Development of a nitric oxide induction assay as a potential replacement for the intracerebral mouse protection test for potency assay of pertussis whole cell vaccines.

The intracerebral mouse protection test (Kendrick test) for the potency assay of pertussis vaccines is a complex and time consuming in vivo test which has a significant intra- and interlaboratory variation. Thus, there is a pressing need to develop a replacement for the Kendrick test. There is now convincing evidence to suggest that Bordetella pertussis can be taken up and survive within macrophages in the lungs and that cell-mediated immunity plays a role in protection. It was hypothesised that murine macrophages could be activated by immunisation with whole cell pertussis vaccines and therefore induce NO production. An alternative in vitro assay based on the determination of reactive nitrogen intermediates produced as a result of macrophage activation has been examined as a possible replacement for the current intracerebral (i.c.) mouse protection test. NO induction was studied in the peritoneal macrophages of female NIH mice immunised with normal and denatured whole cell B. pertussis vaccines respectively. Compared with controls receiving diluent only, macrophages and spleen cells from mice immunised with whole cell pertussis vaccine responded in vitro to selected pertussis antigens by NO synthesis. The production of NO in response to in vitro culture with bacterial antigen was immunisation dose dependent and was correlated with protective immunity in vivo as determined by i.c. challenge. The results suggest that NO production may serve as a marker of macrophage activation in mice immunised with whole cell vaccine, and could form the basis of a potential replacement potency assay.