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.

Introduction

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. The latter application is increasingly relevant: limits to vaccine production and supply have already forced the temporary use of diluted vaccine stocks in emergency mass immunisation campaigns against yellow fever starting 2016–17 [2]; and rapid scalable testing to determine successfully protected subpopulations may become a valuable aspect of epidemic control.

Yellow fever virus (YFV) is an enveloped positive-sense single-stranded RNA virus that has historically been responsible for large disease outbreaks with high morbidity and mortality across Africa and South America [3]. YFV was the subject of high-intensity research from the 1800s onwards, and in 1901 it became the first human virus ever to be isolated [4]. Max Theiler generated the live-attenuated vaccine strain YF-17D in the 1930ies [5], which confers lifelong YFV immunity in more than 95% of vaccinees and remains one of the most effective vaccines ever developed [6]. As the prototypical flavivirus and the first flavivirus against which a highly potent vaccine was developed, YFV has for a long time been a model for systematic patient studies of viral infection, innate and adaptive antiviral immune response, and immunisation [7, 8]. Such research relies on quantitative assays that robustly determine kinetics and magnitude of protective antibody response after exposure or vaccination. Quantitative assays have performance requirements far above those for qualitative diagnostic assays to determine protection status after vaccination.

A protective titre against YFV infection is most commonly measured by neutralisation tests, where the presence of neutralising antibodies results in the reduction of an infection equivalent within the assay. The serum dilution at which patient serum neutralises a set percentage of viral infection is called the neutralising antibody titre. For YFV WHO guidelines require patient sera to show at least 80% neutralisation at 1/10 serum dilution to be considered protective, i.e. the ED80 (effective dilution at 80% neutralisation, also EC80 or NT80) titre should be below 1/10 [9, 10]. For the two most commonly used reduction neutralisation tests (RNTs), i.e. the focus reduction neutralisation test (FRNT) and plaque reduction neutralisation test (PRNT) for YFV, cells are treated with a fixed amount of infectious viral particles inoculated with serially diluted patient serum. After incubation, infection equivalents are quantified, referenced to an assay control and analysed as a function of serum dilution. The resulting dose-response curve allows the reading of the ED80 neutralising antibody titre in serum (or, similarly, e.g. ED50). shows a schematic representation of the three RNTs that are discussed in this work. The assay formats and timelines for YFV-17D PRNT and FRNT depicted here are based on previously published standard protocols adapted by our lab [11-13] and generally depend on the cell lines and virus stocks used for the respective assays.

Comparison of scientific and practical features of the reduction neutralisation assays FluoRNT, PRNT and FRNT.

(A) PRNT, FRNT, and FluoRNT are differentiated in their readout modes. (B) PRNT, FRNT, and FluoRNT are also differentiated in assay workflows and time requirements. FluoRNT has several practical advantages, including (i) avoiding multiple rounds of infection (2nd– 4th rounds of infection in FRNT and PRNT) that otherwise introduce and enhance assay variability; (ii) avoiding CMC addition and removal (PRNT and FRNT), staining (PRNT) or immunostaining (FRNT), and manual counting (PRNT) or image post-processing (FRNT): which are all manual steps that increase the probability to introduce handling errors. Additionally, FluoRNT (iii) is faster overall and has reduced hands-on assay time; (iv) is adaptable for high-throughput use; and (v) its assay progress can be followed noninvasively during run time.

The traditional Plaque RNT (PRNT) counts large plaques that develop in a monolayer of cells after multiple rounds of infection by any cytopathic virus strain, as the viral infection equivalent. YFV PRNT employs a cell monolayer in 6- to 24-well format exposed to virus and diluted test serum, coated with a viscous medium overlay such as carboxymethylcellulose (CMC). The non-neutralised fraction of virus infects cells; and the viscous coating eliminates convective viral transport but allows radiative cytopathic spreading during subsequent rounds of infection. Depending on the virus strain, on the used cell line (e.g. BHK or Vero cells) and on the MOI used, the assay is run for 4–5 days so that multiple rounds of infection give visually detectable plaques in the monolayer, that encircle originally infected cells [14-16]. PRNT readout is then obtained by aspirating the viscous coating and staining live cells to enable counting dead plaques. Determining the percentage of reduction of cytopathic plaque formation for a range of serum dilutions allows the fitting of a dose-response curve to determine the ED80. The reagents and equipment used in PRNT are cheap; but the assay has major drawbacks: it requires much hands-on time, is technically highly variable, and the overall assay run time is long. These and other factors make PRNT prone to numerous sources of variation and error in assay setup and analysis, while additionally being poorly scalable.

The Focus RNT (FRNT) is a 96-well format assay counting much smaller infected cell foci after fewer rounds of infection, visualised by immunostaining; while being similar in setup its throughput can be higher than that of PRNT, and FRNT can also be applied to any cytopathic and non-cytopathic virus for which antibodies exist [12, 17]. Assay setup for the YFV FRNT is similar to that of PRNT, including the need for viscous overlay, but multicellular foci are formed typically after 2–3 days of incubation. After methylcellulose removal and washing, immunostaining has to be performed (primary: anti-virus e.g. 4G2 clone, secondary: typically, enzyme-conjugated for chromogenic staining) [18]. The converse signal-to-background pattern of absorbent foci against an unstained cell layer should enable automated focus counting via a scanning EliSpot-type plate reader. However, in practice, high background from chromogenic staining and low signal depending on the primary antibody makes manual checking of staining results and of image post-processing obligatory in our hands. The cost of the required antibodies also limits its practicality, and the overall savings in time associated with reducing the number of rounds of infection are offset by extra washing and staining steps.

The motivation for the present study has been to develop a robust, quantitative, and scalable assay that avoids the disadvantages affecting plaque and focus reduction neutralisation tests, and better fulfils current higher-throughput needs in basic and applied virology research specifically for YFV, as well as more broadly for other viruses.

Our Fluorescence RNT (FluoRNT) uses a reporter variant of the YF-17D vaccine virus to allow stain-free detection of reporter expression in infected cells after the first round of virus infection, in a 96-well format. There is no need for application and then removal of a viscous overlay; and infected cells can be reliably quantified by flow cytometry based on the yellow fluorescent reporter Venus, without additional staining or immunostaining ().

In this study we focus on evaluating assay performance based on three criteria: (1) reliability, (2) practicability, and (3) data quality. We expect many of the findings to apply not only to neutralisation tests for YFV, but also to neutralisation tests for other viruses.

Methods

Human samples

Human sera before and after vaccination with the YFV Vaccine Stamaril® (Sanofi) were derived from a YF-17D vaccination study, approved by the responsible institutional review board of the Medical Faculty, LMU Munich; (IRB #86–16). In this study, blood was taken from healthy adults directly before the vaccination (d0) as an individual reference and on day 7, 14 and 28 post vaccination (dpv) to determine the titre of neutralising antibodies. Serum was collected in S-Monovettes (Z-Gel; Sarstedt, Nuembrecht, Germany) and separated from whole blood by centrifugation at 2500 × g for 10 min. Samples were frozen and kept at -80°C until use. To probe the reliability of the FluoRNT at later time points after the vaccination blood samples were taken from 15 healthy volunteers with a history of being vaccinated with Stamaril® between 4 month and up to 19 years ago (approved by the IRB of the Medical Faculty, LMU Munich, 24062006GP).

Cells

Vero cells (ATCC CCL-81) and BHK-21 cells (ATCC CCL-10) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS), 1% L-Glutamine 100 IU/mL Penicillin and 100 μg/mL Streptomycin under standard conditions (37°C, 5% CO2, >90% humidity). All cell lines were regularly tested and only used if mycoplasma negative.

YF-17D-Venus production

The YF-17D fluorescent reporter variant YF-17D-Venus plasmid was a kind gift from Charles M. Rice and Margaret MacDonald (The Rockefeller University, New York, USA). pYF17D-5’C25Venus2AUbi encodes a Venus fluorescent protein in frame after the first 25 amino acids of the YFV capsid gene, followed by a foot-and- mouth disease virus (FMDV) 2A peptide to mediate protein cleavage and a ubiquitin (Ubi) monomer followed by the complete YFV polyprotein sequence [19]. YF-17D-Venus genome was cloned into a pACNR backbone using Gibson Assembly Master Mix (New England Biolabs, Ipswich, USA).

For YF-17D-Venus production the construct (5’C25Venus2AUbi) in pACNR was linearized with AflII and in vitro transcribed into sense RNA using the SP6 promotor and the mMessage mMachine Kit (Ambion by life technologies, Carlsbad, USA) according to the manufacturer’s protocol. BHK-21 cells were transfected with YFV-Venus RNA by electroporation. Briefly, 6 × 106 cells in 400 μL ice-cold PBS were mixed with 2 μg in vitro transcribed RNA and transferred into a 0.4 cm gap electroporation cuvette. Electroporation was performed immediately with 1200 V for 100 μs with 2 pulses in 5 s intervals in a Gene Pulser Xcell Electroporation System (Bio-Rad, Hercules, USA). After recovery for 10 min at room temperature cells were added dropwise into 10 cm petri dishes filled with pre-warmed DMEM containing Penicillin/Streptomycin and L-Glutamine. The success of the electroporation was verified with a Leica TCS SP5 confocal system (Leica Microsystems, Wetzlar, Germany). Yellow fluorescent cells could be seen 3–4 days after electroporation, and supernatant was harvested upon onset of cytopathic effect, after 6–7 days. The supernatant was clarified from cellular debris by centrifugation at 400 × g for 5 min and further purified using a 0.45 μm filter. Aliquots of this primary virus culture were stored at -80°C.

Virus stock production

BHK-21 cells were grown in DMEM containing 10% FCS, 1% L-glutamine, 100 U/mL Penicillin, and 100 μg/mL Streptomycin and infected with YF-17D (derived from a Stamaril® vaccine dose) or YF-17D-Venus (see above) at an MOI of 0.1 at a confluency of 80%. Supernatants were collected post-infection when a cytopathic effect was clearly visible (after 48 h for YF-17D and after 72–96 h for YF-17D-Venus). The supernatant was clarified to remove cellular debris by centrifugation at 400 × g for 5 min and further purified using a 0.45 μm filter. Aliquots were then stored at -80°C.

For some experiments an additional purification of the virus stock was performed, adapted from previously described protocols [20]. Briefly, cell culture supernatants from virus producing cells were centrifuged at 2200 × g at 4°C for 15 min. Polyethylene glycol (PEG 8000) was gradually added to the clarified supernatant on ice (final concentration 7% w/v) and rotated at 4°C with low speed for at least 3 h. Supernatant containing PEG was centrifuged in a Sorvall 75006445 Rotor at max speed (~3800 × g) at 4°C for 80 min, the pellet resuspended in cold TNE buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA) and layered on top of a double sucrose cushion of 30% sucrose on top of 60% sucrose (w/w in TNE) in 5 mL open-top thinwall ultra-clear tube (Ref 344057, Beckman Coulter, Brea). Ultracentrifugation was performed at 160,000 × g at 4°C for 2 h in an MLS 50 rotor (Beckman) and resulted in a clear virus band between the two sucrose cushions. Pellet was resuspended in cold TNE buffer and stored at -80°C until use.

FRNT

The focus reduction neutralisation tests for YFV-17D and YFV-17D-Venus were performed similar to previous descriptions [11, 12, 17, 18].

On the first day, Vero cells were seeded in a 96-well plate with 25,000 cells/well and incubated over night at 37°C.

Prior to infection on the second day, human sera were thawed, diluted 1:5 in DMEM without supplements and heat inactivated at 56°C for 30 min. 60 μL of 1:3 serial dilutions of the sera in DMEM supplemented with 0.02% BSA were mixed with an equal volume of YF-17D or YF-17D-Venus diluted in DMEM with 0.02% BSA containing a predetermined amount of infectious units (approximately 100 focus forming units; FFU; or approximately 220 PFU of our YF-17D-Venus stock) per well. Human sera were assayed in two replicates in dilutions reaching from serum dilution log10(ED) = -1.0 to -5.8 for the YF-17D FRNT and from -1.0 to -4.8 for the YF-17D-Venus FRNT. No-serum controls were included using DMEM with 0.02% BSA instead of serum. The mixture was kept at 37°C for 1 h. Afterwards, 50 μL of the inoculum was used to replace the medium of the Vero cells. To block convective viral distribution, after 1 h, 150 μL of pre-warmed methylcellulose (MCS) medium was layered on top of the cells. Briefly, MCS medium contained 1.5% MCS powder (Sigma, M0512-10G, 4000 cP viscosity) in MEM (supplier), 10% FCS, penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (4.5 g/L), and sodium hydrogen carbonate (3.7 g/L) to reach a pH of 7.4 in Millipore water. After infection the cells were incubated at 37°C for 48 h.

On the fourth day, the methylcellulose cover was removed, the cells were fixed in 5% PFA in PBS for 1 h at room temperature, permeabilized for 5 min and blocked for 10 min using 0.5% Triton X-100 and 50 mM Ammonium chloride solution in PBS, respectively. Washing with PBS was included after each step. Foci for the YF-17D FRNT were stained with 1 μg/mL Flavivirus anti-E 4G2 antibody (CG 0042, mouse monoclonal, Clonegene, Atlanta, USA; Antibody Registry AB 2722738) diluted in 0.05% Tween in PBS (15 ± 1 h) at 4°C overnight, followed by an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (7076S Cell Signalling, Danvers, USA; Antibody Registry AB 330924) diluted 1:100 in 0.05% Tween in PBS for 3 h at room temperature on the fifth day. We visualised the foci with a chromogenic reaction using 3,3’- diaminobenzidine (DAB) (D5905, Sigma-Aldrich, St. Louis, USA) in TBS buffer containing approximately 0.3% NiCl solution for 25 min at room temperature.

For the YF-17D-Venus FRNT foci were stained with a rabbit anti-GFP antibody (ab6556, Abcam, Cambridge, UK) diluted 1:2,500 in 0.05% Tween in PBS over night at 4°C and an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1706515, rabbit polyclonal, BioRad, Hercules, California; Antibody Registry AB 11125142) diluted 1:250 in 0.05% Tween in PBS for 1 h at room temperature. Foci were again visualised using 3,3’- diaminobenzidine for 10–15 min at room temperature.

Foci were counted with an EliSpot Reader ELR04 SR (AID, Autoimmun Diagnostika GmbH, Straßberg, Germany). Virus neutralisation was determined as for each dilution. As stated in the figure legends we used either individual pre-vaccination value 0 dpv or the mean of the run-average no-serum controls (NSC) as the reference controls.

Once virus neutralisation results reached zero, they were constrained to zero in all following dilutions. With the results of each dilution, curves with a bottom constraint greater than 0 and a top constraint less than 100 were fitted by nonlinear regression analysis (Four parameter, variable slope dose-response model: Find ECanything; F = 80) using Prism 8 (GraphPad, La Jolla, CA, USA) software. Titres were interpolated at 80% virus neutralisation (ED80).

FluoRNT

For the fluorescence reduction neutralisation test (FluoRNT) the assay setup is as follows:

On the first day, Vero cells were seeded in a 96-well plate with 25,000 cells/well and incubated over night at 37°C.

Prior to infection on the second day, human sera were thawed, diluted 1:5 in DMEM without supplements and heat inactivated at 56°C for 30 min. 60 μL of 1:3 serial dilutions of the sera in DMEM supplemented with 0.02% BSA were mixed with an equal volume of YF-17D-Venus containing a predetermined amount of infectious units to induce an infection rate of 25% ± 5% in the virus quantification, as previously reported [16]. The amount of virus used in the FluoRNT is 22-fold more concentrated than the YF-17D-Venus stock used in FRNT and corresponds to approximately 5000 PFU/well. Human sera were assayed in two replicates in dilutions reaching from serum dilution log10(ED) = -1.0 to -3.9. The mixture was incubated for 1 h at 37°C. No-serum controls as reference and negative controls were included using only DMEM with 0.02% BSA instead of serum. The medium of Vero cells was removed and replaced by 50 μL of the inoculum.

After an incubation for 24 h at 37°C the medium was removed, cells were washed, trypsinised and transferred into a 96 U-well plate. For flow cytometry analysis, cells were stained for 30 min at 4°C with a viability dye using 50 μL of APC-Cy7 Fixable Viability Dye (eBioscience™ Fixable Viability Dye eFluor™ 780, Invitrogen, Carlsbad, USA) diluted 1:5,000 in FACS buffer (PBS with 2% FCS, 2 mM EDTA and 0.1% NaN3). Cells were then washed and the virus was inactivated by 4% PFA in PBS. After incubating for 25 min at room temperature cells were again washed and analysed by FACS by flow cytometry (LSRFortessa Flow Cytometer, BD, San Jose, USA) or stored at 4°C in the dark for analysis on the next day. The infection rate per well was determined as the percentage of Venus-positive cells of all living single cells in one well. The gating strategy is shown in

Virus neutralisation was determined according to the following formula for each dilution:

As stated in the figure legends we used either individual pre-vaccination value from 0 dpv or the mean of no-serum controls (NSC) as the reference values. Once virus neutralisation results reached below zero, they were set as zero as well as the following lower dilutions results regardless whether they reached again above zero or not. Curves with bottom constraint greater than 0 and top constraint less than 100 were fitted by nonlinear regression analysis (Four parameter, variable slope dose-response model: Find ECanything; F = 80) using Prism 8 Software (GraphPad, La Jolla, CA, USA) software. Titres were interpolated at 80% virus neutralisation (ED80).

Statistics

Where shown, the statistical tests used are noted in the figure legends. By default, tests were performed as non-parametric tests, not assuming a normal distribution of the data. P-values smaller than 0.05 were considered significant.

Data analysis

Calculation of 80% neutralising titres (ED80) for both assays is described in the respective paragraphs on assay protocol. All titres are represented as the log10(ED80), starting from the initial 1/10 serum dilution (log10(ED80) = -1), to 1/100 (log10(ED80) = -2), 1/1000 (log10(ED80) = -3) and so on.

The assay dynamic range (ADR) provides the range in which the assays are free from technical scatter and therefore reliable differentiate data points, by taking the technical scatter of bottom and top plateau values into account. The bottom scatter is calculated with primary data values (i.e. foci for the FRNT or Venus+ cells for the FluoRNT) normalised to the run’s average maximum infection values (no-serum controls, NSC). For the top scatter the technical replicates for the maximum protection values (i.e. serum in log10(ED) = -1) are normalised to the run-average NSC, then the greater value is divided by the smaller one. Both scatters ideally show a tight distribution around 1. For visual representation in both scatters were transformed to the same scale. To give the ADR, the separation between the 10th percentile of the bottom scatter and 90th percentile of the top scatter (marked with “d” in ) is divided by the sum of both 10-90th percentile widths (“s1” and “s2” in ). As a numerical example, a near-ideal assay that generates high-quality ED fits could have bottom scatter values with 10th/90th percentiles at 95% / 110% infectious equivalents normalised to run-average NSC and top scatter values with 10th/90th percentiles at 0% / 5% infectious equivalents referenced to run-average NSC, giving a good ADR of (95–5) / ((110–95) + (5–0)) = 4.5. A highly scattered assay that is poorly suitable for ED80 fitting might have the corresponding 10th/90th percentile plateau values at 85% / 125% and 0% / 20%, giving a poor ADR of 1.1.

To analyse assay precision, we tested technical replicates in the range of 55–90% neutralisation for their reproducibility. The area between 55–90% neutralisation is the most relevant region of the dose response fit to define the patient’s ED80 and therefore the titre required as correlate for protection. We therefore paid extra attention to the technical reproducibility of data points in this region. The absolute neutralisation difference ∂ between technical replicates in this area of interest was calculated and weighted as a precision parameter (1-∂)2.

Software

FlowJo Software (FlowJo LLC, BD, San Jose, USA) was used for flow cytometry analysis. Confocal microscopy pictures and live image videos were analysed with FIJI [21] software. Parts of and the graphical abstract were created with BioRender.com.

Results

FluoRNT detects protective titres with equal reliability as the benchmark FRNT assay

To test whether the first-round of infection assay FluoRNT is suitable as an alternative to multiple-round of infection assays for determining YFV protection status according to WHO criteria, we used it to measure neutralising activity after YF-17D vaccination in a cohort of 32 YFV-naive volunteers. Neutralising antibodies are formed following peak viremia and usually do not occur before 8 days post vaccination (8 dpv) [22]. According to the WHO up to 80% of vaccinees demonstrate a neutralising antibody titre by 10 dpv (ED80 ≤ 1/10 serum dilution), and most studies show that > 99% of vaccinees develop a protective titre by 28 dpv [23]. We analysed serum samples pre-vaccination and at 7 and 28 dpv, and compared the resulting antibody titres determined by FluoRNT to those measured by the benchmark assay FRNT. As expected, both assays showed no neutralising titres in the 7 dpv samples, while all volunteers had protective titres at 28 dpv. shows titres measured by both assays, showing a high correlation (r = 0.84, p < 0.0001) in high as well as low titre samples indicating that FluoRNT can detect protection status with equal reliability as FRNT. To probe the applicability of both assays we analysed serum samples from vaccinees 4 month to 19 years after they had been vaccinated with YF-17D (n = 15). Titres of the long-term vaccinees were on average lower than those of the primary study group at 28 dpv. FluoRNT gave a titre median of log10(ED80) = -2.08 for the long-term vaccinees and log10(ED80) = -2.38 for the primary study group. FRNT gave a titre median of log10(ED80) = -2.10 in the long-term study group and -2.54 for the primary study group. Both assays detected protective titres in all of the samples and the high correlation coefficient (r = 0.84) between the results determined by the two assays was preserved ().

FluoRNT reliably detects neutralising activity.

(A) FRNT and FluoRNT yield strongly correlating titres within the same order of magnitude and with similar distributions at 28 dpv (n = 32). (B) FluoRNT has equal reliability to FRNT in determining titres from long-term vaccinees (n = 15). Spearman r. All titres are referenced to NSC. The grey box illustrates that datasets from both assays follow the same general trend.

While the absence or presence of protection is a useful qualitative result, quantitative assay development instead requires determining the actual antibody titre reliably and comparably, with emphasis on assay robustness, data quality, and assay practicability. We first assessed and compared assay practicability between FRNT and FluoRNT.

FluoRNT offers significantly greater assay practicability than FRNT and PRNT

In addition to assay robustness and data quality, it is important for assay selection to evaluate practicability. shows the time line for PRNT, FRNT and FluoRNT. The following features that differentiate the ease of use and implementation of these assays will be considered in more detail in the discussion section. Of note, only FRNT and FluoRNT were evaluated and compared directly experimentally; the comparison to PRNT is from previous experience and literature.

FluoRNT delivers results distinctly faster than FRNT and PRNT (fewer (handling) steps, shorter run time).

FluoRNT readout by flow cytometry is objective and quantitative with the potential for automation

FluoRNT cuts assay costs by 84% compared to FRNT () and scales up with the most affordable lab consumables

FluoRNT can be monitored non-invasively during assay run time

In addition to showing the improved practicability of FluoRNT in its assay setup, we also tested for improvements in data quality delivered by FluoRNT, analysing reproducibility, assay dynamic range and precision compared to the benchmark FRNT.

Assay precision and data quality

FluoRNT yields superior plateau data quality for dose-response analysis

To quantify ED titres reliably, data values must not only be precise (reproducible and free from random scatter) but must also give a high-quality dose-response fit (). This is prototypically a 4-parameter sigmoidal fit, with top and bottom plateaus at high and low serum concentrations respectively, with a characteristic midpoint (ED50) and gradient at this midpoint (Hill slope). The top plateau data points correspond to complete virus neutralisation and therefore maximum protection, i.e. 100% virus neutralisation as defined by the absence of equivalents of infection (number of Foci for FRNT and percentage of Venus-positive cells in FluoRNT) in the presence of a usually less diluted serum sample (e.g. 1/10 serum dilution). The bottom plateau data points correspond to no protection (i.e. 0% virus neutralisation) and are represented by more diluted serum dilutions that approach the number of equivalents of infection of no-serum controls (NSC, no serum or antibodies present) or pre-vaccination serum samples. In our calculations, equivalents of infection of tested serum are referenced to the average NSC run in the same experiment. Values referenced to pre-vaccination samples (0 dpv) can be found in the supplementary information.

In an ideal assay the top values that show maximum protection (in our case meaning the infectious equivalents on 28 dpv in the highest concentration, i.e. log10(ED) = -1.0 (= 1/10 dilution) would give identical technical replicate values. To assess the scatter of the top values we normalise the two replicates to the run-average NSC value, then divide the greater value by the smaller. The FluoRNT and the FRNT both show a tight distribution around 1 indicating that the top scatter is nearly ideal for both assays and allows a confident determination of the top plateaus of the dose response curve ().

To assess the quality of the bottom plateaus, we analysed the NSC values (maximum possible infection equivalents or minimum protection per assay) by plotting the ratios of raw data NSC values to the average NSC counts of infection in all assays that were run simultaneously (). In a high-quality assay, NSC values should show a tight distribution with little deviation arising from technical variability inherent to the assay. The normalised FluoRNT NSCs give a tight distribution, highlighting the reproducibility of the bottom plateaus they define (90% of data points cluster between 0.92 and 1.1 of the normalised NSC average). In contrast, the FRNT NSC values consistently spread over a wider range (90% of data points lie between 0.84 and 1.17), which could make its bottom plateau definition, and thus the subsequent dose-response fitting, problematic. This difference in the data distribution between the NSC values in FluoRNT versus FRNT stayed consistent when we tested different virus batches including batches that were purified or not by a sucrose gradient. (). Therefore, FluoRNT shows a consistently more robust bottom plateau than the FRNT.

FluoRNT yields a greater assay dynamic range for determining ED titre values

To study antibody response quantitatively, ED titres must be interpolated from dose-response curves, which are fitted to neutralisation values intermediate between the top and bottom plateaus. An ideal assay has fully reproducible full- and no-protection plateaus so that any intermediate data values can be reliably used for fitting the intermediate region of the dose-response curve. Fitting to intermediate values becomes increasingly imprecise and inaccurate as data scatter increases. As an extreme example, if top and bottom plateau data values are so scattered that they can be found over the full range of protection values, then confident or meaningful fitting to intermediate data values is not possible, regardless of their precision.

We therefore expected that FluoRNT would allow better and more reliable dose-response titre fits than FRNT. To quantify the quality of data fitting for ED determination that is possible in each assay, we defined an assay dynamic range (ADR). Briefly, the dimensionless ADR is the ratio of the area in which data points suitable for ED fitting can be reliably differentiated from the technical scatter (i.e. area between the bottom and top plateau scatter bands), to the assay-inherent technical scatter (as derived from the scatter of the bottom and top plateaus, ). The formula used can be found in the Methods section.

Fig 3

FluoRNT plateau data quality and reliability is superior to that of FRNT.

If not described otherwise, all plots are box (25%/75%) and whiskers (10%/90%) plots with data outside the whiskers shown as individual data points. (A) Representative FluoRNT dose-response curve for a patient serum at 28 dpv. The “top” represents values of full protection whereas the “bottom” values of the curve show no protection against YF-17D. The grey area bracketing the ED80 is used to calculate the weighted precision of technical replicates (see panel F). (B) Scatter of top plateau: Replicates of infectious equivalents (number of foci or percentage of Venus+ cells) on 28 dpv at log10(ED) = -1 referenced to run-average NSC values, then the greater value is divided by the smaller one (F-test; n = 31). Box and whiskers show minimum and maximum of all data points. (C) Scatter of bottom plateau: infectious equivalents for no-serum controls (NSC) normalised to the run-average NSC (F-test; n = 128;). (D) Assessment of the assay dynamic range (ADR) combining the top and the bottom plateau scatter. Box plot that scatters around 100% displays the bottom scatter as in C (i.e. NSC; n = 128) and box plot that scatters around 0% represents the top plateau scatter as in B brought to the same scale (i.e. replicate ratio of 28 dpv serum at serum dilution log10(ED) = -1; n = 31). The dynamic range is calculated as range between the 10th and 90th percentile of the upper and lower box (see “d”), respectively and referenced to the distribution widths of both (“s1” and “s2”). The ADR of the FRNT assay is 2.4 while that of FluoRNT is 4.2. All values are referenced to run-average NSC. Panels B-D show technical replicates of study participants of the main cohort. (E) Goodness of fit parameter R2 for data fit to a 4-parameter sigmoidal dose-response model (n = 32). Median R2 for FluoRNT is 0.996 and 0.988 for FRNT. Mann Whitney test. (F) Precision of FluoRNT is significantly higher than that of FRNT (58 data points considered for FluoRNT with median of 94.91%, 66 for FRNT with a median of 91.74%). Mann-Whitney test. NSC served as reference for calculation in all panels.

In our hands the ADR for FluoRNT was 4.2, whereas the ADR for FRNT was 2.4 (). Since the ADR reflects expected assay fit quality in a non-linear fashion (see Methods), these ADR differences are substantial. Note that, as the top scatter is tightly distributed around 0% in both assays, the ADR is mainly dependent on the difference between the bottom scatters. We next moved to examine if these better-quality plateau parameters would result in FluoRNT being a more reliable and predictive assay for quantifying antibody titre, when considering the full data sets to be fitted.

FluoRNT data allows superior quality of dose-response fitting for titre determination

Having compared the quality of the assays’ plateaus for dose-response curve fitting by their ADRs, we then analysed the overall quality of the curve fitting that determines patient titre. Both assays allow fitting sigmoidal dose-response curves. However, the goodness of data fit is significantly better for FluoRNT than FRNT (median R2 0.996 vs. 0.988, p = 0.0002; Mann-Whitney test; ). The actual 28 dpv titre values obtained from the fitted curves are similar for FRNT and FluoRNT, although not identical (). This similarity was expected as these assays rely on the same basic principle, but since FluoRNT provides not only better fit quality, but also better data quality, the confidence in its titres should be greater. While referencing FluoRNT and FRNT to individual pre-vaccination serum samples, instead of no-serum controls, did not greatly alter these results (). It should be noted, that the FluoRNT is more robust to different reference samples (i.e. pre-vaccination samples from 0 dpv instead of NSC controls; ).

FluoRNT data yields significantly more precise antibody titre fits than FRNT

Having compared FluoRNT and FRNT for the analytical quality of their dose-response plateaus and their overall curve fits (), we now focused on quantifying the quality of the ED80 titre fits. For this fit, the most important region of the curve is the sloped region that brackets the ED80 (ca. 55–90% neutralisation). Errors in this region most strongly distort the titre since these data have the most weight in ED80 fitting. We therefore analysed the subset of data in the range of 55–90% neutralisation, calculating the absolute neutralisation difference ∂ between technical replicates and weighting it as a precision parameter (1-∂)2.

The median precision of FluoRNT on 28 dpv is at 94.9%, which is significantly higher than the precision of FRNT (91.7% at 28 dpv; p = 0.031, Mann-Whitney test, ). This was validated again in independent assays with different virus preparations, on different patient sub-cohorts handled by different scientists (. This result shows that the precision of FluoRNT is robust to the virus stock preparation used and handling, which are the major laboratory-dependent variables. These results suggest that the greater precision of FluoRNT compared to FRNT arises from both technical aspects and the single-round-of-infection assay design.

Taken together, this shows that the data delivered by FluoRNT have good precision and are robust regardless of whether longitudinal study references or no serum references are used, or whether the YF virus stocks had been purified over a sucrose gradient or not.

Discussion

The live-attenuated vaccine against Yellow Fever is one of the most successful vaccines ever developed. One shot of the vaccine virus YF-17D provides healthy adults with a life-long protection against the disease, and it has been successfully used for immunization for more than eighty years with a very low number of severe adverse events. 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. Recently, the need to efficiently and reliably monitor neutralising antibodies in patients or vaccinees has been highlighted. Reports of diminishing antibody titres after vaccination [24, 25], studies on the effect of booster immunizations, and the need for controlling the efficacy of vaccines that were diluted to accommodate the local need for vaccine—as happened during the 2016 outbreak of Yellow Fever in Angola and the Democratic Republic of Congo [2, 26], and the 2017–18 outbreak in Brazil [27]—are examples that underline the urgent clinical need for implementing efficient testing for neutralizing titres in large patient cohorts.

Here we describe a novel, robust, cost- and time-efficient fluorescence reduction neutralisation test (FluoRNT) utilizing a modified YF-17D virus carrying a Venus-sequence as a fluorescent reporter, to measure neutralising antibodies in human serum samples by flow cytometry. In terms of its assay workflow, FluoRNT is a labour- and time-saving alternative to existing techniques such as the widely used Focus Reduction Neutralisation Test (FRNT) and Plaque Reduction Neutralisation Test (PRNT). In terms of cost-effectiveness, FluoRNT is 10 times cheaper than the commonly used FRNT with an anti-flaviviral antibody, and even 6 times cheaper than a FRNT using the Venus reporter variant of YF-17D with an anti-GFP antibody (). Most importantly however, FluoRNT is also the more precise and robust assay. While the basic diagnostic requirement in accordance with WHO standards (to determine the absence or existence of protection) is as correctly determined by FluoRNT as by the traditional FRNT, FluoRNT offers significant advantages compared to the existing assays when quantitatively determining full patient titre, both in terms of (1) assay performance, and (2) assay practicality:

(1) The primary performance criteria for assays determining neutralising titre are technical robustness and analytical quality. Through its assay design FluoRNT has inherent advantages. A major driver of robustness is its ability to measure infection after only one round of infection, by cytometry-based detection of single infected cells. Many factors other than the content of neutralising antibodies can influence infection efficiency, such as the passage number of the cells to be infected, differences in virus preparation and unspecific serum effects. Measuring reporter expression of the first round of infection restricts the impact of unspecific effects which are otherwise amplified by multiple rounds of infection. FluoRNT’s cytometric detection resolves infection events even when these occur in neighbouring cells, giving its data better reliability. Due to these design features, FluoRNT data are highly reproducible within technical and biological replicates, and is well-suited for automatic dose-response curve-fitting without the requirement for manual checking or error-correction. This results in significantly more accurate and precise ED80 determinations of protective titre in single-timepoint or longitudinal settings.

By comparison, FRNT and PRNT suffer design-dependent drawbacks in respect to precision and accuracy. They rely on multiple rounds of infection and viral release from infected cells to form detectable foci or plaques; any technical error in the assay setup will be potentiated through these rounds of infection and can add up to false-negative and false-positive results, making biologically relevant changes indistinguishable from technical scatter. FRNT and PRNT assays are more dependent on a tight characterization of the viral stock titre and ability of the target cells to form a homogeneous monolayer. The setting of these assays for new virus stocks or cells lines require more rounds of optimization compared to the FluoRNT assay. Due to variable proximity of initially infected cells, plaques or foci can merge over time () making final measurements on plaques/foci with highly variable size and geometry difficult to quantify, both by automatic analysis tools and by eye (deviation from expected sizes or shapes, e.g. by overlap or merging). For the larger plaques in PRNT, manual determination and counting is particularly subjective and prone to experience-dependent bias (there are typically 20–100 plaques per well for the intermediate data points that influence ED80 titres the most). For FRNT the assay readout relies on image post-processing that at least in our experience requires manual checking and interpretation for foci that are out of size specification (very small or very large) or to determine how many counts to assign to fused foci. Even for experienced experimenters it is not trivial to perform these counting/checking steps, and they require significant time commitment and thorough blinding of personnel to give confidence in the analysis.

(2) Assays should be practicable—i.e. observable, fast, safe, and scalable. (a) An ideal assay can be evaluated at the different steps of the assay and sampled during the run-time to detect errors in the assay procedure early. While the appearance of Venus-positive cells can be followed repetitively during the FluoRNT assay via fluorescent microscopy or by sampling aliquots and analysing them in the flow cytometer, neither PRNT nor FRNT can be checked for sufficient number of foci and size before the final readout. This additional quality that brings practical value to FluoRNT is particularly valuable when working with new virus stocks, new cell batches, or samples of unknown quality. In our hands, monitoring during runs greatly simplified assay setup and sped up evaluation loops, since determining whether an assay runs "as expected" is immediately visible and can be done without interrupting or ending the experiment before the final readout. It can hence be achieved without costing significant assay manual handling or equipment time, and without reagent costs. By contrast, FRNT and PRNT require significantly more time and hands-on work to evaluate if an assay ran to completion satisfactorily or should be repeated. (b) Assays benefit from a fast turnaround and low volume needs and a low requirement of the hands-on time of skilled personnel. FluoRNT requires only one round of infection for detection of infection equivalent events (), which saves significant time during incubation: FluoRNT needs 24 h incubation time, compared to minimum times of 48 h for FRNT [12] and 96–120 h for PRNT [14-16]. Furthermore, FRNT and PRNT both require extensive hands-on time (e.g. layering with and removing viscous media, immunocytochemical staining, manual image checking or plaque counting).

FluoRNT’s stain-free readout minimises total assay time by reducing the post-incubation handling steps needed before readout. This saves time of trained personnel, reduces infection risk, and avoids technical errors introduced manually during assay steps. The lower incubation time also reduces occupancy of lab equipment (incubators, fridges) needed to run the assay. Note too, that while FluoRNT can be read out as quickly as 48 h after seeding with adherent cells, it should be feasible to perform the assay on suspension cells instead, which would avoid the cell seeding/adherence/monolayer formation and terminal cell harvesting steps to further reduce the FluoRNT total assay time from setup to readout close to the minimum for first round of reporter expression.

(c) Another major advantage of FluoRNT is the objective and quantitative assay read out by flow cytometry. Its quantification of Venus-positive cells by a cytometer is suitable for automation and it is more reliably quantifiable than FRNT’s immunostaining workup and image analysis, or the slow and subjective manual counting in the PRNT assay, both of which become time- and resource-limiting when considering assay scaleup. Particularly for population studies and for implementation in countries with strained resources, assays should be able to be run and processed in parallel, with only the least expensive assay elements (e.g. cell culture plates and media) scaling linearly to the number of assays run: which is uniquely true for FluoRNT. By contrast, for PRNT/FRNT, it is rather the most expensive elements (trained scientist time for manipulation and manual quantification, and expensive reagents such as antibodies) which scale with assay volume. FRNT’s immunostaining antibody requirements add to assay cost and limits scaleup: adding the primary anti-(flavi)viral protein antibody, secondary HRP-coupled antibody and chromogenic substrate treatment is costly, requires washing after each step, is time consuming and introduces sources for handling errors.

Several considerations will inform decision-making about whether FluoRNT is a practical assay to adopt. Firstly, it requires a two-channel flow cytometer, ideally with a plate autosampler which is less likely to be available in labs performing field testing in some areas. However, for high-throughput analysis as is becoming the norm in virology, this kind of machine is almost certainly accessible. Secondly, as for any neutralisation test, each new virus stock requires validation tests to establish and adapt the number of specific fluorescence-/focus-/plaque-forming units (FFU/PFU) for each virus preparation. Where possible, titres of large cohorts should be measured with only one virus preparation to allow better direct intra-cohort comparisons. Most assays in this study were performed with unpurified virus stocks. When we rechecked the samples with sucrose-gradient purified stocks in a second validation step, we found that FluoRNT is robust against variations in the inoculum. However, where purification via ultracentrifugation is possible, we can recommend using purified virus stocks for any neutralisation test to reduce potential variations in the virus preparation.

In order to calculate the ED80, reference controls are needed. Here we used no-serum controls as an easily feasible control, that does not require naïve pre-vaccination samples. However, it may overestimate the antibody-dependent neutralising effect of the virus, as naïve serum exhibits some neutralising properties, which is not accounted for by no-serum controls. Therefore, we recommend the individual 0 dpv control wherever pre-vaccination controls are available. This control also contains other virus-interfering substances in the serum as well as cross-reacting antibodies and is hence a more meaningful individual control.

Finally, to adapt the FluoRNT protocol to other viruses, the generation and validation of a reporter virus is required. This is likely to be a straightforward step however, as for many viruses such constructs are already described, or can be generated by reverse genetic techniques. Depending on the original virus and the reporter virus biosafety regulations may apply.

Recognising the short-comings of FRNT and PRNT assays, other advances in in neutralisation tests for YFV have been made previously: Whiteman et al. for example assessed the FRNT’s problem of overlapping foci. Instead of error prone manual counting they suggest a fluorescent readout using an imaging cytometer. However, this assay is still dependent on expensive antibody staining and the assay’s progress cannot be checked during the incubation [17]. Incidentally, the same is true for earlier work of Hammarlund et al. who already developed a flow cytometry-based read-out for non-plaque forming strains of YFV, which also relies on antibody staining [28]. Matsuda et al. designed single round infectious particles for different flaviviruses. This neutralisation test is based on a luciferase system that works by the same principle as FluoRNT, using a single round of infection and directly measuring reporter expression, and can therefore be expected to be more precise than conventional neutralisation tests. However, the assay was not compared to FRNT or PRNT and the incubation time of 3 days is no improvement to FRNT or PRNT [29]. The single round of infection principle is also used by Mercier-Delarue et al. Their neutralisation test uses WNV-YF pseudotype VLPs expressing a GFP reporter that can be read out using flow cytometry. However, the test was established in a 48-well format and requires double of FluoRNT’s incubation time [16].

In general, for flavivirus-serology neutralisation assays offer better specificity than assays based on the detection by ELISA of total IgM and IgG antibodies. However, cross-reactivity among flaviviruses has also been documented for neutralisation assays in areas where multiple flavivirus have recently circulated or are endemic [30]. In our YF-17D vaccination cohort assayed with FRNT and FluoRNT about half of the vaccinees have been previously vaccinated against tick-borne encephalitis (TBE) a flavivirus endemic in the south of Germany. When grouped according to TBE-vaccination status neutralisation titres of vaccinees on day 0 or day 28 measured with FRNT and FluoRNT correlated to the same extend as seen for the undivided cohort (data not shown). This argues that towards TBEV and presumably other flaviviruses FluoRNT has the same specificity as seen and described for YFV-FRNT and -PRNT-assays.

Taken together, FluoRNT has numerous advantages. It is a substantially faster, better-scalable, cheaper assay, which is more suited for high-throughput automated implementation than the current FRNT and PRNT. It minimises handling time with infectious material as well as the time needed by trained personnel, and its progress can be estimated non-invasively using a fluorescence microscope or flow cytometer. This enables FluoRNT to measure protective titres more efficiently in terms of sample collection and processing time, as well as cohort management.

Conclusions

FluoRNT is a robust, reproducible method with a range of analytical and practical advantages compared to current methods. We anticipate that it can significantly increase speed and accuracy of monitoring YFV exposure and provide a reliable, scalable, quick-turnaround test for protection status after vaccination with YF-17D. Clinical applications range from field testing of patients with unknown history, to sensitive longitudinal studies determining the effective dose or dilution of new and existing vaccines respectively. This method can be applied in basic research and of systems biology. Beyond research on YF-17D as a model virus, this assay can be adapted to other flaviviruses. We therefore anticipate that the FluoRNT assay could be valuably extended to other viruses, that have up to now been assayed with more traditional RNTs to determine vaccination success, as well as level and kinetics of protective titre.

Notes on the definition, use and inter-comparison of assay titres generated with different assay types.

(DOCX)

Click here for additional data file.

Overview of typical material costs per 96-well plate assay.

Costs are based on one full 96 well plate full of samples (e.g. serum of 0, 7, 14 and 28 dpv) assayed with FRNT against YF-17D or YF-Venus, or FluoRNT.

(PDF)

Click here for additional data file.

Live cell imaging video.

Video shows Venus+ cells 28 to 100 hours after infection. Vero cells were infected with 3,000 FFU YF-17D-Venus and were overlaid with MCS after 1 hour of incubation. Frames were acquired every 30 minutes. Fluorescent cells could already be seen after 28 hours. MCS reduces the viral spread and enables the forming of foci and plaques around the primary infected cells.

(MP4)

Click here for additional data file.

Stills from live cell imaging video.

Stills show Venus+ cells 28, 48, 72 and 96 hours after infection with 3,000 FFU YF-17D-Venus with an MCS overlay. The flow cytometry based FluoRNT is already meaningful as early as 24 hours after infection as it does not rely on foci or plaque forming but on infected cells on single-cell level. Note that foci and plaques in close proximity to each other tend to overlap the more time passes until readout which is therefore less reliable and reproducible as the FluoRNT readout. Image processing was performed to enhance contrast.

(EPS)

Click here for additional data file.

Maximum infection values in different assays.

NSC values normalised to run-average NSC values. “FluoRNT”, “FRNT Venus” and “FRNT 17D” display results from the main cohort of this study, whereas “FluoRNT pure 1” displays the same cohort with a purified virus. The purified virus was again tested for a second cohort (“FluoRNT pure 2” and “FRNT 17D pure”). Box and whiskers plot with 10–90 percentile.

(EPS)

Click here for additional data file.

Superior data quality of FluoRNT gives more robust titres regardless of the reference.

Titres obtained with FluoRNT and FRNT with NSC (A) or pre-vaccination samples 0 dpv (B) as a reference (n = 32). In both cases, FluoRNT and FRNT titres correlate significantly with each other. Spearman r. (C) Goodness of fit for dose-response curves for samples on 28 dpv referenced to pre-vaccination samples 0 dpv. FluoRNT gives a median R2 of 0.996 vs. 0.986 for FRNT (p = 0.0001; Mann Whitney test). (D) Titres referenced to 0 dpv divided by titres referenced to NSC give the titre ratio. FluoRNT is slightly more robust when changing the reference (p = 0.012, Mann Whitney test). Box and whiskers in panel C and D plot with 10–90 percentile.

(EPS)

Click here for additional data file.

FluoRNT shows more precise results in different assays.

Weighted precision of FluoRNT technical replicates for 55–90% virus neutralisation is reproducibly superior to the FRNT data. Assays were performed with purified (“pure”) and unpurified virus by different experimenters. All data are referenced to NSC. Box and whiskers plot with 10–90 percentile.

(EPS)

Click here for additional data file.

Gating strategy for the FluoRNT.

Example of flow cytometry gating strategy for Venus+ cells on live cells in FluoRNT analysis.

(EPS)

Click here for additional data file.

Raw data file.

(XLSX)

Click here for additional data file.

15 Oct 2021

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Reviewer #1: In the manuscript Scheck MK et al., the authors describe a neutralization assay (NT assay) for yellow fever virus (YFV) using a recombinant YFV expressing the fluorescent reporter protein Venus. The authors describe the performance of this NT assay in detail and compare the individual test steps to the protocols for plaque reduction neutralization (PRNT) and focus reduction neutralization assays (FRNT). To validate their fluorescent YFV virus-based assay (named FluoRNT), they used over 30 sera from human YFV vaccines and determined the neutralization titers resulting in 80% reduction in comparison to a focus reduction neutralization assay (FRNT). Detailed statistical analyses and dose response fitting analyses were performed to determine the accuracy and comparability of this assay. According to their data, their assay is comparable or even better with the advantage of being faster in comparison to FRNT assays. Experimental comparison to a plaque reduction neutralization assay (PRNT) was not performed, the procedures where only compared descriptive. To improve the manuscript, the authors should address the following points:

Major points:

1. Line 112 ff: for description of PRNT and FRNT – do the authors only refer to YFV assays or these assays in general? Probably only YFV – but still please specify.

2. The protocols the authors describe for PRNT and FRNT seem to be lab specific protocols (and might also depend on the cell line used) since in other publications for YFV PRNT or plaque assays are done in 3 days compared to 4-5 days as mentioned in the manuscript (line 118). Similar is probably also true for the FRNT, which is described to be done in 96 well plates which seems a rather small format, whereas the PRNT is done in 6-12 well format. Please comment and clarify better in the manuscript (since references for the described assays are missing).

3. L105: ‘The most commonly used NT assays are both reduction neutralization tests’. This sentence does not make much sense since every NT assay is a reduction neutralization assay.

I also would consider PRNT and FRNT rather similar assays (both with infectious virus) just with different read out modalities. Please adjust accordingly.

4. line 194: the authors mention that the yellow fluorescence can be seen after 3-4 days. This seems rather late. What is the titer of the YFV-Venus virus used for the studies? How does it grow in comparison to the wild-type virus?

5. line 334: the authors classify their FluoRNT assays as ‘single-round of infection assay’. This wording is confusing and should not be used since (1) virus release for YFV rather already starts at 8-12 hours post infection resulting in secondary infections until 24 h post infection. Thus ‘single-round of infection assay’ would not be correct. And (2) the term rather implies the use of real single-round infectious particles that really are only able to make one round of infection which were not used in this case.

6. line 337 ff: Description of antibody production after vaccination rather belongs to introduction than to results part.

7. Line 359ff: This whole section is only a descriptive comparison of the assays and not a real comparison with experiments.

This said – the abstract also implies that comparative analyses have been performed for both FRNT and PRNTs. However, PRNTs were not performed in comparison to FRNTs. Either the authors add them or they should make it clearer that only comparison to FRNTs was performed. The whole paragraph should be deleted in the results section and moved to the discussion, where lots of the descriptions (advantage of assay) are repeated anyway.

8. The authors only mention PRNTs and FRNTs as alternative neutralization assays. However, other NT assays have been described as well for flaviviruses like flavivirus pseudotype viruses with GFP as read out or flavivirus single round infectious particles with luciferase as read out. The authors should mention them and compare them to their assay as well in the discussion.

9. What about cross reactivity? Have the authors also tried sera against other viruses in comparison of both assays?

10. Line 591: the authors mention that the FluoRNT is robust against variations in the inoculum when using sucrose-gradient purified stocks. Does it mean that there are variations if using unpurified virus?

Minor points:

1. line 89: which ‘four continents’? Reference?

2. NT80 is more commonly used than ED80 for NT assays

3. List of costs might be relative as other antibodies/self-made antibodies might be used.

4. Is it really an anti-GFP antibody that is used for detection of Venus?

5. Line 594: adaptation to other viruses is mentioned. The authors should comment on the biosafety levels, since for some other reporter flaviviruses BSL3 laboratories might be necessary.

Reviewer #2: The authors present an FNRT method for rapid screening of neutralizing antibodies to the 17D virus. The results are clearly presented and convincing as an alternative method. FNRT has already been used and proposed by other teams for other viruses instead of PFU. Despite this last point the manuscript meets the publication criteria of PLosOne to my point (PLOS ONE does not evaluate manuscripts based on perceived significance or readership) and can therefore be accepted as is.

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5 Dec 2021

Reviewer #1: In the manuscript Scheck MK et al., the authors describe a neutralization assay (NT assay) for yellow fever virus (YFV) using a recombinant YFV expressing the fluorescent reporter protein Venus. The authors describe the performance of this NT assay in detail and compare the individual test steps to the protocols for plaque reduction neutralization (PRNT) and focus reduction neutralization assays (FRNT). To validate their fluorescent YFV virus-based assay (named FluoRNT), they used over 30 sera from human YFV vaccines and determined the neutralization titers resulting in 80% reduction in comparison to a focus reduction neutralization assay (FRNT). Detailed statistical analyses and dose response fitting analyses were performed to determine the accuracy and comparability of this assay. According to their data, their assay is comparable or even better with the advantage of being faster in comparison to FRNT assays. Experimental comparison to a plaque reduction neutralization assay (PRNT) was not performed, the procedures where only compared descriptive. To improve the manuscript, the authors should address the following points:

Major points:

1. Line 112 ff: for description of PRNT and FRNT – do the authors only refer to YFV assays or these assays in general? Probably only YFV – but still please specify.

We refer primarily to YFV and made that clearer in this section of the manuscript. But of course, the same principles and considerations apply similarly to other viruses as stated in lines 113 and 129 ff. PRNT and FRNT described here for YFV work the same way for NTs of any other viruses that form plaques (enabling PRNT) or for which antibodies exist to detect viral infection (enabling FRNT).

2. The protocols the authors describe for PRNT and FRNT seem to be lab specific protocols (and might also depend on the cell line used) since in other publications for YFV PRNT or plaque assays are done in 3 days compared to 4-5 days as mentioned in the manuscript (line 118). Similar is probably also true for the FRNT, which is described to be done in 96 well plates which seems a rather small format, whereas the PRNT is done in 6-12 well format. Please comment and clarify better in the manuscript (since references for the described assays are missing).

Yes, the assay protocols are described as we use them in our lab. Published YFV FRNT/PRNT protocols vary slightly from lab to lab (e.g. both shorter and longer timelines depending on the cell line and the virus stock used); however, the time lines are representative of typical procedures and we have added a more general commentary on this (lines 98ff). Thank you for pointing out the missing references; we have added references for these timelines (lines 120, 131 and 135).

3. L105: ‘The most commonly used NT assays are both reduction neutralization tests’. This sentence does not make much sense since every NT assay is a reduction neutralization assay.

I also would consider PRNT and FRNT rather similar assays (both with infectious virus) just with different read out modalities. Please adjust accordingly.

Yes, thank you for catching it, we have fixed it (line 92 f.); and yes, PRNT and FRNT are indeed similar from the setup, as we say (line 129). We describe them sequentially since (a) they are the most widely used neutralisation tests, so in this way we address the target readership no matter which assay they use; and (b) showing the advances in our paper relies on understanding the influence of readout modality on the precision, accuracy, and practicality of assays: so, a more detailed description of the two assay readouts to us seems adequate.

4. line 194: the authors mention that the yellow fluorescence can be seen after 3-4 days. This seems rather late. What is the titer of the YFV-Venus virus used for the studies? How does it grow in comparison to the wild-type virus?

Thank you for the opportunity to clarify this. After infection with YF-Venus, yellow fluorescence is seen within a day, as we state in e.g. line 576 ff. and illustrated in Supporting Video 1. This should match the reader’s and reviewers’ expectations. Line 194 (now line 191) mentioning “3-4 days” instead referred to electroporating cells with YF-Venus-RNA for YF-Venus virus production, which cannot be compared to how the vaccine strain grows. We edited line 191 to make this clear to the reader.

Please note that each assay type uses different measures of infectivity (e.g. FRNT => FFU, PRNT => PFU), as they operate on different time scales and have different readouts into which the functional infective units have to translate. The relevant descriptor for the virus stock in FluoRNT is the percentage of Venus+ cells obtained 24 hours post inoculation. In our protocol we aim for approximately 25% Venus+ cells (line 269); to achieve that we typically had to use approximately 5000 PFU or 2875 FFU/well/per 25.000 Vero cells of our YF-17D-Venus Stocks. That makes the amount of virus used in the FluoRNT 22-fold more concentrated than in the FRNT (line 270 ff.). As described in the section on virus stock production (lines 195 ff.) the YF-17D-Venus stock used for this study propagates somewhat slower than the YF-17D stock derived from the Stamaril vaccine that was used for this study.

5. line 334: the authors classify their FluoRNT assays as ‘single-round of infection assay’. This wording is confusing and should not be used since (1) virus release for YFV rather already starts at 8-12 hours post infection resulting in secondary infections until 24 h post infection. Thus ‘single-round of infection assay’ would not be correct. And (2) the term rather implies the use of real single-round infectious particles that really are only able to make one round of infection which were not used in this case.

Thank you for the opportunity to clarify what for our paper is a very important point. We have modified the manuscript to define “single round of infection” more precisely (lines 147, 336, 533 ff. and 576 ff.). FluoRNT readout is reachable when the first round of cells has become infected and express sufficient Venus reporter for measurement (see Fig S1 and Video S1). Regarding part (1): Video S1 particularly highlights the time-separation between the waves of reporter expression in the first and second rounds of infection. Given our timeline of measurement at 24 h and the somewhat slower kinetics of the YF-17D-Venus stock compared to the YF-17D stock, we are confident that we are indeed only detecting first-round infected cells. Regarding part (2): yes, technically YF-17D-Venus can progress to multiple rounds of infection, but since conceptually the FluoRNT assay neither requires subsequent rounds of infection, nor runs long enough for them to happen, we agree that “first” is a better description than “single”, implying there could be second, third, fourth rounds of infection, but they are not needed for the assay readout.

6. line 337 ff: Description of antibody production after vaccination rather belongs to introduction than to results part.

Thank you for the suggestion. However, we do feel it is important to mention this in the results part. Understanding the antibody production time course is essential to interpret results from the different post vaccination days. We would therefore rather leave it in the results section than making it a part of the general introduction to NTs and motivation for the FluoRNT assay (now line 339 ff.).

7. Line 359ff: This whole section is only a descriptive comparison of the assays and not a real comparison with experiments. This said – the abstract also implies that comparative analyses have been performed for both FRNT and PRNTs. However, PRNTs were not performed in comparison to FRNTs. Either the authors add them or they should make it clearer that only comparison to FRNTs was performed. The whole paragraph should be deleted in the results section and moved to the discussion, where lots of the descriptions (advantage of assay) are repeated anyway.

Thank you for the constructive suggestions. (1) We have streamlined this section (now line 366 ff.) and incorporated most of it in the discussion part. (2) We have made it clear now, that our direct experimental comparisons were only done between FRNT and FluoRNT, and comparisons with PRNT are purely descriptive (lines 370 ff.). That being said, as the reviewer also stated (item 3), FRNT and PRNT are in many ways such similar assays that we feel this experimental comparison is sufficient to highlight the benefits of FluoRNT compared to both assays: and all the more so, since FRNT is in nearly all practical respects the “better” assay than PRNT (faster, better data quality, less by-eye counting, etc.), so we feel our comparisons to FRNT are the more stringent ones that could be chosen.

8. The authors only mention PRNTs and FRNTs as alternative neutralization assays. However, other NT assays have been described as well for flaviviruses like flavivirus pseudotype viruses with GFP as read out or flavivirus single round infectious particles with luciferase as read out. The authors should mention them and compare them to their assay as well in the discussion.

Correct, we agree and implemented comparisons with other neutralisation assays as suggested (e.g. cited other advances on the PRNT- and FRNT-type assays, including the Luciferase reporter assay published by Matsuda et al. and the pseudotype reporter assay by Mercier-Delarue et al.; lines 631 ff.). We feel that the current state of the manuscript is now fair, by referencing these additional methods appropriately, yet keeping the reader’s focus and reserving the most discussion space for the most widely used neutralisation tests (with which we have previously worked).

9. What about cross reactivity? Have the authors also tried sera against other viruses in comparison of both assays?

A very good point, thank you. Our patient cohort is naïve for all flaviviruses except for TBEV. TBEV is endemic in southern Germany and about half of the vaccinees had been previously vaccinated against TBE. Anti-YFV titres of previously TBEV vaccinated participants do not differ depending on whether they were measured by FRNT or FluoRNT, suggesting, that FluoRNT is as robust to cross-reactivity as FRNT is. Data how previous vaccination influences the immune response to the YF-17D vaccine forms part of a larger study on viral cross-reactivity that will be published elsewhere and is therefore not given in this manuscript. However, in lines 647 ff. we have now included a statement to cross-reactivity and describe this equivalent robustness towards TBEV of FRNT and FluoRNT.

10. Line 591: the authors mention that the FluoRNT is robust against variations in the inoculum when using sucrose-gradient purified stocks. Does it mean that there are variations if using unpurified virus?

An important question. From day to day lab work we know that there are stock-dependent variations for both types of stocks purified as well as unpurified However, conceptually, it is of course easier to claim and show robustness between purified stocks where most contaminants are eliminated, and possibilities for variations are restricted. So, in general, we expect that any assay can benefit from using purified virus if possible. We have made it clearer in the manuscript now, that this is not a FluoRNT-specific recommendation (line 616 ff.). When stock-dependent data for FluoRNT are plotted on the same scale as the same stock-dependent data acquired using FRNT, our conclusions regarding the assay’s precision and data quality stay true regardless whether unpurified or purified virus stocks were used (as shown in Supporting Figures S2 and S4). Therefore, we feel confident to claim that the FluoRNT is essentially robust to the use of unpurified virus, at least in our hands.

Minor points:

1. line 89: which ‘four continents’? Reference?

Yes, this should have been just Africa & South America, changed and referenced (now line 75).

2. NT80 is more commonly used than ED80 for NT assays

We have clarified additionally in line 91, that we write ED for what is also called EC or NT.

3. List of costs might be relative as other antibodies/self-made antibodies might be used.

True; though we find it more helpful to give a typical estimate rather than not to give one, and we consider our estimation quite reasonable; given that cost is just one of the factors we discuss that need to be weighed when choosing an assay, we would like to keep it as it is.

4. Is it really an anti-GFP antibody that is used for detection of Venus?

Yes, since Venus is a variant of GFP, the 4-9 mutations do not affect the antibody binding site.

5. Line 594: adaptation to other viruses is mentioned. The authors should comment on the biosafety levels, since for some other reporter flaviviruses BSL3 laboratories might be necessary.

Yes, thank you, we noted this in the manuscript line 629.

In conclusion, we thank Reviewer 1 for her/his detailed reading and suggestions which we have addressed with the described manuscript changes.

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Reviewer #2: The authors present an FNRT method for rapid screening of neutralizing antibodies to the 17D virus. The results are clearly presented and convincing as an alternative method. FNRT has already been used and proposed by other teams for other viruses instead of PFU. Despite this last point the manuscript meets the publication criteria of PLosOne to my point (PLOS ONE does not evaluate manuscripts based on perceived significance or readership) and can therefore be accepted as is.

We thank the reviewer for this positive evaluation.

Submitted filename: Scheck (Thorn-Seshold)_FluoRNT_Response to Reviewers.docx

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17 Dec 2021

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

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24 Jan 2022

PONE-D-21-23143R1

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

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