Functional and Mass Spectrometric Evaluation of an Anti-Tick Antigen Based on the P0 Peptide Conjugated to Bm86 Protein

Abstract

A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from <i>Megathura crenulata</i> and used as an immunogen against <i>Rhipicephalus microplus</i> and <i>Rhipicephalus sanguineus</i> s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against <i>Amblyomma mixtum</i> and <i>Ixodes ricinus</i> species, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein. SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0-Bm86 conjugate and challenged with <i>R. sanguineus</i> s.l. and <i>R. microplus</i>, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.