Rapid microsphere‐assisted peptide screening (MAPS) of promiscuous MHCII‐binding peptides in Zika virus envelope protein

Abstract

Despite promising developments in computational tools, peptide‐class II MHC (MHCII) binding predictors continue to lag behind their peptide‐class I MHC counterparts. Consequently, peptide–MHCII binding is often evaluated experimentally using competitive binding assays, which tend to sacrifice throughput for quantitative binding detail. Here, we developed a high‐throughput semiquantitative peptide–MHCII screening strategy termed microsphere‐assisted peptide screening (MAPS) that aims to balance the accuracy of competitive binding assays with the throughput of computational tools. Using MAPS, we screened a peptide library from Zika virus envelope (E) protein for binding to four common MHCII alleles (DR1, DR4, DR7, DR15). Interestingly, MAPS revealed a significant overlap between peptides that promiscuously bind multiple MHCII alleles and antibody neutralization sites. This overlap was also observed for rotavirus outer capsid glycoprotein VP7, suggesting a deeper relationship between B cell and CD4+ T cell specificity which can facilitate the design of broadly protective vaccines to Zika and other viruses.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering

“…After cell wall removal, spheroplasts are fixed with varying concentrations of formalin. Formalin concentrations of 0.5%, 1%, 2%, or 4% are commonly used in literature for different types of cells. , Note that other fixation reagents could also be considered. , Formalin-fixed spheroplasts are then permeabilized and stained for housekeeping proteins such as GAPDH and β actin as described above. The fluorescence intensity of housekeeping proteins and the autofluorescence intensity of the isotype control are next evaluated to determine the optimal formalin concentration as illustrated in Figure A.…”

A high-throughput yeast display approach to profile pathogen proteomes for MHC-II binding

“…A wave of higher throughput approaches have been recently developed for studying peptide-MHC interactions, including yeast display ( Jiang and Boder, 2010 ; Liu et al, 2021a ; Rappazzo et al, 2020 ; Wen et al, 2008 ) and mammalian display-based methods ( Obermair et al, 2022 ). Many of these assays rely upon construction of DNA-based libraries ( Jiang and Boder, 2010 ; Obermair et al, 2022 ; Rappazzo et al, 2020 ; Wen et al, 2008 ), although approaches using chemically synthesized peptides have also recently been described ( Liu et al, 2021a ; Smith et al, 2019 ). DNA libraries have been generated either via DNA oligonucleotide synthesis ( Jiang and Boder, 2010 ; Obermair et al, 2022 ; Rappazzo et al, 2020 ) or random fragmentation and insertion of viral genomic material ( Wen et al, 2008 ).…”