Tobacco Mosaic Virus as a New Carrier for Tumor Associated Carbohydrate Antigens

Abstract

Tumor associated carbohydrate antigens (TACAs) are being actively studied as targets for anti-tumor vaccine development. One serious challenge was the low immunogenecity of these antigens. Herein, we report the results on using the tobacco mosaic virus (TMV) capsid as a promising carrier of a weakly immunogenic TACA, the monomeric Tn antigen. The copper(I) catalyzed azide-alkyne cycloaddition reaction was highly efficient in covalently linking Tn onto the TMV capsid without resorting to a large excess of the Tn antigen. The location of Tn attachment turned out to be important. Tn introduced at the N terminus of TMV was immunosilent, while that attached to tyrosine 139 elicited strong immune responses. Both Tn specific IgG and IgM antibodies were generated as determined by enzyme linked immunosorbent assay and a glycan microarray screening study. The production of high titers of IgG antibodies suggested that the TMV platform contained the requisite epitopes for helper T cells and was able to induce antibody isotype switching. The antibodies exhibited strong reactivities towards Tn antigen displayed in its native environment, i.e., cancer cell surface, thus highlighting the potential of TMV as a promising TACA carrier.

Valency and density matter: Deciphering impacts of immunogen structures on immune responses against a tumor associated carbohydrate antigen using synthetic glycopolymers

“…To produce the glycopolymers, the amine bearing Tn antigen 5 25 was covalently linked with polymer 4 through amide bonds as promoted by N , N , N ′, N ′-tetramethyl- O -( N -succinimidyl)uronium tetrafluoroborate (TSTU) and diisopropylethylamine (DIPEA) (Scheme 1a). Glycan analysis showed that this glycopolymer contained on average 4 Tn molecules per chain (designated as 13k-4) (Table 1).…”

“…On day 4, a 96 well MultiScreen ELISPOT plate (Millipore) was coated overnight at 4 °C with a solution of bovine serum albumin-Tn conjugate (BSA–Tn) 25 in PBS buffer (10 μg mL −1 ) and then incubated at 4 °C overnight. On day 5, the ELISPOT plate was washed three times with PBST (0.1%) and blocked with 300 μL/well 5% BSA-PBST at room temperature for 1 hour.…”

“…After incubation with glycopolymers for 4 days, mouse spleen cells (5 × 10 5 cells) were washed twice with FACS buffer (1% BSA + 0.1% NaN 3 /PBS) and incubated with 2 μL of BSA-Tn-Alex Fluor 647 solution (0.5 mg/mL), which was prepared by reacting BSA-Tn 25 with Alexa Fluor 647 NHS ester (Thermo Fisher Scientific A-20006). After incubation for 1 h on ice, cells were incubated with 2 μL mouse Fc block (0.5 mg/mL, BD 55142) for 10 min on ice followed by staining with 2 μL PE rat anti-mouse CD19 (0.2 mg/mL, BD 553786) for 30 min on ice.…”

“…A 96-well microtiter plate was coated with a solution of BSA–Tn 25 in PBS buffer (10 μg mL −1 ) and then incubated at 4 °C overnight. The plate was washed four times with PBST, followed by addition of 1% (w/v) BSA in PBS to each well and incubation at RT for one hour.…”

Carbohydrate antigen delivery by water soluble copolymers as potential anti-cancer vaccines

“…20 Other antigen presenting platforms include dendrimers, 21,22 regioselectively addressable functionalized templates, 23 nanomaterials, 24,25 liposomes and proteoliposomes 26,27 polysaccharides 28 and virus-like particles. 29,30 …”