Figure 3. Human anti-EEEV mAbs recognize three neutralizing antigenic determinants on the EEEV E2 glycoprotein.
(A) Competition-binding groups of neutralizing human anti-EEEV mAbs to recombinant EEEV E2 monomeric glycoprotein as determined through biolayer interferometry. Mouse domain B (magenta) and human mAbs were incubated with EEEV E2 glycoprotein to identify the number of antigenic determinants recognized by these mAbs. The first mAb incubated with E2 is shown in the left-hand column and the second mAb is shown in the top column. Black boxes indicate competition, or reduction in maximum signal for binding of the second mAb to <33%. Grey boxes indicate intermediate competition, or reduction in maximum signal for binding of the second mAb to between 33 to 67%. White boxes indicate no competition, or little to no reduction in maximum signal for binding of the second mAb to >67%. Each mAb is colored based on binding group as defined in Figure 2. IC50 (pM) values for neutralization activity against SINV/EEEV are indicated in parentheses (Figure 1D).
(B) Heat map of critical residues for neutralizing human anti-EEEV mAbs as determined through alanine-scanning mutagenesis library analysis. The average percent binding of each neutralizing human anti-EEEV mAbs is indicated for the critical residues identified (<25% binding of mAb in which at least two mAbs exhibited >70% binding to control for expression; D1-L267) and for the previously characterized murine anti-EEEV mAbs (Kim et al., 2019) and the VEEV-specific human mAb, F5 (Hunt et al., 2010; Porta et al., 2014). The heat map displays average % binding relative to WT EEEV E2 glycoprotein with dark blue (>70%), light blue (25–70%), and light green (<25%). Residues are colored based on E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange). Each mAb is colored based on binding group as defined in Figure 2 and ordered to correspond with the competition-binding groups as defined in Figure 3A. Data represents mean of at least two independent experiments.
(C) Epitope mapping of critical alanine and arginine residues previously identified for neutralizing murine anti-EEEV mAbs binding to the E2 glycoprotein. Critical residues for binding of murine anti-EEEV mAbs as previously determined through alanine and arginine mutagenesis analyses were mapped onto the 4.2 Å cryo-EM reconstruction of EEEV VLP (EMD-22276; PBD ID: 6XO4) for comparison to the critical alanine residues identified for human anti-EEEV mAbs (see Figure 3D). A trimeric top view of the E2 (green) and E1 (red) glycoproteins is shown with critical residues (spheres) for murine anti-EEEV mAbs that recognize the E2 domains A, B, and A/B. Residues are colored based on E2 domain (Domain A - red and Domain B - cyan).
(D) Epitope mapping of critical alanine residues identified for neutralizing human anti-EEEV mAbs binding to the E2 glycoprotein. Critical residues for binding of human anti-EEEV mAbs as identified through alanine-scanning mutagenesis library analyses (Figure 3B) were mapped as described in Figure 3C. Residues are colored based on E2 domain (N-link - purple, Domain A - red, Arch 1 - magenta, Domain B - cyan, and Arch 2 - orange). Yellow spheres indicate the previously identified SINV/EEEV neutralization escape mutants (M68T, G192R, and L227R) (Kim et al., 2019). Each mAb is presented with its respective E2 domain and is colored based on binding group as defined in Figure 2. See Figure S2 for a bar graph representation of the percent binding of each mAb to the alanine residues described in Figure 3B. See Figure S3 for neutralization activity of mAbs against the SINV/EEEV escape mutants (M68T, G192R, and L227R). See Table S1 for critical alanine residues identified for each mAb.