Proc. Natl. Acad. Sci. USAVol. 86, pp. 9657-9661, December 1989Biochemistry

Cloning and expression of a protective antigen from the cattle tickBoophilus microplus

(vaccine development/cDNA/epidermal growth factor-like repeats/membrane glycoprotein)

KEITH N. RAND*, TERRY MOORE*, ALAGACONE SRISKANTHA*, KEVIN SPRING*, Ross TELLAMt,PETER WILLADSENt, AND GARY S. COBON*t*Biotechnology Australia Pty. Ltd., P.O. Box 20, Roseville, New South Wales, 2069, Australia; and tCSIRO Division of Tropical Animal Production, PMB3,Indooroopilly, Queensland, 4068, Australia

Communicated by Rutherford Robertson, August 18, 1989 (received for review June 26, 1989)

ABSTRACT Glycoproteins located on the luminal surfaceof the plasma membrane of tick gut epithelial cells, when usedto vaccinate cattle, are capable of stimulating an immuneresponse that protects cattle against subsequent tick infesta-tion. One such tick gut glycoprotein, designated Bm86, hasbeen purified to homogeneity and the amino acid sequences ofpeptide fragments generated by endoproteinase Lys-C diges-tion have been determined. We report here the isolation andcharacterization of a cDNA that encodes Bm86. The nucleotidesequence of the cDNA contains a 1982-base-pair open readingframe and predicts that Bm86 contains 650 amino acidsincluding a 19-amino acid signal sequence and a 23-amino acidhydrophobic region adjacent to the carboxyl terminus. Themain feature of the deduced protein sequence is the repeatedpattern of6 cysteine residues, suggesting the presence ofseveralepidermal growth factor-like domains. A fusion protein con-sisting of 599 amino acids of Bm86 and 651 amino acids of/3-galactosidase was expressed in Escherichia coli as inclusionbodies. Ticks engorging on cattle vaccinated with these inclu-sion bodies were significantly damaged as a result of theimmune response against the cloned antigen.

The tick Boophilus microplus is a major ectoparasite of cattlein many parts of the world. A single female cattle tick takesup as much as 1.5 ml of bovine blood, increasing its bodyweight to =250 mg. It has been estimated that cattle intropical areas of Australia may become infested with 1000tick larvae per day, resulting in greatly reduced productivity.In addition, B. microplus is the vector of hematoprotozoalparasites such as Babesia bovis. Chemicals have been usedextensively to control ticks and have been partially success-ful, but this approach suffers from certain drawbacks such asenvironmental and residue problems, the high incidence ofacaricide resistance that has developed in tick populations inthe field, the need for frequent administration, and high cost.

Recently it was shown that cattle immunized against amembrane-bound glycoprotein (Bm86) purified from cattleticks are highly resistant to parasite challenge (1). A vaccinebased on Bm86 would be a very attractive alternative toacaricide treatment and would overcome most of the diffi-culties associated with the use of chemicals. Available evi-dence suggests that immunized cattle produce antibodies thatrecognize Bm86 present on the surface of tick gut digest cells(1, 2). After the ticks ingest blood, these antibodies, togetherwith other components of the immune system such as com-plement, cause lysis of the gut epithelial cells, leading eitherto tick death or to damaged ticks exhibiting reduced growthand egg-laying ability. The effects are very striking. As manyas 90% of ticks failed to survive to adulthood on cattle

vaccinated with three doses of only 2 ug of Bm86 (1). How-ever, it was necessary to begin with 40,000-60,000 ticks inorder to purify 20-100 ug of Bm86. In order to produce thelarger quantities of Bm86 that are needed to develop a com-mercial vaccine, we have cloned and expressed the Bm86gene. § Recombinant Bm86, even in the form ofa fusion proteinproduced in Escherichia coli as inclusion bodies, is capable ofinducing a substantial degree of protection against tickinfestation.S

MATERIALS AND METHODSConstruction and Screening of cDNA Library. RNA was

extracted (3) from adult B. microplus (picked from cattle 15days after infestation), cDNA was synthesized from 4 ,ug ofpoly(A)+ RNA (4), and cDNA fragments larger than 800 basepairs (bp) were ligated to Agtll (5) to generate a library of 8x 105 recombinant clones. Oligodeoxynucleotide probes(Table 1) were based on the sequences derived from peptidesgenerated by endoproteinase Lys-C digestion of Bm86 (1).Three nitrocellulose filters were prepared from five 150-mm

plates each containing 105 plaques. After prehybridization in0.6 M sodium pyrophosphate/0.005% heparin (Sigma) at 40°Cfor 4 hr, hybridization was carried out for 16 hr at 40°C in thesame solution with the radioactive oligonucleotide probe. Twoof the filters were hybridized with the 63-mer probe while thethird was hybridized with a mixture of the 50-, 51-, and 72-merprobes. The filters were washed in 0.3 M NaCl/0.03 M sodiumcitrate, pH 7.5/0.1% SDS at 45°C and positive plaques wereidentified by autoradiography. Other molecular biology tech-niques were carried out basically as described (6).DNA Sequencing. The 3.9-kilobase-pair (kb) EcoRI frag-

ment in the Agtll clone was self-ligated to form circles andconcatemers, then sonicated. Fragments were end-repairedand then size-fractionated by electrophoresis in a low-melting-temperature agarose gel. Fragments in the size range200-500 bp were cloned into the phage M13mpl8 and se-quenced (7). The sequence was compiled from all of thesubsequences and analyzed with the programs GEL and SEQ(IntelliGenetics). Some DNA sequence was also obtained bythe exonuclease III/nuclease S1 method (8).

Construction of Expression Vector pBTA708. A Bm86 genefragment was prepared by first inserting a 2058-bp Xmn Irestriction fragment (positions 120-2178) into the Sma I siteof vector M13um3l (International Biotechnologies). Afterdigestion with Sac I and Pst I, a fragment encoding most ofBm86 was ligated into pBTA224 [derived from pUR292 (9) by

Abbreviations: EGF, epidermal growth factor; IPTG, isopropylj3-D-thiogalactopyranoside.4To whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M29321).$E. coli BTA1751, which contains the entire Bm86 gene, has beendeposited in the American Type Culture Collection (ATCC desig-nation 67548).

9657

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Table 1. Oligodeoxynucleotide probesProbe Sequence (5' -* 3')Y50-:mer TTACCAATGGATGTACAAATAGCTTCAAGGACACCATCTTCGTACCACTT51-mer CTTCGACGGATTGGATTCGACGCATCTGCCATAGCTACATTCCCTCGTCTT72-mer TTTAGGTACAACCTCACATTCAGCATTCCTACAAAATTCATTACCGAAATCAAAACAAATACTACTCTCCTT63-mer CTTGCAATGGATTCCATCCTCGGCGACAGTGAAAGCTCTAGGGCAAGTGCACTCATAAGCCTT

eliminating the EcoRI site that lies outside of the P-galactosidase-coding region] to give pBTA708, which wasused to transform E. coli JM101 (10) to give BTA1752.

Expression Tests. BTA1752 was grown in tryptone soybroth(TSB) containing ampicillin (50,ug/ml) overnight, diluted 1:10in fresh TSB, and induced with 1 mM isopropyl ,B-D-thioga-lactopyranoside (IPTG) for 30 hr. The cells were resuspendedin 50 mM Tris, pH 7.5/1 mM EDTA/1 mM phenylmethylsul-fonyl fluoride and disrupted by sonication. Crude inclusionbodies were solubilized in 2.7% SDS/0.8 M urea/1.6% 2-mercaptoethanol), resolved in a SDS/8% polyacrylamide gel,and transferred electrophoretically to nitrocellulose (Schle-icher & Schuell). The membrane was blocked overnight with0.1% gelatin in 10 mM borate buffer (pH 8.0), washed threetimes in 10 mM Tris, pH 8.0/0.1 M NaCl/0.1% Tween 20(TST) and incubated for 1 hr at room temperature in TSTcontaining a 1:500 dilution of serum from cattle 32 and 34,which had been vaccinated with native Bm86 isolated fromticks (1). The membrane was washed three times with TST,incubated for 5 min with a 1:500 dilution in TST of a rabbitanti-bovine antibody conjugated to horseradish peroxidase(Dakopatts, Copenhagen), washed three times with TST, andincubated in a solution of chloronaphthol (0.5 mg/ml) andH202 (0.00025%) in 10 mM Tris (pH 7.5) until color developed.

[35S]Cysteine Labeling. A culture of BTA1752 was grownovernight in minimal medium (11) plus ampicillin (50 Mg/ml)and then diluted 1:20 in TSB and incubated until the opticaldensity at 600 nm reached 0.5. Proteins were induced andlabeled by addition of IPTG and [35S]cysteine to 1 mM and 2,uCi/ml, respectively (1 ,uCi = 37 kBq). Six hours afterinduction, labeled and unlabeled samples were removed andanalyzed by SDS/PAGE. The gel was soaked in Amplify(Amersham), dried, and autoradiographed.

Cattle Vaccination and Challenge Trials. Vaccination andchallenge trials were carried out essentially as described (1).In brief, groups of three cattle received three vaccinations at4-week intervals, the first two intramuscularly in Freund'scomplete adjuvant, the third subcutaneously in 0.9% NaCl.

RESULTS AND DISCUSSIONIsolation ofcDNA Clones. One Agtll plaque that hybridized

with both the 63-mer and the mixture of 50-, 51-, and 72-merprobes was isolated from -5 x 105 recombinants. The clonewas found to contain an insert of 5.6 kb, which could bedigested with EcoRI to give 3.9-kb, 1.5-kb, and 0.2-kbfragments. All four probes hybridized to the 3.9-kb fragmentand not to the others. The sequence (Fig. 1) of nucleotides1-2235 of the 3.9-kb EcoRI fragment encodes Bm86. All ofthe peptide sequences used for designing probes (underlined)as well as the other sequences determined from the endopro-teinase Lys-C peptide fragments (1) can be identified. Thesequence Ser-Gly-Ser at amino acid positions 235-237 isdifferent from that determined by peptide sequencing (Arg-Ala-Phe). The sequence of a second cDNA clone suggeststhat this is due to polymorphism within Bm86.

It is likely that amino acid 20 is the amino-terminal residueof the mature Bm86 protein. (i) The amino-terminal 19-aminoacid segment is hydrophobic, which is a characteristic ofleader sequences (12). (ii) The amino acids at positions -1and -3 of the putative cleavage site are small and nonpolar,which is also characteristic of the amino acids in thesepositions of many signal peptides (12). (iii) Although peptides

were generated from Bm86 by endoproteinase Lys-C diges-tion, only one of the 12 peptide sequences obtained, thatstarting at amino acid -20, is not preceded by a lysine.A polyadenylylation sequence (AATAAA) is present at

position 2203 and 17 nucleotides downstream is a stretch of10 adenines. As the poly(A) stretch was short for a poly(A)tail and there was DNA downstream from this sequence, itwas not clear whether this was the 3' end of the maturemRNA. Another Bm86 cDNA clone was isolated from adifferent cDNA library and was found to terminate in morethan 50 adenines from position 2226. This suggests that anunrelated cDNA fragment was joined to the original Bm86cDNA fragment at some stage in the construction of thelibrary.The carboxyl-terminal 23-amino acid region (amino acids

628-650) is very hydrophobic and resembles segments inother membrane proteins that are removed and then replacedby a glycosyl-phosphatidylinositol anchor (13). If this is thecase, Bm86 has no cytoplasmic domain. The 27 amino acidsthat precede the hydrophobic sequence (amino acids 601-627) contain clusters of serine and threonine residues. This isa characteristic of several membrane-bound proteins, such asthe low density lipoprotein receptor, which may have 0-linked carbohydrate chains in such regions (14). There arealso five sites of possible N-linked carbohydrate addition(Asn-Xaa-Ser/Thr; Fig. 1).Comparison of the Bm86 amino acid sequence with se-

quences in the National Biomedical Research Foundation(January 1989) and Kyoto (July 1988) data banks revealedmany similarities between regions ofBm86 and the epidermalgrowth factor(EGF) precursor and other proteins containingEGF-like repeats. The similarities are mainly due to theconservation of the 6 cysteine residues within the EGF-likeregion. Several regions of Bm86 (Fig. 2) fall into the patternCys-Xaa4-8-Cys-Xaa3-6-Cys-Xaa8_11-Cys-Xaao_1-Cys-Xaa5_15-Cys, where Xaa is any amino acid except cysteine.The pattern in EGF is Cys-Xaa7-Cys-Xaa5-Cys-Xaa1o-Cys-Xaa1-Cys-Xaa8-Cys. In EGF, 5 amino acids precede thefirst cysteine and there are 11 after the sixth, but we showonly the sequence between the cysteine residues in Fig. 2because it cannot be predicted where the regions begin andend in the Bm86 sequence. There are no obvious proteolyticcleavage sites that would release a single EGF repeat fromBm86 in a manner similar to the release of EGF from itsprecursor.

Several other extracellular proteins have been found tocontain EGF-like regions (reviewed in ref. 15). These fall intotwo general categories: those involved in blood coagulationand complement cascades and those associated with theregulation of cell growth. Bm86 clearly resembles the lattergroup, which is characterized by multiple EGF repeats,transmembrane or carboxyl-terminal hydrophobic regions,and location on the extracellular surface. The function ofBm86 is not known but, by analogy with the other membersof this group, one possibility is that Bm86 is a cell membrane-bound ligand transmitting positional or cell-type informationto adjacent cells and perhaps even influencing the cell lineageof those adjacent cells in a manner similar to the function ofthe Drosophila Notch protein (16).There are some striking internal sequence homologies at

the carboxyl terminus of Bm86, which includes the EGF-likeregion H (Fig. 2). There is sufficient sequence similarity to

9658 Biochemistry: Rand et al.

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Biochemistry: Rand et al. Proc. Nati. Acad. Sci. USA 86 (1989) 9659

M Av (y k&Al L Pe Vd Ak AlATO COT GGC ATC aT 10TM C GO 0CCGcT

Ph CysuAg Am Ala (u Cys an Val Val

Ala (Mu Lys Oh Cys (u Tyr Ly Asp T11GC~rG AAG CAA TOC GM TAT MA GACACO

Cys Val Cps (u Ala SCr Asp Amp Las 11TOC arC TGCGAACA TOO GAC GAT CTA ACO

Lys LAs Avg Tr1w Asp MPh 1k (MyQAlMO T1 COCACO GATGGG TIT AUT GCOCA

T11 Cys Ls Avg Pro Asp L11wCysILACO TOT ClT CGT CCC GAC T1G ACC TOC

Ala Cls Ser Ala Ala Pro Po Ala AspGCAAAC TGT TCA 0Cc OCT Ccr CCA GOTOAC

T11 Lys Gin Ala Gly Ph VaI Cys Lys HisACG MA GM GCroGGo TIT GTCTGC MG CAT

Mu MpGly Be Tlb Cys Lys Se Ak SmGAA GAT GGC ATU ACC TGC AA AGT ATr TCO

VA His Lys Gy T11 VaI La Cys an CpuGTG CAC MA GGAACr GTO TrG TGT GAGTGC

His Glu Glu Ph MeAsp Cys Gy VA TyrCACGM GM MTT ATG GAC TOTGGC GTA TAT

k Amu Glu Cys Ls La Am (Mu Tyr TyrAtC MAT GM TGC CTA CrG ATGAG TAT TAC

Axp Ar Vl LaU Gl(u Al 1k Avg T11 Sm.ATr CY1T C.TT .Tr M.A (IM ATA CY.. AM AC.riAI ILU I i I I* Ii* AUUW iAIKtLxjA.UK

Lcu k Al Clu Lys Pro L1w Sm Lys Hiscrc ATA GCA GAGMA CA CMTG TA AA CAC

Lys L Lan 1B Le Ly Am Sm AL 1TiMOG Tr CrG AlT MO MA AAC TCr OCA AcA

Glm Am LyV Cs VALyVA AmpAmacA Ac AAA TGC arCAZam SGAC AMC

Lcs Ag Avg Ser Val Cys Lys Al (y VaICrA CGC COC TCC GTG TOT MAA CT GAGTTAm (My Lys Al Am Cys Gin Cys Pro ProAACGGC AGCGCOT TOC CAATOC OCA CCA

G(n G(u Cys GOh Ap Lys Lys Lcu Gin CysCAAGM TOC CAAGAC OA OCrO GAOTOC

Pro Ala Lys Asp Ser Cys Smr Gia (u AsCCT GCC MAA GAC TCr TGC ATOGAA GAG AT

Lys GI Lv Smr Gin Ala 11w 1w Ala AlaG GAA TCr G Gc AcAAcA ocrGco

VA' Scr Ala Tw Gly LIU Lau Lasu La LGOA tA GCT ACrcrG CrC T1G TrA Cro CrC

Vi Scr LA 1k Val (MIGy T11 Al OhGTT itA CTO AUT OTAGAGGGC ACA

Pro GMy Al (u Asp Asp Ph V Cys Lysa O GCA GAGGAr GATTTIC GTGTOC A

Cys 1wTvr (MaGU Cys Ser Tyr (My ArgTGC AA ACA A0GAOGTOCA0C TATOA CMT

LA1 Gh Cys Lys 1l LwAm Ap Tyr AlaCrA CAATC MA AT MAAAAAT TAC OCA

Th1 Cys Asp Cys (My Oh Tsp (My Ala bMACO TOT GAC TMT aTGATGG GaTrGCG AT

Ap I s (In Am LAu La Gin AvgGA>C CrC TOCGAGAAAACCr C1TCA O

Sm Tyr Cys Smr Pro (My Sm Pro L - (yTCC TAT TOC iTr CMTrGGGAC CCCAAGA

Gly Cys Ai Ser Thr Gly 1y9 Ala Ty (axaGA T7ChAG TCO ACCGCICA GMt TAC Q*

His 11w Val Smr Cys 11w Al Oa Oh LysCAC ACA ar AGC TGC ACT GCT GAGCAA A

Pro Tip Am Gln Hi Lau Val (y Asp 11wCOO TOG MT CAA CAT CTAGTG GGGGAC ACG

Ma Am Avg Oh Smr Cys Tyr Cys Pro TipATG MT CA CA AAGC TOC TAT TOT CCA TGO

Tyr T1 VId Ser Ph 11w Pro A..h...SmTAC ACOGTO TA TlC ACCOCA AAC ATA iTr

Be (My Lys (Mu VId Ph Lys Val Gin laATC GAK A&AMGM aITTr MOG GTT GAG ATA

VaI LAsA1g Lys La Ohn Al Cys au HisGTG Crt AGO A crcA CA GOCA TOCGAG CkT

(Mu Be Oa (Mu (M Am L1 Cys Ap SerGM AlT GM GAAGAGAAC CIT TOC GAC AGT

1= P2e T Per (An Cys Al 4Amp Tcrc T1C Trc CAGT G acr SATGaTOTSmt Cys Am (Mu Am Oh On Ser (a Cys

TOC AAC GM AC GAGCAG TCOGAG TGT

AP T1r Lys Pro (Ry Ohu 1e (y Cys 1kGAC Acr MA CC GGGGAG AT GOCTOC ATUVal Tyr Lys Am Hi Lys Ala Oh Cys GinaGT TAC MA MC CAT AOCA GAM TOC GAGAm(Gy Lys CysO Sr Smt Gly GOl ArgAMT GOT A TOrT CAAACATa GOCAG caT

T1 T11 1rw l1r Lys Al Lys Amp Lys AspACT ACA ACA ACGAA GCGMA GACMOGATAa Ak 1w SCr VA 11w Ak A Smr LaGCA GCT ACT TOA GaC ACCOCA GCA TCG 170

1 cwaACAOcrcr TaroarrcoXcoCsarTAG

Sm Se lb Cys Sm Asp Ph (ly Am GuTCA TCC AU TGC TCAC 1TC 000 AACGAGCys Pso Avg Asp Am Ma Tyr Ph Am AlaTOT COCGA GATMT ATO TAC TrCMT GCrCys VaI Oh Sm A ru2 .M Lys Al SmrTO1 a" AOTACGAAC0 OAOCTlAsp Cys AvgAiAvg (y 1yI la

AOT OAC TOC A AAI CGA OOrACAT GCrT11w Aig Am Cys Vid Pto 11w

AAC ATG ACC ACC CGOAAC Tar GTC CCr ACCATp SAt g Cys Cys Ohn (ly TAm I1GAsp Smr CarTOGTCCAGGG AAc ArA

Pro Amp G Gi Cs i.Am Aa CsLyCco GOcGGAC MTG A T GoCTT A

C k1 Cys Pro Sm Gly Sm 11k VA AlAf"W*Ws n AfrrfUf'rtTr i nrrf'tV

GOh lr Cys Avg Pro 1I G( Amp Cys AvgCAG AM TGCCOC CCAACCGMGAC TOT COT

Cys Be Set Asp Cys Val Asp Lys Lys CysTGC ATA AGT GAT TGC GrCGAC AAO AAA TGC

L SrAsg Lys Pro G(y Pro An ValATC AGmAA CCOG GoC OcA MAT GTC AAC

Phe A Sc Ap His Cys 16n T Tyr anTTT GAT TCrGAC CAT TGCAAA aTLas C (Mu Asp Be Lys Ala ArgCrT AA.C TOCACO CAG AUhAA) GC&AGA

Pro 1k G(y Oh Tip Cys W Ma Tyr ProCCA ATC GGCGAA TGGTGC ATG ATG TAT COG

1As Lm Lyr Ap Ginhu Ala Ala Tyr LysCrG CTCAAG GAT CAG GAA GCT GCC TAC MAA

1w T11 T1r Tyr (u Mat T1 Ara GIy AgAcA AcA A TAC GAGATm ACA CnAO(OTCc

Ala Asp Ly GI Gin S Ph Val Tyr GiuGCr GAC MAGGG CAA ATA TIT GTT TAC GAA

Gs Ag Th1 T11w Cys Am Pro Lys fIkGAG COT ACC ACA TGC AC Ccr AAG ATA

Cys Piro Ap Asp His Ga Cys Tyr Aug GinTGT CCF GAT GAT CAC GAGTGT TAC AGGGAG

Cys Vl Be Qu As Gly Ly Ala Vd CysTGT OTA ATA GAA AAC GGAOG C GTT TGC

PtoAmp Pro (y Lys Scr Sat Ala Ala AlCCA G6r CET GGA AAG TCA AGT GCr GCA GCA

EndTM _GAAGATOtUCAAcnTATACGGAACAG

FIG. 1. The DNA sequence and the derived amino acid sequence of Bm86. Regions-corresponding to the 72-, 51-, 63-, and 50-mer probesand the five sites for potential N-linked glycosylation are underlined.

suggest that the amino acid segment 492-529 is an EGF-likeregion, albeit with only 5 cysteines. Because the strongestfeature of EGF-like regions is the conservation of the cys-teine residues, we considered that a mutation could haveoccurred at some stage in the construction of the cDNAlibrary. We therefore isolated a Bm86 clone from a differenttick cDNA library and determined the DNA sequence in thisregion. Base 1552 in the new clone is a guanine instead of thethymine in the original clone, translating to cysteine insteadof phenylalanine in the predicted amino acid sequence.The significant sequence similarities shown in Fig. 2 sug-

gest that amino acids 576-600 comprise a truncated EGF-like

region that contains only the first 4 cysteines. There are nocysteines after position 600. It has been determined that inEGF and in the EGF repeat of bovine factor X, disulfidebonds are formed between the cysteine pairs 1 and 3, 2 and4, and 5 and 6 (17, 18). If the same occurs in Bm86, the576-600 region could form the first two disulfide bonds of anEGF-like region. There are 4 cysteines between regions Band C (see Figs. 2 and 3). The pattern in this region,Cys-Xaa18-Cys-Xaal-Cys-Xaal2-Cys-Xaal8, falls outside thepattern of Bm86 EGF-like regions. But the region fits thepattern if the cysteine residues are 2, 4, 5, and 6. The disulfidebond that would be missing in this case from this possible

AMINO ACIDPOSITION

A 24 - 65B 71 - 103

113 - 147C 152 - 183D 209 - 246E 255 - 291F 295 - 334

G 492 - 529H 535 - 567

576 - 600

1 2

S D F G N E F -

C K T R E - - - -

cccc

ccc

R P D LK T K E AT A E Q KV D K K -

N E N QKM I

s E D N

T - -

G F VQ T -

-

SQ E -

CCCCC'CC

CCC

4 5

R N A E --E V V P G A E D D FS Y G R - V E S N P S K A S -R N R G G T A K L R T D G F I G AK D L - - - C E K N L L Q R D S RK H G - - - C R S T G K A Y E - -R P T E D - C R V H K G T V L- -H E E F M D C G V Y M N R Q S--

I F*V Y E N G K AN- -QDKlEICIVYK [ NEK AlE --3JSS[3JR j j G VA- -

CCCCCCC

C.CIc

K C P R D NV C E A S DD C G E W G

C Q G W NT C P S G SE C P W N QY C P W K S

QCcP TE C OD H

M Y F N A A E K QD L T L Q ----A M N M T T R N -

T A N-T V A E D G I T -

H L V G D T - - -

R K P G P N V N I

K P G E I G - - -

E - - -

FIG. 2. Comparison of the amino acid sequences of the regions of Bm86 whose sequences fit (or partially fit) the pattern Cys-Xaa4-8-Cys-Xaa3-6-Cys-Xaa8-ll-Cys-Xaao-1-Cys-Xaa5-15-Cys (where Xaa is any amino acid except for cysteine). Dashes were inserted to alignthe cysteine residues (shaded). The regions containing 5 or 6 cysteine residues are labeled A-H for reference (see also Fig. 3). In thecarboxyl-terminal region, residues identical in two or three of the aligned sequences are enclosed in boxed outlines. At amino acid position 507,the asterisk highlights the phenylalanine identified from the sequence of the first Bm86 cDNA clone that is predicted as a cysteine from thesequence of a second cDNA isolated from a separate library.

32

30122

60212

90302

120392

1SO482

180572

210662

240752

270842

300932

3301022

3601112

3901202

4201292

4501382

4801472

5101562

5401652

5701742

6001832

6301922

2014

2126

2235

6

- 1c

- -1c

- -1cc- - C

NEC

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"partial EGF repeat" is that which forms in EGF and bovinefactor X between cysteines 1 and 3. A diagram of Bm86 thatillustrates the above features (Fig. 3) shows that most of theprotein consists of EGF-like regions or regions with only 4cysteines that may be "partial EGFs."

Expression of Recombinant Bm86 in E. coli. pBTA708encodes a fusion protein of 143 kDa consisting of the first 651amino acids of E. coli /3-galactosidase, 599 amino acids ofBm86 (amino acids 31-629), and 19 amino acids that areencoded by other parts of the vector, such as the cloning-siteregions. After induction of cultures of BTA1752 with IPTG,a band of z143 kDa could be seen on polyacrylamide gels(Fig. 4A). Because about 10% of Bm86 consists of cysteineresidues it was expected that Bm86 would be highly labeledby adding [35Slcysteine to the growth medium after induction.A strong band was seen on the autoradiograph in the regionof 143 kDa after induction of cultures with IPTG (Fig. 4B).Further evidence that the 143-kDa band was related to Bm86was obtained by demonstrating that the 143-kDa protein isrecognized by antiserum from a cow vaccinated with nativeBm86 (Fig. 4C) but not by prevaccination serum (data notshown).

Partial Protection of Cattle by Vaccination with 13-Galacto-sidase-Bm86 Inclusion Bodies. Most, if not all, of the /3-galactosidase-Bm86 fusion protein was present as inclusionbodies (19) in the insoluble fraction of the E. coli extract. Acrude preparation of inclusion bodies was used to vaccinatethree cattle which were subsequently challenged with ticks.The most dramatic aspect of the results (Table 2) was the highproportion of damaged ticks that were recovered from thevaccinated cattle. Damage was recognized by the strikingchange in coloration of ticks from the normal gray to red, dueto the leakage of bovine erythrocytes through the damagedtick gut wall into the tick hemolymph. In this experiment,there was only a modest decrease in the number of ticks thatsurvived on the vaccinated animals (24%). However, themajority of the surviving ticks were damaged and the averageweight of ticks dropping from vaccinated animals after en-gorgement was significantly reduced. These damaged ticks

NH2

A

B

Part EGF?

C

D

E

F

G

H

Part EGF?

I

COCH

EGF-like regions164 amino acids

non EGF25 amino acids

EGF-like regions126 amino acids

non EGF157 amino acids

EGF-like regions109 amino acids

Ser/Thr rich 0-linked sugars? 27 amino acidsKEKSEATTAATTIIKAKDKDPDPGKSSMembrane 23 amino acids or phospholipid linkage?AAAVSATGLLLLLAATSVTAASL

FIG. 3. Structure of Bm86. Proposed EGF-like regions are des-ignated A-H. The numbers in the ovals and rectangles refer to thenumber of cysteines in each region; region G is shown as having 5cysteines, the number deduced from the original clone. As describedin the text, a second cDNA clone was found to encode 6 cysteinesin this region. The sites for potential N-linked glycosylation asdefined by Asn-Xaa-Ser/Thr are indicated (-<). The membrane isshaded and possible sites for O-linked carbohydrate addition areshown as short horizontal lines.

were greatly impaired in their egg-laying ability. In thispreliminary experiment, the overall effects of vaccinationwere to reduce the reproductive ability of the ticks on vac-

Am i ii iii ivN1 1 11 1|E X

116 a"9)2.5 go

66.2 i4I

BI ii iii iv

_

CM i ii

FIG. 4. Characterization of recombinantBm86. (A and B) BTA1752 and JM101 contain-ing pBTA224 were grown in tryptone soy broth(A) or in tryptone soy broth containing[35S]cysteine (B). Crude inclusion body prepa-rations were resolved by SDS/8% PAGE andstained with Coomassie brilliant blue (A) orautoradiographed (B). Samples were isolatedfrom JM101/pBTA224 not induced with IPTG(lane i), JM101/pBTA224 induced with IPTG(lane ii), BTA1752 induced with IPTG (lane iii),BTA1752 not induced with lPTG (lane iv). (C)Samples were also transferred to nitrocelluloseand incubated with bovine serum from cattlevaccinated with native Bm86 isolated fromticks. Lane i, JM101/pBTA224; lane ii,BTA1752. Bio-Rad high molecular weight mark-ers were used (lane M, A and C): myosin heavychain, Mr 200,000; ,B-galactosidase, Mr 116,000;phosphorylase b, Mr 92,500; bovine serum albu-min, Mr 66,200; ovalbumin, Mr 45,000.

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Table 2. Results of cattle vaccination and challenge trial

Cumulative Cumulativetick tick Mean tick % ticks Egg/tick Cumulative egg

Animal number weight, g weight, mg damaged weight ratio weight, g

Controls181 979 272 278 1 0.57 155183 1269 319 251 4 0.57 182185 1365 381 279 1 0.57 217Mean ± SD 1204 ± 201 324 ± 55 269 ± 16 2 ± 2 0.57 185 ± 31Total 3613 972 553

Vaccinates180 1191 247 207 81 0.21 52182 662 111 168 80 0.22 25188 880 161 183 74 0.32 52Mean ± SD 911 ± 266 182 ± 84 186 ± 20 78 ± 4 0.25 ± 0.06 43 ± 16Total 2733 519 129

Reduction 24% 47% 31% 56% 77%Three cattle were vaccinated with -400 ,ug of the ,3-galactosidase-Bm86 fusion polypeptide isolated as inclusion bodies

from BTA1752. Due to the high degree of purity of the antigen preparation, control cattle were vaccinated with adjuvantalone. The cattle were subsequently challenged with -1000 tick larvae on each of three successive days. The total numberand total weight of ticks >4 mm (all females) collected from each animal over an 8-day period beginning 18 days after thechallenge commenced was used to calculate the mean weight per tick. The number of ticks that were visibly damaged wasdetermined on each day and the sum of these is expressed as a percentage of the total number of ticks collected from eachanimal. On days 18, 19, 20, and 22 following the commencement of the challenge, 20-60 representative ticks from eachanimal were separated, weighed, and incubated until egg production had ceased. The weight of the eggs produced wasdetermined and used to calculate the proportion of the tick weight that was converted into eggs. This figure was then usedto estimate the total weight of eggs that all of the surviving ticks would have produced under ideal conditions. The percentreduction in the measurements for the experimental groups is indicated relative to the control animals. Differences in totalweight, mean weight, percent damage, and total egg production between the two groups are significantly different (>95%confidence level).

cinated animals by 77% compared with controls (as measuredby the weight of eggs laid by survivors). These results dem-onstrate that protective epitopes are present in the proteinportion of Bm86, and we are confident that it will be possibleto produce an effective recombinant anti-tick vaccine. Math-ematical models (20) predict that if the effects obtained in thispreliminary trial could be maintained in the field for a completetick season, pasture contamination would be reduced to suchan extent that productivity losses due to ticks would beminimal.

We express our appreciation to Drs. Neil Willetts, David Irving,and Vince Atrache for their helpful contributions to the manuscript.We thank Mr. George Riding, Mr. Robby McKenna, and Miss JanineNielsen for assistance during the cattle vaccination and challenge.Mr. Bernie Mclnernie and Miss Megan Ash are also gratefullyacknowledged. This research was supported in part by a grant fromthe Industrial Research and Development Board under the NationalBiotechnology Grant Scheme.

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K. L., Brown, M. S. & Goldstein, J. L. (1984) Cell 37, 577-585.15. Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J.,

Esnouf, M. P., Smith, K. J. & Brownlee, G. G. (1988) EMBOJ. 7, 2053-2061.

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