Validation of the Rapid Fluorescent Focus Inhibition Test for Rabies Virus-Neutralizing Antibodies in Clinical Samples Stefan Kostense, a Susan Moore, b Arjen Companjen, a Alexander B. H. Bakker, a * Wilfred E. Marissen, a Rie von Eyben, a Gerrit Jan Weverling, a Cathleen Hanlon, b and Jaap Goudsmit a Crucell Holland B.V., Leiden, The Netherlands, a and Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas, USA b Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, pro- duced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vacci- nated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentra- tions can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples. R abies occurs worldwide, and more than 3 billion people live in areas in which the disease is enzootic. Every year about 55,000 people die from rabies, with more than 50% in Asia (3, 16). Post- exposure prophylaxis (PEP) against rabies exposure consists of thorough washing of the wound, passive immunization with ra- bies immune globulin (RIG) administered in and around the wound, and active immunization with vaccine (12). The admin- istration of RIG soon after exposure is essential to inhibit viral spread in the interval before sufficient immunity is developed in response to vaccination. Currently, human rabies immune glob- ulin (HRIG) and equine rabies immune globulin (ERIG) are used in PEP. These plasma-derived, polyclonal products are obtained from rabies-vaccinated human donors or horses and can be pro- duced only in limited amounts. Furthermore, the variable quality, low activity, and potential danger of contamination with adventi- tious pathogens warrant replacement with a more optimized product (18). Therefore, the World Health Organization (WHO) strongly encourages the development of alternative products to meet the global demand (17). We have developed an antibody cocktail, CL184, comprising of two monoclonal antibodies that target distinct nonoverlapping epitopes of the rabies virus glyco- protein (1, 5, 10). The CL184 antibody cocktail is currently being tested in clinical trials as a replacement for HRIG in PEP (2). An important requirement of the CL184 antibody combina- tion is that it confers similar rabies neutralizing activity as the comparator HRIG. The rapid fluorescent focus inhibition test (RFFIT) was selected as the pharmacodynamic marker assay. This assay is regarded as the standard rabies virus neutralization assay in diagnostic laboratories, vaccine and biotherapeutic character- ization, and rabies-related clinical studies (9). To demonstrate that this assay is equally well suited for measurement of both poly- clonal HRIG and the monoclonal CL184 combination in clinical serum samples, we conducted an assay validation as described below. The validation plan was based on the regular requirements as stated in the FDA Guidance for Industry (4) and ICH Q2(R1) guidelines (7), taking into account the limitations and variability of cell-based virus neutralization assays. This validation of the assay confirms the suitability and validity of this methodology for the intended purpose. MATERIALS AND METHODS RFFIT protocol. The RFFIT procedure (13) is utilized to measure the level of rabies virus neutralizing antibody activity (RVNA) against the chal- lenge virus standard 11 (CVS-11) strain of rabies virus in human serum samples. Five-fold serial dilutions of heat-inactivated serum samples were incubated with the CVS-11 strain in 8-well tissue culture chamber slides for 90 min at 37°C. Baby hamster kidney (BHK)-21 cells were then added to the serum-virus mixture and incubated for an additional 20 to 24 h at 37°C with 2 to 5% CO 2 . Slides were then acetone fixed and stained with an anti-rabies N-FITC conjugate. Twenty distinct microscopic fields per well were examined using a fluorescence microscope at 160 magnification to score the virus-in- fected cells (foci). The number of positive fields with rabies-infected cells per well was recorded. The neutralization endpoint titer was defined as the highest sample dilution at which 50% of the observed microscopic fields contain one or more infected cells. The RVNA titers are mathematically interpolated using the Reed and Muench method or a Reed and Muench chart for assigning a RFFIT titer (6). The endpoint neutralization titer of the test serum is then transformed into international units (IU)/ml values by calibration against the endpoint neutralization titer of the U.S. Standard Rabies Immune Globulin (SRIG) (lot R-3, 59 IU; first WHO International Standard), which was measured in the same assay run, with an assigned potency value of 2.0 IU/ml. Received 2 December 2011 Returned for modification 29 December 2011 Accepted 24 April 2012 Published ahead of print 30 April 2012 Address correspondence to Stefan Kostense, stefan.kostense@crucell.com. * Present address: Alexander B. H. Bakker, Merus B.V., Utrecht, The Netherlands. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.06179-11 3524 aac.asm.org Antimicrobial Agents and Chemotherapy p. 3524 –3530 July 2012 Volume 56 Number 7