Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

Abstract

With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection.

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

“…On the other hand, while antennal α1,3-fucosylation (Lewis and Lewis-type epitopes) is known from some parasitic nematode species (47), this feature is lacking from C. elegans. However, a fourth fucose residue is apparent in a subset of N-glycans of C. elegans (13,14,22-26) and structural models have suggested that this is attached either directly or indirectly (via galactose) to α-mannose residues of the trimannosylchitobiosyl core (14,22,48,49). However, tetrafucosylation of a trimannosylchitobiosyl core is a structural proposition contradicting not only the known specificity of the enzyme (the FUT-6 α1,3-fucosyltransferase) which modifies the distal core GlcNAc (16), but also GC-MS data indicating that a portion of the core β-mannose residues is 3,4,6-trisubstituted and that 2-substituted galactose is also present in wild-type C. elegans (23).…”

Progress in the development of subunit vaccines for gastrointestinal nematodes of ruminants

“…The potential explanations for the complete lack of success with these recombinant proteins include incorrect folding of the proteins, a lack of/or inappropriate glycosylation of the proteins when expressed in bacteria, yeast or insect cells, the induction of lower avidity antibodies or the protein chosen for recombinant production was not exclusively responsible for protection in the original native fraction. Subsequently, to address the issue of glycosylation, attempts were made to express a recombinant version of H11 in the free‐living nematode, C. elegans . In these studies, although the authors demonstrated that the recombinant protein expressed in C. elegans was enzymatically active, an immunization trial in sheep resulted in no significant reduction in FECs nor worm burdens compared to the challenge control animals .…”

“…Subsequently, to address the issue of glycosylation, attempts were made to express a recombinant version of H11 in the free‐living nematode, C. elegans . In these studies, although the authors demonstrated that the recombinant protein expressed in C. elegans was enzymatically active, an immunization trial in sheep resulted in no significant reduction in FECs nor worm burdens compared to the challenge control animals . As all of the recombinant proteins tested in these trials failed to induce a consistent protective effect against H. contortus , a native vaccine (Barbervax ® , WormVax) based on a gut antigen preparation was developed and launched in Australia recently.…”

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs

“…However, obtaining acceptable protection levels with recombinant antigens has proven difficult. Several recombinant vaccine antigens, expressed in Escherichia coli , insect cells, Pichia pastoris or the free‐living nematode Caenorhabditis elegans , failed to confer protection in vaccine trials (Cachat, Newlands, Ekoja, McAllister, & Smith, ; Geldhof, Meyvis, Vercruysse, & Claerebout, ; Roberts et al., ; Vlaminck et al., ). Current research is focusing on differences in protein folding or secondary modifications, such as glycosylation, between the native and recombinant proteins as possible reasons for the lack of protection (Matthews et al., ).…”

“…Several recombinant vaccine antigens, expressed in Escherichia coli, insect cells, Pichia pastoris or the free-living nematode Caenorhabditis elegans, failed to confer protection in vaccine trials (Cachat, Newlands, Ekoja, McAllister, & Smith, 2010;Geldhof, Meyvis, Vercruysse, & Claerebout, 2008;Roberts et al, 2013;Vlaminck et al, 2011).…”