A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from Megathura crenulata and used as an immunogen against Rhipicephalus microplus and Rhipicephalus sanguineus s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against Amblyomma mixtum and Ixodes ricinus 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 R. sanguineus s.l. and R. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

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A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys 1 pP0 and p64K-βAla 1 pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli , the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys 1 pP0 showed a heterogeneous conjugate compared to p64K-βAla 1 pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys 1 pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-βAla 1 pP0 this ratio was 5-7. Cys 1 pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while βAla 1 pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys 1 pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys 1 pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract

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Traditional vaccines based on killed or attenuated microorganisms or inactivated toxins have disadvantages associated with the risks of pathogenicity reversion, contamination with infectious material, variations between vaccine batches, storage problems, among others. Peptide identification containing important epitopes within antigens could be an attractive possibility to avoid those risks. However, in general, peptides are poorly immunogenic. Adjuvants are a part of the solution to improve the immunogenicity of these peptide based vaccines. Recently, a peptide from the tick P0 acidic ribosomal protein chemically conjugated to the Bm86 protein has been assayed as vaccine candidate against ticks. This antigen formulated in oily preparations containing Montanide TM ISA50 (SEPPIC, France) generated a specific antibody response against the P0 peptide which showed an efficacy around 85% against R. sanguineus ticks when used for dog immunization. This efficacy was positively correlated with the anti-P0 antibody titers. In this paper, the immunogenicity of this chemical conjugate was assayed in dogs when Montanide™ GEL 01 (SEPPIC, France) was used as adjuvant instead of oily Montanide TM ISA 50. The antibody titers against the P0 peptide did not show statistically significant differences between the experimental groups and the evaluation of changes in the inoculation site showed significantly lesser adverse side effects in the group immunized with the antigen in Montanide™ GEL. These results validate the use of this gel as a safer adjuvant for dogs than oily Montanide, encouraging the use of this adjuvant for the technological development of the pP0-Bm86 conjugate as an anti-tick vaccine for dogs.