Insights into the B¬cell response in a natural human infection: high-¬throughput mapping of epitopes using next ¬generation peptide chips

CARMONA SJ; SCHAFER-NIELSEN C; MUCCI J; TEKIEL, V; LEGUIZAMON S; CAMPETELLA, O; BUSCAGLIA, C; NIELSEN M; AGUERO F

Berlin

Congreso; 21st Annual Conference on Intelligent Systems for Molecular Biology / 12th European Conference on Computational Biology; 2013

ISMB/ECCB

Background. The full set of specificities in a human antibody response to a natural infection remains largely unexplored. Here, we used next-generation high-density peptide microarrays to demonstrate for the first time that it is feasible to identify and map hundreds of B-cell epitopes from a complex natural human infection. In this work, we have analyzed the B-cell immune response in humans with Chagas Disease, caused by a protozoan parasite (Trypansoma cruzi).Methods. The peptide chips were synthesized using a mask less photolithographic technique. The chip design contains a tiling array of ~200K 15-mer peptides which in concert cover >500 individual proteins, with a maximal 14-mer overlap. This represents a coverage of ~5% of the proteome, including known antigens, previously uncharacterized proteins selected using a recently published bioinformatic method (Carmona SJ, et al 2012, PLOSOne 7: e50748), randomly selected proteins, and random sequences following parasite's proteome di-peptide composition. The chip was first incubated with IgGs from healthy individuals (negative control), scanned, then assayed with IgGs from Chagas Disease patients, and scanned again. Peptide signals were scaled according to the signal distribution of randomly generated peptides (background). Signal from the negative controls were subtracted from the signal observed in the readout from the positive sample to obtain the disease-specific signal for each peptide. These were used to reconstruct full-length protein antigenicity profiles (Figure 1, left panel). A smoothing procedure showed significant signal to noise ratio improvement. A testing set of Chagas antigens with fine mapped epitopes was used to assess our performance on linear B-cell epitope re-identification.Results. Using these high-density peptide chips, we were able to scan >500 proteins over their entire length. The performance of epitope re-discovery from known antigens was excellent, with ROC AUC = 0.92 (Fig 1). Discrimination of antigens from non-antigens is a more challenging task. Using a threshold of 20u (1u = background interquartile range) and a setup with an antigen-non-antigen ratio of 1%, we were able to detect 20 out of the set of 45 known antigens (44.4%) with a Positive Predictive Value (PPV) of 91% corresponding to 2 false positive predictions. Applying this threshold to the complete set of proteins analyzed on the chip, we detected 78 novel potential antigens, with an average of 1.5 epitopes per protein.Conclusions. In this work we show that high-density peptide chips allow rapid, high-throughput identification of B-cell epitopes from a natural infection, caused by a complex pathogen. With the antigen discovery performance observed in this study, using a single sera pool, we envision the possibility of a complete B-cell response map in a complex human infection