Development of an in vitro antigen‐detection test as an alternative method to the in vivo plaque reduction neutralization test for the quality control of Japanese encephalitis virus vaccine

Abstract

Japanese encephalitis virus (JEV) causes diseases that attack the human central nervous system. Traditionally, the quality control for JEV vaccines, in which the plaque reduction neutralization (PRN) titer is measured by the national control laboratories before the vaccine batches are marketed, has required laboratory animal testing. However, classical animal tests have inherent problems, including the very fact that animals are used, ethical issues, and the possibility of error. In this study, JEV antigen was measured in an in vitro assay to assess the feasibility of replacing in vivo assays that measure the PRN titers of JEV vaccines. We constructed a double‐sandwich enzyme‐linked immunosorbent assay (DS‐ELISA) that could detect JEV envelope (E). Initially, monoclonal antibodies (mAbs) directed against the JEV E protein were generated and characterized. We isolated 18 mAbs against JEV E protein, and most were the IgG1 or IgG2a isotype. The mAbs (5F15 and 7D71) were selected as the most suitable mAb pair to detect JEV E protein. DS‐ELISA with this pair detected as little as approximately 3 μg/mL JEV E protein and demonstrated a relationship between the amount of JEV E protein and the PRN titer. From these results, we surmise that this DS‐ELISA may be useful, not only in terms of measuring the amount of JEV E protein, but also as a substitute for the PRN test for JEV vaccine evaluation.

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice

“…Sera from immunized mice were collected and equal volumes from each mouse (100 μL/mouse) pooled, aliquoted into sterile microtubes, and stored at −70°C until analysis. Neutralizing antibody titers were measured with a 50% plaque‐reduction end‐point assay, as reported previously . Potency was determined by using PRNT in a monolayer of BHK‐21 cells to quantitate JEV neutralizing antibody responses in a group of immunized mice.…”

Novel envelope protein time-resolved fluoroimmunoassay as an alternative in vitro potency assay for quality control of inactivated Japanese encephalitis virus vaccine

“…The detection range of the novel TRFIA was 0.01 U/mL-25 U/mL with the sensitivity was 0.01 U/mL, The intra- and inter-assay coefficients of variation were less than 10 %, and the dilution recovery was between 90 % and 110 %. Compared with previously reported ELISA methods for JE virus E antigen detection, this novel TRFIA provides wider detection range, higher accuracy, excellent precision and application of automation [ 19 , 30 ]. However, the present TRFIA still relied on the traditional 96 well plate for detection.…”

Establishment of the 3rd national standard for lot release testing of the Japanese encephalitis vaccine (Nakayama-NIH strain) in Korea

“…However, there is a recent, active, international movement to transition from in vivo potency testing to more sensitive and reproducible in vitro tests. 8,9,10 Accordingly, the application of in vitro methods for lot release testing will require additional studies to estimate the in vitro potency values for the newly established 3 rd national JE vaccine standard.…”