Conference Abstract| February 01 2002 The H11 group of membrane aminopeptidases; characterisation and expression E.A. Munn; E.A. Munn 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar T.S. Smith; T.S. Smith 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar M. Graham; M. Graham 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar H. Smith; H. Smith 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar W.J. Coadwell; W.J. Coadwell 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar R. Hederer; R. Hederer 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar A. Hutchings; A. Hutchings 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar J. Rocha; J. Rocha 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar S.E. Newton; S.E. Newton 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar J. Sexton; J. Sexton 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar V. Cook; V. Cook 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar M. Walkiewicz; M. Walkiewicz 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar D. Hasse; D. Hasse 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar S. Nikolaou; S. Nikolaou 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar D. Hartman; D. Hartman 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar D. Donald D. Donald 1The Babraham Institute, Babraham, Cambridge CB2 4AT and Victorian Institute of Animal Science, 475 Mickleham Road, Attwood, Victoria 3049, Australia Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2002) 30 (1): A41. https://doi.org/10.1042/bst030a041a Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation E.A. Munn, T.S. Smith, M. Graham, H. Smith, W.J. Coadwell, R. Hederer, A. Hutchings, J. Rocha, S.E. Newton, J. Sexton, V. Cook, M. Walkiewicz, D. Hasse, S. Nikolaou, D. Hartman, D. Donald; The H11 group of membrane aminopeptidases; characterisation and expression. Biochem Soc Trans 1 February 2002; 30 (1): A41. doi: https://doi.org/10.1042/bst030a041a Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2002 Biochemical Society2002 Article PDF first page preview Close Modal You do not currently have access to this content.

Ovine IgG was detected in homogenates of repeatedly washed Psoroptes ovis. Some of the immunoglobulin in the homogenates was fragmented although the host IgG present in mite washings was largely intact. The host immunoglobulin was immuno-localised to the surface or cytoplasm of the gut cells of feeding stages of freshly harvested P. ovis examined by cryosectioning. A similar distribution of rabbit IgG was detected in P. cuniculi. The IgG demonstrated in the mite gut represented partially digested as well as intact immunoglobulin. The presence of intact host immunoglobulin suggests that P. ovis may be susceptible to vaccination by the gut antigen approach, a method used successfully for blood-feeding ectoparasites like Boophilus microplus.

Many parasitic nematodes are developing resistance to chemical treatment, and the search is on to produce commercially viable molecular vaccines. Much progress has been made with highly protective 'hidden antigens', especially for Haemonchus contortus, and recent work with new 'natural antigens' has yielded promising results. Here, Sue Newton and Ed Munn review the most recent advances in these two main approaches to this problem.

Hidden antigens are defined and the general validity of the hidden antigen approach is considered. Approaches to the problem of identifying hidden antigens are offered. The nature of the immune responses induced by injection of hidden antigens and their value in giving protection is considered in the light of the site of the hidden antigen in vivo. Particular attention is given to the value of integral membrane ectoenzymes as protective hidden antigens. The need to generate hidden antigens as recombinant proteins and the possibilities and problems associated with linear, conformational and carbohydrate epitopes are outlined. Finally, concerns about the lack of stimulation of induced immune responses and the risks of resistance developing are addressed.

As part of a systematic examination of the protective epitopes on H11, groups of sheep were vaccinated with preparations of purified H11 used untreated (group A), or progressively denatured (linearized) by incubation with sodium dodecyl sulphate (SDS) (group B) or by boiling with SDS in the presence of dithiothreitol (group C). All the sheep developed antibodies which bound to the untreated H11. When challenged with 10 000 infective larvae of Haemonchus contortus the mean levels of protection relative to the mean values for adjuvant controls were 99.8%, 85% and 79% for faecal egg counts and 95%, 79% and 54% for worm burden at post‐mortem for groups A, B and C respectively. The H11‐specific antibodies inhibited the microsomal aminopeptidase activity of H11 in vitro up to 80%. The levels of inhibition by sera from individual animals correlated with levels of protection with r2, of 0.69–0.87.

To establish for how long protective antibody levels may be maintained, lambs were vaccinated with the gut membrane antigen H11 and challenged with Haemonchus contortus 14, 84, 126 or 168 days later. Compared to controls, mean faecal egg counts of vaccinated lambs were reduced by 97 per cent, 99 per cent, 92 per cent and 86 per cent respectively. Total worm burdens at postmortem five weeks after infection were reduced by 87 per cent, 94 per cent, 92 per cent and 62 per cent respectively. In vaccinated lambs, antibody levels to H11 peaked at about 60 days after the first vaccination and were maintained for the duration of the experiment. There was evidence of secondary antibody responses to H11 following challenge.

Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein.

Pregnant ewes were immunised with a fraction highly enriched in the membrane glycoprotein antigen H11, isolated from the intestinal brush border of adult Haemonchus contortus. Immunity induced by immunisation was able to abolish almost completely (98-99%) the worm egg output from pregnant ewes challenged with ca. 10,000 infective larvae of H. contortus during the last trimester. Furthermore, lambs born and reared on vaccinated ewes had substantial antibody levels to H11 derived from maternal transfer. This antibody conferred moderate protection against a bolus challenge of ca. 3000 infective larvae of H. contortus in 5-week-old lambs.

A detergent extract of adult Haemonchus contortus enriched in the integral membrane protein H11, previously shown to give protective immunity against the parasite, was fractionated by lectin and ion-exchange chromatography. The fractions were evaluated for their ability to immunize Clun Forest and Dorset Horn sheep against experimental haemonchosis. Most of the protective activity was associated with H11. Used in an approximately 95% pure form it gave a mean reduction in parasite egg output of 94.6% and reduced male and female worm numbers by 86.5 and 93.5%, respectively. Level of protection correlated with serum antibody titre to H11.

Haemonchus contortus is an economically important nematode parasite of sheep and the occurrence worldwide of strains resistant to anthelmintic chemicals has emphasized the need to develop a vaccine against it. Here, Ed Munn describes the approach to this problem adopted in his laboratory. The principle devloped should be applicable to other gastrointestinal parasites.

Groups of South African, farm-reared merino lambs about 4 months old were vaccinated with extracts of adult Haemonchus contortus enriched with H11, an integral membrane protein from the parasite's intestinal microvilli, or with proteins soluble in Tween 20. The lambs were challenged with 5000 infective 3rd-stage larvae. Compared to the adjuvant-injected controls, lambs vaccinated with 350 micrograms/kg liveweight of the H11-enriched extract showed an 89% reduction in parasite egg production and an 88% reduction in total worm burden at post-mortem 35 days post-challenge. The H11-enriched extract contained some protein also present in the Tween 20 extract. Lambs vaccinated with 600 micrograms/kg of protein soluble in Tween 20 showed a 40% reduction in faecal egg counts and 51% reduction in total worm numbers 35 days post-challenge. Animals injected with 20 micrograms/kg of a sub-fraction containing H11 obtained from the H11 extract, showed a 71% reduction in egg output and total worm numbers.

An extract of adult Haemonchus contortus enriched in the parasite's intestinal microvillar membrane protein H11 and other integral membrane proteins but free of the protein contortin was evaluated as a potential vaccine in two breeds of sheep. The worm burdens of Clun Forest sheep injected with the extract and challenged with 25,000 infective larvae were reduced 89% by weight compared to the average for the controls. The worm burdens of Dorset sheep (challenged with 10,000 infective larvae) were reduced 72%. In both breeds the reduction in the number of female worms, 92 and 71.8%, respectively, was greater than the reduction in the males (86.5 and 46%). Parasite egg output, determined only for the Dorsets, was reduced 92% protection correlated with serum antibody titre. Most of the antibodies were directed against H11.

A great diversity of gastro-intestinal nematodes parasitise man and his animals. Each nematode is antigenically highly complex with distinct developmental stages. The parasites occupy a range of niches in the host gut and have a variety of feeding habits. Hosts have evolved only partially successful local immune responses. The host and parasite factors modulating these responses are described. Strategies for vaccination are surveyed. It is argued that the best route to successful vaccines will be to seek parasite immunogens which are not normally seen by the host's immune system in the course of infection. Routes to be followed in identifying and characterising such immunogens are proposed and methods for evaluating and maximising the immune response are surveyed. The success of this strategy is illustrated by reference to an experimental molecular vaccine against Haemonchus contortus. Strategies for the use of vaccines and methods for their production in vitro are reviewed.

Zoospores and vegetative growth phases of three cellulolytic rumen chytridiiomycetes, Piromonas, Sphaeromonas and NF1 have been examined by electron microscopy and compared with published and new data on Neocallimastix. The four genera have some 16 distinctive ultrastructural features in common, which collectively may be used to define the group. Some of the common features may individually be sufficient to distinguish these obligate anaerobes from facultative and aerobic chytridiomycetes. These features are the presence of hydrogenosomes at all stages of the life cycle, the presence in rhizoids and sporangia of characteristic crystals coated with hexagonal arrays of particles, and in zoospores the presence of distinct surface layers on the motility organelles and cell body respectively, the organization of the ribosomes into helical and globular arrays and the structures associated with the kinetosomes.

The structure of the kinetosomes of zoospores of the Babraham isolate of Neocallimastix (N. patriciarum, previously described as N. frontalis) both free and in the later stages of zoosporogenesis have been determined from electron micrographs of thin sections. The zoospores have up to 20 flagella. At the base of each, within a circular ridge of the plasma membrane, is a circumflagellar ring. There are no props connecting the triplets of the kinetosome to the plasma membrane, but there are two connections from the skirt around the kinetosome to the circumflagellar ring. In developing zoospores, within sporangia, the circumflagellar ring is perpendicular to the long axis of the flagellum, but in free zoospores it is tilted at an angle of about 60°. The skirt is continued proximally to form a cowl-shaped structure below the kinetosome. The spur has longitudinal and transverse components; microtubules radiate from both. Some microtubules intersect the nucleus, but there is no specific association. The classification of Neocallimastix is discussed.