Discovery of novel serological biomarkers associated to congenital Chagas Disease using a highly-multiplexed discovery platform based on high-density peptide microarrays

CARMONA SJ; SCHAFER-NIELSEN C; A BUSCAGLIA, CARLOS; JUAN MUCCI,; ALTCHEH J; CAMPETELLA O; C FRASCH, ALBERTO CARLOS; NIELSEN M; AGÜERO F

Mar del Plata

Congreso; X Congreso de Protozoología y Enfermedades Parasitarias; 2014

Sociedad Argentina de Protozoología y Enfermedades Parasitaria

The full set of antibody (B-cell) specificities associated with the response to a natural infection remains largely unexplored. We developed a highly-multiplexed discovery platform based on next-generation high-density peptide microarrays and demonstrate for the first time its potential to simultaneously identify and finely map hundreds of B-cell epitopes from a complex natural human infection. The platform consists of a HD tiling peptide array containing ~200K unique 15mer peptides synthesized in situ on a microarray slide, using a maskless photolithographic method. The peptides completely cover the full length of 468 T. cruzi proteins, scanning the protein sequence at maximal resolution (1 residue shift). These proteins include 59 previously described antigens, 50 proteins randomly selected from the proteome, 100 proteins selected using a recently published bioinformatics method (Carmona SJ et al 2012); and 232 surface proteins from a number large gene families. The array also contains 24K 15mers corresponding to ~50 neo-proteins of random sequence to estimate the array background baseline (negative control). Using this plaThe full set of antibody (B-cell) specificities associated with the response to a natural infection remains largely unexplored. We developed a highly-multiplexed discovery platform based on next-generation high-density peptide microarrays and demonstrate for the first time its potential to simultaneously identify and finely map hundreds of B-cell epitopes from a complex natural human infection. The platform consists of a HD tiling peptide array containing ~200K unique 15mer peptides synthesized in situ on a microarray slide, using a maskless photolithographic method. The peptides completely cover the full length of 468 T. cruzi proteins, scanning the protein sequence at maximal resolution (1 residue shift). These proteins include 59 previously described antigens, 50 proteins randomly selected from the proteome, 100 proteins selected using a recently published bioinformatics method (Carmona SJ et al 2012); and 232 surface proteins from a number large gene families. The array also contains 24K 15mers corresponding to ~50 neo-proteins of random sequence to estimate the array background baseline (negative control). Using this platform we have analyzed the B-cell immune response in humans with Chagas Disease, assaying 8 independent HD-arrays with 4 pools of purified IgGs from Chagas Disease patients and negative controls. After defining a very conservative cutoff we identified 1,193 positive 15mers in the set of serologically uncharacterized proteins, which define 98 new antigens. We also identified the location of linear epitopes for a number of known antigens, as well as a number of protein regions which are the target of non-specific antibody binding (putative cross-reactive responses). This high-density peptide array platform now allows high-throughput identification and mapping of B-cell epitopes. In this presentation we will discuss the use of this platform to discover epitopes associated with congenital Chagas Disease. Acknowledgements: Funded by a grant (FITS-Chagas-003) from the National Agency for the Promotion of Science and Technology (ANPCyT, Argentina).