Sugar decoration or glycosylation can be utilized as a customizable design feature of vaccines to direct antigen folding, stability and immunogenicity. Programming glycans onto antigens or carriers can be used to alter pattern-recognition receptor recognition, modulate antigen presentation and abrogate self-tolerance. Glycosylation was previously thought of as merely a post-translational modification.
It's important because pathogens also often mask immunogenic epitopes with glycans, and glycans on host proteins can either enhance or suppress immune recognition. By mimicking or intentionally skewing glycan patterns, vaccines can skew the size, type, and persistence of that response. This lets creators target glycoprotein regions that are conserved and not normally immunogenic, which plain peptides and recombinant proteins don't reveal.
Fig. 1 A brief summary of current chemical and synthetic biology approaches for developing cancer vaccines.1,5
Importantly, the structure of antigens can be controlled by glycans on several levels. Insertion of N-linked glycans into structurally flexible regions can rigidify proteins in their native, protease-resistant conformation, circumventing off-pathway aggregation commonly observed for bacterially expressed subunits. Deglycosylation is also useful for unfolding cryptic surfaces to reveal hidden epitopes necessary for broad neutralisation, as has been shown for influenza HA stem domains where glycan removal improves access to conserved, stalk-directed antibodies. Site-selective glycosylation can also provide a chemical handle for downstream conjugation strategies; incorporation of azido-sugars enables click-based conjugation to liposomes/virus-like particles without perturbing disulfide networks. Glycans also impact the size and valency of antigens. Multivalent presentation of synthetic GM3 or Globo-H on gold nanoparticles transforms low-avidity, monomeric carbohydrate epitopes into potent immunogens capable of overcoming B-cell tolerance. Finally, glycosylation can serve as a self/non-self signal. Tumor-associated antigens such as Tn and sTn are truncated host glycans; when displayed in a clustered, hyper-glycosylated format the immune system can learn to recognize these "self-minus" patterns as dangerous, and several such candidates are now in cancer vaccine pipelines.
Table 1 Structural roles of glycans in vaccine antigens
| Glycan role | Engineering tactic | Functional read-out |
| Conformational lock | N-glycan in flexible loop | Protease resistance |
| Epitope exposure | Sequon removal | Conserved site access |
| Valency boost | Multivalent scaffolds | Avidity increase |
| Click handle | Azido-sugar insertion | Site-specific conjugation |
| Self-minus signal | Truncated Tn/sTn clusters | Tolerance break |
Influence on Immune Recognition and Response
Patterns of glycosylation also determine immune recognition by lectin-mediated interactions with pattern-recognition receptors (PRRs) expressed on antigen-presenting cells. Recognition of mannose-rich glycans through DC-SIGN and the mannose receptor enhances endocytosis and delivery into MHC-I cross-presentation pathways, beneficial for inducing cytotoxic T cells against intracellular microbes. Terminal sialylation motifs ligate Siglec-7/9, providing an inhibitory signal that may be beneficial for self-adjuvanting vaccines designed to avoid unnecessary inflammation. Glycosylation of the antigen itself can modulate the quality of the resulting antibodies: expression of afucosylated glycoforms of the carrier protein biases the response toward Th1 and high-affinity IgG production by enhancing interaction with FcγRIIIa, while hyper-galactosylated conjugates induce Th2 and mucosal immunity-biased responses. Glycan editing has recently been used to qualitatively reshape the antibody response to influenza, with in vitro addition of GalNAc to the receptor binding domain resulting in antibodies that were 30–40 % more effective at neutralization without affecting total IgG titers. Glyco-adjuvants containing TLR7 agonists and mannose ligands conjugated to the same polymeric scaffold function synergistically by targeting uptake through the sugar moieties and maturation through the TLR agonist, allowing antigen to be processed in a pro-inflammatory milieu that enhances both breadth and persistence of immunity with decreased reactogenicity.
Glycosylation can serve two purposes when designing vaccine antigens. It can mask antigenic sites that may be susceptible to neutralization while also exposing conserved regions for neutralization. Scientists can control where glycosylation sites are placed, as well as their density and composition. This can alter protein folding, stability to proteases, and interactions with lectins. Glycosylation was previously thought to simply be an added decoration after protein translation, but is now used as a tool for vaccine design.
Shielding and antigen exposure is driven by how glycans are distributed across the antigen surface. High mannan or complex-type glycan density can mask immunodominant but variable epitopes, skewing immune recognition towards neighboring conserved epitopes; this mechanism has been intentionally leveraged in HIV-1 Env vaccines. Deleting glycans at certain positions (like N332) can unmask the CD4-binding site to improve binding of bnAbs. In contrast, hyper-glycosylation of the receptor-binding domain within SARS-CoV-2 spike reduced antibody binding to spike's variable loops while leaving access to conserved cryptic sites intact, pushing the antibody response towards cross-protective specificities. Steric hindrance is not the only tactic; glycans also tune local electrostatic environments that repel or recruit immune complexes and change how paratopes interface with their targets. By selectively deglycosylating sites using alanine-scanning mutagenesis or chemoenzymatic trimming methods, developers can engineer the glycan shield to create optimal "glycan holes" that funnel broadly reactive B cells. Glycans can shield or direct immune responses based on their neighbors, so teams now use in silico tools to generate rational glycan maps that can be validated by glycopeptide microarrays before in vivo immunization.
Fig. 2 Effector functions of antibodies that are dependent on N-glycans and that are modified during infections.2,5
Glycans additionally determine the physiochemical stability of antigens in their native environment. Attachment of N-linked glycans to structurally dynamic regions serve as molecular shields that prevent aggregation of off-pathway species during protein purification under denaturing conditions such as elevated temperature or desiccation, a principle leveraged to create stable formulations of influenza hemagglutinin (HA) stem nanoparticles that tend to misassemble when expressed in E.coli. On the other hand, removal of glycans in mucin-like regions can alleviate steric bulk, permitting closer packing into viral nanoparticles that can encapsulate greater numbers of antigens per particle for improved B-cell crosslinking. Glycans can also modulate intracellular trafficking pathways of antigens within dendritic cells: unmethylated mannose residues permit uptake by mannose receptor-mediated mechanisms and subsequent trafficking into pathways favoring crosspresentation, leading to stronger CD8+ T cell responses. Antigens terminated with sialic acid are preferentially trafficked to tolerogenic signaling domains of DC-SIGN and have been leveraged in the development of vaccines for allergy desensitization. Chemical stability can be similarly tuned: because sialic acid cap residues can fall off during acidic refrigeration, enzymatic removal exposes underlying galactose residues that serve as a ligand for asialoglycoprotein receptor-mediated clearance. This instability can be circumvented by overexpressing glycans with terminal sialic acid caps or using polyethylene glycol (PEG) homologues introduced through glycan remodeling. Glycan occupancy can also be important for particulation: highly glycosylated antigens are able to maintain structural integrity during stress like spray-drying due to prevention of hydrophobic interactions that lead to aggregation, and allow for antigens to be formulated into stable powders that can withstand tropical climates without requirement for cold-chain storage.
From a vaccine developer's perspective, glycans pose a two-fold problem. Firstly they have the potential to mask protective epitopes. Secondly, host-cell mediated glycosylation is intrinsically heterogeneous which negatively impacts batch-to-batch consistency. Glycans are unique amongst post-translational modifications in that they need to be considered as critical factors that can negatively impact immunogenicity, analytics and invite regulatory questions if not properly considered from the onset of product design.
Accidental glycan shielding takes place when glycosylation targets residues on or near neutralising epitopes. Glycans physically shield antibody access to the underlying peptide and misdirect the immune response towards immunodominant but variable and unprotected surfaces. Coronavirus S-proteins are a classic example: computational models predict that ～40 % of the surface area of the S-protein is glycan-shielded, leaving the receptor-binding domain (RBD) as one of the few large contiguous unglycosylated patches on the protein. This is why vaccines targeting the RBD remain effective even as antigenicity evolves elsewhere on the spike protein. Glycan shielding is not just steric; high-mannose or complex-type glycans impact local electrostatic surface potential making the epitope less attractive to nearby paratopes, decreasing the on-rate of neutralizing antibodies. Glycan positioning can also differ depending on the protein production platform - influenza H3N2 strains adapted to egg manufacturing acquire an additional sequon at residue 158 that glycosylates the globular head of the HA stem. Antisera raised against egg-propagated viruses have decreased breadth compared to those immunized with mammalian-cell produced viruses. Deglycosylation mutants can be rescued if shielding is only mild but the viruses often misfold or aggregate. Glycoengineered cell lines may still glycosylate cryptic sequons. For these reasons rational vaccine design often starts with glycan mapping: crystal structures or cryo-EM maps are combined with glycopeptide LC–MS analysis to predict which sequons are solvent-accessible and mutated out using alanine scanning or replaced with glyco-null sequences. Alternatively, individual glycans responsible for shielding can be chemically trimmed using chemoenzymatic remodeling.
Add to that that an antigen can have vastly different glycosylation patterns depending on the host system it is expressed in (or even between two seemingly identical bioreactors in the same host under only slightly different conditions), and you get complicated batches where influenza HA expressed in eggs has high-mannose, mono-fucosylated glycans while those expressed in HEK293 has tri/tetra antennary heavily sialylated glycans. Terminal galactose can range from 8 % to 66 % between two HEK bioreactors while core fucose is unaffected (. Genes competing for the same nucleotide sugars get influenced by environmental factors such as pH change or dissolved CO2 increase leading to altered glycan expression. The target glycan may only change by one antenna yet be recognized as a completely different glycan by the immune system. Agencies are cracking down on heterogeneity and are now requiring manufacturers to map their product to the glycopeptide level including every antenna, linkage type and sialic acid isomer. Producing such a map can hold up batch release if certain antennae fall out of spec. Ways to combat heterogeneity include using a stable producer cell (such as CHO-FUT8 KO for completely afucosylated), supplementing media with sugar substrates, or even going cell-free with chemoenzymatic synthesis which transfers only one glycoform at a time, allowing for a batch-to-batch cosine similarity of >0.95 and reducing the CMC package to one single intact-mass measurement.
Customizing glycosylation, or adding sugars, to vaccines is becoming an important tool for improving them. The aim is to convert antigens from inconsistently glycosylated, host-cell derived products into engineered immunogens with defined compositions. Programming glycosylation sites, density, and sugar chemistry allows manipulation of epitope accessibility, stability, and biasing of the resulting immune response. This engineering effort seeks to turn glycans from the "wildcard" embellishments they have been considered into a controlled design feature to surpass empirically based vaccines.
Site selection starts with in silico identification of surface availability and B-cell epitope topography. Sequence modifications include addition/deletion of sequons to occlude variable patches or reveal conserved, poorly immunogenic epitopes. Addition of an Asn-X-Ser/Thr sequon to a dynamic loop can constrain a protein antigen in its native conformational fold, minimizing aggregation and protease degradation susceptibility through downstream manufacturing. Alanine substitution of endogenous sequons flanking influenza HA's receptor binding domain unmasked its CD4 binding site, inducing broadly neutralising antibodies in vivo. Spatial density: introducing multiple sequons within 15-aa windows can form a glycan "hedge" that sterically interferes with paratope binding, "forcing" the immune system to target neighboring conserved surfaces. This approach was used for coronaviruses, whereby hyper-glycosylation of mutable loops diverted immunity away from itself and towards the conserved stem. Engineering strategies are cyclical: CRISPR/Cas9 knock-in of sequon acceptor sites into producer cell line DNA, followed by trials of high-mannose or complex-type expression variants. Subsequent glycopeptide LC–MS/MS analysis is used to validate occupancy >95%. When host cell systems fail to achieve the desired occupancy pattern, glycoengineering via a cell-free system allows for in vitro immune priming with a core stub of GlcNAc. The sugar can then be enzymatically trimmed and extended in vitro to incorporate unnatural sugars- azido-GalNAc for click chemistry conjugation pathways, or photo-cleavable fucose for inducible release mechanisms- that cannot be genetically pre-programmed. Site-selection can also impact conjugation valency to carrier-proteins for glycoconjugate vaccines. Positioning a sequon upstream or downstream of a T-cell epitope can increase its MHC presentation capacity, effectively transferring the peptides immunogenic properties to the polysaccharide moiety. This process, referred to as "glycopeptide imprinting" can be leveraged to skew IgG subclass without modifying the glycans itself.
Synthetic approaches can rescue heterogeneity resulting from cellular expression platforms. Glycans are first trimmed to a homogeneous GlcNAc stub with EndoS2 or another endoglycosidase, excising cell-derived heterogeneity. Functionalized oligosaccharide oxazolines pre-synthesized with orthogonal protecting-group strategies are attached in bulk by mutant glycosynthases biased toward the synthetic reaction, pushing occupancy typically >95 %. LacNAc, sialic acid, and fucose are subsequently added in one-pot cascades to complete the oligosaccharide in hours instead of weeks while maintaining native protein folding. Anything tolerated by the enzymes can be incorporated, including unnatural monosaccharides like 6-azido-GalNAc for clickable linkages, photo-cleavable fucose for detachable vaccines, or fluorinated sialic acids for 19F NMR analysis. Microfluidic formats can further modularize the process, dividing priming, extension, and capture into separate wells for live tuning of each reaction zone. This reduces enzyme loading by an order of magnitude compared to stirred-tank reactions. Since each building block is defined by the user, heterogeneity is now encoded in the synthetic sugar donor, not upstream process variation. As such, fermentation drift will not transfer to product release. Regulation also greets the enzymatic polishing step as an extension of protein drug manufacturing, while the synthetic donor is considered a vetted chemical module, partitioning validation efforts. Synthetic carbohydrate scaffolds also facilitate vaccine discovery: diverse chemical donors can be screened against a common trimmed antigen in 96-well format, dramatically reducing weeks of cell-line development into days of combinatorial synthesis and identifying vaccine candidates whose glycans serve as molecular barcodes.
Glycosylation of vaccines should be evaluated as thoroughly as protein sequence as oligosaccharides control antigenicity, stability and batch consistency. Each glycoform is now considered by regulators to be a critical quality attribute requiring orthogonal evidence that all sequons are utilized, all antennae are quantitated and all non-native sugars are accounted for (synthetic origin)—a requirement that presents significant analytical challenges from discovery through commercialization.
Site mapping typically starts with proteolytic release of glycopeptides. Trypsin or IdeS digestion produces fragments which, when mapped back to the parent protein, contain shifts in mass that encode both the peptide sequence as well as the structure of any attached glycans. Enrichment - typically done on a HILIC or lectin column - removes the excess of unmodified peptides from the sample, glycopeptides tend to be quite low in abundance. Enrichment allows one to increase the signal-to-noise by 10-fold or more. LC-ESI-MS/MS fragmentation (EThcD avoids breaking the glycan–peptide bond) produces b/y ions that allow for confident assignment of occupancy to each glycosylation site (aka sequon). For proteins with extensive O-glycosylation such as coronavirus spike protein or influenza HA, one can take advantage of the recently characterized O-protease OpeRATOR, which cleaves N-terminal to glyco-Ser/Thr to release defined short glycopeptides. These are much easier to sequence compared to mucin-like regions. If overlapping peptides are required (ex., to deconvolute crowded regions), one can digest in parallel with chymotrypsin or Asp-N to create a peptide matrix that maps to every residue. Site-specific occupancy can then be determined by comparing the MS1 area of the glycopeptide vs. the deglycosylated peptide after PNGase F treatment. This allows for lot to lot tracing of site occupancy % across tox and commercial lots.
Functional correlation builds on site mapping. Individual glycoforms pulled off by preparative HILIC or lectin affinity are run through FcγR binding, complement consumption or whole-blood ADCC read-outs to create structure–activity maps. Multivariate statistics (PLS regression) associates individual monosaccharide attributes to functional read-outs so that potency can be predicted directly from a glycopeptide map without the need for additional bioassay. For influenza HA, cosine similarity of site-specific glyco-signature between vaccine strain and challenge virus is now incorporated into antigenic cartography, with a score >0.95 across the seven major glycans generally considered acceptable if there are no non-human forms. If chemoenzymatic remodeling is performed, the functional package must also show that the synthetic linker or click handle does not abrogate antigen binding or add immunogenic epitopes; surface plasmon resonance and DC-SIGN activation assays are commonly used to show that the conjugation chemistry does not perturb the neutralizing surface. Stability correlations can be drawn by stressing purified glycoforms at low pH or high temperature; loss of sialic acid or appearance of high-mannose species can be correlated with increased clearance in FcRn or mannose-receptor binding assays to inform formulation and shelf-life decisions. Analytical correlation thus turns glycan readouts into a tool that can inform both release specifications and lifecycle management.
If the native glycosylation pattern on a vaccine antigen does not satisfy immunogenicity or production criteria, or if the target glycoform cannot be expressed by native cell lines, then customized glycosylation support may be needed. If your project demands unnatural sugars, click-ready handles, or controlled epitope exposure, then you'll need to transition to a custom-built chemoenzymatic or cell-free platform. With chemoenzymatic glycosylation, every monosaccharide, linkage and conjugation site is designed.
Targets with large molecule complexity such as viruses requiring non-human glycans for immune evasion, therapeutic antigens needing T cell responses against self epitopes, or multispecifics containing conjugation motifs for antibody drug-like payloads soon deplete internal resources. For large organizations, this may mean repeating cycles of enzyme optimization, protecting group iterations and linker validations longer than most programs will allow. Contract manufacturers have spread these development costs over many clients and can typically provide ready access to libraries of glycosynthases compatible with sterically demanding unnatural C-6 substituted donors and pre-established one-pot reaction cascades capable of building complex glycan structures like LacNAc or even LacNAc-sialic acid-fucose combinations in a chemically defined fashion without intermediate purifications. When the man-hours required to set up screening for a new catalyst, synthesis of tailored donors, and method development for characterization surpass the negotiated payment terms for a custom contract, it often makes financial sense to pay the external price. Expectations also rise from regulators and clients for site-specific glycoforms on therapeutic proteins with >95 % coverage at a designed glycosylation site or sequon, a task that can be readily accomplished by vendors through established LC–MS/MS methods and inclusion of heavy isotope-labeled standards.
Table 2 Complexity indicators favoring external custom solutions
| Project feature | Internal bottleneck | Service-level solution |
| Exotic sugar shields | Enzyme promiscuity gap | Pre-evolved synthase panel |
| Self-minus cancer antigens | Tolerance-break design | Truncated Tn cluster synthesis |
| Multivalent click constructs | Lysine heterogeneity | Azido-oxazoline transfer |
| >95 % site occupancy | Clone screening overhead | Qualified cascade SOP |
| Linkage-specific CMC | Method validation burden | Pre-existing MS library |
Collaboration translates static R&D infrastructure cost into variable spending tied to successful project milestones. It also provides immediate access to databases of previously identified/enzymes, donors and sections of regulatory documentation that would otherwise require weeks or months of internal effort to construct. Many CMOs house integrated service labs offering capabilities in organic chemistry, enzymology and regulatory writing - minimizing the delays associated with translating between multiple vendors. Donor starting materials can be prepared in-house by chemical teams - such as azido Sias, photo-cleavable Fuca, clickable GalNac - with protections strategies already optimized for GMP manufacture of synthetic glycans. Enzyme engineers can work in parallel to create glycosynthases capable of incorporating these unnatural sugars, as both teams are aligned to produce the same glycoform. Analytical groups performing orthogonal downstream assays like intact-glycoprotein LC–MS, glycopeptide mapping and ion-mobility-profiling further separate structure from function in a manner digestible by bioinformatic groups tasked with building glycan composition libraries and similarity matrices that can be submitted along with an IND. Regulatory experts also draft sections of the CMC ahead of drug substance delivery to describe impurity profiles and donor residue tracking methods, paving the way for adherence to ICH Q11 principles of virtue when submitting a batch record and risk assessment summary upon commercialization. Failure or serendipity are mutually owned experiences between partner and sponsor. Strategies that did not produce a crystalline solid for purification or yielded a structure that does not react with known exoglycosidases remain the intellectual property of the collaborator. However, this information can and should be leveraged by the sponsor for the next initiative. External specialization allows clients to borrow scaling and troubleshooting capacity from contract teams without sacrificing their freedom-to-operate.
Glycosylation plays a critical role in shaping antigen structure, epitope exposure, and immune recognition in vaccine development. When precise control over glycan presentation is required to optimize immunogenicity or reduce unwanted immune responses, specialized glycosylation and analytical services provide essential technical support.
Custom glycosylation services enable vaccine developers to design and implement tailored glycosylation strategies based on antigen structure and immunological goals. By selectively introducing, removing, or modifying glycosylation sites and glycan structures, these services help control epitope accessibility, antigen stability, and immune engagement. Such customized approaches are particularly valuable for complex vaccine candidates, including glycoprotein antigens and recombinant subunit vaccines, where uncontrolled glycosylation can significantly impact efficacy.
Accurate evaluation of vaccine glycosylation is essential for understanding how glycan structures influence immune responses. Glycosylation analysis and profiling services provide detailed characterization of glycan composition, site occupancy, and structural heterogeneity, supporting direct correlation between glycosylation patterns and immunogenic outcomes. By integrating glycan profiling into vaccine development workflows, these services help validate glycosylation strategies, ensure batch consistency, and support data-driven optimization of vaccine design.
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