ELSEVIER Molecular and Biochemical Parasitology 67 (1994) 1-10 /'IOLECUI2~ AND BIOCHEMICAL PARASITOLOGY Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata Frank Katzer a, Mark Carrington b, Pamela Knight c, Susanna Williamson d, Andrew Tait c, Ivan W. Morrison e, Roger Hall a,, a Department of Biology, University of York, York, YO1 5DD, UK b Department of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge, CB2 1QW, UK c Wellcome Unit for Molecular Parasitology, University of Glasgow, Bearsden Rd., Glasgow, G61 1QH, UK d Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, Midlothian, Edinburgh, EH25 9RG, UK e AFRC Institute For Animal Health, Compton, Nr. Reading, Berkshire, RG16 ONN, UK Received 19 January 1994; accepted 18 April 1994 Abstract SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed. Keywords: Theileria; Sporozoite; Antigen; Polymorphism; Vaccine 1. Introduction Abbreviations: RFLP, restriction fragment length polymor- phism.; Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank TM database with the accession numbers X78186-X78194. * Corresponding author. Tel: 0904-432864; Fax: 0904-432860; E-mail: frhl@vax.york.ac.uk Antigenic polymorphism is a common theme amongst infectious agents and has serious implica- tions for vaccine development. It is essential when considering molecules as candidates for inclusion in a sub-unit vaccine that an understanding of the na- ture and degree of variation in their structure is achieved. Ideally the aim is to define an immunolog- 0166-6851/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSD1 0166-6851(94)00086-3