In silico characterization of peptides for major surface protein 2, rhoptry-associated protein 1, and the vitellogenin receptor as potential vaccine candidates against babesiosis and anaplasmosis

Abstract

Background: Bovine babesiosis and anaplasmosis caused by Babesia bovis and Anaplasma marginale respectively pose significant threats to the livestock industry, particularly in regions where both parasites co-exist. Current live vaccines against these two diseases have limitations, including safety risks and the potential introduction of new strains. Methods: This study aimed to explore safer alternatives by use of subunit vaccines by an in silico approach. Three key proteins were targeted which are rhoptry-associated protein 1 (RAP-1) of Babesia bovis, Major Surface Protein 2 (MSP-2) of Anaplasma marginale, and the vitellogenin receptor of their vector tick Rhipicephalus microplus. Consensus sequences of these three proteins were generated to capture the most conserved regions across multiple strains. The B and T cell epitope predictions were performed where several B-cell epitopes demonstrated high antigenicity and surface accessibility. Results: For RAP-1, regions 111-122, 240-255, 294-443, and 543-560 stood out as notable B-cell epitopes, with region 111-122 exhibiting the highest antigenicity. For MSP-2 , though lower in antigenicity, region 161-173 was closest to the threshold. The vitellogenin receptor had several regions, including 35-164 and 529-544, which showed high surface accessibility and immunogenic potential. Physiochemical analysis revealed that RAP-1 epitopes displayed significant variability in molecular weight, pI values, and GRAVY scores, with most regions being hydrophilic. MSP-2 epitopes exhibited a similar trend, with region 501-519 being the most hydrophilic. Most predicted epitopes were nonallergenic and non-toxic, with only a few exceptions in RAP-1 for regions 68-92 and 111-122 as well as the vitellogenin receptor for regions 166-205 and 1247-1279. MHC-I epitopes were predicted across nine BoLA alleles, RAP-1 showed a higher number of binding peptides compared to MSP-2, thus a stronger CD8+ T cell response. Molecular docking results for MSP-2, the peptide AGAFYHKVL from the BoLA-AW10 allele had the highest though predicted as a probable allergen. On the other hand, SAFAGAFYH from BoLA-T5 showed strong binding and was non-allergenic and non-toxic. RAP-1 peptides like AKFFNRFSF showed strong binding across multiple alleles, reinforcing their importance as potential vaccine targets. Conclusion: This study identified conserved, non-toxic, and non-allergenic B and T cell epitopes from the proteins, providing a strong foundation for the development of multi-target subunit vaccines. These peptides could address the co-infections of the parasites and tick control.