Utilizing Machine Learning to Improve Neutralization Potency of an HIV-1 Antibody Targeting the gp41 N-Heptad Repeat

Filsinger Interrante, Maria V.; Tang, Shaogeng; Kim, Soohyun; Shanker, Varun R.; Hie, Brian L.; Bruun, Theodora U. J.; Wu, Wesley; Pak, John E.; Fernandez, Daniel; Kim, Peter S.

doi:10.1021/acschembio.5c00035

Utilizing Machine Learning to Improve Neutralization Potency of an HIV-1 Antibody Targeting the gp41 N-Heptad Repeat

Journal Article · Fri Jun 20 00:00:00 EDT 2025 · ACS Chemical Biology

DOI:https://doi.org/10.1021/acschembio.5c00035· OSTI ID:2583445

^[1]; Tang, Shaogeng ^[2]; Kim, Soohyun ^[3]; Shanker, Varun R. ^[1]; Hie, Brian L. ^[4]; ^[1]; Wu, Wesley ^[5]; Pak, John E. ^[5]; Fernandez, Daniel ^[6]; Kim, Peter S. ^[7]

Stanford University School of Medicine, CA (United States); Stanford University, CA (United States)
Stanford University, CA (United States); Stanford University School of Medicine, CA (United States); Yale University, New Haven, CT (United States)
Stanford University, CA (United States); Stanford University School of Medicine, CA (United States)
Stanford University, CA (United States); Stanford University School of Medicine, CA (United States); Arc Institute, Palo Alto, CA (United States)
Chan Zuckerberg Biohub─San Francisco, CA (United States)
Stanford University, CA (United States)
Stanford University, CA (United States); Stanford University School of Medicine, CA (United States); Chan Zuckerberg Biohub─San Francisco, CA (United States)

The N-heptad repeat (NHR) of the HIV-1 gp41 prehairpin intermediate (PHI) is an attractive potential vaccine target with high sequence conservation across diverse strains. However, despite the potency of NHR-targeting peptides and clinical efficacy of the NHR-targeting entry inhibitor enfuvirtide, no potently neutralizing NHR-directed monoclonal antibodies (mAbs) nor antisera have been identified or elicited to date. The lack of potent NHR-binding mAbs both dampens enthusiasm for vaccine development efforts at this target and presents a barrier to performing passive immunization experiments with NHR-targeting antibodies. To address this challenge, we previously developed an improved variant of the NHR-directed mAb D5, called D5_AR, which is capable of neutralizing diverse tier-2 viruses. Building on that work, here we present the 2.7Å-crystal structure of D5_AR bound to NHR mimetic peptide IQN17. We then utilize protein language models and supervised machine learning to generate small (n < 100) libraries of D5_AR variants that are subsequently screened for improved neutralization potency. We identify a variant with 5-fold improved neutralization potency, D5_FI, which is the most potent NHR-directed monoclonal antibody characterized to date and exhibits broad neutralization of tier-2 and −3 pseudoviruses as well as replicating R5 and X4 challenge strains. Additionally, our work highlights the ability of protein language models to efficiently identify improved mAb variants from relatively small libraries.

View Accepted Manuscript (DOE)

Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); Stanford University Medical Scientist Training Program; USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 2583445

Journal Information:: ACS Chemical Biology, Journal Name: ACS Chemical Biology Journal Issue: 7 Vol. 20; ISSN 1554-8937; ISSN 1554-8929

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (58)

MolProbity: More and better reference data for improved all-atom structure validation: PROTEIN SCIENCE.ORG Williams, Christopher J.; Headd, Jeffrey J.; Moriarty, Nigel W. Protein Science, Vol. 27, Issue 1 https://doi.org/10.1002/pro.3330	journal	November 2017
Inhibiting HIV-1 Entry Eckert, Debra M.; Malashkevich, Vladimir N.; Hong, Lily H. Cell, Vol. 99, Issue 1 https://doi.org/10.1016/S0092-8674(00)80066-5	journal	October 1999
HIV Entry and Its Inhibition Chan, David C.; Kim, Peter S. Cell, Vol. 93, Issue 5 https://doi.org/10.1016/S0092-8674(00)81430-0	journal	May 1998
A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice Qi, Zhi; Pan, Chungen; Lu, Hong Biochemical and Biophysical Research Communications, Vol. 398, Issue 3 https://doi.org/10.1016/j.bbrc.2010.06.109	journal	July 2010
Leveraging Uncertainty in Machine Learning Accelerates Biological Discovery and Design Hie, Brian; Bryson, Bryan D.; Berger, Bonnie Cell Systems, Vol. 11, Issue 5 https://doi.org/10.1016/j.cels.2020.09.007	journal	November 2020
A Meta-analysis of Passive Immunization Studies Shows that Serum-Neutralizing Antibody Titer Associates with Protection against SHIV Challenge Pegu, Amarendra; Borate, Bhavesh; Huang, Yunda Cell Host & Microbe, Vol. 26, Issue 3 https://doi.org/10.1016/j.chom.2019.08.014	journal	September 2019
Broadly neutralizing antibodies against HIV-1 and concepts for application Gruell, Henning; Schommers, Philipp Current Opinion in Virology, Vol. 54 https://doi.org/10.1016/j.coviro.2022.101211	journal	June 2022
Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1 Sarzotti-Kelsoe, Marcella; Bailer, Robert T.; Turk, Ellen Journal of Immunological Methods, Vol. 409 https://doi.org/10.1016/j.jim.2013.11.022	journal	July 2014
Inference of Macromolecular Assemblies from Crystalline State Krissinel, Evgeny; Henrick, Kim Journal of Molecular Biology, Vol. 372, Issue 3 https://doi.org/10.1016/j.jmb.2007.05.022	journal	September 2007
Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics Nelson, Josh D.; Kinkead, Heather; Brunel, Florence M. Virology, Vol. 377, Issue 1 https://doi.org/10.1016/j.virol.2008.04.005	journal	July 2008
Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth Gustchina, Elena; Louis, John M.; Frisch, Christian Virology, Vol. 393, Issue 1 https://doi.org/10.1016/j.virol.2009.07.019	journal	October 2009
Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers Sliepen, Kwinten; Medina-Ramírez, Max; Yasmeen, Anila Virology, Vol. 486 https://doi.org/10.1016/j.virol.2015.08.002	journal	December 2015
HIV-1 gp41 Six-Helix Bundle Formation Occurs Rapidly after the Engagement of gp120 by CXCR4 in the HIV-1 Env-Mediated Fusion Process Gallo, Stephen A.; Puri, Anu; Blumenthal, Robert Biochemistry, Vol. 40, Issue 41 https://doi.org/10.1021/bi0155596	journal	September 2001
Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry Kilby, J. Michael; Hopkins, Sam; Venetta, Thomas M. Nature Medicine, Vol. 4, Issue 11 https://doi.org/10.1038/3293	journal	November 1998
Antibody neutralization and escape by HIV-1 Wei, Xiping; Decker, Julie M.; Wang, Shuyi Nature, Vol. 422, Issue 6929 https://doi.org/10.1038/nature01470	journal	March 2003
Challenges in the development of an HIV-1 vaccine Barouch, Dan H. Nature, Vol. 455, Issue 7213 https://doi.org/10.1038/nature07352	journal	October 2008
Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors Kutner, Robert H.; Zhang, Xian-Yang; Reiser, Jakob Nature Protocols, Vol. 4, Issue 4 https://doi.org/10.1038/nprot.2009.22	journal	March 2009
Enfuvirtide LaBonte, Jason; Lebbos, John; Kirkpatrick, Peter Nature Reviews Drug Discovery, Vol. 2, Issue 5 https://doi.org/10.1038/nrd1091	journal	May 2003
Structural basis for HIV-1 neutralization by a gp41 fusion intermediate–directed antibody Luftig, Micah A.; Mattu, Marco; Di Giovine, Paolo Nature Structural & Molecular Biology, Vol. 13, Issue 8 https://doi.org/10.1038/nsmb1127	journal	July 2006
Advancing an HIV vaccine; advancing vaccinology Burton, Dennis R. Nature Reviews Immunology, Vol. 19, Issue 2 https://doi.org/10.1038/s41577-018-0103-6	journal	December 2018
Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies Haynes, Barton F.; Wiehe, Kevin; Borrow, Persephone Nature Reviews Immunology, Vol. 23, Issue 3 https://doi.org/10.1038/s41577-022-00753-w	journal	August 2022
Efficient evolution of human antibodies from general protein language models Hie, Brian L.; Shanker, Varun R.; Xu, Duo Nature Biotechnology https://doi.org/10.1038/s41587-023-01763-2	journal	April 2023
A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope Miller, M. D.; Geleziunas, R.; Bianchi, E. Proceedings of the National Academy of Sciences, Vol. 102, Issue 41 https://doi.org/10.1073/pnas.0506927102	journal	October 2005
Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates Bianchi, E.; Joyce, J. G.; Miller, M. D. Proceedings of the National Academy of Sciences, Vol. 107, Issue 23 https://doi.org/10.1073/pnas.1004261107	journal	May 2010
Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d -peptide HIV entry inhibitor Nishimura, Yoshiaki; Francis, J. Nicholas; Donau, Olivia K. Proceedings of the National Academy of Sciences, Vol. 117, Issue 36 https://doi.org/10.1073/pnas.2009700117	journal	August 2020
Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences Rives, Alexander; Meier, Joshua; Sercu, Tom Proceedings of the National Academy of Sciences, Vol. 118, Issue 15 https://doi.org/10.1073/pnas.2016239118	journal	April 2021
The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat Montefiori, David C.; Filsinger Interrante, Maria V.; Bell, Benjamin N. Proceedings of the National Academy of Sciences, Vol. 118, Issue 3 https://doi.org/10.1073/pnas.2018027118	journal	January 2021
A new lease on life for an HIV-neutralizing antibody class and vaccine target Burton, Dennis R. Proceedings of the National Academy of Sciences, Vol. 118, Issue 7 https://doi.org/10.1073/pnas.2026390118	journal	January 2021
HIV-1 prehairpin intermediate inhibitors show efficacy independent of neutralization tier Bell, Benjamin N.; Bruun, Theodora U. J.; Friedland, Natalia Proceedings of the National Academy of Sciences, Vol. 120, Issue 8 https://doi.org/10.1073/pnas.2215792120	journal	February 2023
Stabilized trimeric peptide immunogens of the complete HIV-1 gp41 N-heptad repeat and their use as HIV-1 vaccine candidates Wu, Chengwei; Raheem, Izzat T.; Nahas, Debbie D. Proceedings of the National Academy of Sciences, Vol. 121, Issue 22 https://doi.org/10.1073/pnas.2317230121	journal	May 2024
Covalent Trimers of the Internal N-terminal Trimeric Coiled-coil of gp41 and Antibodies Directed against Them Are Potent Inhibitors of HIV Envelope-mediated Cell Fusion Louis, John M.; Nesheiwat, Issa; Chang, LengChee Journal of Biological Chemistry, Vol. 278, Issue 22 https://doi.org/10.1074/jbc.M301627200	journal	May 2003
Prophylactic HIV vaccine: vaccine regimens in clinical trials and potential challenges Pitisuttithum, Punnee; Marovich, Mary Anne Expert Review of Vaccines, Vol. 19, Issue 2 https://doi.org/10.1080/14760584.2020.1718497	journal	February 2020
A Broadly Neutralizing Human Monoclonal Antibody against gp41 of Human Immunodeficiency Virus Type 1 Purtscher, Martin; Trkola, Alexandra; Gruber, Gerhard AIDS Research and Human Retroviruses, Vol. 10, Issue 12 https://doi.org/10.1089/aid.1994.10.1651	journal	December 1994
UniRef: comprehensive and non-redundant UniProt reference clusters Suzek, B. E.; Huang, H.; McGarvey, P. Bioinformatics, Vol. 23, Issue 10 https://doi.org/10.1093/bioinformatics/btm098	journal	March 2007
HIV subtype diversity worldwide Bbosa, Nicholas; Kaleebu, Pontiano; Ssemwanga, Deogratius Current Opinion in HIV and AIDS, Vol. 14, Issue 3 https://doi.org/10.1097/COH.0000000000000534	journal	January 2019
Broadly neutralizing antibodies for HIV treatment and cure approaches Frattari, Giacomo Schmidt; Caskey, Marina; Søgaard, Ole Schmeltz Current Opinion in HIV and AIDS, Vol. 18, Issue 4 https://doi.org/10.1097/COH.0000000000000802	journal	May 2023
Language models enable zero-shot prediction of the effects of mutations on protein function Meier, Joshua; Rao, Roshan; Verkuil, Robert https://doi.org/10.1101/2021.07.09.450648	preprint	November 2021
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/S0907444904019158	journal	November 2004
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
Towards automated crystallographic structure refinement with phenix.refine Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4 https://doi.org/10.1107/S0907444912001308	journal	March 2012
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L. Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10 https://doi.org/10.1107/S2059798319011471	journal	October 2019
Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy Wei, X.; Decker, J. M.; Liu, H. Antimicrobial Agents and Chemotherapy, Vol. 46, Issue 6 https://doi.org/10.1128/AAC.46.6.1896-1905.2002	journal	June 2002
Broadly Neutralizing Monoclonal Antibodies 2F5 and 4E10 Directed against the Human Immunodeficiency Virus Type 1 gp41 Membrane-Proximal External Region Protect against Mucosal Challenge by Simian-Human Immunodeficiency Virus SHIVBa-L Hessell, A. J.; Rakasz, E. G.; Tehrani, D. M. Journal of Virology, Vol. 84, Issue 3 https://doi.org/10.1128/JVI.01272-09	journal	November 2009
Design of a Potent D-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance Welch, B. D.; Francis, J. N.; Redman, J. S. Journal of Virology, Vol. 84, Issue 21 https://doi.org/10.1128/JVI.01339-10	journal	August 2010
Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies Seaman, M. S.; Janes, H.; Hawkins, N. Journal of Virology, Vol. 84, Issue 3 https://doi.org/10.1128/JVI.02108-09	journal	November 2009
A Derivative of the D5 Monoclonal Antibody That Targets the gp41 N-Heptad Repeat of HIV-1 with Broad Tier-2-Neutralizing Activity Rubio, Adonis A.; Filsinger Interrante, Maria V.; Bell, Benjamin N. Journal of Virology, Vol. 95, Issue 15 https://doi.org/10.1128/JVI.02350-20	journal	July 2021
Peptides Corresponding to the Heptad Repeat Motifs in the Transmembrane Protein (gp41) of Human Immunodeficiency Virus Type 1 Elicit Antibodies to Receptor-Activated Conformations of the Envelope Glycoprotein de Rosny, Eve; Vassell, Russell; Wingfield, Paul T. Journal of Virology, Vol. 75, Issue 18 https://doi.org/10.1128/JVI.75.18.8859-8863.2001	journal	September 2001
Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion Gallo, Stephen A.; Reeves, Jacqueline D.; Garg, Himanshu Retrovirology, Vol. 3, Issue 1 https://doi.org/10.1186/1742-4690-3-90	journal	December 2006
Characterization of resistance to a potent d-peptide HIV entry inhibitor Smith, Amanda R.; Weinstock, Matthew T.; Siglin, Amanda E. Retrovirology, Vol. 16, Issue 1 https://doi.org/10.1186/s12977-019-0489-7	journal	October 2019
Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41 Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo PLoS ONE, Vol. 8, Issue 11 https://doi.org/10.1371/journal.pone.0078187	journal	November 2013
Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41 Gustchina, Elena; Li, Mi; Louis, John M. PLoS Pathogens, Vol. 6, Issue 11 https://doi.org/10.1371/journal.ppat.1001182	journal	November 2010
Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41 Sabin, Charles; Corti, Davide; Buzon, Victor PLoS Pathogens, Vol. 6, Issue 11 https://doi.org/10.1371/journal.ppat.1001195	journal	November 2010
The Mosaico HIV Vaccine Study: A Step Back or a Stepping Stone for Future Vaccine Development? Wallace, Stephaun E. Infectious Diseases, Vol. 2, Issue 1 https://doi.org/10.17925/ID.2023.2.1.2	journal	January 2023
Major Scientific Hurdles in HIV Vaccine Development: Historical Perspective and Future Directions Ng’uni, Tiza; Chasara, Caroline; Ndhlovu, Zaza M. Frontiers in Immunology, Vol. 11 https://doi.org/10.3389/fimmu.2020.590780	journal	October 2020
HIV-1 Entry and Membrane Fusion Inhibitors Xiao, Tianshu; Cai, Yongfei; Chen, Bing Viruses, Vol. 13, Issue 5 https://doi.org/10.3390/v13050735	journal	April 2021
Preventive HIV Vaccines-Leveraging on Lessons from the Past to Pave the Way Forward Sobia, Parveen; Archary, Derseree Vaccines, Vol. 9, Issue 9 https://doi.org/10.3390/vaccines9091001	journal	September 2021
Gaussian Processes for Machine Learning Rasmussen, Carl Edward; Williams, Christopher K. I. The MIT Press https://doi.org/10.7551/mitpress/3206.001.0001	book	January 2005

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