Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoscreening of peptide arrays with sera from mice infected with different T. cruzi strains.(A) Sera from un-infected mice; sera from mice infected with Colombiana (TcI) (B), Y (TcII) (C), or CL Brener (TcVI) (D). (E) Venn diagram showing the specific and shared epitopes among T. cruzi strains.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814679&req=5

pntd-0002524-g001: Immunoscreening of peptide arrays with sera from mice infected with different T. cruzi strains.(A) Sera from un-infected mice; sera from mice infected with Colombiana (TcI) (B), Y (TcII) (C), or CL Brener (TcVI) (D). (E) Venn diagram showing the specific and shared epitopes among T. cruzi strains.

Mentions: A total of 50 conserved, 50 polymorphic Esmo-specific, and 50 polymorphic Non-Esmo-specific peptides with high epitope prediction scores were selected for the construction of peptide arrays. The reactivity of the peptides was tested using a pool of sera from six C57BL/6 mice chronically infected with Colombiana (TcI), Y (TcII), or CL Brener (TcVI) strains and un-infected mice as the control group (Figure 1). The quantification of the reactivity was performed by densitometric analysis (Supplementary Table S3). A peptide was considered reactive and antigenically conserved if its intensity signal with all T. cruzi strains was two times higher than its signal with the sera from un-infected mice. A peptide was considered reactive and antigenically polymorphic if its intensity signal with a specific strain was two times higher than the values with the other two strains and the un-infected mice. A total of 36 peptides were considered reactive with at least one strain (Figure 1E). A conserved peptide with the highest reactivity with all T. cruzi strains (C6_30_cons) and three polymorphic peptides specific for Colombiana (A6_30_col), Y (B2_30_y), and CL Brener (B9_30_cl) were selected for soluble synthesis, and their reactivity was validated by ELISA.


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)

Immunoscreening of peptide arrays with sera from mice infected with different T. cruzi strains.(A) Sera from un-infected mice; sera from mice infected with Colombiana (TcI) (B), Y (TcII) (C), or CL Brener (TcVI) (D). (E) Venn diagram showing the specific and shared epitopes among T. cruzi strains.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002524-g001: Immunoscreening of peptide arrays with sera from mice infected with different T. cruzi strains.(A) Sera from un-infected mice; sera from mice infected with Colombiana (TcI) (B), Y (TcII) (C), or CL Brener (TcVI) (D). (E) Venn diagram showing the specific and shared epitopes among T. cruzi strains.
Mentions: A total of 50 conserved, 50 polymorphic Esmo-specific, and 50 polymorphic Non-Esmo-specific peptides with high epitope prediction scores were selected for the construction of peptide arrays. The reactivity of the peptides was tested using a pool of sera from six C57BL/6 mice chronically infected with Colombiana (TcI), Y (TcII), or CL Brener (TcVI) strains and un-infected mice as the control group (Figure 1). The quantification of the reactivity was performed by densitometric analysis (Supplementary Table S3). A peptide was considered reactive and antigenically conserved if its intensity signal with all T. cruzi strains was two times higher than its signal with the sera from un-infected mice. A peptide was considered reactive and antigenically polymorphic if its intensity signal with a specific strain was two times higher than the values with the other two strains and the un-infected mice. A total of 36 peptides were considered reactive with at least one strain (Figure 1E). A conserved peptide with the highest reactivity with all T. cruzi strains (C6_30_cons) and three polymorphic peptides specific for Colombiana (A6_30_col), Y (B2_30_y), and CL Brener (B9_30_cl) were selected for soluble synthesis, and their reactivity was validated by ELISA.

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