AU688949B2 - Antigen for inclusion in a vaccine against blowfly strike - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Actual Inventors: Address for Service: Invention Title: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH

ORGANISATION

Ross Lindsay Tellam Sandra Anna-Maria Schorderet Craig Harold Elsemann CULLEN CO., Patent Trade Mark Attorneys, 240 Queen Street, Brisbane, Qld. 4000, Australia.

ANTIGEN FOR INCLUSION IN A VACCINE AGAINST BLOWFLY STRIKE Details of Associated Provisional Applications: No. PM8452 filed on 29 September 1994 The following statement is a full description of this invention, including the TECHNICAL FIELD This invention relates to a protein antigen for inclusion in a vaccine which when administered to sheep results in the induction of an immune response. The immune response is capable of retarding the growth of blowfly larvae feeding on the vaccinated sheep thereby restricting or limiting the effects of blowfly strike thereon. The invention also relates to DNA encoding the antigen, a method for the production of commercial quantities of the antigen, as well as methods for administering vaccines comprising the antigen.

BACKGROUND

Blowfly strike, cutaneous myiasis, in sheep is caused by fly larvae feeding on the tissue and tissue fluids of the sheep. The problem is of significant economic importance to the Australian sheep industry and it is estimated that up to three million sheep per annum, or approximately 2% of the Australian flock, are killed by blowfly strike despite blowfly control practices. The major species of blowfly which initiates 80-90% of all primary strikes in Australia is Lucilia cuprina while the closely related blowfly Lucilla sericata initiates most strikes in other locations su :h as Europe.

The background relating to the problem of blowfly strike in sheep is described in detail in Australian Patent Application No. 29,716/92 (Tellam, Elsemann, Elvin and East "Flystrike antigen and vaccine and method for preparation") as well as in East and Eisemann (Immunology and Cell Biology 71, 453-462; 1993) and East et al. (International Journal for Parasitology 23, 221-229; 1993). The patent application also gives a comprehensive review of the state of the art existing at that time in relation to vaccination against L. cuprina and other insects which feed on mammalian hosts. In particular, the patent application describes the production of a vaccine which can be used in sheep to alleviate the effects of blowfly strike.

Australian Patent Application No. 29,716/92 describes effective antigens for inclusion in a vaccine as being those arising from the peritrophic membrane (PM) of larvae of L. cuprini or whole peritrophic membrane itself. The application discloses the purification and identification of three specific protein antigens all derived from peritrophic membrane of L. cuprina larvae. These antigens, PM44, PM90 and can be used as vaccine components either individually or in combination.

The application also describes the gene sequence and deduced amino acid sequence of PM44. This information enables the production of much larger quantities of this particular vaccine antigen by the production of corresponding recombinant proteins in bacterial cells, insect cells or other suitable expression systems or by the synthesis of peptides representing particular regions in these antigens.

European Patent Application EP 0381427 A2 (Meeusen, Brandon and Bowles "Vaccine composition") discloses a general method for the production of antibodies to parasites which include L. cuprlna. However, this patent application does not describe how to produce antibodies which identify L. cuprina antigens after natural infestations of L. cuprlna larvae in sheep that is, the disclosure does not teach the identification of any L.

cuprina antigens. More importantly, there is no indication that this approach will isolate protective antigens from L. cuprlna. Rather, at best, the procedure will only identify general parasite proteins. Further, previous studies have clearly demonstrated that natural infestations of L. cuprina do not induce any significant level of naturally acquired immunity in sheep which would be expected if the parasite antigens which come into contact S 20 with sheep during natural infestations were protective (Sandeman et al., International Journal for Parasitology 16, 69-75; 1986 see also Eisemann et al., International Journal for Parasitology 20, 299-305; 1990). Thus, apart from the lack of any evidence that this procedure will work, the S. information provided in EP 0381427 is not sufficient to enable production of 25 a vaccine against L. cuprina.

International Patent Publication Number WO 94/02169 (Smith, Smith, Murray, Liddell and Knox "Vaccines against metazoan parasites") describes the use of an antigen isolated from a helminth as a potential protective antigen for vaccination against L. cuprina. However the naltre of the helminth antigen bears no relationship with the L. cuprina antigen which will be described below. Nor are there any details relating to said antigen from L. cuprina or any data relating to protection of sheep against L. cuprlna using that antigen.

The invention described below relates to a novel L. cuprina peritrophic membrane protein, PM48, which when injected into sheep induces an immunme response. The immune response inhibits the growth of larvae of L.

cuprina which subsequently feed on those sheep at the site of a fly strike or on their sera. This antigen can therefore be used in a vaccine which protects sheep from blowfly strike. However, as it is not commercially viable to isolate this vaccine component directly from peritrophic membrane produced by culture of L. cuprina larvae. Indeed, only 1-2 mg of PM48 can be isolated in pure form from 20 g of peritrophic membrane obtained by larval culture. This 4uantity of antigen is only sufficient for vaccination of three to fou r sheep. Twenty grams of peritrophic n.embrane is obtained from approximately 630,000 larvae cultured over a total period of 5 weeks.

The logistical and scientific difficulties associated with the scaling-up of this process for the production of commercial quantities of these antigens are prohibitive. Furthermore, the scale-up of larval culture is economically not viable. Consequently, the commercial production of a vaccine against blowfly strike in sheep using the antigen PM48 will require the artificial production of large quantities of this antigen. This may be achieved by producing the antigen as a recombinant protein in bacteria, yeast or insect cells. Fragments of the whole antigen may also be similarly produced or by peptide synthesis. Essential to this process is the determination of the 20 cDNA and deduced arnino acid sequences of the vaccine antigen which allows ma'i.plation of the gene coding for this protein or antigen and its production L. artificial expression systems.

SUMMARY OF THE INVENTION An object of the present invention is to provide DNA encoding the PM48 L. cuprina antigen to allow the determination of the encoded amino acids and the large scale production of a commercial vaccine which may be used to combat blowfly larvae infestations of vaccinated sheep.

According to a first embodiment of this invention, there is provided PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant thereof.

According to a second embodiment of this invention, there is provided an isolated DNA comprising a sequence encoding PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant thereof.

According to a third embodiment of this invention, there is provided an expression vector which includes a DNA sequence encoding PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant or immunogenic fragment thereof.

According to a fourth embodiment of this invention, there is provided a method of producing PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue, variant or immunogenic fragment thereof, the method comprising the steps of: introducing into a host cell DNA which includes a DNA sequence encoding said PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof in conjunction with elements for the expression of polypeptide encoded by said DNA; culturing said host cell under conditions which allow expression of the encoded polypeptide; and isolating the expressed PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof.

According to a fifth embodiment of this invention, there is provided a vaccine for the prophylaxis or treatment of blowfly strike in sheep, the vaccine comprising PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant or immunogenic fragment thereof.

According to a sixth embodiment of this invention, there is provided a method of prophylaxis or treatment of blowfly strike in sheep, the method comprising administering to sheep an antigen according to the first embodiment or a vaccine according to the fifth embodiment.

The invention also provides methods for isolating PM48 antigen from peritrophic membrane.

The blowflies to which the present invention is applicable include not only Lucilla cuprlna but also related flies causing mylasis in their hosts.

These flies are characterised by the ability of their larval stages to parasitise their vertebrate hosts (typically cattle or sheep) by feeding on host tissue or tissue fluid and are generally related and belong to the family Calliphoridae and sub-families Calliphorlnae and Chrysomyinae. While not exclusive, the following list represents the range of flies applicable to this patent: Lucilla cuprlna, Lucilla sericata, Calllphora augur, Calliphora stygla, Calliphora noclva, Calliphora alblfrontalls (also called Calliphora australls or Calliphora maryfullert), Calliphora hilll, Calliphora vicna (also called Calliphora erythrocephala), Chrysomya ruflfacles, Chrysomya varipes, Chrysomya bezziana, Chrysomya albiceps and Cochllomyla homlnlvorax. However, it is recognised that the vaccine, or variations thereof, based on the same or similar effective antigen, may have general application for the control of Dipteran insects or their larvae which have a similar peritrophic membrane structure to L. cuprina and which feed on the tissue, tissue fluids or blood of vertebrate hosts Haematobta exigua irrltans and Haematoba irritans irritans and related species).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a protocol for purification of PM48 and the results of SDS-PAGE analysis of purified antigen.

Figure 2 depicts indirect immunofluorescence localisation of PM48 on peritrophic membrane from L. cuprlna.

Figure 3 is a photograph of a stained electrophoresis gel of DNA fragments amplified using the polymerase chain reaction with PM48-specific oligonucleotide primers.

Figure 4 presents the nucleotide and deduced amino acid sequences of the PM48 DNA fragment produced by PCR.

Figure 5 presents the nucleotide and deduced amino acid sequences of PM48 determined from cDNA.

Figure 6 shows the result of SDS-PAGE analysis of purified recombinant PM48 protein. Lane 1, molecular weight standards; lane 2, purified hexa-his-PM48 The gel resulting from SDS-PAGE was silver 25 stained for detection of protein bands.

:DETAILED DESCRIPTION OF THE INVENTION Abbreviations listed hereafter are used throughout the following description.

AEBSF 4-(2-aminoethyl)-benzene sulfonyl fluoride ATP Adenosine triphosphate BSA Bovine serum albumin DIG Digoxigenin DNaseI Deoxyribonucleose I dNTP Deoxynucleotide triphosphate DTT Dithiothreitol ~0 b oc r c r e c i r r EDTA Ethylenediaminetetraacetic acid Endo Lys C Endoproteinase Lys C Elisa Enzyme-linked immunosorbtion assay HFBA Heptafluorobutyric acid HPLC High performance liquid chromatography Ig Immunoglobulin G IPTG Isopropylthiogalactoside LB Luria-Bertaini medium mRNA Messenger RNA NNTA Nickel-nitrilo-tri-acetic acid resin PBS Phosphate-buffered saline PCR Polymerase chain reaction pfu Plaque forming units SDS Sodium dodecylsulfate SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis Tris Tris (hydroxymethyl) aminomethane TBS Tris-buffered saline X-Gal 5-bromo-4-chloro-3-indolyl-B-D-galactoside DNA DNA The one-letter code for nucleotides in DNA and the one- and threeletter codes for amino acid residues conform to IUPAC-IUB standards described in The Biochemical Journal 219. 345-373 (1984).

As indicated above, the present inventors have identified a hitherto unknown protein which induces an immune response in vaccinated animals. The antigenic protein, designated PM48, is from the peritrophic membrane of the fly L. cuprina and can be used as the principal active component of a vaccine against fly strike in animals such as sheep.

DNA encoding PM48 is advantageously identified by screening cDNA or genomic DNA libraries of L. cuprlna or related organisms with oligonucleotides complementary to coding regions of the PM48 gene.

Alternatively, such oligonucleotides can be used as primers for amplification of DNA encoding PM48 by PCR. The sequences of suitable ollgonucleotides can be established from a consideration of amino acid sequence data for all or portions of PM48.

To provide protein or peptides for sequencing, antigen can be purified from blowfly peritrophic membrane. A procedure for the isolation of PM48 from L. cuprlna peritrophic membrane is detailed below.

Purified antigen is typically digested with proteases to provide a number of peptides so that different portions of the polypeptide making up the antigen can be sequenced. Suitable proteases are well known in the art and include trypsin, chymotrypsin, Endoproteinase Lys-C, papain and V8 protease from Staphylococcus aureus. Peptides are isolated and sequenced by manual or automated means.

Suitable oligonucleotides are designed from the peptide amino acid sequences. Oligonucleotides preferably comprise sequences having no secondary structure and minimal degeneracy. Labelled oligonucleotides can be used as probes for the PM48 gene in genomic or cDNA libraries or oligonucleotides can be used as PCR primers for amplification of the desired DNA. Preferably, the amplified DNA is cDNA. It will be appreciated by one skilled in the art that the gene coding for PM48 may also be isolated by screening a L. cuprina cDNA expression library with antisera to PM18 or antisera raised to peritrophic membrane or crude extracts thereof.

DNA encoding PM48 can be incorporated into a vector for expression of the antigen, or a portion or derivative thereof, in a suitable host cell. The 20 expression host can be prokaryotic or eukaryotic and include bacteria, yeast cells and insect cells. Typically the expression host is eukaryotic so that the expressed antigen is glycosylated. A preferred eukaryotic expression host is a baculovirus-infected insect cell.

The expression vector used can include elements which result in the 25 PM48 antigen being expressed as a fusion protein. The fusion protein typically comprises a leader or signal peptide linked at the amino terminal end of the PM48 polypeptide or fragment thereof. Expression of the antigen as a fusion protein can be exploited to deliver the protein to the exterior of the host cell and in the purification of the protein. For example, the leader peptide can be an affinity ligand which allows affinity purification of the fusion protein after which the leader peptide can be cleaved from the PM48 antigen or fragment thereof.

Antigen produced by the recombinant DNA-based method of the invention can be used alone or in combination with other immunogens in a vaccine against flystrike in sheep. Both glycosylated and non-glycosylated forms of antigen can be used as immunogens. Homologues of the effective antigen either from the same or different insect species can also be used. In addition, the PM48 antigen can be used in conjunction with other peritrophic membrane antigens: for example, the PM44, PM90 and antigens described in Australian Patent Application No. 29,716/92, the entire disclosure of which is incorporated herein by cross-reference.

The vaccine typically contains in addition to PM48 antigen and/or other immunogens a suitable adjuvant. Such adjuvants include, but are not limited to, Montanide/Marcol, saponin, Quill A, ISCOMS, alum, aluminium phosphate, poly-anions (dextran sulfate for example) and chitin or derivatives thereof or combinations of suitable cytokines.

Vaccines of the invention can be administered by intramuscular injection, subcutaneous injection, intraperitoneal injection or infusion techniques. Dosage and frequency of injection are factors which can be optimised using ordinary skills in the art.

Synthetic peptides based on specific regions of the PM48 antigen or carbohydrate antigens from a suitable source can be included as Immunogens in a vaccine against flystrike. These antigens will induce an immune response upon injection into sheep or other animals which are capable of recognising the effective of the PM48 antigen. The effective component of the vaccine can also comprise at least one anti-idiotypic antibody capable of inducing a protective immune response by mimicking the PM48 antigen or immunogenic domains thereof.

The invention also includes within its scope antibodies raised against PM48 antigen. Such antibodies can be isolated and directly infused into sheep to afford passive immunisation against blowfly strike. The antibodies can be polyclonal or monoclonal antibodies and can be obtained from a vaccinated animal or produced by phage or in any other expression system.

It will be appreciated that there may be homologues of PM48 which will be equally as effective as vaccine components. Thus, the invention includes within its scope these homologues produced as either native proteins or as recombinant proteins. The homologues could be derived from allelic variants or alternative genes in L. cuprlna or from different species of insects. From the foregoing, therefor it will be appreciated that the invention includes within its scope, DNA sequences and/or recombinant antigens corresponding to the sequences shown in Figure 5 as well as hybrids or antigenic fragments derived from these sequences or synthetic peptides. Also included within the scope of the invention are structural homologues of these sequences having one or more of th' fobllowing properties: at least 50% identity compared to the DNA sequence shown in Figure 5 and/or; structural homologues of the amino acid sequence shown in Figure 5 having at least 70% homology (identical plus conserved positions) and/or; structural homologues having a Z score (Lipman and Pearson, Science 227. 1435-1441; 1985) greater than 3.0 (a statistical measure of probable similarity) and/or; structural homologues which contain one or more of a polypeptide domain consensus sequence present five times in PM48 i.e.

C 12 20 XCX-CXigCXio 0 4

CX

4 6

C

(where x is any amino acid residue); e) protein structural homologues which bind chitin or N-acetyl glucosamine polymers and/or; 20 protein structural homologues which when injected into an animal induce an immune response which recognises PM48 and/or; peritrophic membrane proteins which bind to PM48.

The invention is further described in the following examples which are illustrative of the invention but In no way limiting on its scope.

MATERIALS AND GENERAL METHODS Laboratory equipment and chemicals were obtained from the suppliers listed hereafter.

Trade Name Supplier AEFSF ICN Biochemicals AmpliTaq Perkin Elmer Cetus Aquapore RP-300 C-8 Waters Centricon Amicon DIG-11 -dUTP Boehringer Mannheim DNA ligase (T4) Promega E. coll XL -blue Stratagene Endoproteinase Lys C Boehringer Mannheim Freund's Complete Adjuvant Commonwealth Serum Laboratories Freund's Incomplete Adjuvant Commonwealth Serum Laboratories IPTG Boehringer Mannheim LambdaSorb Promega Magic PCR Prep Promega Mono Q Pharmacia Newborn calf serum Commonwealth Serum Laboratories pGEM7ZII+) Promega Rota Vac Savant Smal Promega Streptavidin-peroxidase Amersham Superose 12 Pharmacia X-GAL Boehringer Mannheim zwittergent 3-14 Calbiochem Other laboratory chemicals were purchased from Sigma Chemical Company (St. Louis MO, Acids and -olvents were analytical grade or HPLC grade and purchased from Ajax Chemical Company (Auburn NSW, Australia). Unless otherwise specified, laboratory chemicals were of analytical grade.

Sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) Protein samples were analysed by SDS-PAGE on 6-18% gradient gels. All gels included molecular weight standards (Pharmacia) and were stained with silver using the method of Morrissey (Analytical Biochemistry 117, 307-310; 1981).

Immunofluorescence localisations Sera from sheep vaccinated with purified native PM48 and from control sheep were diluted 1:2000 in phosphate-buffered saline (PBS).

Pieces of peritrophic membrane collected from larval culture and lengths of peritrophic membrane freshly dissected from the midguts of third instar larvae of L. cuprina fed on an artificial larval rearing medium (Singh and Jerram, New Zealand Journal of Zoology 3, 57-58; 1976), were incubated overnight at 7 0 C in each of the diluted sera. After 4 washes in PBS (over 1 h at room temperature), the peritrophic membrane was incubated with a 1:50 dilution of fluorescein isothiocyanate-labelled rabbit anti-sheep Ig serum in I- I PBS for 2 h at room temperature. After 4 washes in PBS (over 1 h at room temperature), the samples of peritrophic membrane were mounted on slides, examined in a fluorescence microscope and photographed. Slides were photographed at 55x magnification.

Sheep Experimental animals were 6-12 months old merino ewes. These animals had not previously suffered flystrike and were maintained in pens on a diet of lucerne pellets. Animals were randomly assigned to various treatment groups.

Vaccination The purified peritrophic membrane antigen PM48 was homogenised with an equal volume of Freund's Complete Adjuvant. The first injecti a was intramuscular, given half into each rear leg. The second injectio-) days later) used Freund's Incomplete Adjuvant and was given intramuscular in the neck region. All animals were bled from the jugular vein prior to each e injection. Two weeks after the second injection, the effect of vaccination was assessed by in vitro larval growth assay as described by Etsen"rann et al.

(International Journal for Parasitology 20, 299-305; 1990). The in vitro assay consisted of allowing first instar larvae to feed on an agar-based 20 medium containing 75% serum from vaccinated animals. The number of surviving larvae and their weights were measured after 20 h. An in vivo bioassay (Eisemann et al., Australian Veterinary Journal 66, 187-189; 1989) o' was also used for measuring the growth and survival of L. cuprina larvae on the back of sheep. Antibody titres were assessed by Eltsa as described by 25 Elsemann et al. (1990 supra).

Protein Concentration Determinations Protein concentration determinations were made using the Pierce BCA kit with bovine serum albumin as a standard.

Establishment of an L. cuprlna colony Laboratory populations of L. cuprina, which had originated from flystruck sheep, were maintained on an artificial medium (Singh and Jerram, supra) for up to 10 generations. Eggs were collected by placing small trays of minced liver covered with fine nylon gauze inside cages of adult L. cuprina for 4-5 h. The eggs were then incubated overnight at 16 0

C

and 100% relative humidity before surface sterilisation.

Preparation of PM48 Peritrophic membrane was obtained from L. cuprina larvae cultured in vitro (East et al., International Journalfor Parasitology 23, 221-229; 1993).

The peritrophic membrane was progressively extracted with: water; 0.1 M Tris-HCl, pH 7.5 containing 150 mM NaCI, 5 mM EDTA and 5 mM benzamidine; 20 mM Tris-HC, pH 7.4. 140 mM NaCI (TBS) containing 2 Zwittergent 3-14; and TBS containing 6 M urea and 100 uM 4-(2aminoethyl)-benzene sulfonyl fluoride. The 6 M urea extract of peritrophic membrane was concentrated in a Centricon 30 cell (Amicon) and applied to a 150 ml Superose 12 gel permeation column equilibrated with TBS containing 6 M urea. 5 mM benzamidine and 100 ,uM p-arinoethylbenzene sulfonyl flaoride run at a flow rate of 0.5 ml/min (250C) and monitored by absorbance at 280 nm. Two peaks of protein were eluted. The second, a later eluting peak, contained the majority of eluted protein. Fractions of 1 S 15 ml were collected from the Superose-12 column and analysed by SDS- PAGE under reducing conditions, which demonstrated the presence of 2 predominant proteins in the later eluting peak, a 44,000 Da protein (PM44, a pre. frusly identified antigen: Australian Patent Application No. 29,716/92) and a 48,000 Da protein, PM48. Fractions containing PM48 were pooled S 20 and dialysed against 20 mM Tris-HCl, 4 M urea, 1 mM EDTA, 1 mM benzamidine, pH 8.5 before being subjected to anion exchange chromatography on a Mono Q column (Pharmacia) equilibrated with the dialysis buffer and eluted with a non-linear gradient of NaCI (0-250 mM) in the same buffer. Fractions containing PM48 (as determined by SDS-PAGE 25 analysis) were pooled. This procedure separated PM48 from PM44.

Figure 1(a) shows a diagrammatic representation of the purification of PM48 from L. cuprina larvae peritrophic membrane. An SDS-PAGE profile of the purified protein is shown in Figure l(b) in which the following material was analysed: lane 1, molecular weight standards; lane 2, purified PM48 (1 ig); and lane 3, purified PM48 (2 Immunoblots using antisera raised to the purified peritrophic membrane proteins PM44, PM90 and PM95 or their recombinant protein counterparts demonstrated no immunoreactivity with purified PM48. This information indicated firstly that PM48 was pure and secondly that there was no cross-immunoreactivity between these previously identified antigens

I

and PM48. It is recognised that PM48 can be isolated by a number of alternative procedures such as combinations of Isoelectric focussing, preparative SDS-PAGE, chitin affinity chromatography, lectin affinity chromatography or ion exchange chromatographies, for example.

Molecular biology Many of the standard procedures used for the cloning and sequencing of PM48 are described by Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press; 1989). Other methods are described in the text.

EXAMPLE 1 Demonstration of anti-larval effects in the sera of sheep vaccinated with purified PM48 Sheep were injected with purified PM48 twice 4 weeks apart. The adjuvant was Frenmd's Complete Adjuvant and Freund's Incomplete Adjuvant, respectively. Each sheep was injected with a total of 200 ug of PM48. Ten days after the second injection the sheep were bled and their sera used in an in vitro feeding bioassay (Eisemann et al.; 1990 supra). In this bioassay, the ovine serum was introduced into an agar-based diet on which first instar L. cuprina larvae were fed. The weights of the larvae after 20 20 h of feeding were then recorded. Results are presented in Table 1.

Table 1 Effect of sera from sheep vaccinated with PM48 on the weight of first instar L. cuprina larvae which subsequently fed on that sera Animal No. Group Mean Larval Wt. (mg) 25 2504 Control 3.86 0.23 2515 Control 3.66 0.30 2724 Control 3.74 0.33 2525 Control 4.14 0.37 Gp. Mean 3.85 0.26 2435 PM48 2.42 0.25 (37.1%) 2440 PM48 2.25 0.37 (36.4%) 2548 PM48 2.63 0.33 (31.7%) 2983 PM48 3.39 0.40 (11.9%) Gp. Mean 2.67 0.50 (30.6%) The standard deviation associated with each measurement presented in Table 1 is listed adjacent to the measurement. For individual sheep, changes in the weight of larvae are expressed as the percentage reduction of the mean larval weight compared to the mean larval weight from the control group (bracketed). The difference between the means of the larval weights for each group is significant at p<0.005.

The sera from all 4 of the vaccinated sheep inhibited growth of first instar L. cuprina larvae compared to control experiments which used sera from sheep vaccinated with adjuvant and PBS alone. The larval weight was reduced by 12-38% compared to the mean control weight. Three of the four sera gave larval weight reductions in excess of 32%. The mean larval weight for larvae grown on the sera from sheep vaccinated with PM48 was significantly less than that for the corresponding control group at p<0.005.

Therefore, it is concluded that vaccination of sheep with PM48 induced an immune response which inhibited the growth of larvae of L. cuprina which subsequently fed on that sera. The mean weight of larvae feeding on the sera from the control sheep group were not significantly different from the mean weight of larvae feeding on the prevaccination sera from the group subsequently vaccinated with PM48.

20 An in vivo bioassay (Eisemann et al.; 1989 supra) was also used to assess the efficacy of vaccinations with PM48. In this bioassay, larvae were introduced into an enclosed area on the back of sheep and allowed to feed for 20 h on the sheep after which larval weights were measured. The results of this bioassay are shown in Table 2.

25 Table 2 Weight of first instar larvae of L. cuprina feeding directly on the back of sheep as measured by an in vivo bioassay Animal No. Group Mean Larval Wt. (mg) 2504 Control 1.35 0.18 2515 Control 1.63 0.16 2724 Control 1.16 0.23 2525 Control 1.01 0.17 Gp. Mean 1.29 0.27 2435 PM48 0.98 0.11 (24.1%) 2440 PM48 0.74 0.02 (42.6%) 2548 PM48 0.97 0.11 (24.8%) 2983 PM48 1.30 0.19 Gp. Mean 1.00 0.23 (22.5%) The standard deviation associated with each measurement presented in Table 2 is listed adjacent to the measurement. For individual sheep, the weight of the larvae is also expressed as the percentage reduction of the larval weight compared with the control group (bracketed). The difference between the means of the larval weights from each group is significant at p<0.1.

Three of the four sheep vaccinated with PM48 returned larvae which were significantly smaller than those returned from the control sheep. (One sheep (#2983) gave no effect in this assay system and also a very poor effect in the In vitro bioassay (Table 1) this sheep was later shown to be ill). The larval weights after feeding on these three sera were reduced by 25-43% compared to the mean control weight. The mean weight of larvae feeding on sheep vaccinated with PM48 was significantly smaller than the control group Inclusion of the mean larval weight from the nonresponding sheep (No. 2983) gave a mean larval weight for the vaccinated group which was still significantly less than the control group This bioassay demonstrated that larval growth was inhibited when L. cuprlna larvae fed directly on sheep vaccinated with PM48.

The antib,. titre in the serum from each of these sheep was measured by Elisa and the results obtained are presented in Table 3.

Table 3 Antibody ttres in the sera of sheep vaccinated with PM48 as measured by Elisa Animal No. Group Ig titre 2504 Control <1,000 2515 Control <1.000 2724 Control <1,000 2525 Control <1,000 Gp. Mean <1,000 2435 PM48 4,000,000 2440 PM48 4,000.000 2548 PM48 1,000,000 2983 PM48 1,000,000 Gp. Mean 2,500,000 Antibody titres were measured by Elisa as described by Eisemann et al. (1990 supra). The antigen used on the Elisa plate was 1 ug of 6 M urea extracted peritrophic membrane protein.

All 4 of the sera from sheep vaccinated with purified PM48 showed strong antibody titres (mean antibody titre 2,500,000) compared with the sera from the control group (mean antibody titre <1000) or sera from the pre-vaccination sera of the sheep subsequently injected with PM48 (mean antibody titre <1000). Thus, injection of PM48 into sheep induced a humeral immune response which is associated with inhibition of the growth and development of larvae of L. cuprina.

15 EXAMPLE 2 Enhanced inhibition of the growth of larvae of L. cuprina when feeding on ovine serum with increased concentrations S. of Ig from sheep vaccinated with native PM48 Immunoglobulin (Ig) was isolated using method E of Mostratos and 20 Beswick (Journal of Pathology 98, 17-24; 1969) from the sera of two of the strongest responding sheep which had been vaccinated with native PM48 and also from pooled sera of 12 control sheep which had been injected with adjuvant and PBS only. The percentage yields of Ig obtained from the sera were estimated using Elisa. With the PM48 sera, this was performed on a range of dilutions of both the original sera and of the isolated Ig using procedures described previously (Eisemann et al., 1990 supra). An antigen capture Elisa was performed for the control sera and the isolated Ig. Wells of microtitre plates were coated with rabbit anti-sheep Ig serum and a range of dilutions of the original serum or isolated Ig were then added.

Subsequent steps were essentially those described by Eisemann et al. (1990 supra). Relative concentrations of Ig in the original sera and in isolation were estimated in each case by comparing curves obtained by plotting log Elisa optical densities against log dilution. The Ig samples were concentrated by vacuuri dialysis against PBS. Aliquots of each Ig solution were added to 4 ml of pooled control serum to give Ig concentrations equal to once, twice and four times those in the original sera. The total volume was adjusted to 5 ml with PBS. It was necessary to supplement the Ig preparations with the control serum to provide appropriate nutrition for satisfactory larval growth.

The 5 ml preparations so obtained were formulated into diets and larvae grown on them as in the in vitro feeding assays described above. The results of an in vitro growth experiment using these antibody-enriched sera are shown in Table 4. Sham control experiments were performed using immunoglobulin isolated from pre-vaccination serum.

Table 4 Effect of increased concentrations of immunoglobulin from the sera of sheep vaccinated with PM48 on the weight of L. cuprina larvae which had fed on Ig-enriched serum Serum tested Mean larval Wt. Reduction larval Returned (mg) weight larvae Control serum 4.59 0.22 48 Control x 1 Ig 3.87 0.34 49 Control x 2 Ig 3.45 0.27 46 Control x 4 Ig 2.00 0.27 49 PM48 serum 1.92 0.20 58.2 49 PM48 x 1 Ig 1.67 0.13 56.8 44 PM48 x 2 Ig 0.90 0.18 73.9 48 PM48 x 4 Ig 0.40 0.07 80.0 36 This experiment demonstrated a greater retardation of larval growth when larvae fed on immune sera enriched with Ig isolated from the serum of sheep vaccinated with native PM48. The experimental results showed that larval growth inhibition changed from 57% of the corresponding control at Ixlg to 74% of the corresponding control at 2xig and 80% at 4xlg. The very high immunoglobulin concentrations caused some inhibition of larval growth in the controls as well. However, this effect was small in comparison with the effects observed with the immune sera. For example, while the control larval weight change was 48 at 4xlg compared with lxIg, the corresponding anti-PM48 immune serum reduced larval weights by 76 compared with lxlg. Significant, although variable, increases in mortality were associated with the larvae fed on the serum enriched 4 fold with Ig from the serum of a sheep vaccinated with PM48. This experiment demonstrated that the retardation of the growth of larvae feeding on these vaccinated sheep was mediated by antibody and the extent of the effect depended on the concentration of relevant antibody in the serum. The latter can be manipulated using various adjuvants and combinations of antigens in the vaccine using procedures which are in common use by persons skilled in the art.

EXAMPLE 3 Localisation of native PM48 in L. cuprina Sera from sheep vaccinated with native PM48 were used to localise this protein to peritrophic membrane by indirect immunofluorescence techniques. Peritrophic membrane was obtained by fresh dissection of second or third instar L. cuprina larvae. The results of indirect immunofluorescence localisation are depicted in Figure 2 in which panel (a) is a control using pre-vaccination serum and panel is immunofluorescence localisation of PM48 using ovine antiser,'nu 4i.tsed to purified PM48.

Pre-vaccination serum did not react with this peritrophic membrane preparation (Fig. Serum from sheep vaccinated with native PM48 showed strong immunofluorescence on peritrophic membrane (Fig. 2(b)) indicating the presence of PM48 in freshly dissected peritrophic membrane.

Thus, PM48 is an antigen which can be isolated from cultured peritrophic membrane and is also present on the pentrophic membrane from freshly dissected larvae.

EXAMPLE 4 The production, purification and amino acid sequences of peptides from PM48 The isolation and purification of native PM48 from peritrophic membrane derived from L. cuprina larvae cultured in vitro is described above. However, this method of production is not commercially viable.

Sufficient quantities of this antigen can be produced artificially as recombinant proteins in appropriate bacterial or eukaryotic expression systems. To this end, peptides from PM48 were isolated and their amino acid sequences determined as a first step in the process of making recombinant antigens.

Internal peptides from PM48 were prepared by the following procedure. The protein (50 /ug) was mixed with 100 l of 0.1 M Tris-HC1, pH 8.3 containing 20 mM dithiothrettol and 2% SDS and then incubated for 30 min at 56°C. The solution was cooled to room temperature and sodium lodoacetamide added to a final concentration of 0.14 M. After min in the dark, cold methanol was added in a ratio of 9:1 methanol/sample The sample was stored at -200C overnight, centrifuged, the supernatant removed and the pellet dried. The pellet was dissolved in 76 pl of 0.1 M Tris-HCI buffer, pH 8.5 containing 4 M urea and then 4 ul of Endoproteinase Lys C (6 U/ml) was added. After 2 h at 37 0

C,

another 4 ul of the protease was added and the digestion continued for a further 17 h.

The protein digest was applied directly to an Aquapore RP-300 C-8 column in 0.1% trifluoroacetic acid and peptides were eluted in a linear gradient from 0-60% acetonitrile/water in 0.1% trlfluoroacetic acid by HPLC. If necessary, peptides were rechromatographed in the same solvent system using an Aquapore C-18 column or on a C-8 column in a similar solvent system in which the trifluoroacetic acid was replaced by 20 heptafluorobutyric acid (HFBA). Peptides were collected and concentrated to 50-100 1l by rotary desiccation in a Rota Vac. The amino acid sequences of the peptides were determined using an Applied Biosystems 471A amino acid sequencer. In addition, the amino-terminal amino acid sequence of PM48 was directly determined.

25 The results of amino acid sequence analysis are presented in Table The one letter code for amino acids has been used.

Table Peptide amino acid sequences from PM48 Peptide Amino Acid Sequence 1 (K)IGTLMPSMISCQDYY 2 GICLGNLVYDTK 3 (K)CGANTVFDK N-term(4) GYNVAKYCELVKIGTLMPSMISCQ

(E)FGKPQLMDCPPNTYFPY

The amino-terminal bracketed residues are the expected amino acids based on the cleavage specificity of the protease used for the digestion of PM48 endoproteinase Lys C: E, endoprotelnase Glu "N-term" refers to the amino-terminal sequence of PM48 which overlaps with peptide 1.

The amino acid sequences of the peptides presented in Table 5 were used to search the NBRF Proteins (National Biomedical Research Foundation, Washington, USA) and SWISS-PROT (European Molecular Biology Laboratory, Heidelberg, Germany) amino acid sequence databases.

No significant similarities were found indicating the novelty of the identified antigen.

EXAMPLE Molecular cloning of the gene coding for PM48 Oligonucleo tide synthesis The amino acid sequences described in Example 4 (Table 5) were used to design suitable oligonucleotide primers that could be used in the polymerase chain reaction (Saiki et al., Science 239, 487-491; 1988) to amplify DNA fragments of the PM48 gene from L. cuprina first instar cDNA.

Degenerate oligonucleotide primers were designed and synthesised on a Pharmacia LKB Gene Assembler Plus oligonucleotide synthesiser 20 according to the manufacturer's instructions. The synthesised oligonucleotide primers and their peptide of origin were: sense primer TA(TC)AA(TC)GT(TCAG)GC(TCAG)AA(AG)TA(TC)TG(TC)G (derived from the amino-terminal peptide sequence); anti-sense primer TT(TAG)CC(TC)A(AG)(AG)CA(TA)AT(TAG)CC(TC)TT 25 (derived from peptide 2).

Preparation of cDNA from L. cuprina first instar larvae.

Total RNA was isolated from 1 g of L. cuprina first instar larvae using a modification of the method of Chomczynski and Sacchi (Analalytical Biochemistry 162, 156-159; 1987). The fresh larvae were ground on ice in 20 ml of 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH n-lauroyl sarcosine and 0.1 M 2-mercaptoethanol in a Dowuce homogeniser. Two ml of 2 M sodium acetate pH 4.0 were then added, followed by 20 ml acid phenol and 4 ml chloroform:iso-amyl alcohol (49:1), the solution being shaken between each addition. The solution was cooled on ice for 15 min and centrifuged for 20 min at 12000xg The aqueous phase was recovered, extracted twice with an equal volume of phenol:chloroform:iso-amyl alcohol (50:49:1) and the- _eracted once more with chloroform:lso-amyl alcohol The nucleic acids were precipitated from solution by the addition of 2 volumes of absolute ethanol. This precipitate was resuspended in diethylpyrocarbonate-treated water and an equal volume of 8 M LICI was added to precipitate the mRNA and rRNA. The RNA was monitored for integrity by agarose gel electrophoresis. Poly A+ RNA was isolated by oligo-dT chromatography using the method of Sambrook et at. (supra). The integrity of the poly A+ RNA was monitored by denaturing gel electrophoresis before being used as a template for cDNA synthesis (Sambrook et al. (supra). Double stranded cDNA was synthesised from 5 mg of L. cuprina first instar poly A+ RNA using an oligo-dT primer with the Riboclone cDNA Synthesis System: Oligo (dT) 15 Primer (Promega).

This cDNA was used for all PCR reactions and for the construction of a L.

cuprina first instar cDNA lambda gtll11 library.

Construction of L. cuprina first Instar larval cDNA lambda gtll library.

A cDNA library was constructed from 250 ng of L. cuprina first instar larval cDNA in the bacteriophage lambda insertion vector, lambda gtll, using a Riboclone EcoRI Adaptor Ligation System and a Packagene Lambda 20 DNA Packaging System (both from Promega). The primary library contained 270,000 recombinant pfu and was subsequently amplified for storage on E.

coll Y1090 plating cells.

Preparation of a probe for screening the L. cuprina first instar larval cDNA library.

25 A PM48-specific double-stranded DNA probe was prepared using PCR. The procedure was based on that described by Saiki et al. (supra) and used a recombinant form of Taq DNA polymerase obtained from Perkin Elmer Cetus (Amplitaq). PCR was performed on cDNA that had been purified as described in section above. The reaction mixture contained ng of cDNA, 500 ng of each of the sense and anti-sense oligonucleotide primers listed in section 0.2 mM of each dNTP. 2 mM MgCl 2 2.5 U of Taq DNA polymerase and in 100 jil of buffer (10 mM Tris-HCl pH 8.3, mM KC1). Each reaction was overlain with 100 ;l of mineral oil.

Amplification was performed for 35 cycles in a Hybaid Omnigene thermal cycler using the following conditions: 1 cycle of 2 min at 95°C, 1 min at 0 C and 2 min at 72°C; 33 cycles of 1 min at 95 0 C, 1 min at 50°C and 2 min at 72°C; and 1 cycle of 1 min at 95°C, I min at 50 0 C and 5 min at 72 0 C. Appropriate controls, as described by Salkl et al. (supra), were also included.

Samples of the PCR reaction (10 1l) were analysed on 1 agarose gels at the end of the reactions. The results of the analysis are shown in Figure 3 in which lane 1 are standards and lane 2 is the PCR-amplified

DNA.

The single band of approximately 500 bp amplified with this combination of oligonucleotide primers (Fig. 3) was purified using Magic PCR Preps (Promega). Ten percent of the purified 306 bp DNA was ligated to 100 ng of pGEM-T DNA (Promega) in 30 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 mM DTT, 1 mM ATP and 2.5 units of T4 DNA ligase. 0.1 volume of the ligation was transformed into 50 /d of E. coll XL1-blue competent cells, and 15 plated on LB agar plates in the presence of 50 mg/ml of ampicillin, 480 mg of IPTG and 1 mg of X-gal. White colonies were screened for the presence of the appropriate insert by PCR using the oligonucleotide primers and conditions detailed above for the isolation of the original DNA product.

Colonies representing the correctly sized DNA fragment were cultured into 20 ml of LB media containing 50 mg/ml ampicillin. Plasmid DNA was isolated by the alkaline lysis method (Sambrook et al., supra). A 0.3 volume of each plasmid insert was sequenced (on both strands) using a Sequenase version 2.0 DNA sequencing kit (United States Biochemicals) in the presence of either SP6 or T7 promoter primers.

25 The nucleotide sequence of the DNA fragment is presented in Figure 4.

In the figure, identified peptide amino acid sequences are underlined. The broken lines denote the position of the oligonucleotide primers used to isolate this DNA fragment while cysteines are in bold type and potential Nlinked glycosylation sites are boxed.

The sequence presented in Figure 4 has a single open reading frame of 504 bp with no indication of the 5' or 3' ends of the coding region (lack of an initiating methionine residue or a predicted signal sequence at the 5' end and the lack of a stop codon or a poly A tail at the 3' end). The deduced sequence of 168 amino acids extended the regions used for the design of the oligonucleotide primers into regions which agreed with the determined peptide amino acid sequences. The translated amino acid sequence contained the sequences of 4 of the peptides derived from native PM48 (Table The location of these peptide amino acid sequences in the deduced amino acid sequence indicated that this DNA corresponded to a fragment of the gene coding for PM48. The most striking feature of the deduced amino sequence was the abundance of cysteine residues (16 cysteine residues in 168 amino acids).

Cloning of a full-length PM48 cDNA.

An L. cuprina first instar cDNA lambda gtl. library was screened using the 506 bp DNA fragment amplified from L. cuprina first instar cDNA using PCR (see section above). The DNA probe was labelled using PCR in the presence of DIG-11-dUTP (Boehringer Mannheim) and the oligonucleotide primers, using conditions previously described. The cDNA library was transferred in duplicate to Hybond N+ positively charged nylon membrane (Amersham). The membrane was denatured and neutralised using standard conditions, and alkali-fixed in a modification of the manufacturer's instructions. Hybridisations were performed at 42 0 C for 16 h in 50% formamide, 5 x SSC (1 x SSC is 0.15 M NaC1, 0.015 M tri-sodium citrate, pH 0.1% n-lauroyl sarcosine, 0.2% SDS and 2% blocking 20 reagent (Boehringer Mannheim) after a prehybridisation step of 4 h. Filters S were washed twice each for 5 min in 2 x SSC, 0.1% SDS at room temperature and in 1 x SSC, 0.1% SDS for 15 min at 58 0 C. Eighteen positive plaques were detected after 2 rounds of screening, using antidigoxigenin Fab fragment conjugated to alkaline phosphatase (Boehringer 25 Mannheim) and Fast Violet stain (West et al., Analytical Biochemistry 190, 254-258; 1990). Phage DNA was prepared from a 10 ml plate lysate of clone lambda PM48#5.2 using LambdaSorb phage adsorbent (Promega). A small fraction (0.0002 volume) of the phage DNA was amplified in a PCR reaction using forward and reverse-specific lambda gtll primers with the following conditions (see section 1 cycle of 2 min at 95°C, 1.5 min at 69°C. and 3 min at 72°C; 33 cycles of 1.5 min at 95 0 C, 1.5 min at 69°C, and 3 min at 720C: and 1 cycle of 1.5 min at 950C. 1.5 min at 69°C. and min at 72 0 C. The amplified DNA sample was analysed on a 1% agarose gel. A single band of approximately 1200 bp was amplified, and purified using Magic PCR Prep (Promega). A fraction (0.005 volume) of this preparation was used In a new PCR reaction using the sense and anti-sense ollgonucleolide primers listed in section and the conditions mentioned In section 0.01 volume of the 1200 bp fragment was ligatcd to 100 ng of pGEM-T DNA (Promega) as described in section 0.1 volume of the ligation was transformed into 50 dl of E. colt XL1-blue competent cells and plated on LB agar plates in the presence of 50 mg/ml of ampicillin, 480 mg of IPTG and 1 mg of X-gal. One colony which contained the correctly sized fragment was cultured into 250 ml of LB media containing 50 mg/ml ampicillin. Plasmid DNA was isolated by the alkaline lysis method (Sambrook et al.. supra). Plasmid PM48#5.2 was completely sequenced on both strands using a Sequenase version 2.0 DNA sequencing kit (United States Biochemicals) in the presence of SP6, T7 and four sense and four anti-sense internal primers. The insert from PM48#5.2 was 1129 bp in length (adaptors and primers removed).

15 Sequence of cDNA coding for PM48.

Alignment of the DNA sequence derived from the cDNA clone with that derived directly from the PCR.-generated DNA fragment showed that PM48#5.2 had a 63 nucleotide deletion. This deletion explains the difference in size between DNA products amplified by the same 20 oligonucleotide primers from cDNA (approximately 500 bp) and PM48#5.2 (approximately 440 bp). This deletion in PM48#5.2 was confirmed by the sequencing of a new PCR-generated DNA fragment directly amplified from L.

cuprina first instar larval cDNA using the sense oligonucleotide primer described in section and an anti-sense oligonucleotide primer based on 25 the 3' end sequence of PM48#5.2 TTA CT AGC TGA TGG TTT TGT TG). In addition, the sequence of the gene coding for PM48 derived from genomic DNA also confirmed this deletion in the cDNA clone. A consensus nucleotide sequence for PM48 was obtained from part of PM48#5.2, the two PCR-generated DNA fragments amplified from cDNA and the genomic DNA sequence of PM-,8. The sequence is presented in Figure 5 in which identified peptide amino acid sequences are underlined. The broken lines denote the positions of the oligonucleotide primers used for amplification of the PCR fragment. Cysteines are in bold type and potential N-linked glycosylatlon sites are boxed. A potential poly-adenylation signal sequence is twice underlined with a broken line.

The consensus sequence of Figure 5 contains contained a single open reading frame of 1065 nucleotides which codes for a polypeptide 355 amino acids in length. Four peptides derived from purified PM48 were located within this deduced amino acid sequence. One peptide sequence (peptide was not located and was probably derived from a contaminating protein in one of the two preparations of PM48 used for the production of peptides.

Peptides derived from the preparation of PM48 which was also used in the vaccination trial were all located within the deduced amino acid sequence of PM48 indicating that this preparation did not contain any of the putative contaminating protein. The 3' untranslated region contains a consensus signal sequence for polyadenylation (AATAAA: Proudfoot and Brownlee, Nature, 263, 211-214; 1976) but there is no suggestion of a poly-A tail.

The consensus sequence contains the complete nucleotide and deduced amino acid sequence of mature FM48 as verified by the aminoterminal amino acid sequence of PM48. The mature polypeptide is 355 amino acids in length and has a calculated molecular weight of 38,934 Da and a calculated pi=4,2. The amino acid sequence contains 32 cystelnes which are arranged in 5 sub-domains each containing 6 cystelnes as well as 2 additional cysteines at the carboxy-terminal end of the deduced amino 20 acid sequence. There is only limited sequence homology between these subdomains, the characteristic feature of which is the spacing of the 6 cysteine residurs which conform to the following consensus sequence: CX,2- 2 oCX 5 CX9 IoCXio-I4CX4I 6 C (where x is any amino acid residue except cysteine) The cysteines are probably involved in extensive intramolecular 25 disuDhide bonding (Thornton, Journal of Molecular Biology 151, 261-287; 1981). There are 2 potential N-linked glycosylation sites (NX(S/T)X, where X is not proline Gavel and von Heijne, Protein Engineering 3, 433-442; 1990) present within the coding sequence. PM48 reacts with biotin-labelled lentil lectin, alrectly confirming the presence of glycosylation which is of the high mannose type.

EXAMPLE 6 Expression of recombinant PM48 In this example, the expression of PM48 via recombinant DNA means is demonstrated. There are many expression systems which will allow the production of recombinant PM48. Further, recombinant PM48 can be produced as a full length protein, a fusion protein or a truncated protein.

The following is but one example of the production of full length recombinant PM48 but it will be recognised that recombinant PM48 can be produced in a variety of expression systems such as those specific to bacteria, insect cells, fungi and i- ammalian cell lines.

Production of plasmid vector for expression of recombinant PM48.

The plasmid, pQElO (Diagen) was employed to express recombinant PM48 in the cytoplasm of E. coll. A sense oligonucleotide primer gtcggatcctGAATACAATGTGGCCAAGTATTG was designed from DNA sequence coding for the amino-terminal region of the mature PM48 polypeptide. This primer incorporated a BamHI restriction site at the 5' end (lower case letters). A reverse-sense oligonucleotide primer ggccggatccTTAAACTGGTGAATTTAGCATGCAA- was designed from within 15 the 3' non-coding region of the PM48 cDNA sequence. A BamHI restriction site was also incorporated into the 5' end of this primer (lower case letters).

PCR was performed using first strand cDNA prepared as described in Example The PCR consisted of 100 ul of 10 mM Tris-HC1, pH 8.3, mM KC1 containing 0.25 mM of each dNTP, 3 mM MgC1 2 and 1.25 U 20 AmpliTaq DNA polymerase (Perkin Elmer Cetus). Amplification was performed for 40 cycles in a Hybaid Omnigene thermal cycler under the following conditions for each cycle: 2.5 min at 94°C, 2.5 min at 60 0 C and min at 720C. The "1,100 bp DNA fragment generated by this procedure was purified through a Magic PCR Preps column (Promega) into H 2 0 and was 25 subsequently ligated into pGEM-T vector (Promega). The resulting plasmid was transformed into E. coll XL-1 strain (Stratagene) and the sequence of the DNA insert verified as Identical to that shown in figure 5 for the mature protein. 51ig of this plasmid was digested with BamHI (Promega) to liberate the insert which was subjected to electrophoresis on a 1.5% low melting point agarose gel containing 10 jig of ethidium bromide. The DNA insert was excised from the gel and purified through a Magic PCR Preps column into H 2 0 and ligated into the pQE-10 vector digested with BamHI. The resulting plasmid (pQEPM48#1) was transformed inrto E. coll XL-1.

Production of recombinant PM48 protein.

Ten ng of pQEPM48#1 was transformed into E. coll M15 cells (Diagen) for the production of recombinant protein. Three colonies were examined for protein. Colonies were picked into 1.5 ml of LB media containing 100 ug/ml ampicillin and 25 pg/ml kanamycin and grown at 37 0 C with vigorous shaking overnight. 500 ul of each overnight culture was then sub-cultured into 1.5 ml of fresh, warmed LB media containing 100 pg/ml ampicillin and pg/ml kanamycin and grown at 37 0 C for 30 min to ensure an even cell density. The cultures were induced by the addition of 2 mM IPTG and grown at 37 0 C for 5 h. 1 ml of each culture was centrifuged for 1 min to pellet the cells which were resuspended in 40 pl of PBS. An equal volume of 2 x SDS-PAGE sample buffer (reducing; Laemmli, Nature 227, 680; 1970) was added to the pellet, mixed and heated to 95 0 C for 5 min prior to freezing at -20 0 C. An un-induced control was also included. 2 pl of each sample was run on two identical 10 SDS-PAGE gels. One gel was stained for protein with Coomassie Blue while the other was electro-blotted to 15 nitrocellulose for an immuno-blot which was processed in the following manner. After 60 min incubation at 37 0 C in TBS containing 2 gelatin, the nitrocellulose was incubated for 2 h at 37 0 C with a 1/1000 dilution of S"sheep anti-native PM48 serum in TBS containing 0.1 Tween-20 (TBS- Tween). The nitrocellulose was washed in the same buffer (3 x 10 min) and 20 incubated with a 1/2000 dilution of horse raddish peroxidase-labelled rabbit anti-sheep IgG in TBS-Tween for 1 h. After washing the nitrocellulose as before, it was incubated with 3.3 mM 4-chloro-l-naphthol and 0.02 H 2 0, in TBS to detect the presence of recombinant PM48. All three cultures expressed recombinant hexa-his-PM48 at approximately the 25 same level as detected by the specific immuno-blot and by examination of the induced recombinant protein on the Coomassie Blue-stained gel. One of these cultuxes was then selected for production of larger quantities of recombinant hexa-his-PM48.

Large scale bacterial cultures expressing recombinant hexa-his-PM48 were prepared in the following manner. Colonies of E. coll M15 cells containing the expression plasmid pQEPM48#1 were picked into 4 x 5 ml aliquots of LB media containing 100 pg/ml anpicillin and 25 p/g/ml kanamycin and grown at 37°C with vigorous shaking, overnight. Each 5 ml of culture was sub-cultured into 250 ml of fresh, warmed LB media containing 100 pg/ml ampicillin and 25 p/g/ml kanamycin and grown at 37"C for 2 h. The cultures were induced as described above and grown at 37 0 C for 5 h. The cultures were then centrifuged for 10 min at 4,200xg and the resulting pellets stored at -20°C until required. The immunoreactive recombinant hexa-his-PM48 remained with the cell pellet after freeze/thawing and subsequent sonication indicating that the recombinant protein was in the form of a bacterial inclusion body.

Purification of recombinant PM48.

One of the features of the pQE10 expression plasmid is the addition of a small amino acid sequence containing 6 histidine residues at the aminoterminus of the recombinant protein. This hexa-histidine addition facilitates the purification of the recombinant protein because of its affinity for a nickel-nitrilo-tri-acetic acid resin (NNTA; Qiagen Inc, USA) even in the presence of strong denaturants such as high concentrations of urea and guanidine HCI which are used to solubilize recombinant proteins from 15 inclusion bodies. Cells from one litre of E. colt M15 transformed with plasmid pQEPM48#1 and induced with IPTG were pelleted and washed with TBBS (50 mM Tris-HCl, 50 mM NaCI, 1 mM EDTA, pH 7.8) containing 1 mM benzamidine, 10 uM AEBSF and 12 mM 2-mercaptoethanol. The cell pellet was resuspended in 30 ml of the same buffer but containing 100 mg 20 lysozyme and incubated for 10 min at 4 0 C (with stirring) to lyse the cells.

The solution was then made 0.1 in Triton X-100, sonicated (3x15 s) at 4°C and centrifuged (12,000xg, 4 0 C, 25 min). The resulting pellet was washed by centrifugation in the above buffer plus 0.5 Triton X-100 and then made up to 30 ml with TBBS containing 20 mM MgCl 2 1 mM 25 benzamidine, 10 /uM AEBSF and 10 /~g/ml DNaseI. The solution was incubated for 30 min at 37°C (with stirring) and centrifuged (12,000xg, min, 4 0 C) to pellet the recombinant protein. After washing the pellet with TBBS, the recombinant protein was extracted with 8 ml of 6 M guanidine HCI, 5 mM 2-mercaptoethanol, 0.1 M NaH 2

PO

4 and 10 mM Tris-HCl, pH 8.0 for 60 min at 25 0 C (with stirring). The solution was centrifuged as before and the supernatant collected for addition to the NNTA affinity resin.

The recombinant hexa-his-PM48 protein was purified using this affinity resin essentially according to the manufacturer's instructions (Qiagen Inc.

USA). Figure 6 shows an SDS-PAGE profile of the purified protein stained with silver. Molecular weight standards are in lane 1 and lane 2 contains

I

purified recombinant hexa-his-PM48 protein (3 ug).

Refolding of recombinant hexa-his.-PM48.

The presence of glycosylation and probably extensive disulphide bonding in the native PM48 protein are not conducive to expression of a correctly folded recombinant hexa-his-PM48 in the cytoplasm of bacteria.

This is a result of the inability of bacteria to glycosylate recombinant proteins and also the general inability of the bacterial cytoplasm to form correctly linked disulphide bonds in recombinant proteins. Consistent with this, recombinant hexa-his-PM48 was produced as an insoluble inclusion body within E. coll. Therefore, it is desirable to chemically refold recombinant hexa-his-PM48 such that disulphide bonding occurs In a manner similar to, or preferably identical with, that present in native PM48.

Purified recombinant hexa-his-PM48 was refolded in the following manner.

The affinity purified recombinant hexa-his-PM48 was diluted into 20 mM 15 Tris-HCl, 0.1 Tween-20, 0.25 M NaCI, pH 8.5 to a final protein concenL-ation of 100 /pg/ml and final urea concentration of 3 M. The solution was then made 1 mM in reduced glutathione and 0.1 mM in oxidized glutathione, the pH adjusted to 8.5 and then stirred for 20 h at 4 0

C

and for 1 h at 22 The refolded protein was then concentrated. The final 20 yield of refolded recombinant hexa-his-PM48 was 20 mg.

EXAMPLE 7 Vaccination of sheep with refolded recombinant hexa-his-PM48 A vaccination trial in sheep was undertaken to assess the ability of refolded recombinant hexa-his-PM48 to induce an immune response which 25 inhibited the growth of larvae of L. cuprlna. Four sheep were each vaccinated with a total of 500 ig of refolded recombinant hexa-his-PM48.

The adjuvant (2.5 ml) was Montanide CSA50 (Seppic). An identical injection occurred 4 weeks after the first injection. Each injection was intramuscular, given half into each rear leg for the first injection and in the neck for the second injection. A control group of 4 sheep were vaccinated with adjuvant alone. The sheep were bled from the jugular vein prior to vaccination and two weeks after the second injection. The sera from these sheep were then tested for their ability to inhibit growth of first instar larvae of L. cuprlna in an in vitro feeding assay (Eisemann et al., 1990, supra). Briefly, 10 larvae wer- allowed to feed on an agar medium containing 75 serum in a small 31 plastic container. A total of 5 of these containers 50 larvae) were used to measure mean larval weights for each serum. After 20 h of feeding, the number of surviving larvae and their weights were measured. Antibody titres were measured by elisa as described by Eisemann et al. (1990, supra).

The results of this vaccination trial are shown in Table 6. The mean larval weight was 4.64±0.11 mg for the control group and mg for the group of sheep vaccinated with recombinant hexa-his-PM48. The reduction in larval weights is highly significant Assessment of the specific immune response by elisa indicated that there was a strong antibody titre to recombinant hexa-his-PM48 in each of the vaccinated sheep. These results indicated that recombinant hexa-his-PM48, when injected into sheep, induced a strong antibody response which inhibited the growth of L. cuprina larvae feeding on the sera from these sheep.

Table 6 S 15 Effect of sera from sheep vaccinated with refolded recombinant hexa-his-PM48 on the weight of first instar L. cuprina larvae which subsequently fed on that sera as measured by an in vitro feeding bioassay Animal No. Group Mean Larval Wt. (mg) 20 3212 Control 4.49 0.44 3214 Control 4.65 0.10 3227 Control 4.69 0.23 3231 Control 4.74 0.38 Gp. Mean 4.64 0.11 3206 hhPM48' 3.65 0.27 3207 hhPM48 3.53 0.47 3208 hhPM48 3.25 0.47 3209 hhPM48 3.05 0.27 Gp. Mean 3.37 0.27 hhPM48, recombinant hexa-his-PM48 The mean larval weight was measured by an In vivo feeding bioassay. The standard deviation associated with each measurement is listed adjacent to the measurement. The difference between the means of the larval weights from each group is significant at p<0.01.

Claims (26)

1. PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant thereof.

2. An isolated DNA comprising a sequence encoding PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant thereof.

3. An expression vector which includes a DNA sequence encoding PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant or immunogenic fragment thereof.

4. Expression vector according to claim 3, which vector is pQEPM48#1, as herein defined. A host cell transformed with the expression vector of claim 3 or claim 4.

6. Host cell according to claim 5, wherein said host cell is a prokaryotic 5 or a eukaryotic cell.

7. Host cell according to claim 6, wherein said host cell is a bacterial cell.

8. Host cell according to claim 6, wherein said host cell is a yeast cell.

9. Host cell according to claim 6, wherein said host cell is a baculovirus-infected insect cell. 1: 0. A method of producing PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant or immunogenic fragment thereof, the method comprising the steps of: introducing into a host cell DNA which includes a DNA sequence encoding said PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof in conjunction with elements for the expression of polypeptide encoded by said DNA; culturing said host cell under conditions which allow expression of the encoded polypeptide; and isolating the expressed PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof.

11. Method according to claim 10, wherein said host cell is a prokaryotic or a eukaryotic cell.

12. Method according to claim 11, wherein said host cell is a bacterial cell.

13. Method according to claim 11, wherein said host cell is a yeast cell.

14. Method according to claim 11, wherein said host cell is a baculovirus-infected insect cell. Method according to any one of claim 10 to 14, wherein said elements for the expression of polypeptide encoded by said DNA include an element for the extracellular expression of said polypeptide.

16. Method according to any one of claims 10 to 15, said method comprising the further step of purifying PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof from isolated expressed PM48 antigen.

17. Method according to claim 16, wherein said purification includes at least one affinity purification step.

18. Method according to claim 17, wherein said affinity purification is by immuno-affinity purification or lentil lectin affinity purification. S 15 19. A vaccine for the prophylaxis or treatment of blowfly strike in sheep, the vaccine comprising PM48 antigen having an amino acid sequence depicted in Figure 5, or an allele, homologue or variant or immunogenic :fragment thereof.

20. Vaccine according to claim 19, which further includes an adjuvant.

21. Vaccine according to claim 19 or claim 20, wherein said PM48 antigen, or allele, homologue or variant or immunogenic fragment thereof is glycosylated.

22. Vaccine according to any one of claims 19 to 21, which further includes at least one additional peritrophic membrane immunogen.

23. Vaccine according to claim 24, wherein said additional peritrophic membrane immunogen is selected from PM44 and PM95, as herein defined.

24. A method of preventing or treating blowfly strike in a sheep, said method comprising administering to said sheep an effective amount of a vaccine according to any one of claims 19 to 23.

25. Method according to claim 24, wherein said blowfly strike is due to a species of blowfly from the genera Lucilla, Calliphora, Chrysomya or Cochlimyla.

26. Method according to claim 25, wherein said blowfly strike is due to Lucilla cuprina.

27. Method according to claim 25, wherein said blowfly strike is due to I 34 Chrysomya ruflfacles.

28. Method according to any one of claims 24 to 27, wherein said vaccine is administered intramuscularly, subcutaneously, intraperitoneally or by infusion.

29. Host cell harbouring an expression vector according to claim 3 or claim 4 which host cell is substantially as hereinbefore described with reference to Example 6. A method of producing PM48 antigen having an amino acid sequence depicted in Figure 5, which method is substantially as hereinbefore described with reference to Example 6.

31. A vaccine for the prophylaxis or treatment of blowfly strike in sheep, which vaccine is substantially as hereinbefore described with reference to Example 7. DATED THIS 20TH DAY OF SEPTEMBER 1995 15 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By their Patent Attorneys CULLEN CO 4* o- ABSTRACT This application relates to DNA encoding the PM48 antigen of Luclla cuprina. Utilisation of 'he DNA for high level expression of PM48 antigen is also described as well as vaccines comprising the antigen. Vaccines comprising PM48 antigen are efficacious for the prophylaxis or treatment of blowfly strike in sheep. The vaccine is effective against blowfly strike due to blowflies from genera including Luctlia, Calliphora, Chrysomya and Cochliomyla. C oa I