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. Author manuscript; available in PMC: 2021 Dec 23.
Published in final edited form as: Cell. 2020 Dec 9;183(7):1884–1900.e23. doi: 10.1016/j.cell.2020.11.011

Figure 2. Neutralizing human anti-EEEV mAbs bind to SINV/EEEV particles and/or recombinant EEEV E2 glycoprotein.

Figure 2.

(A) Binding ratio of neutralizing human anti-EEEV mAbs to SINV/EEEV particles versus recombinant monomeric EEEV E2 glycoprotein. A dotted line indicates 32 pM EC50 values for binding, revealing distinct binding patterns of human anti-EEEV mAbs to SINV/EEEV particles and EEEV E2 glycoprotein. Neutralizing human anti-EEEV mAbs are labeled with the anti-EEEV mAb name and are colored according to binding group (Group 1 [red] = virus>protein binding; Group 2 [green] = strong (SINV/EEEV EC50 = <32 pM) virus » protein binding; Group 3 [purple] = weak (SINV/EEEV EC50 = >32 pM) virus » protein binding; Group 4 [orange] = protein>virus binding).

(B) EC50 values (pM) for binding of neutralizing human anti-EEEV mAbs to SINV/EEEV particles or EEEV E2 glycoprotein. Neutralizing human anti-EEEV mAbs are listed in order of binding group and increasing EC50 value for binding to SINV/EEEV particles. EC50 value in pM is indicated by the blue heat map (<32 [dark blue], 32.01 to 100 [medium blue], 100.01 to 320 [light blue], <10,000 [lightest blue]). Ratio of binding to SINV/EEEV particles versus EEEV E2 glycoprotein is indicated as the ratio of EC50 values, corresponding to Figure 2A. Increasing depth of green color indicates lower ratios (<0.1 [dark green], 0.1 to 1.0 [medium green], 1.01 to 2.0 [light green], >2.0 [lightest green]), suggesting recognition of a quaternary epitope on virion particles.

(C) Representative binding curves of neutralizing human anti-EEEV mAbs to four different antigens. Binding curves of neutralizing human anti-EEEV mAbs to SINV/EEEV particles (green) and EEEV E2 glycoprotein (blue), with mAb concentration (nM) on the x-axis and optical density at 405 nm on the y-axis. Binding to EEEV E1 (purple) or CHIKV E1 (pink) glycoproteins was not detected.

Data in A–C represent mean ± SD of technical triplicates and are representative of three independent experiments. See also Figure S1 for recombinant IgG1, IgA1, or Fab binding reactivity to SINV/EEEV particles or EEEV E2 glycoprotein.