Abstract
Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.
Highlights
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We developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi
We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins
Summary
To assess the performance of combined data from different experiments, the normalized and negative sample-subtracted peptides values of different chip/assays were averaged and ROC analysis performed as described. The intensity correlation between all pairs of peptides derived from the same protein (but physically located at random microarray positions) was high for highly overlapped peptides (PCC of 0.86 Ϯ 0.02 (S.D.), 14 residue overlap, i.e. with single residue shifts) and monotonically decreases for lower sequence overlaps (down to a PCC Ͻ0.25 for pairs of peptides with a six residue overlap) This analysis was performed with a subset of 105 reactive and nonrepetitive proteins (i.e. excluding those proteins where Ͼ20% of sequence is composed of internal tandem repeats, as detected by the TRUST repeat detection method [36]). The complete set of peptides was submitted to the Immune Epitope Database Resource [5] under submission ID 1000642
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