RESUMO
Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN-) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.
Assuntos
Babesia bovis , Babesiose , Doenças dos Bovinos , Animais , Anticorpos Neutralizantes , Antígenos de Protozoários , Babesiose/prevenção & controle , Bovinos , Epitopos de Linfócito T , Imunidade Humoral , Proteínas de ProtozoáriosRESUMO
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.