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Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

ABSTRACT: The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

SUBMITTER: Brown WC

PROVIDER: S-EPMC174227 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

Brown W C WC McElwain T F TF Ruef B J BJ Suarez C E CE Shkap V V Chitko-McKown C G CG Tuo W W Rice-Ficht A C AC Palmer G H GH

Infection and immunity 19960801 8

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is high ...[more]

PMID: 8757873

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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:A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.

Project description:Babesiosis is an important haemoparasitic disease, caused by the infection and subsequent intra-erythrocytic multiplication of protozoa of the genus Babesia that impacts the livestock industry and animal health. The distribution, epidemiology and genetic characterization of B. bigemina, B. bovis, and B. ovata in cattle in China as well as the prevalence of these protozoan agents were assessed.A total of 646 blood specimens from cattle, dairy cattle and yaks from 14 provinces were collected and tested for the presence of the three Babesia species via a specific nested PCR assay based on the rap-1 and ama-1 genes. The PCR results were confirmed by DNA sequencing. Gene sequences and the genetic characterization were determined for selected positive samples from each sampling area.Of a total of 646 samples, 134 (20.7 %), 60 (9.3 %) and 10 (1.5 %) were positive for B. bovis, B. bigemina and B. ovata infections, respectively. Mixed infections were found in 7 of 14 provinces; 43 (6.7 %) samples were infected with B. bovis and B. bigemina. Three samples (0.5 %) exhibited a co-infection with B. bovis and B. ovata, and 6 (0.9 %) were infected with all three parasites. The rap-1a gene of B. bovis indicated a high degree of sequence heterogeneity compared with other published rap-1a sequences worldwide and was 85-100 % identical to B. bovis rap-1a sequences in Chinese isolates. B. bigemina rap-1c and B. ovata ama-1 genes were nearly identical, with 97.8-99.3 % and 97.8-99.6 % sequence identity, respectively, in GenBank.Positive rates of B. bovis and B. bigemina infection are somewhat high in China. The B. bovis infection in yaks was first reported. The significant sequence heterogeneity in different variants of the rap-1a gene from Chinese B. bovis isolates might be a great threat to the cattle industry if RAP-1a protein is used as immunological antigen against Babesia infections in China. The data obtained in this study can be used to plan effective control strategies against babesiosis in China.

Dataset Information

Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

Publications

Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

Similar Datasets

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