Cryo-EM structures of antibodies elicited by germline-targeting HIV MPER epitope scaffolds

Applying cryoelectron microscopy (cryo-EM) to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa human immunodeficiency virus (HIV) membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryo-EM by complexing them with 10E8 or Fabs derived from MPER-GT-immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryo-EM maps. Mutagenesis screening revealed that off-target monoclonal antibodies (mAbs), which do not compete with 10E8, bind non-MPER epitopes, and the binding of two most dominant epitopes were verified by cryo-EM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 heavy chain complementarity-determining region 3 (HCDR3) loop. Additionally, we demonstrate that high-resolution maps can be generated from heterogeneous samples with pooled competing Fabs. Overall, our findings will facilitate the optimization of MPER-GT antigens and push the size limit for cryo-EM-based epitope mapping with smaller antigens and heterogeneous antibody mixes.