IDENTIFICATION OF CANDIDATE VACCINE ANTIGENS OF BOVINE HEMOPARASITES THEILERIA-PARVA AND BABESIA-BOVIS BY USE OF HELPER T-CELL CLONES

Current vaccines for bovine hemoparasites utilize live attenuated orga nisms or virulent organisms administered concurrently with antiparasit ic drugs. Although such vaccines can be effective, for most hemoparasi tes the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selectio n of potentially protective antigens has traditionally made use of ant ibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predict s the ability of an antigen to confer protective immunity nor correlat es with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immuno gens should be used. Through the elaboration of cytokines, T helper 1- (Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an impor tant role in protective immunity against bovine hemoparasites. CD4(+) T cell clones specific for soluble or membrane antigens of either Thei leria parva schizonts or Babesia bovis merozoites were therefore emplo yed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatog raphy, or a combination thereof, and fractions were tested for the abi lity to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. par va prot eins of approximately 10 and 24 kDa. Antisera raised against the purif ied 24 kDa band reacted with a native schizont protein of approximatel y 30 kDa. Babesia bovis-specific Th cell clones tested against fractio nated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separate d by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Stu dies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasi te membranes as well as from the cytosolic fraction of B. bovis merozo ites. The Th cell clones reactive with these different hemoparasites e xpressed either unrestricted or Th1 cytokine profiles, and were genera lly characterized by the production of high levels of IFN-gamma. A com prehensive study of T cell and macrophage responses to defined parasit e antigens will help elucidate the reasons for vaccine failure or succ ess, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.