Functional capacities of clonal antibodies to Haemophilus influenzae type b polysaccharide

Abstract

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.