The highly abundant, conserved and surface exposed Lancefield Group A Carbohydrate (GAC) is of interest as a GAS specific vaccine candidate due to evidence of protective properties of anti-GAC antibodies. However, it is necessary to conjugate polysaccharides to protein carriers to improve immune memory responses.
This work investigates chemical conjugation of GAC and rhamnose polymers to classical protein carrier Tetanus Toxoid (TT) and GAS protein antigen SpyAD. Either wildtype GAC or GAS_Rha (GAC extracted from mutant ΔgacI NCTC-8198 strain devoid of autoimmunogenic GlcNAc epitopes) was enzymatically extracted from GAS cells and conjugated to proteins using carbodiimide crosslinker chemistry. Three glycoconjugate vaccines were successfully generated; SpyAD conjugated to GAC as a “double-hit” GAS vaccine, and TT conjugated to either GAC or GAS_Rha.
GAC and protein IgG antibody production was investigated by ELISA after three subcutaneous immunisations of BALB/c mice with the produced glycoconjugate vaccines. Anti-GAC antibody responses were significantly elevated in mice immunised with TT-GAC, but not with SpyAD-GAC (p = 0.037 and 0.467 respectively), in comparison with the adjuvant only group. In contrast, the TT-GAS_Rha glycoconjugate containing rhamnose epitopes only, failed to induce anti-GAC antibody production above baseline. Meanwhile, SpyAD-GAC produced GAS protein specific opsonic antibodies, inducing significant opsonophagocytosis of GAS cells demonstrating that SpyAD remains immunogenic following conjugation. Although both proteins were successfully conjugated to GAC, TT showed superior anti-GAC IgG titres compared to SpyAD, however demonstrated less GAS specific killing activity. These are important observations in the progress towards adopting optimal vaccine antigen selection and glycoconjugate manufacturing methods.