MSACL 2025 Abstract

DeGlyPHER-ing Site-Specific Distribution of N-Glycans on Candidate Vaccines Against HIV

Baboo S.(1), Diedrich J.(1), Steichen J.(2), Schiffner T.(2), Rantalainen K.(2), Swanson O.(2), Cottrell C.(2), Kalyuzhnyi O.(2,3), Liguori A.(2,3), Schief W.(2,3,4,5), Paulson J.(1,2), Yates J.(1)
(1) The Scripps Research Institute, Molecular Medicine, La Jolla, CA, United States (2) The Scripps Research Institute, Immunology and Microbiology, La Jolla, CA, United States (3) The Scripps Research Institute, IAVI Neutralizing Antibody Center, La Jolla, CA, United States (4) The Ragon Institute of Mass General, MIT, and Harvard, Cambridge, MA, United States (5) Moderna Inc., Cambridge, MA, United States

John Yates, PhD (Presenter) Scripps Research Institute

Presenter Bio: John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. He received a B.A in Zoology and an M.S. in Chemistry from the University of Maine at Orono. He obtained his Ph.D. in Chemistry at the University of Virginia in the laboratory of Donald F. Hunt with a dissertation entitled Protein Sequencing by Tandem Mass Spectrometry. He performed postdoctoral research in the laboratory of Leroy E. Hood at California Institute of Technology. At the University of Washington, he obtained the rank of Associate Professor with tenure before moving to The Scripps Research Institute in LaJolla, CA. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS, the 2019 HUPO Award in Discovery, and the 2024 Pittsburgh Society Award in Analytical Chemistry. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List of Most Highly Influential Biomedical Researchers, 1996-2011 (European J. Clinical Investigation 2013, 43, 1339-1365) and the Clarivate List of Highly Cited Scientists in 2015 and 2019-2024. He has published over 1000 scientific articles with >183,000 citations, and an H index of 211 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Site-specific N-glycosylation on many highly glycosylated candidate vaccines must be determined. This helps to estimate the impact of changing glycosylation on antigenicity and immunogenicity when developing vaccines and provides a measure for monitoring the consistency of GMP-grade vaccine production. With substantial number of vaccines being designed, it is essential to rapidly select candidates with glycan landscapes that most closely resemble a naturally-occurring immunogen. DeGlyPHER (Deglycosylation-dependent Glycan/Proteomic Heterogeneity Evaluation Report) is developed as a highly sensitive, rapid, and reproducible approach, that perfectly fits these needs.

METHODS:
Using Proteinase K we generate overlapping peptides mapping to every N-glycosylation site, sequentially deglycosylate these peptides with Endo H and then PNGase F, hence creating residual mass signatures that are identified by LC-MS/MS, thus quantifying the degree of glycan occupancy, and the broad characterization of glycan processing – constituting the transformation of high mannose forms into complex forms owing to mannose residues being replaced by other "apical" monosaccharides.

RESULTS:
DeGlyPHER has evaluated N-glycan distribution on >200 immunogens that are being developed as vaccines against HIV, SARS-CoV-2, Influenza and Ebola. We have DeGlyPHER-ed >150 vaccines being developed using the highly-promising germline-targeting approach against every important immunogenic epitope on HIV Env/spike-protein that elicits broadly-neutralizing antibodies (bNAb). E.g., [1] we scanned boost-vaccine candidates against the V3-glycan epitope that elicits BG18-like antibodies, to inform V1-loop design to cause high glycan occupancy, potentially limiting off-target responses; [2] we discovered that in 10E8 priming-immunogens comprising of membrane proximal external region (MPER) epitope grafted into a protein-scaffold to create a multi-valent nanoparticle, the nanoparticle component has much higher glycan occupancy than the epitope scaffold; and [3] we confirmed the plugging of the N276 "glycan-hole" as part of a "prime-boost" strategy based on VRC01 epitope. We also found that HIV Env gp41 is significantly better shielded with N-glycans when HIV Env is membrane-tethered – its native state on intact virus and in most mRNA vaccine strategies, thus promising a means to reduce unwanted base effects stemming from partial glycan-shielding of gp41. All these findings have contributed substantially to downstream non-human primate (NHP) studies and clinical trials.

CONCLUSION:
DeGlyPHER is informing HIV vaccine development in real time, being a much more rapid, sensitive, and robust quantitative alternative to "intact-glycan" glycoproteomic approaches, with excellent potential for adapting it to high-throughput platforms for bulk glycoprotein analysis.

Funding:
This work was supported by the NIH grants P41GM103533, UM1AI100663, UM1AI144462, and R01/R56AI113867.