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. Author manuscript; available in PMC: 2022 Aug 19.
Published in final edited form as: Cell. 2021 Aug 19;184(17):4430–4446.e22. doi: 10.1016/j.cell.2021.07.033

Figure 4. Acidic pH-dependent or -independent binding reactivity of human E1-specific mAbs to virus-like particles (VLPs) or recombinant E1 glycoproteins.

Figure 4.

A to C. Representative binding curves of acidic pH-dependent EEEV-specific (green labels) and cross-reactive (‘pan-alphavirus’ [purple labels] and ‘broadly-reactive’ [orange labels]) mAbs to EEEV (green), VEEV (dark blue), or WEEV (orange) VLPs, recombinant EEEV (light purple), CHIKV (magenta), or MAYV (purple) E1 glycoproteins, and recombinant EEEV E2 (blue) glycoprotein at 1× DPBS pH 5.4 (open circles) or 1× DPBS pH 7.4 (open triangles) with mAb concentration (μg/mL) on the x-axis and optical density at 450 nm on the y-axis. Binding curves are ordered by decreasing dependence on pH 5.4 for binding and grouped according to epitope and binding reactivity (DI (A) [EEEV-specific], DII (B) [EEEV-specific], and fusion loop (C) [EEEV-specific (C.1.), ‘broadly-reactive’ (C.2.), and ‘pan-alphavirus’ (C.3.)]. D to G. Representative binding curves of pH-independent EEEV-specific (green labels) and cross-reactive (‘New World’ [magenta labels], ‘EEEV-VEEV’ [blue labels]) mAbs to respective antigens as described in A. Binding curves are ordered numerically and grouped according to epitope and binding reactivity (DII (D) [EEEV-specific], DIII (E) [EEEV-specific (E.1.) and ‘EEEV-VEEV’ (E.2.)], a quaternary epitope including DI, DII, DIII, DI/DII hinge region, and the DI/DIII linker (F) [‘New World’], or an unknown epitope (G). H and I. Representative binding curves of a pH-independent positive control neutralizing, EEEV E2-specific mAb (rEEEV-129 IgG [H]; Williamson et al., 2020) and the negative control DENV-specific mAb (rDENV-2D22 IgG [I]; Fibriansah et al., 2015). Data in A to I represent mean ± SD of technical triplicates and are representative of 2 independent experiments. See Figure 1 for mAb binding by ELISA in the presence of the nonionic detergent Tween®20. See Figures 3 and S4 for epitope summary. See Table S4 for acidic pH-dependent or pH-independent binding summary.