Towards high-throughput immunomics for infectious diseases: use of next-generation peptide microarrays for rapid discovery and mapping of antigenic determinants

SANTIAGO CARMONA; MORTEN NIELSEN; CLAUS SCHAFER-NIELSEN; JUAN MUCCI; JAIME ALTCHEH; VIRGINIA BALOUZ; VALERIA TEKIEL; ALBERTO C. FRASH; OSCAR CAMPETELLA; CARLOS A. BUSCAGLIA; FERNAN AGUERO

MOLECULAR & CELLULAR PROTEOMICS

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2015

1535-9476

Complete characterization of antibody specificities associated to natural infections is expected toprovide 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 peptidemicroarrays to map these specificities in Chagas Disease, an exemplar of a human infectious diseasecaused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarraycontaining more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptideswere synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteinswith fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified frominfected patients and healthy donors demonstrated both a high technical reproducibility as well asepitope mapping consistency when compared with earlier low-throughput technologies. Using aconservative signal threshold to classify positive (reactive) peptides we identified 2,031 diseasespecificpeptides and 97 novel parasite antigens, effectively doubling the number of known antigensand providing a tenfold 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 ofdisplayed peptides can increase the protein space covered in a single chip by at least ~3 fold withoutsacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discoveryof pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for highthroughputbiomarker discovery screenings and proteome-wide studies of immune responses againstpathogens.