Margaret Graham

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

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 conortus

An extract of adult Haemonchus contortus enriched in the parasites 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.

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.

Vaccination of young Dorset lambs against haemonchosis.

Summary Six Dorset Horn lambs were each vaccinated at age 7 weeks and 9 weeks with 50 μg of glycosylated integral membrane proteins, particularly enriched in the protein H11 from the intestinal brush border of adult Haemonchus. At 11 weeks of age the lambs were infected with 10 000 infective third stage Haemonchus larvae. Compared with the average for the control group the vaccinated group of lambs had a 78% reduction in parasite egg output over the patent period, with four of the six better than 93% protected. At autopsy 35 days post‐infection the mean total worm burden of the vaccinated lambs was 83% reduced compared with the controls. The serum antibody titres to H11 correlated with the degree of protection.

Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus

Summary Clun Forest lambs were injected with a fraction containing integral membrane glycoproteins derived from the intestinal microvilli of Haemonchus contortus in two equal doses of 0.5, 5, 50 or 500 μg protein and then challenged with 10 000 infective larvae. The time‐course of serum specific antibody responses were determined. Compared to the adjuvant control group, the animals injected with 1000 μg protein were better than 84% protected and those injected with 100 μg. better than 95% protected by all criteria. For these two groups parasite egg output was reduced 99% on average over the patent period. Two of the animals injected with 10 μg protein were partially protected, with 86% reduction in egg output. Two animals in the group injected with 1 μg protein also showed partial protection. Antibody level correlated with protection.

A Natural Hypomorphic Variant of the Apoptosis Regulator Gimap4/IAN1

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a “delayed” apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency (∼30%). This suggests an adaptive significance for this hypomorphic allele.

Characterization of multiple alleles of the T-cell differentiation marker ART2 (RT6) in inbred and wild rats

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2a2 and Art2b2. Monoclonal antibody Gy12/61 specifically reacted with Art2a2 but not Art2a1 lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2a but not the Art2b alleles in all rats studied. Analysis of Art2a1 and Art2b2 genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection.