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Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

Mendes TA, Reis Cunha JL, de Almeida Lourdes R, Rodrigues Luiz GF, Lemos LD, dos Santos AR, da Câmara AC, Galvão LM, Bern C, Gilman RH, Fujiwara RT, Gazzinelli RT, Bartholomeu DC - PLoS Negl Trop Dis (2013)

Bottom Line: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs.Four peptides were tested against a panel of chagasic patients using ELISA.A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

ABSTRACT

Background: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

Methodology: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

Findings and conclusions: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

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Reactivity and affinity of sera from T. cruzi-infected mice against conserved and polymorphic epitopes.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. The dotted line represents the cutoff value. The solid gray line represents the mean values. C-, un-infected mice. Colombiana (TcI), mice infected with the Colombiana strain. Y (TcII), mice infected with the Y strain. CL Brener (TcVI), mice infected with the CL Brener strain. *p<0.05 and **p<0.005.
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pntd-0002524-g002: Reactivity and affinity of sera from T. cruzi-infected mice against conserved and polymorphic epitopes.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. The dotted line represents the cutoff value. The solid gray line represents the mean values. C-, un-infected mice. Colombiana (TcI), mice infected with the Colombiana strain. Y (TcII), mice infected with the Y strain. CL Brener (TcVI), mice infected with the CL Brener strain. *p<0.05 and **p<0.005.

Mentions: Because immunoblotting assays are semi-quantitative techniques, we validated the results with quantitative ELISA and affinity ELISA assays using individual sera from six C57BL/6 mice chronically infected with the Colombiana (TcI), Y (TcII), or CL Brener (TcVI) strains and the un-infected mice as a control group. For the conserved peptide C6_30_cons, no significant difference in the reactivity among the sera from animals infected with different T. cruzi strains was observed (Figure 2A). The sera from mice infected with the Colombiana strain had a higher antibody titer against the A6_30_col peptide compared to the sera from mice infected with the Y strain. More importantly, the affinity antibodies discriminated Colombiana infection from those caused by the other two strains (Figure 2B). An expected recognition profile was also observed for the peptide B2_30_y (Figure 2C): sera from mice infected with the Y strain had a significantly higher antibody titer than those from mice infected with Colombiana, and the highest affinity antibodies generated by the Y strain discriminated its infection from those caused by Colombiana and CL Brener. With regard to peptide B9_30_cl, conventional ELISA was able to discriminate CL Brener infection from that caused by Y and Colombiana (Figure 2D).


Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

Mendes TA, Reis Cunha JL, de Almeida Lourdes R, Rodrigues Luiz GF, Lemos LD, dos Santos AR, da Câmara AC, Galvão LM, Bern C, Gilman RH, Fujiwara RT, Gazzinelli RT, Bartholomeu DC - PLoS Negl Trop Dis (2013)

Reactivity and affinity of sera from T. cruzi-infected mice against conserved and polymorphic epitopes.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. The dotted line represents the cutoff value. The solid gray line represents the mean values. C-, un-infected mice. Colombiana (TcI), mice infected with the Colombiana strain. Y (TcII), mice infected with the Y strain. CL Brener (TcVI), mice infected with the CL Brener strain. *p<0.05 and **p<0.005.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814679&req=5

pntd-0002524-g002: Reactivity and affinity of sera from T. cruzi-infected mice against conserved and polymorphic epitopes.(A) Peptide C6_30_cons. (B) Peptide A6_30_col. (C) Peptide B2_30_y. (D) Peptide B9_30_cl. The dotted line represents the cutoff value. The solid gray line represents the mean values. C-, un-infected mice. Colombiana (TcI), mice infected with the Colombiana strain. Y (TcII), mice infected with the Y strain. CL Brener (TcVI), mice infected with the CL Brener strain. *p<0.05 and **p<0.005.
Mentions: Because immunoblotting assays are semi-quantitative techniques, we validated the results with quantitative ELISA and affinity ELISA assays using individual sera from six C57BL/6 mice chronically infected with the Colombiana (TcI), Y (TcII), or CL Brener (TcVI) strains and the un-infected mice as a control group. For the conserved peptide C6_30_cons, no significant difference in the reactivity among the sera from animals infected with different T. cruzi strains was observed (Figure 2A). The sera from mice infected with the Colombiana strain had a higher antibody titer against the A6_30_col peptide compared to the sera from mice infected with the Y strain. More importantly, the affinity antibodies discriminated Colombiana infection from those caused by the other two strains (Figure 2B). An expected recognition profile was also observed for the peptide B2_30_y (Figure 2C): sera from mice infected with the Y strain had a significantly higher antibody titer than those from mice infected with Colombiana, and the highest affinity antibodies generated by the Y strain discriminated its infection from those caused by Colombiana and CL Brener. With regard to peptide B9_30_cl, conventional ELISA was able to discriminate CL Brener infection from that caused by Y and Colombiana (Figure 2D).

Bottom Line: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs.Four peptides were tested against a panel of chagasic patients using ELISA.A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

ABSTRACT

Background: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

Methodology: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

Findings and conclusions: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Show MeSH
Related in: MedlinePlus