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The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

ABSTRACT: In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

SUBMITTER: Mercado-Uriostegui MA

PROVIDER: S-EPMC9144247 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

Mercado-Uriostegui Miguel Angel MA Castro-Sánchez Luis Alberto LA Batiha Gaber El-Saber GE Valdez-Espinoza Uriel Mauricio UM Falcón-Neri Alfonso A Ramos-Aragon Juan Alberto JA Hernández-Ortiz Ruben R Kawazu Shin-Ichiro SI Igarashi Ikuo I Mosqueda Juan J

Pathogens (Basel, Switzerland) 20220518 5

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed ...[more]

PMID: 35631112

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Project description:Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Project description:BackgroundBabesia 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.ResultsThe 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.ConclusionsThe data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

Project description:Among important candidates for babesial vaccines are apical complex proteins, including rhoptry-associated protein 1 (RAP-1) from Babesia bovis and B. bigemina, which have been shown to induce partial immunity. Four variant B. bigemina rap-1 transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-alpha1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes in B. bigemina RAP-1alpha1 protein (I. Hötzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89-99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains of B. bigemina, raising the possibilities that the T-cell epitopes may vary antigenically and that CT-1 may be differentially expressed with respect to the other RAP-1 CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains of B. bigemina. We further show that the RAP-1alpha1-immunized cattle were protected against homologous B. bigemina challenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.

Dataset Information

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

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The GP-45 Protein, a Highly Variable Antigen from <i>Babesia bigemina</i>, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

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