Elucidating structural features of an entirely carbohydrate cancer vaccine construct employing circular dichroism and fluorescent labeling

Abstract

The zwitterionic polysaccharide PS A1 from anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 is known to elicit a T-cell-dependent, major histocompatibility complex class II (MHCII) immune response through a correspondingly similar protein-antigen-based mechanism/pathway. The biological activity of PS A1 is known to arise from alternating charged motifs on adjacent monosaccharides comprising a tetrameric repeating oligomeric unit creating an alpha-helical secondary structure. However, we have learned that this alpha-helical structural characteristic may not play a role in immune activation. Paradoxically, our current knowledge of structure - activity relationships (SARs) with electrostatically charged polysaccharides has become more clearly defined, yet a lack of tools/probes for measuring dynamic structural changes hinders progress in carbohydrate-based vaccine development. Site- and region-specific structural modifications of PS A1, followed by conjugation with a known carbohydrate cancer antigen, the Thomsen-nouveau (Tn = alpha-D-GalNAc-OSer/Thr) antigen, does not alter antibody isotype switching ability and leads to specific IgG3 antibodies in C57BL/6 mice. Circular dichroism (CD) and studies using fluorescently labeled PS A1, described herein, reveal information pertaining to structure - activity relationships and the nature of Tn conjugation to chemically modified PS A1. The CD spectra of a Tn-PS A1 construct at 8.5 ≥ pH ≤ 3.5 illustrates complete loss of alpha-helical character while spectra obtained in the 3.6 ≤ pH ≥ 8.4 range denotes minimal alpha-helicity in comparison to naturally occurring PS A1. Temperatures exceeding 60 °C reveal complete loss of helical character. Two methods for Alexa Fluor488® fluorescent labeling studies of chemically oxidized PS A1 have given rise to percent conjugation values (% loading) calculated to be on average 35 Tn molecules bound. Combined, our results argue that altering the structure of PS A1, without chemically modifying the electrostatic charge character, does not alter immune response/recognition in mice. These findings have important implications for the design of entirely carbohydrate-based vaccine constructs.

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

“…3-5 The polymer, isolated from B. fragilis , has also recently been used as a carbohydrate antigen to replace classic antigenic proteins, like cationic BSA and KLH, for vaccine development. 6, 7 CPSA is made up of the four-sugar repeating unit →3) α -D-2- N -acetylamido-4-amino-galactopyranose (AADGal)-(1→4)-( β -D-galactofuranose (Galf)-(1→3)) α -D- N -acetylamido-galactopyranose (GalNAc)-(1→3)-(4,6-O-pyruvate)- β -D-galactopyranose (PyrGal)-(1→ (Figure 1a). 89 Exceptional work has been done in understanding the biological mechanisms by which the relatively unique CPSA zwitterionic capsule is associated with the symbiotic effect of the organism on its mammalian host.…”

Carbohydrate‐based vaccines for oncotherapy

“…Robust Tn‐specific immune response was introduced, which implied that chemical modification does not abrogate immunogenicity of PS A1. In addition, similar results were obtained from adjuvant‐free vaccinated mice, which also implied that PS A1 acts as not only a carrier, but also an adjuvant . They were further inspired to synthesize numerous ZPSs‐based vaccine candidates employing their linker‐free antigen synthesis strategy .…”

“…In addition, similar results were obtained from adjuvant-free vaccinated mice, which also implied that PS A1 acts as not only a carrier, but also an adjuvant. [167][168][169][170] They were further inspired to synthesize numerous ZPSs-based vaccine candidates employing their linker-free antigen synthesis strategy. [171][172][173] With immune response directed toward a linker itself, immune suppression to the target antigen could occur.…”

Fluorescently labelled glycans and their applications

“…For more complex carbohydrates, preparation of carbohydrate-fluorophore conjugates through a variety of chemistry has been developed, which is the subject of a later section of this review. There has been literature precedence, however, for successful site-specific fluorescent labelling of complex glycans, such as zwitterionic polysaccharide from Bacteroides fragilis [15].…”