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.

Keywords: B cell epitopes; CD4+ T cell epitopes; MHC class II; Zika virus; peptide binding prediction; promiscuous peptide; rotavirus; vaccine.

FIGURE 1

MAPS involves three phases: prepare, load, and analyze. The preparation phase involves (1a) purifying and biotinylating a panel of diverse human MHCII alleles and (1b) synthesizing an overlapping DNP-tagged peptide library. The loading phase involves (2) loading the DNP-tagged peptides from the library onto each MHCII allele via peptide exchange, and (3) loading the biotinylated, peptide-exchanged MHCII on streptavidin-coated microspheres. The analysis phase involves (4) staining the loaded microspheres for the DNP-tagged peptide and (5) analyzing resulting signal using flow cytometry. DNP, dinitrophenyl; MAPS, microsphere-assisted peptide screening

FIGURE 2

Validation of MAPS strategy with DR1-HA_306–318 interaction. (a) SDS-PAGE analysis of the SDS-stability of various DR1 complexes. After thrombin cleavage of the CLIP peptide in the DR1-CLIP protein, the target HA_306–318 peptide was added to allow DR1-HA_306–318 complex formation through peptide exchange, or no peptide was added (empty DR1) as a control. (b) Flow cytometry dot plots of DNP-signal detected on microspheres loaded with: empty DR1 (left), HA_306–318 peptide alone (middle), or DR1-HA_306–318 (right). (c) Relative MAPS signal plotted with respect to the relative position of the HA_306–318 PBR within a 20mer peptide. The peripheral flanking residues were mutated to alanine. Relative MAPS signal represents the MAPS signal of each peptide normalized by the MAPS signal observed for the peptide with a relative PBR position of 1. DNP, dinitrophenyl; MAPS, microsphere-assisted peptide screening; PBR, peptide binding register

FIGURE 3

ROC analysis of MAPS strategy compared to IEDB peptide-MHCII binding predictors for the AhpC reference peptide library. The AUC of each curve is provided in the legend. AUC, area under curve; IEDB, Immune Epitope Database and Analysis Resource; MAPS, microsphere-assisted peptide screening; ROC, receiver operating characteristic

FIGURE 4

MAPS analysis of overlapping peptides from Zika virus E protein for four human MHCII alleles. (a) MAPS signal of each ZikVE peptide. Data are shown as the mean of two independent experiments. Error bars signify standard deviation. Dashed lines represent the peptide-binding threshold of MAPS signal of 5. For the heat map, orange and red indicate MAPS signals of 5–30 and >30, respectively. (b) Pie-charts representing the percentage of ZikVE peptides that bound to each MHCII allele. Colored slices represent the fraction of binders. (c) A pie-chart representing the fractions of ZikVE peptides binding 0, 1, 2, 3, or 4 MHCII alleles. MAPS, microsphere-assisted peptide screening

FIGURE 5

Structural analysis of MAPS-identified promiscuous MHCII-binding peptides in the Zika virus E protein. (a) MAPS signal for each promiscuous MHCII-binding E protein peptide. Dashed lines represent the peptide-binding threshold of MAPS signal of 5. E protein structural analysis of (b) the protein domains, (c) the five promiscuous MHCII-binding peptides as well as the quasi-promiscuous peptide ZikVE_391–410 identified by MAPS and (d) the B cell epitopes reported previously.^, In (d), the residues of the B cell epitopes overlapping with the promiscuous MHCII-binding peptides are in black, and the nonoverlapping residues are in teal. In (c) and (d), only EDIII is shown in the image on the left for clarity. MAPS, microsphere-assisted peptide screening

FIGURE 6

MAPS analysis of overlapping peptides from VP7 protein for four human MHCII alleles. (a) MAPS signal of each VP7 peptide. Data are shown as the mean of two independent experiments. Error bars signify standard deviation. Dashed lines represent peptide-binding threshold of MAPS signal of 5. For the heat map, orange and red indicate MAPS signals of 5–30 and >30, respectively. (b) Pie-charts representing the percentage of VP7 peptides that bound to each MHCII allele. Colored slices represent the fraction of binders. (c) A pie-chart representing the fraction of VP7 peptides binding 0, 1, 2, 3, or 4 MHCII alleles. MAPS, microsphere-assisted peptide screening

FIGURE 7

Structural analysis of MAPS-identified promiscuous MHCII-binding VP7 peptides. (a) MAPS signal for each promiscuous MHCII-binding VP7 peptide. VP7 structural analysis (b) of the promiscuous MHCII-binding peptides VP7_81–100, VP7_111–130, and VP7_211–230, and (c) the B cell epitopes (BCEs) reported previously. In (c), the residues of the BCEs overlapping with the promiscuous MHCII-binding peptides VP7_81–100, VP7_111–130, or VP7_211–230 are in black, and the nonoverlapping residues are in teal. (d) Amino acid sequence overlaps between the three dominant BCEs on the VP7 protein and the promiscuous MHCII-binding peptides VP7_81–100, VP7_111–130, and VP7_211–230. Overlapping residues are indicated in red. MAPS, microsphere-assisted peptide screening