Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, which can be greatly enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development. Progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret.
We have developed an opsonophagocytic assay (OPA) for GAS that utilises DMF-differentiated HL-60 cells and baby rabbit complement. As such, we have removed the major sources of variation in current assays. We have standardised the OPA for several clinically relevant isolates including emm-types 1, 6, 12, 53, 75, 89 and 100. In the presence of both baby rabbit complement and differentiated HL-60 cells, we have shown antibody-specific killing for each strain using a series of M-protein-specific sera. Antisera was generated through the immunisation of rabbits with recombinant full-length M-protein. This antibody-specific killing can be blocked through pre-incubation of the antisera with homologous antigen. This OPA assay has the potential to provide a reliable and reproducible platform to assess GAS vaccine candidates.