Structure of hydrated immunoglobulins and antigen-antibody complexes. Electron microscopy of spray-freeze-etched specimens.

The structure of spray-frozen IgG and IgM, either free in solution or specifically bound to bacterial flagella is compared after freeze-etching with the corresponding preparations negatively stained. The freeze-etched IgG is readily detected; most of the hydrated particles appear approximately spherical with an average diameter of about 12 nm. About 20% are triangular with sides of about 13-14 nm. Antibodies attached to flagella are clearly seen on the top as well as the exposed edges. The technique can thus be used for antigen localization on freeze-etched macromolecules and also on the surface of larger structures. The dimensions and shape of freeze-etched bound IgM confirm the earlier interpretations of the structure of this antibody based on negative staining.

Isolation and characterisation of goat C-reactive protein.

A pentraxin was isolated from acute phase goat serum by its calcium-dependent affinity for agarose, and although it did not bind to phosphorylcholine immobilised on Sepharose, its binding to agarose was reversed by exposure to fluid phase phosphorylcholine. It was identified as goat C-reactive protein on the basis of its immunochemical cross-reactivity with human and bovine C-reactive protein. The molecule was composed of five identical, glycosylated, non-covalently associated subunits, each of molecular weight approx. 24,000. Acute phase serum levels in a small number of samples were not significantly different from normal levels (means 72 and 55 micrograms/ml, respectively), suggesting that goat C-reactive protein is not a major acute phase reactant. No other pentraxin was detected in goat serum.

Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

Rational design of nematode vaccines: hidden antigens.

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.

Vaccination of sheep against haemonchosis with H11, a gut membrane-derived protective antigen from the adult parasite: prevention of the periparturient rise and colostral transfer of protective immunity.

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.

Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contortus.

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.

The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus contortus.

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 helical, polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells.

The structure of the intestinal cells of the parasitic nematode Haemonchus contortus is described. The cells have numerous microvilli about 0-09 micron in diameter; most being 5-5-7-5 micron in length. The microvillar (plasma) membrane is coated with a layer of amorphous material (glycocalyx) about 60 A thick which is electron dense in sectioned preparations. Associated with the surface of this material, and filing the spaces between the microvilli, are filaments in the form of helices about 400 A in diameter and of variable pitch. The helices appear to be flexible but they are aligned approximately with the long axes of the microvilli. There are up to ten helices per microvillus; they extend beyond the tips of the microvilli and are up to 10 micron long. The material has been obtained nearly pure in small amounts. It is primarily protein and it is proposed that it should be called contortin. The monomeric form (of molecular weight about 60,000) has been identified with a Y-shaped structure with arms about 45 A long and 25 A wide seen in negatively stained preparations. The helical filament appears to be formed by lateral polymerization of patirs of these units.

Fine structure of basal bodies (kinetosomes) and associated components of Tetrahymena.

Basal bodies (kinetosomes) and adjoining structures, notably the rootlets (kinetodesmal fibres) and a component of the terminal plate, the rosette, have been examined in the electron microscope in sections of whole Tetrahymena and in negatively stained fragments from homogenates. The previously described structure of the basal bodies has been confirmed and certain details, not apparent in sectioned preparations, have been revealed by negative staining. The rosette is a filamentous structure with nine-fold symmetry lying between the fibres of the basal body at the level of the circumciliary ring. The rootlets are composed in the main of a parallel array of filaments with regularly spaced thickenings which, being in register, give rise to a transverse banding with a repeat period of about 320 A. The rootlets are differentiated at one end for attachment to the basal bodies. Some of the structure revealed by negative staining has been analysed by means of optical diffraction.

The ultrastructure and possible relationships of four obligate anaerobic chytridiomycete fungi from the rumen of sheep.

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.

Localisation and characterisation of ovine immunglobulin within the sheep scab mite, Psoroptes ovis.

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.

Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11.

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.

Strategies for vaccination against gastro-intestinal nematodes.

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.

Development of a vaccine against haemonchus contortus.

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 developed should be applicable to other gastrointestinal parasites.

Surface binding and uptake of polycationic ferritin by neonatal piglet intestinal epithelium.

Pieces of small intestine from newborn, I- and 6-day-old piglets were incubated in vitro and ligated segments were incubated in vivo with polycationic ferritin (PCF) to determine the distribution and possible role in protein uptake of anionic sites on membranes at the luminal surfaces of the epithelial cells. Most PCF binding to the absorptive cells occurred on the upper third or half of the villi and to some non-absorptive cells (tuft cells) throughout the length of the villi. Pieces of intestine which were fixed before incubation had PCF on microvilli and apical invaginations of absorptive cells, but none in the sub-apical tubules. When samples were incubated with PCF in vitro before fixation PCF was bound to the surfaces of microvilli of absorptive cells and to the membranes lining the apical invaginations, some sub-apical vesicles and some sub-apical tubules in all age-groups. In vivo experiments with longer incubation times resulted in a similar distribution, with increased amounts of PCF in the sub-apical tubules in samples from newborn and 1-day-old piglets only. In the newborn there were small vesicles containing PCF which had apparently moved further into the cells. At the same concentration (2 mg/ml) ferritin did not enter the sub-apical tubules; but it did enter and was taken up by the cells in the presence of 4% (w/v) serum albumin. It is concluded that the mechanism of protein uptake does not involve the microvillar membrane and that non-specific interaction with the invaginated plasma membrane, as a function of charge-density, leads to opening of the sub-apical tubular system to protein. Movement further into the cell depends on other factors.

The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus.

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.