FluoRNT: A robust, efficient assay for the detection of neutralising antibodies against yellow fever virus 17D

Abstract

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016–2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials

“…Specifically, a study on PRNT for dengue virus that served as a model to our study, showed similar performances for the 4 dengue serotypes 18 . Beyond the PRNT methods using live virus, other types of YF PRNT have been developed and evaluated to measure neutralizing antibodies such as Focus Reduction Neutralization Test (FRNT) and the Fluorescence Reduction Neutralization Test (FluoRNT) which showed respective precision performances of 91.7% and 94.9% in 28 days postvaccinated subjects 27 . Taking together, these data showed that the PRNT we developed here for YFV is consistent and reliable enough for use in diagnostic and evaluation of vaccine efficacy 28 …”

“…18 Beyond the PRNT methods using live virus, T A B L E 5 Accuracy/dilutability of PRNT90 yellow fever virus. 27 Taking together, these data showed that the PRNT we developed here for YFV is consistent and reliable enough for use in diagnostic and evaluation of vaccine efficacy. 28 In addition to these assay performance evaluations, our PRNT presents other advantages including a shorter turn-around time, 4-5 days, compared with others YF PRNT protocols that take 6-7 days.…”

“…These tests are based on recombinant proteins or virus‐like particles and would offer a solution to biosafety issues such as the requirement for BSL3 facilities and the need for well‐trained technical staff 31 . Moreover, the FluoRNT recently published for YF is also positioned as a powerful alternative tool to avoid inconveniences associated with the traditional PRNT technique as it is easier to use and more time and cost‐effective 27 …”

“…31 Moreover, the FluoRNT recently published for YF is also positioned as a powerful alternative tool to avoid inconveniences associated with the traditional PRNT technique as it is easier to use and more time and cost-effective. 27…”

Immuno-Colorimetric Neutralization Test: A Surrogate for Widely Used Plaque Reduction Neutralization Tests in Public Health Virology

“…There are methods in place to characterize functional Nt-Ab responses to the Poliovirus and Yellow fever virus, for which well-tested and proven vaccines are available [2,11,19,49,67]. Different types of NTs based on either viral plaque or focus staining procedures have been developed for the VPDs, i.e., Measles virus (MeV), Mumps virus (MuV), Rubella (RuV) and Varicella Zoster virus [7,10,13,16,21,22,[38][39][40][41][42][43][44][45]55,56,65,68].…”