AU639682B2 - Polypeptides, antigens or vaccines protective against babesiosis - Google Patents

Description

o%0968,2 Tils document cunuins the amendments a tn,,c.i u-r Section 83 by the Supervising Examiner on and is correct for prin'ing COMMONWEALTH OF AUSTRALIA The Patents Act 1952 Name of Applicant(s): Address of Applicant(s): Actual Inventor(s): Address for Service: COMMONWEALTH SCIENTIFIC INDUSTRIAL RESEARCH

ORGANIZATION

LIMESTONE AVENUE, CANBERRA, AUSTRALIAN CAPITAL TERRITORY,

AUSTRALIA

KEVIN RICHARD GALE DAVID JOHN WALTISBUHL IAN GORDON WRIGHT G.R. CULLEN COMPANY, Patent Trade Mark Attorneys, Dalgety House, 79 Eagle Street, Brisbane, Qld. 4000, Australia.

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COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: S. a Ge «e

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o POLYPEPTIDES, ANTIGENS OR VACCINES PROTECTIVE AGAINST

BABESIOSIS

The following statement is a full description of the invention including the best method of performing it known to us: THIS INVENTION relates to DNA sequences, recombinant DNA molecules and processes for producing polypeptides or antigens eliciting antibodies protective against Babesia parasitaemia and babesiosis when administered to suitable animals such as Bos taurus cattle. This invention also relates to such polypeptides and antigens as well as vaccines produced from these polypeptides or related polypeptides or antigens which vaccines are highly protective against Babesia infection, or infection with any other parasite species.

More particularly, the invention relates to DNA sequences expressed in appropriate host organisms. The recombinant DNA molecules disclosed herein are characterised by DNA sequences that code for polypeptides which have the immunological activity of a polypeptide of B. bovis or any other species. A particular example of such a polypeptide is designated as antigen W11C5.

Antigen W11C5 (Ag W11C5) is an antigen reactive with the monoclonal antibody W11C5 (MAb W11C5) secreted by hybridoma W11C5, which hybridoma was deposited with the ee S European Collection of Animal cell cultures on 16 December 1988 under accession number 88121501 W11C5.1.A5.F10.A2.

Accordingly, this invention further relates to hybridoma W11C5, the monoclonal antibody secreted by hybridoma 2 W11C5 and antigens specifically reactive with this monoclonal antibody.

However it will be appreciated that the invention includes within its scope any monoclonal antibody derived from antigens related to antigen W11C5 eliciting antibodies ,GER 'protective against babesiosis and that therefore the invention However it will be appreciated that the invention includes within its scope any monoclonal antibody derived from antigens related to antigen W11C5 elicitinj antibodies

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protective against babesiosis and that therefore the invention is not restricted to the monoclonal antibody secreted by hybridoma W11C5. The invention also includes within its scope any hybridoma capable of producing the abovesaid monoclonal antibodies exemplified by hybridoma W11C5 Babesia bovis is the major causative agent of tick fever morbidity in domestic cattle ranged on tropical and subtropical pastures. Early work on the immunity conferred S. on infected animals which survived indicated that the immune response was humoral, since immunity was conferred to calves by colostrum immunoglobulins from infected mothers.

Unresolved antibodies from animals immune to B. bovis were subsequently shown to elicit immunity when transferred to non-immune animals.

Hitherto vaccines have been produced which protect ~cattle against the severe clinical manifestations of B. bovis 0 parasitaemia. The vaccines used to date include those using killed B. bovis, live vaccines using B. bovis attenuated by either irradiation or rapid passage in splenectomised calves and vaccines derived from in vitro culture supernatant antigens. Although such vaccines are to some extent protective, they all have the inherent problem of containing 4 many antigens. Vaccination with such vaccines therefore elicits an immune response which places the immune system of the vaccinated animal under considerable stress. Also, multi-antigenic vaccines may contain antigens which elicit a strong response activity but which are not especially protective due to the location or chemistry of the antigen in the virulent field strains. This strong response may mask or otherwise inhibit the development of immunity based on antibody response to the presence of a less reactive antigen capable in isolation of conferring protective immunity.

Where live attenuated vaccines are used, contamination of the vaccine with other pathogens may occur.

Animals vaccinated with the live vaccines are subclinically infected carriers of the parasite and the potential for breakthrough infection due to de-attenuation is present.

Refrigerated vaccine has a shelf life of only seven days.

In order to identify specific babesial antigens which confer protective immunity, babesial proteins from e infected erythrocytes have been fractionated and assayed for P, immunoprotective potential. Protective antigens have been found in the soluble protein fraction obtained from lysis of babesia-infected erythrocytes as described in Australian patent specification No. 553779. Monoclonal antibodies have 0 Q* been raised against the protective soluble babesia protein fractions, and IFA, ELISA and Western blotting techniques used to identify clones producing monoclonal antibodies against specific babesial antigens. Antigens were then aiinity purified with the monoclonal antibodies to yield single antigens for vaccination testing.

Whilst purified antigen may be produced from babesial lysates by using the monoclonal antibody to that antigen, this method is not suitable for large scale production of vaccination grade product. The requirement therefore arises for improved commercial scale processes for the manufacture of antigen and polypeptides having like immunogenic activity. Attention has therefore been focused o0*S on the use of recombinant DNA techniques to transform nonbabesial hosts with babesial genes.

Kemp et al. (Mol. Biochem. Parasit., 12 (1984) 61- 67) discloses that babesia genes can transform the eukaryotic yeast host Saccharomyces cerevisiae, and that the transformed host produces poly A' RNAs corresponding to the transforming genes. However, there is no data to indicate that the polypeptides corresponding to the poly A- mRNA obtained from K-strain B. bovis-infected erythrocytes were protective. The library was amplified in ~-gt10, cleaved, radiolabelled, fractionated, and re-ligated into an ampicillin-resistanceconferring expression vector \-amp3. Phage were packaged in vitro and plated out on E. coli BTA282 on ampicillincontaining media, and colonies producing cDNA detected by colony hybridisation. Co".onies expressing babesia antigens were detected by autoradiography using bovine antiserum to K strain B. bovis and 25 I-labelled anti-bovine Ig. One clone was selected for further study (designated K Bbl). This clone produced a fusion protein of P-galactosidase and a babesial antigen 5-10 kDa larger than native 3-galactosidase.

Anti-K antisera were fractionated against the fusion protein to yield affinity-purified anti-K Bbl antibodies. These antibodies were used to detect a 220 kDa antigen, correspondina to the dominant 220 kDa antigen detected by unfractionated serum. Immunofluorescent assay indicated that anti-K Bbl reacts only with the Babesia parasite and not with the surrounding erythrocytic cytoplasm in vitro. By contrast, unfractionated anti-K serum reacts with both the infected erythrocyte cytoplasm and the parasite.

However, vaccines formulated from the K Bbl/Bgalactosidase fusion protein did not exhibit significant protective immunisation of animals against virulent heterologous B. bovis Challenge (Timms et al., unpublished results, Animal Research Institute, Queensland Department of Primary Industries). Indeed, the results indicated that the K Bbl/P-galactosidase vaccines were less efficacious than live S. 0 S S K vaccines.

The whole approach adopted by Kemp in regard to location of polypeptides protective against babesiosis however was a "shotgun" approach and was not based on an approach which could be used to provide protective native as well as recombinant polypeptides.

Reference also may be made to Cowman described in Manipulation and Expression of Genes in Eukaryotes (1983) 185-188 wherein a gene coding for avirulence associated polypeptides of B. bovis were isolated by differential colony hybridisation.

Poly A* RNA was isolated from K-avirulent (K-A) strain by extraction from parasitised cattle erythrocytes and oligo-dT cellulose chromatography. The RNA was copied into double-stranded cDNA which was inserted in pBR322.

Transformed cells were plated on nitrocellulose filters and replica filters were hybridised with labelled cDNA probes synthesised from poly(A)'RNA of K-A and the virulent K geographical isolate cDNA plasmids were selected representing RNAs of differing abundance. Increased levels of RNA molecule were found to correlate with avirulence.

*oo* However, the main thrust of this reference was to identify cDNAs corresponding to RNAs which-hybridise in greater abundance with probes synthesised from poly A* mRNA of the avirulent Ka strain. This reference did not disclose however preparation of polypeptides.

*e The present invention in one aspect resolves at 9e least some of the problems associated with the prior art by providing a process which serologically determines a protective antigen and also raising a monoclonal antibody to that antigen. The process also includes cloning DNA sequences that code for polypeptides at least partially homologous with that antigen and using said monoclonal antibody as a probe to isolate a cDNA clone expressing the desired polypeptide. The said process thereby provides DNA sequences, recombinant DNA molecules and methods for use of those sequences and molecules in the production of polypeptides displaying at least some of immunogenic activity of that antigen.

By virtue of this invention, it is possible to obtain polypeptides displaying an immunological activity of the W11C5 antigen, for use in protective immunisation of Bos Staurus against clinical Babesiosis. This recombinant DNA *0 produced polypeptide may also be used for the purpose of immunoassay and immunodiagnosis. This invention allows the production of a novel polypeptide or polypeptides derived, modified or otherwise produced from the novel polypeptide in amounts and by methods hitherto not available.

e00 As will be appreciated from the disclosure to follow, the DNA sequences and recombinant DNA molecules of

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the invention are capable of directing the expression of a polypeptide displaying at least some of the immunological activity of the native W11C5 antigen. Replication of these DIIA sequences and recombinant DNA molecules in appropriate hosts also provides a means of amplifying DNA to yield quantities of DNA coding for the polypeptide in hitherto 9 unobtainable quantities. The molecular structure of these genes may thus be readily determined. The polypeptide and corresponding DNA is useful, either as produced in the host or after appropriate derivatisation or modification, in compositions and methods for detecting and improving the production of these products themselves and for use in immunoprotective immunodiagnostic and immunoassay agents and methods.

The sequences of this invention are further characterised in that they permit the production of W11C5-like polypeptides in non-babesial hosts.

In another aspect of the present invention there is provided antigen W11C5 (Ag W11C5) purified by immunoreactivity with monoclonal antibody W11C5 (MAb W11C5).

The invention also includes within its scope an anti-idiotypic antibody displaying at least some of the immunogenic activity of WilC5 antigen.

In yet another aspect of the present invention there is provided DNA sequences coding for polypeptides having at S least some of the immunoreaotivity of Ag W11C5 wherein the sequences comprise cDNA sequences corresponding to babesial mRNAs having substantial homology which at least part of the B. bovis gene or genes coding for antigen W11C5.

Also within this aspect of the present invention are S. DNA sequences which hybridise to any of the foregoing DNA sequences, DNA sequences from whatever source including natural, synthetic or semisynthetic sources, related by mutations including single or multiple-base substitutions, deletions, insertions, inversions and 3' or 5' additions to any of the foregoing DNA sequences or inserts, and DNA sequences comprising sequences of codons which on expression code for a polypeptide displaying similar immunogenic properties to a polypeptide produced on expression of any of the foregoing DNA sequences.

In another aspect of the present invention are provided compositions comprising any of Ag W11C5, MAb and polypeptides expressed as above together with an appropriate vehicle such as an adjuvant, which compositions are variously useful as vaccines, immunoassay reagents and immunodiagnosis reagents.

The process used to generate the subject hybridoma is described fully hereinafter. However it should be recognised that the hybridoma may have been derived by any Sother protocol known to the art. A broad description .2a encompassing at least some of the available protocols involves

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the steps of: 2 s: preparing nucleic acids from babesia infected erythrocytes depleted of leucocytes; (ii) forming a cDNA or genomic library from nucleic acids obtained in step (iii) screening said library formed in step (ii) with a suitable probe to identify clones of interest; (iv) obtaining DNA from clones of interest identified in step (iii); inserting DNA from step (iv) into an expression vector; (vi) propagating a recombinant expression vector made in step in an appropriate host; and (vii) obtaining a recombinant polypeptide from the host harbouring the recombinant expression vector, wherein the recombinant polypeptide has the immunological activity of the W11C5 antigen and is protective against babesiosis.

The stroma of Babesia-infected erythrocytes may be prepared in any suitable manner. For example, any of the following protocols may be employed osmotic lysis (ii) mechanical disruption through proliferation of parasites 0*0*

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or (iv) cell lysis using lysins or antibodies.

Preferably the cell stroma are prepared by protocol and in particular by any of the methods referred to in Mahoney (1967) Exp. Parasitol. 20, 232-41.

The parasites so prepared may be separated from the cytosol, resuspended and disrupted, preferably by sonication and the sonicate preferably ultracentrifuges to yield a supernatant containing soluble babesial antigens. This supernatant may be harvested to provide the crude mixture of soluble babesial antigens.

Monoclonal antibodies to the soluble babesia antigens prepared above may be prepared by any known method, including that described in Australian Patent number 553779.

Preferably, the crude antigen mixture is administered to a suitable animal, for example, mice such that the animal elicits antibody-producing cells such as B-lymphocytes or splenocytes. The antibody-producing cells may be harvested.

However, since the harvested cells age and die in culture, the cells are generally fusea with immortal cells from the same species such as myeloma cells. This yields hybrid cells which excrete antibodies and are able to be cultured indefinitely.

It is particularly preferred to use BALB/c mice as the source of antibody producing cells, and to fuse the B-lymphocytes or splenocytes harvested therefrom with a mouse myeloma cell line such as P3-NSI-Ag4-1.

After fusion, the hybrid cells may be diluted out in a suitable medium such as RPMI1640 supplemented with IgG depleted foetal calf serum, and the diluted cells placed in

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for example a microtltre plate having a plurality of wells.

After a suitable growth time, the supernatant from each of the wells may be screened to identify wells producing antibody which is preferentially immunoreactive with infected erythrocytes, bireening may be performed by any known procedure including immunofluorescent assay (IFA) or enzymelinked immunosorbent assay (ELISA).

25 The cells in the wells so identified may be recloned any number of times by limiting dilution with screening to yield a hybrid cell line producing a monoclonal antibody having the desired immunoreactivity with infected erythrocytes.

By using the specific protocol described hereinafter, and in accordance with the foregoing, the present applicant derived the subject hybridoma W11C5.

The monoclonal antibody produced by the selected clone is then preferably classified to determine its immunotype. The W11C5 monoclonal antibody proved to be an IgG type immunoglobulin which was extremely stable on storage at The hybrid cell is generally grown up in culture to yield quantities of monoclonal antibody suitable for purification and further uses.

:5 After harvesting the culture supernatant, the antibody may be purified by concentration by ultrafiltration followed by affinity chromatography.

~The purified monoclonal antibody is then available for immunodiagnosis, immunoassay and production of purified antigen as is known in the art.

Antigen may be purified from the crude mixture of soluble babesial antigens obtained from infected erythrocyte stroma by any means known in the art of affinity purification. For example the antigen may be purified by immunoadsorption onto the monoclonal antibody mounted on a o suitable support such as agarose beads, acrylic, celllosic or nitrocellulosic supports. The antibody may be coupled via ligands. The supported antibody may be packed in an affinity chromatography column and equilibrated with a suitable buffer. Optionally the column may also be treated to inactivate any nonspecific binding sites. The crude mixture of soluble babesial protein antigens may be passed through the column to effect binding of the antigen specified by the monoclonal antibody. The bound antigen may be eluted from the column using a suitable elutic. buffer. The eluted antigen may be further purified and/or concentrated such as by dialysis and/or ultrafiltration.

The purified antigen may be used, in conjunction with a suitable adjuvant, as a vaccine, and suitable vaccination trials conducted.

35 In the present circumstances, antibody purified 0 antigen was used to raise bovine antisera for serological analysis of the efficacy of the antigens produced so fermentatively as is described hereinafter. The affinity purified antigen may also be used for immunodiagnosis of unknown sera.

Whilst affinity purified antigen may form an acceptable vaccine, in terms of industrial scale production this means of production is insufficient compared to fermentative processes using recombinant DNA technology.

This inefficiency arises from the need to prepare large quantities of infected erythrocytes to source the crude antigen mixture.

Accordingly it is desirable to identify babesial genes coding for polypeptides having at least some of the immunogenicity of the antigen of interest such that the genes may be cloned and expressed in appropriate hosts.

There exists many ways by which the gene of interest may be identified. For example, the purified antigen may be partially amino acid sequenced, and corresponding DNA probes synthesised to screen a genomic or cDNA library to identify clones containing homologous DNA sequences.

The said DNA sequences through further manipulation may be expressed to give polypeptides exhibiting the desired immunological characteristics of the antigen.

S. Alternatively, the babesial gene may be identified by screening of a cDNA expression library constructed from the reverse transcription of babesial poly A' mRNA using an o; antibody probe to identify clones expressing antigenic gene product. The library may then be used to provide inserts for expression vectors which are in turn used to transform appropriate hosts. The transformed host may be screened for other expression of the gene of interest.

In the process of the invention any suitable expression vector may be utilised. Examples of suitable 25 vectors include bacteriophages such as the derivatives of .bacteriophage X or pasmids such as derivatives of E. coi bacteriophage or plasmids such as derivatives of E. coli 16 plasmids pUR, pUC or p-GEX series). Where the particular polypeptide of interest is most ably expressed. by a eukaryotic host, it may be more appropriate to use the derivatised plasmids of a yeast such as Saccharomyces cerevisae.

In the process of the invention any suitable host for the expression vector may be used. Examples of suitable hosts include yeast cells, mammalian cells, insect cells and bacterial cells. Transformants may be isolated by any appropriate means, for example, growth on selective 'nedia.

The transformed hosts may then be screened, either by detection of the desired expressed polypeptide using immunoassay or by probing with DNA or RNA probes.

In the preferred case where monoclonal antibodies have been raised against the antigen of choice, immunological screening of expressing recombinants is preferred.

The vector/host combination may be chosen such that the expressed polypeptide is just the translation product of the cDNA transcript of the poly A' mRNA. Alternatively, and more easily, the translation of the cDNA insert of the expression vector is placed under the control of a strong promoter for expression of a host protein by insertion of the cDNA (in the correct reading frame and crientation) within the gene coding for that host protein. The result is that 25 the host strongly expresses a fusion protein comprising the 6 o 6 0 106 17 translation product of the cDNA insert coupled to at least part of the host protein.

The selected transformant may be grown up to yield sufficient quantities of expression product having the desired immunogenic properties for use in immunodiagnosis, immunoassay or vaccines.

It will also be appreciated in relation to this invention that one or more "reverse engineering" steps could be utilized in the inventive process wherein in some cases the antigen W11C5 or related antigens may be obtained from sera from vaccinated animals or infected animals using a B.

bovis expression library which may be cDNA or genomic in origin. In this. case the sera would be used as a probe to screen the expression library to isolate W11C5 antigen or 5 related antigens in a similar manner as described above.

In another aspect of the invention it will also be appreciated that the vaccine containing the antigen of interest could also be utilized as a starting material to eventually isolate the W11C5 antigen or related antigen. In this procedure the antigen from the vaccine could be o* sequenced to obtain a partial amino acid sequence and *t oligonucleotide probes constructed from the amino acid sequence could be used to screen a B. bovis cDNA or genomic library to isolate a gene coding for a related antigen. By 25 using recombinant technology one could then prepare the S S antigen in large amounts.

18 The invention also includes within its scope a test kit for diagnosis of habesiosis which includes the monoclonal antibody MAb W11C5 or related monoclonal antibodies as well as selected reagents for detection of babesiosis antigen in body fluids such as blood or lymph. In this test kit any suitable label may be used to detect the antigen-antibody reaction and thus this may be IFA, RIA or more suitably enzyme immunoassay such as ELISA. A method of immunoassay using monoclonal antibody MAb W11C5 is also included within the scope of the invention.

It will also be appreciated that a monoclonal antibody similar or related to MAb W11C5 (ie. not necessarily reactive with the same epitope) may be obtained by using antigen W11C5 or a similar antigen or related polypeptide instead of the SPA antigen (Soluble Protein Antigen) obtained from a lysate of B. bovis infected erythrocytes as described herein.

The invention will be further described hereinafter with reference to the accompanying figures.

too 0 oo oo*o *e *o oo 19 DETAILED EXPERIMENTAL PROTOCOLS 1. Preparation of Babesia bovis Soluble Protein Antigen (SPA) Blood was obtained from a hyperinfected splenectomised calf (10 15% parasitaemia) infected with the (Samford) strain of B. bovis. Erythrocytes were pelleted by centrifugation (3000 rpm, 20 minutes, room temperature) in a Sorvall GSA rotor. Pelleted erythrocytes were resuspended in five volumes of PBS (phosphate buffered saline) pH 7.2 by inversion of the centrifuge bottle. PBS-washed erythrocytes were centrifuged as before and the washing procedure repeated.

To prepare 100% infected cells, washed erythrocytes were pelleted as described above and small samples of the erythrocytes treated with 5 volumes of NaCl solutions ranging S, from 0.15 to 0.85% NaCI to determine the concentration SCOr o* effecting lysis of uninfected cells preferentially to *a infected cells. In this case, the concentration was *6*6 determined as 0.35% NaCl.

The remaining erythrocyte pellet was resuspended in 5 volumes of 0.35% NaCl and after 15 minutes at room *400 temperature the whole (infected) erythrocytes were pelleted as before. The supernatant containing red cell "ghosts" and cell debris was discarded.

25 The infected erythrocyte pellet was resuspended in

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S* one volume of PBS pH 7.2 and the suspension passed through a 0 oe Whatman CF11 cellulose column (pre-equilibrated with PBS pH 7.2) to remove white blood cells and platelets. The highly visible infected erythrocyte band was eluted with 2 3 column volumes of PBS.

The infected erythrocytes were centrifuged as before and the resulting pellet resuspended in PBS pH 7.2 to give a 50% suspension of 100% infected erythrocytes.

Lysis of infected erythrocytes was accomplished by the addition of 5 volumes of distilled water. After minutes at room temperature with mixing, the lysate was centrifuged (10,000 rpm, 30 minutes, 4°C) in a Sorvall GSA rotor and the supernatant containing the erythrocyte cytosol discarded. The pelleted parasites and cell debris were resuspended in one volume of PBS pH 7.2 and the suspension sonicated (100 Watts, 3 5 minutes) to disrupt and solubilise parasites and infected red cell stroma.

The sonicate was centrifuged (105,000 x g, minutes, 20°C) in a Beckman Ti 60 rotor to sediment the cell debris. The supernatant containing the soluble protein babesial antigen (SPA) and soluble red cell material was retained and stored in 10 ml aliquots at 2. Identification of SPA Immunoprotective Fraction To a gel filtration column (BIO-RAD, BIOGEL equilibrated with isotonic saline was added 1 quarter column volume of the crude soluble protein antigen (SPA) fraction prepared as above. The column was run with 5 volumes of saline yielding two visible bands, the first pigmented band corresponding to haemoglobin complexed with babesial and erythrocytic proteins and the second pigmented band corresponding to normal oxyhaemoglobin.

As the first pigmented band eluted from the column it was collected (half column volume) and was concentrated to the original volume by dialysis against polyethylene glycol (CARBOWAX, MW 20,300).

500 microlitre aliquots of this fraction were electrophoresed agarose gel, type C, Pharmacia AB) in pH 8.6, ionic strength 0.1 veronal acetate buffer for approximately 2 hours at 150 volts to separate the soluble proteins. The typical 3 fraction haemoglobin complex band 5 may be visualised in the gel by its pigmentation and this pigmented band was exised from the gel.

oo* Analysis of this gel slice (the P fraction) by immunoelectrophoresis and haemagglutination assay showed that the majority of the soluble babesial antigens migrated with the P fraction haemoglobin complex. It is surmised that the haemoglobin exhibits non specific binding affinity to many ~proteins including the majority of the subject basesial antigens as a consequence of lipid peroxidation.

The excised gel slice was comminuted to a particle 25, size suitable for use directly as a vaccine in a tissue *o o* grinder with one volume of Freund's Complete Adjuvant (FCA).

22 The crude vaccine so derived corresponds to 2 ml of 100% infected red cells (packed volume).

The crude vaccine so produced was used to vaccinate cattle and the cattle so vaccinated were homologously challenged with Samford strain Babesia bovis. The crude vaccine was subsequently found to be protective against heterologous challenge also.

3. Production of Hybridoma W11C5 Balb/C mice were injected subcutaneously with 100 microlitres of the gel slice P fraction described above together with 100 microlitres of FCA each month for four successi!e months and then injected on four successive days *ee with the gel slice P fraction alone. Splenic cells were .1'5 harvested from the immunised mice and were fused with myeloma cells from P3-NSI-AG4-1 mice.

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Hybrid supernatants were screened b immunofluorescent assay (IFA) and positive wells were cloned and recloned twice by limiting dilution. Clones were than stored in the vapour phase of liquid nitrogen.

The clone designated W11C5 was selected by the following criteria.

By IFA the antibodies secreted by the W11C5 clone stained the infected erythrocyte preferentially with only @e minor staining of the parasite and with no staining of uninfected erythrocytes (see Figure This criteria for

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selection was used on the basis that antibody against the infected erythrocyte should be more effective than antibody raised against the parasite itself, since the parasite and its antigenic epitopes are masked by virtue of the fact that the parasite resides within the infected erythrocyte.

Antigen prepared using the monoclonal antibody excreted by hybridoma W11C5 produced a unique ladder of bands by immunoblotting, starting at approximately 50 kilodaltons (KDa) and increasing in approximately 10 KDa increments. In addition, with some antigen batches, it produced 1 to 4 distinct bands between 200 and 300 KDa (see Figure 2).

The antigen was also detected by the monoclonal antibody in a heparin fraction of B. bovis infected S* erythrocytes. This fraction has recently been shown to be T5 protective in vaccination studies (Goodger et al., Int. J.

Oe S Parasitol. 17 (1987) 935).

**a The mc.noclonal antibody was classified as an IgG 1 and has been shown to be extremely stable on storage at- 2: 4. Purification of MAb W11C5 and W11C5 Antigen The W11C5 monoclonal antibody was purified by affinity chromatography utilising a column containingp fraction antigens bound to CNBr-Sepharose 4R (5 mg 8-fraction bound to 1 ml of Sepharose beads). Purified antibody was eluted from the column with 0.2 M glycine-HCl pH 2.8 containing 5% NaCl. The pH was restored to neutrality and .4

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24 the eluate stored at W11C5 monoclonal antibody purified as above milligrams) was coupled to CNBr-Sepharose. 20 ml of oxyhaemoglobin depleted B. bovis antigen (SPA) was reacted with the column which was then washed with PBS until the eluate gave zero absorbance at 280 nanometres.

The column was then washed with 2 column volumes of 0.025 M NaBorate, 0.01 M Boric acid, 0.1% Tween 20, 1 M NaCl to remove non-specifically bound proteins. The bound antigen was then eluted with 0.2 M glycine-HC1 pH 2.8 containing NaCl. The neutralised eluate was tested for the presence of W11C5 antigen by enzyme linked immunosorbent assay (ELISA).

Elution was continued until no antigen was detected in the eluate. The positive eluate fractions were pooled, *5 concentrated back to 20 ml by ultrafiltration and used for analysis and vaccination of Bos taurus animals.

Vaccination Trial Using Antigen Purified by the W11C5 Monoclonal Antibody Four splenectomised calves (free of Babesia spp) were injected twice at 1 month intervals with 2 ml of the e W11C5 antigen obtained above in FCA. Six weeks later the calves and a control group of four splenectomised calves, were challenged with the homologous Samford strain of Babesia 025 bovis.

The vaccinted calves, compaative to the control The vaccinated calves, comparative to the control animals, had significantly lower parasitaemias throughout the challenge. The probability value was at the level of 0.006 significance.

Antisera from the vaccinated animals prior to challenge produced similar IFA and Western blotting patterns to those obtained with the W11C5 MAb. (Infected erythrocytes staining was observed in IFA and a characteristic "ladder" was evident on Western blots of B. bovis SPA).

The W11C5 antigen has also been shown to be present by positive IFA and ELISA results with W11C5 MAb against infected erythrocytes and SPA from strains of B. bovis other than Samford, including Lismore and Ka.

By the same methods, cross-reactive antigen has also been detected in erythrocytes infected with other species of Babesia, namely B. ovis and B. equi. It is surmised that homologous Cntigens may also be present in other parasite species. Accordingly it is surmised that the W11C5 antigen or the corresponding analagous antigens isolated from other parasite species and/or strains may ?N provide protection against infection of animals by other species in addition to B. bovis.

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4 Cloning and Expression in E. coli of W11C5 Antigen 6. Preparation of B. bovis Poly A' RNA a) Purification of total RNA Ten ml of a 50% suspension of 100% infected erythrocytes bovis Samford strain) was prepared as described previously. To this was added 10 ml of guanidinium isothiocyanate stock (GI mix) at (GI mix consists of 100 g of guanidinium isothiocyanate (Merck) dissolved in 100 ml of double distilled water plus 10.6 ml 1 M Tris-HCl pH 7.6, 4.24 ml 0.5 M EDTA pH 8.0, 21.2 ml 20% Nlauryl sarcosine, 2.1 ml B-mercaptoethanol, filtered through 0.22 4m Nalgene nitrocellulose sterilising filter).

The infected erythrocyte/GI mixture was maintained at 60°C with gentle mixing until the solution became highly viscous, indicating lysis of cells and subsequent release to solution of the babesial nucleic acids. The mixture was sonicated (100 W, 1 minute) to reduce viscosity. Twenty-five ml of phenol (pre-equilibrated with 1 M Tris-HCl pH 7.4) was added to the mixture at 60°C with thorough mixing. Twenty-five ml of NaAc buffer (0.1 M NaAc pH 5.2, 10 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0) at ,25c 60°C was then added with thorough mixing. Twenty- Sof chloroform/isoamyl alcohol (24:1) was five ml of chloroform/isoamyl alcohol (24:1) was added and the mixture shaken vigorously for 10 minutes at 60°C. After cooling on ice for minutes, the mixture was centrifuged (15 minutes, 5,500 rpm, 4°C) in a Sorvall GSA rotor and the aqueous (top) phase carefully recovered.

The aqueous phase was re-extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) at 20°C for 5 minutes. After centrifugation (10 minutes, 10,000 rpm, 20°C) in a Sorvall SS34 rotor, the aqueous phase was again recovered. Extraction as above was repeated using an equal volume of chloroform/isoamyl alcohol and S, the aqueous phase recovered.

0*e Two volumes of absolute ethanol at -20°C was i5 added to precipitate the nucleic acids. After overnight incubation at -20°C, precipitated nucleic acids were pelleted (20 minutes, 10,000 rpm, 4°C, SS34 rotor) and redissolved in 12 ml of sterile distilled water. Cesium chloride (4.8 g, *0 Boehringer Mannheim molecular biology grade) was then dissolved in the solution and cleared of insoluble material by centrifugation (10 minutes, 10,000 rpm, 20°C, SS34 rotor).

To a 12 ml polypropylene centrifuge tube was added 4 ml of 5.7 M CsCl and the cleared nucleic

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acid/CsCl solution layered carefully on top. The tube was centrifuged (16 hours, 30,000 rpm, 17°C) in a Kontron TST 41 swing-out rotor to pellet the

RNA.

The RNA pellet was redissolved in 400 4l of sterile water and centrifuged to remove any insoluble material (5 minutes, 14,000 rpm, 4°C, Eppendorf centrifuge). Purified RNA was stored in aliquots at Approximate yield and purity of the RNA solution was determined by measurement of optical absorbance of the solution at 260 and 280 nanometers wavelength (Varian U.V.

•0 ,spectrophotometer). The absorbance at 260 nm is o, used to quantitate the RNA using the approximation: 1: 1 absorbance unit is equivalent to 40 ig/ml RNA concentration. For pure RNA the ratio of absorbance at 260 nm divided by the value at 280 nm The integrity of the purified RNA was checked by agarose gel electrophoresis of a 2 g sample (BRL H6 minigel, 1% agarose, 70 volts, 1 hour, Trisacetate electrophoresis buffer containing mg/litre ethidium bromide). The ribosomal bands appeared intact, thus establishing the integrity of 25 the purified RNA.

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b) Selection of poly A- RNA Stock solutions for selection of poly A' RNA were as follows: Loading buffer: 20 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 500 mM NaC1, 0.1% sodium dodecyl sulphate (SDS).

2 x loading buffer: as above, double concentration.

Elution buffer: 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.05% SDS.

Oligo-dT-cellulose (Bcehringer Mannheim, 0.2 g) was equilibrated with elution buffer (10 ml) and used to form a column. The column was washed with 15 10 volumes (5 ml) 1 x loading buffer, then 3 volumes (1.5 ml) each of sterile water, 0.1 M NaOH/5 mM EDTA and sterile water, followed by ml 1 x loading buffer.

The purified RNA (400 4l as prepared previously, was heated (65°C, 5 minutes), chilled oo* on ice, diluted with 400 4l of 2 x loading buffer and brought to room temperature The RNA was applied to the column followed by an equal volume of loading buffer. The void material was 2V reloaded followed by 4 ml of loading buffer. The bound RNA was eluted using 2 n.l of elution buffer.

This eluate was then made 500 mM with respect to NaCI, heated and ,ooled as before and re-applied to the column whic'.i had been regenerated as per the preceding paragraph. The column was washed with 4 ml of loading buffer and the bound RNA eluted with 2 ml of elution buffer. The eluted RNA (poly A* RNA) was made 200 mM with respect to NaCI and mixed with 2.5 volumes of absolute ethanol at After overnight incubation at -20°C, precipitated poly A' RNA was pelleted (20 minutes, 10,000 rpm, 4"C, Sorvall HB4 swing-out rotor) and the vacuumdried pellet redissolved in 50 .l of sterile distilled water. Purified poly A' RNA was stored in 10 l 1 aliquots at -80°C. Approximate concentration of the RNA was determined by absorbance as described previously.

6 Integrity of the RNA was gauged by agarose gel electrophoresis as described previously.

The poly A' RNA showed good size distribution, with most RNA migrating behind the small ribosomal RNA subunit.

7. Construction ofXgtll cDNA Expression Library a) cDNA Synthesis cDNA synthesis was performed using a kit Se o4 system (Amersham International plc, cDNA Synthesis 31 System) containing standardised Amersham reagents.

First strand cDNA synthesis: To an Eppendorf tube on ice were added: 10 p. 5 x first strand buffer, 2.5 .1 Na-pyrophosphate, 2.5 .l RNase inhibitor, 5 1 dNTP (deoxynucleosidetriphosphate) mix, 2.5 pl oligo-dT solution, 1.25 4 1/12.5 pCi a 32 P-dCTP, 10 41/5 4g B. bovis strain) poly A* RNA (as prepared above) and 13 .1 water. After gentle mixing, 4 .1 (100 units) of reverse transcriptase was added and the mixture incubated at 42°C for 40 minutes, and then placed on ice. 1 pl was removed for analysis purposes.

Second strand synthesis: To the remaining reaction mixture on ice were added: 93.5 pl second strand buffer, 12.5 4l (125 4Ci) a P-dCTP, 5 il RNase H, 5 p.1 DNA polymerase I and 151 pi1 water. This b* mixture was incubated at 12°C for 60 minutes, 22°C for 60 minutes, 70'C for 10 minutes, and then chilled on ice. T4 DNA polymerase (2.5 4p1/10 a* Z* units) was then added and the tube incubated at 37°C for 10 minutes. 12..5 p 1 0.5 M EDTA pH and 25 pl 10% SDS were then added, mixed, and a 1 p.

aliquot removed for analysis purposes. The reaction mixture was then phenol extracted by .25 mixing with 300 .1 of phenol:chloroform:isoamyl S e 4( alcohol (25:24:1) for 5 minutes prior to centrifugation (5 minutes, 14,000 rpm, Eppendorf centrifuge) to separate phases. The aqueous phase -was recovered and the extraction repeated. To the recovered aqueous phase was added an equal volume of 4 M ammonium acetate. After mixing, 2 volumes of absolute ethanol at -20°C was added and the mixture snap frozen (15 minutes on dry ice). The mixture was thawed to room temperature with gentle mixing and centrifuged minutes, 14,000 rpm, 4"C, Eppendorf centrifuge) to pellet cDNA. The cDNA pellet was redissolved in l of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH An equal volume of 4 M ammonium acetate was Smixed with the cDNA solution and two volumes of a absolute ethanol at -20°C added to reprecipitate Sa the cDNA. After freezing, thawing and centrifugation as above, the cDNA pellet was washed with 200 pl of absolute ethanol at dried under vacuum and redissolved in 20 il of TE.

The cDNA was stored at Calculation of cDNA synthesis efficiency and yield: The 1 4i aliquots previously put aside from a the first and second strand synthesis reactions were diluted to 20 4l with water. For each sample, 2 2 pl of solution was spotted onto two 1 cm filter discs (Whatman DE-81 cellulose) labelled 'A and discs (Whatman DE-81 cellulose) labelled and 33 The filters were washed six times with M Na H PO 4 (5 minutes per wash, 20°C) and rinsed twice in water and then twice in absolute ethanol. All four filters were then dried and counted in a scintillation counter (LKB rack B).

The results for the first strand synthesis were that filter produced 20,594 cpm and filter returned 929 cpm, equivalent to incorporation of label. Knowing that the total dCTP utilised was 25 nanomoles, we therefore deduce that the total dCTP incorporated was 1.13 nanomoles and therefore that the total dNTP incorporation is 4.51 nanomoles (assuming equal uptake of all dNTP's). From this we can deduce that the weight a i of the cDNA synthesised was 1.58 micrograms of .6 single stranded DNA. Since 5 micrograms of poly A- RNA was the starting material, the percentage of RNA transcribed was 31.6%.

On the second strand analysis and by equivalent calculation, the total percent incorporation was 8.74%, and knowing that the total dCTP was 25 nanomole- we know that 1.09 nanomoles incorporated dCTP was the corresponding figure, and that the amount of total dNTP incorporated in the S. second strand synthesis is therefore 4.36

S

nanomoles. This relates to an equivalent amount >f 1.53 micrograms of second strand cDNA synthesised.

As a measure of the efficiency of double stranding of the first strand cDNA the calculated percent of second strand transcribed off the first strand is the percentage ratio of the relative weights, and this leads to a 97% efficiency for production of double stranded DNA from the single strand. By calculation, the total double stranded cDNA produced is therefore 3.06 micrograms. This cDNA provided the source for the cDNA library.

b) Cloning of cDNA into Xgtll Reagents from the Amersham XgtlO cloning system together with prepared Xgtll phage DNA were em C. S* 1 used to prepare the cDNA for cloning, ligation into vector and in vitro packaging of recombinant phage St

DNA.

.*S

Methylation of B. bovis cDNA: 1 i 1 of Sadenosyl methionine (SAM) was diluted to 100 pl with water. To an Eppendorf tube on ice was added 1 p g (6 41) of B. bovis cDNA, 4 pl methylase buffer, 2 il freshly diluted SAM and 6 41 water. After mixing, 2 9 1 (20 units) of EcoRI methylase was added and mixture incubated at 37°C for 60 minutes. The tube 25 was further' incubated at 70°C for 10 minutes to inactivate the enzyme and then placed on ice.

inactivate the enzyme and then placed on ice.

Linker addition: To the methylated cDNA on ice was added 3 p1 of Linker buffer, 2 li of EcoRI linkers and 3 41 of water. After mixing, 2 p1 units) of T4 DNA ligase was added and the mixture incubated overnight at 15°C. The mixture was further incubated at 70°C for 10 minutes to inactivate the enzyme and then placed on ice.

EcoRI digestion to cleave excess linkers: To the ligation mix on ice was added 10 p)1 of EcoRI buffer and 58 4l of water. After mixing, 2 4l (120 units) of EcoRI restriction endonuclease was added and the mixture incubated overnight at 37°C. The reaction was then heated (70°C, 10 minutes) and placed on ice.

Removal of excess linkers: A gel filtration column (as supplied by Amersham in cDNA cloning o o* kit) was equilibrated with STE buffer (100 mM NaCl TE). One hundred 4i of the EcoRI digest prepared previously was loaded onto the column and washed-in with 100 4i of STE buffer. Two hundred 41 o, fractions were collected (nine in total) and counted by scintillation to determine the cDNAcontaining fractions. These fractions (3 and 4) were pooled. To the pooled fractions ws added a 0.1 volume of 3 M sodium acetate pH 4.8, and after mixing, 2.5 volumes of ethanol at -20°C. After 5 overnight incubation at -20°C, the precipitated cDNA was pelleted (10 minutes, 14,000 rpm, 4°C) in an Eppendorf microfuge and the resulting pellet dried under vacuum. The dried cDNA was redissolved in 10 pl of TE buffer. This cDNA solution was used as insert DNP in subsequent cloning steps.

Preparation of Xgtll vector DNA: One hundred micrograms (300 4l) of Xgtll DNA prepared from standard stock phage was added to 4041 10 x EcoRI buffer (1 x EcoRI buffer 10 mM MgCl 2 100 L:M Tris-HC1 pH 7.5, 50 mM NaC1), 40 pl HO and 20 til (200 u) EcoRI restriction endonuclease (Boehringer Mannheim). The mixture was incubated at 37°C for 2 hours and then phenol 9 e 9 S oo S

S

S S S S extracted, chlor iform extracted and ethanol precipitated as described previously. The precipitated, restricted, phage DNA was washed with 400 pl of 70% ethanol at -20°C, dried under vacuum and redissolved in 40 4.1 of water. To this solution was added 4.8 4Li of 10 x calf intestinal alkaline phosphatase (CIP) buffer (1 x CIP buffer mM Tris-HC1 pH 7.5, 10 mM MgCl,) plus 4 i4 of CIP enzyme (88 units, Boehringer Mannheim). After incubation (37°C, 2 hours), the phosphatase reaction mixture was phenol extracted twice, chloroform extracted once and ethanol precipitated as before. After washing with 70% ethanol as described above, the DNA was dried under vacuum and redissolved in 40 pl of TE. The concentration of 0 a5 a this vector solution was 2 mg/ml.

o. Ligation of cDNA insert and Xgtll vector DNA: gtll vector (10 Gg/5 41) plus 0.5 p4 0.1 M MgCl were mixed and incubated at 42°C for 60 minutes to anneal the phage cohesive termini.

Four tubes labelled to were filled as follows: Tube 2 4g (1 4p) annealed vector 3.3 4 (approx. 1204g) cDNA Tube 4 4g (2 pl) 3.3 41 Tube C~ 0 Clg k4 [U) 3. 3 Il it UI Tube 1 g (0.5 pl) only Water was added to each tube to give a total volume of 8 p 1. To each tube was added 1 l 10 x ligation buffer (1 x ligation buffer 50 mM Tris- HC1 pH 7.4, 10 mM MgC1 2 10 mM dithiothreitol, 1 mM spermi 1 mM ATP (adenosine triphosphate), 100 4 j/ml BSA (bovine serum albumin)) and 1 p4 (6 un of T4 DNA ligase (Boehringer Mannheim). Ligation was performed overnight at In vitro packaging of recombinant phage: Amersham in vitro packaging mixes were used. Four packaging mixes were used for each ligation reaction to 'C One packaging mix was used to @000 package one quarter of r 'ion For each ligation mixture, the four packaging reactic s (incubated for 2 hours at 20°C) were diluted to 2 ml with TM buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl,) and a drop of chloroform added as a preservative. The packaging reaction from was diluted similarly. A furthet packaging reaction was performed using 100 mg of uncut Xgtll phage DNA and diluted as above. Packaging efficiencies were 4900•• o C determined by plating a further dilution of each phage stock produced by in vitro packaging as e a described above.

*0 Appropriately diluted phage in TM buffer was added to 200 il of E. coli Y1088 plating cells (plating cells were grown to an O.D. (at 600 nm wavelength) of 1.0, centrifuged: 3,000 rpm, minutes, 20°C, MSE benchtop centrifuge, and resuspended in 0.1 volumes of TM buffer) to which was added 60 .1 IPTG (isopropylthiogalactoside, 24 mg/ml, sterile), 4 ml molten top agar Bactotryptone, 0.5% Bacto-yeast extract, 1% NaC1, 0.75% agar) at 50°C and 60 pl of X-gal (5-bromo-4chloro-3-indolyl-galactopyranoside, 20 mg/ml in dimethylformamide) prior to plating on a 37°C prewarmed 9 cm diameter L-agar plate (Luria agar, as for top agar, 1.5% agar). After setting and brief drying, plates were incubated at 42'C overnight and the phage plaques counted. In the *presence of IPTG (inducer) and X-gal (substrate), wild-type Xgtll plaques which express functionals- 0.

galactosidase are blue, whereas recombinant phage which express B-galactosidase fusion proteins or truncated B-galactosidase are colourless.

In this experiment, the titre of total plaque-forming units (pfu) from ligations 'C' was 106 pfu/ml.

The titre of clear plaques only, was 7.7 x 10 s pfu/ml The total number of recombinant 0* phage obtained was 5 x 10 6 pfu. Packaging efficiency of uncut gtll was 6 x 10' pfu/ tg.

Packaging of (vector only) yielded 3.85 x 10 s pfu/tg (blue plaques).

8. Screening of B. bovis Xqtll cDNA expression library using W11C5 MAb Hybridoma W11C5 was grown in cell culture to yield a supernatant containing the W11C5 MAb. The supernatant was collected and the antibody purified by affinity chromatography on a babesial antigen-cellulose column.

Twenty-five ml of purified 11C5 MAb (300 [ig/ml protein) was diluted to 100 ml in TBS (Tris-buffered saline: 50 mM Tris- HC1 pH 7.5, 300 mM NaC1) made 0.5% with respect to gelatin.

**ea "915 Two hundred thousand recombinants from the cDNA

S

Slibrary as prepared above were plated onto four 14 cm diameter L. agar plates (no X-gal or IPTG) using 500 pl of E.

coli Y1090 plating cells (prepared as for Y1088 plating cells above) and 10 ml of molten top agar per plate. The plates were incubated at 42°C until small plaques were just visible (approx. 3 hours) and then overlaid with 13.5 cm nitrocellulose discs (Millipore, 0.45 Itm pore size) S impregnated with 1 mM sterile IPTG to induce galactosidase/fusion protein expression. Orientation marks were made through the nitrocellulose discs using a needle S dipped in scripting ink. After a further 3 hours incubation dipped in scripting ink. After a further 3 hours incubation 0 41 at 42°C, the filters were removed and the plates stored at 4°C. The filters were incubated overnight at 42°C in 1% gelatin/TBS to block non-specific antibody binding sites.

Filters were then incubated with the diluted W11C5 MAb at room temperature for 2 hours. Filters were washed three times in TBS/0.1% Tween 20 (15 minutes, room temperature) to remove unbound MAb. Filters were then incubated fzr 1 hour at room temperature with 1:300 diluted biotinylated ovine anti-mouse immunoglobulin antiserum (Amersham) in gelatin. Unbound antibody was washed off as described previously. Filters were then incubated with streptavidinhorseradish peroxidase conjugate (strep-HRP, 1:300 dilution in TBS/0.5% gelatin, Amersham) for 30 minutes at room temperature. Unbound conjugate was removed (3 washes in TBS, JJ5 5 minutes per wash, room temperature). Filters were then S. incubated in substrate solution (prepared by dissolving 30 mg of 4-chloro-1-napthol (Sigma) in 10 ml of methanol, then adding 10 ml of 10 x TBS, 80 ml water and 100 il of 30% H O (BDH AnalaR)). All blocking steps, antibody incubations, washes and substrate incubations were done on a rocking platform.

After 15 minutes, filters were removed from the S substrate solution, rinsed in TBS and air-dried. Ten clones immunoreactive with MAb W11C5 were clearly visualised as 25 purple spots on the nitrocellulose filters.

So @0 42 9. Generation of pure phage stock and growth of recombinant phage The ten positive clones obtained previously were picked and re-screened at low plaque density through a further two rounds to yield pure, low titre phage stocks.

Eight stocks so obtained were plated onto E. coli Y1088 at high density (100,000 pfu per 9 cm plate) to give plate lysates. Each plate was then flooded with 5 ml of TM buffer and incubated overnight on a rocking platform at room temperature. The TM was then centrifuged (5,000 rpm, minutes, 4°C, SS34 rotor) to clear. Fifty il of chloroform was added as a preservative and the phage titre determined.

This yielded a high titre phage stock (approximately 1010 pfu/ml), stable when stored at 4°C for several years. The high titre stock of one W11C5-positive clone (designated S* 11C5.1) was used to construct a lysogenic cell-line in E.

S*

coli Y1089. To 10 pl of Y1089 plating cells (8 x 10' cells) was added 27 il of 11C5.1 high titre stock (4 x 10' pfu), to give a multiplicity of infection of 5. The bacteria/phage mixture was incubated at 32°C for 15 minutes (preadsorption). Lysis of Xgtll is suppressed at 32°C. This *so* mixture was diluted to 1 ml with L-broth (Luria broth: as for L-agar, minus agar) and 10 4l of this further diluted to 1 ml with L-broth. One hundred 41 of the second dilution was plated onto an ampicillin-containing L-agar plate .s (50 4g/ml ampicillin) and incubated overnight at 32°C.

S 4 43 Approximately 70 colonies were obtained. Ten of these were replicated using sterile tooth-picks onto two fresh L-agar/ampicillin plates. One plate was incubated at 42°C and the other at 32°C. One colony, which grew at 32°C but not 42°C (a lysogen) was picked from the 32°C plate and a small culture prepared (50 ml L-broth containing 50 pg/ml ampicillin was inoculated with the colony and incubated overnight at 32°C with mixing).

This culture was used to prepare glycerol stocks (equal volumes of culture plus sterile glycerol were mixed and stored at -20°C and -80°C in duplicates) and to inoculate larger cultures. Two 250 ml L-broth/amp- cultures were inoculated with 2.5 ml of the overnight culture and grown at 32°C to an O.D. 600 nm of 0.6. Cultures were induced at o15 for 20 minutes (shaking water bath) and IPTG added to 1 mM.

Cultures were further incubated for 1 hour at 380C (with 0e mixing). Longer incubation periods at 38°C resulted in cell lysis and thus loss of induced fusion protein to culture 0 medium.

The cells were centrifuged (7,000 rpm, 5 minutes, Sorvall GSA rotor) and the pellets promptly resuspended oooo in a total of 10 ml cf buffer (50 mM Tris-HC1 pH 7.5, 2 mM EDTA, 0.1 mM dithiothreitol). The suspension was snap frozen in liquid nitrogen and thawed to effect cell lysis, as indicated by high viscosity. The lysate was sonicated (100 W, 1 minute, or until viscosity was low) and centrifuged *0 9 44 minutes, 15,000 rpm, 0°C, Beckman J2-21 rotor). The supernatant was decanted, glycerol added to 15% and stored in aliquots at -20°C. The pellet was resuspended in 2 ml of buffer, glycerol added and stored as for the lysate.

Total protein concentrations of lysate and pellet were determined by the Bradford assay. 5 i1 of each was diluted to 100 4l with water and mixed with 3 ml of Bradford reagent (prepared by dissolving 100 mg of Coomassie G250 dye (Aldrich) in 50 ml of absolute ethanol to which was added 100 ml of 85% phosphoric acid and 850 ml water). After 5 minutes at room temperature the absorbance of the solution at 595 nm was determined. This value was converted to protein concentration using a standard (BSA) calibration curve. The protein concentrations of the lysate and pellet were 5 mg/ml **e i and 9 mg/ml respectively.

9..

Identification of 11C5-8-galactosidase fusion protein by SDS- PAGE and Western blotting: 25 Lg each of lysate and pellet as prepared above was added to an equal volume of 2 x gel loading buffer SDS, 10% B-mercaptoethanol, 20% glycerol, 0.2% bromophenol blue), heated (100°C, 2 minutes) and loaded o* onto a Biorad Mini Protean II 5% acrylamide SDSpolyacrylamide gel with a 3% stacking gel. (Laemmli, U.K.

(1970) Nature 277, 680 685).

25 After electrophoresis (1 hour, 100 V) the gel (0.75 0* mm thick) was stained in Coomassie in 10% acetic 0e

S

methanol) for 1 hour at 42°C and destai,.d in the above mixture minus Coomassie for 2 3 hours with several changes of destaining solution.

A protein band of approximately 280 KDa (kilodaltons) was visible in the"llC5.1 lysogen lysate which was not present in\gtll wild-type induced lysate (see Figure The approximately 120 KDa 0-galactosidase band present in the Xgtll wild-type lysate was absent from the .11C5.1 lysate. Thus the clone )11C5.1 expresses a 280 KDa fusion protein consisting of approximately 160 KDa of 11C5 antigen fused with E. coli 0-galactosidase. Distribution of fusion protein between lysate and pellet fractions indicated fusion protein was present in E. coli cells in a predominantly soluble form.

O**

Western transfers of p-galactosidase-11C5 fusion bS S* protein in E. coli lysate and appropriate controls were made using a Biorad Mini Protean II apparatus with blotting module 0* as per the manufacturers directions. Five percent SDSpolyacrylamide gels were prepared as described previously and blotted for 1 hour at 100 V onto Amersham Hybond C nitrocellulose. Western blots were blocked in 1% gelatin in 0 TBS overnight at 37°C. Antibody screening was performed as Q described previously for library screening with the exception 0 that HRP-labelled second antibody was used directly rather than a biotinylated second antibody in conjunction with streptavidin-HRP. HRP labelled second antibodies were S9 O S* S i °e obtained from KPL inc. and were used at 1:1,000 dilution.

The 11C5-3-galactosidase fusion protein was shown to react specifically with W11C5 MAb, bovine antisera from naturally immune animals (1:600 dilution) and with antisera from cattle immunised with SPA of the lysate of infected cattle (1:600 dilution) (Figure 4 for example). No significant reaction with these antibody probes was obtained for any natural E. coli proteins, including B-galactosidase.

The pattern of bands obtained on the 11C5-0galactosidase Western (a 'stepladder' of bands of lower molecular weight than the main protein band) is characteristic of this fusion protein and is also observed on Western blots of naturally derived B. bovis antigen probed with an anti-11C5 antibody.

5 S, 10. Vaccination trials using 11C5-B-galactosidase fusion protein To investigate the efficacy of the 11C5-3galactosidase fusion protein as a protective antigen, a vaccination trial was undertaken using five groups of six intact adult Bos taurus cattle. Cattle were immunised on day 0 and day 28 each with 10 |jg of the fusion protein either as an acrylamide slurry cut from an SDS-P.A. gel, or in total cleared lysate (500 g total E. coli protein containing 10 pg fusion protein). Freunds complete adjuvant (FCA, 2 ml) was emulsified with an equal volume of the antigen prior to S intramuscular injection. Control vaccination groups received either FCA/PBS only, FCA plus 10 ig B-galactosidase in acrylamide slurry, or FCA plus 500 itg IPTG-induced gtll wild-type cleared lysate coli proteins, includingBgalactosidase). Animals were challenged on day 56 with homologous strain) B. bovis parasites injected intravenously. Vaccination groups are summarised below: Group 1: Naive controls (2 ml FCA 2 ml

PBS).

Group 2: polyacrylamide gel band.

Group 3: (500 gg total protein).

Group 4: polyacrylamide gel band.

-galacto s idase 10 4 g) P-galactosidase cleared lysate W11C5-0-galactosidase (10 pg)

VMS

0a Group 5: W11C5-P-galactosidase cleared *a lysate (10 g in 500 pg total .a protein).

Vaccination groups 4 and 5 (which received gelpurified and crude W11C5-P-galactosidase fusion protein respectively) developed high titres of antibody against the W11C5 antigen as assayed by ELISA using crude W11C5-Bse galactosidase antigen. None of the control groups developed a significant anti-W11C5 titres. Figure 5 summarises ELISA results for the sera of vaccination groups 1 4 and 5. Sera from group 4 and group 5 animals reacted by IFA and B. bovis *a 9 48 infected erythrocytes. The pattern of staining obtained by IFA was similar to that obtained using the W11C5 MAb. A serum dilution of 1:1,000 was sufficient to give good IFA staining. Similarly 1:1,000 diluted serum gave a strong signal on Western transfers of parasite and E. coli expressed W11C5 antigen and the pattern of immuno-reaction obtained was identical for W11C5 fusion protein antiserum and W11C5 MAb.

On parasite challenge, animals in groups 2 to exhibited immediate elevated temperatures. This is indicative of a hypersensitivity reaction. No such reaction was observed in group 1 naive control animals. Animals in group 4 developed parasitaemias significantly lower than in both the naive controls (P 0.014) and the gel-purifiedpgalactosidase control group (P 0.034). Animals in group developed significantly lower parasitaemias than those in the corresponding control group which received E. coli lysate/3galactosidase (P 0.028), although the value obtained by comparison with group 1 naive animals (P 0.059) is not statistically significant (0.05 cut-off). Parasitaemia and survival results are summarised in Figures 6 8.

One animal of six in each of the W11C5 vaccinated groups failed to recover naturally after infection (terminally ill animals were drug-treated before death occurred).

Four animals in the naive control group, five in the 0-galactosidase gel-band control group and all six animals in the P-galactosidase crude lysate control group failed to survive untreated Sera from animals in control groups 1 3 appeared highly jaundiced from day 6 7 onwards (indicating impaired liver function and/or haemolysis) whereas sera from groups 4 and 5 vaccinated animals remained clear.

The high degree of protection conferred by the E.

coli expressed B. bovis antigen used in this vaccination trial is comparable with the protection conferred using crude B. bovis antigen.

It was concluded that a pure antigen may be required to abolish the hypersensitivity reaction observed in this vaccination trial. Also it was decided that a heterologous challenge experiment was required to determine e whether cross-strain protection was elicited by W11C5 recombinant antigen derived from the Samford strain of B.

bovis.

11. Patterns of hybridisation of the cDNA insert from 11C5.1 on Southern and Northern blots of B. bovis genomic DNA and poly A' RNA The size or sizes of nucleic acid fragments detected on Southern or Northern blots using a specific DNA S201 sequence as a probe is a distinct characteristic of that particular DNA sequence.

particular DNA sequence.

Southern Blotting: The following genomic DNAs were digested to completion using EcoRI: Bos taurus, B. bigemina (Lismore), Anaplasma marginale, Theileria orientalis and B. bovis (Samford). Each reaction consisted of DNA (4 gg) 3 41 10 x EcoRI buffer 1.5 L 1 (7.5 units) EcoRI HO0 to 30 4l Digestions were performed for 3 hours at 37°C. Digested DNAs were then size fractionated on a 0.8% Agarose gel (BRL H4 apparatus, 40 V, 16 hours) using X/Hind III and SPPI/RI molecular weight markers (BRESA). Positions of marker bands were marked by injecting the DNA fragments with scripting ink whilst the gel was transilluminated over U.V. The gel was denatured for 40 minutes in 0.5 M NaOH/1.5 M NaCl and **s neutralised in 0.5 M Tris-HCl/3 M NaC1 pH 7.0 for 1 hour.

0 p The single-stranded DNA was transferred to nitrocellulose

I*

(Amersham Hybond C) using 20 x SSC as the transfer solution (1 x SSC 0.15 M NaC1, 0.015 M NaCitrate). After marking the position of the wells and DNA standards using pencil, the nitrocellulose filter was baked (2 hours, 80°C). The baked filter was prehybridised overnight, 42°C, (to block nonspecific DNA binding sites) in a solution containing: deionised formamide, 3 x SSC, 50 pg/ml denatured sheared herring sperm DNA, 10 jg/ml yeast tRNA, 50 mM HEPES buffer, o* pH 7.0, 5 x Denhardts solution (1 x Denhardts solution 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin).

The \11C5.1 cDNA insert was radiolabelled using the Amersham 'Multiprime' Kit. DNA was radiolabelled in A reaction mixture containing denatured\11C5 insert DNA: ng, Multiprime buffer: 10 [il, oligonucleotide primer: 5 pl, DNA polymerase I Klenow fragment: 2 41, a 32 P dCTP (50 4Ci, 3,000 Ci/m mol, Amersham): 5 Li. Incubation was performed overnight at room temperature. Labelled DNA was separated from unincorporated nucleotides by gel filtration using a Pharmacia 'NICK-column' run in T.E. buffer. The labelled DNA was counted (LKB Rackbeta scintillation counter) and the specific activity of the synthesised DNA probe (40 ng) calculated to be 10 9 dpm/ig. The probe was denatured by boiling for 5 minutes prior to addition to 10 ml of prehybridisation solution (as above) and incubation with the 1 ,5 prehybridised filter overnight at 42°C. Unbound probe was washed off the filter (3 x 15 minute washes in 0.1 x SSC at room temperature) and the dried filter autoradiographed

*OO*

overnight at -75°C using a Kodak intensifying screen and Kodak X-Omat AR film. The resulting autoradiogram (Figure 9) shows that 11C5 cDNA specifically hybridises with B. bovis DNA (not B. bigemina, Theileria, Anaplasma, or host DNA) in the form of two EcoRI bands of approximately 15.0 Kb (Kilobase pairs of DNA) and 1.25 Kb in length, as judged from calibration data.

0 2 A further Southern blot (using a BRL minigel apparatus) of DNA from three B. bovis strains: Samford, apparatus) of DNA from three B. bovis strains: Samford, Lismore and Ka. (Figure 10) shows that with EcoRI digestion of the DNA, there is no variation in banding pattern between the three different strains.

Northern Blotting Running buffer for RNA gels:20 mMNaMOPS, 5 mM NaAc, 1 mM EDTA.

Gel: 1% Agarose, 2.2 M formaldehyde, in running buffer.

Sample buffer: 50% formamide, 2.2 M formaldehyde in running buffer.

Total cellular RNA (2 4g) from B. bovis (Samford), B. bigemina, Anaplasma marginalis, Theileria orientalis and bovine liver was dissolved in 10 il of sample buffer, heated at 55°C for 15 minutes and run on a 1% Agarose-formaldehyde gel with size standards coli rRNA and BRL RNA ladder).

The BRL minigel apparatus was run at 70 V for 1 hour.

The gel was soaked in 500 ml of HO for 30 minutes (to remove formaldehyde) prior to staining in ethidium bromide (500 pg/litre) for 30 minutes. The positions of standards were marked as for Southern blotting. The gel was then denatured for 30 minutes in 50 mM NaOH, 100 mM NaCl and neutralised for 30 minutes in 20 x SSC (see Southern blotting). Tranfer of RNA to nitrocellulose (Amersham Hybond C) was performed using 20 x SSC as the transfer solution. After marking the positions of wells and size standards, the filter was baked (80°C, 2 hours) and prehybridised, hybridised with 11C5 probe, washed and autoradiographed as described for Souther:. blotting. The resulting autoradiogram (Figure 11) shows that for the RNA samples used, 11C5 cDNA hybridises specifically to B. bovis RNA, in the form of a major 10 Kb RNA band plus a minor 3.4 Kb RNA band.

1.

2.

*Oe*

C

L.

C S 300 bp.

Sizes of BRESA Molecular Weight Markers Used DNA digested with Hind III: 23130, 9416, 6557, 4373, 2322, 2027, 564 and 125 bp.

Phage SPPI digested with EcoRI: 7840, 6960, 5860, 4690, 3370, 2680, 1890, 1800, 1450, 1330, 1090, 880, 660, 480 and 380 bp.

BRL RNA Ladder: 9500, 7500, 4400, 2400, 1400 and References: Southern, E.M. (1979) Standard procedure for transfer of DNA from Agarose gels. Methods in Enzymology 68, 158 159.

Maniatis, T. et al. (1982) in Molecular Cloning: A Laboratory Manual pp 202 203. Cold Spring Harbor Laboratory Publications.

2Q 0000 see.

0 be C e 0 0@ S S.2 *r

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12. Partial DNA Sequencing of cDNA Insert from \11C5.1 The cDNA insert of clone \11C5.1 was isolated as follows. Whole clone DNA was prepared from \11C5.1 by the liquid lysate method. E. coli Y1088 plating cells (250 p 1) were mixed with 1IIC5.1 phage (from high titre stock described previously) to give a m.o.i. of 0.01. After minutes at 42°C, this mixture was added to 250 ml of L-broth supplemented with glucose to 0.1% and made 10 mM with respect to MgCl 2 After overnight incubation at 42°C with vigorous mixing, 2.5 ml of chloroform was added and the incubation continued for a further 15 minutes to complete cell lysis.

Cell lysate wab decanted from the chloroform and centrifuged (8,000 rpm, 15 minutes, 4°C, GSA rotor) to pellet cell debris. Phage were precipitated from the supernatant by the addition of 8.75 g of NaCl and 62.5 g of polyethylene glycol 8000 (PEG, Sigma) and incubation overnight at 4°C.

Precipitated phage were pelleted (7,500 rpm, 20 minutes, 4°C, GSA rotor) and the drained pellets resuspended in 5 ml of TM buffer. After centrifugation (10,000 rpm, 10 minutes, 4°C, SS34 rotor) to remove debris, the phage suspension was layered onto a stepped CsCl gradient (2 ml steps of 1.7 g/ml, g/ml and 1.3 g/ml CsCl in a 12 ml polypropylene centrifuge tube) and centrifuged (32,000 rpm, 2 hours, 18°C, Sorvall TST41 rotor). The pale blue phage band visible between the 1.5 and 1.7 g/ml steps was collected using a S needle and syringe. After dialysis to remove CsCl (twice against 2 litres of 25 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM MgCl RNase and DNase (both supplied by Sigma) were added a. to final concentrations of 50 pg/ml and 10 pg/ml respectively.

Si After incubation (30 minutes, 20°C) the suspension was made mM with respect to EDTA, 1% SDS and heated (65°C, minutes). After cooling to 37°C, proteinase K (Sigma) was added to 50 4g/ml and the mixture incubated for 1 hour at 37°C. The mixture was then phenol extracted twice and chloroform extracted once as described previously and then dialysed three times against 2 litres of TE buffer.

Purified DNA was checked by Agarose gel electrophoresis and quantitated by U.V. adsorption (260 nm) using 1 absorbance unit 50 g/ml DNA. The yield was 1.0 mg.

Purified 11C5.1 phage DNA (20 Gg) was digested to completion with EcoRI (80 4l DNA, 20 pl 10 x EcoRI buffer, pl/50 units EcoRI (Boehringer), 95 il water, 2 hours, 37°C) and size fractionated through 1% low melting Agarose (BRL 0041 6006 Utra Pure) by gel electrophoresis as described previously.

.e The cDNA insert fragment (approximately 3.1 Kb) was excised using a scalpel and extracted from the Agarose by the method of Langridge et al. (Analytical Biochemistry 103, 264 271, 1980). This method relies on the partitioning of the DNA into an organic phase (butanol) in the presence of a nonionic detergent: hexadecyl-trimethylammonium bromide, (C- TAB, Sigma), then re-partitioning into an aqueous phase free *be from contaminants. 0.6 4g of purified insert (approximately recovery) was obtained.

Ten I g of M13 mpl8 (Pharmacia AB) double stranded 25 DNA (20 4l), 20 4i 10 x EcoRI buffer, 160 4i H 2 O and *l (17.5 units) EcoRI were incubated at 37°C for 1 hour to digest the DNA. The digested mixture was phenol extracted, ethanol precipitated, spun down and the pellet redissolved in .l TE buffer.

Two p1l of insert DNA (approximately 25 ng), 1 p1 digested M13 mpl8 DNA (0.25 2 p1 T4 DNA ligase (5 units), 1 1 10 x T4 ligase buffer and 4 yl HO0 were incubated overnight at room temperature. The ligation mixture was used directly to transform E. coli JM109. Transformation was performed as for plasmid DNA (see following section: Plasmid expression of p-galactosidase-11C5 fusion protein) Approximately 4,000 plaques were obtained. All were blue (non recombinant) except for 20 clear (recombinant) plaques designated mpl8-11C5.1 to 20. One plaque was selected (mpl8- 11C5.4) for further study and preparation of further 11C5coding insert DNA. A high titre stock of mpl8-11C5.4 recombinant phage was prepared by inoculation of a 50 ml culture with a single clear plaque, followed by incubation overnight at 37°C. The cells were spun down and the collected supernatant was the high titre stock containing approximately 10'" phage per ml.

A 250 ml culture of E. coli JM109 was grown to ODo 1 ml of high titre stock of mpl8-11C5.4 clone was added and the culture grown overnight at 37°C. The cells were spun down in 250 ml GSA bottles (7,000 rpm, 10 minutes, and the pellet drained. The pellet was resuspended in 4 m of 50 mM glucose, 25 mM Tris-H pH 7.5, 0 mM EDTA, 4 ml of 50 mM glucose, 25 mM Tris-HCl pH 7.5, 10 mM EDTA, mg/ml fresh lysozyme and incubated at 20°C for 10 minutes.

Eight ml of 0.2 M NaOH/1% SDS was added with gentle mixing and incubated for 10 minutes on ice. Four ml of 3 M K Acetate pH 4.8 was added, the mixture vortexed thoroughly and incubated for 30 minutes at 0°C. The mixture was centrifuged (7,000 rpm, 10 minutes, 4'C) and the supernatant carefully decanted. A 60% volume of isopropanol was added and the mixture incubated for 20 minutes at -20°C. The mixture was centrifuged (10,000 rpm, 10 minutes, 20°C) and the resulting pellet redissolved in 13 ml of TE buffer. 13.7 g of CsCl (Analar) and 1.3 ml of 10 mg/ml ethidium bromide was added to give a density of 1.56 g/ml. Clarification was performed by centrifugation (10,000 rpm, 10 minutes, 9:00 and the supernatant centrifuged (45,000 rpm, 40 hours, 0 to band the DNA (Beckman 70 Ti fixed angle rotor). By *006 S backlighting the centrifuge tube with long wavelength U.V., it was possible to remove the lower of the two DNA bands visible using a syringe. This contained supercoiled replicative form DNA whereas the upper band contained nicked R.F. DNA and E. coli chromosomal DNA. Ethidium bromide was removed by extracting three times with isoamyl alcohol. The DNA solution was then dialysed three times

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against TE buffer to remove CsC1. The insert for further cloning experiments was prepared by EcoRI digestion and 0o 29 subsequent extraction of the insert DNA band from low melting Agarose as described previously. Two other M3 clones (mpl 0 Agarose as described previously. Two other M13 clones (mpl8- 11C5.3 and were grown and analysed as above. These were found to contain the 11C5 cDNA insert in each of the two possible orientations. The presence of a Smal site approximately 0.4 Kb from one terminus of the11C5 insert was used to identify the two orientations. The SmaI site was subsequently found to be 0.4 kilobases from the 3' end of the expressed cDNA fragment of the 11C5 gene. M13 mpl8 clone numbers 3 and 8 were partially sequenced by the dideoxy method as per Sanger et al. (1977). A Biorad sequencing kit was used. This gave the DNA sequence from both ends of the molecule. DNA sequence further into the DNA insert is currently being determined by sequencing particularly deleted clones, constructed as per the Stratagene Cloning Systems o, Exonuclease/Mung Bean Nuclease Kit. Such deleted clones also ?S enable the DNA sequence to be determined for both strands to give an accurate sequence.

Parial nucleotide sequence and derived amino-acid sequence of the cDNA insert of)llC5.1 is shown in Figure 12.

13. Plasmid expression of B-galactosidase-11C5 fusion protein Plasmid pUR288 DNA was digested to completion with EcoRI (5 gg DNA, 5 41 10 x RI buffer, 2.5 4y EcoRI en2yme (12.5 units, Boehringer Mannheim), HO to 50 4l final volume; hours, 37°C). The digestion mixture was phenol extracted and

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ethanol precipitated as described previously. The dried DNA pellet was redissolved in TE buffer. Twenty l of digested pellet was redissolved in TE buffer. Twenty ji of digested 59 DNA (1.6 1 il calf intestinal alkaline phosphatase (CIP, 22 units Boehringer Mannheim), 4 4 10 x CIP buffer and 15 4i HO were mixed and incubated for 1.25 hours at 37°C, then minutes at 65 0 C to inactivate the enzyme. The phosphatase reaction mixture was then phenol extracted and ethanol precipitated as before.

Ligation of plasmid and insert DNA: Fifty nanograms of digested, phosphatased pUR288 DNA from above (2 41), 5 i isolatedX11C5 insert DNA from the M13 sequencing construct mpl8 11C5.4 (150 ng DNA), 1.5 ~l 10 x ligase buffer, 1.0 4l T4 DNA ligase (2.5 units, Boehringer Mannheim) and 5.5 pl HO 2 were incubated at room temperature for 4 hours before being used to transform E. coli JM101.

The transformation protocol used is similar to the

I

1 method originally developed by Mandel Higa (1970, Journal 9V** of Molecular Biology, 53, p15 4 Two 10 ml L-broth cultures of the host cells were grown to an O.D. 600 nm of 0.6 with good aeration at 37°C.

The inoculum used was from fresh growth on L-agar. Cells *2 were centrifuged (3,000 rpm, 5 minutes, MSE benchtop a centrifuge) and resuspended in 2 x 5 ml of 0.1 M MgCl 2 on S ice. Cells were re-centrifuged as before and gently resuspended in a total of 1 ml of 0.1 M CaCl on ice. After 30 minutes on ice, competent cells were added to DNA in a S# S 2 small volume of TE or ligation buffer 5 il) with 10 |ig of tRNA (transfer-RNA) carrier. The competent cells/DNA mixture was incubated on ice for 40 minutes, heat-shocked (42°C, 2 minutes) and then re-incubated on ice for a further minutes. For M13 RF DNA transformations, transformed cells were mixed with plating cells and plated directly (as for plating phage described previously). For plasmid DNA transformations, transformed cells were diluted to 1 ml with L-broth and incubated at 37°C for 50 minutes (to allow initiation of expression of antibiotic resistance) prior to plating on antibiotic-containing L-agar (eg 50 gg/ml ampicillin).

Nine transformants obtained ly this method using the pUR288/11C5 ligation mixture were used to inoculate 1.2 ml L-broth/ampicillin cultures. After three hours at 37°C, S cultures were induced by the addition of IPTG to 1 mM and the 0 P, incubation cbntinued overnight.

*Ofu 2.1, Induced cells were pelleted (5 minutes, 10,000 rpm, 0 ~20°C, Eppendorf microfuge) and resuspended in 50 il of 'P' weoo buffer. Fifty l of 2 x gel loading buffer was then added and the samples boiled for 2 minutes. Twenty 4p oi each *i sample was applied to a 5% SDS-polyacrylamide gel and electrophoresed (100 V, 1 hour), stained and destained as described previously. This identified one clone (designated pUR-11C5) which strongly expressed a 280 KDa fusion protein equivalent to that expressed by clone 11C5.1. The advantages of pUR-directed expression of the B-galactosidase- 0G 11C5 fusion protein over 111C5.1 lysogen-directed expression UO Z oNTo Sec. C A TIT 0 are: no temperature induction is necessary, all culturing may be performed at one temperature plasmid may be stably maintained in the presence of ampicillin and longer induction times may be used to give high fusion protein yields (no spontaneous lysis of cells). Titration of IPTG used to induce cultures indicates that 0.1 mM IPTG was sufficient to induce expression of fusion protein.

Larger-scale growth of pUR-11C5 directed fusion protein: 250 ml L-broth/ampicillin (50 jig/ml) cultures were grown to OD 600 nm 1.0, IPTG added to 0.1 mM and then cultured overnight at 37°C. Cells were harvested (5,000 rpm, minutes, 4°C, GSA rotor), resuspended in 5 ml of 'P' buffer, sonicated (100 W, 1 minute, to lyse cells and shear 0** DNA) and then centrifuged (15,000 rpm, 20 minutes, 4°C, JA21 A, 1 rotor). Cleared lysate was made 17% with respect to glycerol e e and stored at -20°C. Pellet material was resuspended in 2 ml of buffer and stored similarly. Comparison of lysate and pellet material by SDS-PAGE indicated that the fusion protein remained predominantly soluble when expressed at a high level ."*260 from the pUR-11C5 construct (see Figure 3).

14. Subcloning of 11C5 cDNA Insert into pGEX-1 The plasmid expression vector pGEX-1 was selected for further expression of theXllC5 cDNA insert due to the t a 2 possible ease of purification of the resulting antigenglutathione-S-tr ansferase (GST) fusion protein using 0 Sec.

77 glutathione-Agarose beads (Smith, D.B. and Johnson, K.S.

(1988) Gene 67, 31 pGEX-1 plasmid DNA was digested with EcoRI, phenol extracted, phosphatased, re-extracted with phenol an. ethanol precipitated as before for pUR288. One hundred ng of prepared pGEX-1 vector DNA and 100 ng ofAllC5 cDNA insert DNA (as described previously) were ligated in a 20 ILi ligation reaction using 2.5 units of T4 DNA ligase (Boehringer Mannheim) overnight at room temperature. The ligation mixture was used to transform E. coli HB101. Twelve randomly picked transformants were used to inoculate 1 ml Lbroth/ampicillin vials and incubated at 37°C for 3 hours.

IPTG was added to 1 mM and the cultures grown overnight.

Induced cells were pelleted and resuspended in 50 pl of 'P' buffer. Fifty 41 of 2 x gel loading buffer was then added, the samples boiled, and 20 4l run on a 10% SDS-PA gel as described previously. Two transformants displayed protein 0 S" staining profiles with no band at 26 KDa (native GST), but strong bands of approximately 190 KDa (11C5-GST fusion). The 11C5-GST fusion protein was subsequently found to react specifically on Western blots with W11C5 MAb and anti-11C5 bovine polyvalent antisera. One of the two transformants was selected and glycerol stocks prepared as described previously.

25 The glycerol stock was streaked out on L- S0o agar/ampicillin (50 4g/ml), grown at 37°C overnight and used

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o Sec. 77 l U v I AT to inoculate two 250 ml cultures. At OD 600 nm 1, cultures were induced using IPTG (0.1 mM) and incubated for a further 4.5 hours. Cells were harvested by centrifugation (5,000 rpm, 10 minutes, 4°C, GSA rotor), resuspended in 10 ml of buffer and lysed by sonication (1 minute, 100 The lysate was cleared by centrifugation (10,000 rpm, 15 minutes, 4°C, SS34 rotor) and the resulting lysate and pellet analysed by SDS-PAGE to confirm high levels of expression (approximately 20%) of a predominantly soluble fusion protein (see Figure 3).

Purificatio of 11C5-GST Fusion Protein The fusion protein as produced by the pGEX-11C5 transformant selected previously was purified from E. coli lysate as follows.

Glutathione-Agarose beads (GA, sulpher linkage, SSigma) were preswollen overnight at 4°C in TBS and washed S three times (centrifuged 2,000 rpm, 10 seconds, 200C, MSE 00 benchtop centrifuge, resuspended in 50 ml TBS). Two -1 of GA beads thus prepared were mixed with cleared lysate (10 ml, as prepared above) for 1 hour at room temperature. The beads we-e then centrifuged as above and the supernatant removed and stored for further extractions. The beads were then washed three times with 50 ml of TBS/0.1% Tween 20/0.1% Triton X-100/1 mM dithiothreitol (DTT). Bound fusion protein was eluted from the GA beads by mixing the washed beads with 2 volumes of freshly prepared 5 mM reduced glutathione 0 64 mM Tris-HCl pH 8.0 (final pH Purified protein yield was determined using the Bradford assay as described previously. The yield of fusion protein purified from 10 ml of total lysate (50 mg total protein) containing approximately 20% fusion protein was 500 gg (approximately recovery). GA beads were regenerated by washing three times with 3 M NaCl followed by three times with TBS. Reextraction of lysate was repeated several times with little reduction in efficiency. Purity and integrity of fusion protein was determined by SDS-PAGE. (Figure Also, an

J

endotoxin assay was performed (Limulus assay, CSL laboratories) which indicated less than 2.5 endotoxin units were present in each ml of purified protein.

*5 15. Vaccination trial using W11C5-GST fusion protein 0 Four groups, each of five adult steers, were used.

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Animals were injected twice (day 0 and day 28) subcutaneously with antigen in 2 ml of PBS emulsified with 2 ml of FCA.

Animals were challenged intravenously on day 56 with 10 7

B.

bovis Lismore strain parasites.

Group 1: 10 pg purified GST Group 2: 10 pg W11C5-0-galactosida 00

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0 01 0

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004 *4 se

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@6 0* o @0 0@ 00 fusion protein (in crude lysate) Group 3: 10 ig W11C5-GST fusion protein (in crude lysate) Group 4: 10 Ig purified W11C5-GST fusion protein Animals were bled on weeks 0, 2, 4, 6 and 8 and sera analysis by both IFA and ELISA was performed at a standard 1:500 serum dilution and 1:500 crude lysate B. bovis strain) antigen dilution. ELISA readings were ranked 0 10 using a hyperimmune serum from a natural infection as the maximum 10 value. Negative sera values ranked 0 1.

All animals in groups 2 ard 4 showed uniformly strong serological responses to the W11C5 antigen after four weeks, whilst those in group showed relatively weak responses, even after eight weeks. The data are detailed in Figure 13.

The IFA pattern of infected erythrocyte staining using sera from W11C5-GST immunised animals was similar to that obtained using the W11C5 MAb. Sera from those animals o Salso reacted avidly on Western blots of B. bovis-derived and Srecombinant W11C5 antigen and gave the characteristic 'stepladder' pattern.

Upon challenge, all vaccinated animals (groups 2- 4) showed an immediate increase in temperatures, indicating a hypersensitivity and/or an anaphylactic shock reaction. This was not observed in the GST-vaccinated control group animals.

All group 2 4 animals had significant delays in the onset of parasitaemias, as shown in Figure 14 16. Parasitaemias were significantly different from the controls during this period (P>0.05 P>0.001).

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66 Mean maximum parasitaemias were observed on day 8.

These were: Group 1 controls: 2680 1438 per pl blood Group 2 "104 34 Group 3 "61.8 28 Group 4 "76.2 49 One of the group 4 animals died on day 12, whilst one of the control animals also required treatment. All controls were severely affected (inappetance, ataxia, high fever). This was not generally the case with the vaccinates. The mean haematocrit fall in the controls was whilst in the vaccinates it was A post-mortem examination of the group 4 animal which died showed that the internal organs were heavily congested with unparasitised red blood cells. This animal showed signs of cerebral involvement (impaired balance) prior to death, although its maximum parasitaemia was just 51 parasites per of blood.

(Normally, infected erythrocytes are responsible for cerebral babesiosis/organ capillary congestion).

All vaccinated animals had a rapid rebound in their haematocrits by day 11 whereas those of the controls were still depressed.

This vaccination data shows that the W11C5-GST purified fusion data elicits antibody titres which are comparable with those obtained using the W11C5-Pgalactosidase fusion protein. Parasitaemias in these two 67 groups (2 and 4) were also similar; one animal in each group had no detectable parasites.

The crude W11C5-GST fusion protein induced weaker antibody titres and vaccinated animals exhibited a larger haematocrit fall on challenge than the other two vaccination groups although the parasitaemia data were comparable for all three vaccination groups.

All three vaccinated groups (2 controlled parasitaemias to levels observed in the previous W11C5-Bgalactosidase and crude B. bovis vaccination trials, although differences were not statistically significant throughout this trial due to the lower parasitaemias observed in the control animals.

This vaccination trial demonstrates the ability of W11C5 recombinant antigen derived from the B. bovis Samford

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strain to elicit protection against challenge with the heterologous Lismore strain of B. bovis.

It was concluded from this vaccination trial that further work was required to determine the dose of W11C5 antigen, vaccination regime and adjuvant suitable to elicit protective immunity against B. bovis without the induction of a hypersensitivity reaction.

We also advise that a sample of the E.coli HB101 containing the pGEX-11C5 construct was deposited at the Australian Government Analytical Laboratories on 15 December, 1988 under accession number 88/39601.

*O e 4 0S So 68 Sequencing of W11C5 cDNA The cDNA insert of clone )11C5 was sub-cloned into the plasmid sequencing vector pGEM blue as described previously for sub-cloning into pUR322. Sets of nested deletions were generated from both ends of the sub-cloned W11C5 cDNA insert using the exonuclease III/mung bean nuclease system (Bluescript Exo/Mung DNA Sequencing System, Stratagene Cloning Systems, San Diego). Undeleted pGEM-11C5 and deleted derivatives were sequenced using the dideoxy sequencing method (K/RT Sequencing Systems, Promega Biotech, Madison). DNA sequence data was compiled using the Macintosh 'DNA Inspector' computer software.

The W11C5 cDNA structure is shown diagramatically in Figure 16. The cDNA (approximate length 3100bp) is composed of a repetitive 5' region (containing approximately 19 copies of a 90bp repeated DNA sequence in a head-to-tail 0 0 "O tandem array) and a unique 3' region 1414bp in length, which 4* terminates with a row of 13 residues. The unique region DNA sequence and the derived amino acid sequence which terminates with a TGA 'stop' condon at position 1285 in the unique region DNA sequence are shown in Figures 17 and 18 *e respectively.

The DNA sequence of a copy of the 90bp repeat and the derived amino acid sequence are shown in Figure 19. From the amino acid sequence, it is apparent that the 4 repeated sequence consists of two sub-repeats of 10 and 4* e 69 amino acids in length. From amino acid sequence homology between the two sub-repeats, it is evident that the 20 amino acid repeat may have been generated by an insertion of a DNA sequence block (30bp in length) into a copy of the 10 amino acid sub-repeat coding sequence. The 10 amino acid subrepeat is encoded by DNA residues 1-30 in the DNA sequence as shown in Figure 19 and the 20 amino acid repeat by residues 31-90. The proposed inserted sequence is from positions to 75 in the 90bp repeat DNA sequence. The region from position 54 to 60bp in the 90bp repeat is highly variable between different copies of the repeat. Minor variations in other regions of the repeat may occur in a small number of the repeat copies.

The W11C5 cDNA begins at a nucleotide position 2 in a copy of the 90bp repeat preceded by a residue from the 04 O EcoRI linker used for cloning the cDNA. The repeat region of the cDNA (approximately 1700bp in length) ends at position 21

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Sin a copy of the 90bp repeat. The total length o' the W11C5 6:09 cDNA open reading frame is approximately 3000bp (encoding approximately 1000 amino acid residues). The estimated size of the W11C5 antigen portion of the W11C5 fusion protein o expressed in clone 11C5 is therefore 120KDa as judged by DNA sequence data.

0 0 0 0 Immuno-electron Microscopy Staining of Monoclonal Antibody W11C5 to Babesia bovis The cerebral cortex of cattle acutely infected with Babesia bovis was dissected out, fixed and prepared for immunoelectron microscopy. Portions of cerebral cortex were placed in 1% glutaraldeyde, 1% paraformaldehyde, 0.1Mphosphate buffer, 0.08M- sucrose, 0.1 mM- CaCl ,pH 7.4 at 4°C, trimmed to 2 mm 3 transferred co fresh fixative at for 16h, washed x3 in fixative buffer, dehydrated in ascending concentrations of ethanol, infiltrated with at least 3 changes of fresh London White Resin (LRW) (1:1 soft hard) over 24 h placed in fresh resin in gelatin capsules No.001 which were polymerised 18h 500C.

Rabbit antiserum to mouse IgG was prepared by the immunization of rabbits with mouse IgG (Gell Coombs, 1963- Se e in "Clinical aspects of Immunology" 1st. Edition p.7.

Blackwell, Oxford) isolated using Protein A Sepharose 4B (Pharmacia). Protein-A gold (9 nm) was prepared (Slot Geuze, 1985 in European Journal of Cell Biology 38:87-93).

Gold sections were cut on an LKB ultramicrotome using a glass knife and stained by the sequential application of Monoclonal e* Antibody W11C5, rabbit antiserum to mouse IgG and Protein-A gold (Slot Geuze, 1984; "Immunolabelling for electron microscopy" Edited by J.P. Polack I.M. Vardell, pp. 129- 142, Elsevier Science Publishing, Amsterdam). Staining protocol consisted of: 0.02 M- glycine, 10 min; wash X 2 PBS,

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71 1%BSA, 5 min; Mab W11C5 diluted 1/20 in PBS, 1% BSA, 60 min, and then washed X 5 PBS, 1%BSA, 5 min; rabbit anti-mouse Ig diluted 1/100 in PBS, 1%BSA, 60 min; wash X 5 PBS, 1%BSA, min; ProteinA-Gold diluted 1/20 PBS, 1%BSA, 30 min; wash X PBS, 1% BSA, 5 min; wash X 4 distiled water, 5 min. Sections were then stained with 2% aqueous uranyl acetate lead citrate (Imin) (Reynolds, 1963; Journal of Cell Biology 17:208-000) and viewed in a Phillips S300 transmission electron microscope at an accelerating voltage of 60 keV.

The relative distribution of gold particles was measured from electron micrographs exposed at magnification setting 9 (X16000) to produce prints of whole erythrocytes present within an area of the capilla-y lumen. Gold particles were counted on the parasite: surface, nucleus, spherical body, rhoptry, other; erythrocyte: membrane, cytoplasm, debris; and capillary lumen for each erythrocyte infected with B. bovis. The number of gold particles in each category was summed and expressed as a percentage of the total count. A minimum of 25 erythrocytes infected with B.

bovis were counted and the average and standard error calculated. The gold count was recorded as "Not Present" and S..0 not as "zero" when an organelle was absent from the photographic print of the parasite.

Electron micrographs of immunostained sections showed that gold stained mostly a spherical or mitochondrion like organelle at the anterior of the parasite (see lower solid arrow) as well as the membrane of the infected erythrocyte. The gold staining of the erythrocyte membrane appeared to be greater on the stellate projections of the membrane (see solid arrow pointing to right). Other organelles of the parasite and the erythrocyte cytoplasm showed minimal staining (FIG 20). This shows that the monoclonal antibody W11C5 recognises a protein within the above organelle as well as a protein located on the surface of the infected erythrocyte.

The invention also includes within its scope the cDNA structure shown in FIG 16 as well as the sequences shown in FIGS 17, 18 and 19. The invention also includes within its scope sequences substantially homologous thereto (i.e sequences having greater than 40% homology over a length of *000 100 nucleotides or longer in the case of a DNA sequence and sequences having greater than 40% homology over a length of

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30 amino acids or greater in the case of a protein). The term "substantially homologous thereto" may also include

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within its scope DNA sequences showing cross hybridisation with the W11C5 cDNA under standard hybridisation conditions.

The antigens of the invention are also useful in

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~this invention when used as a vaccine to be protective against babesiosis particularly against homologous challenge *(challenge by the same strain or isolate of Babesia) as well as heterologous challenge (challenge by different strains or isolates of Babesia). The antigens may also be protective 73 not only against B bovis but also B. ovis, B bigemina, B canis and other strains of Babesia.

The recombinant antigen when expressed as a fusion protein was expressed at a relatively low level in \gtll.

When subcloned into pUR322 higher levels of expression were obtained. 'When subcloned into pGEX 1 relatively high levels of expression were obtained to facilitate purification.

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Claims (24)

1. A process for preparation of an antigen including the steps of: preparing nucleic acids from babesia infected erythrocytes depleted of leucocytes; (ii) forming a cDNA or genomic library from nucleic acids obtained in step (iii) screening said library formed in step (ii) with a suitable probe to identify clones of interest; (iv) obtaining DNA from clones of interest identified in step (iii); inserting DNA from step (iv) into an expression vector; (vi) propagating a recombinant expression vector made in step in an appropriate host; and (vii) obtaining a recombinant polypeptide from the host harbouring the recombinant expression vector, Swherein the recombinant polypeptide has the immunological activity of the W11C5 antigen as herein described and is protective against babesiosis.

2. A process as claimed in Claim 1, wherein in step (ii) a ODNA library is formed from reverse transcription of babesial poly A* mRNA.

3. A process as claimed in Claim 1 or 2, wherein the probe is selected from monoclonal antibodies or polyclonal antisera raised against babesiosis infected erythrocytes to identify immunoreactive clones. \v

4. A process as claimed in Claim 3, wherein said cDNA library is produced in Igt 11 expression vector and propagated in an E. coli host. A process as claimed in Claim 4, wherein a fusion protein is obtained comprising a babesial antigen fused with P galactosidase.

6. A process as claimed in Claim 1, wherein the expression vector is pUR322.

7. A process as claimed in Claim 2, wherein the expression vector is PGEX1 to thereby provide a fusion protein comprising a babesial antigen coupled to glutathione-S- transferase.

8. An antigen having the immunological activity of the W11C5 antigen as herein described inducing protective immunity against homologous or heterologous challenge with B. bovis which is immunoreactive with a monoclonal antibody or polyclonal antisera recognising a protein located on the surface of babesia-infected erythrocytes and within a spherical or mitochondrion like organelle. 9

9. An antigen as claimed in Clatm 8, wherein the 6** monoclonal antibody is MAb W11C5 as herein described from hybridoma 88 121501-W11C5.1.A5.F10.A2 as herein described.

10. An antigen as claimed in Claim 8, obtained as a genetically engineered polypeptide in the form of a fusion S25 protein.

11. An antigen as claimed in Claim 10, wherein the antigen is coupled to P-galactosidase.

12. An antigen as claimed in Claim 10, wherein the 7i antigen is coupled to glutathione-S-transferase. 76

13. A babesial antigen immunoreactive with MAb W11C5 as herein described or with antisera raised against W11C5 antigen as herein described or antisera raised against recombinant W11C5 antigen as herein described.

14. A babesial antigen derived from a gene showing a DNA sequence substantially homologous with at least part of W11C5 cDNA as herein described encoding the W11C5 antigen as herein pG-C \\c's described as contained in the plasmid pGEXAl-G5-of the AGAL deposit 88/39601 as herein described.

15. A recombinant antigen having the immunological activity of the W11C5 antigen as herein described which is expressed as a fusion protein with P-galactosidase or with glutathione-S-transferase and which is protective against heterologous or homologous challenge with B. bovis in cattle. i16. An antigen capable of inducing protective immunity against homologous or heterologous challenge with Babesia and having the immunological activity of the W11C5 antigen as S herein described, wherein said antigen is encoded by the DNA sequence shown in FIG 16 and which has a unique region having '0 the amino acid sequence shown in FIG 18 and DNA sequences *a* substantially homologous thereto and a repeat region having the amino acid sequence shown in FIG 19 and DNA sequences substantially homologous thereto.

17. Monoclonal antibody MAb W11C5 as herein described .25 obtained from hybridoma 88 121501-W11C5.1.A5.F10.A2 as herein age described.

18. A monoclonal antibody reactive with an antigen having the immunological activity of the W11C5 antigen as Sp *herein described inducing protective immunity against w r 77 homologous or heterologous challenge with B. bovis and recognises a protein located on the surface of babesia infected erythrocytes and within a spherical or mitochondrion- like organelle.

19. A DNA sequence which when expressed in E. coli produces a protein which is protective against babesiosis when administered as a vaccine and which elicits antibodies showing similar immunological reactivity with MAb W11C5 as herein described or antibodies raised against native or recombinant W11C5 antigen as herein described. A DNA sequence as claimed in Claim 19 which is a cDNA insert of the plasmid pGEX-11C5 of the AGAL deposit 88/39601 as herein described.

21. cDNA encoding W11C5 antigen as herein described having the structure shown in FIG 16 and structures cross hybridisable therewith under standard hybridisation 6:61 conditions. *0

22. A DNA sequence as shown in FIG 17 and sequences substantially homologous thereto. '0 23. A polypeptide having an amino acid sequence as shown in FIG 18 and sequences substantially homologous thereto.

24. A DNA sequence as shown in FIG 19 and sequences substantially homologous thereto. S 25. An amino acid sequence as shown in FIG 19 and 0 sequences substantially homologous thereto. 0. 26. A DNA sequence having a repetitive component of approximate unit length 90 base pairs followed by a unique component of approximately 1414bp. which when expressed as a polypeptide has the immunological activity of the W11C5 Z I, 78 antigen as herein described which induces immunity against babesiosis.

27. A vaccine containing an antigen as claimed in any one of claims 8-16 in combination with an adjuvant.

28. A test kit for the detection of babesiosis including a monoclonal antibody as claimed in claim 17 or claim 18.

29. A vaccine containing an antigen derived from a DNA sequence or DNA structure as claimed in any one of claims 19, 21, 22, 24 or 26 in combination with an adjuvant.

30. A vaccine containing an antigen containing an amino acid sequence or polypeptide as claimed in claim 23 or 25 in combination with an adjuvant. DATED this fourteenth day of December 1992 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION By Their Patent Attorneys CULLEN CO e* *o *N o* *0 *e *eo