Characterization of a 35‐kDa carbohydrate larval antigen (CarLA) fromTrichostrongylus colubriformis; a potential target for host immunity

Abstract

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.

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

“…Nine of the 35 unique scFv ELISA positive clones (representing 70% of the ELISA positive clones) produced the same recognition pattern in Western blots of larval extracts, a major band migrating at 35 kDa atop a diminishing ladder of lower MW species (Figure 1). This banding pattern is highly characteristic of T. colubriformis CarLA [9] and virtually identical to the pattern recognized by PAB-1 [10], an anti-CarLA mAb (Figure 1). …”

“…Clearly, the identification of promising vaccine targets benefits from better understanding of the host immune effectors that are involved in protective immunity and the mechanisms deployed by the parasites to evade these effectors. We previously reported identification of a carbohydrate larval antigen (CarLA) that is found on the surface of infectious L3 stage strongylid nematodes [9]. Levels of anti-CarLA mucosal antibodies strongly correlate with rapid immune rejection of infecting Trichostrongylus colubriformis [10] and purified anti-CarLA antibodies are able to elicit the rapid rejection of T. colubriformis L3 infection in naïve sheep [10],[11].…”

Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes

“…For instance, the high association between IgA response and T. circumcincta infection observed by Strain et al (2002) suggests the role of this antibody (Ab) as a possible biomarker for resistance to GIN infections. Sheep challenged with GIN were able to increase IgA levels against carbohydrate larval surface antigen (CarLA; Harrison et al, 2003a;2003b). The widespread presence of CarLA in various nematode species has been suggested as a biomarker that off ers practical, rapid, and easy diagnostic possibilities for identifying GIN resistance in sheep (Shaw et al, 2012).…”

A Phosphorylcholine-Containing Glycolipid-like Antigen Present on the Surface of Infective Stage Larvae of Ascaris spp. Is a Major Antibody Target in Infected Pigs and Humans

“…The 35 kDa carbohydrate antigen (CarLA) detected on L3 of the sheep parasite Trichostrongylus colubriformis [38] shows characteristics similar to As12 as it is also very resistant to multiple enzymatic and chemical treatments, except for those that targeted carbohydrate structures. Although no glycans were detected following PNGase-F and -A digestion or chemical β-elimination, monosaccharide analysis indicated a simple composition suggesting a more polysaccharide nature with repeating units composed of GalNAc and GlcNAc.…”

“…However, unlike As12, the CarLA antigen does not appear to possess any PC group [38]. This might explain why the As12 antigen could not be recognized by immune sheep sera or monoclonal antibodies directed against CarLA.…”