A Theileria Annulata sporozoite surface antigen as a potential vaccine for tropical Theileriosis

Abstract

The aim of this study was to identify potentially protective antigens of the infective, sporozoite, stage of Theileria annulata and to clone the relevant gene(s) to obtain one or more of these antigens as a recombinant protein with which to perform immunisation trials.

Theileria annulata and the disease it causes, tropical theileriosis, were described with particular reference to literature concerning characteristics of the sporozoite stage, immunity and immunisation. The application of biotechnology to the production of recombinant DNA vaccines was reviewed and relevant examples were detailed.

To this end, antisporozoite monoclonal antibodies (Mabs) were raised and those giving positive fluorescence of formalin fixed sporozoites by the indirect fluorescent antibody test (IFAT) were selected. Nineteen such Mabs were screened for the ability to neutralise sporozoite infectivity for bovine peripheral blood mono¬ nuclear cells in vitro. Two antisporozoite Mabs, 1A7 and 4B11, which demonstrated surface immunofluorescence of live sporozoites, exhibited a significant degree of sporozoite inhibition. These were chosen for further investigation. The in vitro assay was also used to detect sporozoite neutralising activity in hyperimmune bovine sera, sera from calves exposed to irradiated sporozoites and serum from rabbits on which infected ticks had fed.

Using SDS-PAGE Western blotting, Mabs 1A7 and 4B11 were shown to identify different epitopes on the sporozoite surface. Mab 1A7 specifically recognised an epitope present on several sporozoite V proteins of approximate molecular weights 85, 72, 63 and 54 kilodaltons (kdal) in Western blots. This was believed to reflect pro¬ cessing of a high molecular weight precursor. Mab 4B11 recognised a low molecular weight protein of 17-20 kdal, also located on the sporozoite surface. Some immune bovine sera and antisporozoite rabbit serum also detected these two sporozoite epitopes.

Large amounts of one of the epitopes were made available as a recombinant protein by cloning and expressing the relevant theilerial gene fragment in Escherichia coli (E. coli) . To achieve this a λgtl 1 expression library, constructed using genomic DNA from T. annulata piroplasm DNA, was screened with Mabs 1A7 and 4B11. Two recombinant clones, λ gtl1-SR1 and λgtll-SR2, were obtained, both of which contained the gene sequence coding for the epitope recognised by Mab 1A7. The theilerial DNA insert of clone Agtl1-SRl was 330 base pairs in size and hybridised to three DNA bands in EcoRl digested genomic DNA from an uncloned parasite stock. These bands were segregated to single copies of the gene sequence in the DNA from cloned parasite material. Northern blot analysis using RNA from infected tick salivary glands showed expression of the λgtll-SRl insert to be stage specific, occurring only in sporoblast and sporozoite stages and not in macroschizonts or piroplasms, nor in uninfected tick salivary glands.

The recombinant DNA cloned in λgtll-SRl and λgtll-SR2 was expressed in E. coli as B-galactosidase fusion proteins of 135 and 147 kdal respectively. These proteins reacted specifically with Mab 1A7 and also with antisporozoite rabbit serum and certain sera from calves exposed to live or irradiated sporozoites. Immunisation trials using the purified 135 kdal protein from λgtl1-SRl, were performed in mice, rabbits and calves. Inoculation of rabbits and calves with this fusion protein combined with Freund's adjuvant elicited a strong and specific antibody response. These antibodies alpo recognised the native sporozoite epitope when assessed by IFAT and Western blotting, the latter revealing exactly the same multiple reactivity of sporo¬ zoite proteins with anti λgtll-SRl sera as observed with Mab 1A7. Significantly, the same sera also neutralised sporozoite infectivity in vitro very effectively, while control sera failed to do so. On challenge with live virulent sporozoites, the calves immunised with theλgtll-SRl protein became infected and underwent clinical reactions which were not significantly different from those observed in control B-galactosidase immunised or unimmunised calves. Discussion of these results concentrated on whether this failure to stimulate protective immunity reflected an inadequate method of antigen presentation, or whether antisporozoite immunity alone is insufficient to protect otherwise fully susceptible calves.