Background:
Streptococcus pyogenes vaccine development requires from broadly applicable immunoassays to measure vaccine immunogenicity. The reliability of current functional assays including whole bacteria is limited by diversity across S. pyogenes isolates available to labs. The Australian Strep A Vaccine Initiative (ASAVI) has undertaken to establish a collection of reference isolates to advance the development of internationally standardised immunoassays, enabling comparison and interpretation of data from different research sites.
Methods:
Selection of isolates representing 4 different S. pyogenes emm-types was guided by molecular epidemiology, clinical relevance, and previous laboratory characterisation: M1, M12, M53 and M75. Bacterial growth kinetics were studied in standard broth and animal-free media, and gene expression of key virulence factors measured by RT-qPCR. Reference genomes were generated, and analysed for phylogeny. Genomic stability with serial passage were also assessed. Functional characteristics were assessed using adhesion and internalisation assays, flow cytometry, and Western blot.
Results:
Each isolate was phylogenetically representative of their emm-type. Growth was fastest for all isolates in animal-free media. Relative expression of spyCEP, spyAD and slo varied between isolates, growth medium, and over time. These data were used to generate high density master cell banks according to Good Laboratory Practice principles. All processes and batch manufacturing records were reviewed and approved by an independent bio-regulatory expert to assure high quality.
Conclusion:
We have manufactured and characterised a set of 4 well-characterised S. pyogenes reference isolates banks to streamline global vaccine development efforts. These master cell banks are ready to use and will be freely available for research purposes worldwide.