Adsorbed Diphtheria-purified Pertussis-tetanus-inactivated polio(Sabin strain)Combined Vaccine(Minimum Requirements for Biological Products)

Bordetella pertussis produces pertussis toxin (PT) as a major virulence factor; detoxified PT is an essential antigen for acellular pertussis vaccines. The toxicity of PT (residual toxicity and reversion to toxicity) in pertussis vaccines has long been assessed using the mouse histamine sensitization test (HIST). In this study, as an alternative to animal safety testing-based HIST, a 96-well in vitro assay (fetuin-ADP-ribosylation assay), which assesses both the ADP-ribosyltransferase of the S1 subunit and the carbohydrate-binding activities of the B-oligomer in PT, was developed and evaluated for its applicability to the final products of acellular pertussis combination vaccines, DPT, DPT-IPV, and DPT-IPV-Hib. The fetuin-ADPR assay is based on a two-step process: (i) recovery of PT antigen with retained carbohydrate-binding activity from vaccine samples using fetuin-coated magnetic beads, and (ii) measurement of ADP-ribosyltransferase activity of the recovered PT antigen using a 96-well microplate immobilized with the target molecule, Gα_i3C20 peptide. In the assay system, the standard curve of the PT reference NIBSC 90/518 exhibited a linear response up to 320 ng/mL (R² ≥ 0.98). The fetuin-ADPR assay showed that the activatable PT content in most final vaccine products stored at 4 °C was <20 ng/mL (below the detection limit), whereas that in vaccines stored at 37 °C for 28 days ranged from <20 to 159.7 ng/mL. The assay successfully detected the reversion to toxicity of detoxified PT. Validation of the fetuin-ADPR assay showed a day-to-day reproducibility of 27.5 % coefficient of variation and 44-50 % accuracy as assessed by spike recovery. The fetuin-ADPR assay has the advantage of being performed in a 96-well plate with standard laboratory equipment, and is a promising alternative to in vivo HIST for assessing PT toxicity in the final products of acellular pertussis combination vaccines.