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Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

Maass DR, Harrison GB, Grant WN, Hein WR, Shoemaker CB - PLoS Pathog. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Science and Research Ltd, Porirua, Wellington, New Zealand.

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.

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Immunofluorescence of living T. colubriformis L3 larvae probed with selected anti-CarLA scFvs individually or in combination.Live T. colubriformis exsheathed L3 larvae were probed with Tc.C1 scFv alone, Tc.C2 scFv alone, Tc.C3 scFv alone, or all three in combination as indicated. Bound scFv was stained with anti-E-tag/FITC conjugated mAb and detected by fluorescence microscopy.
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ppat-1000597-g004: Immunofluorescence of living T. colubriformis L3 larvae probed with selected anti-CarLA scFvs individually or in combination.Live T. colubriformis exsheathed L3 larvae were probed with Tc.C1 scFv alone, Tc.C2 scFv alone, Tc.C3 scFv alone, or all three in combination as indicated. Bound scFv was stained with anti-E-tag/FITC conjugated mAb and detected by fluorescence microscopy.

Mentions: The bacterial expressed scFvs were incubated individually with living T. colubriformis L3 larvae and bound scFv was visualized with anti-E-Tag/FITC. All of the nine anti-CarLA scFvs bound to the surface of the exsheathed T. colubriformis nematode although, surprisingly, none were able to stain all the larvae in the population. As seen in Figure 4, members of epitope E1 and E2 groups bound to approximately 40–50% of the population whereas members of epitope group E3 bound about 10–20% of the larvae. When the scFvs in the different epitope groups were incubated with exsheathed T. colubriformis L3 larvae in combinations, the percentage of the population exhibiting binding increased proportionally to the amount that each individual scFv epitope group member were able to bind singularly. As seen in Figure 4, greater than 95% of the larvae in the population were stained when scFvs for all three anti-CarLA epitope classes, E1, E2 and E3, were combined. Although completely unstained worms were observed occasionally, our results indicate that these three classes of CarLA represent the vast majority of all T. colubriformis L3 larvae in our worm population.


Intraspecific epitopic variation in a carbohydrate antigen exposed on the surface of Trichostrongylus colubriformis infective L3 larvae.

Maass DR, Harrison GB, Grant WN, Hein WR, Shoemaker CB - PLoS Pathog. (2009)

Immunofluorescence of living T. colubriformis L3 larvae probed with selected anti-CarLA scFvs individually or in combination.Live T. colubriformis exsheathed L3 larvae were probed with Tc.C1 scFv alone, Tc.C2 scFv alone, Tc.C3 scFv alone, or all three in combination as indicated. Bound scFv was stained with anti-E-tag/FITC conjugated mAb and detected by fluorescence microscopy.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742895&req=5

ppat-1000597-g004: Immunofluorescence of living T. colubriformis L3 larvae probed with selected anti-CarLA scFvs individually or in combination.Live T. colubriformis exsheathed L3 larvae were probed with Tc.C1 scFv alone, Tc.C2 scFv alone, Tc.C3 scFv alone, or all three in combination as indicated. Bound scFv was stained with anti-E-tag/FITC conjugated mAb and detected by fluorescence microscopy.
Mentions: The bacterial expressed scFvs were incubated individually with living T. colubriformis L3 larvae and bound scFv was visualized with anti-E-Tag/FITC. All of the nine anti-CarLA scFvs bound to the surface of the exsheathed T. colubriformis nematode although, surprisingly, none were able to stain all the larvae in the population. As seen in Figure 4, members of epitope E1 and E2 groups bound to approximately 40–50% of the population whereas members of epitope group E3 bound about 10–20% of the larvae. When the scFvs in the different epitope groups were incubated with exsheathed T. colubriformis L3 larvae in combinations, the percentage of the population exhibiting binding increased proportionally to the amount that each individual scFv epitope group member were able to bind singularly. As seen in Figure 4, greater than 95% of the larvae in the population were stained when scFvs for all three anti-CarLA epitope classes, E1, E2 and E3, were combined. Although completely unstained worms were observed occasionally, our results indicate that these three classes of CarLA represent the vast majority of all T. colubriformis L3 larvae in our worm population.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Science and Research Ltd, Porirua, Wellington, New Zealand.

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.

Show MeSH
Related in: MedlinePlus