Development of a Glycoconjugate Vaccine Against Group A Streptococcus (GAS)

Group A Streptococcus (GAS) is responsible for superficial infections, systemic disease, and autoimmune complications globally. Despite extensive research no commercial GAS vaccine currently exists. The highly abundant, conserved surface exposed Lancefield Group A Carbohydrate (GAC) is of interest as a vaccine candidate due to evidence of protective properties of anti-GAC antibodies. However, for effective vaccines it is necessary to conjugate polysaccharides to protein carriers to improve immune memory responses. This thesis investigates glycoconjugate production as an approach for the development of GAS vaccines, comparing chemical linkage and an E. coli cell based (“biological”) glycoengineering system as distinct manufacturing methods. For chemical conjugation reactions, 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. Biological conjugates were built in E. coli strain CLM24 (W3110 ΔlpxM, ΔwaaL) through recombinant polyrhamnose and protein expression, conjugated by chromosomally encoded Campylobacter jejuni PglB oligosaccharyltransferase. Three chemical glycoconjugate vaccines were successfully generated; GAS protein antigen SpyAD conjugated to GAC as a ‘double hit’ GAS vaccine, and classical carrier protein Tetanus Toxoid (TT) conjugated to either GAC or GAS_Rha. One biological glycoconjugate vaccine was also generated with an optimised Streptococcal carrier protein conjugated to recombinant polyrhamnose. 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, in comparison with the adjuvant only group. In contrast, the TT-GAS_Rha chemical conjugate and Streptococcus specific biological conjugate, both 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 methods successfully generated GAS glycoconjugates, in this study chemical linkage of wildtype GAC produced a more robust immune response than rhamnose variants conjugated either chemically or using PglB glycoengineering technology. Observations and technological advances developed in this thesis contribute to optimal GAS specific glycoconjugate vaccine design and production.