Molecular and Biochemical Parasitology 107 (2000) 309 – 314 www.elsevier.com/locate/parasitology
Short communication
Molecular characterisation of a Theileria lestoquardi gene encoding a candidate sporozoite vaccine antigen Robert A. Skilton a,*, Anthony J. Musoke a, Vishvanath Nene a, Delia P.S. Wasawo a, Clive W. Wells a, Paul R. Spooner a, Richard P. Bishop a, Julius Osaso a, Catherine Nkonge a, Abdulla Latif b, Subhash P. Morzaria a b
a International Li6estock Research Institute (ILRI), PO Box 30709, Nairobi, Kenya Faculty of Veterinary Science, Department of Parasitology, Uni6ersity of Khartoum, Khartoum, Sudan
Received 17 August 1999; accepted 21 December 1999
Keywords: Theileria lestoquardi; Sporozoite antigen; Sub-unit vaccine
Theileria are tick-transmitted, haemoprotozoan parasites infecting wild and domestic ungulates throughout many areas of the world [1]. The most economically important species are T. par6a and T. annulata which are pathogenic to cattle, and T. lestoquardi (syn. hirci ) which is pathogenic to sheep and goats. The infective sporozoite stage of Theileria develops in the tick salivary gland and is transmitted to the host as the tick feeds. Sporozoites invade specific sub-populations of blood mononuclear cells, where they differentiate into a multinucleate schizont stage. Host cell entry of T. par6a and T. annulata sporozoites is likely to be dependent upon interaction between molecules of Note: Nucleotide sequence data reported in this paper are available in the GenBank™ database under the accession number AF128526 * Corresponding author. Tel.: +254-2-630743; fax: + 2542-631499. E-mail address:
[email protected] (R.A. Skilton)
the parasite surface coat and those of the host cell surface as determined by the activity of antibodies that inhibit invasion [2–4]. Monoclonal antibodies (mAbs) to sporozoite stage-specific surface proteins of T. par6a and T. annulata, known as p67 and SPAG-1, respectively, neutralise sporozoite infectivity for host cells in vitro [2–4]. These proteins contain conserved B-cell epitopes and exhibit considerable sequence similarity indicating that they belong to one gene family [5]. Native and recombinant p67 also inhibit T. par6a sporozoite infectivity supporting the hypothesis that p67 and SPAG-1 play a major role in mediating host cell infection [6]. In laboratory trials, immunisation with recombinant p67 induces protection against T. par6a sporozoite challenge in approximately 65% of vaccinated cattle [7–9]. Also, immunisation of cattle with recombinant SPAG-1 provides some degree of protection against T. annulata sporozoite chal-
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lenge [10,11]. In this report we describe the cloning and analysis of the gene encoding the p67/SPAG-1 homologue of T. lestoquardi. This protein, which we call SLAG-1 (s6 porozoite l6 estoquardi a6 ntig6 en 16 ), is predicted to have potential as a sporozoite-neutralisation antigen for inclusion in a sub-unit vaccine against T. lestoquardi infection of sheep and goats. Hyalomma anatolicum anatolicum adult ticks collected from sheep in Sudan, north of Khartoum, were applied to a susceptible sheep at ILRI and a schizont-infected lymphocyte cell line, SON 3, was established from a lymph node biopsy taken during acute infection. Genomic DNA from SON 3 was subjected to PCR with degenerate oligonucleotide primers IL2734 (forwards primer 5%-TTC ANT TTC CNA CAC TGA ACG ATG3%) and IL2840 (reverse primer 5%-TTA KTK TTA GTG GAC KAT GCT G-3%) that were based upon conserved sequences of the 5% and 3% termini, respectively, of the SPAG-1 and p67 genes (underlined sequences represent the start and stop codon of the sporozoite antigen genes, respectively). The nucleotide sequence of both DNA strands of the SLAG-1 gene was determined directly from the PCR product using the PCR primers and additional primers derived from the acquired sequence. The extreme 5% and 3% ends of the SLAG-1 gene were sequenced after PCR products were cloned into a plasmid T-vector. The SLAG-1 PCR product contains a 2229 bp sequence with an open reading frame from bases 22 – 2220. The SPAG-1 gene sequence is known to contain an in-frame 30 bp intron [12] and a sequence of similar length was identified within the SLAG-1 gene sequence at bases 941 – 970. That this sequence is an intron was confirmed following sequencing of a sporozoite-derived reverse transcriptase (RT)-PCR product that encompassed this region of the SLAG-1 gene (data
not shown). The SLAG-1 and SPAG-1 introns have 87% sequence identity. The p67 gene sequenc contains an intron of 29 bp [3] which has 67% identity with the intron of SLAG-1 (data not shown). The SLAG-1 gene sequence encodes a predicted protein of 723 amino acids (Fig. 1) with a calculated molecular mass of 76285. This predicted mass resembles that of p67 (75367–79544) [3,9,13] but differs from the predicted mass of SPAG-1 (91880) [14]. The deduced SLAG-1 protein sequence contains a predicted signal sequence with cleavage between G17 and G18 [15] and a C-terminal region of hydrophobic amino acids (residues I700 –V722) which is predicted to be a trans-membrane anchor sequence [16]. Similar features are also associated with the p67 and SPAG-1 proteins and are consistent with a surface location of SLAG-1 within the parasite [3,14]. A feature that is unique to SLAG-1 is a 13 amino acid peptide (GNGTEGKDQQPAS) that is repeated contiguously three times near the C-terminus (Fig. 1). The SLAG-1 sequence contains six predicted Nlinked glycosylation sites, three of which occur within the peptide repeat. SLAG-1 exhibits 42 and 58% amino acid identity with p67 and SPAG-1, respectively, as compared to 40% identity between p67 and SPAG-1. The results of Southern blotting indicate that a single copy gene within the T. lestoquardi genome encodes the SLAG-1 (data not shown). Similarly, the genes encoding p67 and SPAG-1 are also considered to be single copy [3,12]. The closer sequence relationship between SPAG-1 and SLAG-1 is consistent with previous studies in that phylogenetically T. lestoquardi is more closely related to T. annulata than it is to T. par6a [17]. T. lestoquardi and T. annulata exhibit strong serological cross-reactivity [18], and T. lestoquardi and T. annulata also share a common tick vector [1].
Fig. 1. Alignment of the deduced amino acid sequence of T. lestoquardi SLAG-1 with those of T. annulata SPAG-1 (GenBank™ accession number X78194) and T. par6a p67 (GenBank™ accession number M67476). Identical residues are indicated by black boxes. Gaps were introduced to maximise the alignment and are shown as dashes. The following features within SLAG-1 are shown: an arrowhead denotes the predicted site of signal sequence cleavage; a bold line denotes amino acids PSLVI, the conserved mAb 1A7 sporozoite-neutralising epitope; a double arrow denotes the GNGTEGKDQQPAS repeat; a dotted line denotes the predicted hydrophobic trans-membrane helix. A star denotes an amino acid, the codon of which is interrupted by an intron in the each of the SLAG-1, SPAG-1 and p67 genes.
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Fig. 1.
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Fig. 2. Serological cross-reactivity between SLAG-1, SPAG-1 and p67. (A) Recombinant proteins separated by SDS-PAGE and stained with Coomassie Blue: HisTag-SLAG-1 (lane 1); HisTag-p67 (lane 2); HisTag-SPAG-1 (lane 3); irrelevant HisTag protein, a 22 kDa recombinant protein derived from Rhipicephalus appendiculatus salivary glands (lane 4). Molecular weight markers for sizes indicated (lane M). (B) Proteins from an identical gel to (A) were transferred to a nitrocellulose filter and probed with rat antiserum to HisTag-SLAG-1 (1:1000 dilution) using methods described previously [3]. The antiserum showed cross-reactivity between sporozoite antigens (lanes 1, 2 and 3) but did not react with an irrelevant HisTag protein (lane 4). Pre-immunisation serum from the same rat showed no reactivity with any of the proteins (data not shown).
Regions of highest sequence identity between p67, SPAG-1 and SLAG-1 are located in the first 100 amino acids from the N-terminus and also in the C-terminal half of the proteins (Fig. 1). This is consistent with allelic polymorphisms of p67 and SPAG-1 which are primarily located in the central region of the proteins [5,12]. Mapping of antibody epitopes on p67 and SPAG-1 has revealed the presence of conserved antigenic determinants in the N and C terminal domains [5]. MAb 1A7 that neutralises both T. par6a and T. annulata sporozoite infectivity binds to the core peptide sequence of PSLVI in the C-terminal region [5]. This peptide sequence is also present in SLAG-1 (Fig. 1)
but the prediction that 1A7 will inhibit infectivity of T. lestoquardi sporozoites remains to be confirmed. Conservation of N and C terminal sequences in these proteins implies functional significance, perhaps in facilitating the sporozoite entry process. A fragment of SLAG-1, (D319 –G704) encoding the C-terminal end but lacking the predicted hydrophobic trans-membrane sequence, was produced as a recombinant histidine tag (HisTag) fusion protein using the pQE-30 bacterial expression system (Qiagen, Hilden Germany) (Fig. 2A). A rat antiserum to affinity-purified HisTagSLAG-1 was produced. In immunoblot analysis,
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this antibody cross-reacts with recombinant p67 and SPAG-1 (Fig. 2B) confirming the presence of conserved antibody epitopes amongst all three proteins. Due to the current unavailability of T. lestoquardi sporozoites we have not been able to analyse native SLAG-1. However, by immunoelectron microscopy the rat antisera to SLAG-1 was shown to bind to the surface of T. par6a (Muguga) sporozoites (Fig. 3) and is thus likely to contain sporozoite neutralising activity. These results indicate immunological cross-reactivity between SLAG-1, p67 and SPAG-1. Studies are in progress to express full length recombinant SLAG-1 and to determine the extent of antigen polymorphism between different stocks
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of T. lestoquardi. Work is ongoing to produce T. lestoquardi sporozoites for in vivo experiments and to confirm the predicted vaccine potential of SLAG-1 against T. lestoquardi infection in sheep and goats. Immunisation of cattle with recombinant p67 or SPAG-1 affords a degree of crossprotection against T. annulata and T. par6a challenge, respectively [11] (Hall, Musoke and Morzaria, unpublished data). Thus it is tempting to speculate that it may be possible to combine components of p67, SPAG-1 and SLAG-1 into a single immunogenic preparation for priming protective immune responses against theilerial infections of livestock.
Acknowledgements We wish to thank Dr Nicky Boulter of University of York, UK, for providing HisTag-SPAG-1. We gratefully acknowledge the excellent technical assistance of Luka Juma, Jackson Makau, Lucy Gichuru, Tahiya Twaha, Stephen Wanyoni, Stephen Mwaura and staff of the ILRI tick unit. For art work we thank Dave Elsworth, Joel Mwaura and Francis Shikhubari. This is ILRI publication number 990143.
References
Fig. 3. Cross-reactivity of antiserum to SLAG-1 with T. par6a sporozoites. Immunogold electron microscopy demonstrated that rat antiserum to recombinant HisTag-SLAG-1 reacted predominantly to the surface membrane of T. par6a sporozoites. This reactivity was specific since it could be abrogated by preabsorbing the serum with HisTag-SLAG-1 (data not shown). ImmunoEM followed methods described previously [19]. Bar, 0.5 mm.
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