AU7505198A

AU7505198A - Parasite vaccine

Info

Publication number: AU7505198A
Application number: AU75051/98A
Authority: AU
Inventors: Vernon Morrison Bowles; Malcolm Roy Brandon; Elizabeth Cornish
Original assignee: University of Melbourne; Australian Wool Research and Promotion Organization AWRAP
Current assignee: University of Melbourne; Australian Wool Research and Promotion Organization AWRAP
Priority date: 1997-07-04
Filing date: 1998-07-06
Publication date: 1999-01-14
Also published as: NZ330913A; GB9814470D0; GB2327346A; AUPO767497A0

Description

kA P/00/ I I Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE

SPECIFICATION

STANDARD

PATENT

Invention Title: PARASITE

VACCINE

Applicants: THE UNIVERSITY OF MELBOURN'.E .and AUSTRLIAN WOOL RESEARCH AND PROMOTION

ORGANISATION

The following statement is a full description of this invention, including the best method of performing it known to me: p7 2 PARASITE VACCINE The present invention relates to antigens for preventing or reducing parasite infection and disease, their use in the formation of vaccine compositions and methods for identifying such antigens end vaccine compositions.

The sheep blowfly Lucilia cuprina is the most important ectoparasite of sheep in Australia. It is estimated to cost the industry in excess of 250-300 million dollars per annum in lost production and methods of control. Eggs of the sheep blowfly hatch on the skin of sheep where the newly hatched larvae actively feed using powerful proteases and rasping mouthparts to degrade the skin. They create a wound within 8-12 hours and then progress through 3 moults before leaving the sheep after 3-4 days. Approximately 3 million sheep/year die due to flystrike in-Australia as a result of septicaemia and shock. Traditional methods of control include both radical surgical procedures mulesing) and repeated application of insecticides. Resistance to insecticides is now a major problem in the industry and surgery is becoming less acceptable. New and improved methods of control are being sought with vaccination being identified as a worthwhile goal.

Considerable effort has been devoted in the prior art to the development of vaccines to control parasite infections of animals including livestock, cats, dogs and aquatic species. The identification of protective antigens in parasite infections in animals has remained the principal stumbling block to the generation of vaccines.

One of the principal reasons for this is the enormous complexity of for example ectoparasites which may have up to 10% of the genetic information of a mammal, and as a consequence have the ability to produce an enormous array of products at various stages of their life cycle, only a few of which may be important in developing an effective vaccine. In most instances, the researcher is confronted by hundreds of potential antigens. The central puzzle still remaining is which are the parasite antigens that elicit host-protective immune responses.

Reliable methods of testing potential protective antigens are also crucial in the development of parasite vaccines. In general terms, the prior art describes two types of bioassay to evaluate pp. I ~-llOC- the efficacy of potential ectoparasite vaccines against the sheep blowfly. The first is an in vitro feeding system in which larvae are fed on the sera from vaccinated animals, and the second is an in vivo bioassay system in which larvae are placed in rings attached to the skin of sheep. The skin is scarified before the larvae are added. The use of these methods to evaluate two purified serine proteases from L. cuprina larval excretory and secretory material is described in Tellam et al.

(1994) Int. J. Parasitol. 24:757-764. Whilst the serine proteases induced an immune response, the bioassays used indicated that this response did not protect against blowfly strike. Tellam et al. (1994) concluded that serine proteases from the secretory and excretory material of L. cupnna were not good candidates for inclusion into an anti-blowfly strike vaccine. However, applicants further studies suggest that this is not the case as 4 antigens including a chymotrypsin-like protease have been shown to be important in the defence of sheep against the sheep blowfly (see International Patent Application PCT/AU95/00078 the entire disclosure of which is incorporated herein by reference).

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties and deficiencies related to the prior art.

Surprisingly, applicants have found that the use of larvae in some bioassays to evaluate potential ectoparasite vaccines may not be appropriate.

Accordingly, in a first aspect of the present invention there is provided a method for identifying an antigen for preventing or reducing parasite infection or disease in an animal, which method includes administering to the animal an effective amount of an antigen and challenging the animal with an invasive stage of the parasite to mimic natural challenge.

For example in the case of an ectoparasite infection, the animal may be challenged with ectoparasite eggs, in contradistinction to larval challenge.

The eggs may be at a suitable stage of development, e.g. at or near the onset of expression of the antigen. For example, the eggs may be approaching the larval stage.

Whilst the applicant does not wish to be restricted by theory, it is postulated that the use of, for example, L. cupnna eggs or an equivalent invasive stage of other parasites more properly mimics the situation when an animal is infected in 6 -i the field. Accordingly, a number of antigens previously identified and rejected on the basis of inappropriate bioassays may in fact be suitable for the production of successful vaccines.

In a further aspect of the present invention, there is provided an antigen for preventing or reducing parasite infection identified by the above method.

The parasite may be an ectoparasite. The ectoparasite may be, for example, a blowfly or other fly, flea, tick, mite or louse species.

Further examples of fly species include the Calliphoridae family including sub families Calliphorinae spp and Chrysomyinae, and the Haematobia spp.

Further examples of flea species include Ctenocephalides canis and Ctenocephalides fe!is.

S Further examples of tick species include Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Amblyomma spp, Dermacentorspp, Ixodes spp or Hyaloiima spp.

The antigen may be administered by any suitable route. For example, it may be administered orally or may be administered parenteraily (for example by intramuscular, intradermai, subcutaneous or intravenous injection).

In a particularly preferred embodiment the antigen is administered subcutaneously.

The amount of antigen required will vary with its antigenicity. Simple -experimentation will easily establish the required amount. Typical initial doses may be approximately 0.0001-1 mg active ingredient/kg body weight. Multiple doses may be used. For example, the antigen may be administered in an initial dose, optionally followed by at least one further immunisation at a later time, for example after 4 to 8 weeks.

In a preferred embodiment of this aspect of the invention, the method is particularly applicable to the identification of antigens for prevention or reduction of blowfly strike in sheep.

SApplicant has cloned and sequenced a number of antigens which have been identified as protective against blowfly strike by the method described above.

S" Accordingly, in a further aspect of the present invention there is provided an antigen for preventing or reducing parasite infection in an animal, the antigen being a protease; functionally active mutants, derivatives and fragments thereof.

The protease antigen may be a serine protease, in particular a chymotrypsin-like protease.

The antigen may be identified by the identification method described above.

The antigen may be a natural or synthetic antigen or derivative thereof.

Accordingly, in a still further aspect of the present invention there is provided an antigen for preventing or reducing parasite infection or disease in an animal, said antigen having the nucleotide sequence shown in Figure 1A, functionally active mutants, derivatives, fragments thereof and family members or related members of the serine protease family.

In a further aspect of the present invention there is provided an antigen for preventing or reducing parasite infection or disease in an animal, said antigen having the amino acid sequence shown in Figure 1B, functionally active mutants, derivatives, fragments thereof and family members or related members of the serine protease family.

The parasite infection may be an ectoparasite infection.

The nucleotide fragment may include a conserved region or regions of a nucleotide sequence, for example the sequence of Figure lA.

Furthermore, the protein fragments may include a conserved region or regions of an amino acid sequence, for example the sequence of Figure 1 B.

In a preferred embodiment of this aspect of the invention, the antigen is particularly applicable to the prevention or reduction of blowfly strike in sheep.

The antigen may be isolated by a method as described in International Patent Application PCTIAU95100078 referred to above.

Accordingly in a preferred embodiment of this aspect of the invention there is provided a recombinant antigen for preventing or reducing parasite infection or disease in an animal, said antigen having the nucleotide sequence shown in Fig.

1A, functionally active mutants, derivatives and fragments thereof or an amino acid sequence shown in Fig. 1B, functionally active mutants, derivatives, fragments thereof and family members or related members of the serine protease family.

The recombinant antigen may be expressed in any suitable manner. For

I

6 example, expression systems including prokaryotic, eukaryotic or viral expression systems may be used. For example, the pET series of prokaryotic vectors may be used. For eukaryotic expression systems, yeast, mammalian or baculovirus expression vectors may be used. The protein may be expressed with or without a fusion partner.

The nucleotide or protein fragment may include a conserved region or regions of the sequence of Figure 1A or B, respectively.

In a still further aspect of the present invention there is provided an antigen or group of antigens for preventing or reducing ectoparasite infection or disease in an animal, said antigen(s) including a protease, a functionally active mutant, derivative or fragment thereof and additionally including an N-terminal amino acid sequence(s) selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and

GYNEDEYXQ;

antigenic mutants and fragments thereof.

Preferably the protease is a serine protease. More preferably it may be a chymotrypsin-like protease.

In a still further aspect of the present invention there is provided an antigen for preventing or reducing parasite infection or disease in an animal, said antigen encoded by a nucleotide sequence including a nucleotide sequence which encodes an N-terminal amino acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and

GYNEDEYXQ;

antigenic mutants and fragments thereof, 30 and a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family.

GY.EYQ

The N-terminal portion and the protease component of the above referenced combined antigen may be expressed singularly or in combination.

In a still further aspect of the present invention there is provided an antigen for preventing or reducing parasite infection or disease in an animal, said antigen having an amino acid sequence having an N-terminal amino acid sequence(s) selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and

GYNEDEYXQ;

antigenic mutants and fragments thereof, and an amino acid sequence of Figure 1B, a functionally active mutant, derivative or fragment thereof and family members or related member of the serine protease family.

The antigen(s) according to this aspect of the invention may be such that when the animal is immunised with the antigen(s) immunity of the animal to the ectoparasite is boosted by natural challenge of the animal with the ectoparasite.

o The antigen(s) may have an N-terminal amino-acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ; and having a molecular weight of approximately 70 to 100 kd and an isoelectric point of between approximately 2.5 and 5 pH units.

The antigen(s) may be isolated, characterised and sequenced by a method as described in International Patent application PCT/AU95/00078, referred to above.

In a further aspect of the present invention there is provided a further antigen from the surface of Lucilia cupnna larvae, a functionally active mutant, 4 derivative or fragment thereof. The further antigen may have an approximate molecular weight of 50-65kD. It may also have an isoelectric point of between

I

8 approximately 2.5 and 5.0 pH units. Preferably the further antigen is a first stage larvae antigen of Lucilia cuprina. The further antigen may be isolated, characterised and sequenced by a method as described in International Patent Application PCT/AU95/00078, referred to above.

In a still further aspect of the present invention there is provided a monoclonal antibody to the further antigen from the surface of Lucilia cuprina as described above.

This monoclonal antibody may react with the surface of Lucilia cuprina larvae. The monoclonal antibody may be used to probe a Western blot of the different larval stages. The major reactivity was associated with first stage larvae (Figure The monoclonal antibody may be prepared by any process known to the skilled addressee.

In a still further aspect of the present invention there is provided a vaccine composition for the prevention or reduction of parasite infection or disease in an animal, said composition including a prophylactically effective amount of an antigen or monoclonal antibody as hereinbefore described, optionally together with one or more of a pharmaceutically acceptable carrier, adjuvant or cytokine.

The antigen included in the vaccine composition may be a protease. for example a chymotrypsin-like protease, an antigen from the surface of Lucilia S 20 cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ;or any combination thereof.

Preferably, the antigen is encoded by a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, or is encoded by an amino acid sequence of Figure 1B, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, an antigen from the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid 9 sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ;or any combination thereof.

In a preferred embodiment of this aspect of the invention, the vaccine composition is particularly applicable to the prevention or reduction of blowfly strike in sheep.

The vaccine composition may be administered by any suitable route. For example, it may be administered orally or may be administered parenterally (for example by intramuscular, intradermal, subcutaneous or intravenous injection).

In a particularly preferred embodiment the vaccine composition is administered subcutaneously.

The amount of antigen required may be approximately 0.0001-1 mg active :ingredient/kg body weight.

The vaccine composition may further include a pharmaceutically acceptable carrier, diluent or excipient thereof. Preferably the active ingredient may be suspended or dissolved in a carrier. The carrier may be any solid or solvent that is non-toxic to the animal and compatible with the active ingredient. Suitable carriers include liquid carriers, such as normal saline or other non-toxic salts and solid carriers, such as talc or sucrose.

The adjuvant, when present, may be of any suitable type. Simple adjuvants of the aluminium hydroxide-type may be used. Adjuvants such as the Montanide group of adjuvants (SEPPIC, Division Cosmetique Pharmacie, Paris), and Freund's adjuvant, complete or incomplete have also been found to be suitable.

Other adjuvants including MAP (multiple-antigen peptide), KLH (Keyhole Limpet Hemacyanin), RAS (Ribi Adjuvant System), MPL and TDM or TPL and TDM and CWS (Monophosphoryl Lipid A and Trehalose Dicorynomycolate and cell wall skeleton) alone or in admixture may be included.

The cytokine, when present, may be of any suitable type. Whilst applicants do not wish to be restricted by theory, it is thought that cytokines enhance the ~pr~Jrmu; ir~ oll- antigenicity of the antigen. For example, a cytokine according to PCT/AU91100358, the entire disclosure of which is incorporated herein by reference, may be used. ILl-p is particularly preferred.

When used for administering via the bronchial tubes, the vaccine composition is suitably present in the form of an aerosol.

The vaccine composition may preferably take the form of slow release systems and/or implants.

Accordingly, in a preferred embodiment of this aspect of the invention, there is provided a vaccine composition for the prevention or reduction of ectoparasite infection or disease in an animal, said composition including a prophylactically effective amount of an antigen or monoclonal antibody as hereinbefore described; and a cytokine, IL1-p.

The antigen included in the vaccine composition may be a protease, for 15 example a chymotrypsin-like protease, an antigen from the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ; or any combination thereof.

Preferably, the antigen is encoded by a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, or is encoded by an amino acid sequence of Figure 1B, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, an antigen from the surface of Lucilia cuprina, andlor an antigen having an N-terminal amino acid sequence selected from the group including: 11

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ; or any combination thereof.

The vaccine composition may further include any suitable adjuvant, e.g. of the Montanide-type.

Preferably the Montanide is montanide In a still further aspect of the present invention there is provided a method for preventing or reducing ectoparasite infection or disease in an animal, which method includes administering to the animal an effective amount of an antigen or a vaccine composition as hereinbefore described.

The vaccine composition may preferably take the form of slow release systems and/or implants. A mini-pellet system may be used.

In a further aspect of the present invention there is provided a DNA sequence coding for an antigen(s) as described above, functionally active.

mutants, derivatives or fragments thereof for preventing or reducing a parasite infection or disease in an animal.

The DNA sequence may code for. a protease antigen, for example a chymotrypsin-like protease, an antigen from the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ; or any combination thereof.

Preferably, the antigen is encoded by a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, or is encoded by an amino acid sequence of Figure 1B, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, an antigen from "Ago a 12 the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including:

GYNEXEYXQ;

GYNVXEYXQ;

GYNVDEYXQ; and GYNEDEYXQ; or any combination thereof.

The DNA sequence may be utilised directly or indirectly to immunise an animal via DNA injection. Also, the DNA sequence may be included in combination with cytokine DNA in a suitable vector, for example a mammalian expression vector, viral or bacterial vector.

The present invention will now be more fully described with reference to the following examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

SIn the Figures: Figure 1A shows the nucleotide sequence of the expressed chymotrypsinlike protease and variations thereof. The sequence corresponding to the expressed chymotrypsin is in bold script. Nucleotide variations in other family members are indicated by the substituted nucleotide below the main sequence.

Figure 1B shows the predicted amino acid sequences of Lucilia cuprina chymotrypsin-like proteases. The sequence corresponding to the expressed protease is in bold script. Differences in the amino acid sequence between family members are indicated below the main sequence.

Figure 2 shows a Western blot of different larval stages from eggs of 3rd stage larvae of Lucilia cuprina larvae protease with a monoclonal antibody to a surface antigen of Lucilia cuprina larvae. The area of specific activity is indicated.

Figure 3 shows a Southem blot of Lucilia cuprina genomic DNA probed with the chymotrypsin-like protease (Lc4). Genomic DNA was digested with either Ssp I or Hind III subjected to agarcse gel electrophoresis and transferred to nylon membrane. Similar membrane strips were hybridised with different concentrations of formamide at 45°C with the radioactive probe. DNA molecular -J 13 size standards (kb) are shown. The addition of formamide in the hybridisation solution increases the relative hybridisation temperature and therefore affects the probe's binding capacity with homologous DNA. Fragments hybridising with the probe at 45 0 C and 50% formamide will share approximately 100% homology with the sequence of the probe. At 35%, 22% and 7% formamide the homology between the probe and target DNA will be at least 90%, 80% and respectively. The Southern blot indicates that there are at least thirteen gene copies of the chymotrypsin-like protease exhibiting greater than 70% homology with the probe. This suggests the existence of a gene family of which any one member could serve as a vaccine antigen.

Figure 4 shows a Northern blot of total RNA from different developmental stages of Lucilia cuprina larvae probed with the chymotrypsin-like protease (Lc4).

RNA was extracted from eggs (Lanes 1st (Lanes 7-22), 2nd (Lanes 24-28) and 3rd (Lane 30) instar larvae. Samples were collected every hour for the first 15 20 hours then at six hourly intervals. RNA molecular weight markers are in lanes 6, 23 and 29. First appearance of the chymotrypsin-like protease mRNA is <1 hour before hatch. Chymotrypsin-like protease mRNA was not detected in either 2nd or 3rd instar larvae.

Figure 5 shows an immunoblot of excretory-secretory products from Lucilia cuprina reacted with anti-recombinant Lc4 sera. Excretory/secretory material was collected hourly starting four hours prior to egg hatch and then hourly after that.

The numbers 1, 2 and 3 in Figure 5 refer to first, second and third stage Lucilia cuprina larvae. Native Lc4 was first detected approximately 2 hours after egg hatch (arrowed).

Figure 6 shows an immunoblot of 1st instar L. cuprina excretory/secretory products reacted with sera from sheep which were immunised with the adjuvant Montanide ISA-25+ 50 ig rov IL-11 (Lanes recombinant Lc4 (Lanes 10-11, 13-14, 16 and 20) or L. cuprina Lc4 (Lanes 15, 17-19 and 21-22). Dilution of sheep serum was 1:250.

EXAMPLE 1 Cloning and expression of Lc4 The protease (Lc4) is part of a multigene family of chymotrypsin proteases from L.

14 cuprina which comprises several family members. Three family members have been cloned, sequenced and expressed. These family members are known as variant 161(v161), v164 and v108. These three proteases share significant homology at the amino acid and nucleotide level. In addition, there is a degree of crossreactivity between the antisera raised against these three recombinant proteases.

Methods N-terminal sequence data was obtained for Lc4 and data base searches indicated significant homology with chymotrypsin-like proteases.

Oligonucleotides primers were designed and an RT-PCR reaction performed using Taq polymerase (Perkin-Elmer).

S: The amplitied product was cloned into a commercial T-vector PROMEGA) and sequenced (Figure 1A and Figure 1B).

Lc4 cDNA was ligated into the prokaryotic expression vector pET 23b which S 15 contained a vector derived histidine tag.

Recombinant Lc4 was produced as insoluble inclusion bodies which were then solubilised in 0.2% SDS and then purified on an immobilised metal affinity column (IMAC) and eluted with 6M urea and 0.2M sodium hydroxide in the absence of any reducing agent.

The urea was removed by dialysis which resulted in the production of both a soluble form and an insoluble form of Lc4.

Both soluble and insoluble Lc4 were then freeze-dried and reconstituted in sterile saline at the appropriate concentration for administering to sheep in vaccination trials- refer Examples 2 and 3.

Genomic Southern analysis (Figure 3) indicates the presence of several chymotrypsin-like protease genes.

Northern blot analysis (Figure 4) shows the mRNA expression from the time of hatch through to third instar. The hybridisation signal appeared strongest in the first stage larvae compared to the second and third stage larvae.

Detection of native Lc4 in the excretory/secretory products of L. cuprina first stage larvae (Figure Native Lc4 was first detected approximately 2 hr after egg hatch.

Use of a purified recombinant and native Lc4 in a vaccine trial. Recognition of native Lc4 by animals immunised with either recombinant or native Lc4 (Figure 6).

EXAMPLE 2 Vaccination Trial Recombinant Lc4 v. Native Lc4.

The recombinant Lc4 was prepared according to Example 1.

Protocol 3 Vaccinations, week 0, 2 and 6.

Challenge week 8.

Adjuvant Montanide ISA-25 50pg rov-IL-1 1/sheep Route Intradermal Recombinant Lc4 100 pg/sheep and Adjuvant Native Lc4 10-20 jig/sheep and Adjuvant Control adjuvant only.

Challenge S. 200 eggs of L. cuprina placed on wet skin and left for 48 hours 2 Sites per sheep After 48 hours, sites examined for presence of larvae and a wound. Larvae present were weighed.

Results Group No. of No. of Red in Sheep Infections Sheep Growth Wound Size Infected Inhibition Native Lc4 6 1/12 8 21 86 Recombinant 6 0/12 0 100 100 Lc4 Control 8 6116* Four Control sheep had one site infected. Two of those four sheep had both sites infected.

Inhibition of growth or reduction in wound size of vaccinates are compared to controls.

16 Mode of protection is as yet unidentified, but is postulated to be through a combination of cellular responses in the skin as well as through antibody leakage onto the skin, thus blocking the combined action of the family of proteases which are believed to be involved in wound formation.

EXAMPLE 3 Vaccination Trial Results This example represents pooled data from 4 sheep trials and refers to use of three separate chymotrypsin variants. The sequences of the antigens are contained in Figures 1A and lB.

The antigen used was one of 3~forms of recombinant Lc4. The recombinant Lc4 was prepared according to Example 1.

Protocol Vaccinations, week 0, 2 and 6.

Challenge week 8 15 Adjuvant Montanide 1SA-25 and 50 pg rov-IL-l1/sheep Route Subcutaneous Vaccinates Recombinant Lc4 100 pg/sheep plus adjuvant Controls Adjuvant only Challenge 300 L. cuprina eggs were placed on wet skin and left for 48 hours on 2 sites/sheep After 48 hours, sites were examined for presence of larvae. Larvae were recovered, counted and weighed.

Results Group No. of sheep Total no. of Av. no larvae Av. larval challenged larvae recoveredlsheep weight (mg) recovered challenged Vaccinates 113 8985 78* 6.3±2.3** Control 71 6489 91 7.0±3.1 S P<0.135 S P<0.294 17 Example 3 represents pooled data from 4 separate vaccine trials using the recombinant chymotrypsin protease. Three Lc4 variants (v161, v164 and v108) were tested in separate trials and the results pooled. Each of these variants have been shown to provide similar levels of protection in sheep. There was a 10% reduction in the average weight of the larvae recovered from vaccinated sheep compared to unvaccinated controls. In addition, there was a 14% reduction in the average number of larvae recovered from the vaccinated sheep compared to the control sheep.

Finally, it is to be understood that various other modifications andlor 10 alterations may be made without departing from the spirit of the present invention as outlined herein.

01

Claims

1. A method for identifying an antigen for preventing or reducing parasite infection or disease in an animal, which method includes administering to the animal an effective amount of an antigen and challenging the animal with an invasive stage of the parasite to mimic natural challenge.

2. A method according to claim 1 wherein the parasite infection or disease is an ectoparasite infection or disease.

3. A method according to claim 2 wherein the ectoparasite is a blowfly or other fly, flea, tick, mite or louse species.

4. A method according to claim 3 wherein the fly species is selected from the group including Calliphoridae family including sub families Calliphonnae spp and Chrysomyinae, and the Haematobia spp; the flea species is selected from the group including Ctenocephalides canis and Ctenocephalides felis; and the tick species is selected from the group including Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Amblyomma spp, Dermacentorspp, Ixodes spp or Hyalomma spp. A method according to any one of claims 1 to 4 wherein the parasite is Lucilia cupnina.

6. A method according to any one of claims 1 to 5 wherein the challenging of the animal with the invasive stage of the parasite includes challenging with a parasite egg.

7. An antigen for preventing or reducing parasite infection identified by the method according to any one of claims 1 to 6.

8. An antigen for preventing or reducing parasite infection in an animal wherein the antigen is a protease, functionally active mutant, derivative or fragment thereof.

9. An antigen according to claim 8 wherein said protease is a serine protease, in particular a chymotrypsin-like protease. An antigen for preventing or reducing parasite infection or disease in an animal, said antigen having a nucleotide sequence shown in Figure 1A, a functionally active mutant, derivative, or fragment thereof and family members or related members of the serine protease family. __77M

11. An antigen for preventing or reducing parasite infection or disease in an animal, said antigen having an amino acid sequence shown in Figure 1B, a functionally active mutant, derivative, or fragment thereof and family members or related members of the serine protease family.

12. An antigen according to any one of claims 8 to 11 wherein said parasite infection is an ectoparasite infection.

13. A recombinant antigen for preventing or reducing parasite infection or disease in an animal, said antigen having a nucleotide sequence shown in Fig. 1A, a functionally active mutant, derivative or fragment thereof or related members of the serine protease family or an amino acid sequence shown in Fig. 1B, a functionally active mutant, derivative, or fragment thereof and family members or related members of the serine protease family.

14. An antigen or group of antigens for preventing or reducing ectoparasite infection or disease in an animal, said antigen(s) including a protease, a 15 functionally active mutant, derivative or fragment thereof and additionally including an N-terminal amino acid sequence(s) selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ; or an antigenic mutant, derivative or fragment thereof. An antigen for preventing or reducing parasite infection or disease in an animal, said antigen encoded by a nucleotide sequence including a nucleotide sequence which encodes an N-terminal amino acid sequence selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ; antigenic mutants and fragments thereof, "a- i 4 a and a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family.

16. An antigen for preventing or reducing parasite infection or disease in an animal, said antigen having an amino acid sequence having an N-terminal amino acid sequence selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ; antigenic mutants and fragments thereof, and an amino acid sequence of Figure 1B, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family.

17. An antigen according to any one of claims 14 to 16 having an N-terminal amino acid sequence selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ; antigenic mutants and fragments thereof, and having a molecular weight of approximately 70 to 100 kd and preferably an isoelectric point of between approximately 2.5 and 5 pH units.

18. An antigen according to claim 7 derived from the surface of Lucilia cupinna, a functionally active mutant, derivative or fragment thereof.

19. An antigen according to claim 18 having an approximate molecular weight of

50-65 kD and preferably an isoelectric point of between approximately 2.5 and pH units. An antigen according to claim 19 which is a first stage larvae antigen of Lucilia cuprina. 21. A monoclonal antibody to an antigen according to any one of claims 18 to C a~ Ip A 21 22. A vaccine composition for the prevention or reduction of parasite infection or disease in an animal, said composition including a prophylactically effective amount of an antigen according to any one of claims 8 to 20 or a monoclonal antibody according to claim 21, optionally together with one or more of a pharmaceutically acceptable carrier, adjuvant or cytokine. 23. A vaccine composition according to claim 22 wherein the antigen is a protease, particularly a chymotrypsin-like protease, an antigen from the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ;or any combination thereof. 24. A vaccine composition according to claim 23 wherein the antigen is encoded by a nucleotide sequence of Figure 1A, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, or is encoded by an amino acid sequence of Figure 1B, functionally active mutant, derivative, fragment thereof or a family member or related member of the serine protease family, an antigen from the surface of Lucilia cuprina, and/or an antigen having an N-terminal amino acid sequence selected from the group including: GYNEXEYXQ; GYNVXEYXQ; GYNVDEYXQ; and GYNEDEYXQ; or any combination thereof. A vaccine composition according to any one of claims 22 to 24 wherein the parasite infection or disease is blowfly strike. 26. A vaccine composition according to any one of claims 22 to 25 wherein the cytokine is ELL1B. 27. A method for preventing or reducing ectoparasite infection or disease in an 22 animal, which method includes administering to the animal an effective amount of an antigen according to any one of claims 7 to 20 or a vaccine composition according to any one of claims 22 to 26. 28. A DNA sequence coding for an antigen or a functionally active mutant, derivative or fragment thereof according to any one of claims 7 to 20 or a combination thereof for preventing or reducing a parasite infection or disease in an animal. 29. An antigen according to claim 8 substantially as hereinbefore described with reference to the examples. DATED: 6 July, 1998 S 15 PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE UNIVERSITY OF MELBOURNE and AUSTRALIAN WOOL RESEARCH AND PROMOTION ORGANISATION