CryoEM Structures of Antibodies Elicited by Germline-Targeting HIV MPER Epitope-Scaffolds

Huang, J.; Swanson, O. M.; Rantalainen, K.; Fernandez-Quintero, M. L.; Loeffler, J. R.; Tingle, R.; Georgeson, E.; Phelps, N.; Ozorowski, G.; Schiffner, T.; Schief, W. R.; Ward, A. B.

doi:10.1101/2025.08.19.671101

bioRxiv · 10.1101/2025.08.19.671101

CryoEM Structures of Antibodies Elicited by Germline-Targeting HIV MPER Epitope-Scaffolds

Huang, J.·Swanson, O. M.·Rantalainen, K.·Fernandez-Quintero, M. L.·Loeffler, J. R.·Tingle, R.·Georgeson, E.·Phelps, N.·Ozorowski, G.·Schiffner, T.·Schief, W. R.·Ward, A. B.

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Abstract

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. HighlightsO_LIElucidate the structural basis of increased 10E8 affinity to GT12 over GT10.2 C_LIO_LICryoEM epitope mapping reveals three main binding sites on MPER-GT immunogens C_LIO_LIProof of concept for application of CryoEM epitope mapping to small immunogens C_LIO_LIStructural characterization and MD analysis confirms 10E8-like interactions of the MPER-specific Fabs C_LI

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Huang, J., Swanson, O. M., Rantalainen, K., Fernandez-Quintero, M. L., Loeffler, J. R., Tingle, R., Georgeson, E., Phelps, N., Ozorowski, G., Schiffner, T., Schief, W. R., Ward, A. B.. 2025-08-19. CryoEM Structures of Antibodies Elicited by Germline-Targeting HIV MPER Epitope-Scaffolds. https://doi.org/10.1101/2025.08.19.671101

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