Virus reduction neutralization test and LI-COR microneutralization assay bridging and WHO international standard calibration studies for respiratory syncytial virus.

Background: Respiratory syncytial virus (RSV) vaccine is an unmet medical need. The virus reduction neutralization test (VRNT) was developed to replace the LI-COR microneutralization assay to measure RSV neutralization titers. Methods: A bridging study using selected V171 phase I samples and calibration studies using the WHO international standard antiserum to RSV were performed to compare VRNT and LI-COR. Results: From the bridging study, we showed good concordance between VRNT and LI-COR titers, and similar post-/prevaccination titer ratios. From the calibration studies, we can convert VRNT and LI-COR titers into similar IU/ml. Conclusion: The VRNT and LI-COR microneutralization assay correlate well and the titers can be standardized as similar IU/ml, enabling direct comparison of titers from different assays.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7-25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.