Vaccine 25 (2007) 6872–6881 A sensitive cell-based assay for the detection of residual infectious West Nile virus M.H. Koldijk a , J.A. Bogaards a , S. Kostense a , M. de Vocht a , L. Gijsbers a , M. ter Haak a , C. Ophorst a , J.P.J. Brakenhoff a , G.J. Weverling a , J.Y. Guichoux a , F. UytdeHaag a , J. Lewis b , J. Goudsmit a , G. Marzio a,∗ a Crucell Holland BV, PO Box 2048, 2301CA, Leiden, The Netherlands b AecselaBiologics, Little Compton, RI, USA Received 2 March 2007; received in revised form 31 May 2007; accepted 10 July 2007 Available online 27 July 2007 Abstract Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus. © 2007 Elsevier Ltd. All rights reserved. Keywords: Virus; Inactivation; Assay 1. Introduction Since the licensing of the trivalent, formalin-inactivated polio vaccine [1], vaccines based on inactivated virus have played a critical contribution in the control of infectious disease. In addition to poliomyelitis, the list of inactivated vaccines currently available includes influenza, hepatitis A, Japanese encephalitis, rabies and tick born encephalitis. Although new technologies are likely to play a major role in vaccine development in the 21st century, well established as well as novel inactivated vaccine products will continue to be key tools for maintenance of public health in the foreseeable future (for a comprehensive review see [2]). ∗ Corresponding author. Tel.: +31 71 519 9249; fax: +31 71 519 9800. E-mail address: giuseppe.marzio@crucell.com (G. Marzio). The inactivation process is a critical step in the manufac- turing of inactivated vaccines. In the past failure to detect incomplete inactivation resulted in vaccine recipients devel- oping poliomyelitis [3] and rabies [4]. In order to minimize the risks of releasing products containing residual infectious virus, a two tier approach is currently recommended [5] con- sisting of validation of the inactivation kinetics [6,7] and bulk testing of final product [8–10]. Kinetics of inactivation are routinely studied for example by means of endpoint dilution assays [11,12]. The usefulness of these assays, however, is limited by the amount of material that can be tested. Conse- quently, the absence of residual virus is to be confirmed by an assay that allows testing of large volumes of final product. Sensitive methods for detection of residual virus have been described based on amplification in cells and scoring for the appearance of cytopathic effect (CPE) [9,10,13] or detection of viral antigen positive cells [14,15]. Alternatively, 0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.07.011