Theileria annulata is an economically important protozoan parasite that threatens an estimated 250 million cattle with the disease tropical theileriosis. Development of a defmed subunit vaccine is one means of trying to develop control measures against the disease. To this end we have characterized a surface antigen complex of the infective stage (sporozoite), by using a monoclonal antibody that neutralizes sporozoite infectivity in vitro. We have cloned the gene coding for this complex and have demonstrated that a fusion protein expressed from a fragment of this gene elicits strong neutralizing antibodies. Furthermore we provide data on the structure and expression of this gene. In particular we show that the region of the gene, expressed in one clone, codes for a protein segment relatively rich in proline residues. Also we demonstrate that expression of this gene appears to be stage specific, transcripts being present only in the sporoblast and sporozoite stages. The relevance of these rmdings to the production of a defined subunit vaccine is discussed.Tropical theileriosis threatens an estimated 250 million cattle in the Mediterranean littoral, North Africa, the Middle East, the Indian subcontinent, and Central Asia and acts as a major constraint on livestock production and improvement in many developing countries (1). The disease is caused by a tickborne protozoan, Theileria annulata, which enters the bovine host as the infective sporozoite stage in the saliva of a feeding tick. These sporozoites rapidly invade mononuclear cells in which the macroschizont stage develops; subsequently these parasites are liberated, whereupon they invade erythrocytes producing the piroplasm stage of the life cycle (2). Present methods of vaccination depend upon establishing active infection (3) and thus incur both the risk of causing clinical disease and the problems of handling live parasite material under tropical conditions. For these reasons we are actively engaged in identifying parasite antigens capable of inducing a protective immune response (4-6) using similar approaches to those followed by investigators working on Theileria parva, the cause of East Coast fever (7-11). Here we describe an antisporozoite monoclonal antibody (mAb) that exhibits significant sporozoite neutralizing activity and provide data on the nature of the polypeptides recognized by this antibody. In addition we describe the isolation of two recombinant DNA clones that express the epitope defined by this mAb and we report our findings on the structure and expression of this gene(s). § MATERIALS AND METHODSParasite Material. Three uncloned isolates of Theileria annulata were used in this study: they are called Ankara, from Turkey (12); Hissar, from India (13); and Gharb, from Morocco (14). Sporozoites for mAb production, immunofluorescence tests, inhibition assays, and immunoblotting were obtained from Theileria annulata-infected adult ticks (Hyalomma anatolicum anatolicum). These ticks were fed on rabbits for 3 days to stimulate sporozoite m...

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SummaryThe major sporozoite surface antigen of Theileria annulata (SPAG-1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG-1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both ␤-galactosidase and hepatitis B core antigen fusions or as a full-length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freund's, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG-1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.keywords Theileria annulata, sporozoite antigen (SPAG-1), vaccine, protection correspondence Roger Hall,