CHARACTERIZATION OF AN INSECT CELL-DERIVED THEILERIA-PARVA SPOROZOITEVACCINE ANTIGEN AND IMMUNOGENICITY IN CATTLE

Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoi te surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural prote in of influenza virus, and a linker DNA sequence. NS1-p67 lacked react ivity with TpM 12, a monoclonal antibody to native p67, and had an est imated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attem pt to express this parasite protein in a native form and thereby incre ase the protective capacity of the antigen. However, Spodoptera frugip erda SF21AE cells infected with recombinant virus expressed p67 as a 1 00-kDa molecule. The host cells exhibited a limited capacity to glycos ylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule o f about 70 kDa. Since the bulk of insect cell-derived p67 was not expr essed in an appropriate form, we tested the immunogenicity of these pa rtially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals devel oped high levels of anti-p67 antibodies that neutralized sporozoite in fectivity in vitro, but antigen-specific T-cell proliferative response s were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the mo lecule of choice for the development of vaccines against East Coast fe ver.