[Evaluation of pertussis toxin/toxoid by in vitro assays ]

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Abstract

Mechanisms were studied initially to develop an in vitro safety test for detectingpertussis toxintoxicity in acellular pertussis vaccines based on the histamine sensitisation test. Maximal contractions and sensitivities to different agonists and adrenoceptor-induced contractions in Ca2+-free medium of isolated rat small mesenteric resistance arteries were significantly reduced by in vivo [30 µg/kg, intravenously (i.v.), day 5] or in vitro (10 µg/ml, 2 h)pertussis toxinpretreatment. Pertussis toxin -induced decrease in sensitivity of small mesenteric resistance arteries to noradrenaline was endothelium-dependent. N?-nitro- -arginine methyl ester ( -NAME) (100 µM, 20 min) did not reestablish the sensitivity to noradrenaline. In vivo -NAME treatment (0, 1, 10 or 30 mg/kg) of pertussis toxin-pretreated (15 µg/kg) rats did not reduce pertussis toxin-induced enhancement of the histamine-induced decrease in blood pressure and histamine (10, 30, 100 or 300 mg/kg) induced mortality. Finally, in vivopertussis toxinpretreatment sensitises rats for sodium nitroprusside (50 µg/kg/min). We conclude that pertussis toxin-induced histamine sensitisation is caused by an interference ofpertussis toxinwith the contractile mechanisms of vascular smooth muscle of resistance arteries which indicates only an indirect role for histamine in the histamine sensitisation test.

Evaluation of pertussis toxin/toxoid by in vitro assays

1. Introduction

Pertussis toxin, one of the major toxic components of Bordetella pertussis whole-cell vaccines, is very important for the development of immunity to whooping cough (Cherry, 1996; Edwards and Karzon, 1990 and Pittman, 1979). Acellular pertussis vaccines contain detoxified antigens, i.e. detoxifiedpertussis toxinand pertussis toxoid (Anderson et al., 1994; Robinson and Funnell, 1992 and Wardlaw, 1992). Regulatory authorities require safety testing of acellular pertussis vaccines in order to confirm absence of residualpertussis toxintoxicity. Currently, the histamine sensitisation test is the only test considered by the regulatory authorities to be suitable for this purpose (EP, 2003 and WHO, 1998). The test is based on the principle that mice vaccinated with biologically activepertussis toxinare sensitised for histamine, resulting in a decrease of the lethal dose of histamine (Parfentjev and Goodline, 1948). The test gives rise to major animal welfare concern. Therefore, we started with the development of an in vitro safety test for detecting biologically activepertussis toxin in acellular pertussis vaccines based on mechanisms involved in the histamine sensitisation test. Ultimately, we want to reproduce a mechanistic effect of in vivopertussis toxinwith in vitropertussis toxintreatment.

Mechanisms were studied initially to clarify which receptors, signal transduction systems and physiological (cardiovascular and pulmonary) systems are involved in pertussis toxin-induced histamine sensitisation. Previous studies had shown that vaccination of rats with biologically activepertussis toxindecreases diastolic blood pressure, enhances histamine-induced decrease in mean arterial blood pressure, decreases the lethal dose of histamine and that this histamine sensitisation mainly involved histamine H1 receptors (Vleeming et al., 2000a). In addition, we found that in vivopertussis toxinpretreatment of male Wistar rats reduced maximal noradrenaline- or KCl-induced contraction of isolated small mesenteric resistance arteries, decreased sensitivity to noradrenaline of isolated rat small mesenteric resistance arteries and did not affect histamine- or acetylcholine-induced relaxations (Van Meijeren et al., 2004). It is known that vascular contraction induces ...