Vaccination remains one of the most effective public health interventions against bacterial pathogens. Carbohydrate-based vaccines, particularly glycoconjugate vaccines, have proven highly successful in combating pathogens. However, traditional approaches that depend on the extraction and purification of polysaccharides from bacterial cultures face significant challenges, including biosafety concerns, culture methods and scalability limitations. To meet the growing demand for consistent and precisely defined antigens, synthetic methods have emerged as powerful alternatives. This paper discusses the foundations and synthetic strategies driving the next generation of carbohydrate-based vaccines, with emphasis on the design and synthesis of well-defined oligosaccharide antigens and their conjugation into effective glycoconjugate vaccines.
Introduction
Proteins, as thymus-dependent antigens, are the preferred antigens in subunit vaccines because they elicit robust and long-lasting immune responses, which is essential for long-term protection. However, many pathogens are covered by a dense and complex glycocalyx that shields potential antigenic epitopes, masking them to the immune system. Moreover, this glycocalyx is often the first structure encountered by host immune defenses. For these reasons, bacterial polysaccharides (PS) began to be explored as alternative vaccine components. [1]
However, PS are T-independent antigens that do not effectively activate neonatal B cells, resulting in weak and short-lived immune responses in infants and the elderly, two of the most vulnerable populations. To address these limitations and enhance the immunogenicity of carbohydrate antigens, polysaccharides are conjugated to carrier proteins. Upon uptake by dendritic cells, the glycoconjugate is processed and presented to T-helper cells via major histocompatibility complex (MHC) molecules. This conjugation converts the immune response from T-independent to T-dependent, enabling the activation of T-helper cells and the formation of immunological memory. The development of glycoconjugate vaccines has led to a substantial reduction in the incidence of invasive bacterial diseases worldwide and reduced the morbidity and mortality associated with these infections.[2].
Figure 1. Structure of a model Gram-negative bacteria pathogen cell wall.
Glycoconjugate vaccine design workflow
The design and development of conjugate vaccines is an inherently complex and multidisciplinary process, involving numerous experimental and analytical stages. Despite this complexity, the general workflow can be outlined as follows (Fig. 2).[3] Initially, the target pathogen and its various pathogenic strains must be accurately identified and characterized. Subsequently, carbohydrate antigens are isolated via chemical hydrolysis or enzymatic approaches.
To determine the precise antigenic structures suitable for vaccine formulation, polysaccharides from the pathogens must be accurately characterized employing techniques likes mass spectrometry and nuclear magnetic resonance. Then, sera from infected or convalescent individuals are analyzed using glycan microarray technology, which enables the identification of the preciseimmunodominant epitopes recognized by immuno-relevant antibodies.
Finally, the selected antigenic determinants are then conjugated to a carrier protein to enhance immunogenicity and elicit a robust immune response. [4]
Figure 2. Preclinical vaccine development workflow.
Although this workflow can be summarized in simplified form, the practical implementation of each step involves numerous challenges. These include difficulties in strain identification, limitations in pathogen culturing, the complexity of PS extraction and purification, structural instability of polysaccharides, epitope identification, antigen degradation, variability in immune protection, and formulation stability issues, just to name few. Each of these factors represents a critical obstacle that must be systematically addressed to achieve effective and safe vaccine development.
Mapping the Molecular Diversity of Polysaccharides
Pathogens produce surface capsules that serve as protective barriers, shielding underlying cell surface antigens from components of the innate immune system.[5] Cell surface glycans are synthesized through modular biosynthetic pathways from oligosaccharide precursors, typically resulting in repeating unit structures. To identify the epitopes that confer protective immunity, it is essential to examine the precise structure of these polysaccharides in detail.
The structural characterization of these carbohydrate antigens is achieved by isolating cell-surface glycans from cultured bacterial cells, followed by controlled chemical degradation and comprehensive analysis using physicochemical techniques such as nuclear magnetic resonance (NMR) spectroscopy on the intact PS.[6] These methods provide detailed information regarding the composition, size, and configuration of the repeating units.
However, the structural diversity of carbohydrate antigens across pathogens is exceptionally high, making their characterization particularly challenging. Bacteria, fungi, and protozoan parasites are often enveloped by layers of capsular polysaccharides (CPS), lipopolysaccharides (LPS), and related glycoconjugates, which can adopt linear or branched architectures and incorporate both common and rare monosaccharides (Fig. 3). In contrast to human cell surface glycans, which are constructed from only nine distinct monosaccharides, the bacterial glycome comprises hundreds of potential sugar building blocks. Covalent attachment of substituent groups such as acetate, phosphate, pyruvate, or glycerate further contributes to the structural heterogeneity of these molecules. [7]
Figure 3. Schematic representation of model lipopolysaccharide structure.
This vast structural heterogeneity is further intensified by serotype variability within a single bacterial species, where distinct strains may express different PS. Such diversity poses significant challenges for the development of broadly protective vaccines. For example, in Streptococcus pneumoniae, more than 90 distinct CPS repeating unit structures have been identified and classified into corresponding serotypes. [3]
Asparia Glycomics, leveraging extensive expertise in complex glycan analysis, provides advanced analytical services for the characterization of polysaccharides, utilizing state-of-the-art instrumentation and a team of highly experienced scientists. Our capabilities include:
- LPS extraction and purification: enzymatic treatment (DNase, RNase, and protease) followed by separation using various gel permeation chromatography methods and purity assessment.
- Quali-quantitative analysis: Hydrolysis of polysaccharides into individual monosaccharide residues to determine stoichiometric ratios and fatty acid composition analysis via GC-MS.
- Absolute configuration: monosaccharide identification by pure chiral alcohol under acidic conditions to form O-alkyl glycosides. The resulting compounds are acetylated and analyzed by GC-MS and compared against standards of known configuration.
- Linkage and sugar ring analysis: Determination of glycosidic linkage positions and ring sizes of individual sugar residues.
- In-depth profiling:
- Anomeric configuration (NMR)
- Determination of non-carbohydrate appendages (MALDI/ESI-MS or NMR)
- Full structural details of the repeating unit (MALDI/ESI-MS, NMR)
Once this information has been established, all residues can be assembled into a carbohydrate chain to reveal the complete structural details of the repeating unit or the oligosaccharide moiety. Identification of non-carbohydrate structures is also achieved through a combination of the analytical approaches described above, allowing full elucidation of the target molecule’s structure.
Glycotope Identification: Challenges
Once the structure of the is determined, the next step is to identify the antigenic epitopes.
This process involves determining the optimal antigen length, defining the repeating unit frameshift, polysaccharide chain length, and terminal monosaccharide identity (Fig. 5), as these parameters can profoundly influence antigenicity and immunogenicity, as well as evaluating the role of covalent modifications such as acetylation or phosphorylation.[3]
Precise identification of glycotope requires the use of synthetic oligosaccharides, as isolation through extraction, purification, and chemical degradation often fails to yield well-defined fragments. In cases where labile functional groups are present, structurally more stable analogues must be synthesized and evaluated to ensure molecular integrity and reproducibility.
Moreover, the benefits of a synthetic approach in vaccine development compared to the traditional purification procedure are significant: it enables the creation of more drug-like constructs compared to long polysaccharide chains, ensures well-defined vaccine compositions with reduced batch-to-batch variability, allows the conjugation of multiple antigens to a single carrier to target diverse serotypes, and facilitates scalable, endotoxin-free antigen production.
Once, all fragments and synthesized and immobilized onto the microarray, sera from infected or recovered individuals is used to identify the key immunodominant epitopes and PS fragments recognized by protective antibodies, guiding the selection of candidates for in vivo studies. (Fig 4)
Figure 4. Schematic representation of glycotope identification. The process involves identifying the specific repeating unit, chemically synthesizing polysaccharides of varying lengths, frameshifts, and modifications, followed by glycan array analysis and antigen selection.
At Asparia Glycomics, our expert scientists possess extensive expertise in complex glycan synthesis and the design of tailored glycan arrays, enabling the acceleration and de-risking of vaccine discovery and development programs through rigorous structural and immunological characterization.
Over the years, we have established advanced expertise in the synthesis of defined fragments, repeating units or frame shifted fragments. Such synthetic approaches also enable systematic investigation of how specific structural features, such as branching patterns or modifications like pyruvylation and methylation, affect the immunogenicity of the resulting fragments.
Glycoconjugate preparation and formulation
Once the antigens are selected, these polysaccharides are conjugated to carrier proteins to induce T-cell-mediated immunity. Currently marketed vaccines use carrier proteins such as diphtheria toxoid (DT), tetanus toxoid (TT), or detoxified variants like CRM197.[8] More recent strategies, including the use of virus-like particles, are now being used as innovative carrier alternatives.[9]
The choice of linker is critical to ensuring conjugate performance. It must minimize undesired immunogenic responses, react under mild reaction conditions, maintain the stability of the conjugate, and allow precise control during the coupling process.[10] In addition, the antigen-to-carrier protein ratio must be carefully controlled and consistent to ensure product reproducibility and efficacy. For example, in the case of CRM197, typically four to ten of the forty available lysine residues are conjugated to an oligosaccharide. [3]
Figure 5. Considerations during glycoconjugate preparation.
Our scientists can also prepare defined glycoconjugates tailored to specific research or vaccine development needs. This includes the selection and optimization of carrier proteins, linker chemistry, and antigen-to-protein coupling ratios to achieve the desired immunological profile and manufacturing consistency. Through advanced analytical characterization, MALDI-TOF MS, SDS-Page and high-field NMR, and formulation expertise, we ensure that each glycoconjugate exhibits high structural integrity, reproducibility, and stability, meeting the standards required for preclinical development.
Vaccine efficacy in vivo
Glycoconjugates are subsequently formulated with an adjuvant to enhance the immune response. The most used adjuvant in humans is alum; however, other options include oil-in-water emulsions, liposomes containing saponins, and various novel formulations.[11] Selecting the optimal adjuvant for human use is crucial for achieving a strong and balanced immune response, yet this evaluation and testing typically occur in the later stages of vaccine development.
The immunogenicity of glycoconjugates is typically evaluated in mice or rabbits. Mice are cost-effective and easy to handle, while rabbits provide immune responses more comparable to humans. [12] After animal injection, antibody responses (IgG, IgM, IgA) are monitored via glycan microarrays, which also assess antibody specificity and epitope mapping. During this step, the carrier protein is also included on the microarray to measure antibody responses directed against both the carrier protein and the spacer moiety. To gain deeper insight into the nature of the humoral immune response to the epitopes; various adjuvants are commonly be used.
Finally, to evaluate the efficacy of a vaccine candidate, immunized animals are challenged with the target pathogen. Pathogen loads are measured at defined time points to assess the protective effects of vaccination on infection and bacterial growth.[13] Effective immunization should significantly, or almost completely, prevent the bacteremia observed in non-immunized control animals. Assessing the induction of long-lasting immunity and the potential need for booster doses is also essential. Strong and durable immune responses, along with the ability to boost immunity upon re-exposure, are key positive indicators supporting the preclinical and clinical advancement of carbohydrate-conjugate vaccine candidates.
Accelerate your vaccine pipeline
Asparia Glycomics brings over two decades of specialized expertise in complex glycan synthesis and glycan array development, two technologies that are fundamental to the creation of next-generation glycoconjugate vaccines. Our scientific team has collaborated extensively with leading pharmaceutical companies, biotechnology firms, and academic institutions on vaccine research and development programs targeting a wide range of bacterial pathogens.
Figure 6. Overview of Asparia Glycomics’ capabilities across the vaccine development pipeline. The company’s expertise relies of the foundations of chemical and enzymatical synthesis of complex oligosaccharides and custom glycan array development. We support preclinical in vitro and in vivo studies providing high quality polysaccharide synthesis and characterization analyses to support your research.
With our integrated expertise, collaborative approach, and commitment to scientific excellence, Asparia Glycomics serves as a strategic partner for accelerating the development of innovative and effective glycan-based vaccines.
We offer synthetic carbohydrate structures, comprehensive analytical services, and glycan microarray technology to support vaccine development throughout both the discovery and preclinical stages, delivering precision, reliability, and the highest quality at every step.
Conclusions
Current vaccines are predominantly based on isolated glycans, and although they have achieved remarkable medical and commercial success, the development of glycoconjugate vaccines remains both labor-intensive and costly. By integrating advances in immunology and synthetic chemistry, synthetic glycans enable the design of safer antigens, free from contamination, reduced batch-to-batch variability and culturing issues, and allow the creation of stable, non-natural epitopes capable of eliciting broad and protective antibody responses. However, still, one of the major challenges that remains is the synthesis and preparation of the highly complex and diverse polysaccharide structures required for effective vaccine design.
To date, several synthetic and semisynthetic glycoconjugates have demonstrated strong protective efficacy in animal models and are advancing through preclinical and clinical development, however Quimi-Hib remains the only approved vaccine produced using a synthetic carbohydrate approach.[14] Fully synthetic vaccines show significant promise, offering the potential to streamline and reduce production costs. Looking ahead, both semisynthetic and fully synthetic glycoconjugates are expected to play key roles as stand-alone vaccines or as components of multivalent formulations that combine natural and synthetic glycans.
Download Whitepaper
Download the document and discover how Asparia’s experience and expertise in glycan synthesis and glycan microarray development can help accelerate your research.
"*" indicates required fields
References
- Del Bino, L.; Østerlid, K. E.; Wu, D.-Y.; Nonne, F.; Romano, M. R.; Codée, J.; Adamo, R. Synthetic Glycans to Improve Current Glycoconjugate Vaccines and Fight Antimicrobial Resistance. Chem. Rev. 2022, 122 (20), 15672–15716. https://doi.org/10.1021/acs.chemrev.2c00021.
- Kay, E.; Cuccui, J.; Wren, B. W. Recent Advances in the Production of Recombinant Glycoconjugate Vaccines. Npj Vaccines 2019, 4 (1), 16. https://doi.org/10.1038/s41541-019-0110-z.
- Seeberger, P. H. Discovery of Semi- and Fully-Synthetic Carbohydrate Vaccines Against Bacterial Infections Using a Medicinal Chemistry Approach. Chem. Rev. 2021, 121 (7), 3598–3626. https://doi.org/10.1021/acs.chemrev.0c01210.
- Lockhart, S. Conjugate Vaccines. Expert Rev. Vaccines 2003, 2 (5), 633–648. https://doi.org/10.1586/14760584.2.5.633.
- Yother, J. Capsules of Streptococcus Pneumoniae and Other Bacteria: Paradigms for Polysaccharide Biosynthesis and Regulation. Annu. Rev. Microbiol. 2011, 65 (Volume 65, 2011), 563–581. https://doi.org/10.1146/annurev.micro.62.081307.162944.
- Mulloy, B. High-Field NMR as a Technique for the Determination of Polysaccharide Structures. Mol. Biotechnol. 1996, 6 (3), 241–265. https://doi.org/10.1007/BF02761706.
- Adibekian, A.; Stallforth, P.; Hecht, M.-L.; Werz, D. B.; Gagneux, P.; Seeberger, P. H. Comparative Bioinformatics Analysis of the Mammalian and Bacterial Glycomes. Chem. Sci. 2011, 2 (2), 337–344. https://doi.org/10.1039/C0SC00322K.
- Pichichero, M. E. Protein Carriers of Conjugate Vaccines: Characteristics, Development, and Clinical Trials. Hum. Vaccines Immunother. 2013, 9 (12), 2505–2523. https://doi.org/10.4161/hv.26109.
- Mohsen, M. O.; Bachmann, M. F. Virus-like Particle Vaccinology, from Bench to Bedside. Cell. Mol. Immunol. 2022, 19 (9), 993–1011. https://doi.org/10.1038/s41423-022-00897-8.
- Huang, Y.-L.; Wu, C.-Y. Carbohydrate-Based Vaccines: Challenges and Opportunities. Expert Rev. Vaccines 2010, 9 (11), 1257–1274. https://doi.org/10.1586/erv.10.120.
- Pulendran, B.; S. Arunachalam, P.; O’Hagan, D. T. Emerging Concepts in the Science of Vaccine Adjuvants. Nat. Rev. Drug Discov. 2021, 20 (6), 454–475. https://doi.org/10.1038/s41573-021-00163-y.
- Orme, I. M. The Use of Animal Models to Guide Rational Vaccine Design. Microbes Infect. 2005, 7 (5), 905–910. https://doi.org/10.1016/j.micinf.2005.03.008.
- Oberli, M. A.; Tamborrini, M.; Tsai, Y.-H.; Werz, D. B.; Horlacher, T.; Adibekian, A.; Gauss, D.; Möller, H. M.; Pluschke, G.; Seeberger, P. H. Molecular Analysis of Carbohydrate−Antibody Interactions: Case Study Using a Bacillus Anthracis Tetrasaccharide. J. Am. Chem. Soc. 2010, 132 (30), 10239–10241. https://doi.org/10.1021/ja104027w.
- Habig, W. H. Potency Testing of Bacterial Vaccines for Human Use. Vet. Microbiol. 1993, 37 (3), 343–351. https://doi.org/10.1016/0378-1135(93)90033-4.
- Zasłona, M. E.; Downey, A. M.; Seeberger, P. H.; Moscovitz, O. Semi- and Fully Synthetic Carbohydrate Vaccines against Pathogenic Bacteria: Recent Developments. Biochem. Soc. Trans. 2021, 49 (5), 2411–2429. https://doi.org/10.1042/BST20210766.