Combined use of two separate but protective vaccine antigens provides protection against Taenia ovis infection in lambs in the presence of protective maternal antibody.

Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

Antibodies to surface epitopes of the carbohydrate larval antigen CarLA are associated with passive protection in strongylid nematode challenge infections.

Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody-coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode-naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc-120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti-CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.

The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

Vaccines against veterinary helminths.

The majority of attempts to develop commercial vaccines for veterinary helminths have focussed on identifying protein antigens, which could be formulated as protective vaccines. Notable successes have been achieved for some cestode parasites, where recombinant proteins have been developed into highly effective vaccines. Although effective protection can also be obtained using some nematode proteins in their native forms, it has not yet been possible to formulate commercially successful vaccines for other helminth parasites of veterinary significance. Increasing evidence suggests that parasite glycan moieties may provide an alternative source of vaccine antigens, and increased attention is now being given to this class of compounds. In addition to identifying candidate protective antigen(s), an increased research effort is needed to develop appropriate strategies for the formulation and delivery of helminth vaccines.

Echinococcus granulosus: use of an intermediate host mouse model to evaluate sources of protective antigens and a role for antibody in the immune response.

A Balb/cJ mouse model was used to determine which stage of the E. granulosus life cycle possessed the most potent protective antigens. Mice were immunized with crude extracts of protoscoleces, brood capsules, cyst fluid, adult worm tissue, eggs or oncospheres and then challenged intraperitoneally with 600 activated oncospheres. Sonically disrupted oncospheres induced the highest levels of protection (greater than 90%) at doses greater than or equal to 10(3) oncosphere equivalents per mouse. High levels of protection were maintained when these preparations were solubilized in SDS. Immunization with Taenia ovis or T. hydatigena oncosphere preparations induced a maximum of 62 and 40% cross-protection, respectively. In passive transfer experiments, serum from triple-infected immune donors that were completely resistant to subsequent challenge induced 69% protection in naive recipients (P less than 0.01). Serum from mice that had been immunized with oncosphere sonicates that were shown to be highly immune, failed to induce statistically significant protection in recipients. A sheep trial confirmed the protective ability of prior infections. Immunization of sheep with a SDS solubilized oncosphere preparation produced 91% protection (P less than 0.01).

Echinococcus granulosus: development of an intermediate host mouse model for use in vaccination studies.

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.

Identification of host-protective antigens of Taenia ovis oncospheres.

Sheep were fully protected against challenge infection following immunization with a homogenate of T. ovis oncospheres. Ultracentrifugation of sonicated oncospheres either alone or in the presence of a range of detergents did not reduce the immunogenicity of the extracts. Solubilization of oncosphere extracts in non-ionic detergents or sodium dodecyl sulphate (SDS) enabled analysis of host-protective antigens by isoelectric focusing (IEF) and electrophoresis in polyacrylamide gels (SDS-PAGE), respectively. Immunoblotting analysis of oncosphere antigens with immune sheep sera identified predominantly two groups of antigens with relative mobilities of 31-34 kDa and 47-52 kDa with a common isoelectric point of 5.8. The immunogenicity of these antigens was confirmed in vaccination trials using appropriate fractions cut from SDS-PAGE gels and agarose IEF gels. Affinity-purified antibodies prepared against the candidate antigens were used to select the corresponding recombinant DNA-derived polypeptides, one of which was subsequently found to be host-protective.

Cross-reacting antibodies to Sarcoptes suis, Chorioptes bovis and Notoedres cati and anti-P. ovis IgE in sera from sheep infested naturally with Psoroptes ovis.

For the development of immunodiagnostic tests to detect mange mite infections in man and animals, it is necessary to know about antigen structure and cross-reactivity between different mite species and other arthropods in contact with the host. Sera from sheep infected with Psoroptes ovis (sheep anti-P. ovis sera) showed positive reactions in dot blots to P. ovis antigen and cross-reactivity to crude antigen extracts from Sarcoptes suis, Notoedres cati and Chorioptes bovis. Using sheep Specific Pathogen Free (SPF) sera in dot blots, weak reactions were seen to all but the Ch. bovis antigen. In SDS PAGE-separated P. ovis antigens at least 35 different proteins could be distinguished. In western blots, at least 24 out of these 35 were recognized as antigens by sheep anti-P. ovis immunoglobulins. At least 13 were recognized by sheep anti-P. ovis IgE. One of these, at 19 kDa, was recognized only with sheep anti-P. ovis IgE, the other 12 also with anti-P. ovis immunoglobulins. Cross-reactive antigens were recognized by sheep anti-P. ovis immunoglobulins in SDS PAGE-separated, and nitrocellulose transferred mite extracts in western blots as follows: 13 antigens in S. suis extracts, 9 in N. cati and 8 in Ch. bovis. Sheep SPF sera recognized an antigen at 67 kDa in each of these 4 mite species. Rabbit anti-sheep immunoglobulins in the P. ovis antigen control bound to a protein at 28 kDa which may be sheep IgG light chain taken up by P. ovis feeding on sheep. Despite the antigenic similarities between mange mites, sufficient differences were apparent to make immunodiagnostic tests for mange feasible.

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres.

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18-12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants.

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.

Relationship of nematode cholinesterase activity and nematode burdens to the development of resistance to trichostrongyle infections in sheep.

The changes in nematode cholinesterase (ChE) activities were examined in relation to the development of resistance in (1) a flock of young grazing sheep, (2) grazing and penned sheep treated with dexamethasone and (3) penned sheep receiving a single mixed infection. Nematodes from grazing sheep with high faecal egg counts (FECs) had higher ChE activities than those from sheep with low FECs. Female nematodes tended to have higher ChE activities than males, and ChE activities in both tended to decline with increasing age of the sheep. The decline in female Trichostrongylus colubriformis ChE activity was associated with a decline in both worm length and in utero egg count. No decline in nematode ChE activity was observed when grazing sheep were treated with dexamethasone. ChE activity of T. colubriformis established in immunosuppressed penned sheep declined 10-20 fold 8 weeks after cessation of treatment. Nematode burdens in the small intestine and abomasum of grazing sheep were significantly correlated, and in individual species they were also correlated with ChE activities. The development of resistance in sheep and the elimination of adult nematode burdens is discussed in relation to gastrointestinal mucosal globule leucocyte numbers, mucus antiparasite activity and the impairment of nematode metabolic function.

Relationship of gastrointestinal histology and mucus antiparasite activity with the development of resistance to trichostrongyle infections in sheep.

Changes in the numbers of globule leucocytes, mast cells, eosinophils and goblet cells in the gastrointestinal mucosa were examined in relation to the development of resistance and elimination of nematodes in grazing sheep in their first year of life. Sheep immunised against Trichostrongylus colubriformis, and sheep treated with dexamethasone were also examined. A strong association between resistance to infection and the presence of globule leucocytes was found. In contrast, the numbers of mast cells or goblet cells were not correlated with resistance. Globule leucocyte and eosinophil numbers were also correlated with antiparasite activity in mucus. Immunising infections of T. colubriformis given to 10-month-old sheep, their duration limited by thiabendazole treatment, gave rise to considerable immunity to homologous challenge infections. Larvae that developed to the 4th stage were as effective at stimulating immunity as those that developed to the 5th stage. Dexamethasone treatment abrogated resistance to trickle challenge infection with T. colubriformis and reduced mucosal globule leucocyte and mast cell numbers. After cessation of drug treatment, the re-establishment of resistance and adult worm elimination were associated with repopulation of the mucosa with large numbers of globule leucocytes and high antiparasite activities in mucus.

Relationship of histamine in tissues and antiparasitic substances in gastrointestinal mucus to the development of resistance to trichostrongyle infections in young sheep.

The development of resistance to nematode infection and self-cure in a flock of young grazing sheep were examined in relation to changes in the levels of histamine in tissues and levels of antiparasitic substances in gastrointestinal mucus. Analysis of faecal egg counts showed that when the sheep were ranked according to individual mean monthly egg counts there was a significant trend to similar rankings in successive months. Sheep with high and low egg counts were slaughtered at monthly intervals for examination and comparison. Histamine levels in blood and intestinal content fluids were similar in both groups of sheep and were highest during maximum challenge by larval nematodes. Antiparasite activity of the intestinal mucus was significantly higher in sheep with low egg counts than those with high counts, between January and May, and was associated with significantly lower burdens of fourth stage larvae.

Maternal transfer of protection from Echinococcus granulosus infection in sheep.

Although passive immunity against larval metacestodes of the genus Taenia is well established, the transfer of immunity against Echinococcus granulosus infection has not been demonstrated convincingly. The immune status of newborn lambs born to ewes that had been infected or immunised with E granulosus eggs or oncospheres was investigated. The ewes and their six- to eight-day-old lambs were subsequently challenged. Lambs born to triply infected ewes were 80 per cent protected from a challenge infection whereas lambs born to simply infected ewes were only 45 per cent protected. Lambs born to ewes that had been immunised with preparations of sonicated oncospheres had the lowest levels of immunity. The infected ewes were challenged intramuscularly with activated oncospheres and showed some degree of immunity. Ewes which had received oncospheres solubilised in sodium dodecylsulphate or sonicated oncospheres were protected from subsequent oral challenge by 60 to 66 per cent. Radial immunodiffusion revealed that lambs with statistically significantly lower levels of IgG were more susceptible to challenge. However, the degree of protection did not show a simple relationship to the titre of antibody, as determined by an ELISA using a solubilised antigen.

Cotranslocational insertion of apolipoprotein B into the inner leaflet of the endoplasmic reticulum.

Apolipoprotein (apo) B100 is required for the distribution of hepatic triglyceride to peripheral tissues as very-low-density lipoproteins. The translocation of apo B100 into the endoplasmic reticulum (ER) and its subsequent assembly into lipoprotein particles is of particular interest as the protein is both very large (relative molecular mass 512,000) and insoluble in water. It has been proposed that apo B translocation occurs in discrete stages and is completed post-translationally. Several sites of arrest of translocation were reported to be present in apo B15 (the N-terminal 15% of the protein). We have re-examined this question by in vitro translation coupled with translocation into microsomes, and find no evidence for transmembrane segments in truncated apo B proteins. Translocated apo B17 is strongly associated with the membrane of the ER, being only partially releasable with alkaline carbonate, and remaining bound to the microsomes following disruption with saponin. The efficient binding of short segments of apo B, despite the absence of transmembrane domains, suggests that apo B is cotranslationally inserted into the inner leaflet of the ER. This will obviate problems caused by the size and insolubility of apo B100, because the growing hydrophobic protein chains will never exist in a lipid-free form during translocation. From the inner leaflet, apo B in association with membrane-derived lipid can bud into the lumen of the ER to form nascent lipoprotein particles.

Parasite vaccine development: large-scale recovery of immunogenic Taenia ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies.

Genetically modified Escherichia coli expressing the Taenia ovis fusion protein GST-45W(B/X) as inclusion bodies were grown in volumes ranging up to 1000 l. Bacteria were inactivated by heat or chemical treatment without affecting immunogenicity. The fusion protein was recovered in a highly immunogenic form from washed inclusion bodies and from urea-solubilised inclusion bodies. The fusion protein was found to be stable in solution after storage at 4 degrees C for up to 2 years. Vaccines formulated with fusion protein from urea-soluble inclusion bodies gave consistently high protection (89-100%) against challenge infection. The methods described enabled the production of sufficient vaccine for large field trials. These trials generated the data required for product registration and manufacture of a vaccine to prevent T. ovis infection in sheep.

Systematic expression of the complete coding sequence of apoB-100 does not reveal transmembrane determinants.

We have investigated the hypothesis that apolipoprotein B undergoes a regulated process of translocation into the endoplasmic reticulum (ER) which causes the protein to adopt a transmembrane configuration. Protein segments representing the complete coding sequence of apolipoprotein B were first expressed by in vitro translation of transcripts from seven overlapping transcripts. Two regions were identified (located at residue 2425 and between residues 4149 and 4348) that can undergo incomplete translocation into pancreatic microsomes. Ribosome pausing at these sites uncoupled translation from translocation, leading to the synthesis of large cytoplasmically oriented segments of protein. In contrast, when these two regions were expressed by transfection in cultured cells, transmembrane structures were not detected. Endogenous apolipoprotein B-100 synthesis in HepG2 cells generates a spectrum of nascent chains, indicating that ribosome pausing can also occur in intact cells. However, the cellular pause products were cotranslationally translocated. While endogenous apolipoprotein B-100 in HepG2 cells was fully translocated, discrete proteolytic fragments were generated from the amino terminus of the protein when proteases gained access to the lumen of permeabilized microsomes. These products were similar in size and sequence to apoliprotein B proteolytic fragments previously ascribed as the luminal domains of transmembrane apoB-100 molecules (Du, E. Z., Kurth, J., Wang, S. L., Humiston, P., and Davis, R. A. 1994. J. Biol. Chem. 269: 24169-24176).

Studies on the translocation of the amino terminus of apolipoprotein B into the endoplasmic reticulum.

Apolipoprotein (apo) B is either co-translationally assembled into lipoproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded. It has been proposed that apoB undergoes a novel process of translocation which generates cytoplasmically exposed apoB in the ER of hepatic and non-hepatic cells. Transmembrane forms of apoB can also be generated by in vitro translation (Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21), which might explain the origin of untranslocated apoB in vivo. Here we have investigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts. We observe that apoB can become transmembrane at sites of ribosome pausing and be held in this configuration by persistence of tRNA on the peptide chains. Ribosome pausing also occurs at these same sites in the absence of acceptor microsomes. Transmembrane topology can be generated at sites of ribosome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence. Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology. Chimeric constructs with engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with termination codons. Transmembrane forms of apoB and cyclin were not generated during translocation into the ER in transfected COS cells.