Babesia bovis enolase is expressed in intracellular merozoites and contains predicted B-cell epitopes that induce neutralizing antibodies in vitro

Authors: CARDENAS-FLORES, ALMA - Autonomous University Of Querétaro; CAMACHO NUEZ, MINERVA - Universidad Autonoma De Ciudad Juarez; Ueti, Massaro; HIDALGO-RUIZ, MARIO - Autonomous University Of Chiapas; RODRIGUEZ-TORRES, ANGELINA - Autonomous University Of Queretaro; HERNANDEZ SILVA, DIEGO JOSIMAR - Autonomous University Of Queretaro; GOMEZ-SOTO, JOSE GUADALUPE - Autonomous University Of Queretaro; ASADA, MASAHITO - Obihiro University; KAWAZU, SHIN-ICHIRO - Obihiro University; TAMAYO-SOSA, ALMA - Obihiro University; RUIZ-MANZANO, ROCIO ALEJANDRA - Universidad Nacianal Autonoma De Mexico; MOSQUEDA, JUAN - Autonomous University Of Queretaro

Submitted to: Vaccines
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2025
Publication Date: 7/31/2025
Citation: Cardenas-Flores, A., Camacho-Nuez, M., Ueti, M.W., Hidalgo-Ruiz, M., Rodriguez-Torres, A., Hernandez Silva, D., Gomez-Soto, J., Asada, M., Kawazu, S., Tamayo-Sosa, A.R., Ruiz-Manzano, R., Mosqueda, J. 2025. Babesia bovis enolase is expressed in intracellular merozoites and contains predicted B-cell epitopes that induce neutralizing antibodies in vitro. Vaccines. 13(8). Article 818. https://doi.org/10.3390/vaccines13080818.
DOI: https://doi.org/10.3390/vaccines13080818

Interpretive Summary: Cattle tick fever poses a significant threat to livestock globally, with Babesia bovis as the primary culprit. Recent advancements in vaccine development using immuno-bioinformatics and reverse vaccinology have identified promising antigens, including enolase—an enzyme involved in glucose metabolism with intriguing "moonlighting" properties, though its role in B. bovis is still underexplored. In our research, we sequenced the enolase gene from two B. bovis isolates and confirmed its transcription in intra-erythrocytic parasites. We identified two peptides with important B-cell epitopes from the enolase protein and administered these synthetic peptides to rabbits to produce hyperimmune sera. The antibodies generated effectively recognized intra-erythrocytic merozoites. In in vitro neutralization assays with B. bovis blood cultures, antibodies against peptide 1 showed no impact on parasitemia, while those against peptide 2 resulted in an impressive reduction in parasitemia. Our findings suggest that B. bovis enolase contains B-cell epitopes that can induce neutralizing antibodies, marking it as a promising candidate for vaccine development.

Technical Abstract: Bovine babesiosis is a leading important tick-borne disease in cattle worldwide; Babesia bovis is the species that causes the most serious clinical disease. The development of vaccines involving tools like immunobioinformatics and reverse vaccinomics includes the discovery of novel antigens. Enolase is a "Moonlight" enzyme of glucose metabolism, that has been shown to have potential as a vaccine against various pathogens. However, this protein has not been studied in B. bovis. In this study, the enolase gene of two B. bovis isolates was sequenced. The gene consisting of 1,366 base pairs was identified and its transcription in intra-erythrocytic parasites (IEP) was confirmed by RT-PCR. The predicted sequence of 438 amino acids was obtained, and two peptides containing predicted B-cell epitopes were determined. Synthetic peptides administered to rabbits obtained hyperimmune sera, with antibody titers up to 1:256,000. The specific antibodies recognized intra-erythrocytic merozoites by confocal microscopy and bound to a protein of approximately 47 kDa from B. bovis erythrocytic culture by western blot. A neutralization assay was performed with the specific anti-sera in a B. bovis in vitro culture. The antibodies generated against peptide 1 had no effect, while those against peptide 2 reduced the parasitemia by 71.99%. We conclude that B. bovis enolase contains B-cell epitopes that induce neutralizing antibodies and can be considered as a vaccine candidate.