An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

Abstract

Background: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable.

Methods: Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described.

Results: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed.

Conclusions: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

Figure 1

A)Schematic diagram of constructed recombinant RSV (rRSV-X)expressing EGFP gene in the first(E1-rRSV-X)and the seventh(E7-rRSV-X)position in the virus genome and the positions of the restriction enzymes in the RSV-X genome used for insertion of EGFP. Blue: RSV gene; green: EGFP gene. B) Replication kinetics of recombinant RSV viruses E1-rRSV-X, E7-rRSV-X and the parental rRSV-X in Vero and Hep-2 cells. Cells were infected at a multiplicity of infection (MOI) of 0.1 with the rRSVs. At the indicated time points, cells and supernatants were collected and virus titers were determined. Closed lines represent growth on Vero cells; dashed lines represent growth on Hep-2 cells.

Figure 2

Fluorescent images of Vero cells infected with E7-rRSV-X over time. Initial input is 115 PFU per well. The images show plaques produced by RSV expressing EGFP. At the indicated time points, plaques were detected after 27 hrs, 48 hrs and 72 hrs. in a fluorescence Elispot reader, manual counted (upper panel) and counted using the AID EliSpot Software 'algorithm C' with emphasis settings were set on ”big” (middle panel) or “tiny” (lower panel). Each yellow ‘x’ represents one plaque counted by the imager software.

Figure 3

Correlation of automated and conventional plaque counting. Scatter plots of counted plaques by the automatic counting of fluorescent plaques (X-axis) and manual counting of the same plaque immunostained (Y-axis). Plaques were counted 48 hrs after infection using the ‘big’ settings.

Figure 4

Sample graph of neutralization assay results with human reference sera. Virus neutralizing graphs by polyclonal anti-human reference sera (high, medium and low control) [15] obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH in the absence (A) or presence (B) of 10% guinea pig serum. As negative control, control serum from naive cotton rats was included because of the lack of availability of negative human reference serum. Plaque reduction titers were calculated by regression analysis of the inverse dilution of serum that provided a 60% plaque reduction titer compared to control wells in the absence of serum. Results are means ± SD (triplicate). Settings of fluorescence reader are as described for Figure 3.

Figure 5

Fluorescent virus neutralizing antibody plaque titers.A) Scatter plots of virus neutralization (VN) titers obtained by automatically counted plaques of fluorescence E7-rRSV versus manually coloured and counted plaque reduction of the parental rRSV. B) Serum antibody neutralization titers of cotton rat, mouse and human serum samples obtained with the fluorescent virus neutralizing antibody assay. Serum neutralizing titers from different experiments are depicted. Animals were immunized with RSV vaccine candidates [16]. Pre-immune serum was obtained at day 0 (black bars) and were at the lower limit of detection. Mice were immunized at day 0 and day 28 with 10^6 RSV- ΔG and serum was obtained at day 28 (light gray) and at day 51 (gray) (n=6 per group). Cotton rats were immunized at day 0, day 14 and day 28 with 10^5 RSV-ΔG and serum was obtained at day 28 (light gray) and at day 51 (gray) (n=5 per group). As reference, serum from animals immunized with formalin-inactivated RSV (white) are included (n=5 per group). The human reference human sera: high (green), medium (blue) and low (orange) were included as standards [15]. Error bars indicate standard deviation.