Intact Reducing Glycan Promotes the Specific Immune Response to Lacto-N-neotetraose-BSA Neoglycoconjugates

Abstract

The mammalian immune system responds to eukaryotic glycan antigens during infections, cancer, and autoimmune disorders, but the immunological bases for such responses are unclear. Conjugate vaccines containing bacterial polysaccharides linked to carrier proteins (neoglycoconjugates) have proven successful, but these often contain repeating epitopes and the reducing end of the glycan is less important, unlike typical glycan determinants in eukaryotes, which are shorter in length and may include the reducing end. Here we have compared the effects of two linkage methods, one that opens the ring at the reducing end of the glycan, and one that leaves the reducing end closed, on the glycan specificity of the vaccine response in rabbits and mice. We immunized rabbits and mice with bovine serum albumin (BSA) conjugates of synthetic open- and closed-ring forms (OR versus CR) of a simple tetrasaccharide lacto-N-neo-tetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and tested reactivity to the immunogens and several related glycans in both OR and CR versions on glycan microarrays. We found that in rabbits the immune response to the CR conjugate was directed toward the glycan, whereas the OR conjugate elicited antibodies to the reducing end of the glycan and linker region but not specifically to the glycan itself. Unexpectedly, mice did not generate a glycan-specific response to the CR conjugate. Our findings indicate that the reducing end of the sugar is crucial for generation of a glycan-specific response to some eukaryotic vaccine epitopes, and that there are species-specific differences in the ability to make a glycan-specific response to some glycoconjugates. These findings warrant further investigation with regard to rational design of glycoconjugate vaccines.

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins

“…A major drawback of using DAP, AEAB, 2-AA, and 2-AB is that their conjugation reaction relies on reductive amination which, although highly efficient, opens the sugar ring at the reducing end monosaccharide (Figure 1). This destroys the reducing end integrity of the glycan (Prasanphanich et al, 2015) and potentially results in non-natural presentation of glycans on the array. This would affect the binding affinities of smaller glycans, and particularly those with modifications in the core region, such as core Fuc on N-glycans (Prasanphanich et al, 2015).…”

“…This destroys the reducing end integrity of the glycan (Prasanphanich et al, 2015) and potentially results in non-natural presentation of glycans on the array. This would affect the binding affinities of smaller glycans, and particularly those with modifications in the core region, such as core Fuc on N-glycans (Prasanphanich et al, 2015). We, therefore, have been developing new strategies to overcome this weakness while retain the merits mentioned above.…”

Peer Review #3 of "Application of built-in adjuvants for epitope-based vaccines (v0.1)"

“…In addition, a short peptide (UPK3A 65-84) from Uroplakin 3A (UPK3A) covalently coupled with KLH and CpG as adjuvant was found to be an immunotherapeutic vaccine for bladder cancer (Izgi et al, 2015). In addition, BSA is often used as a carrier protein for small antigens in glycoconjugate vaccines (Prasanphanich et al, 2015). For example, Cai et al combined a synthesized MUC1 glycopeptide with BSA or three different T-helper cell epitopes of TTox and demonstrated a beneficial effect (Cai et al, 2013).…”

Glycan microarrays of fluorescently-tagged natural glycans

“…Even if the reducing end is preserved, the next step to immobilize these glycans often requires ring-opening tagging, which significantly affects the structural integrities of the O-glycans due to their relatively smaller size compared to N-glycans. The reducing end of glycans is often critical to their recognition by antibodies and other molecules [52]. For O-glycans the reducing end GalNAc often serves as a branching point for O-glycans.…”