Development of a novel subunit vaccine against East Coast fever based on the Theileria parva sporozoite surface protein p67

Theileria parva is an intracellular protozoan parasite and the causative agent of a lethal cattle disease, called East Coast fever (ECF). This disease poses a major constraint on improvement of cattle production in Eastern, Central and Southern Africa , especially for smallholder farmers. The protozoa are transmitted to cattle in the form of sporozoites by the brown-ear tick, Riphicephalus appendiculatus . The sporozoites invade lymphocytes, where they develop into schizonts. In addition, they induce a large-scale uncontrolled proliferation of the lymphocytes, leading to severe clinical symptoms, like weight loss, pyrexia, anaemia, terminal respiratory distress and finally death ensues within two to three weeks, if the animal is not treated. The disease can be cured with antibiotics, and this is the basis for the current method of vaccination, called "Infection and Treatment", where animals are injected with T. parva sporozoites and are, simultaneously, treated with antibiotics over a longer period of time. This vaccination method, however, is far from convenient, since the production of large amounts of sporozoites is very time consuming and a cold environment is needed to keep the sporozoites alive. In addition, the use of a live vaccine has pertinent risks, especially when the instructions for antibiotic treatment are not strictly followed and the immunity engendered is strain-specific. The research described in this thesis was aimed at the possibilities of developing a subunit vaccine against East Coast fever, based on the production of T. parva sporozoite surface major protein p67. This protein is present on the outside of sporozoites and plays a crucial role in the entry of sporozoites into lymphocytes and is the major antigen producing neutralising antibodies. The first objective was to produce large amounts of p67 in a near-authentic conformation. Production of recombinant p67 in bacterial expression systems had failed to produce correctly processed protein and large amounts were needed to achieve a reasonable (70 %) level of protection. The baculovirus-insect cell expression system forms a valuable alternative for the expression of large amounts of near-authentic and immunologically active proteins. Previous attempts, however, to produce p67 in insect cells resulted in low levels of recombinant protein, which had a conformation different from the native p67 protein. Again large quantities were needed to protect cattle against ECF. In the research described in thesis several types of novel baculovirus vectors were constructed to produce different regions of p67 in insect cells. In the first set of vectors, various domains of p67 were expressed as separate entities, but this resulted in low levels of expression. For the second set, domains of p67 were fused to the carboxy-terminus of the "green fluorescent protein" (GFP), a visible marker, leading to a considerable increase in yield of recombinant p67. In addition, GFP:p67 fusion polypeptides were recognised by a monoclonal antibody (TpM12), which was raised against native p67 and capable of neutralising sporozoites. On the contrary, only a small portion of full length, non-fused p67 expressed in insect cells was recognised by this antibody. Fusion to GFP, thus, appeared to increase the stability of p67 and to result in a more native configuration of the recombinant protein. In a third set of baculovirus vectors, N and C terminal domains of p67 were fused to the baculovirus envelope protein GP64. This resulted in the display of recombinant p67 on the outside of insect cells as well as on the surface of budded baculovirus particles. The TpM12 epitope was also conserved when p67 was fused to GP64. P67 could also be expressed as a secreted soluble protein. The rationale behind this experiment was to ultimately facilitate the purification of the recombinant protein. This was achieved by removal of a putative transmembrane domain and fusion of p67 to a specific signal peptide derived from honeybee melittin. Deletion of the viral genes, chitinase and v-cathepsine from the baculovirus genome enhanced the integrity and increased the stability of this secreted p67 protein. Unfortunately, the secreted form was no longer recognised by TpM12, and hence, had a conformation different from p67 in sporozoites. Therefore, the secreted p67 was not tested in further immunological studies. In order to select the best recombinant p67 products for extensive vaccine trials, the various fusion proteins combining domains of p67 with GFP or GP64 were tested in mice for their immunogenicity and, especially, the ability to induce neutralising antibodies. In mice, the p67 molecule, lacking both its signal peptide and transmembrane region, and fused to GFP (GFP:p67ΔSS) gave the best humoral immune response, followed by the p67 C-terminal domain coupled to GP64 (GP64:p67C). These two immunogens were tested in cattle, in combination with a water-in-oil or a saponin-based adjuvant. Also in cattle, a high level of sero-conversion was obtained using a total of 100 µg recombinant p67 for immunisation divided over two needle injections. Moreover, the antisera raised in mice and cattle neutralised the infectivity of T. parva sporozoites in an in-vitro assay. Subsequently, in Kenya Boran cattle were vaccinated with GFP:p67 D SS or with GP64-p67C. After a primary immunisation followed by a single booster, T. parva stabilated sporozoites were injected to test whether the vaccines protected the animals from ECF. Eighty five percent of the animals was protected from the lethal disease (ECF) using a much lower dose of recombinant protein than was used in the earlier studies. The research described in this thesis exploited the versatility of the baculovirus-insect cell expression system and showed that an ECF subunit vaccine based on recombinant p67, in a better conformation and formulated in an optimal adjuvant, can be used effectively in a vaccination program. Both of the proteins tested are good candidates for the development of a commercial ECF subunit vaccine and may contribute substantially to improvement in cattle productivity and poverty alleviation in sub-Saharan Africa