Molecular profile and B-cell epitope mapping of trypsin inhibitor-like proteins of Hyalomma anatolicum and Rhipicephalus linnaei as potential anti-tick vaccine candidates

Ticks are important ectoparasites of livestock that cause major economic losses and transmit a wide range of pathogens. Trypsin inhibitor-like (TIL) proteins are involved in blood feeding and modulation of the host immune responses, making them promising targets for anti-tick vaccine development. However, studies on TIL proteins in Hyalomma anatolicum and Rhipicephalus linnaei remain limited. This study provides the first molecular characterization and B-cell epitope mapping of TIL proteins from these tick species as potential anti-tick vaccine candidates. Hyalomma ticks were collected from cattle in Pakistan, while the R. linnaei specimens were obtained from a laboratory colony in Brazil. Hyalomma species identification was confirmed using fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) sequencing. TIL coding sequences were retrieved from corresponding species genomes, primers were designed, and target genes were amplified from synthesized cDNA. Bioinformatics analyses were performed to determine sequence similarity, conserved domains, phylogenetic relationships, antigenicity, allergenicity, secretory properties, and linear B-cell epitopes. Hyalomma ticks were morphologically identified as H. anatolicum and confirmed by cox1 sequences that showed 100% identity with previously reported sequences. The amino acid sequences of HaTIL905 from H. anatolicum showed 100% identity, whereas RsTIL401 amino acid sequence from R. linnaei exhibited 96.3% identity with similar amino acid sequences derived from the same species. HaTIL905 and RsTIL401 contained one and three conserved cysteine-rich TIL domains, respectively. Phylogenetic analysis showed that both sequences clustered within the clades of similar proteins from their respective species. Both TIL amino acid sequences were predicted to be antigenic, non-allergenic, and secretory. Four and fifteen potential linear B-cell epitopes were identified in HaTIL905 and RsTIL401, respectively. These findings highlight the potential of TIL proteins as candidates for future anti-tick vaccine development and provide a foundation for further functional and immunological studies to control tick infestations.