Identificação e validação de epítopos potencialmente antigênicos de Babesia bovis por abordagens de vacinologia reversa, genética molecular e imunologia experimental

Abstract

Bovine Parasitic Sadness (BPS) is a complex of hemoparasitic diseases caused by Babesia bovis, Babesia bigemina, and Anaplasma marginale, with B. bovis being the agent associated with the most severe clinical manifestations of BPS. Its control involves management of the tick vector, chemoprophylaxis, and the use of live vaccines, strategies that present limitations related to acaricide resistance, maintenance of enzootic stability, and the risks and practical difficulties associated with the use of viable infectious agents. In this context, the development of vaccines based on defined antigens represents a promising alternative, and reverse vaccinology, integrated with bioinformatics and immunoinformatics tools, enables the identification and rational prioritization of candidates based on genomic information. Therefore, this study aimed to identify and evaluate candidate epitopes of B. bovis through the integration of reverse vaccinology, molecular genetics, and experimental immunology. Initially, the predicted proteome derived from the reference genome of the B. bovis T2Bo strain, comprising 3,959 protein-coding genes, was analyzed in silico, and application of the selection criteria allowed the prioritization of nine plasma membrane proteins with VaxiJen antigenicity scores above 0.7, from which 31 candidate epitopes were predicted. The gene regions corresponding to the selected candidates were evaluated in Brazilian B. bovis isolates by PCR, sequencing, and alignment analysis. High conservation was observed in the analyzed regions of the BBOV_IV000770 gene, which encodes the predicted protein XP_001609243.1 and contains five of the selected epitopes. Based on this validation, the peptides CNPGL-Bbovis-5E2, CNPGL-Bbovis-5E3, and CNPGL-Bbovis-5E5 were synthesized, initially conjugated to the carrier protein KLH. The conjugated peptides were evaluated for immunoreactivity by indirect ELISA using sera from cattle experimentally infected with B. bovis, and for their effects on the metabolic activity of peripheral blood mononuclear cells (PBMCs) using cells obtained from animals previously exposed to natural B. bovis infection. The peptides were also evaluated in their free form using ELISA and the resazurin reduction assay. The peptides conjugated to the carrier protein KLH showed no IgG recognition attributable to the peptides and did not consistently differ from KLH alone in terms of PBMC metabolic activity. In their free form, the peptides CNPGLBbovis-5E3 and CNPGL-Bbovis-5E5 showed serological IgG recognition in a subset of B. bovis-positive animals, with heterogeneity among samples, while the three peptides exhibited distinct profiles of modulation of PBMC metabolic activity. CNPGL- Bbovis-5E3 stood out by showing effects associated with peptide concentration and incubation time. Overall, the results enabled the prioritization of B. bovis candidates based on evidence of conservation, serological recognition, and functional activity, highlighting CNPGL-Bbovis-5E3 as a promising antigenic candidate. Further studies specifically addressing immunogenicity and protection are required to determine its vaccine potential and, additionally, to assess the applicability of these candidates in immunodiagnostic strategies.