Mapping of neutralizing anti-MAYV mAbs to sites within E1 and E2 proteins. (A–C) Solvent-exposed residues on the MAYV E2 B domain were changed to the indicated amino acids. WT and mutant MAYV B domain proteins were purified, and binding to anti-MAYV mAbs was tested by ELISA. Regions were divided into groups A (blue), B (red), and C (green) based on patterns of mutations that resulted in loss of binding of mAbs. Representative mAbs from each group are shown (MAY-117, group A [A]; MAY-125, group B [B]; and MAY-139, group C [C]), with the remainder of the data in Fig. S4. (D–F) 293T cells were transfected with a C-E3-E2-6K-E1 plasmid containing alanine mutations in the B domain of E2 and tested for binding with anti-MAYV mAbs by flow cytometry. Additional arginine or glutamic acid changes were made to residues in two loops in the B domain (residues 179–186 and 212–218). Representative mAbs from three binding groups are shown (MAY-117, group A [D]; MAY-125, group B [E]; and MAY-139, group C [F]), with the remainder of the data in Fig. S4. Critical residues were defined as those with ≤25% binding to an individual mAb but ≥75% binding to an oligoclonal pool of anti-MAYV mAbs. Data are from three experiments performed in triplicate. Error bars represent SD within one experiment. (G) Alignment of the B domain of E2 of MAYV, UNAV, CHIKV, RRV, and ONNV with the critical interaction residues identified for each mAb marked. Residues mapped by structure-guided mutagenesis and ELISA (circles), alanine scanning mutagenesis and flow cytometry (triangles), or both (squares) are marked. Boxes around amino acids 179–186 and 212–218 indicate loops in the B domain. Bars above the B domain indicate regions used to define binding groups A (blue), B (red), and C (green). (H–J) Neutralization escape mutants were generated by serial passage of MAYV (strain CH) in the presence of MAY-115 or MAY-131. Sequence-confirmed mutations were introduced into an infectious cDNA clone of the parental MAYV strain and tested for neutralization by MAY-117 (H), MAY-115 (I), or MAY-131 (J). Data are representative of two experiments performed in triplicate. Error bars represent SD within on experiment. (K) Key residues necessary for mAb engagement are highlighted on the surface representation of CHIKV (Basore et al., 2019; left; PDB: 6NK5) and depicted as balls and sticks on a ribbon diagram of the predicted structure of MAYV E2-E1 monomer generated using Phyre2 (right). Inset: Zoomed-in view of a trimeric spike. The E1 glycoprotein is in light gray, with the FL in green and the B-, C-, and D-strands of domain II in cyan. The E2 glycoprotein is in dark gray, with the B domain in yellow. Epitope-mapped residues in E2 domain B and E1 domain II are colored in blue and magenta, respectively.