The Role of Glycopeptides in Modern Vaccine Development

Why Glycopeptides Matter in Vaccine Design?

(1) Glycan-based Antigen Presentation

Glycopeptides are of high importance to vaccine design as glycans can affect antigen presentation and immune responses. Glycans are potential ligands to many receptors expressed on APCs (e.g., DCs). Binding of glycopeptides to such receptors on APCs can improve uptake and presentation of co-delivered or associated antigens. This strategy has been widely applied in CLR targeting. As mentioned before, CLRs such as DC-SIGN and Langerin have specificities to certain carbohydrate motifs. Glycan-modified nanoparticles or liposomes were used to target CLRs on DCs to enhance cross presentation and activation of CD8+ T cells. Targeting DC-SIGN, Langerin and other CLRs with glycans as ligands is one of the most studied strategies for antigen delivery, due to the multivalent presentation of glycans. In the presence of mannosylated liposomes, immunization of mice with protein antigen led to enhanced uptake of the antigen by DCs and activation of CD8+ T cell responses. Binding of glycodendrimers to DC-SIGN, and the enhanced uptake and cross presentation of associated antigens by DCs, led to activation of CD8+ T cells. Activation of CD8+ T cells using antigens associated with glycans has also shown great potential for cancer immunotherapy.

(2) Enhanced Immunogenicity via Glycosylation

The glycosylated form of the protein expressed in mammalian system demonstrated more immunogenicity than the vaccine candidate expressed in bacterial expression systems. The glycan protein-conjugated vaccine against the tick-borne disease was developed by the researchers in 2006. The recombinant proteins Bm86 and Bm95 were expressed in Pichia pastoris, and the resulted protein was found to be more immunogenic than the nonglycosylated one, and hence it was proved to be a potential immunogen. Tumor cells demonstrate high frequency for occurrence of O-linked glycosylation on their surface. Tn, sTn, and T antigens were synthesized with the help of T-synthase (gluconyltransferase), and it (T synthase) folds appropriately with the help of Cosmc, an essential molecular chaperon. The overexpression of these tumor-associated carbohydrate antigens (TACA; Tn, Sn, T) having aberrant O-linked glycosylation promotes metastasis, which in turn leads to adverse or poor condition in cancer patients. Now-a-days, these TACA have been targeted for the development of anticancerous vaccines. In this regard, the researchers in 2012, have designed a synthetic Tn-based anticancer vaccine. The designed construct contains LAA (Lipoamino acid; TLR-2 stimulant), Tn antigen B-cell epitopes, CD4+ T cell epitope from poliovirus and CD8+ T-cell epitope from ovalbumin. After that, they checked the immunogenicity of the designed construct in BALB/c mice, which showed that the vaccine candidates without any immunostimulatory agent were able to induce a tremendous immune response.

Fig. 1 Schematic representation of the immune response to polysaccharides (A) and glycoconjugates (B).^1,2

Comparing Glycopeptides with Traditional Peptides in Vaccines

(1) Structural Complexity vs Functional Precision

Glycopeptides and traditional peptides represent two different approaches to vaccine design. Most peptides are considered to be either linear or cyclic in structure. The primary structure of peptide antigens remains devoid of any complex structural modifications from carbohydrate attachment. Glycans are known to be involved in a variety of biological processes that can modulate protein function in several ways. Glycans can be ligands for a variety of receptors on immune cells, such as dendritic cells, that may lead to potentiating of presentation of the underlying antigen or the peptide attached to the glycan. Due to the increased structural complexity of glycopeptides, there is an increased potential for precision in engineering the ability to target specific subsets of receptors. This would then lead to more potent and selective immune responses. A glycopeptide antigen may be designed to specifically target a given C-type lectin receptor (CLR) that is known to be involved in antigen uptake and presentation, such as DC-SIGN. Glycosylation may also affect physiochemical properties of a peptide, such as solubility and stability. Changes in stability of peptides in peptide-based therapeutics such as vaccines, for example, may alter the bioavailability of the vaccine and thus the immune potency of the vaccine. Attachment of glycans may also protect peptides from enzyme degradation, thus increasing their stability. Glycosylation can also improve their ability to cross the blood-brain barrier (BBB).

(2) Safety, Stability, and Bioavailability

Traditional peptides perform less effectively when compared to glycopeptides used in vaccines. The design of vaccine candidates must take into account a wide variety of factors, including safety, stability, and bioavailability. Safety is not likely to be an issue with traditional peptides, but both stability and bioavailability can be potential factors. Peptides are not membrane-permeable and can be readily degraded by proteases. Glycopeptides may have improved stability, and bioavailability, in part due to the fact that the glycans protect against enzymatic degradation. This can lead to an increased half-life and a prolonged immune response, both of which are desirable properties for vaccine applications. Glycopeptides also have higher bioactivity compared to traditional peptides. Glycosylated peptides have been found to be more readily able to cross the BBB than non-glycosylated peptides, making glycopeptides a promising vaccine candidate for diseases of the central nervous system. Glycosylated peptides have also demonstrated an increase in receptor binding affinity. Glycosylation of a peptide produced higher binding affinity which led to enhanced glycopeptide effectiveness. There are some caveats that should be taken into consideration as well for glycopeptide development.

Real-World Applications of Glycopeptides in Vaccines

(1) Cancer Vaccines (e.g. MUC1, Tn antigen)

Multiple protein carriers, such as BSA and KLH, have been used to conjugate MUC1 glycopeptide in order to induce an immune response, because the protein carriers are immunogenic and have many epitopes. T or Tn antigen conjugated to BSA was synthesized by MUC1 and immune responses were investigated. They noted that there was an enhancement in the anti-MUC1 immune response. The same authors then synthesized MUC1 conjugated to STn or 2,6-STn on BSA and used this to immunize BALB/c mice. They noted a strong IgG response that could bind to MCF-7 tumor cells, while they and others noted low immunogenicity in their previous work with the BSA vaccine containing MUC1-STn. Reseachers then synthesized a MUC1 glycopeptide conjugated either to BSA or to 3 different T-helper cell epitopes of TTox and immunized mice with the conjugate in either buffer solution or Freund's adjuvant. They noted that they often found a stronger immune response to the vaccines in buffer solution compared to the vaccines in Freund's adjuvant, particularly with regard to tumor binding antibodies and complement dependent cytotoxicity (CDC) activation. Freund's adjuvant is no longer thought to be safe for use in humans due to severe side effects. Researcher synthesized a MUC1 glycopeptide-BSA conjugate vaccine containing 3'-fluoro-TF antigen, which generated anti-MUC1 mouse antibodies with specific binding to TF antigen. This group also noted synthesis of a 4'-fluoro-TF-MUC1-TTox/BSA conjugate vaccine, which produced IgG antibodies that bound to MUC1 epitopes on MCF-7 cancer cells.

(2) Bacterial Polysaccharide Conjugate Vaccines

A number of glycoconjugate vaccines (chemical conjugation of a polysaccharide antigen to a carrier protein) have been licensed over the past 40 years to prevent infections caused by Haemophilus influenzae type b (Hib), 10-20 serotypes of Streptococcus pneumoniae (Sp), serogroups A, C, W, Y of Neisseria meningitidis (Nm) and Salmonella typhi Vi (Vi). Clinical studies are ongoing to develop pneumococcal and meningococcal vaccines that will provide protection against the serogroups that are not included in licensed vaccine formulations. The plain polysaccharide (T-cell-independent antigen) vaccines are generally well tolerated and safe, but have some important limitations with regard to immunogenicity (e.g. no immune memory, hyporesponsiveness) especially in some populations (e.g. naive subjects, as infants). It is worth noting that infants are not only (most of the times) naive subjects, but have an immature immune system responding differently than other ages to vaccination stimulus. On the other hand, the glycoconjugate vaccines, in which a capsular polysaccharide is chemically conjugated to a protein usually referred to as a 'carrier protein', elicit T-cell-dependent responses and thus immunological memory. Infants, differently from the plain polysaccharide vaccines, are also able to induce IgG response when vaccinated with glycoconjugates. In addition, glycoconjugates may result in improved responses (vs plain saccharides) also in the elderly and in immunocompromised (and depending on the endpoint also in immunocompetent adults).

How Our Glycopeptide Products and Services Can Help?

(1) Custom Synthesis for Vaccine Candidates

Our customized synthesis service for glycopeptide vaccine candidates is tailored to the specific requirements of researchers and developers in the field of vaccine design. We specialize in the synthesis of highly defined and homogeneous glycopeptides, which are essential for generating robust and specific immune responses. By combining advanced chemical and chemoenzymatic approaches, we can introduce glycans at precise locations on proteins, allowing for the design of specific epitopes and the enhancement of immunogenicity. Using chemical and chemoenzymatic methodologies, we can achieve site-specific glycosylation of proteins, resulting in homogeneous glycoforms and well-defined epitope presentation. We are able to synthesize glycopeptides of up to 150 amino acids in length and use both native chemical ligation (NCL) and expressed protein ligation (EPL) methods to generate fully defined vaccine candidates. Our glyco-engineered in vivo expression platform can also be used to produce glycoproteins with defined glycosylation and can therefore also be exploited for vaccine generation. Custom synthesis services are of particular interest for vaccines against complex antigens such as cancer or bacterial antigens, where defined glycosylation is required for immune recognition.

(2) QC and Analytical Support (e.g. LC-MS, HPLC)

We provide comprehensive scale quality control (QC) and analytical support for glycopeptides so that the glycopeptides we provide for you will be of the highest purity and quality and will be consistent lot to lot for your vaccine candidates. Analytical services we offer include, but are not limited to state of the art techniques such as LC-MS and HPLC to ensure the quality of our product. Glycopeptide identity verification is possible through chromatographic and electrophoretic techniques along with immunological methods.

Peptide Synthesis Services at Creative Peptides

References

1. Image retrieved from Figure 1 "Schematic representation of the immune response to polysaccharides (A) and glycoconjugates (B)," Stefanetti G.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Stefanetti G.; et al. "Immunobiology of carbohydrates: implications for novel vaccine and adjuvant design against infectious diseases." Frontiers in cellular and infection microbiology, 2022, 11: 808005.
3. Hossain M K, Wall K A. Immunological evaluation of recent MUC1 glycopeptide cancer vaccines[J]. Vaccines, 2016, 4(3): 25. https://doi.org/10.3390/vaccines4030025.
4. van der Put R M F, Metz B, Pieters R J. Carriers and antigens: new developments in glycoconjugate vaccines[J]. Vaccines, 2023, 11(2): 219. https://doi.org/10.3390/vaccines11020219.
5. Berti F, De Ricco R, Rappuoli R. Role of O-acetylation in the immunogenicity of bacterial polysaccharide vaccines[J]. Molecules, 2018, 23(6): 1340. https://doi.org/10.3390/molecules23061340.