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.
Highlights
In light of recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), it has become urgent to develop quick, reliable and scalable assays to determine neutralising antibodies indicative of protection against infection
We analysed serum samples pre-vaccination and at 7 and 28 dpv, and compared the resulting antibody titres determined by fluorescence reduction neutralisation test (FluoRNT) to those measured by the benchmark assay focus reduction neutralisation test (FRNT)
YF-17D is extensively used as a model in systems biology and vaccinology studies, which require the longitudinal determination of neutralising antibody titres in a large number of samples
Summary
In light of recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), it has become urgent to develop quick, reliable and scalable assays to determine neutralising antibodies indicative of protection against infection. These assays should be suitable for testing large numbers of patient samples, using automated and objective evaluation methods, which can be applied for diagnostics [1]. Such assays are important for a range of applications ranging from fundamental systems biology research on model viruses to practical testing of protection status after vaccination. Quantitative assays have performance requirements far above those for qualitative diagnostic assays to determine protection status after vaccination
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