Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies

TLDR

Hybridomas H3 and D3 were generated to produce monoclonal neutralizing antibodies against poliovirus type 1, and toluene‑2,4‑diisocyanate was used to covalently cross‑link their F(ab) fragments to the virus, revealing that VP1 contains both epitopes. The two monoclonal antibodies recognize distinct, non‑overlapping epitopes on capsid protein VP1, with H3 binding virions, 80S, and 14S subunits and D3 binding only virions and empty capsids, and escape variants arise at a frequency of ~10⁻⁴ during cell‑culture passage.

Abstract

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.