Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

Abstract

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes. Strongylid nematode worm parasites currently infect hundreds of millions of people, and most farmed animals, causing enormous morbidity and economic loss. These parasites commonly produce chronic gastrointestinal infections that are highly refractory to immune clearance mechanisms. Mucosal antibodies against a carbohydrate surface antigen (CarLA) can cause rapid expulsion of incoming larval nematodes. Sheep develop strong anti-strongylid immunity following long-term grazing on contaminated pasture. From these sheep, we identified and characterized recombinant antibodies that recognize CarLA on living L3 stage infective larvae of the strongylid parasite, Trichostrongylus colubriformis. The selected antibodies are specific only to larvae of the T. colubriformis species and, surprisingly, recognize only a subset of these worms. Three different anti-CarLA antibody classes were found and each recognizes different, non-overlapping subsets of worms which, together, comprise virtually the entire population. These results are the first demonstration of “intraspecific epitopic variation” within strongylid nematodes and suggest that these parasites have a mechanism that permits the surface presentation of at least three different antigenic forms of CarLA to avoid immune clearance.