Abstract:
Acute rheumatic fever (ARF), caused by Group A Streptococcus (GAS), as well as COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are both infectious diseases associated with significant and inequitable health outcomes in Aotearoa New Zealand. Laboratory assays that accurately measure serological antibody responses to multiple antigens are critical tools for infectious disease management and research, including diagnosis of previous infection and investigation of immune responses. This project aimed to create novel bead-based multiplex immunoassays to study antibody responses to GAS and SARS-CoV-2.
A triplex bead assay was first developed that simultaneously measured antibodies to GAS antigens SLO, DNaseB and the novel antigen, SpnA, from very small volumes of patient sera, with increased precision and efficiency compared to traditional serological methods. In a large cohort of ARF patients, anti-SpnA antibodies showed similar diagnostic sensitivity to SLO and DNaseB but appeared to wane quicker, suggesting anti-SpnA has utility in identifying a recent infection.
The triplex assay was then expanded to a robust and reproducible 8-plex assay, that also quantified antibodies to five prospective GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD and the group A carbohydrate). The assay was compatible with serum, plasma and eluted dried blood, and validated in a large cohort of children with ARF, GAS pharyngitis/skin infections and healthy controls. Multi-antigen analysis revealed striking differences in the serological profile of children with ARF, supporting the hypothesis that ARF pathogenesis is triggered by repeated precursor infections, including GAS skin infections.
A SARS-CoV-2 triplex assay was developed that enabled antigen-specific isotypes and subclasses to be quantified against spike, nucleocapsid and receptor-binding domain antigens, from serum and dried blood. Multi-antigen analysis of COVID-19 patients enabled a
comprehensive view of the composition of SARS-CoV-2 antibodies and showed responses were durable over a period of 8 months, albeit with antigen-specific differences in kinetics. The methodologies and assays developed were further utilised in a nationwide serosurvey and a vaccine response study.
This thesis demonstrates how multiplex immunoassay technology can be used to provide novel insight into current and emerging infectious diseases that are of major importance in Aotearoa New Zealand.