A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein

Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines.