Babesia bovis RON2 contains conserved B-cell epitopes that induce an invasion-blocking humoral immune response in immunized cattle

Abstract

Background: Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis, the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis.

Results: The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis. Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas.

Conclusions: The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Keywords: Babesia bovis; Bovine babesiosis; CLAG domain; Invasion process; Tight junction.

Fig. 1

B. bovis ron2 identification. a Synteny map for B. bovis and B. bigemina. b A representation of B. bovis RON2, including the signal peptide (SP) located from aa 1 to 19 in different strains of B. bovis and the CLAG domain that is located from aa 727 to 1168. Both are depicted as gray boxes. Shaded boxes represent the transmembrane regions and black boxes represent the sequences of the selected peptides

Fig. 2

Transcription and expression analysis of B. bovis ron2. a Bioinformatics transcription analysis. The results on the y-axis are shown by robust multi-array average (RMA) normalized values (log₂). The comparison of the expression level of ron2, ama-1 and sbp-tc9 (spherical body protein 2 truncated copy 9) genes between attenuated (white) and virulent (gray) strains is shown on the x-axis. b WB expression analysis of RON2. Lane 1: proteins of B. bovis iRBC incubated with post-immunization sera anti-RON2; Lane 2: proteins of B. bovis iRBC incubated with pre-immunization sera anti-RON2; Lane 3: proteins of nRBC incubated with post-immunization sera anti-RON2. The molecular weight marker is shown in kDa

Fig. 3

Indirect immunofluorescence of B. bovis blood stages detected with antibodies against RON2. Smears of B. bovis-infected merozoites were incubated with bovine antiserum against P2 (a), P3 (c) or serum from naturally infected cattle (e). No signal was observed in the pre-immunization serum of cattle immunized with P2 (b), P3 (d) or cattle immunized only with adjuvant (f) used as the negative control. Scale-bars: 10 μm

Fig. 4

Neutralization assay. The results on the y-axis are shown as percentages of the parasitemia inhibition. The evaluation of the inhibition generated by different antibodies is shown on the x-axis. Abbreviations: PC, positive control; AC, adjuvant control (PBS + ADJ); P1, anti-Peptide1 antibodies; P2, anti-Peptide2 antibodies; P3, anti-Peptide3 antibodies; P4, anti-Peptide4 antibodies; P2+P3, a mix of the anti-Peptide2 and anti-Peptide3 antibodies. There were significant differences between the pre- and post-immunization serum samples (*P < 0.05)