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.

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.

Characterization of a 35-kDa carbohydrate larval antigen (CarLA) from Trichostrongylus colubriformis; a potential target for host immunity.

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.

Immune rejection of Trichostrongylus colubriformis in sheep; a possible role for intestinal mucus antibody against an L3-specific surface antigen.

Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti-larval activity in their intestinal mucus and high-speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti-larval activity of immune mucus is antibody against a 35-kDa L3-specific cuticular antigen. The anti-larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS-PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB-1 recognizes the 35 kDa T. colubriformis larval antigen and also cross-reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22-kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB-1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host-protective immunity against nematode infections.

Intestinal infusion of soluble larval antigen stimulates rejection of heterologous nematode larvae by immune sheep.

Sheep immunised by multiple truncated infections with Trichostronglyus colubriformis were highly resistant to subsequent challenge with homologous exsheathed larvae, administered via a surgically implanted duodenal cannula. The duration of immunity after truncated infections was 12-14 weeks against challenge with T. colubriformis or Cooperia curticei, but there was little cross-protection against challenge with Nematodirus spathiger. When immune sheep were given booster doses of T. colubriformis larvae before challenge with N. spathiger, there were 97% fewer N. spathiger larvae in the first 5 m of small intestine, and an overall 79% reduction of N. spathiger larvae in immunised sheep, compared with naive controls. Boosting T. colubriformis immune sheep with killed T. colubriformis larvae plus soluble T. colubriformis L3 antigen, or with soluble antigen alone, also caused significant displacement of N. spathiger challenge larvae (98% and 100% respectively), indicating a non-specific expulsion process. These results indicate that N. spathiger can be used as an indicator species in T. colubriformis immune sheep, to quantify the effects of stimulating mucosal immunity with specific antigens, which may lead to identification of the antigens required for immunisation against nematodes.