Development of an assay for detecting the residual viable virus in inactivated rabies vaccine by enzyme-linked immunosorbent assay

Abstract

Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test; however, the current protocol conducted in Japan requires a large number of mice. The development and introduction of animal-free alternative assays are essential from the perspective of the 3Rs (reduction, refinement, and replacement) of animal testing. Here, we propose a novel inactivation assay for confirming the complete inactivation of the viable rabies virus using cultured Neuro-2a cells and an enzyme-linked immunosorbent assay (ELISA). The detection ability of ELISA was similar to that of a direct immunofluorescence assay, with the detection limit of ELISA being as low as 0.014 focus forming units/test. These results suggest that the assay could be used as a viral inactivation test. In comparison with a traditional in vivo assay, this assay has a higher detection ability, an objective interpretation, and would shorten the test duration from 25 days to 8 days.

Introduction

Rabies is a lethal zoonotic disease that is prevalent worldwide and is a grave problem, especially in Asia and Africa. Infection with rabies virus is estimated to cause approximately 60,000 deaths annually [1]. Currently, the rabies vaccine is used in the pre- and post-exposure settings to prevent disease. Human rabies vaccines contain inactivated rabies viruses produced in cell cultures or embryonated eggs [2]. Each lot of rabies vaccine must be tested for complete inactivation to ensure that there are no residual infectious virions [3]. The current test in Japan for confirming viral inactivation is performed using suckling or adult mice [4]. As such, from the viewpoint of the 3Rs (reduction, refinement, and replacement) of animal testing as well as cost and efficiency, an alternative means of confirming viral inactivation is desired [5].

The World Health Organization (WHO) has stipulated a method using cell cultures [6]. Briefly, bulk vaccines or vaccine products are inoculated into cells, which are cultured for 21 days. Then, the cells are assayed by a direct immunofluorescence assay (DIFA) or the culture fluids are inoculated into weanling mice to confirm the presence of the viral antigens or infectious virus. The European Pharmacopoeia has already excluded in vivo assays and the provided method is almost the same as that of the WHO [3]. However, conducting these assay takes more than 21 days, and there is no detailed information about the detection ability.

We previously developed a sensitive in vitro inactivation assay for detecting residual infectious virions in the rabies vaccine [7]. This assay, which uses DIFA, is more sensitive and convenient and has a shorter test duration than the mice-based inactivation tests. Although DIFA is specific and sensitive, it requires fluorescein isothiocyanate (FITC)-labeled antibodies and fluorescence microscopy. DIFA also requires experience to determine the intensity and pattern of fluorescence, and the interpretation of the results depends on the skill of the technician. In contrast, an advantage of the enzyme-linked immunosorbent assay (ELISA) is that the results are obtained as optical density (OD) values and are easier to interpret than those of DIFA. To provide a more convenient and reliable method, we developed a novel ELISA-based inactivation test for detecting infectious rabies virions in rabies vaccines.