Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein

Abstract

A rapid and sensitive neutralization assay is required to evaluate alternative smallpox vaccines. Here we describe the development and use of a 96-well plate, semi-automated, flow cytometric assay that uses a recombinant vaccinia virus expressing enhanced green fluorescent protein and which would be applicable to other viruses.

FIG. 1.

Detection of GFP-expressing cells by flow cytometry. HeLa cells were mock infected or infected with dilutions of purified WR-GFP in a 96-well plate. After 17 h at 37°C, the cells were analyzed by flow cytometry. Live cells were gated for forward and side scatter and subsequently for GFP expression. The number of GFP-expressing cells (upper right corner of each box) was determined by using a bivariate plot of fluorescence versus side scatter, with the gate set by using uninfected cells. Virus titers, used to indicate the multiplicity of infection (MOI) at the bottom of each panel, were determined by plaque assay.

FIG. 2.

Dose-response curve. HeLa cells were infected with WR-GFP in triplicate as described for Fig. 1. Bars indicate standard deviations.

FIG. 3.

Neutralization of vaccinia virus by hyperimmune rabbit serum. Purified WR-GFP was incubated with dilutions of hyperimmune rabbit serum in a 96-well plate. After 1 h at 37°C, HeLa cells were added and the incubation was continued for 17 h. The numbers of GFP-expressing cells (upper right corner of each box) were enumerated as for Fig. 1; the serum dilution is shown in the lower right corner of each box.

FIG. 4.

Comparison of the flow cytometric and plaque reduction neutralization assays. Purified WR-GFP was incubated with dilutions of hyperimmune rabbit serum (A and B) or human VIG (C and D) in duplicate as for Fig. 3 for the flow cytometric assay (A and C) or as described in the text for the plaque reduction assay (B and D). Experiments were done in duplicate and bars indicate standard deviations. The percentage of neutralized virus was determined by comparing the numbers of GFP-expressing cells or plaques in the presence and absence of serum.

FIG. 5.

Stability of cells expressing GFP. Flow cytometric neutralization assays were carried using hyperimmune rabbit serum 8191 (A) or human VIG (B). After the incubation period, the cells were fixed with paraformaldehyde and stored at 4°C for up to 45 days before flow cytometric analysis. The number of days of storage of fixed cells is indicated in each inset.