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CryoEM Structures of Antibodies Elicited by Germline-Targeting HIV MPER Epitope-Scaffolds
Jiachen Huang1, Olivia M Swanson1,2,3,4, Kimmo Rantalainen2,3,4
1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
Applying cryoEM to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa HIV membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryoEM by complexing them with 10E8 or Fabs derived from MPER-GT immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryoEM maps. Mutagenesis screening revealed off-target mAbs that do not compete with 10E8 bind non-MPER epitopes, and the two most dominant epitopes were verified by cryoEM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 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 cryoEM-based epitope mapping with smaller antigens and heterogeneous antibody mixes.
Applying cryoEM to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa HIV membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryoEM by complexing them with 10E8 or Fabs derived from MPER-GT immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryoEM maps. Mutagenesis screening revealed off-target mAbs that do not compete with 10E8 bind non-MPER epitopes, and the two most dominant epitopes were verified by cryoEM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 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 cryoEM-based epitope mapping with smaller antigens and heterogeneous antibody mixes.
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01:10Antibody Structure
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