| Technological advancements in glycobiology have added a novel dimension to immunotherapy when carbohydrates are considered as key targets in developing safe and effective vaccines to combat cancer, viral infections, bacterial infections etc. Carbohydrates have long been known to be T cell independent and only elicit the innate immune responses and weak adaptive immune responses via cross-linking with B cell receptors. In order to achieve T cell dependent immunity, carbohydrates are generally conjugated with immunogenic carrier proteins. However, carrier proteins, being self-immunogenic, can lead to increased peptide specific antibody production resulting in the suppression of immunity towards the targeted carbohydrate antigen. Therefore, as an alternative to carrier proteins, we have utilized a zwitterionic polysaccharide (PS A1), isolated from the commensal anaerobe Bacteroides fragilis ATCCRTM 25285(TM)/NCTC RTM 9343(TM), which is known to bind with the MHC class II molecule and invoke T cell dependent immunity.;We hypothesize that covalent attachment of carbohydrate antigens with PS A1 will lead to the generation of carbohydrate antigen specific robust humoral and cellular responses. To validate this idea, we have synthesized a number of carbohydrates or glycopeptide antigens and conjugated these antigens with PS A1. Initially, a stereoselective glycosylation strategy has been developed to synthesize a complete set of tumor associated Thomsen-nouveau (Tn) antigen, D-GalNAc-O-Ser/Thr (Tn).5 Phthaloyl protected amino acid acceptors played a key role in generating alpha- or beta- anomers in excellent yields (72-96%) and selectivity (~100%) when appropriate C-2 substitution was installed. Protected Tn antigens were divergently transformed into Tn-based carboxylates or hydrazides by hydrogenolysis followed by hydrazine hydrate treatment or hydrazine hydrate treatment alone.;alpha-L-Thr-Tn hydrazide was coupled to PS A1 utilizing sodium metaperiodate (NaIO4) mediated oxidation of PS A1 in acetate buffer (pH 5.0), followed by overnight incubation with the antigen in 1X PBS buffer (pH 7.2). Structural modification of PS A1 can modulate immunogenicity of PS A1. Therefore, it is required to ascertain that the oxidation and conjugation condition will not alter the intrinsic biological nature of PS A1. As it is challenging to precisely identify the reaction outcome from a polysaccharide, we have synthesized 1-propyl-D-galactofuranose and 1-thiophenyl-4,6-O-pyruvate acetal-D-galctopyranose, which mimics two monosaccharides present in the core structure of PS A1. These two saccharide moieties can be affected by the reaction conditions employed for conjugation. In order to evaluate the specific oxidation of the vicinal-diol containing a primary alcohol over the trans-diol, 1-propyl-D-galactofuranose was subjected to NaIO4 mediated oxidation with a relatively harsher conditions (more concentrated NaIO 4 solution, longer reaction time) and experimental outcomes revealed that only the vicinal-diol containing a primary alcohol was oxidized to form an aldehyde even under harsh reaction conditions. This observation suggests that a regioselective conjugation of antigen to the PS A1 is possible under the reaction conditions. Again, an NMR based stability study was performed with 1-thiophenyl-4,6-O-pyruvate acetal-D-galctopyranose and it was determined that the pyruvate acetal is very stable at pH 5.0. Therefore, the zwitterionic charge character of PS A1 remains intact in the reaction conditions that is generally employed for antigen-PS A1 conjugation.;L-Thr-alpha-Tn antigen was coupled with poliovirus derived immunogenic peptide (PV), which was further conjugated to oxidized PS A1 to access a glycopeptide-PS A1 based vaccine (Tn-PV-PS A1). We were under the assumption that the Tn-PV-PS A1 construct would elicit Tn antigen specific robust cellular and humoral responses via both MHC I and MHC II dependent pathways. To validate our hypothesis, we injected our vaccine and Tn-PV in C57BL/6 mice and a series of assays, including ELISA, FACS, anti-proliferation, T cell proliferation, IFNgamma production and JAM assay, were performed to decipher the immunogenicity of the vaccine giving mechanistic insight as well. It was revealed that the vaccine candidate (Tn-PV-PS A1) was able to generate more anti-Tn IgG antibodies than Tn-PV alone and the induced antibodies were able to recognize Tn expressing tumor cell lines. Mice immunized with our vaccine construct showed higher antigen specific CD4+ T cell proliferation than the Tn-PV immunized group. Again, better Tn specific CD8+ T cell responses were also induced with the Tn-PV-PS A1 construct than Tn-PV. Considering the aforementioned observations, it could be concluded that PS A1 is an unique immunogen which is suitable to use for generating carbohydrate specific CD4+ and CD8+ T cell dependent immunity. |
