Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus

Molecular and Biochemical Parasitology, 61 (1993) 179 188

179

© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00 MOLBIO 02033

Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus M a r y - H e l e n Binger a'~, Denis H u g b, Gilbert W e b e r c, Eugene Schildknecht d, M a r k u s H/imbelin ~ and Luis P a s a m o n t e s b* aVitamin Research, VRD/F, F. Hoffmann-La Roche Ltd., Basel, Switzerland," bVitamin Research, Biotechnology 64/7, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland, CResearch Center for Animal Nutrition and Health, Soci~t~ Chimique Roche Ltd., Village Neuf France," and dAnimal Science Research Experiment Station, Hoffmann-La Roche Inc., Wrightstown, N J, USA (Received 7 January 1993; accepted 16 June 1993)

A rabbit serum raised against Eimeria tenella merozoites was used to screen a 2gtl 1 c D N A library made from merozoite m R N A of E. tenella. The insert of the phage clone 2Mz 5-7 revealed an open reading frame consisting of 945 nucleotides, encoding a 33-kDa protein. This size is consistent with the size of a protein translated in vitro from merozoite m R N A and immunoprecipitated with monospecific anti-Mzp 5-7 antibodies. A smaller protein of 24 kDa, located on the surface of the parasite, also reacted with the monospecific antiserum and is the potential processed form of the Mzp 5-7. Furthermore, a recombinant vaccinia virus expressing the Mzp 5-7 antigen was constructed and used to immunize chickens. Key words: Polymerase chain reaction; Coccidiosis; Eimeria tenella; Merozoite; Recombinant vaccinia virus

Introduction

Avian coccidiosis, a disease caused by protozoan parasites of the genus Eimeria, represents a severe problem for the poultry industry. Although losses from mortality with most of the species are negligible, growth and performance of the birds are heavily impaired. Today, the disease is controlled by preventive medication using polyether ionophores or chemical agents as anti coccidial drugs. In the past few years, prophylactic chemotherapy has lost some of its efficacy due to the appearance ~ponding 45.

author. Tel.: (061) 688 78 38; Fax: (061) 688 16

1Present address." Clinical Oncology/Hematology, HoffmannLa Roche Inc., Nutley, NJ 07110, USA. Note. Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number L08257.

of resistant strains of the parasites [1]. As the resistance problem is getting worse, it is a matter of time until it will no longer be possible to effectively control chicken coccidiosis with the current drugs. Vaccination has been proposed as an alternative for chemoprophylaxis. Live vaccines consisting of virulent parasites (Coccivac; Sterwin Lab. Inc., Immunocox; Cynamide) have been available for many years to control infections in breeder chickens and egg layers. Vaccines based on attenuated parasites have also been developed. For several reasons these vaccines do not offer a practical solution. Major drawbacks are (i) the difficulty of controlled administration; (ii) producing mass quantities needed to supply the world poultry industry; and (iii) potential reversion to virulence [2,3]. Therefore, subunit vaccines containing important parasitic antigens and administered as protein or via a vector (e.g., viruses or bacteria)

180 have been envisaged as an alternative to the virulent or attenuated vaccines mentioned above [4]. The chicken immune response to coccidiosis involves both, the humoral and the cellular immune system. The cellular immune mechanisms as well as host genetics factors are thought to play a major role in the protection against coccidiosis. The contribution of the humoral response to such an immune status is rather minor, according to the literature, but still unclear [5]. Antibodies directed against the surface antigens of the parasite are usually •found in the serum of a convalescent host. Monoclonal antibodies that inhibit E. tenella sporozoite invasion, in vitro, have been described [6]. Proteins of the surface coat of parasites have therefore always been a preferential target for the development of subunit vaccines against Eimeria spp. and other parasites, e.g. Plasmodium falciparum [7], Theileria parva [8]. Recently a number of groups have described the molecular cloning of Eimeria spp. genes from different parasite stages and their subsequent use as a subunit vaccine [9-13]. This report describes the cloning and characterization of a polypeptide of 33 kDa, designated Mzp 5-7, isolated from a merozoite stage cDNA library of E. tenella. In addition, the use of recombinant vaccinia viruses as a delivery model to present the Mzp 5-7 antigen to the immune system of the chicken is discussed.

Materials and Methods

Virus and cells. The wild-type vaccinia virus (strain WR) was obtained from the American Type Culture Collection. The temperaturesensitive vaccinia virus mutant ts7 [14] was obtained from R. Wittek, University of Lausanne, Switzerland. African green monkey kidney cells (CV-1) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% fetal calf serum (FCS). Human thymidine kinase negative ( T K - ) 143 cells were grown in DMEM supplemented with

8% FCS and 25 mg ml -~ 5-bromodeoxyuridine. Chicken embryo fibroblasts (CEF) were prepared as described elsewhere [15] and grown in DMEM supplemented with 10% FCS, 1% chicken serum and Biotin. Infected CEF were grown at 39°C or at 41°C. Parasites. Procedures for the isolation and maintenance of oocysts [16] and for the isolation and purification of sporozoites merozoites have been described elsewhere [17,18]. Construction and immunoscreening of a E. tenella merozoite cDNA library. A cDNA expression library in 2gtll was constructed using Poly(A)+-selected RNA, isolated and purified from frozen merozoite pellets (second generation) containing 1 x 109-1 x 10 l° parasites, as described elsewhere [19,20]. Double-stranded cDNA was synthesized from 6 #g poly(A) + merozoite RNA by the method of Gubler and Hoffmann [21]. Recombinant 2 clones were packaged using the Packagene kit and following the procedures supplied by the manufacturer (Promega Biotech). The cDNA library contained approximately 90% recombinant phages. The library was screened essentially as described elsewhere [20] with 1:100 diluted E. tenella merozoite specific rabbit antisera, previously preabsorbed to Y1090 cells. Antisera against merozoites of E. tenella were generated by immunizing rabbits with glutaraldehyde-fixed merozoites. One positive clone, designated 2Mz 5-7, was further characterized. Expression of the Mzp 5-7 protein in Escherichia coli. The EcoRI insert from the ~ phage Mz 5-7 was isolated from an agarose gel. The ends were filled in with Klenow polymerase in the presence of dATP and dTTP, and BamHI linkers were ligated to both ends. The modified fragment was inserted into the BamHI site of each of the three expression vectors pDS56/ RBSII (0;-1;-2) [22]. Transformants of the E. coli M15 containing the insert in the right orientation were isolated and expression induced by IPTG. The cell lysates were analyzed by Western blotting using the rabbit anti-

181

merozoite serum, preabsorbed to bacterial extract, as probe. Parasite DNA. DNA was isolated from sporozoites. The parasite material was suspended in 0.5 M EDTA, pH 8.0, 0.5% Sarcosyl and digested with proteinase K (Boehringer-Mannheim) at 0.1 mg ml-1 for 2 h at 50°C, then with RNase (10 #g ml-1) for 1 h at 37°C, and again with proteinase K for 1 h at 50°C. The DNA was purified by extractions with phenol, followed by a phenol/chloroform extraction and ethanol precipitation. In vitro translation of mRNA. Between 0.1 and 0.5 #g mRNA was used to program in vitro protein synthesis in a nuclease treated rabbit reticulocyte lysate (Amersham or Promega-Biotec) supplemented with 10--20 #Ci [35S]methionine per 20/~1 reaction. The in vitro translation products were visualized by fluorography after immunoprecipitation and SDSpolyacrylamide gel electrophoresis (SDSPAGE). Immunoprecipitation. Samples for immunoprecipitation were diluted in 0.25% NP40/ 0.15 M NaCI/20 mM Tris-HC1, pH 7.5, precleared by incubation for 20 min on ice with Staph-A protein (Pansorbin, Calbiochem) and centrifugation. The supernatant was incubated for several hours at 40°C with 5-10 /21 antiserum. After a second incubation with Staph-A protein, the antibody complexes were collected by centrifugation, eluted by heating to 100°C for 5 min in SDS-PAGE sample buffer [23] and analyzed by SDS-PAGE. DNA sequence analysis. The Mz 5-7 cDNA was cloned into M13 and the sequence determined by the dideoxy chain termination method [23]. Both strands were completely sequenced and the sequence analyzed using the GCG sequence analysis software package (Version 7) by Genetics Computer, Inc. [25]. 125I-surface labelling. Freshly purified merozoites were labeled with 125I using Iodobeads (Pierce Chemical Co.), collected by centrifuga-

tion and solubilized in 0.1% NP40 in PBS before dialysis against PBS at 4°C. PMSF (phenylmethylsulfonyl fluoride) was added to 5 mM. Labeled merozoites were subjected to immunoprecipitation with affinity selected rabbit anti-Mzp 5-7 antibodies, and separated by SDS-PAGE under reducing conditions using established procedures [23]. Affinity selected antibodies against the Mzp 5-7 recombinant E. tenella antigen were obtained essentially as described elsewhere [26] using 2 phage Mz 5-7 plaque lifts. Western blotting. Proteins separated by SDSPAGE were electrophoretically transferred to nitrocellulose filters [27]. The filters were incubated with rabbit anti-Mzp 5-7 antibodies or chicken sera diluted 1:100 in 20% goat serum in Buffer A (20 mM Tris pH 8.0, 150 mM NaC1) for 2 h or overnight at room temperature. After washing, bound antibody was reacted with either affinity-purified goatanti-rabbit IgG (H + L) peroxidase conjugate (Bio-Rad) or affinity purified goat-anti-chicken IgG (H + L) peroxidase conjugate (KPL) at a 1:1000 dilution in Buffer A and 5% non-fat milk powder. The blots were washed once more and bound peroxidase conjugate was detected by reacting the membrane with 4chloro-l-naphthol and H202. In the experiments where the serum from rVV R3 immunized chicken was tested, the filters were pre-incubated with rabbit anti vaccinia serum (1:10 dilution in Buffer A for 2 h) to block the antigenic sites of the vaccinia proteins. Plasmid constructs. The EcoRI fragment encoding the merozoite 5-7 gene was excised from the clone 2Mz 5-7 and subcloned into the unique EcoRI site of the recombination vector pUC8-TK-7.5K. The recombination vector consists of the pUC8 plasmid carrying the vaccinia virus (VV) TK gene disrupted by the VV 7.5K promoter [27]. Multiple cloning sites are Positioned downstream of the promoter which allow the introduction of the gene to be expressed. A recombination vector was propagated which contained the Mz 5-7 EcoRI

182

fragment in the correct orientation. This construct had an in-frame start codon situated 97 nucleotides upstream of the natural start codon of the merozoite Mz 5-7 gene. To delete this upstream ATG the plasmid was digested with the restriction enzymes SmaI and BglII. After filling in the BglIl site with Klenow enzyme in the presence of the four deoxyribonucleotides, the plasmid was religated and the construct pR3, carrying the correct deletion, was propagated and used for recombination into the vaccinia virus.

Recombinant vaccinia viruses. Recombinant vaccinia virus (rVV) was constructed according to described methods [14,29]. Putative recombinants were isolated by plaque purification on the basis of their T K - phenotype using human TK-143 cells and bromodeoxyuridine as the selective agent. Recombinant plaques were analyzed by PCR as described elsewhere [30]. Analysis of expressed proteins from recombinant vacciniavirus. Usually 1 x 106-1 x 107CV-1 or CEF ceils were infected with wild-type or recombinant vaccinia virus at about 10 pfu per cell. After 48 h the cells were harvested in SDSPAGE sample buffer. The proteins were separated by SDS-PAGE and analyzed in a Western blot (approximately 5 x 105 cells/lane). Immunization of chickens with r VV R3. Cockerels of the layer breed Warren were supplied by the hatchery E. Wuethrich in Belp (Switzerland). On day 17, the chickens were inoculated with 3 x 108 pfu of the recombinant vaccinia virus rVV R3 in 100 #1 of PBS, whereof 50 #1 were injected subcutaneously into the wing web and the other 50 #1 were given intramuscularly into the breast. At intervals of one week, this procedure was repeated twice. One week after the last immunization all chicks were bled for analytical purposes. Results

A cDNA expression library from poly(A) +

selected merozoite RNA of E. tenella was constructed in order to isolate potentially immunoprotective antigens. Screening with rabbit anti-merozoite serum resulted in a number of positive clones. One such positive recombinant phage, designated 2Mz 5-7, contained a 1.2-kb insert. To verify that the insert originated from E. tenella, PCR amplifications were performed using Mz 5-7 derived primers 1 and 2 (Fig. 1) and genomic DNA from E. tenella, Gallus gallus and E. coli. The latter two genomic DNAs are potential contaminants in the merozoite preparation used to isolate the mRNA. Only the PCR reaction containing E. tenella DNA gave the expected fragment of 103 bp. The PCR reactions using E. coli DNA and G. gallus DNA gave no amplification product. The protein encoded by the 2Mz 5-7 clone was identified by immunoprecipitation of in vitro translation products of merozoite mRNA. One major specific product of 33 kDa was precipitated with affinity-selected anti-Mzp 5-7 antibodies (Fig. 2A, lane I) but not with control antibodies (Fig. 2A, lane 2). Western blot analysis of merozoite extracts with the affinity-purified antibodies (not shown) reacts strongly with one major protein band of approximately 29 kDa and very weakly with a number of protein bands, within the range of 28-35 kDa. Western blot analysis of sporozoite extracts with affinity purified anti Mzp 5-7 antibodies revealed 5 major protein bands between 28 kDa and 35 kDa. The extent to which these bands belong to different sporozoite proteins sharing epitopes with Mzp 5-7 or to degraded/processed forms of a single protein has not been determined. The protein pattern seen for sporozoites resembles the faint pattern observed for merozoites. The complete nucleotide sequence of the 2 Mz 5-7 insert consists of 1196 bases. The longest open reading frame which is also in frame with the j%galactosidase fusion protein starts with the first ATG initiation codon at base pair 67 and ends with a TGA stop codon at position 1012 (Fig. 1). A second in-frame ATG codon is found only 10 nucleotides

183

I

CTTTTGCGTCGGAGATAGTCGTTGTGTGTTTGCGCGATCACCCGCGAACTTCTCTACCAACTGAAAATGG CTAAGTCTATGCTTTCTGGAATTGTTTTTG 1

M

A

K

S

M

L

S

G

I

V

F

A

E

101

201

CTGGTCTTGTTGCTGCTGCAG CGGCCAGTTCGGCCAACAGCGCCGCCAACGTCTCCGTTTTGGAGAGTGGGCCCG C T G T G C A G G ~ G T G CCAGCGCGCAC G L V A A A,~ A A S S A N S A A N V S V L E S G P A V 0 E V P A R T J. 4 . . . . . . . . . . GGT~A~AG~T~G~TGGCGA~G~CTTTG~TGCTT~TTT~TG~T~TTG~T~GA~TTTGGC~GCAGCTTTCCT~GTTTTGCAATG~TT~ATCAT~TC~ V T A R L A K P L L L L S A L A A T L A A A F L V L O C F N I I S

301

AGCAAC~d~CCAGCA.~CCA(~CGTCAGGAG,%CTGGCCGCCGG&GGTGC&TGCGGAGATG&GGAAGATGeAGATGAGGG~CTTCACAGCAGGCCAGCCGGA s N N Q Q T S v a a L A X G a A e G o E E D x D Z ~ T S Q 0 A S a a

401

GGAGGAGAA~CTGATACCCCTGCAGCAGATAAATACGATTTTGTTGGCGGAACT~CAGTTTCGGTCACTGAGC~G~TGTTGATG~GT~CTTAT~A R

R

K

P

D

T

P

A

A

D

K

Y

D

F

V

G

G

T

P

V

S

V

T

E

P

N

V

D

E

V

L

I

Q

501 ~ T T A G A A A T A A A ~ A T C T T T T T G ~ G ~ C C C & T G G A ~ T G G A C ~ G ~ G ~ G T T ~ T A G T A C T G G & A C G & ~ A A A G T ~ G ~ C ~ C A T T C T G A T T G T G I R N K Q I F L K N P W T G Q E E Q V L V L E R Q S E E P I L X V 601

GCG&GGA~AAG&~A~ACTTG~GG&TATCTTGGTAGTC~G~T~TTG~ACAGGACGG&~GA~TG~TAAAG~GAG~M%GTTG~GGAGG~T~ A

701

R

T

L

E

G

Y

L

G

S

Q

A

L

A

Q

D

G

K

T

A

K

E

E

K

V

E

G

G

K

T

H

R

Y

K

V

K

S

S

D

P

G

Y

G

F

P

Y

T

T

V

L

D

G

V

P

V

G

T

D

E

D

G

Y

V

E

V

L

M

K

T

G

P

H

G

G

V

D

M

M

T

S

T

A

S

Q

G

K

F

C

G

V

L

M

D

D

G

AAAGGAXACCTAGT~GATGGA~AAGGGAG~TTA~GCCGTTAT~GG~ATG~T~TC~GGATAC~GAGTTTAG~GCGGA~AGGAGA~GACG K

1001

Q

~GT~G~GTTCTTATGA~A~GGAC~CCAT~GAGGA~TCGACATGATGACTAGCACAGCAT~AC~GGAAAATTCTGCGG~GTGCTTATGGATGACGGA V

901

R

A~&G~GAT~AAAGT~GAGCAG~G&C~CAGGATATGGATTCCCATACA~CA~GGTG~TCGA~GGGGTTC~TGTGGGAACAGACG~G&CGGATA~GT R

801

T

G

N

L

V

D

G

Q

G

R

K

I

T

A

V

I

G

M

L

T

Q

P

D

T

E

F

R

S

G

P

G

D

D

E

AGGA~GA~GAGTGAGTGAG~GGAGTTGG~TTTTGT~CCTGTTGATGcCGTTGC~CA~TTTCGCAGCTTGCTTGTTTC~TGGG~TTGC~TGTG~CG~GACA D

D

E

*

1101 T G C G ~ T T ~ G C ~ T T ~ G ~ T ~ A ~ T T C T T T C G G A ~ T G T T T T ~ C T T T T ~ T T C A T T T T ~ T A ~ T G ~ G C A A A A A A A A A A A A ~ ~ % A A A

Fig. 1. Nucleotide sequence of the 2 p h a ~ Mz 5-7 insert. The amino acid sequence translated ~om the open ~ading ~ame (nucleotides 67-1014) is depicted by the one letter code. Arrows indicate the site of the primers 1 and 2 used ~ r the PCR amplifications in Fig. 2.

downstream from the first. The open reading frame (945 nucleotides) encodes a protein of 315 amino acid residues with a predicted molecular weight of 33375. This size is consistent with the size of the in vitrotranslated and immunoprecipitated protein (Fig. 2A, lane 1). However, there is an inframe TAG stop codon at position 16 of the cDNA, interrupting the fl-galactosidase fusion protein predicted from the 2Mz 5-7 clone. It appears, therefore, that the Mzp 5-7 protein translation is initiated at a Shine-Delgano sequence positioned on the cDNA insert. This hypothesis is supported by the observation that the expression of the 1.2 kb cDNA in all three reading frames of the vector pDS56/RBSII gave independently of the reading frame, a protein that had an apparent size of about 30 kDa, and which reacted with the rabbit antimerozoite serum (not shown). The presence of an in frame TAG stop codon upstream of the

predicted start codon suggests that the fulllength cDNA has been cloned. Furthermore, the first ATG within the cDNA insert (position 67) matches the Kozak consensus sequence [31] (nucleotide A in position - 3 and G in + 4) suggesting that this might be the translation start site. Although the message contains a poly(A) tail, no conventional polyadenylation signal, AATAAA [32], is detectable in the 3' untranslated sequence. A GenBank database search (release 73.1) revealed no significant sequence similarity to known proteins. The encoded polypeptide is negatively charged ( - 1 0 ) and the hydropathicity plot [33] of the Mzp 5-7 amino acid sequence (not shown) reveals 2 highly hydrophobic domains within the first 77 amino-terminal residues. The first region, a putative prokaryotic signal sequence [34], spans from the initiation methionine to the serine at position 22, with

184

1

A 97

2

1 2

B

_

68_

m

97 68

43--

43

26_

26

18_

18

Fig. 2. (A) Immunoprecipitation of in vitro translation products from merozoite mRNA using monospecific and control antisera. Poly(A) + mRNA from E. tenella merozoite was translated in vitro and the 35S-labeled translation products were precipitated with affinity selected rabbit anti-Mzp 5-7 antibodies (lane 1) and control antibodies selected by adsorption of rabbit anti merozoite serum to non-recombinant 2gtl 1 plaques (lane 2). The immunoprecipitated polypeptides were resolved by SDS-PAGE and visualized by fluorography. (B) Immunoprecipitation of 125Isurface labeled proteins from E. tenella merozoites using affinity selected rabbit anti-Mzp 5-7 antibodies. Lane l, the control precipitation using normal rabbit serum adsorbed to non-recombinant 2gtl I plaques. Lane 2, affinity purified rabbit anti-Mzp 5-7 antibodies. Proteins were resolved by SDS-PAGE.

a potential cleavage site between amino acids 22 and 23. The second region, from Leu 54 to Phe 74, may represent a transmembrane helix that may function as an anchor segment [35,36]. The protein has a potential Nglycosylation site at amino acid Asp 28. In addition, there are 3 cysteine residues in the sequence available for disulfide bridges. To confirm the predicted surface location of the Mzp 5-7 protein, 125I-labeled merozoites were immunoprecipitated with the affinityselected anti-Mzp 5-7 antibodies. Only one polypeptide of approx. 24 kDa was immunoprecipitated (Fig. 2B, lane 2) which was not seen in the control reaction (Fig. 2B, lane 1), in which affinity selected antibodies on nonrecombinant phages were used. This protein

is approx. 9 kDa smaller than the polypeptide from the in vitro translation (Fig. 2A, lane 1). This indicates that the 33-kDa polypeptide may be the precursor of the 24-kDa surface antigen. CV-1 cells infected with the recombinant vaccinia virus rVV R3, carrying the Mz 5-7 gene, expressed two distinct proteins of 33 kDa and 28 kDa, as detected by western blot analysis using rabbit anti-merozoite serum (Fig. 3A, lane 1). CV-1 cells infected with wild-type WR vaccinia virus did not react with the immune serum (Fig. 3A, lane 2). The larger 33-kDa protein compares with the theoretical value for the polypeptide encoded by the Mz 57 gene and with the protein immunoprecipitated from the in vitro translation products by affinity selected anti-Mzp 5-7 antibodies. The nature of the 28-kDa protein is not yet clear (see Discussion). Both recombinant merozoite proteins (33 kDa and 28 kDa) expressed in CV-1 were recognized in a Western blot assay by immune serum from chickens naturally infected with E. tenella oocysts (Fig. 3B, lane 2). The use of vaccinia virus as a suitable vector for the expression of recombinant proteins in birds was investigated by infecting chicken cells (CEF) with the rVV R3 followed by incubation at 41°C. Expression of the Mzp 5-7 protein was monitored by Western blot analysis 48 h after infection. Like CV-I cells, the chicken cells also expressed two immunoreactive proteins of 33 kDa and of 28 kDa respectively (not shown). This result indicates that the recombinant proteins induced by the rVV R3, should be properly expressed in chickens although the ceiling temperature for vaccinia virus growth is identical with the chicken body temperature (41°C). The confirmation of this latter assumption was obtained by immunizing chickens with rVV R3 and testing their serum for the presence of anti-Mzp 5-7 antibodies. The western blot analysis shows that both recombinant proteins expressed in rVV R3-infected CV-1 cells were detected by the immunechicken serum (Fig. 3C, lane 1). No reaction is seen with wild type VV-infected cells (Fig.

185

A

¢

B 2

106 80

_

106

--

80

_

49

_

32

_

27

_

18

49.

32.

27.

18.

Fig. 3. (A) Expression of the recombinant Mzp 5-7 protein in CV-1 cells. CV-I cells were infected with the recombinant vaccinia virus rVV R3 (lane 1), control infection using wild type (WR) vaccinia virus (lane 2), and the recombinant Mzp 5-7 protein was detected with affinity selectedrabbit anti-Mzp 5-7 antibodies, in a Western blot analysis. (B) Identification of the recombinant Mzp 5-7 antigen by immune chicken serum. CV-1 cells infected with wild type (WR) vaccinia virus (lane 1) or infected with rVV R3 (lane 2) were run on a SDS-polyacrylamidegel (12.5%) under reducing conditions, transferred to a nitrocellulose membrane and reacted with serum from immune chicken. (C) Immune reactivity of serum from rVV R3 vaccinated chicken. CV-1 cell lysates were separated by SDS-PAGE (12.5%) under reducing conditions and transferred to nitrocellulose. Lane 1, CV-1 cells infected with rVV R3; lane 2, CV-1 cells infected with wild type vaccinia virus. The Western blot was pre-incubated with rabbit anti-vaccinia serum before reaction with the chicken serum. 3C, lane 2).

Discussion

In host-parasite interactions, cell surface molecules play an important role and surface antigens are therefore logical targets for vaccine development. An effective vaccine against coccidiosis might include merozoite antigens or antigens common to most stages of the parasite life cycle. Implication of the merozoite as the most immunogenic stage of coccidia parasites has been suggested by Rose [37]. Therefore, detection and characterization o f antigens present on the surface of merozoites are of considerable interest. Mzp 5-7 described in this report is a surface antigen present on the merozoite stage of E. tenella. The fact that immune chicken sera obtained from naturally infected convalescent birds, reacted with the Mzp 5-7 recombinant polypeptide (Fig. 3B, lane 2) indicates that this antigen is recognized by the immune system of

the chicken during the parasite infection. Affinity-selected anti-Mzp 5-7 antibodies, purified on recombinant proteins from plaques generated by the 2 phage Mz 5-7, precipitate a 24-kDa protein of E. tenella merozoites (Fig. 2B, lane 2). The accessibility of this protein to iodination suggests that it is located on the surface of the parasite. This prediction correlates with the computer analysis of the Mzp 5-7 amino acid sequence which shows a potential signal sequence (aa 1-21) and a helical transmembrane segment (aa 5474), both located at the N-terminus of the Mzp 5-7 polypeptide. These features are characteristic for surface proteins. The same affinity-selected antibodies immunoprecipitate a 33-kDa product of in vitro translated merozoite m R N A (Fig. 2A), suggesting that this polypeptide might be the precursor of the putative mature 24 k D a form of the Mzp 5-7 antigen. The nature of the processing events leading from the 33-kDa precursor to the 24-kDa mature form are unknown. Interesting is the detection of two

186

protein products, of 28 kDa and 33 kDa, when the Mz 5-7 gene is expressed in rVV R3 infected CV-1 cells. The origin of the 28-kDa polypeptide is still unclear. One could speculate, that the correct parasite processing signals contained within the Mzp 5-7 amino acid sequence are not recognized by the mammalian cell. Different Eimeria spp. antigens have been described lately by several groups (for review see ref. 38). LPMC-61 is one of those antigens expressed on E. tenella merozoites [39]. Furthermore, a 35-kDa portion of the p250 immunodominant merozoite surface antigen of Eimeria acervulina [40] has been cloned and characterized and used in vaccination trials with some success [12]. In contrast to the amino acid repeats seen in a number of genes isolated from different Eimeria spp. (for review see ref. 4) and other protozoan parasites, the Mzp 5-7 protein contains no relevant stretches of such repeats. The impact of those repeats on the host immune response is not clear. The success of a recombinant vaccine will ultimately depend on a suitable vehicle for the presentation of the antigen to the immune system of the chicken [4,41]. Presentation of the antigen to the mucosal system using E. eoli [11] or Salmonella spp. [42,43] as delivery vectors seems to be the most logical approach. But viral vectors based on poxviruses might turn out to be more suitable vaccine candidates due to the ease of generation and application, the large capacity for foreign DNA and the possibility to use naturally host-specific viruses which are non-pathogenic for humans. In our studies we used vaccinia, a member of the poxvirus family, as a model system to express E. tenella antigens in chickens. We have demonstrated that the recombinant antigen is properly expressed in chicken cells despite the fact that the body temperature of the chicken (41°C) coincides with the maximal temperature at which viral replication in cell culture occurs. The Mzp 5-7 protein expressed in CV-1 cells was detected by western blot analysis using serum from naturally infected chickens (Fig. 3B, lane 2), implying that the recombinant

product successfully mimicks antigenic sites of the native surface protein. Experiments are in progress to evaluate the protective capacity of the recombinant Mzp 5-7 antigen in chickens.

Acknowledgements The authors would like to thank Dr. D. van Loon and Dr. J. Schwager for critical reading of the manuscript and K. Becker for the figures.

References 1 Chapman, H.D. (1993) Resistance to Anticoccidial Drugs in Fowl. Parasitol. Today 9, 159-162. 2 Shirley, M.W. (1989) Development of a live attenuated vaccine against coccidiosis of poultry. Parasite Immunol. 1l, 117-24. 3 Shirley, M.W. and Long, P.L. (1990) Control of coccidiosis in chickens: immunization with live vaccines. In: Coccidiosis of Man and Domestic Animals (Long, P.L., ed), pp. 321-341. CRC-Press, Boca Raton, FL. 4 Ellis, J. and Tomley, F. (1991) Development of a genetically engineered vaccine against poultry coccidiosis. Parasitol. Today 7, 344~346. 5 Lillehoj, H.S. and Trout, J.M. (1993) Coccidia: a review of recent advance on immunity and vaccine development. Avian Pathol. 22, 3-31. 6 Crane, M.S.J., Murray, P.K., Gnozzio, M.J. and MacDonald, T.T. (1988) Passive protection of chickens against Eimeria tenella infection by monoclonal antibody. Infect. Immun. 56, 972 976. 7 Cox, F.E.G. (1991) Malaria vaccines progress and problems. Trends Biotechnol. 9, 389-394. 8 Dotterty, P.C. and Nussenzweig, R. (1985) Tropical diseases: progress on Theileria vaccine. Nature 316, 484-485. 9 Crane, M.S.J., Goggin, B., Pellegrino, R.M., Ravino, O.J., Lange, C., Karkhanis, Y.D., Kirk, K.E. and Chakraborty, P.R. (1991) Cross-protection against four species of chicken coccidia with a single recombinant antigen. Infect. Immun. 59, 1271-1277. 10 Brothers, V.M., Kuhn, I., Paul, L.S., Gabe, J.D., Andrews, W.H., Sias, S.M., TcCanan, M.T., Dragon, E.A. and Files, J.G (1988) Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli. Mol. Biochem. Parasitol. 28, 235-248. 11 Jenkins, M.C., Castle, M.D. and Danforth, H.D. (1991) Protective immunization against the intestinal parasite Eimeria acervulina with recombinant coccidial antigen. Poultry Sci. 70, 539-547. 12 Kim, K.S., Jenkins, M.C. and Lillehoj, H.S (1989) Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protection against coccidiosis.

187 Infect, Immun. 57, 2434-2440. 13 Miller, G.A., Bhogal, B.S., McCandliss, R., Strausberg, R.L., Jessee, E.J., Anderson, A.C., Fuchs, C.K., Nagle, J., Likel, M.H., Strasser, J.M. and Strausberg, S. (1989) Characterization and vaccine potential of a novel recombinant coccidial antigen. Infect. Immun. 57, 2014-2020. 14 Drillien, R. and Spehner, D. (1983) Physical mapping of vaccinia virus temperature-sensitive mutations. Virology 131,385-393. 15 Silim, A., El Azhary, M.A.S.Y. and Roy, R.S. (1981) A simple technique for preparation of chicken-embryoskin cell cultures. Avian Dis. 26, 182-185. 16 Long, P.L., Joyner, L.N., Millard, B.J. and Norton, C.C. (1976) A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Fol, Vet. Lat. 6, 201217. 17 Wisher, M.H. and Rose, M.E. (1984) The large-scale preparation of purified sporozoites of Eimeria spp. by metrizamide density gradient centrifugation. Parasitol. 88, 515-519. 18 Danforth, H.D. (1986) Development and use of hybridoma antibodie~ directed against Eirneria acervulina merozoites for cross-reactive and ferritin-labeling studies. Avian Dis. 31, 99-104. 19 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20 Huynh, T.H., Young, R.A. and Davis, R.W. (1985) Construction and Screening cDNA libraries in 2gtl0 and 2gtl 1. In: DNA Cloning. A Practical Approach (Glover, D.M., ed.), pp. 49-78. IRL Press, Oxford, England. 21 Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. 22 Bujard, H., Gentz, R., Lanzer, M., Stiiber, D., Mfiller, M., Ibrahimi, I., Hiiuptle, M.T. and Dobberstein, B. (1987) Methods Enzymol. 155, 416-433. 23 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 6804585. 24 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 25 Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. 26 Hall, R., Hyde, J.E., Goman, M., Simmons, D.D., Hope, I.A., Mackay, M., Scaife, J., Merkli, B., Richle, R, and Stocker, J. (1984) Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria. Nature 311,379-385. 27 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacryla-

28

29

30 31 32 33 34 35

36 37 38

39

40

41 42 43

mide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA, 76, 4350-4354. Venkatesan, S., Baroudy, B.M. and Moss, B. (1981) Distinctive nucleotide sequences adjacent to multiple initiation and termination sites of an early vaccinia virus gene. Cell 25, 805-813. Mackett, M., Smith, G.L. and Moss, B. (1985) The construction and characterization of vaccinia virus recombinants expressing foreign genes. In: DNA Cloning. A Practical Approach (Glover, D.M., ed), pp. 191-211. IRL Press, Oxford. Pasamontes, L. Gubser, J., Wittek, R. and Viljoen, G.J. (1991) Direct identification of recombinant vaccinia virus plaques by PCR. J. Virol. Methods 35, 137-141. Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292. Nevins, J.R, (1983) The pathway of eukaryotic mRNA formation. Annu. Rev. Biochem. 52, 441-446. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R. (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179, 125-142. Klein, P., Kanehisa, M. and De Lisi, C. (1985) The detection and classification of membrane-spanning proteins. Biochim. Biophys. Acta 815, 468-476. Rose, M.E. (1982) Host immune responses. In: The Biology of the Coccidia (Long, P.L., ed.), pp. 329-371. University Park Press, Baltimore, MD. Danforth, H.D. and Augustine, P.C. (1990) Control of Coccidiosis: Prospects for subunit vaccines, In: Coccidiosis of Man and Domestic Animals (Long, P.L., ed.), pp. 343-348. CRC Press, Boca Raton, FL. Ko, C., Smith II, C.K. and McDonell, M. (1990) Identification and characterization of a target antigen of a monoclonal antibody directed against Eimeria tenella merozoites. Mol. Biochem. Parasit. 41, 53-64. Jenkins, M.C., Strohlein, D.A., Lillehoj, H.S. and Danforth, H.D. (1988) Recombinant DNA cloning of genes encoding surface proteins of Eimeria acervulina sporozoites and merozoites. 77th Annual Meeting of the Poultry Science Association, 67, p. 100. Murray, P.K. (1987) Prospects for molecular vaccines in veterinary parasitology. Vet. Parasitol. 25, 121-133. Griffin, H.G. (1991) Attenuated Salmonella as live vaccines: prospects for multivalent poultry vaccines. World Poultry Sci. J. 47, 129-140.48. Tomley, F. (1991) Recombinant vaccines for poultry. Vaccine 9, 4-5.