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

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.

Measurement of Cellular Immune Response to Viral Infection and Vaccination.

Combined cellular and humoral host immune response determine the clinical course of a viral infection and effectiveness of vaccination, but currently the cellular immune response cannot be measured on simple blood samples. As functional activity of immune cells is determined by coordinated activity of signaling pathways, we developed mRNA-based JAK-STAT signaling pathway activity assays to quantitatively measure the cellular immune response on Affymetrix expression microarray data of various types of blood samples from virally infected patients (influenza, RSV, dengue, yellow fever, rotavirus) or vaccinated individuals, and to determine vaccine immunogenicity. JAK-STAT1/2 pathway activity was increased in blood samples of patients with viral, but not bacterial, infection and was higher in influenza compared to RSV-infected patients, reflecting known differences in immunogenicity. High JAK-STAT3 pathway activity was associated with more severe RSV infection. In contrast to inactivated influenza virus vaccine, live yellow fever vaccine did induce JAK-STAT1/2 pathway activity in blood samples, indicating superior immunogenicity. Normal (healthy) JAK-STAT1/2 pathway activity was established, enabling assay interpretation without the need for a reference sample. The JAK-STAT pathway assays enable measurement of cellular immune response for prognosis, therapy stratification, vaccine development, and clinical testing.