A contribution to the development of anti-tick vaccines

Abstract

Ticks and tick-borne diseases seriously affect animal and human health worldwide with the highest economic losses occurring in livestock production in the developing world. The control of ticks and the diseases they transmit depends mainly on chemical tick control using acaricides. The development of acaricide resistance, concerns about environmental pollution and pesticide residues in food products result in the need for alternative tick control methods such as anti-tick vaccines. Commercial vaccines based on the recombinant antigen Bm86 have been developed for the control of Rhipicephalus (Boophilus) microplus infestations on cattle. Although they can make an important contribution to an integrated tick control strategy, more efficacious and ideally stand-alone vaccines are required to control multiple tick species in wide geographical areas. There is thus a need for improved vaccine formulations and for the discovery of new tick-protective antigens. This identification of tick-protective antigens is a pivotal step in vaccine development. To study gene function and to identify genes which are important to the tick’s function and survival, RNA interference (RNAi) can be a valuable tool. In this thesis, a strategy for gene silencing by RNAi in the one-host tick R. microplus is outlined and a novel RNAi application for the transovarial silencing of genes in ticks is introduced. Subolesin, a protein which was identified as a tick-protective antigen is further characterized and evidence of the role of subolesin in gene expression in ticks is provided. Cross protection of Bm86 vaccines against tick species other than R. microplus has been reported, but Bm86 vaccines do not protect against Rhipicephalus appendiculatus infestations. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. To measure the Bm86 gene expression in a reliable and accurate manner, normalization of gene expression data against reference genes is essential and the expression stability of commonly used reference genes was evaluated in both species. The most stable reference genes were subsequently used for normalization of the Bm86 expression profile in all life stages of these ticks to examine the role of antigen abundance in Bm86 vaccine susceptibility. Partial protection of the Bm86 vaccine against other Rhipicephalus (Boophilus) and Hyalomma tick species suggests that the efficacy of a Bm86-based vaccine may be enhanced when based on the orthologous recombinant Bm86 antigen. We therefore identified the Bm86 homologues from species representing the main argasid and ixodid tick genera. A novel protein from metastriate ticks with multiple Epidermal Growth Factor-like domains which is structurally related to Bm86 was discovered and named ATAQ. The vaccine potential of ATAQ proteins against tick infestations is to be evaluated. The expression and purification of Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) as secreted products from Pichia pastoris is also described. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery and purity which is likely to result in a more reproducible conformation closely resembling the native protein and increase the efficacy of these vaccines.