Quantitative measurement of varicella-zoster virus infection by semiautomated flow cytometry

Abstract

Varicella-zoster virus (VZV; human herpesvirus 3) is the etiological cause of chickenpox and, upon reactivation from latency, zoster. Currently, vaccines are available to prevent both diseases effectively. A critical requirement for the manufacturing of safe and potent vaccines is the measurement of the biological activity to ensure proper dosing and efficacy, while minimizing potentially harmful secondary effects induced by immunization. In the case of live virus-containing vaccines, such as VZV-containing vaccines, biological activity is determined using an infectivity assay in a susceptible cellular host in vitro. Infectivity measurements generally rely on the enumeration of plaques by visual inspection of an infected cell monolayer. These plaque assays are generally very tedious and labor intensive and have modest throughput and high associated variability. In this study, we have developed a flow cytometry assay to measure the infectivity of the attenuated vaccine strain (vOka/Merck) of VZV in MRC-5 cells with improved throughput. The assay is performed in 96-well tissue culture microtiter plates and is based on the detection and quantification of infected cells expressing VZV glycoproteins on their surfaces. Multiple assay parameters have been investigated, including specificity, limit of detection, limit of quantification, range of linear response, signal-to-noise ratio, and precision. This novel assay appears to be in good concordance with the classical plaque assay results and therefore provides a viable, higher-throughput alternative to the plaque assay.

FIG. 1.

Measurement of VZV infection using flow cytometry. (a) FSC (x axis) and red fluorescence intensity channel (RED; y axis) correlation dot plot showing selective gating (gray events) on 7-AAD-negative events (live cells and nonnucleated debris). (b) RPE-conjugated gH staining intensity (yellow fluorescence intensity channel; x axis) and FSC (y axis) correlation dot plot. Selective gating on cellular-sized events is indicated in gray, while subcellular debris (black) are excluded from the analysis. This plot is logically gated on the plot in panel a to include only live events. (c) The RPE-conjugated gH staining intensity histogram, logically gated on plots a and b, displays a characteristic biphasic frequency distribution corresponding to uninfected cells (gray) and infected, high-fluorescence, gH-positive cells (black). The marker bars 1 and 2 are used to calculate the fraction of infected cells in each sample or well.

FIG. 2.

Progression of VZV infection in MRC-5 cells measured by flow cytometry. (a) Characteristic RPE-conjugated gH-staining intensity (yellow fluorescence intensity channel; x axis) and FSC (y axis) correlation contour plots illustrating the course of an infection with a CA VZV inoculum in MRC-5 cells. (b) Fraction of gH-positive cells (open circles) as a function of hours postinfection compared to the appearance of signs of CPE (closed circles). Mock-infected (closed inverted triangles) and isotype-staining controls (open triangles) are included to demonstrate the specificity of the staining. Error bars represent 1 standard deviation for cytometric measurements (n = 3 experiments), while the CPE curve is representative of a single experiment. (c) Infection kinetics of CA (closed circles) and CF (closed squares) samples in MRC-5 cells, using the flow cytometry assay. Control infections with heat-inactivated (CF/Δ; open squares) and UV-inactivated (CF/UV; open triangles) CF samples demonstrating that the signal observed is dependent on the presence of live attenuated virus.

FIG. 3.

Detachment of MRC-5 cells and preservation of surface VZV glycoproteins. (a) Recovery and viability of MRC-5 cells from tissue culture-treated 96-well plates, using a panel of different detachment reagents. Measurements for cell concentration (gray bars) and cell viability (closed circles) are performed using the Guava VIAcount Flex kit. Cell recovery data are plotted as averages over 24 wells with associated 95% confidence intervals. Guava CDR (cell dispersal reagent) is an accessory reagent used to disaggregate cells for viability measurements. (b) Measurement of the amount of cells remaining on the substrate after cell detachment using crystal violet staining. Yield of detachment is indicated as a percentage in each bar (control is set at 0.0%). Data are plotted as average optical densities at 570 nm (OD 570 nm) with associated 95% confidence intervals (over 16 wells). (c) Stability of the measured fraction of gH-positive cells as a function of exposure time to trypsin-EDTA (closed circles) or Accumax (open circles) cell detachment reagents. Data are plotted as averages of four wells with associated 95% confidence intervals. (d) Overlay of fluorescence intensity histograms for RPE-conjugated VZV gH after 6 min treatment with trypsin-EDTA (dark gray) and Accumax (light gray). The black histogram represents staining for mock-infected cells.

FIG. 4.

Cell readherence and plate effect. (a) Observed acquisition rates normalized to the first well acquired (well A1) in a 96-well format assay stained and acquired in a U-bottom tissue culture plate at low mixing setting (closed circles) and a U-bottom non-tissue culture plate and acquired in a hydrogel low-cluster flat-bottom plate at low mixing setting (open squares) or at high mixing setting (open diamonds). Data are presented as an average over an entire dilution curve (12 wells) with associated 95% confidence intervals. (b) Mean fluorescence intensity (M.F.I.; closed circles) of gH staining and fraction of positive cells (open circles) for a 96-well plate infected with a single dilution of the same VZV test article.

FIG. 5.

Assay dynamic range, background, and discrimination. (a) Assay dynamic response over 17 twofold dilutions of a VZV test article, spanning 5 orders of magnitude of infected-cell-to-target-cell ratio (PFU/cell). Signal is measured at 48 h postinfection (closed circles), and nonspecific background is estimated at 2 h postinfection (open circles). Data are presented as averages over 12 dilution curves with associated 95% confidence intervals. (b) Discrimination illustrated by infection with the same test article at various predilution factors (closed circles, 1×; open circles, 1.25×; closed triangles, 1.5×; open triangles, 2×; and closed squares, 4×) to demonstrate recovery of small differences in infectivity. Data are presented as averages of three representative curves at each predilution level with associated 95% confidence interval. The data are fitted using a four-parameter logistical function.

FIG. 6.

Assay concordance plot. Bivariate scatter correlation plot between the infectivity of VZV-containing test articles measured using the flow-based infectivity assay (x axis) and the plaque assay (y axis). The solid line is the ideal linear curve with slope of 1. The dashed line is the observed concordance curve (LnPlaque = 0.68 × LnFlow + 3.62).