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The dissemination of Theileria parva immortalized bovine lymphoblasts into lymphoid and nonlymphoid tissues results in East Coast Fever – a disease that continues to ravage cattle herds owned by resource poor small holder farmers and pastoralists throughout eastern, central, and southern Africa. The shortcomings of available control options are exemplified by failing acaricides and chemotherapeutics, and the technical and operational inadequacies of the live vaccination regime termed infection and treatment method (ITM). These constraints have provided impetus towards the development of subunit vaccines intended to render genetically diverse out-bred cattle populations immune to challenge by antigenically distinct parasites in the field throughout the endemic areas.
Schizont antigens complexed with cattle class I MHC induce CD8+ cytotoxic T-lymphocytes (CTLs) that are the major effectors for immune control of T. parva. However, at present, there is insufficient information to predict the constraints to recombinant vaccine development based on induction of protective CTLs that may be imposed by functional divergence within cattle class I MHC genes. The first of the two broad studies described herein, utilized amplicon-based next generation sequencing combined with rigorous read processing algorithms to obtain reliable class I MHC genotypes from a field population of African native Bos taurus (Ankole). The study then leveraged progress in 'reverse immunology' to infer the extent of functional difference among the class I MHC alleles expressed by Ankole cattle as well as by exotic (Holstein) cattle. Finally, the study sought to ascertain if the dissimilarities seen in the in silico predictions of class I MHC peptide binding specificities between the two taurine lineages could be corroborated by ex vivo tests assaying cytokine responses to defined T. parva antigens.
The major findings from the first study included: (i) the identification of 18 novel cattle class I MHC allelic sequences in Ankole cattle, (ii) the evidence of positive selection for sequence diversity including in residues that predominantly interact with peptides in Ankole class I MHC, (iii) the evidence from in silico functional analysis of peptide binding specificities that are largely distinct between the two breeds and (iv) the demonstration that CD8+ T-cells derived from Ankole cattle that were seropositive for T. parva did not recognize vaccine candidate antigens originally identified in Holstein cattle breeds. This includes the immunodominant Tp1 which is currently the main focus of ongoing ECF recombinant vaccine trials. Taken together, the data clearly demonstrates that overlap between the peptide binding specificities of cattle class I MHC molecules is likely to be largely confined to alleles belonging to the same breed. These differences have the implication that a number of different antigens/epitopes will need to be incorporated in a CD8+ T-cell based recombinant antigen cocktail vaccine to provide broad coverage.
In addition to the cellular responses, a contribution of humoral responses in mediating immunity against the sporozoite stage of the parasite has been documented. The closest approach to an effective anti-sporozoite vaccine to date has used a recombinant version of p67, the major sporozoite surface antigen of T. parva that is the target of antibodies that can potentially neutralize the establishment of infection. However, exposure of p67 immunized cattle to field tick challenge has resulted in very limited protection relative to experimental laboratory trials. The second study sought to shed light on the possible impacts of heterologous parasite challenge on the protection afforded to p67 immunized cattle. The concern was that whereas the gene encoding p67 is conserved in all cattle-derived populations of T. parva, it remained unclear whether parasites that originate from buffalo, and capable of causing severe disease in co-grazing cattle, contain diverse or invariant p67 genotypes. The primary analysis involved examining allelic variation, principally length polymorphisms and amino acid diversity in the closely juxtaposed B cell epitopes mapped to the central region of the p67 protein. The study also sought to shed light on the mechanisms that underpin molecular evolution of the p67 gene.
The findings included: (i) the identification of ten discrete p67 allelic sequences with an overall DNA polymorphism of 19.6%. This contrasts with the complete conservation of nucleotides at the p67 locus, including introns and the third base in codons, in cattle-transmissible isolates, (ii) the demonstration that the p67 allelic sequences described herein varied widely in size principally due to the 129 and 174 base pair deletions in the central region of the gene, (iii) the identification of an in-frame deletion and nonsynonymous substitutions in the two closely juxtaposed B cell epitopes in the central region of the p67 protein respectively, (iv) the demonstration of an evolutionary pattern within the T. parva p67 locus that is consistent with the effects of positive selection. In sum, these findings suggest that a p67 based vaccine suitable for field use, particularly where a wildlife reservoir of infection is present, should incorporate polymorphic epitopes as an improvement to the current conserved p67 vaccine antigen.
The totality of the data presented here provides a more informed picture of the development needs of subunit vaccines satisfying both the efficacy and coverage criteria. The key recommendation is that priority should be given to grouping cattle class I MHC alleles into functional ‘supertypes’, once extensive polymorphism data is collated so as to better inform the feasibility of generating CD8+ T-cell based vaccines with a high population coverage regardless of 'breed' differences. It could also be helpful to re-assess the field performance of the p67 vaccine based on simultaneous immunization with vaccine constructs expressing the allelic variants described herein.