Molecular and Biochemical Parasitology, 46 (1991) 45-52 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100037D

45

MOLBIO 01502

Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes Carlos E. Suarez, Guy H. Palmer, Douglas P. Jasmer, Stephen A. Hines, Lance E. Perryman and Terry F. McElwain Department of Veterinary Microbiology and Pathology, WashingtonState University, Pullman, WA, U.S.A. (Received 30 August 1990; accepted 7 November 1990)

A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain ofBabesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specificallyimmunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [3SS]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia 'L', and Texas strains ofB. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia 'L' B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis. Key words: Babesia bovis; Recombinant protein; Surface exposed epitopes; cDNA library; DNA sequence

Introduction

Epitopes exposed on the merozoite surface of Babesia bovis are potential candidates for vaccine development due to their accessibility to the immune system of the host and their involvement in the mechanism of host erythrocyte recognition and invasion. In previous work, we identified a 60-kDa merozoite surface polypeptide on the B. bovis Mexico strain by surface specific radioiodination [1]. Serum from cattle protected against challenge with the Mexico strain bound a 60-kDa polypeptide

[ 1,2]. A panel of four monoclonal antibodies (mAb) to the 60-kDa polypeptide bind the live merozoite surface in a polar immunofluorescencepattern [3]. This binding pattern is similar to previously described apical complex binding antibodies in the related hemoprotozoa Plasmodium spp.[4]. Significantly, the surface exposed epitopes recognized by the mAbs are conserved among B. bovis strains examined to date (unpublished data). In this paper, we describe the cloning and expression of the gene encoding this 60-kDa polypeptide. Materials and Methods

Correspondence address: Carlos E. Suarez, Department of Veterinary Microbiologyand Pathology, Pullman, WA 991647040, U.S.A. Abbreviations: mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; pBv60, plasmid encoding recombinant Bv60; rBv60, recombinant Bv60 protein; IFA, immunofluorescence;

Parasites. A Mexico strain of B. bovis was obtained in 1979 from a Boophilus microplus tick-induced infection of cattle [ 1]. The cloned Mexico strain of B. bigemina (JG-29) employed was described elsewhere [5]. Australia 'L' strain of B. bovis [6] was a gift of Ian Wright (CSIRO, Aus-

46 tralia), and the B. bovis Texas strain was a gift of USDA Animal Disease Research Unit, Pullman WA. The Mexico strain of B. bovis (Mo-7) was cloned by limiting dilution as described [7]. All strains were maintained as cryopreserved stabilates in liquid nitrogen [8]. Organisms of each strain were grown in long term culture by previously described techniques [9].

B. bovis cDNA expression library. Poly(A) ÷ RNA was obtained by oligo(dt) cellulose chromatography [10] from cultured B. bovis-infected erythrocytes washed three times in Puck's saline G and stored frozen in liquid nitrogen. Purified poly (A)+RNA was used to prepare a blood stage cDNA library in )~ZAP II (Stratagene, La Jolla, CA, U.S.A.) by a modified Gubler and Hoffman method using EcoRI adapters (Pharmacia LKB, Piscataway, NJ, U.S.A.) [11]. The cloned insert in plaquepurified )~ phage was subcloned into Bluescript SK(-) phagemid using the in vivo exsion capabilities of)~ZAP II [12]. Immunoscreening. Screening was done with mAb 23/53.156 which binds a conserved surface exposed epitope of the 60-kDa polypeptide of B. bovis (Bv60) [3]. Plaque lifts onto isopropyl thiogalactopyranoside soaked nitrocellulose were screened using mAb 23/56.156 followed by rabbit antibody against murine immunoglobulin, and 125Iprotein A. Reactive plaques were identified by autoradiography [13]. Recombinant phagemid excised from positive, plaque-purified )~ phage was tested for expression by a similar method using colony lifts from transformed, ampicillin-resistant Escherichia coli (XL 1-Blue strain) [ 13 ]. This plasmid was designated pBv60, DNA sequencing. CsCl-purified pBv60 was sequenced as double-stranded DNA in both directions using sequentially derived primers initiating dideoxy chain reactions [14], (Sequenase Version 2.0 Kit, USB, Cleveland, OH, U.S.A.). T7 and T3 promotor specific primers were used in the initial sequencing reactions followed by synthesis of new oligonucleotide primers based on the sequences obtained. Prediction of antigenic sites, signal peptide [15], regulatory signals, and sequence similarity with other genes (GenBank, release 65) was done mbp0905502

using the Genetics Computer Group (GCG) package of the University of Wisconsin, version 6.2 [ 16] on a Vax 11/785 computer.

Antisera against recombinant Bv60 polypeptide. Recombinant bacteria, washed three times in phosphate-buffered saline (PBS), were lysed either in PBS or in 0.1 M sodium deoxycholate by sonication. The resulting bacterial lysate was cleared by ultracentrifugation at 100 000×g for 1 h, the supernatant filtered through a 0.22 ~tm membrane and sonicated again before freezing in aliquots at -70°C. Two rabbits each were immunized four times with either 0. l ml of PBS or sodium deoxycholate crude extract emulsified in 1 ml of complete Freund's adjuvant for the first injections, and incomplete Freund's adjuvant for the next three. Control antibody was produced in the same manner against a lysate of XL1-Blue bacteria with Bluescript phagemid encoding an unrelated Anaplasma marginale polypeptide (pAm36--4). The ability of the rabbit antisera to bind the live merozoite surface was determined by indirect immunofluorescence as previously described [3,17]. Radioimmunoprecipitation and SDS-PAGE analysis. Parasite antigens were metabolically labeled in vitro with [35S]methionine and processed as described [ 17,18]. For labeling of recombinant antigens, purified pBv60 was linearized by digestion with BamHI and used to direct in vitro transcription and translation with a prokaryotic DNAdirected translation kit from Amersham (Amersham Corporation, IL, U.S.A.). Radiolabeled antigen (106 protein bound cpm) was incubated with either 5 ~tg ofmAb or 5 txl of serum for I h at room temperature. 100 ~tl of a 20% suspension of recombinant protein G-Sepharose (Gammabind Plus, Genex, Gaithersburg, MD, U.S.A.), blocked overnight with washing buffer (0.01 M sodium phosphate, pH 7.0/0.15 M NaC1/0.01 M EDTA/ 1% NP-40) containing 1 mg m1-1BSA, were added for 30 rain at 4°C. Immunoprecipitates were washed twice in washing buffer containing 1 mg ml -1 BSA, four times with washing buffer containing 2.0 M NaC1, and twice with washing buffer alone, and were eluted by boiling for 3 min in 100 ~tl of SDS-PAGE sample buffer. Immunoprecipitated polypeptides were electrophoresed on continuous 14-03-91 08:30:25

47 7.5-17.5% gradient SDS-polyacrylamide gels and the gels were fixed and processed for autoradiography as described [ 18].

not by control mAb. mAb 23/56.156 did not react with control recombinant bacteria expressing an unrelated B. bovis polypeptide (data not shown).

Depletion of By60 polypeptide from native antigen. [3YS]methionine-labeled B. bovis antigen (106 protein bound cpm/tube) prepared as above for immunoprecipitation was incubated with 5 ktl of rabbit antiserum against recombinant polypeptide Bv60 (rBv60), (R 179), or 5 ~1 of rabbit antiserum against A. marginale pAm36-4. Following immunoprecipitation of the immune complexes with protein-G-Sepharose as described above, both supernatant antigens were transferred to new tubes and incubated with another 5 ~tl of the same antiserum. Immune complexes were again precipitated with protein G, and the same cycle was repeated six times. Supernatants from the last cycle were incubated with 5 ktg of mAb 23/56.156. Immune complexes were precipitated with protein G, washed, eluted, and examined after SDS-PAGE and fluorography as above.

Characterization and DNA sequence of the insert. Linearized recombinant phagemid migrates in agarose electrophoresis with a size of 4.9 kb. An insert of 1.9 kb was recovered in a double digest using Hind11I and Pstl, both of which cut in the multiple cloning site of ~,Zap 1I but do not cut inside the coding sequence. The DNA sequence of the insert is shown in Fig. 1. Starting with methionine in position 122, a single open reading frame (ORF) of 1695 nucleotides is present. Two stop signals precede the long ORF at nucleotide positions 74--76 and 116-118, and the ORF is not in frame with I]galactosidase. However, it is possible that B. bovis sequences preceding the long ORF direct transcription and translation of the Bv60 insert in E. coli. A region compatible with prokaryotic promotor consensus sequences are located at positions -10 and -35, including an A+T-rich sequence upstream of the-35 region [20] (Fig. 1). In addition, a possible prokaryotic ribosome binding site defined by the consensus sequence GGA preceded by an adeninerich stretch, starts at position -16 from the first ATG codon of the ORF (Fig. 1). Computer aided searching of the GenBank data base (release 65) revealed no significant similarity to known sequences. The ORF encodes a polypeptide of 565 amino acid residues with a calculated size of 64.9 kDa. At the N-terminus of the polypeptide there is a hydrophobic stretch of 22 residues, including a consensus cleavage site between residues 21 and 22 (Fig. 1), that may function as a signal peptide. Two additional hydrophobic stretches (residues 102-106 and 163-180) are present. A conserved sequence with a tandemly repeated 23-residue periodicity, starting as PTK(X)F, occurs seven times from residue 317 to 477 (Fig. 2). The sequence PTKEFFREAPQATKHFL is exactly duplicated once (residues 386-402 and 409-425). Variation among the additional repeats usually involves conservative substitutions. A degenerate version of the repeats also occurs between residues 478-500 and 512-534 (Fig. 2).

Southern blot hybridization. A [32p]RNApBv60 probe prepared using T7 RNA Polymerase (Riboprobe System, Promega, Madison, WI, U.S.A.) was hybridized as described [19] to undigested or restriction enzyme-digested B. bovis genomic DNA, bovine leukocyte DNA, B. bigemina genomic DNA or pBv60. The membranes were washed at room temperature with buffer 2xSSC (0.30 M NaCI/0.03 M trisodium citrate, pH 7.0), 2×SSC/ 0.1% SDS, 0.5xSSC/0.1% SDS, 0.1xSSC/0.1% SDS and with 0.1 xSSC/1% SDS at 65°C [ 19]. Results Identification of a cDNA clone encoding an epitope of the Bv60 polypeptide. A cDNA library prepared from a cloned Mexico strain ofB. bovis containing 1.3x10 s recombinant plaque forming units was immunoscreened using the mAb 23/56.156, specific for the Bv60 polypeptide. A positive clone (designated ~,Bv60), reactive with 23/56.156 but not with the isotype control mAb, was identified and plaque-purified. ~,Bv60 was converted to pBv60 by in vitro excision of the ~ZAP II phagemid. Bacteria containing pBv60 expressed a product specifically recognized by mAb 23/56.156 but

Characterization of the recombinant product.

In

I 61 121

GACGGATAGTATTTTACATATACATTTGTCGACTTTTATATATAGCAGTGCTATAGAC~ P P AC~TACACAGAT~TCTTTAGATACT~GTTC~T~TATTACGGACATATTG~AC RBS ~TGAG~TCATTAGCGGCGTTGTCGGTTGCCTTTTCTTGGTGTTTTCACACCATGTGTC M R I I S G V V G C L F L V F S H H V S

60 120 180

181

TGCTTTTCGCCAC~TCAGAGAGTAGG~GTCTCGCTCCAGCTG~GTGGTAGGTGATTT A ~ F R H N Q R V G S L A P A E V V G D L

240

241

~CCTCCACATTGGAAACAGCTGATACTTTGATGACTCTCCGTGACCACATGCAC~CAT T S T L E T A D T L M T L R D H M H N I

300

301

TACT~GGATATGAAACATGTTTTGAGC~TGGTCGTGAGCAGATTGTA~TGATGTTTG T K D M K H V L S N G R E Q I V N D V C

360

361

CTCT~TGCTCCTGAGGACTCC~CTGTCGTGAGGTAGTT~C~TTATGCTGACCGTTG S N A P E D S N C R E V V N N Y A D R C

420

421

TGAAATGTACGGATGCTTTACGATTGAC~TGTCAAATATCCGTTGTATC~GAGTACCA E M Y G C F T I D N V K Y P L Y Q E Y Q

480

481

ACCTCTATCTCTTCCAAACCCTTACCAGTTGGATGCTGCGTTCAGATTGTTC;tAAGAGAG P L S L P N P Y Q L D A A F R L F K E S

540

541

TGCATCG~CCCTGCC~G~CAGCGTAAAACGcG~TGGTTGCGTTTCAGA~TGGAGC A S N P A K N S V K R E W L R F R N G A

600

601

G~CCaTGGTGATTACCACTACTTCGTCACTGGTCTGTTG~C~C~TGTTGTGCACGA N H G D Y H Y F V T G L L N N N V V H E

660

661

GG~GG~CTACCGATGTTG~TATCTTGTC~C~GGTACTCTATATGGCTACCATG~ E G T T D V E Y L V N K V L Y M A T M N

720

721

CTAC~GACTTATTTGACAGTAAACAGTATG~CGCC~GTTCTTC~CAGATTCAGCTT Y K T Y L T V N S M N A K F F N R F S F

780

781

CACTACAAAGATATTCAGTCGTCGTATTAGGCAAACATTGAGTGATATCATCAGGTGG~ T T K I F S R R I R Q T L S D I I R W N

840

841

TGTTCCTG~GATTTTG~GAAAGGAGCATCG~CGTATCACTC~CTTACTAGCAGCTA V P E D F E E R S I E R I T Q L T S S Y

900

901

CG~GATTACATGTTGACCCAGATTCC~CTCTTTCC~GTTTGCACGTCGTTATGCTGA E D Y M L T Q I P T L S K F A R R Y A D

960

961

CATGGTG~G~GGTTCTGCTCGGTAGCTTGACCTCGTACGTTG~GCTCCTTGGTAC~ M V K K V L L G S L T S Y V E A P W Y K

1020

1021

~GATGGATA~GA~TTCAGAGACTTTTTCTCTaaa~CGTTACCC~CCTACA~G~ R W I K K F R D F F S K N V T Q P T K K

1080

1081

GTTCATCGAGGATACT~CG~GTTACCA~CTATCTGAAAGCC~TGTTGCTGAGCC F I E D T N E V T K N Y L K A N V A E P

1140

1141

CACTA/~GTTTATGCAGGACACTCACGAAA~CCAAAGGCTATCTG~J~AGAG~TGT T K K F M Q D T H E K T K G Y L K E N V

1200

1201

AGCCG~CCTACT~GACTTTTTTC~GGAGGCTCCTC~GTCACCAAACACTTCTTCGA A E P T K T F F K E A P Q V T K H F F D

1260

1261

TGAG~CATTGGCC~CCCACC~GGAGTTTTTCAGGG~GCTCCCC~GCCACT~aCA E N I G Q P T K E F F R E A P Q A T K H

1320

1321

TTTCCTAGACGAAAACATCGGTC~CC~CC~GGAGTTCTTCAGGGAGGCTCCTC~GC F L D E N I G Q P T K E F F R E A P Q A

1380

1381

CACT~GCACTTcCTAGGCGAG~TATTGCTC~CCTACTA~G~TTTTTC~GGATGT T K H F L G E N I A Q P T K E F F K D V

1440

1441

CCCTC~GTCACC~G~GGTTAT~CTGAG~CATTGCTC~CC~CT~GGAGTTCCG P Q V T K K V I T E N I A Q P T K E F R

1500

1501

GAGGGAGGTTCCTCATGCTACCATGAAAGTCTTG~TGA/%AACATTGCTC~CCTGCC~ R E V P H A T M K V L N E N I A Q P A K

1560

1561

GGAAATCATACATGAGTTTGGTACAGGCGCC~G~TTTCATTTCCGCAGCCCATG~GG E I I H E F G T G A K N F I S A A H E G

1620

1621

TACT~GCAGTTCTTAAACGAAACTGTTGGCC~CCTACA~GG~TTCCTG~CGGAGC T K Q F L N E T V G Q P T K E F L N G A

1680

1681

TTTAGAAACTACTAAAGACGCATTACACCATCTGGGTAAATCATCAG~G~GCC~CCT L E T T K D A L H H L G K S S E E A N L

1740

1741

TTATGATGCCACGGAAAATACCACTCAGGCT~CGACTC~CTACTTCC~CGGTG~GA Y D A T E N T T Q A N D S T T S N G E D

1800

1801

CACCGCCGGATACCTCTGATGAGATGCGTTTAT~TGGCACAAACTC~CA/%ATGATGTA T A G Y L

1860

1861

TCGTCATCTGATCCATCGGTTTTC~TATTGTATTGGATGC~TATCTG~TGCATATGA

1920

1921

TGCGACAGTTTCCATCATCGGGTGCCG~TCGT~CTCTCAT~CACCATTTT~GTTAT

1980

1981

GCTCGTGCCG

1990

Fig,I. Nuclcotidc sequence of pBv60 and deduced amino acid scqucncc of the coding region.~ e stop codons m c indicatedby an asterisk~ d potential N-glycosylation sites me underlined. ~tativc prokmyotic promotor rcgion (-10 ~ d -35 regions) ~ d ribo-

some binding sequences me indicated by 'P' or 'RBS' respectively. ~tative signal ~ptide cleavage site is indicated by ~ a~ow.

49 294 VEAPWY K R W I K K F R D F F S _KIh'VTO 317 PTKKFI E D T N E V T K N Y L KAy_ AE 3 4 0 PTKKFM Q D T H E K T K G Y L ~__NVAE 3 6 3 PTKT_FF K~A~QVT_KKHFF DENIGQ 386 PTKEFF R~A_PQATKHFL DEN~,GQ 409 PTKE_FF R~A_PQAT_KHFL G E N I A Q 4 3 2 PTKE_FF KDV__PQ~__KKKVI T E N I A Q 4 5 5 PTKEFR RE V_PHA_TMKVL N E N I A Q 478 PAKEII HEFGTGAKNFI SAAHEG 501 TKQFL N E T V G Q 512 PTKEFL N G A L E T T K D A L HHLGKS Fig. 2. Amino acid sequence of Bv60 from residue 317 to 534. Highly conserved residues are denoted by an underline.

order to compare recombinant polypeptide to native Bv60 polypeptide, the [35S]methioninelabeled products of pBv60-directed transcription and translation were immunoprecipitated with mAb 23/56.156 (Fig.3). The mAb immunoprecipitated polypeptides ranging in size from 60 to 30 kDa and the 60-kDa recombinant polypeptide comigrated with the 60-kDa native polypeptide. Furthermore, mAb 23/56.156 did not precipitate any labeled polypeptide after incubation with an irrelevant in vitro translated product, and mAb which immunoprecipitated a 42-kDa metabolically labeled B. bovis polypeptide failed to precipitate any polypeptide when incubated with the products of in vitro translated pBv60 (Fig. 3). The specificity of antibodies raised against rBv60 was tested by immunoprecipitation of [35S]methionine metabolically labeled B. bovis merozoites. The results are shown in Fig. 4A. Rabbit immune sera raised against both PBS and sodium deoxycholate extracted rBv60 are able to specifically immunoprecipitate a 60-kDa polypeptide from native B. bovis. No polypeptides were detected when pre-immune sera were used in identical immunoprecipitations. In addition, the rabbit antibodies against rBv60 bound to the live merozoite surface in a polar immunofluorescence pattern while antibodies in the pre-immune sera were unreactive (data not shown). To further verify that the antibodies produced by the recombinant polypeptide recognized the same 60-kDa native polypeptide as mAb 23/56.156, antibodies from a rabbit immunized with rBv60 were used to deplete native 60-kDa polypeptide from a [35S]methionine metabolically labeled preparation ofB. bovis, mAb 23/56.156 failed to immunoprecipitate any native polypeptide after depletion with

1

2

345

2 0 0 k D ,,-

9 2 . 5 kD " 6 9 kD " 4 6 kD ,.-

3 0 k D ,'-

14.3 kD "-

Fig. 3. Characterizationof [35S]methionine-labeledproducts of pBv60directedin vitrotranscriptionandtranslation.Immunoprecipitationof in vitro [35S]methionine-labeledmerozoites (lanes 1and 2), pBv60directedin vitrotranscriptionand translation(lanes4 and5), or controlplasmidDNAdirectedin vitro transcription and translation (lane 3) with mAb BABB-35 (lane 1 and 4), mAb 23/56.156 (lane 2,3 and 5). Molecular weightstandardsare on the left. antiserum against rBv60, but was able to immunoprecipitate the 60-kDa native polypeptide after depletion with control rabbit serum pAm36-4, prepared against an unrelated A. marginale recombinant antigen (Fig. 4B).

Characterization of the gene encoding the Bv60 polypeptide. To determine the number of parasite gene copies encoding the polypeptide Bv60, purified genomic DNA from a cloned Mexico strain of B. bovis was digested with XbaI, which cuts outside the coding sequence, and the products analyzed in Southern blots with a pBv60 [3zp]RNA probe. The probe strongly hybridized with only one fragment of the digested B. bovis genomic DNA, and did not hybridize to digested B. bigemina nor bovine leukocyte DNA (data not shown). Southern blotting of genomic DNA from the cloned Mexico, uncloned Mexico, and Texas strains digested with SspI, which cuts just inside the 5' and 3' ends of the coding sequence, produced bands of 1.3 kb and 0.45

50

1 2

34

5

6

78

9 10

1 2 3 4 5 2 0 0 kD ~

9 2 . 5 kD ~

69 kD ~ 4 6 kD ~

3 0 kD ~

2 0 0 kD

9 2 . 5 kD ,.6 9 kD ,'4 6 kD ,"

30 kD " 14.3 kD ~

14.3 kD ,'Fig.4. ImmunogenicityofrecombinantBv-60.(A) Immunoprecipitationof [35S]methionine-labeledmerozoiteswithrabbitpre-immune serum (lanes 14), rabbit serum after immunization with PBS extract (lanes 5 and 6) or sodium deoxycholate extract (lanes 7 and 8), mAb 23/56.156 (lane 9); or mAb BABB-35 (lane 10). (B) Immunoprecipitation of [35S]methionine-labeledmerozoites with mAb 23/56.156 (lane 1), control rabbit pAm36--4 antiserum (lane 2), rabbit rBv60 antiserum (lane 3), and mAb 23/56.156 after 6 cycles of depletion with rabbit rBv-60 antiserum (lane 4) or control rabbit pAm36-4 antiserum (lane 5). Molecular weight standards are indicated on the left.

kb, exactly the same pattern observed after SspI digestion of pBv60 (Fig. 5). Southem blotting of an SspI digest of Australia 'L' strain resulted in hybridization with 1.28-kb and 0.45-kb fragments (Fig. 6). Discussion

Despite the clear demonstration of protective homologous and heterologous immunity following recovery from B. bovis infection with live merozoites, the mechanisms and targets of immunity remain poorly defined. Passive transfer of protection with immune serum [21, 22] leads us and others to hypothesize that protective immunity is mediated by antibody directed against the merozoite surface.

We have focused here on the 60-kDa polypeptide based on the following criteria: (i) epitopes exposed on the live merozoite surface [ 1,3]; (ii) apical complex-like immunofluorescence pattern [3] and (iii) conservation of surface exposed epitopes among diverse geographic isolates (data not shown). The cloning and characterization of the gene encoding this surface polypeptide provides a defined source of antigen for experimental immunization and for detailed comparison among heterologous isolates of B. bovis. Several lines of evidence indicate that the cloned sequence described here encodes the entire 60-kDa surface antigen: (i) the 1.9-kb insert appears to be a faithful replica of the gene, which is likely present in the B. bovis genome as a

51

1 2345

1.35kb,-

0.45kb,"

Fig. 5. Southernblot with a 32P-labeledRNA probe prepared frompBv60. SouthernblotafterdigestionwithSspIof: pBv60 (lane 1), genomicDNAfromunclonedMexicostrain (lane2), Australia 'L' strain (lane 3), Texas T2B strain (lane 4), and clonedMexicostrain(lane5). The sizesof the majorhybridizationfragmentsare indicatedon the left. single copy and without introns; (ii) the size of the ORF is compatible with the native 60-kDa polypeptide; (iii) pBv60-directed transcription and translation produces a recombinant polypeptide that comigrates with the native polypeptide and is specifically reactive with mAb 28/56.120; (iv) antibodies raised in rabbits against rBv60 recognize the same 60-kDa surface exposed polypeptide defined by mAb 23/56.120 and (v) rBv60 elicit antibodies which react with live merozoites in a polar immunofluorescence pattern. These data indicate that rBv60 is a faithful immunological replica of the native Bv60 polypeptide and suggest that the surface exposed epitopes present in the recombinant polypeptide are highly immunogenic. The Bv60 polypeptide bears surface epitopes which are conserved among B. bovis strains from disparate geographic isolates, including all strains examined in this paper. Hybridization experiments described here also indicate significant structural conservation of this gene among the Texas and Mexico strains. The small 450-bp SspI fragment was conserved among all strains tested. However,

the estimated 50-bp variation in the size of the major fragment obtained after SspI digestion of the Australia 'L' strain indicates some heterogeneity in this region of the gene. Interestingly, the panel of mAbs (including 23/56.156) that immunoprecipitate the 60-kDa native polypeptide in the Mexico and Texas strains, also immunoprecipitate a 58kDa native polypeptide from the Australia 'L' strain (data not shown). The 50-bp discrepancy in the large SspI fragment approximately corresponds to the observed difference in molecular size of the native polypeptide between strains. Although the expression of the surface exposed epitope recognized by mAb 23/56.156 remains unaffected, the biological significance of gene and polypeptide size variation in the Australian 'L' strain as compared to the American strains is unknown. The presence of a hydrophobic 22-amino-acid stretch, including a cleavage site in the amino terminus, is characteristic of a signal peptide. This sequence may target the 60-kDa polypeptide to the external membrane, a feature consistent with the surface localization previously determined by immunofluorescence and surface radioiodination [ 1, 3]. Inclusion of this sequence in a eukaryotic expression vector may be critical for membrane targeting to enhance immunogenicity of an experimental vaccine. Computer aided secondary structure analysis predicts the presence of at least two additional hydrophobic stretches (residues 102-106 and 163-180) which may help to anchor the Bv60 polypeptide into the membrane. However, neither a C-terminal hydrophobic transmembrane segment nor a common signal sequence for attachment of a glycosyl-phosphatidylinositol anchor have been found. In addition, Bv60 is not posttranslationally modified by incorporation of myristic acid or glucosamine [7]. The polypeptide Bv60 also contains a large region of repeated amino acid sequences which accounts for 32% of the total number of amino acids. The region from amino acid 317 to 477 may be considered as seven tandem repetitions of 23 residues following a pattern as depicted in Fig. 2. Computer analysis of the polypeptide sequence indicates that the pattern present in this region has a secondary structure with a predicted high antigenicity. We are presently determining if this predicted antigenic region is surface exposed, allowing us to directly test

52 the h y p o t h e s i s that a n t i b o d i e s a g a i n s t c o n s e r v e d and surface exposed merozoite epitopes can block i n f e c t i v i t y for host e r y t h r o c y t e s .

Acknowledgements T h e a u t h o r s w i s h to a c k n o w l e d g e T e r e s a H a r k ins, B e v H u n t e r , D a v i d Jones, P a t M a s o n , K a y M o r r i s , a n d C a r l a R o b e r t s o n for e x c e l l e n t t e c h n i c a l assistance. T h e s o f t w a r e c i t e d is p a r t o f the V A D M S C e n t e r , a c o m p u t e r r e s o u r c e at the W a s h i n g t o n State U n i v e r s i t y . T h e w o r k w a s s u p p o r t e d b y grants f r o m the A g e n c y for I n t e r n a t i o n a l D e v e l opment DAN-4178-A-00-7056-00, US Department of Agriculture Binational Agricultural Research and Development Program US 4080-86, and the I N T A A r g e n t i n a F e l l o w s h i p P r o g r a m s p o n s o r e d b y the I n t e r - A m e r i c a n D e v e l o p m e n t B a n k and c o o r d i n a t e d b y W i n r o c k I n t e r n a t i o n a l .

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