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.