal/mol/RT). The Gibbs energy of attachment is 11.7 $\pm$ 0.1 kcal/mol. The non-aggregating antibody mAb12 dramatically reduced the ability of HRV14 to attach to HeLa cells in a concentration-dependent manner. In contrast, mAb1, a strongly-aggregating antibody, enhanced attachment at Ab/V ratios below $\sim$70 and had no effect on attachment under antibody excess. A hypothesis is presented which quantitatively explains the attachment data in terms of the types of antibody:virus binding interactions present: bivalent binding of antibody to virus prevents the attachment of HRV14 to HeLa cells whereas univalent binding has no effect. Comparison with infectivity data suggests that mAb12 neutralizes primarily by blocking attachment to cellular receptors while mAb1 neutralizes by aggregation. This work demonstrates the value of using a comprehensive, quantitative approach to studying viral netralization mechanisms.