AU638728B2

AU638728B2 - Anthelmintic non-living vaccine

Info

Publication number: AU638728B2
Application number: AU59614/90A
Authority: AU
Inventors: Ben Adler; Duncan James Mcgillivery; George Gerald Riffkin; Weng Kwong Yong
Original assignee: Daratech Pty Ltd
Current assignee: Agriculture Victoria Services Pty Ltd
Priority date: 1989-07-21
Filing date: 1990-07-17
Publication date: 1993-07-08
Anticipated expiration: 2010-07-17
Also published as: WO1991001150A1; AU5961490A; EP0486507A1; EP0486507A4

Description

ANTHELM1NTIC NON-LIVING VACCINE

FIELD OF THE INVENTION

This invention relates to vaccines and in particular to anthelmintic veterinary non-living vaccines. The invention also relates to methods of treating animals.

BACKGROUND OF THE INVENTION

Gastrointestinal roundworms (nematodes) are parasites which are found in farm animals and in man, and are the cause of a variety of diseases. Infestation with gastrointestinal roundworms and in particular Ostertagia circumcincta is a major cause of parasitic disease and production losses in the sheep industry, especially in the winter rainfall areas of Australia and other similar climatic areas around the world. Chemical drenches have been extensively used to control this gastrointestinal roundworm parasite since the 1960s with the consequence that drench-resistant parasites are now wide spread and threatening the viability of this industry. The development of an effective anthelmintic vaccine is regarded as one of very few alternative options to save the industry.

Previous workers have developed anthelmintic vaccines which have proven unsatisfactory in practise. One such vaccine is described in a United States Patent No. 3,395,218 (Silverman), granted July 30, 1968. This patent describes the preparation of a non-living vaccine produced by in vitro incubation of third-stage infective nematode larvae into histotrophic stages in an aqueous medium, and then removing the larvae and lyophilising the used aqueous medium. Similarly, a UK Patent Specification 1580539 granted 22 June

1977 describes the preparation of metabolic antigens excreted/secreted by either infective Trichinella spiralis or Haemonchus contortus larvae cultured in a synthetic medium. These antigens are claimed to be useful as an oral vaccine against T. spiralis and other helminth parasites of mammals.

SUBSTITUTE SHEET PCT Application (No PCT/AU85/00282, International Publication No. WO86/02839) by Biotechnology Australia Pty Ltd describes a vaccine comprising a suspension, homogenate or extract of a nematode species which is non-parasitic to mammals or birds. This preparation is claimed to be useful for vaccination against nematode species which are parasitic to mammals and birds.

It is now well known that such preparations contain a great number of different substances, and are therefore antigenetically complex (Anders, Howard & Mitchell, 1982). Only a very small minority of the many antigens present in this prior art preparation may be capable of promoting host-protective immune responses, while the majority generally induce inconsequential or pro-parasitic immune responses which may enhance the worm's ability to survive and/or mediate and produce the pathological lesions responsible for disease. Therefore, a vaccine prepared from such a mixture may not even protect the host from a subsequent challenge infection, let alone be an efficacious vaccine.

In the last few years, considerable efforts have been made towards the identification of individual proteins or groups of proteins which are identical in certain physio-chemical characteristics present in roundworm parasites and which may induce responses in the host animal to protect it from re-infection. A sodium-deoxycholate soluble 41 kilodaiton tropomyosin - like molecule extracted from third stage Trichostronαylus colubriformis larvae was shown to induce 43-51% protection in guinea pigs following immunisation (O'Donnell, Dineen, Wagland, Letho, Werkmeister and Ward 1989). In international patent application _.

WO 89/00163 filed in respect of this molecule, a claim was made that sheep and other mammals can also be protected against parasitic nematode infections when they were immunised with this 41 kilodaiton molecule. In a recent report, a 94 kilodaiton excretory- secretory product of exsheathed third stage T. colubriformis larvae was claimed to induce a mean level of protection of 46% in guinea pigs (O'Donnell, Dineen, Wagland, Letho,

Dopheide, Grant and Ward 1989). However, the ability of this latter molecule to protect sheep and other animals is not known.

PCT Application No. WO 88/00835 (Munn, E.A.) describes a protein doublet (H110D) isolated from the plasma membrane of the intestinal microvilli of Haemonchus contottus. H110D has a molecular weight of about 110 kilodaltons, and this material is claimed to protect lambs against haemonchosis when animals are injected with either components of H110D or the whole protein doublet H100D.

SUBSTITUTE SHEET However in all of these reports, the protection observed with each preparation is marginal. In all cases, the molecule is identified initially as being the most prominent protein component when the crude parasite preparation is analysed. It is obvious that there are other parasite components, including those which are not among the major components of any parasite preparation, that also play a significant role in conferring the near absolute protection one generally observes in the natural host-parasite relationship where immunological resistance is induced by a normal infection. In this latter situation the host can be exposed to a series of parasite proteins and therefore respond accordingly as the parasite grows and develops.

There is therefore a need to identify parasite antigens which are differentially recognised by the immunological mechanism of resistant animals compared with susceptible animals. These antigens are the protective antigens and should form the basis of any vaccines against nematode parasite infections in sheep, cattle and other domesticated animals.

Accordingly, it is an object of the present invention to provide an effective anthelmintic vaccine.

SUMMARY OF THE INVENTION

This invention provides in one form a non-living anthelmintic vaccine comprising an immunologically effective amount of a proteinaceous antigen of molecular weight about 31 kilodaiton as determined by SDS-PAGE extracted from species of gastrointestinal nematode parasites belonging to the phylum Nemathelminthes, class Nematoda, order Strongyloidea, together with a veterinary diluent or carrier therefore.

Preferably the species is selected from the group comprising Ostertagia circumcincta. Haemonchus contortus. and Trichostronαylus colubriformis.

More preferably the species is Ostertagia circumcincta

More preferably the species is Ostertagia circumcincta at the third larval stage.

Preferably the antigen is an excretory, secretory or metabolic product of Ostertagia circumcincta.

More preferably the antigen is an intracellular, somatic and membraneous extract of Ostertagia circumcincta.

In a preferred embodiment the antigen is an alkyl phenol ethoxylate extract, especially iso-

SUBSTSTUTE SHEET octylphenol ethoxylate.

More preferably the antigen includes 5-15% sugar content as measured by phenol/sulphuric acid assays for hexose sugars.

More preferably the sugar content is composed of inositol, mannose, galactose and hexosamine as measured by GC-mass spectrophotometry.

Preferably the vaccine further comprises adjuvants, especially saponin.

In an alternative form the invention provides a method of treating ruminants against the occurrence of nematode parasites by administering an effective dose of the vaccine as described above, thereby eliciting an immune response.

Preferably the ruminants are sheep. Whilst the vaccine of this invention has most economic value with ruminants it is useful for other mammals as well.

Preferably the method comprises administering a low dose of said vaccine.

Preferably the dose level is less than 400^g of the said antigen or its individual components thereof.

The molecular weight of the proteinaceous antigen which may consist of a number of different molecules according to the invention is approximately 31 kilodaiton. The preferred method of measuring molecular weight is that of Laemmli (1970) which uses polyacryiamide gel mixed with 10% sodium dodecyl sulphate (SDS). This technique has been found- to provide consistent results ± 1000 daltons but it should be realised that the molecular weights may vary by two or more thousand daltons. In gel fractionation studies there is evidence that the proteinaceous antigen may be a protein doublet. The term proteinaceous antigen is used in a broad sense and the term includes glycoprotein as a component of the gel fraction.

It is believed that the proteinaceous antigen of the present invention comprises a mixture of proteins of similar molecular weight of about 31 kilodaiton. These proteins may be fractionated by two dimensional SDS-poiyacrylimide gel electrophoresis into at least 10 components with different apparent pi values.

The vaccines according to the present invention may be administered parentally or orally.

Preferably the vaccine further comprises molecules derived from the group comprising Ostertagia circumcincta. Haemonchus contortus. Trichostronoylus colubriformis and other

SUBSTITUTE SHEET I nematode species. It is likely that other molecules, unrelated to the said proteinaceous antigen may also induce a protective immune response in ruminants and that a cocktail vaccine comprising these other molecules together with the proteinaceous antigen or Its components may also be an effective vaccine.

The invention will be further described by reference to a preferred EΞxample.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are reproductions of SDS-PAGE and immunoblot profiles (Figures 1-5 and 8), photomicrographs of immuno-stained worm sections (Figures 6 and 7), Elisa antibody responses of sheep (Figure 9), and comparison of faecal egg outputs between immunised and control sheep (Figure 10).

FIGURE 1 illustrates protein and antigen profiles of third stage infective larvae and adult Ostertagia circumcincta Triton X-100 sonicates. Protein profiles (lanes A & C) are revealed by staining with Coomassie Brillant Blue after SDS-PAGE separation. Antigen profiles (lanes B & D) are revealed using pooled sera from Ostertagia circumcincta infected sheep with the immunoblot technique (see Materials and Methods section). Numbers on the left margin are molecular weight standards expressed in thousands.

FIGURE 2 illustrates the immunoblot identification of antigens in Triton X-100 sonicates from third stage (lanes B, D & F) and adult (lanes A, C & E) Ostertagia circumcincta using sera from experimentally infected resistant sheep (lanes A & B), infected susceptible sheep (lanes C & D), and uninfected worm-free sheep (lanes E & F). Note the strongly stained 31 kDa molecule (arrow) from third stage larval extract revealed by sera from resistant sheep. Numbers on left margin are molecular weight standards expressed in thousands.

FIGURE 3 illustrates the profiles of the 31 kilodaiton (kDa) proteinaceous antigen (OC31) of third stage Ostertagia circumcincta larvae on a 10% (lane A) and a 15% (lane B) SDS- PAGE gel after staining with coomassie brillant blue. Note the doublet appearance of this protein after separation on a 15% gel. The protein doublet consists of two very closely associated antigenic bands as revealed by immunoblotting using monospecific OC31 rabbit antiserum (lane C). Molecular standards are indicated (kDa).

SUBSTITUTE SHEET FIGURE 4 illustrates the appearance of antibodies to the 31 kDa proteinaceous antigen (OC31) in resistant sheep following experimental infection as detected by the immunoblot technique.

FIGURE 5 illustrates the effects of Proteinase K or Periodate oxidation treatments on the antigenicity of OC31 molecule in Triton X-100 extracts of third stage O. circumcincta larvae as detected by the immunoblot technique. The left lane of each treatment is the extract without the respective treatment

FIGURE 6 illustrates the localisation of proteinaceous antigen OC31 using indirect fluorescent antibody staining technique. Transverse sections of third-stage larvae of Ostertagia circumcincta were reacted with monospecific rabbit antiserum against OC31, washed to rid excess antibodies, and the reaction was then detected with a flouresein conjugated anti-rabbit immunoglobulin antiserum. The image was obtained using a laser scanning microscope with confocal imaging system. Note interal location of fluorescence. Scale bar is 3_/ιm.

FIGURE 7 illustrates the localisation of proteinaceous antigen OC31 using immunoelectron micrography:

a. Transverse section of third-stage Ostertagia circumcincta taken from the anterior pharyngeal region reacted with OC31 monospecific rabbit antisera and protein A-gold labelled conjugate. Note triradiate lumen of the oesophagus and darkly stained secretory organelles. Scale bar is 3_/ m.

b. Higher magnification of one of the darkly stained secretory organelles (arrow). Scale bar is 0.3μm.

FIGURE 8 illustrates an autoradiograph of an immunoblot, probed with resistant sheep serum and developed using protein G ¹²⁵l (Amersham), showing the predominant 31 kDa molecule OC31 (arrow) of third stage Ostertagia circumcincta (lane A) present in Triton X-

100 extracts of third stage larvae of TrichostrongyJus colubriformis (lane B) and Haemonchus contortus (lane C), but not present in second stage larvae of Toxocara canis (lane D).

FIGURE 9 illustrates the ELISA antibody responses between immunised and control sheep.

FIGURE 10 illustrates the faecal nematode egg outputs between immunised and control sheep.

SUBSTITUTE SK EET MATERIALS AND METHODS

a. Parasite Materials

The Ostertagia circumcincta strain used in this study has been maintained at the Regional Veterinary Laboratory, Hamilton since 1984. It originated from a naturally infected sheep reared in Western Victoria. Approximately 500 live male and female adult Ostertagia circumcincta were recovered from this sheep at necropsy and surgically transplanted into the abomasum of a worm-free sheep. The parasite life cycle was maintained by serial passage of fresh infective larvae (L3) through worm-free lambs every 4 months. All lambs used for the production of parasite materials were born and reared indoors in a worm-free environment using elevated wire-bottomed cages. Lambs were infected when 3-6 months old with L3 Ostertagia circumcincta. by oesophageal intubation. For the production of nematode eggs, ram lambs were infected with three doses of approximately 50,000 L3 given on alternate days. Faeces were collected for nematode egg recovery after the pre-patent period. L3 were obtained after hatching the eggs and culturing the larvae in vitro for about 10 days (Denham, 1969). Adult Ostertagia circumcincta were recovered from the mucosal scrapings of these animals necropsied about 10 weeks after infection. Fourth (L4) and fifth (l_5) stage larvae were obtained from abomasal digests of ewe lambs infected with single doses of approximately 500,000 L3 Ostertagia circumcincta and then necropsied between 5-10 days, and 15-20 days respectively, after infection (Herlich, 1956).

b. Extraction of antigens

The extraction buffer was 10mM Tris-HCI, pH 8.0 containing 150mM NaCI, 2mM phenyl- methylsulphanyl fluoride (Sigma, USA), 1mM EDTA (Sigma, USA), 50/ιg/ml L-1-Tosyiamide- 2-phenylethychloro-methyl ketone (Sigma, USA) and 25 μg/ml 2-p-TosyI-L-lysine chloromethyl ketone (Sigma, USA) (Clark, Philip & Parkhouse, 1982; Simpson, James & Sher, 1983). Detergents were added separately at the following concentrations: zwitterionic

- Empigen BB-AU (1% v/v) (Albright & Wilson, Australia) and 3-3-Cholamidopropyl- dimethyiammonio-1-propane-sulfonate (CHAPS) (0.25% w/v) (Sigma, USA); anionic - sodium desoxycholate (1% w/v) (Sigma, USA), N-lauroylsarcosine (1% w/v) (Sigma, USA) and sodium dodecyl sulphate (SDS) (1% v/v) (BDH Chemicals, UK); cationic - cetyitrimethylammonium bromide (CTAB) (0.5 w/v) (Sigma, USA); and nonionic -Triton X-

100 (1 % v/v) (Iso-octylphenolethoxylate [10 units]) [Triton is a Trade Mark of Rohm & Haas Co.] (Boehringer Mannheim, W. Germany) and Nonidet P-40 (0.20% v/v) (Sigma, USA) (Pritchard, Crawford, Duce & Behnke, 1985). Larvae (10,000) or adult worms (100) were washed three times in 10mM phosphate buffered saline (PBS) pH 7.4 and then 0.5 ml of extraction buffer, containing the appropriate detergent, was added. Control sonicates of

Ostertagia circumcincta contained no detergent. Samples were sonicated 10-15 times at

SUBSTITUTE SHEET 4°C for 30 seconds each time at an amplitude of 21 μm when greater than 40% of the worms were broken when an aliquote was examined microscopically. Sonicates were then left overnight at 4°C, vortexed for 30 seconds and centrifuged at 10,000g for 3 minutes. The protein content of each supernatant was then determined by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.

c. Gel eiectrophoresis and immunoblot procedures

Samples standardised for protein content (25 μg/well) were electrophoresed on 1.5mm, 10% (or 15%) SDS poiyacryiamide gels (SDS - PAGE) using the discontinuous buffer system of Laemmli (1970). Following eiectrophoresis at 60 mA constant current for 3.5 hours, gels were either stained with 0.25% Coomassie Brillant Blue R-250 (BDH Chemicals,

UK) in water: isopropanol: acetic acid at a ratio of 68:25:7 (v/v), or electroblotted onto 0.45 urn nitrocellulose membrance (Schleicher and Schull, West Germany) in a Trans-Blot cell (Bio-Rad Laboratories, USA) at 60 V for 3 hours using the buffer (diluted 1/2) of Towbin, Staehelin & Gordon (1979). The nitrocellulose membrane was then immunostained by first blocking with 5% skim milk containing 0.05% Tween 20 (Sigma, USA) (Batteiger, Newhall

& Jones, 1982) in Tris-buffered saline for 20 minutes. Sheep serum, affinity purified rabbit anti-sheep immunoglobulins (Kirkegaard & Perry Laboratories inc., USA) and peroxidase- conjugated rabbit-antisheep immunoglobulins (Bio-Rad Laboratories, USA) were appropriately diluted in the blocking buffer and consecutively incubated with the blots for 2 hours at 37°C. Blots were washed with four 10 minute changes of blocking buffer after each incubation and developed with 4-chloro-1-napthol (Sigma, USA) (Hawkes, Niday & Gordon, 1982). Where required, blots were treated before the blocking step with either 10 mM periodic acid (BDH Chemicals, UK) (Woodward, Young & Bloodgood), 1985) or Tritirachium album proteinase k (Protease-type XI, Sigma, USA) at a concentration of 100 μg/m in PBS for 24 hours at 37°C.

Two dimensional eiectrophoresis was performed by the method of O'Farrell (1975). For the first dimension, isoelectric focusing was performed in glass tubes using a 1 :1 mixture of pH 5-7 and pH 7-9 ampholytes (Pharmacia, Uppsala, Sweden). SDS-PAGE, using 13% acrylamide slab gels, was used for the second dimension. The gels were stained and fixed in 0.05% w/v Coomassie blue R250 in 50% (v/v) methanol and 10% (v/v) acetic acid for

20 minutes, destained with 5% methanol and 7% acetic acid, then dried under vacuum before autoradiography.

SUBSTITUTE SHEE^" d. Silver staining of gels

On occasion, eiectrophoresis gels were sliver stained by the method of Morrissey (1981). In brief, the gels were rinsed in H₂0 and soaked in 50% methanol (10% acetic acid fixative for 3 minutes. After a 5 minute immersion in 5% methanol/7% acetic acid solution, the gel was treated with 10% glutaraldehyde for 30 minutes. At this stage the gel was left overnight in a large volume of H₂0. Following a further wash (30 minutes) in H₂0, the gel was immersed in a fresh 0.1% AgN0₃ solution for 30 minutes and then rinsed once in H₂0 and twice in developer solution (3% Na₂C0₃, 0.05% formalin). The gel was then stained with the developer solution until the desired intensity of staining was achieved. The reaction was arrested by the addition of 2.3 M citric acid (5 ml per 100 ml of developer).

e. Purification of O. circumcincta proteinaceous antigen OC31

The proteinaceous antigen OC31 was purified by preparative eiectrophoresis on a 15% SDS-PAGE gel. Briefly, larval lysates were separated on a 15% SDS-PAGE gel. The gel strip containing the proteinaceous antigen OC31 was excised and the proteins eluted by incubation in PBS at room temperature for 10 hours. The protein content and purity of the 31 kDa extract were assessed by coomassie blue and silver staining after it was re- electrophoresed on another SDS-PAGE gel.

f. Sheep Sera

Sera from sheep carrying monospecific infections with Ostertagia circumcincta were collected from 22 animals bred specifically for their resistance or susceptibility to Ostertagia circumcincta as described previously (Riffkin & Yong, 1984). Briefly, these animals were born and reared indoors under worm-free conditions and at 6 months of age they were assayed for their lymphocyte blastogenic responses to a crude L3 Ostertagia circumcincta antigen preparation in the in vitro micro whole blood lymphocyte culture test (Riffkin & Yong, 1984). They were then infected with a total of 50,000 L3 Ostertagia circumcincta.

Nematode egg outputs were monitored from 3 weeks after infection and total adult worm burdens were recovered at post mortem 10 weeks after infection. Eleven of the 22 sheep were designated as "resistant" because they had high lymphocyte blastogenic indices (S.l.>2.0), low mean egg counts (>200 eggs per gram faeces) and low total adult worm burdens (>30C). The remaining 11 sheep designated as "susceptible" had low lymphocyte blastogenic indices (S.I. >1.2), high mean egg count (> 1,500 e.p.g.) and high total worm burdens (> 1,000) (Yong & Riffkin, 1986). For negative controls, sera were obtained and pooled from 9 uninfected six month old worm-free sheep. AH blood samples were collected

SUBSTITUTE SHEET in silicone coated vacutainer tubes (Becton Dickinson, USA) and after separation from the clot, the sera were stored at 20°C.

g. FPLC Profiling

Approximately 300 mg of the purified proteinaceous antigen OC31 were reduced in the presence of 1% w/v SDS, 10mM DTT, in 100mM Tris (pH 8.0) for 60 minutes to 58°C. On cooling to ambient temperature, iodoacetamide was added to a final concentration of 22mM and carboxyamidomethylation proceeded for 15 minutes at RT. Protease was added to 1-

2% (w/w), and the mixture precipitated at -20°C (18 hours) in 10 volumes of acetone

(Aristar, BDH). The pelleted material was washed with 2 changes of acid-ethanol and once in ethanol. The pellet was air dried and resolubilized in the buffer of choice. In the case of trypsin digest, the OC31 pellet was taken up in 200μl 1% v/v trimethylamine (pH 8.0), and a further 7μg trypsin (Worthington, Freehold, USA) added. Digestion occurred overnight at 37°C. The chymotrypsin digest was prepared by addition of 200μ 0.1 M

NH₄HC0₃ pH 7.8, (C0₂) and 10μg chymotrypsin (Worthington) and proteolysis conducted at 37°C for 4 hours. Digestions were arrested by storage at -20°C.

The ensuing peptides were separated by reverse phase chromatography using an organic/aqueous gradient delivered by an FPLC system (Pharmacia). Complete digests were primarily resolved with a 0-92.5% v/v acetonitrile (AcN) gradient in 15-20mM ammonium formate, pH 4.0 (C0₂) applied over 46 minutes, onto a Pro PRC 5/10 C_1/c8 reverse phase column (Pharmacia). The elution was monitored at 214nm.

h. Immunofluoresceπt labelling

Cryostat sections 5-1 Oμm thick of L3 Ostertagia circumcincta were mounted on alcohol- cleaned slides and fixed by immersion in acetone for 5 minutes. After drying at 37°C for 5 minutes, the sections were incubated with 50μl of rabbit anti-OC31 antisera for 1 hour at 37°C in a humidified box. The sections were washed three times in phosphate buffered saline (PBS), incubated with 50μl of a 1/10 dilution of fluorescent anti-rabbit immunoglobulin (Wellcome P/L, Beckenham) for 30 minutes at 37°C,washed again in PBS before mounted with PBS-glyceroI (1 :9) for examination by a lasersharp MRC-500 Scanning Microscope (Bio Rad Laboratories Pty Ltd, Oxfordshire, UK).

SUBSTITUTE SHEET i. Immunoelectron microscopy

L3 Osterta^gia circumcincta were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde, dehydrated (3-5 minutes) in successive concentrations of alcohol (30, 50, 75, 95 and 100%) and infiltrated (2 hour, room temperature) with LR White resin (Timms, 1986). Sections (60 nm) were cut with a diamond knife on a rotary ultramicrotome (Reichert-Jung, Austria) and specimens were picked up onto formvar coated gold (400 mesh) grids (Bio Rad Laboratories Pty Ltd, Oxfordshire). Sections were incubated with rabbit anti-OC31 antiserum (1/5 dilution) for 30 nm at room temperature, washed and then incubated again with a 1/20 dilution of 5 nm gold labelled protein A (Sigma Chemical Company, USA) for 5 minutes, followed by successive treatment with 2.5% glutaraldehyde in PBS for 10 minutes; saturated aqueous uranyl acetate for 5 minutes; and finally lead citrate for 3 minutes. Sections were then washed in 0.2M NaOH and water, dried and examined with an electron microscope (Joel JEM 1005, Japan).

RESULTS

Protein and immunoblot analysis of antigens extracted with different detergents

^• The addition of detergents in the extraction buffer generally resulted in a higher yield of protein from L3 and adult Ostertagia circumcincta compared with sonicates extracted without detergents (Table 1). The detergent which yielded the highest protein content under identical extraction conditions from L4 (p<0.01) and adult worm (p<0.025) was Triton X- 100. Similarly, extraction of proteins from sonicates of L3 and audit Ostertagia circumcincta with CHAPS, N-lauroylsarcosine and CTAB was also less efficient than Triton X-100 (data not shown). SDS-PAGE analysis of Triton X-100 sonicates of L3 (lane A, Figure 1) and adult worm (lane C, Figure 1) of Ostertagia circumcincta revealed few differences in protein profiles between these two life cycle stages of the parasite. However, immunoblot analysis using pooled sera from Ostertagia circumcincta infected sheep revealed that there were more antigens recognised in L3 (lane B, Figure 1) than in adult worm (lane D, Figure 1) extracts.

Antigens differentially recognised bv sera from "resistant" sheep

In order to determine whether there were any Ostertagia circumcincta antigens differentially recognised by sera from sheep which had been identified as resistant to this parasite by both immunological and parasitological parameters (Yong & Riffkin, 1986), the antigens in Triton X-100 extracts of L3 Ostertagia circumcincta were probed with sera from 11 resistant and 11 susceptible sheep. A major antigenic band of 31 kilodaiton (kDa) from L3 extract was identified by antibodies in 8 out of eleven sera from resistant sheep (lane b, Figure 2).

SUBSTITUTE SHEET In experimentally infected resistant animals bled at various times after infection, antibodies to the 31 kDa antigen band appeared in the serum as early as 32 days after infection and persisted until at least 70 days after infection (Figure 3). On the other hand, only 2 out of eleven sera from susceptible sheep reacted with this antigen band, the reaction intensity was much weaker, and detected with sera obtained from animals at post-mortem 70 days after infection compared to those from resistant sheep (lane D, Figure 2). This 31 kDa antigen band did not react with pooled sera from uninfected worm-free sheep (lane F, Figure 2).

TABLE 1

PROTEIN YIELDS FROM Ostertagia circumcincta EXTRACTED USING VARIOUS DETERGENTS

DETERGENT INFECTIVE LARVAE ADULT WORM (ug/ul) (ug/ul)

Triton X-100 0.57 +.0.16* 0.69 ± 0.211

Empigen BB-AU 0.22 ± 0.17 0.49 ± 0.15

Sodium desoxycholate 0.18 ± 0.25 0.16 ± 0.19

Nonidet P-40 0.40 ± 0.12 0.49 ± 0.21

SDS 0.10 ± 0.11 0.60 ± 0.16

No detergent 0.11 + 0.07 0.09 + 0.12

P value + >0.01 > 0.025

mean ± standard deviation of data from 7 experiments, mean ± standard deviation of data from 4 experiments, statistical analysis was carried out using Analysis of Variance.

CHARACTERISATION OF THE 31 kDa ANTIGEN

The 31 kDa antigenic material on a 10% SDS-PAGE is an unusually broad band, and occasionally it has the appearance of a closely associated doublet in certain experiments.

Confirmation of the double banding pattern of this material was obtained by separating the larvae extract on a higher resolution SDS-PAGE gel containing 15% acryiamide and staining the separated proteins with Coomassie blue or probing the proteins with sheep antisera after electroblotting onto a nitrocellulose membrane in the upper and lower bands (Figure 4 ). FPLC profiles of tryptic digests of the reduced and S-carboxymethylated proteins confirm that the two bands are closely related.

SUBSTITUTE SHEET The protein doublet 0C31 can readily be stained with carbohydrate silver stain which suggested that it is a glycoprotein. However, the sugar content of 0C31 is only 5-15% of the total molecular complex as indicated by phenol/sulphuric acid assay for hexose sugars. GC mass spectrometric analysis identified the presence of inositol, mannose, galactose and hexosamine residues in the carbohydrate moiety. In addition Boehringer gylcan detection kit (Boehringer Mannheim, W. Germany) revealed the presence of giycan when the glycan detection kit was used in immunoblot. Neither proteinase K nor periodate oxidation treatments abolished the immunological activity of 0C31 suggesting the presence of epitopic structures other than those normally conferred by peptides (Figure 5).

Apart from its detection in L3 preparations, the protein doublet 0C31 antigen is not found in Triton X-100 sonicates of L4, L5 and adult Ostertagia circumcincta (lane A, Figure 2).

Extracts of L3 Ostertagia circumcincta^" probed with sera from Osterta^gia circumcincta infected sheep were more immunoreactive than those of adult Osterta^gia circumcincta (Figure 1). In contrast to these findings Maizels, Meghji & Ogilvie, (1983) reported that Nippostronoylus braslliensis adults had the greatest number of antigens recognised by immune sera compared to larval stages. Parkhouse & Clark (1983) also found few major antigens in L3 and many antigens in immunoprecipitates of adult Trichinella s^piralis. It would appear therefore, that the predominant antibody response elicited by a nematode is stage specific and that the most immunogenic stage differs between nematodes.

LOCALISATION OF OC31

The internal distribution of the proteinaceous antigen OC31 was observed in cryostat sections of L3 Ostertagia circumcincta immunostained with rabbit anti-OC31 antibodies and fluorescent anti-rabbit immunoglobulin. Both the cuticular and hypodermal layers were non- fluorescent areas and were readily seen (Figure 6). To further define the structures responsible for fluorescence, serial sections of L3 Ostertagia circumcincta were examined under an electron microscope after immuno-staining. Specific deposition of electron-dense gold particles was seen in "secretory organelles" of transverse sections taken from the anterior pharyngeal region of the larvae (Figure 7). No gold particles were detected in similar sections of L3 Ostertagia circumcincta which had been exposed to normal rabbit sera.

SUBSTITUTE SHEET The internal location of the immunogold label to the secretory organelles was highly specific and reproductible. The biological function of OC31 is not known but the secretory organelles in the oesophageal glands are thought to discharge via the lumen of the oesophagus (Lee, 1968, 1970). The localisation of OC31 in the secretory organelles had further suggested that these molecules could be excreted/secreted by the larvae.

Metabolic labelling of L3 Ostertagia circumcincta and immunoprecipitation analysis of the excreted/secreted material provided evidence that OC31 is probably an excretory/secretory product. Other low molecular weight antigens secreted by living worms have been described (Lightowlers & Rickard, 1988).

SPECIES DISTRIBUTION OF OC31

In order to determine whether the proteinaceous antigen OC31 was unique to L3 Osterta^gia circumcincta. Triton X-100 extracts of infective larval stages of other nematode species were probed with serum from Ostertagia circumcincta infected resistant sheep. Figure 8 is an autoradiograph of an immunoblot, probed with resistant sheep serum and developed using protein G ¹²⁵l (Amersham), showing the predominant 31 kDa band is a group of molecules

(arrow) of third stage Ostertagia circumcincta (lane A) present in Triton X-100 extracts of third stage larvae of Trichostrongylus colubriformis (lane B) and Haemonchus contortus (lane C), but not well represented in second stage larvae of Toxocara canis (lane D).

The OC31 antigen (Figure 8 lane A) was found to be present in extracts of infective larvae of Trichostrongylus colubriformis (Figure 8 lane B) and Haemonchus contortus (Figure 8 lane C), but not in Toxocara canis (Figure 8, lane D). Similar results were obtained with monospecific rabbit anti-OC3l serum. It is well established that the stimulation of specific immune responses, by a given species of nematode parasite, may protect the host against infection by other nematode species. For example, Blanchard & Wescott (1985) have recently shown that previous infection with Ostertagia circumcincta enhances the resistance of lambs to Haemonchus contortus infection. While it is possible that these observations are accounted for by the stimulation of non-specific mechanisms, OC31 could well be an important cross-reactive or common antigen involved in the protective immune response of sheep to other gastrointestinal nematode species. Milner, Beall & Orwat (1987) have indicated that there may be considerable homology between antigens of Trichostrongylus colubriformis and Ostertagia circumcincta. they stated that the majority of proteins detected were shared by each species, but failed to specifically identify any particular antigens for meaningful comparisons.

SUBSTITUTE SHEET VACCINATION TRIAL WITH THE NATIVE PROTEINACEOUS ANTIGEN OC31

a. Experimental Design

Eleven 6 month old lambs, reared indoors in a worm free environment from birth, were randomised on the basis of pre-immunisation ELISA titres to the proteinaceous antigen OC31 into treatment (N=6) and control (N=5) groups respectively.

The treatment group were given an initial intradermal injection of 100 μg of the purified proteinaceous antigen OC31 in Quil A adjuvant (Harcros International Chemicals, Australia). Identical booster injections were given 3 weeks later and thereafter at weekly intervals for two weeks. Each immunisation and booster injection consisted of 100 μg of the OC31 containing 250 μg/ml of Quil A in 10 mM phosphate buffered saline, pH 7.4 in a final volume of 2 ml. Control animals received the same regimen of immunisation and booster injections excluding only the purified proteinaceous antigen OC31.

Both control and treated groups were infected with three doses of 14,000 L3 Ostertagia circumcincta given on alternate days (total /animal 42,000 larvae) commencing 6 weeks after the initial immunisation injection (one week after the third and final booster immunisation).

b. Faecal egg counts

Faecal egg counts (F.E.C.) were carried out using the quantitative floatation method described by Gibson (1965). Briefly, faecal samples were collected daily, in the morning, from each animal. 2.0g samples were thoroughly mixed with 10 ml of water and allowed to stand for 5 minutes. Saturated salt (NaC1, S.G. 1.20) was then added to 60 ml. The suspension was stirred thoroughly and aliquots were then transferred to McMaster egg counting slides.

c. Collection of blood samples

Blood samples from sheep were collected prior to commencement of immunisation and thereafter prior to each immunisation injection at the day of challenge and at weekly intervals thereafter until necropsy at 10 weeks post immunisation.

d. Immunblots and enzyme linked immunosorbent assay (ELISA)

immunoblot procedures have been previously described. Briefly, L3 Ostertagia circumcincta Triton X-100 sonicate was resolved by sodium dodecyi sulfate-polyacryiamide gel eiectrophoresis (Laemmli, 1970) and electrophoretically transferred to nitrocellulose paper (Schleucher Schull, West Germany) as described by Towbin et al (1979). Blocking

SUBSTITUTE SHEET and wash buffer consisted of 5% non-fat milk powder containing 0.05% Tween 20 (Sigma Chemical Company, Missouri) in Tris-buffered saline, and colour development was carried out using 4-chIorc-1-naptho! (Sigma Chemical Company, Missouri) (Hawkes §!§! 1982).

5 EUSAs were carried out using flat bottomed microtitre trays (Immunlon, Dynatech

Laboratories) coated with 10 μg/ml (0.5 μg/well) of purified OC31 antigen in carbonate- bicarbonate buffer, pH 9.6, by incubation at room temperature for 1 hour. Sheep antiserum diluted 1:500 in PBS, pH 7.4 containing 1% bovine serum albumin (Sigma Chemical Company, Missouri) was added. After incubation at room temperature for 2 hours, the trays

10_. were washed three times with PBS containing 0.05% Tween 20. Bound sheep OC31 antibody was then reacted with HPRO conjugated rabbit antisheep immunoglobulins (Kirekgaard & Perry Laboratories Inc., USA) and incubated at room temperature for 2 hours. After 3 washes in PBS followed by 3 washes in distilled water, ABTS colour development substrate was added (100 ul/well) and the colour reaction developed for 1 hour at room

₁5 temperature. The absorbance (OD) of the enzyme substrate reaction product was measured at 450 nm in an automatic micro ELISA reader (Model MR580; Dynatech Laboratories). Uninfected sheep serum was used as a negative control in each test tray.

e. Lymphocyte blastogenic indices

Lymphocyte blastogenic responses of control and vaccinated sheep to the purified OC31 20 antigen were assayed in an in vitro micro whole blood lymphocyte culture test described by Riffkin & Yong (1984). Briefly, 0.2 mi blood samples were collected from each sheep and immediately added to 2.4 ml of sterile HEPES (25 mM) buffered RPMI 1640 culture medium supplemented with 4% heat inactivated foetal calf serum, 100 units/ml sodium penicillin and 100 μg/ml streptomycin sulphate.

25 0.1 ml aliquots of diluted whole blood were dispensed into round bottomed microtest plates (Nunclon, A/S Nunc Denmark). 25 μl of mitogens, antigens and plain medium (control) were then added to triplicate wells. The plates were sealed with polypropylene tape (Dynatech), mixed for 15 seconds on a microplate shaker and incubated at 37°C until the cells were harvested 4 days later. Sixteen hours before harvesting each culture received

30 3.7 kBq tritiated thymidine (specific activity 185 G Bq dispensed in 25 μl RPM1 1640). The cultures were then mixed and harvested onto glass fibre filter paper discs using a multiple cell harvester (Flow Lab). Erythrocyte lysis (15 seconds distilled water), bleaching (10 seconds H₂0₂), dehydration (10 seconds methanol) and drying (20 seconds in air) were carried out before drying filter paper discs in a 60°C oven for 10-15 minutes. Finally each

35 disc was placed into 2m! of toluene based scintillation fluid and then counted in a liquid scintillation spectrophotometer.

SUBSTITUTE SHEET f. Total worm counts

For total worm counts, the abomasum was dissected out at post mortem after clamping the duodenum just below the pyloric sphincter. The greater curvature of the abomasum was slit and opened flat. The contents were washed with PBS pH 7.2, and collected together with the mucosal scrapings. The combined washes and scrapings were then concentrated and cleaned by repeated refilling and shaking in a 600 ml screw top jar (with a 317 μm sieve inserted in the lid) until the discharged water was clear. Adult worms were individually picked and counted with the aid of a dissecting microscope. Pepsin digestion of the abomasa after scraping was carried out to determine the number of immature worms. Pepsin digestion was carried out in PBS containing 2% HCI and 1.6% pepsin (Sigma

Chemical Company, Missouri, USA), at 42°C for 3 hours, with frequent shaking. Immature worms were collected from the abomasal digest by washing through a 38 μ m sieve.

g. Histopathology

Formalin fixed, paraffin embedded tissues from the abomasa of control and vaccinated sheep were obtained at necropsy, stained by Haematoxyiin and Eosin (H & E) and examined by light microscopy.

RESULTS

Evaluation of mmunisation

Humoral OC31 antibody levels in vaccinated and control sheep were monitored by EUSA and immunoblot analyses throughout the course of the experiment. Cell mediated immune responses were assayed using peripheral lymphocyte stimulation indices. Evaluation of the course of parasite infection was determined by monitoring faecal egg counts on a daily basis from day 28 after challenge until the day before necropsy. After necropsy the total worm counts were carried out.

The ELISA antibody responses of sheep immunised with OC31 are shown in Figure 9.

Elevated levels of OC31 antibodies were detected as early as two weeks after the first immunisation dose in vaccinated sheep. Compared with the control animals OC31 antibody titres rose steadily in the vaccinated group and remained at a high level throughout the duration of the experiment. Immunoblots probed with serum taken from vaccinated sheep during the course of the experiment demonstrated and confirmed the appearance of OC31 antibodies four days after the first booster immunisation. In control animals no OC31 antibodies were detected in immunoblots throughout the course of the experiment.

| SUBSTITUTE SHEET immunisation with the OC31 antigen resulted in a marked and specific cell mediated response of peripheral lymphocytes (Table 2) as detected by the in-yrtro whole blood lymphocyte culture assay. A heightened response during the preinfection immunisation program was still significant at post-infection but tailed off by the end of the experiment. Examination of the H & E sections indicated that in vaccinated animals there were more larvae present in the dilated crypts compared to control animals and these larvae were surrounded with eosinophils. in H & E sections from vaccinated animals, a massive infiltration of lymphocytes in the lamina propria and oedema in the sub-mucosa and muscle layers were observed. TABLE 2

CELL-MEDIATED RESPONSE OF VACCINATED AND CONTROL SHEEP

IMMUNISED WITH THE PROTEINACEOUS ANTIGEN OC31 OF

L3 OSTERTAGIA CIRCUMCINCTA

Positive stimulation index is defined as 2 or above.

Statistical analysis was carried out using a split plot (animals split for time) analysis of variance. Treatment was allocated to six animals at random, the remaining animals were controls.

# C = control; V= vaccinated

Nematode Burden

The faecal egg outputs of immunised sheep were significantly lower (P<0.05) than those of the control sheep (Figure 10). Similarly the total worm counts were significantly lower

SUBSTITUTE SHEET (P< 0.005) from immunised animals than from control animals as shown in TABLE 3.

TABLE 3

Ostertagia circumcincta ADULT WORM BURDENS IN THE

ABOMASA OF VACCINATED AND CONTROL SHEEP AT POST MORTEM

Group df# mean range P value*

control + 4 6047 3860-9900 vaccinated 5 2555 1175-3647 P< 0.005

+ adult worm count for one of the control sheep was unable to be calculated.

* statistical analysis was carried out using the Pitman randomisation test rather than analysis of variance because the necessary assumptions of normality and homogeneity of variance were not satisfied, even under log transformation.

# degree of freedom

These experiments indicate an immune response after administering unusually low levels of the vaccine according to the invention. It is not known why the present vaccine is so efficacious, but it may be as a result of using a relatively pure fraction of the active material, thereby avoiding blocking effects.

Since modifications within the spirit and scope of the invention may be readily effected by persons skilled in the art, it is to be understood that the invention is not limited to the particular embodiments described, by way of example, hereinabove.

SUBSTITUTE SHEET REFERENCES

ANDERS, R.F, HOWARD, R.J and MITCHELL, G.F 1982. In: Immunolo^gy of Parasitic Infections. S Cohen & K S Warren, Editors, Blackwell Scientific Publications, Oxford, p.28.

BATTEIGER, B., NEWHALL, W.J. and W.J. and JONES, R.B. 1982. J Immunol. Methods 55: 297

BLANCHARD, J.L and WESCOTT, R.B. 1985. Am J. Vet. Res. 46:2136

BRADFORD, M.M. 1976. Anal Biochem 72:248

CLARK, N.W.T., PHILIPP, M. and PARKHOUSE, R.M.E. 1982. Biochem. J 206:27

DENHAM, D.A. 1969. J. Helminth 43:299

GIBSON, T.E. 1965. Manual of Parasitological Techniques. Ministry of Agriculture, Fisheries and Food Central Veterinary Laboratory, Weybridge, England

HAWKES, R.E., NIDAY, E. and GORDON J. 1982. Anal. Biochem. 119:142

HERLICH, H. 1956. Proc. Helminth. Soc. Wash. 23:102

LAEMNLI, N.K. 1970. Nature 227:680

LEE. D.L 1968. J. Zool 154(b) :9

LEE, D.L. 1970. Tissue and Cell 2:225

LIGHTOWLERS, M.W. and RICKARD, M.D. 1988. Parasitoloov 96:123

MAIZELS, R.M., MEGHJI, M. and OGILVIE, B.M. 1983. Immunology 48:107

MILNER, A.R., BEALL, J.A. and ORWAT, A. 1987. Parasit. Immunol. 9:615

MORRISSEY. J.H. 1981. Anal Biochem 117:307

SUBSTITUTE SHEET O'DONNELL, I.J., DINEEN, J.K., WAGLAND, B.M., LETHO, S., WERKMEISTER, J and WARD, C.W. 1989. Int. J. Parasitol. 12:327

O'DONNELL, I.J., DINEEN, J.K., WAGLAND, B.M., LETHO, S. DOPHEIDE, T.A.A., GRANT, W.M. and WARD, C.W. 1989. Int. J. Parasitol. 19:793

O'FARRELL. P.H.. 1975. J.Biol. Chem. 250:4007

PARKHOUSE, R.M.E. and CLARK, N.W.T. 1983. Mol. Biochem. Parasitol. g:319

PRITCHARD, D.I., CRAWFORD, C.R., DUCE, I.R. and BEHNKE, J.M. 1985. Parasit. Immunol 7:575

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OUTTERIDGE, P.M., EDITORS, Australian Wool Corporation and CSIRO Division of Animal Health, Australia, p.30.

SIMPSON, A.J.G., JAMES, S.L and SHER, A. 1983. Inf. Imm. 41:591

TIMMS, B.G. 1986. Am. J. Anat. 175 2:267

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76:4350

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DARATECH PTY LTD by its Patent Attorney David V Gibson, 17 July, 1990.

SUBSTITUTE SHEET i

Claims

1. A non-living veterinary vaccine comprising a proteinaceous antigen OC31 of molecular weight about 31 kilodaiton as determined by SDS-PAGE extracted from species of gastrointestinal nematode parasites belonging to the phylum Nemathelminthes, class Nematoda, order Strongloidea.

2. A vaccine as defined in claim 1 wherein the antigen is an excretory/secretory and metabolic product of Ostertagia circumcincta.

3. A vaccine as defined in claim 1 wherein the antigen is an intracellular, somatic and membraneous extract of Ostertagia circumcincta.

4. A vaccine as defined in either claim 2 or 3 wherein the antigen is produced by Triton X-100 extraction with or without any other detergents from infective third stage larvae or Ostertagia circumcincta.

5. A vaccine as defined in claim 1 wherein the antigen is an excretory/secretory and metabolic product of Haemonchus contortus.

6. A vaccine as defined in claim 1 wherein the antigen is an intracellular, somatic and membranous extract of Haemonchus contortus.

7. A vaccine as defined in claim 1 wherein the antigen is produced by Triton X-100 extraction or by extraction with or without any other detergents from infective third stage larvae of Haemonchus contortus.

8. A vaccine as defined in Claim 1 wherein the antigen is an excretory/secretory and metabolic product of Trichostrongylus colubriformis

9. A vaccine as defined in Claim 1 wherein the antigen is an intracellular, somatic and membranous extract of Trichostrongylus colubriformis.

10. A vaccine as defined in Claim 1 wherein the antigen is produced by Triton X-100 extraction or by extraction with or without any other detergents from infective third stage larvae of Trichostrongylus colubriformis.

11. A vaccine as defined in any one of Claim 1-10 further comprising an adjuvant.

12. A method of treating animals by administering an effective dose of a vaccine as defined in any one of Claims 1-11.

SUBSTITUTE SHEET