(590d) Microsphere-Assisted Peptide Screening (MAPs): High-Throughput Identification of Promiscuous MHCII-Binding Peptides for T-Cell Epitope Vaccine Designs
AIChE Annual Meeting
Wednesday, November 1, 2017 - 4:09pm to 4:27pm
To address some of the limitations of current experimental systems used to screen peptide-MHC binding, we developed a novel high-throughput screening method to rapidly identify promiscuously binding peptides. Microsphere-assisted peptide screening (MAPs) coupled with our modular baculovirus-MHCII expression system allows one to screen large peptide libraries for binding to many MHCII alleles. The MAPs approach consists of performing peptide-exchange reactions in which an epitope-tagged peptide is loaded on to one of four common MHCII alleles. These exchanged-pMHC complexes are loaded to the surface of microspheres and analyzed using flow cytometry to determine the relative peptide binding. We validated the MAPs system by screening a 20mer peptide library with known binding data and analyzed the results using receiver operating characteristic (ROC) curves. This ROC analysis demonstrated the MAPs system is rapid and accurate with an area under curve greater than 0.90. We then applied the MAPs approach to successfully identify four promiscuously-binding peptides within a 26-member peptide-library of the rotavirus capsid protein VP7. Interestingly, structural analysis of the rotavirus VP7 protein revealed that these four promiscuously-binding peptides share a number of residues with known antibody neutralization sites on the native VP7 trimer. Finally, the complete peptide-binding dataset for the VP7 library screen was compared to peptide binding predictions by computational methods within the IEDB Analysis Resource including SMM-align, NN-align, and NetMHCIIpan. The predictive-algorithms captured many of the peptide-binders observed using the MAPs system; however, the computational predictions had a false-negative rate of roughly 20% including the four promiscuously binding peptides discovered using MAPs. Therefore, in addition to serving as a robust experimental platform for identifying promiscuously binding peptides for potential T cell epitope vaccines, MAPs should also contribute to the improvement of computational systems that predict peptide-MHCII binding in silico.