AU694179B2 - Recombinant blowfly strike antigen - Google Patents

Description

_i.

1

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT 0q44 00 *0 Name of Applicant: Actual Inventors: Address for Service: COMMONWEALTH SCIENTIFIC INDUSTRIAL RESEARCH ORGANISATION Ross Lindsay Tellam Rosanne Elena Casu Craig Harold Eisemann CULLEN CO., Patent Trade Mark Attorneys, 240 Queen Street, Brisbane, Qld. 4000, Australia.

.0,0*0 *I 0 Invention Title: RECOMBINANT

ANTIGEN

BLOWFLY STRIKE Details of Associated Provisional Application: No. PM5235 filed on 22 April 1994 The following statement is a full description of this invention, including the best method of performing it known to us: I~ ,I 2 TECHNICAL FIELD This invention relates to the production of an antigen for inclusion in a vaccine which when administered to sheep results in the induction of an immune response which is capable of retarding the growth of blowfly larvae feeding on the vaccinated sheep thereby restricting or limiting the effects of blowfly strike. In particular, the invention describes a method for the production of large quantities of an antigenic component of the vaccine as well as a preferred method of administering the vaccine.

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.

15 The major species of blowfly which initiates 80-90% of all primary strikes in Australia is Lucila cuprina while the closely related blowfly Lucilla serlcata initiates most strikes in other locations such as Europe.

Australian Patent Application No. 29,716/92 describes the production of a vaccine which can be used in sheep to alleviate the effects of blowfly S 20 strike. The principal components of the vaccine are described in that application as antigens from the peritrophic membrane (PM) of larvae of L.

cuprina or whole peritrophic membrane itself. Australian Patent Application No. 29,716/92 also describes the purification and identification of three specific protein antigens derived from peritrophic membrane of L. cuprina larvae. These antigens, designated PM44, PM90 and PM95, can be used either individually or in combination as immunogenic components of a vaccine against blowfly strike.

Australian Patent Application No. 29,716/92 also disclox.a the gene sequence and deduced amino acid sequence of one peritrophic membrane antigen, PM44. The latter information enables the production of large quantities of this particular antigen through expression of PM44 from recombinant DNA in bacterial cells, insect cells or other suitable expression systems or by the synthesis of peptides representing particular immunogenic regions of PM44.

As alluded to above, data included in Australian Patent Application No.

3 29,716/92 demonstrate that serum from sheep vaccinated with PM90 or can retard the growth and development of L. cuprina larvae. This indicates that the antigens can be used in a vaccine to reduce the severity and/or incidence of blowfly strike. However, 29,716/92 does not disclose a method for the large-scale production of PM90 and PM95. In particular, a characterisation of PM90 or PM95, other than a method of preparing small quantities of antigen, is not included in 29,716/92.

It is not commercially viable to isolate vaccine antigens directly from peritrophic membrane produced by culture of L. cuprina larvae. Indeed, only 1-2 mg of PM95 or PM90 can be isolated in pure form from 20 g of peritrophic membrane obtained by larval culture. This quantity of antigen is only sufficient for vaccination of three to four sheep. Approximately 630,000 larvae cultured over a total period of 5 weeks is required to produce 20 g of peritrophic membrane. The logistical and scientific difficulties associated S. 15 with the scaling-up of this process for the production of commercial quantities of PM90 and PM95 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 these antigens must rely on the artificial production of sufficient quantities of the antigens.

Such quantities are most easily produced by recombinant DNA means in bacteria, yeast or insect cells or as synthetic peptides representing fragments of the whole antigens. There is thus a need to identify, and determine the sequences of, the DNAs encoding PM90 and PM95 and/or to deduce the amino acid sequences of these antigens.

SUMMARY OF THE INVENTION An object of the present invention is to provide DNA encoding the or PM95 L. cuprina antigens, DNA encoding immunogenic fragments of the or PM95 antigens, or the amino acid sequences of the PM90 or antigens, to allow the large scale production of a commercial vaccine which may be used to combat blowfly larvae infestations of vaccinated sheep.

Further objects of the invention are to provide expression vectors comprising the foregoing DNA, host cells harbouring the expression vectors and capable of expressing antigen or fragments thereof, and vaccines containing the antigen or fragments thereof as the principal immunogenic component.

The present inventors have surprisingly found that a single DNA can be 4 isolated from L. cuprina which encodes both the PM90 and PM95 :antigens and the protein expressed from that DNA is effective in a vaccine against blowfly strike.

According to a first embodiment of this invention, there is provided an isolated DNA comprising a sequence encoding PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant thereof.

According to a second embodiment of this invention, there is provided an expression vector which includes a DNA sequence encoding PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant or immunogenic fragment thereof.

According to a third embodiment of this invention, there is provided a host cell harbouring an expression vector according to the second embodiment.

According to a fourth embodiment of this invention, there is provided a method of producing PM95 antigen having an amino acid sequence depicted in Figu 2 8, 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 PM95 antigen, or allele, homologue or variant or immunogenic fragment thereof in conjunction with elements for the 1 expression of polypeptide encoded by said DNA; culturing said host cell under conditions which allow expression of the encoded polypeptide; and isolating the expressed PM95 antigen, or allele, homologue or U variant or immunogenic fragment thereof.

According to a fifth embodiment of this invention, there is provided a jii .vaccine for the prophylaxis or treatment of blowfly strike in sheep, the vaccine comprising PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant or immunogenic fragment thereof.

According to a sixth embodiment of this invention, there is provided a method of preventing or treating blowfly strike in a sheep, the method comprising administering to said sheep an effective amount of a vaccine according to the fifth embodiment.

_~LLII.I_ i. i. 1~ The blowflies to which the present invention is applicable include not only L. cuprina but also related flies causing myiasis in their hosts. These flies are characterized by the ability of their larval stages to parasitise their vertebrate hosts, typically cattle or sheep, by feeding on host tissue or tissue fluid. These flies are generally related and belong to the family Calliphoridae and sub-families Calllphorlnae and Chrysomyinae. While not exch ,ive, the following list represents the range of flies applicable tu this invention: Lucilia cuprina, Lucilta sericata, Calliphora augur, Calliphora stygia, Calliphora nociva, Calllphora albifrontalls (also called Calliphora australls or Calliphora maryfullert), Calliphora hill, Calliphora vicina (also called Calliphora erythrocephala), Chrysomya ruflfacies, Chrysomya varipes, Chrysomya bezziana, Chrysomya albiceps and Cochlliomyla hominivorax. However, it is recognised that the vaccine, or variations thereof, based on the same or I' similar effective antigens, may have general application for the control of 15 Dipteran insects or their larvae which have a similar peritrophic membrane structure to L. cuprina and which feed on the tissue or tissue fluids of vertebrate hosts for example, Haematobia exlgua irritans and related Sspecies).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the effect of sera and various concentrations of isolated anti-PM90 immunoglobulin on growth of L. cuprina larvae in vitro.

Figure 2 presents indirect immuno-fluorescence and immuno-gold localisations of PM90 on peritrophic membrane from L. cuprina.

Immuno-fluorescence localisations are shown in Figures 2(a) and 2(b) while immuno-gold localisations are shown in Figures 2(c) and 2(d).

Figure 3 presents results of SDS-polyacrylamide gel electrophoresis of purified PM90 and Figure 4 depicts amino acid sequences of peptides from PM90 and Figure amino acid sequences of peptides from PM90; Figure amino acid sequences of peptides from PM95; and, Figure similarities between and PM95 peptide amino acid sequences.

Figure 5 shows gel analysis of DNA fragments amplified using the polymerase chain reaction with PM90 and PM95-specific oligonucleotide primers.

Figure 6 depicts the location of a common restriction site in several DNA 6 6 fragments amplified using PCR with PM90 and oligonucleotide primers. Results of gel analysis are shown in Figure 6(a) while a restriction map is shown in Figure 6(b).

Figure 7 presents nucleotide and deduced amino acid sequences of the DNA fragment produced by PCR using oligonucleotide primers 2 and 7.

Figure 8 presents nucleotide and deduced amino acid sequences of determined from cDNA.

Figure 9 depicts the binding of biotin-labelled wheat germ lectin to purified PM95. Separated protein detected by silver-staining and with biotin-labelled wheat germ lectin after SDS-PAGE are shown in Figure 9(a) and Figure 9(b) respectively.

Figure 10(a) and Figure 10(b) respectively depict the results of SDS-PAGE and immuno-blotting of purified recombinant hexa-his-PM95 proteins.

1 5 Figure 11 depicts indirect immunofluorescence localization of PM95 on 15 peritrophic membrane from larvae of L. cuprina using anti-serum to refolded recombinant hexa-his-PM95. Figure 11(a) is the result of a control experiment using pre-vaccination serum while Figure 11(b) is the result of immunofluorescence localization using anti-serum to refolded recombinant Figure 12 is an immunoblot showing the production of recombinant in a baculovirus/insect cell expression system.

t Figure 13 shows the result of SDS-PAGE analysis of purified full-length protein. The gel resulting from SDS-PAGE was silver stained for detection of protein bands.

DETAILED DESCRIPTION OF THE INVENTION The following abbreviations are used throughout this description: AEFSF p-Aminoethylbenzene sulfonyl fluoride AMP Ampicillin BSA Bovine serum albumin DIG Digoxigenin dNTP Deoxynucleotide triphosphate DTT Dithiothreitol EDTA Ethylenediamine tetra acetic acid Endo Lys C Endoproteinase Lys C i 35 Endo Glu C Endoproteinase Glu C 7 ELISA Enzyme-linked immunoabsorbtion 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 PTH-amino acid Phenylthiohydantoin-amino acid rRNA Ribosomal RNA SDS Sodium dodecylsulfate 15 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 I The one-letter code for nucleotides in DNA and the one- and threet letter codes for amino acid residues conform to IUPAC-IUB standards described in The Biochemical Journal 219, 345-373 (1984).

In accordance with the invention, PM90 and PM95 antigens can be produced by expression in suitable host cells from DNA encoding these antigens. The present inventors have found that the PM90 and antigens are the product of the same gene. The difference i' size between the two antigens is probably due to post-translational modification.

DNA encoding PM95 is advantageously identified by screening cDNA or genomic DNA libraries of L. cuprina or related organisms with oligonucleotides complementary to coding regions of the PM95 gene.

Alternatively, such oligonucleotides can be used as primers for amplification of DNA encoding PM95 by PCR. The sequences of suitable oligonucleotides can be established from a consideration of amino acid sequence data for all or portions of PM95 or ~LIIWII.-11I._ _I 8 To provide proteins or peptides for sequencing, antigen can be purified from blowfly peritrophic membrane. Procedures for the isolation of and PM95 from L. cuprina peritrophic membrane are described in Australian Patent Application No. 29,716/92, the entire disclosure of which is incorporated herein by cross-reference. A brief description of one procedure follows. Peritrophic membrane is obtained from L. cuprina larvae cultured in vitro. The peritrophic membrane is progressively extracted with: water; 0.1 M Tris-HCl, pH 7.5 containing 150 mM NaCl, 5 mM EDTA and 5 mM benzamidine; 20 mM Tris-HCl, pH 7.4, 140 mM NaC (TBS) containing 2 Zwittergent 3-14; and TBS containing 6 M urea. The 6 M urea extract of peritrophic membrane is concentrated in a Centricon 30 cell (Amicon) and o fractionated by preparative SDS-PAGE (Model 491 Prep Cell; Biorad) on a oo 10% polyacrylamide gel. This procedure is carried out according to the manufacturer's instructions. Fractions collected from this procedure are 15 subjected to analytical SDS-PAGE and proteins stained with silver.

Fractions containing proteins of Mr=90,000 (PM90) and Mr=95,000 are independently pooled and concentrated in a Centricon 30 cell for further analysis. The foregoing method for purification of native PM90 and PM95 is illustrative but not limiting for example, PM95 can also be isolated from the 6 M urea extract of peritrophic membrane by lectin affinity chromatography, gel permeation chromatography, anion exchange Schromatography or other methods in common use by those skilled in the art.

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 i .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 prefer-ably comprise sequences having no secondary structure and minimal degeneracy. Labelled oligonucleotides can be used as probes for the PM95 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 9 skilled in the art that the gene coding for PM95 may also be isolated by screening a L. cuprlna cDNA expression library with anti-sera to PM95 or anti-sera raised to peritrophic membrane or crude extracts thereof.

DNA encoding PM95 can be incorporated into a vector for expression of the antigen, or a portion or derivative thereof, in a suitable host cell. The 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 prefered eukaryotic expression host is a baculovirus-infected insect cell.

The expression vector used can include elements which result in the PM95 antigen being expressed as a fusion protein. The fusion protein typically comprises a leader or signal peptide linked at the amino terminal o% end of the PM95 polypeptide or fragment thereof. Expression of the antigen I as a fusion protein can be exploited to deliver the protein to the exterior of 15 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 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 S.vaccine against flystrike in sheep. Both glycosylated and non-glycosylated forms of antigen can be useu as immunogens. Homologues of the effective antigen either from the same or different insect species can also be used. In addition, the PM95 antigen can be used in conjunction with other peritrophic membrane antigens: for example, the PM44 antigen described in Australian Patent Application No. 29,716/92.

The vaccine typically contains in addition to PM95 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 combination 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 optimized using ordinary skills in the art.

I I I Synthetic peptides based on specific regions of the PM95 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 is capable of recognising the effective antigens PM90 and PM95. 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 PM95 antigen or immunogenic domains thereof.

The invention also includes within its scope antibodies raised against PM95 antigen. Such antibodies can be isolated and directly infused into I 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 PM95 which 15 are equally as effective in a vaccine against flystrike. Thus, the invention includes within its scope these homologues produced as either native proteins or as recombinant DNA-based proteins. The homologues can be derived from allelic variants or alternative genes in L. cuprina or from different species of insects.

From the foregoing, therefore, it will be appreciated that the invention includes within its scope DNA sequences or recombinant antigens corresponding to the sequences shown in Figure 8 as well as h yrids 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 the following properties: at least 50% identity compared to the DNA sequence shown in Figure 8 and/or; structural homologues of the amino acid sequence shown in Figure 8 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 PM95 i.e.

CX

12 20

CX

5

CX

9 19

CX

10 14

CX

4 4 6

C;

protein structural homologues which bind chitin or N-acetyl glucosamine polymers and/or; protein structural homologues which when injected into an animal induce an immune response which recognises PM95 and/or; peritrophic membrane proteins which bind to The invention is further described in the following examples which are illustrative of the invention but in no way limiting of its scope.

Materials and General Methods The following chemicals and reagents were obtained from the indicated sources: Name Supplier AEFSF ICN Biochemicals Aquapore RP-300 C-8 Waters 15 Biotinylated Lectins Vector Laboratories Inc or Pierce Chemical 99 en, a I 4.

4 9.9.

.9 99 Centricon DNA ligase (T4) E. coi XL 1-blue EcoRI EcoRV Endo Lys C Endo Glu C Freund's Complete Adjuvant Gentamicin

IPTG

Magic PCR Prep Newborn calf serum pGEM7Zfl+) Rota Vac SinaI Streptavidin-peroxidase Superose 12

X-GAL

Zwittergent 3-14 Company Amicon Promega Stratagene Prornega Promega.

Boehringer Mannheim Boehringer Mannheim Commonwealth Serum Laboratories (CSL)

CSL

Boebringer Mannheim Promega,

CSL

Promega.

Savant Promega Amersham Pharmacia.

Boehringer Mannheim Calbiochem ;1 I: .i 12 Unless otherwise specified, other laboratory chemicals were of analytical grade and purchased from Sigma Chemical Company (St. Louis, or Ajax Chemical Company (Auburn, Australia).

Sodium dodecyl sulphate gel-electrophoresis (SDS-PAGE).

Protein samples were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis on 6-18% gradient gels. All gels included molecular weight standards (Pharmacia) and were stained with silver using the method of Morrissey (Analytical Blochemistry 117, 307-310, 1981). Glycoproteins were detected using biotinylated lectins. Briefly, proteins were separated by SDS-PAGE and then electroblotted onto nitrocellulose using an LKB semi-dry NOVA blotter (used according to the manufacturer's instructions). The nitrocellulose was blocked for 1 h at 37°C o* in TBS (20 mM Tris-HCl, pH 7.3, 140 mM NaCI) containing 2.5% gelatin, I incubated with a 1/500 dilution of the appropriate biotinylated lectin for 1 h at 37 0 C, washed (x3) in TBS containing 0.1% Tween 20, incubated with a 3/1000 dilution of streptavidin-peroxidase for 30 min at room temperature and then developed with chloronapthol/HO, substrate according to the manufacturer's instructions. Immuno-blots were performed essentially according to methods described by Richardson et al. (Insect Molecular Biology 1, 139-147, 1993).

Immunofluorescence and immunogold localis-ations.

C Sera from sheep vaccinated with purified native PM95 or PM90 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 J overnight at 7°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 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. The magnification of the photographs was For immunogold localizations, second instar larvae of L. cup.!na were i- 13 dissected open in 4% paraformaldehyde in phosphate-buffered saline and allowed to fix in this solution for 1 h at room temperature. The samples were dehydrated through a series of alcohol washes and embedded in Epon-Araldite resin (Mollenhauer; Stain Technology 39, 111-114, 1964).

Sections were cut on an LKB Nova Ultramicrotome and collected on coated copper grids.

The sections were processed on the grids by inverting them on drops of liquid at room temperature. They were first placed on fresh 0.5 M NH 4

C

in PBS containing 0.5 ovalbumin and 0.1 Tween 20 (buffer A) for 2 10 min before washing in the same buffer (x3, each 5 min). The samples were transferred to 10% heat-inactivated normal horse serum (0.5 h, 56 0

C),

°o followed by introduction of a 1:500 dilution of either sheep serum or normal sheep serum (pre-vaccination control) for 1.5 h. After washing in 5 changes of buffer A (each 5 min), the sections were transferred to a 1:100 dilution of donkey anti-sheep antibody conjugated to 10 nm too colloidal gold particles (Biocell Res. Lab., Cardiff, for 1.5 h. This was I t followed by several further washes in buffer A and distilled water and then the sections were stained lightly (approx. 2 min) in 2 aqueous uranyl acetate and 0.08 M lead citrate and examined and photographed in a 20 Phillips EM 300 transmission electron microscope.

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 native antigens PM90 or were each homogenised with an equal volume of Freund's Complete Adjuvant. The first injection was intramuscular, given half into each rear leg. The second injection (28 days later) used Freund's Incomplete Adjuvant and was given intramuscularly in the neck region. All animals were bled from the jugular vein prior to each injection. Fourteen days after the second injection, the effect of vaccin ition was assessed by in vitro larval growth assay (Eisemann et al.; International Journal for Parasitology 20, 299-305, 1990). The in vitro assay consisted of allowing first instar larvae to feed on VVJ:F 7-1 14 an agar-based medium containing 75% serum from vaccinated animals.

The number of surviving larvae and their weights were measured after 20 h.

Antibody titres were assessed by ELISA as described by Eisemann et al.

(supra).

Protein Concentration Determinations.

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

Establishment of a L. cuprina colony.

Laboratory populations of L. cuprina, which had originated from S 10 flystruck sheep, were maintained on an artificial medium (Singh and Jerram, supra) for up to 10 generations. Eggs were collected by placing I 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°C and 100% relative humidity before surface sterilisation.

Preparation of PM90 and The purification of PM90 and PM95 from peritrophic membrane derived from L. cuprina larvae cultured in vitro is described in Australian Patent Application No. 29,716/92 and is briefly reiterated above. The determination of amino-terminal and internal peptide amino acid sequences for PM90 and PM95 is described in the text below. Evidence is presented which indicates that PM90 is the same as PM95 except for a post-translational modification of the former.

Molecular biology.

Many of the standard procedures used for the cloning and sequencing of PM95 are described by Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989). Other methods are described in the text.

Example 1 Enhanced retardation of the growth of larvae of L. cuprina when feeding on ovine serum with increased concentrations of Ig from sheep vaccinated with native PM90 or Immunoglobulin (Ig) was isolated using method E of Mostratos and Beswick (Journal of Pathology 98, 17-24, 1969), from the sera of two of the strongest responding sheep which had been vaccinated with native and also from pooled sera of 12 control sheep which had been injected with i ,i i adjuvant only. The percentage yields of Ig obtained from the sera were estimated using ELISA. With the PM90 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., 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. (supra). Relative concentrations of Ig in the original sera and in isolation were estimated in each I 10 case by comparing curves obtained by plotting log ELISA optical densities against log dilution. The Ig samples were concentrated by vacuum 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.

S' 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 20 are shown in Figure 1 in which the stippled bars represent the mean weight P of the larvae for each modified serum. The fully shaded bars represent sham control experiments in which immunoglobulin isolated from prevaccination sera was reconstituted at one-fold, two-fold and four-fold enrichments in control ovine serum. The unshaded portions of the error bars denote 1 S.D.

This experiment demonstrated a greater retardation of larval growth Swhen larvae fed on immune sera enriched with Ig isolated from the serum of sheep vaccinated with native PM90. The experimental results showed larval growth changed from 30% of the corresponding control at Ixlg to 14% of the corresponding control at 2xlg and 2% 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 was reduced by 48 at 4xIg compared with IxIl the corresponding anti-PM90 immune serum was reduced by 95 compared L i_ i I-- LIS~-- E~ 16 with IxIg. Significant, although variable, mortality is associated with the larvae feeding on the 4xIg-enriched serum. This experiment demonstrated that the retardation of the growth of larvae feeding on these vaccinated sheep is mediated by antibody and the extent of the effect depends 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 2 10 Localization of native PM90 in L. cuprina Sera from sheep vaccinated with native PM90 were used to localize this protein to peritrophic membrane by indirect immuno-fluorescence techniques and immuno-gold techniques. Peritrophic membrane was obtained by fresh dissection of second or third instar L. cuprina larvae.

Pre-vaccination serum did not react with this peritrophic membrane preparation (Fig. Serum from sheep vaccinated with native I showed strong immuno-fluoresence on peritrophic membrane (Fig. 2(b)) indicating the presence of PM90 in freshly dissected peritrophic membrane.

Identical results were also obtained when anti-serum to native PM95 was 20 used for these immuno-localizations. Anti-serum to PM90 also strongly Sreacted with peritrophic membranes isolated from Haematobia exigua irritans (a blood-feeding fly) and the larvae of the plant-feeding insect Hellothis armigera. These results indicate that antigens similar to PM90 are present in the peritrophic membranes from insects not closely related to L.

cuprlna.

Figures 2(c) and 2(d) show the immuno-gold localization of PM90 on peritrophic membrane for serum from control sheep and sheep vaccinated with native PM90, respectively. The distribution of gold particles on the peritrophic membrane, arrowed in Figure localizes PM90 to peritrophic membrane There is a uniform distribution of PM90 on peritrophic membrane but not to the bacterium in the gut lumen. Thus, PM90 is an antigen which can be isolated from cultured peritrophic membrane and is also present on the peritrophic membrane from freshly dissected larvae.

Identical results were obtained with anti-serum to PM95. The magnificati-n in Figure 2 is 45,000 fold.

antigen or iragni a ilt uLi ,L o The present inventors have surprisingly found that a single DNA can be 17 Example 3 The production, purification and amino acid sequences of peptides from PM90 and The isolation and purification of native PM90 and PM95 from peritrophic membrane derived from L. cuprina larvae cultured in vitro is described in Australian Patent Application No. 29,716/92 and briefly reiterated above. Native PM90 and PM95 were obtained from peritrophic membrane produced by culture of L. cuprina larvae. However, this method of production is not commercially viable. Sufficient quantities of these 10 effective antigens (PM90 and PM95) can be produced artificially as to recombinant proteins in appropriate bacterial or eucaryotic expression systems. To this end, peptides from PM90 and PM95 were isolated and their amino acid sequences determined as a first step in the process of making recombinant antigens. Figure 3 shows an SDS-PAGE profile of the purified native antigens, PM90 and PM95 which were used for the production of internal peptide amino acid sequences as well as amino-terminal amino acid sequences. The following samples were analysed: lanes 1 and 2, molecular Sweight standards; lane 3, 1 fug PM90; and lane 4, 1 1 ug PM95. Samples were denatured by heating and reduced with 5 mM DTT prior to electrophoresis.

20 Internal peptides from both PM90 and PM95 were prepared by the 4 9 following procedure. The protein (50 L/g) was mixed with 100 ul of 0.1 M Tris-HC1, pH 8.3 containing 20 mM dithiothreitol and 2% SDS and then incubated for 30 min at 56 0 C. The solution was cooled to room temperature and sodium iodoacetate added to a final concentration of 0.14 M. After 45 min in the dark, cold methanol was added in a ratio of 9:1 methanol/sample The sample was stored at -20 0 C overnight, centrifuged, the supernatant removed and the pellet dried. The pellet was dissolved in 76 l of 0.1 M Tris-chloride buffer containing 4 M urea, pH and then 4 ul of Endoproteinase Lys C (6 units/ml) or 4 ul of Endoproteinase Glu C (EC 3.4.21.19; 6 units/ml) were added. After 2 h at 37°C, another 4 #1 of the appropriate 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% trifluoroacetic acid 2 i l I i 18 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 that replaced the trifluoroacetic acid by heptafluorobutyric acid (HFBA). Peptides were collected and concentrated to 50-100 il by rotary desiccation in a Rota Vac. The amino acid sequences of the peptides were determined using an Applied Biosystems 471A amino acid sequencer.

Figure 4 shows the determined peptide amino acid sequences from and PM95. The one letter code for amino acids has been used. Blank 10 cycles denoted could be due to phenyl thiohydantoin-derivitized carboxymethyl cysteine or due to glycosylated amino acids (Williams et al.; Today's Life Science 4, 50-60, 1992). Question marks denote uncertainty I associated with the identification of amino acids at some positions while refers to an amino-terminal sequence. The amino-terminal bracketed residue is the expected amino acid based on the cleavage specificity of the respective proteases used for the digestion of PM90 and viz. K, Endoproteinase Lys C; E, Endoproteinase Glu C.

Of particular note are peptides 90P201, 90P110 and 95P406 which have near identical amino-terminal sequences except for a few uncertain 20 positions. These peptides were derived from both PM90 and Further, the PM95 peptide was obtained by an Endoproteinase Glu C protein digest while the PM90 peptides were obtained from an Endoproteinase Lys C protein digest. This information can only be reconciled if these peptide sequences represent a common amino-terminal sequence in both proteins indicating that PM90 and PM95 are related.

Indeed, the amino-termini of both PM90 and PM95 were sequenced directly and shown to be identical to the appropriate regions in these peptide amino Sacid sequences (Fig. In addition, the PM90 peptides 90P081 and 90P210 are identical with the end of the amino-terminal peptide 95P406 derived from PM95 and the PM90 peptide, 90P206, shows overlap with the peptides, 95P405 and 95P213a. This peptide sequence information strongly indicates that PM90 and PM95 are derived from a single gene. The difference in size between PM90 and PM95 is probably due to differential post-translational modifications such as glycosylation or proteolytic processing. The amino acid sequences of these peptides have been used to C

L

primers.

Figure 6 depicts the location of a common restriction site in several DNA 19 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.

Example 4 Molecular cloning of the gene coding for The amino acid sequence identities of peptides derived from PM90 and and the common amino-terminal sequences of these proteins indicated that both were probably derived from one gene and thus all of the 10 peptide amino acid sequence information from both PM90 and PM95 was used to isolate the gene coding for these proteins.

Oligonucleotide synthesis.

The amino acid sequences described in Example 3 (Fig. 4) were used to design suitable oligonucleotide probes that could be used in the polymerase chain reaction (Saiki et al.; Science 239, 487-491, 1988) to amplify DNA fragments of the PM95 gene from L. cuprina first instar cDNA.

Degenerate oligonucleotide primers were designed and synthesised on a Pharmacia LKB Gene Assembler Plus oligonucleotide synthesiser according to the manufacturer's instructions. The synthesised 20 oligonucleotide primers and their peptide of origin are listed hereafter with bracketed nucleotides showing alternatives at a specific position.

Primer 2 derived from peptides 90P081, 90P210, 95P406, 90P201 and 90P110 (sense): 5'-C(TCA)AC(TCA)GG(TCA)AC(TCA)AA(AG)TT(TC)CC(TCA)AG-3' Primer 5 derived from peptides 95P311a, 95P214 and 95P215 (antisense): 5'-CC(AGT)GA(AG)TT(AGT)GG(AGT)AT(AGT)AT(TC)TT(AG)TT-3' Primer 6 derived from peptides 95P311a, 95P214 and 95P215 (antisense): 5'-CC(AG)CT(AG)TT(AGT)GG(AGT)AT(AGT)AT(TC)TT(AG)TT-3' Primer 7 derived from peptides 95P311a, 95P214 and 95P215 (antisense): 5'-C(AG)AA(AG)TA(AGT)AT(AGT)GA(AG)AA(AGT)AC(AGT)CC-3' Primer 10 derived from peptides 90P41 and 90P101 (anti-sense): 5'-C(AG)TC(AG)TA(AG)TA(TC)TC(AGT)CC(AG)TT(TC)TC-3'

I

Endo Lys C Endo Glu C Endoproteinase Lys C Endoproteinase Glu C -i I In particular, primer 2 (sense primer) was derived from common peptide sequences which represented the amino-terminal regions of both and PM95 peptides 90P081, 90P210, 95P406, 90P201 and 90P110). This sense primer was used in conjunction with anti-sense primers (primers 7 and 10) derived from internal peptide amino acid sequences (95P311a, 95P214, 95P215 and 90P41, 90P101 respectively).

Preparation of cDNA from L. cuprina first instar larvae.

Total RNA was isolated from 2 g of L. cuprina first instar larvae using a modification of the methods of Chirgwin et al. (Biochemistry 18, 5294-5299, 1979) and Sambrook et al. (supra). The fresh larvae were ground in 35 ml of 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5 n-lauroyl sarcosine and 0.1 M 2-mercaptoethanol in a Dounce homogeniser at 650C. Phenol (35 ml), equilibrated with TS (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), was added, followed by 17.5 ml of 0.1 M CH 3 COONa, 10 mM Tris-HCl, 1 mM EDTA, pH 4.8. 35 ml of chloroform:iso-amyl alcohol (24:1) was then added and the solution shaken vigorously at 65°C for 15 min, cooled on ice and then centrifuged for 15 min at 3600xg The aqueous phase was recovered and extracted twice with phenol:chloroform:iso-amyl alcohol (25:24:1) and then extracted once more with chloroform:iso-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 LiCl 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 al. (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 pg 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 gtl 1 library.

Construction of L. cuprina first instar cDNA lambda gtl I library.

A cDNA library was constr',e, i d from 250 ng of L. cuprina first instar ii 1: i r 21 cDNA in the bacteriophage lambda insertion vecior, lambda gtll, using a Riboclone EcoRI Adaptor Ligation System and a Packagene Lambda DNA Packaging System (both from Promega). The primary library contained 270,000 recombinant pfu and was subsequently amplified for storage on E.

colt Y1090 plating cells.

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

A PM95-specific double-stranded DNA probe was prepared using the polymerase chain reaction (PCR). The procedure was based on that 10 described by Saiki et al. (supra) and used a recombinant form of Taq DNA polymerase obtained from Perkin Elmer Cetus (Amplitaq). PCR was .o performed on cDNA that had been purified as described in section above.

oThe reaction mixture contained 5 ng of cDNA, 500 ng of each of the sense and anti-sense oligonucleotide primers listed in section 2 mM of each dNTP, 4 mM MgC12, 2.5 U of Taq DNA polymerase and in 100 1 of buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1). Each reaction was overlain with 100 of mineral oil. Amplification was performed for 35 cycles in a Hybaid Omnigene thermal cycler using the following conditions: 1 cycle of 5 min at 95°C, 1 min at 50 0 C and 5 min at 72°C; 33 cycles of 1 min at 95°C, 1 min at 500C and 5 min at 72°C; and 1 cycle of 1 min at 950C, 1 min at and 10 min at 720C. Appropriate controls, as described by Saiki et al.

(supra), were also included.

Samples of the PCR reaction (10 were analysed on 0.8 agarose gels at the end of the reactions. The results of this analysis are shown m Figure 5: lanes 1 and 6, DNA fragment standards; lane 2, DNA fragment amplified using primers 2 and 5; lane 3, DNA fragment amplified using primers 2 and 6; lane 4, DNA fragment amplified using primers 2 and 7; and, lane 5, DNA fragment amplified using primers 2 and 10. Single bands were visualised for oligonucleotide primer combinations 2/5 (574 bp), 2/6 (574 bp), 2/7 (595 bp) and 2/10 (668 bp). Anti-sense primers 5 and 6 were nearly identical except for the differing nucleotides coding for a serine position in the peptide. The combination of sense primer 2 with either of the anti-sense primers 5 and 6 yielded a single DNA product of 574 bp. Primer 7 (anti-sense) was designed from a different portion of the same peptide sequences (95P311a, 95P214, 95P215) from that was used to design primers be used as probes for the PM95 gene in genomic or CUNA lDrares ui 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 21 cDNA in the bacteriophage lambda insertion vector, lambda gtll, using a Riboclone EcoRI Adaptor Ligation System and a Packagene Lambda DNA Packaging System (both from Promega). The primary library contained 270,000 recombinant pfu and was subsequently amplified for storage on E.

colt Y1090 plating cells.

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

A PM95-specific double-stranded DNA probe was prepared using the polymerase chain reaction (PCR). The procedure was based on that 10 described by Saiki et al. (supra) and used a recombinant form of Taq DNA fo. 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 5 ng of cDNA, 500 ng of each of the sense and anti-sense oligonucleotide primers listed in section 2 mM of each dNTP, 4 mM MgC1 2 2.5 U of Taq DNA polymerase and in 100 p, of buffer (10 mM Tris-HC1, pH 8.3, 50 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 5 min at 95°C, 1 min at 50*C and 5 min at 72 0 C; 33 cycles of 1 min at 95°C, 1 20 min at 50 0 C and 5 min at 72 0 C; and 1 cycle of 1 min at 95 0 C, 1 min at 50 0 C and 10 min at 72°C. Appropriate controls, as described by Saiki et ai.

(supra), were also included.

Samples of the PCR reaction (10 ld) were analysed on 0.8 agarose gels at the end of the reactions. The results of this analysis are shown in Figure 5: lanes 1 and 6, DNA fragment standards; lane 2, DNA fragment amplified using primers 2 and 5; lane 3, DNA fragment amplified using primers 2 and 6; lane 4, DNA fragment amplified using primers 2 and 7; and, lane 5, DNA fragment amplified using primers 2 and 10. Single bands were visualised for oligonucleotide primer combinations 2/5 (574 bp), 2/6 (574 bp), 2/7 (595 bp) and 2/10 (668 bp). Anti-sense primers 5 and 6 were nearly identical except for the differing nucleotides coding for a serine position in the peptide. The combination of sense primer 2 with either of the anti-sense primers 5 and 6 yielded a single DNA product of 574 bp. Primer 7 (anti-sense) was designed from a different portion of the same peptide sequences (95P311a, 95P214, 95P215) from that was used to design primers 22 and 6 in order to produce a slightly longer product. Indeed, combination of primers 2 and 7 yielded a 595 bp DNA product which is slightly longer than that produced by the primer 2/5 and primer 2/6 combinations.

Primer 2 and primer 10 yielded a 660 bp DNA product. Tlhe DNA products produced using primer 2 as the sense primer and primers 5, 6, 7 and 10 as anti-sense primers should be related.

The relationship between the DNA products referred to in the previous paragraph was proven by the presence of a common EcoR-V restriction site 400 bp from one end of all of these DNA products (see Fig. Figure 6(a) t 10 depicts gel analysis of DNA fragments amplified by PCR using a single sense primer (primer 2) and a number of different anti-sense primers and then digested with EcoRV: lane 1, primers 2 and 5; lane 2, primers 2 and 5 and digested with EcoRV; lane 3, primers 2 and 6; lane 4, primers 2 and 6 and digested with EcoRV; lane 5, primers 2 and 7; lane 6, primers 2 and 7 and digested with EcoRV; lane 7, primers 2 and 10; lane 8, primers 2 and and digested with EcoRV; lane 9, DNA fragment standards. The arrow indicates the common restriction fragment. Figure 6(b) is a restriction map of the fragments amplified using PCR with primer 2 in combination with primers 5, 6, 7 or 10. The common EcoRV site is located 390 bp from the 20 end of all fragments indicating that they are related.

,"The DNA product generated using oligonucleotide primer combination 2/7 was purified using Magic PCR Preps (Promega). A tenth volume of the purified 595 bp DNA was ligated to 100 ng of pGEM7Zf(+) DNA linearized with Smal in 30 mM Tris-HCl, pH 7.8, 10 mM MgC12, 10 mM DTT, 1 mM ATP and 2.5 units of T4 DNA ligase. A tenth volume of the ligation was transformed into 50 /l of E. colt XL1-blue competent cells, and plated on LB agar plates in the presence of 480 u/g of IPTG and 1 mg of X-gal. White colonies were screened for the presence of the appropriate insert by PCR utsing oligonucleotide primers 2 and 7 and conditions detailed above for the isolation of the original DNA product. Colonies representing the correctly sized DNA fragment were cultured into 5 ml of LB media containing /ug/ml ampicillin. Plasirid DNA was isolated by the alkaline lysis method (Sambrook et al., supra). A 0.3 volume sample of each plasmid insert was sequenced (both strands) using a Sequenase version 2.0 DNA sequencing kit (United States Biochemicals) in the presence of either SP6 or T7 promotor probable similarity) and/or; structural homologues which contain one or more of a polypeptide domain consensus sequence present five times in PM95 i.e.

I

23 pimers.

The nucleotide sequence of the DNA fragment is shown in Figure 8 in which double underlining denotes identification of peptide amino acid sequences, single underlining shows the position of oligonucleotide primers used to isolate the fragment by PCR, and broken lines show the extension of peptide amino acid sequences into regions which were not used to design the oligonucleotide primers.

The DNA fragment has a single open reading frame of 595 bp with no indication of the 5' or 3' ends of the coding region (lack of an initiating S 10 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 198 amino acids extended the regions used for the design of the oligonuleotide primers into regions which agreed with the determined peptide amino acid :io° sequences. The translated amLno acid sequence contained the sequences of 10 of the peptides derived from native PM90 and PM95 (90P081, 90P210, 95P406, 90P201, 90P110 and 95P311a, 95P214, 95P215, 95P213b, 90206).

Many of these peptides are either identical or overlapping. The location of these peptide amino acid sequences in the deduced amino acid sequence indicated that this DNA corresponds to a fragment of the gene coding for PM95. The most striking feature of the deduced amino sequence was the S* abundance of cysteine residues (17 cysteine residues in 198 amino acids).

Cloning of a full-length PM95 cDNA.

A L. cuprina first instar cDNA lambda gtl 1 library was screened using the 595 bp DNA fragment amplified fror L. cuprina first instar cDNA using oligonucleotide primers 2 and 7 (see section above). The DNA probe was labelled using PCR in the presence of DIG-11-dUTP (Boehringer Mannheim) and primers 2 and 7, 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. iybridisations were performed at 42 0 C for 16 h in 50% formamlde, 5 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M tri-sodium citrate, pH 0.1% n-lauroyl sarcosine, 0.2% sodium dodecyl sulphate (SDS) and 2% .blocking reagent (Boehringer Mannheim) after a prehybridisation step of 4 h. Filters were washed in 2 x SSC, 0.1% SDS for 24 min at room temperature and in 1 x SSC, 0.1% SDS for 15 min at 58 0

C.

7 positive plaques were detected after 2 rounds of screening, using anti-digoxigenin Fab fragment conjugated to alkaline phosphatase (Boehringer 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 lambdaPM95.11 using LambdaSorb phage adsorbant (Promega). Phage DNA (0.17 volume) was incubated with the restriction enzyme EcoRI in the presence of 90 mM Tris-HCl, pH 7.5, 10 mM MgC12 and mM NaCI to release the DNA insert. This restriction digest was S 10 incubated with 100 ng of pGEM7Zf(+) DNA linearized with EcoRI and extracted with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1). The ligation was performed in the presence of 30 mM Tris-HCl, pH 7.8, 10 mM MgC12, 10mM DTT, 1 mM ATP and 2.5 units of T4 DNA ligase. A tenth volume of the ligation was transformed into 50 ul of E. coli XL1-blue competent cells and plated on LB agar plates in the presence of 480 pg of IPTG and 1 mg of X-gal. White colonies were cultured into 5 ml of LB media containing 50 ug/ml ampicillin. Plasmid DNA was isolated by the alkaline lysis method (Sambrook et al., supra). A three tenths volume of one example of plasmid pM95.11 was partially sequenced using a Sequenase 20 version 2.0 DNA sequencing kit (United States Biochemicals) in the presence G f of either SP6 or T7 promotor primers to verify the identity of the insert.

Uni-directional deletions were generated for both strands of pM95.11 using an Erase-a-Base system (Promega) and the insert was completely sequenced on both strands using a Sequenase version 2.0 DNA sequencing kit (United States Biochemicals). The insert from pM95.11 was 1616 bp in length (adaptors removed).

Sequencing of cDNA coding for The sequenced DNA contained a single open reading frame of 1440 nucleotides which coded for a polypeptide 481 amino acids in length see Figure 8 in which the numbers at the end of each line refer to nucleotides (upper) and amino acids (lower). All peptides from PM90 and PM95 were located within this deduced amino acid sequence and are shown by single underlining. The 3 untranslated region contains a consensus signal sequence for polyadenylation (AATAAA; Proudfoot and Brownlee; Nature 263, 211-214, 1976) which is shown by double underlining but there is no magnification of the photographs was For immunogold localizations, second instar larvae of L. cup, ina were indication of a poly-A tail.

The deduced amino acid sequence of PM95 contains an amino-terminal signal sequence (Briggs and Gierasch; Advances in Protein Chemistry 38, 109-180, 1986) of 24 amino acids in length, italicised in Figure 8, with the mature peptide being 457 amino acids in length. The cleavage site for the removal of the signal sequence to generate the mature protein was directly verified from the amino-terminal amino acid sequence of both PM90 and PM95. The mature protein has a calculated molecular weight of 50,100 Da. The amino acid sequence contains 30 cysteines, S 10 shown in bold type in the figure, which are arranged in 5 domains each containing 6 cysteines. There is only limited sequence homology between these domains, the characteristic feature of which is the spacing of the 6 cysteine residues. The latter are probably involved in extensive intramolecular disulphide bonding (Thornton; Journal of Molecular Biology 151, 261-287, 1981). There are 4 potential N-linked glycosylation sites where X is not proline; Gavel and von Heijne; Protein Engineering 3, 433-442, 1990) present within the coding sequence. These sites are boxed in Figure 8. However, the potential N-linked glycosylation site located 3 residues from the amino-terminus of the protein is not glycosylated as 20 peptide amino acid sequences from this region did not show a characteristic blank cycle for the relevant asparagine during amino acid sequencing which would be expected if this site was glycosylated (Williams et al., supra).

The protein reacts with biotin-labelled wheat germ lectin, directly confirming the presence of glycos-ylation (Fig. Samples analysed in Figure 9 were as follows: molecular weight standards in lane 1 and 3 jig of purified PM95 in lane 2; and, blue molecular weight sta-dards in lane 1 and 3 ,g of purified PM95 in lane 2.

Adjacent to the 5 cysteine-rich domains is a hydrophilic proline- and threonine-rich carboxy-terminal domain of 140 amino acids containing several repeated structures. Notably, there are no cysteine residues in this domain thereby further emphasising the unique nature of this domain in The presence of the proline and threonine residues in this domain suggests that it may be extensively involved in O-linked glycosylation (Williams et al., supra). This possibility, as well as the prevalence of proline residues in the domain, probably accounts for the difference in molecular injection, the effect of vaccin tion was assessea Dy in uLau i*vmu 6vwLA assay (Eisemann et al.; International Journal for Parasitology 20, 299-305.

1990). The in vitro assay consisted of allowing first instar larvae to feed on 26 weight between the native form of PM95 (Mr=95,000) and the calculated molecular weight of the mature protein (50,100 Da) derived from the deduced amino acid sequence. The genomic sequence of PM95 is only slightly larger than the cDNA sequence, this difference being due to a small intron in the former. Further, the correspondence between the sizes of the cDNA and genomic sequences coding for PM95 indicates that the difference between the size of the native protein and that calculated from the amino acid sequence of the mature protein is real and probably attributable to the factors listed above. Finally, a 95 kDa recombinant PM95 protein has been 10 expressed in a baculovirus/insect cell system directly verifying the aberrant S' Mr of PM95 measured by SDS-PAGE.

Comparison of the nucleotide sequence of the PCR fragment with the equivalent region in the cDNA sequence reveals a few minor sequence differences. Excluding the regions used for design of the oligonucleotide 15 primers (which were intentionally degenerate), there are 12 nucleotide e* Sdifferences which translate into 11 amino acid changes out of a total of 198 amino acids. Many of these amino acid changes are conservative substitutions which may reflect allelic variations. It is unlikely that these minor differences would significantly effect the immunogenicity of the protein when injected into sheep.

SExample Expression of recombinant PM95 in bacteria There are many bacterial expression systems which will allow the production of recombinant PM95 which can be produced in the cytoplasm or in the periplasm of bacteria depending on the particular expression system employed. Further, recombinant PM95 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 recombinant PM95 in bacteria.

Production of plasmid vector for expression of recombinant The plasmid, pQE1O (DIAGEN) was employed to express recombinant in the cytoplasm of E. coll. A sense oligonucleotide primer (Bex.for; was designed from DNA sequence coding for the amino-terminal region of the mature PM95 polypeptide. This primer also incorporated a BamHI restriction site at the 5' end. A reverse-sense oligonucleotide primer (Bact.rev; AATTAGTGCTGCTTGACTGAAG-3') was designed from within the 3' non-coding region of the PM95 cDNA sequence. A HLndIII site was incorporated into the 5' end of the latter primer. Five of the plasmid pM95.11 was digested with EcoRI and subjected to electrophoresis on low melting point agarose (NuSieve GTG FMC Bioproducts) containing pg of ethidium bromide. The DNA insert was excised from the gel and added to 100 ul of TE buffer. PCR was performed using 10 p, of the DNA insert (melted at 75°C for 3 min) and the Bex.for and Bact.rev primers in the presence of 100 pl of 10 mM Tris-HCl, pH 8.3, 50 mM KC1 containing mM of each dNTP, 4 mM MgC1, and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer Cetus). Amplification was performed for 35 cycles in a Hybaid Omnigene thermal cycler under the following conditions: 1 cycle of 2 min 0 at 95°C, 1 min at 50 0 C and 2 min at 72°C; 33 cycles of 1 min at 950C, 1 min at 50 0 C and 2 min at 72°C; 1 cycle of 1 min at 950C, 1 min at 50 0

C

and 10 min at 720C. The 1,413 bp DNA fragment generated by this procedure was purified through a Magic PCR Preps column (Promega) into HO and was subsequently ligated into pGEM-T vector (Promega). The resulting plasmid was transformed into E. colt XL-1 strain (Stratagene) and the sequence of the DNA insert verified. Five pg of this plasmid was digested with BamHI (Promega) and HindIII (Promega) to liberate the insert which was subjected to electrophoresis on a 1.5% low melting point agarose gel containing 10 pg of ethidium bromide. The DNA insert was excised from the gel and purified through a Magic PCR Preps column into HO and ligated into the pQE-10 vector digested with BamHI and HindIII. The resulting plasmid (pQEPM95#1) was transformed into E. colt XL-1 strain and characterised by restriction enzyme analysis.

Production of recombinant PM95 protein.

Ten ng of pQEPM95#1 was transformed into E. colt M15 cells (DIAGEN) for the production of recombinant protein. Three colonies were examined for protein expression (all three colonies gave identical expression of recombinant PM95). Colonies were picked into 1.5 ml of LB media containing 100 pg/ml ampicillin and 25 pg/ml kanamycin and grown at 370C with vigorous shaking overnight. A 500 pl portion of each overnight culture was then sub-cultured into 1.5 ml of fresh, warmed LB media containing 100 pg/ml ampicillin and 25 pg/ml kanamycin and grown at I i- 28 37°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°C for 5 h. One ml of each culture was centrifuged for 1 min to pellet the cells which were resuspended in 40 ul of PBS. An equal volume of 2 x SDS-PAGE sample buffer (reducing; Laemmli; Nature 227, 680-685, 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. Two 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 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°C with a 1/1000 dilution of sheep anti-native serum in TBS containing 0.1 Tween-20 (TBS-Tween). The nitrocellulose was washed in the same buffer (3 x 10 min) and 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 2 in TBS to detect the presence of recombinant A major immuno-reactive band of Mr=30,000 was visualized as well as a less abundant band of Mr=50,000. These immuno-reactive proteins were not present in the un-induced cells. The minor band (Mr=50,000) was the correct size for full length recombinant PM95. The more abundant, but smaller recombinant PM95 proteins (Mr" 30,000) may be the result of premature termination during synthesis or proteolysis. The corresponding Coomassie Blue-stained gel did not show any obvious over-expression of Large scale bacterial cultures expressing this recombinant protein were prepared in the following manner. Colonies of E. colt M15 cells containing the expression plasmid pQEPM95#1 were picked into 4 x 5 ml aliquots of LB media containing 100 pg/ml ampicillin and 25 pg/ml kanamycin and grown at 37 0 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 pg/ml kanamycin and grown at 37°C for 2 h. The cultures were induced as described above and grown at 37°C for 5 h. The cultures were then centrifuged for 10 min at 4,200xg i; 1, i i i also present on the peritrophic membrane from freshly dissected larvae.

Identical results were obtained with anti-serum to PM95. The magnification in Figure 2 is 45,000 fold.

29 (4 0 C) and the resulting pellets stored at -20°C until required. The immuno-reactive protein remained with the cell pellet after freeze/thawing and subsequent sonication indicating that the recombinant protein was probably in the form of an insoluble bacterial inclusion body.

Purification of recombinant One of the features of the pQE10O expression plasmid is the addition of a small amino acid sequence containing 6 histidine residues at the amino-terminus 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 wich are used to solubilize recombinant proteins from inclusion bodies. Cells from one litre of E. colt M15 transformed with plasmid pQEPM95# 1 and induced with IPTG were pelleted and washed with TBBS (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.8) containing 1 mM benzamidine, 10 UM AEFSF and 12 mM 2-mercaptoethanol. The cell pellet was resuspended in 30 ml of the same buffer but containing 100 mg 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 20 4°C and centrifuged (12,000xg, 4 0 C, 25 min). The resulting pellet was benzamidine, 10 M AEFSF and 10 pg/ml DNasel. The solution was incubated for 30 min at 37 0 C (with stirring) and centrifuged (12,000xg, 25 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 HC1, 5 mM 2-mercaptoethanol, 0.1 M NaHEPO 4 and 10 mM Tris-HC1, pH for 60 min at 25 0 C (with stirring). The solution was centrifuged as before and the supernatant collected for addition to a NNTA affinity resin.

The recombinant hexa-his-PM95 was purified using this affinity resin essentially according to the manufacturer's instructions (QIAGEN Inc, USA).

The recombinant hexa-his-PM95 was eluted from the affinity column with a linear gradient (0-250 mM) of imidazole.

Figure 10 shows an SDS-PAGE gel stained with silver and also the corresponding immuno-blot of the affinity-purified recombinant I; .'icuinbde.Clsfo n ir fE oiM5tasomdwt 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% trifluoroacetic acid I I i so (the latter probed with an anti-serum to native Samples analysed by SDS-PAGE were as follows: molecular weight standards (lane 11 ug of purified hexa-his-PM95 (lane 5.5 1 g of purified hexa-his-PM95 (lane and coloured molecular weight standards (lane 5.5 pg of purified hexa-his-PM95 (lane There was good correspondence between the proteins in the silver-stained gel and those shown in the immuno-blot indicating that the recombinant was pure.

Refolding of recombinant The presence of glycosylation and probably extensive disulphide bonding in the native PM95 protein are not conducive to expression of a correctly folded recombinant hexa-his-PM95 in the cytoplasm of bacteria.

t: to This is a result of the inability of bacteria to glycosylate recombinant proteins and also the general inability of the bacterial cytoplasm to form S 15 correctly linked disulphide bonds in recombinant proteins. Consistent with Sthis, recombinant hexa-his-PM95 was produced as an insoluble inclusion body within E. coll. Therefore, it is desirable to chemically refold recombinant hexa-his-PM95 such that disulphide bonding occurs in a manner similar to, or identical with, that present in native .t 20 Purified recombinant hexa-his-PM95 (containing the 30 kDa and kDa recombinant proteins) was refolded in the following manner. The affinity purified recombinant hexa-his-PM95 was diluted into 20 mM Tris-HC1, 0.1 Tween-20, 0.25 M NaCI, pH 8.5 to a final protein concentration 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°C and for 1 h at 22°C. The refolded protein was then concentrated. The final J yield of refolded recombinant hexa-his-PM95 was 5 mg.

Amino-terminal amino acid sequence of recombinant The amino-terminal amino acid sequences of the purified 30 kDa and kDa recombinant hexa-his PM95 proteins were determined to confirm that the recombinant protein was correctly constructed. Purified recombinant hexa-his-PM95 (10 pg) was subjected to SDS-PAGE under reducing conditions and the protein was then transferred by electroblotting to a ProBlott membrane (Applied Biosystems). The 30 kDa and 50 kDa 31 recombinant proteins were excised and directly sequenced in an Applied Biosystems 471A amino acid sequencer according to standard protL issued by the manufacturer.

The determined amino-terminal sequences for both the 50 kDa and 30 kDa recombinant proteins compared with the sequence expected from the design of the expression vector follow where is the 50 kDa hexa-hissequence, the 30 kDa hexa-his-PM95 sequence, and the expected sequence. The underlined portion of each sequence denotes the coding region for the amino-terminus of mature, native

MRGSHHHHHHTDPGYNVTS

MRGSHHHHHHTDPGYNVTS

MRGSHHHHHHTDPGYNVTS

Both recombinant protein sequences show an exact match to the expected sequence. Since both the 30 kDa and 50 kDa recombinant 15 hexa-his-PM95 proteins have identical amino-terminal sequences, the differences in their sizes must be due to differences at their carboxy- Sterminal ends.

Example 6 Vaccination of sheep with refolded recombinant 20 Vaccination trial.

A vaccination trial in sheep was undertaken to assess the ability of refolded recombinant hexa-his-PM95 to induce an immune response which inhibited the growth and development of larvae of L. cuprina. Four sheep were each vaccinated with a total of 500 pg of refolded recombinant 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 Itg for the first injection and in the neck for the second injection. A control group of 4 sheep was 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 istar larvae of L. cuprina in an in vitro feeding assay (Eisemann et al., supra). Briefly, larvae were allowed to feed on an agar medium containing 75 serum in a small plastic container. A total of 5 of these containers 50 larvae) were 32 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. (supra). The results of this vaccination trial are shown in Table I.

Table I Sheep vaccination trial using refolded recombinant 4#*O 4 0** Le 1 10 Animal No. Group Mean Larval Wt.

(mg) 2803 Control 4.43 0.33 2777 Control 3.96 0.19 2708 Control 4.15 0.43 2711 Control 4.11 0.35 Gp. Mean 4.16 0.20 2714 Hexa-his-PM95 3.95 0.23 (19%) 2800 Hexa-his-PM95 3.03 0.63 (28%) 2731 Hexa-his-PM95 3.01 0.63 (28%) 2737 Hexa-his-PM95 2.56 0.16 (39%) Gp. Mean 3.14 0.58 Data for "Control" group of Table I was obtained from larvae allowed to feed on serum from sheep vaccinated with adjuvant alone. Bracketed numbers in the table show the percentage reduction in larval weight compared with the corresponding pre-vaccination controls. A Students ttest analysis indicated that the difference between the group and control group is significant at p<O.01.

The sera from 3 of the 4 vaccinated sheep reduced larval weights by 30-40 compared with the appropriate pre-vaccination control sera. The serum from the one remaining vaccinated sheep reduced the mean larval weight by 20 This result is highly significant (p<O.01) when a direct comparison is made between the mean larval weights of the refolded recombinant hexa-his-PM95 vaccination group compared to the control group. Thus, even though the majority of the recombinant protein was not full length, it still induced an immune response in sheep which inhibited larval growth. Table II shows the corresponding ELISA titres for these sera.

I, 33 There were strong anti-hexa-his-PM95 titres in the sera from sheep vaccinated with hexa-his-PM95 indicating that a strong immune response in the sheep had been generated.

Table II Serology from the sheep vaccination trial using refolded recombinant 9009 4 4* 0 4990 9t *1 15 Animal No. Group Elisa Titre 2803 Control 256 2777 Control 256 2708 Control 256 2711 Control 256 Gp. Mean 256 2714 Hexa-his-PM95 256,000 2800 Hexa-his-PM95 160,000 2731 Hexa-his-PM95 160,000 2737 Hexa-his-PM95 340,000 Gp. Mean 229,000 87,000 Immuno-fluorescence localization of native PM95 on L. cuprina larval peritrophic membrane using serum from sheep vaccinated with refolded S 20 recombinant Figure 11 shows the indirect immuno-fluorescence localization of native PM95 on L. cuprina larval peritrophic membrane using serum raised to refolded recombinant hexa-his-PM95. Figure 11(a) is a control using a 1:1000 dilution of pre--vaccination serum while Figure 11(b) depicts indirect immuno-fluoresence localisation of PM95 on peritrophic membrane from larvae of L. cuprina using a 1:1000 dilution of anti-serum to refolded recombinant The strong fluorescence was evenly distributed throughout the peritrophic membrane. There was no fluorescence when pre-vaccination or control sera were used. This result, as well as the effect observed in the in vitro feeding assay indicates that refolded recombinant contains epitopes which are identical to the native PM95 protein. Thus, at least part of the recombinant protein probably has been correctly folded.

l L I 34 Example 7 Expression of recombinant PM95 in baculovirus-infected insect cells Production of baculovirus transfer vector.

Two oligonucleotide primers were designed specifically for expression of PM95 in baculovirus-infected insect cells. The sense p, er (Bac.for; GGCCGGATCCGCAATGGGTGTTTCATCTCAAG-3') was based on the first 19 nucleotides in the coding sequence for PM95 and also incorporated a BamHI site and 3 extra nucleotides im.nediately prior tr the initiating ATG. The reverse primer (Bac.rev; 5'-GGCCGGATCCTAATTAGTGCTTGACTGAAG-3') was based on sequence located within the 3' untranslated region of the gene (begining 19 bp from the stop codon) and also incorporated a BamH! site at its 5' end.

9 99 The baculovirus transfer vector pAcYM1 was used for cloning. Five upg of pM95.11 was digested with EcoRI, and electrophoresed on a 1.5 low melting point agarose gel containing 10 pg of ethidium bromide. The insert was excised from the gel and diluted with 100 1 of TE buffer. PCR was performed using 10 /l of the pM95.11 fragment (melted at 75 0 C for 3 min), the Bac.for and Bac.rev oligonucleotide primers (each 1 pM) in the presence 20 of 10 mM Tris-HC1, pH 8.3, 50 mM KCL, 0.5 mM of each dNTP, 4 mM MgCl 2 and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer Cetus). DNA amplification was performed for 35 cycles in a Hybaid Omnigene thermal Scycler under the following conditions: 1 cycle of 2 min at 95 0 C, 1.5 min at and 2 min at 72*C; 33 cycles of 1.5 min at 95 0 C, 1.5 min at 72 0

C;

1 cycle of 1.5 min at 95 0 C and 5 min at 72 0 C. The PCR reaction containing the resulting DNA fragment, was purified using Magic PCR preps (Promega). The insert was digested to completion with BamHI and cloned into pAcYM1 that had been linearized with BamHI and dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim). The resulting plasmid was transformed into E. coll XL-1 strain and examples characterised by restriction enzyme analysis. The DNA inserts from three clones (designated pAcPM95#21, pAcPM95#22 and pAcPM95#26) which were in the correct orientation were used in all subsequent manipulations.

The DNA insert coded for all of PM95 including its amino-terminal signal sequence.

anti-sense primers 5 and 6 yielded a single DNA product of 574 bp. Primer 7 (anti-sense) was designed from a different portion of the same peptide sequences (95P31 la, 95P214, 95P215) from that was used to design primers 5 -sqec 9 Expression of recombinant PM95 in insect cell culture.

Plasmids pAcPM95#21, pAcPM95#22 and pAcPM95#26 (1 ug each) were each co-transfected with 100 ng of linearized AcPAK6 baculovirus DNA in the presence of an equal volume of 66% lipofectin (Gibco-BRL), onto x 106 Spodoptera frugiperda Sf9 cells in TC-100/10% foetal calf serum (FCS; both from Gibco-BRL) and incubated at 28°C under humid conditions for four days. Recombinant baculovirus was plaque-purified from these incubations in the presence of 5-bromo-4-chloro-3-indolyl-B-galactoside (X-gal; 100 1 g/ml) (Promega). Fifteen recombinant plaques (five for each construct) were picked. Each plaque was transferred into 500 ,ul which TC-100/10% FCS to make a viral suspension from 50 ul was t inoculated onto 5 x 10 4 Sf9 cells in two 96-well plates and incubated under humid conditions at 28 0 C. One plate was used for the amplification of virus (incubated for six days), the other for the determination of the presence of PM95 DNA within the recombinant baculoviruses (incubated for three days).

The latter was achieved by dot blot hybridisation of the -suspended lysed cells from t.i plate to a positively charged nylon membrane (Boehringer Mannheim) using digoxigenin-labelled pM95.11 DNA insert as the probe.

Hybridisations were performed at 37 0 C for 16 h in 50% formamide, 5 x SSC S 20 1 x SSC is 0.15 M NaCI, 0.015 M tri-sodium citrate, pH 0.1% n-lauroyl sarcosine, 0.02 SDS and 2 blocking reagent (Boehringer Mannheim) after a prehybridisation step of 2 h. Filters were washed in 2 x SSC, 0.1% SDS for 5 min at room temperature and in 1 x SSC, 0.1% SDS for 5 min at 60°C. Recombinant baculovirus containing PM95 DNA was detected using an anti-digoxigenin Fab fragment conjugated to alkaline phosphatase (Boehringer Mannheim), naphthol-AS-phosphate (Sigma) and Fast Violet stain (ICN Biochemicals) (West et al., supra).

Small scale production of recombinant PM95 was initiated for 12 i positive plaques (three for each clone) from the remaining 96-well plate.

Viral suspensions were transferred to a fresh 96-well plate. Ten pl of each viral suspension was added to 1 x 106 Sf9 cells in TC-100 media/10% FCS in a 24-well plate and incubated for 6 days at 28°C under humid conditions. A 100 pl portion of each resulting viral suspension was added to 1.5 x 106 Sf9 cells in TC-100 media/10% FCS in a 35 mm culture dish and incubated at 28 0 C for 2 days under humid conditions. The media was i Pg/I II MtU Plll I L. I .o I (Sambrook et al., supra). A 0.3 volume sample of each plasmid insert was sequenced (both strands) using a Sequenase version 2.0 DNA sequencing kit (United States Biochemicals) in the presence of either SP6 or T7 promotor 36 then removed from each dish and the cell monolayer washed twice with PBS. Each monolayer was resuspended in 1 ml of PBS and centrifuged at 12,OOOxg for 30 s. Each pellet was resuspended in 100 Il of PBS to which was added 100 pl of 2 x SDS-PAGE sample buffer. After heat denaturation, 20 dp of each sample was run on duplicate 10 SDS-PAGE gels. Controls consisting of only AcPAK6 in Sf9 cells and Sf9 cells were also run. One gel was stained with Coomassie Blue, the other was electroblotted onto a ,iitrocellulose membrane for an immuno-blot. The gel stained with Coomassie Blue did not show any obvious indication of the over-production of recombinant The immuno-blot (Fig. 12) was processed as described for the detection of recombinant hex-his-PM95 (see Example The following samples were analysed: lanes 1 and 13, coloured molecular weight standards; lane 2, construct #21.2 after 24 h post-infection; lane 3, 15 construct #21.3 after 30 h post-infection; lane 4, construct #21.2 after 48 h o infection; lane 5, construct #22.1 after 24 h post-infection; lane 6, construct S. #22.1 after 30 h post-infection; lane 7, construct #22.1 after 48 h postinfection; lane 8, construct #26.1 after 24 h post-infection; lane 9, construct #26.1 after 30 h post-infection; lane 10, construct #26.1 after 48 h post- 20 infection; lane 11, AcPAK6 vector alone after 48 h post-infection; lane 12, Sf9 insect cells alone. The arrow indicates the presence of full-length recombinant PM95 in lane 4.

An immuno-reactive band of Mi=95,000, as well as three lower molecular weight bands (Mr=30,000-37,000), were visualised for all of the recombinant clones (see Fig. 12). The production of a 95 kDa recombinant suggests that this recombinant protein is full length, appropriately glycosylated and folded in a manner similar to native PM95. The other lower molecular weight immuno-reactive bands represent truncated forms of recombinant Recombinant PM95 was purified from the baculovirus-infected insect cells by immuno-affinity chromatography of the cult, supernatant on an ovine anti-hexahis-PM95 antibody affinity column. The latter was 6constructed from affinity-purified antibodies to purified Equally efficient for the purification of full length recombinant PM95 from the insect cell culture supernatant are lentil lectin affinity chromatography 37 or a combination ion exchange and gel permeation chromatographies.

Figure 13 shows a silver-stained SDS-PAGE analysis of the purified full length recombinant PM95 protein (bvPM95). Lane 1 contains molecular weight standards while lane 2 shows purified bvPM95. The purified was then used for analysis of its amino-terminal sequence.

Direct analysis of the amino-terminal sequence of bvPM95 revealed that the signal sequence amino acids 1-24) had been removed as is expected for a secreted protein, i.e. the amino acid sequence of began at amino 25. Consequently, the amino-termini of native PM95 and the full length recombinant PM95 protein produced hi insect cells were j identical.

ci'i Example 8 2 Vaccination trial with purified recombinant PM95 expressed in baculovirus-infected insect cells A vaccination trial in sheep was performed with purified recombinant expressed in baculovirus-infected insect cells (bvPM95). An in vitro feeding bioassay was used to assess the ability of this antigen to induce anti-larval effects in the sera from the vaccinated sheep. The design of the experiment was identical to that described in Example 6. The result of the 20 in vitro feeding bioassay and the corresponding analysis of serum antibody titres are shown in Tables 3 and 4, respectively. The sera from all sheep vaccinated with this antigen significantly reduced larval weights by a mean of 22.5% compared with the control group (which received adjuvant alone).

Table 4 demonstrates that all sheep vaccinated with bvPM95 induced antibody responses to native Table III Vaccination trial with purified recombinant PM95 expressed in baculovirus-infected insect cells Animal No. Group Mean Larval Wt' (mg 2871 Control 3.94 .69 2874 Control 3.80 0.23 2875 Control 4.01 0.66 2891 Control 3.73 0.59 2876 2887 2888 2889 Gp Mean bvPM95 2 bvPM95 bvPM95 bvPM95 Gp Mean 3.87 3.50 3.02 2.80 2.70 3.00 0.13 0.35 0.24 0.37 0.63 0.36 0 ff 0 eee 4 *4*4 .4.

4 I 4 44 i ft 9 ae 4 rt c «i ~ii ii Ii '4 i Measured by an in vitro feeding bioassay.

2 bvPM95 is full length recombinant PM95 expressed in baculovirus-infected insect cells.

Table IV Serology for vaccination trial with purified recombinant expressed in baculovirus-infected insect cells Aniri.al No. Group ELISA Antibody Titre' 2871 Control <250 2874 Control <250 2875 Control <250 2891 Control <250 2876 bvPM95 1,600,000 2887 bvPM95 2,000,000 2888 bvPM95 2,500,000 2889 bvPM95 32,800,000 Measured against native Example 9 Sera from sheep vaccinated with Lucilia cuprina PM95 inhibit the growth of larvae of Chrysomya rfifacies.

Sheep were vaccinated with purified Lucilla cuprina native PM95 or purified hexahis-PM9,S in a manner identical to that described in Example 6. The sera from these sheep were then used in an in vitro feeding bioassay with larvae of Chrysomya ruflfacies. The results are shown in Table L li IL -i I CY' 39 Table V Sheep vaccination trial using L. cuprina antigens and Chrysomya ruflfacies larvae.

I,

0 0.0* 4r rI 4 04

IIQ

.44441 44 4 Li Animal No. Group Mean Larval Wt' (mg) 1793 Control 2.69 0.25 2257 native PM95 0.83 0.47 (31%) 2737 hexahisPM95 2.03 0.07 Bracketed percentages are the mean percentages of the control weights.

The mean larval weights were measured by an in vitro feeding bioassay.

This experiment demonstrates that the sera from sheep vaccinated with 15 Lucilla cuprina antigens native PM95 and hexahis PM95) inhibited the growth of larvae of Chrysomya ruffacles. Consequently, the vaccine will not only be efficacious against L. cuprina larvae (a primary strike fly) but also against a wide range of species of other flies whose larvae cause cutaneous myiasis in their mammalian hosts.

Example Evidence for an anti-larval effect caused by antibodies to oligosaccharides attached to Antibodies specific to oligosaccharides on PM95 were purified in the following manner. The Zwittergent 3-14 extract from L. cuprina peritrophic membrane (which does not contain any PM95) was reduced, alkylated and digested with endolys-C. The resulting peptides were passed across a wheatgerm lectin-Sepharose affinity column from which affinity-purified small glycopeptides were eluted. The latter were attached to Sepharose 4B and used to affinity-purify antibodies to PM95 oligosaccharides. This was performed by passing total Ig from serum of a sheep vaccinated with native across the glyco-peptide affinity column. The bound antibodies represented those specific to oligosaccharides attached to PM95. These antibodies were then reconstituted back into control serum and tested in an in vitro feeding bioassay. That bioassay indicated a specific reduction in larval weight of 30 2% compared to an appropriate control group. This indicates that oligosaccharides on PM95 are at least partially responsible for inducing the anti-larval effects in sheep vaccinated with -11 1-1 1. -1.1 1 c Wr It will be appreciated that many changes can be made to the invention as exemplified above without departing from the broad ambit and scope thereof, which ambit and scope is to be limited only by the appended claims.

a a 6 ao a ts e a ea a 0 CC a e. a t I, t e ff €l i i I rr I i II

Claims (25)

1. An isolated DNA comprising a sequence encoding PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant having the immunogenic properties of said PM95 antigen.

2. An expression vector which includes a DNA sequence encoding PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant having the immunogenic properties of said PM95 antigen, or immunogenic fragment thereof.

3. Expression vector according to claim 2, which vector is PQEPM95#1, as herein defined.

4. Expression vector according to claim 2, which vector is selected from pAcPM95#21, pAcPM95#22 or pAcPM95#26, as herein defined.

5. A host cell transformed with the expression vector of any one of claims 2 to 4.

6. Host cell according to claim 5, wherein said host cell is a prokaryotic or a 3 15 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.

10. A method of producing PM95 antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant having the immunogenic properties of said PM95 antigen, or immunogenic fragment thereof, the method comprising the steps of: introducing into a host cell DNA which includes a DNA sequence encoding said J 25 PM95 antigen, or allele, homologue or variant or immunogenic fragment thereof in conjunction with elements for the expression ofpolypeptide encoded by said DNA; i culturing said host cell under conditions which allow expression of the encoded polypeptide; and isolating the expressed PM95 antigen, or allele, homologue or variant or immunogenic fragment thereof.

11. Method according to claim 10, wherein said host cell is a prokaiyotic or a eukaryotic cell. J 12. Method according to claim 11, wherein said host cell is a bacterial cell. s I 37°C for 2 h. The cultures were induced as described above and grown at 370C for 5 h. The cultures were then centrifuged for 10 min at 4,200xg 42

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 PM95 antigen, or allele, homologue or variant or immunogenic fragment thereof from isolated expressed PM95 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- fl.# affinity purification or lentil lectin affinity purification.

19. A vaccine for the prophylaxis or treatment of blowfly strike in sheep, the vaccine comprising as the expression product of a DNA according to claim 1 j antigen having an amino acid sequence depicted in Figure 8, or an allele, homologue or variant having the immunogenic properties of said PM95 antigen, or immunogenic fragment thereof. Vaccine according to claim 19, which further includes an adjuvant.

21. Vaccine according to claim 19 or claim 20, wherein said PM95 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 imnuunogen is PM44, as herein defined. S24. 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. Method according to claim 24, wherein said blowfly strike is due to a species of blowfly from the genera Lucilia, Calliphora, Chrysomya or Cochlimyia.

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

27. Method according to claim 25, wherein said blowfly strike is due to Chrysomya I I 43 rufifacies.

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 accord;ng to any one of claims 2 to 4, which host cell is substantially as hereinbefore described with reference to Example or Example 7. A method of producing PM95 antigen having an amino acid sequence depicted in Figure 8, which method is substantially as hereinbefore described with reference to Example 5 or Example 7.

31. A vaccine for the prophylaxis or treatment of blowfly strike in sheep, which vaccine is substantially as hereinbefore described with reference to Example 6. DATED this first day of June 1998 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By their Patent Attorneys CULLEN CO. t a r I ABSTRACT This application relates to DNA encoding the PM95 antigen of Lucilla cuprina. Utilisation of the DNA for high level expression of PM95 antigen is also described as well as vaccines comprising the antigen. Vaccines comprising PM95 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 Luclla, Calliphora, Chrysomya and Cochllomyla. o a o o I o o ea *i t .j i