Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.
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 15 mer 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 15 mers (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.
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Santiago J
SJ
Carmona
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina; §Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark;
Morten
M
Nielsen
¶Schafer-N ApS, 2100 Copenhagen, Denmark;
Claus
C
Schafer-Nielsen
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Juan
J
Mucci
‖Servicio de Parasitología y Chagas, Hospital de Niños Ricardo Gutiérrez, Ciudad de Buenos Aires, Argentina.
Jaime
J
Altcheh
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Virginia
V
Balouz
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Valeria
V
Tekiel
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Alberto C
AC
Frasch
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Oscar
O
Campetella
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina;
Carlos A
CA
Buscaglia
From the ‡Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús, Universidad de San Martín - CONICET, Sede San Martín, B 1650 HMP, San Martín, Buenos Aires, Argentina; fernan@unsam.edu.ar.
Fernán
F
Agüero
D000953:Antigens, Protozoan / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D014355:Chagas Disease / Q000276:immunology*; D030562:Databases, Protein; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D010455:Peptides / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D000953:Antigens, Protozoan; D018985:Epitopes, B-Lymphocyte; D010455:Peptides