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.

Figure 1

Genomic organisation and developmental expression of H. contortus H11 isoforms. (A)H. contortus overlapping genomic scaffold sequences indicating tandem arrangement of all five H11 isoforms. Arrows indicate the direction of transcription from each scaffold. (B) Semi-quantitative RT-PCR of the five H. contortus H11 genes in infective L3 larvae (striped bars) and in adult worms (blue bars). Expression of each gene is shown relative to constitutively expressed control gene Hc-sod-1. Mean of triplicate PCRs is shown, with error bars showing the standard error of the mean.

Figure 2

Expression and purification of recombinant H11-4, H11-1 and H11-5 expressed in C. elegans. rH11 proteins purified by cobalt chelation chromatography were separated by 4-15% SDS-PAGE and stained with Coomassie blue or transferred to PVDF membrane and probed with anti-His (C-terminal) antibody or antiserum to native H11-enriched extract (Anti-nH11). Antiserum from animals prior to H11-extract immunisation showed no reactivity to rH11 isoforms.

Figure 3

Aminopeptidase activity of C. elegans-expressed recombinant H11 proteins. (A) Aminopeptidase activity of rH11 proteins was measured as the change in absorbance at 405 nm using l-leucine p-nitroanilide as substrate. Activity was measured at pH5.5 (striped bars) and pH7 (blue bars) for singly expressed rH11 proteins, rH11-4 and rH11-5 co-expressed in C. elegans (rH11-4/5) and native H11-enriched extract. 500 ng total protein was added per assay. The results of triplicate assays are shown with error bars representing the standard error. (B) Reduction in aminopeptidase (AP) activity of rH11-4/5 co-expressed proteins (striped bars) and native H11 extract (blue bars) was measured in the presence of amastatin or bestatin, both at 10 μM final concentration. Assays were carried out in duplicate and % reduction calculated relative to pre-incubation in buffer alone.

Figure 4

C. elegans-expressed rH11-4 protein is modified by N-linked glycosylation and phosphorylcholine. (A) Purified rH11-4 protein, rH11-4 protein in which the three potential N-glycosylation sites were mutated by site directed mutagenesis (rH11-4 ΔN-Gly) and native H11-enriched extract were separated by 4-15% SDS-PAGE, transferred to PVDF membrane and probed with HRP-labelled Con A lectin. (B and C) MALDI-TOF mass spectrometry (MS) profiles of N-linked glycans from rH11-4 protein. N-glycans of purified rH11-4 were released from tryptic glycopeptides by digestion with PNGase F (B) or PNGase A (C). Profiles of N-glycans are from the 50% MeCN fraction from a C₁₈ Sep-Pak (Materials and Methods). All molecular ions are [M + Na]⁺. Putative structures are based on composition, tandem MS and biosynthetic knowledge. Structures that show sugars outside of a bracket have not been unequivocally defined. (D) Western blot of rH11-4 and rH11-4 ΔN-Gly probed with phosphorylcholine (PC) antibody TEPC-15.

Figure 5

Faecal egg output, adult worm burdens and ELISA antibody responses following vaccination with rH11-4/5 co-expressed proteins or with native H11-enriched extract. Faecal egg count (FEC) (A) was monitored from days 14–34 post-challenge infection and adult worm burdens (B) counted at necropsy on day 35. The mean group count from lambs vaccinated with rH11-4 /5 proteins, native H11-enriched extract or Vax Saponin adjuvant are shown, with error bars representing the standard error of the mean. (C) ELISA OD values of antisera (1/800 dilution) reactivity to rH11-4/5 co-expressed proteins or native H11 extract following immunisation with the homologous antigen. Lambs were vaccinated on days 0, 21 and 42 and challenged with 5000 L3 on day 42, as indicated. (D) Ig isotype responses (1/50 dilution) to native H11-enriched extract measured by ELISA.

Figure 6

Antibody reactivity to glycan epitopes on C. elegans recombinant or native H11 proteins. Western blots showing recognition of rH11-4, rH11-4ΔN-Gly, native H11-enriched extract and native H11-enriched extract treated with mild periodate, by antibody to native H11 extract (anti-nH11; panel A) or to rH11-4/5 proteins (anti-rH11; panel B). Reactivity of anti-His antibody confirmed equal loading of rH11-4 protein (panel C). Proteins in addition to H11 are observed in the nH11 extract.