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. 2004 Mar;11(2):406-10.
doi: 10.1128/cdli.11.2.406-410.2004.

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus

Antonio Cosma  1 , Silja Bühler, Rashmi Nagaraj, Caroline Staib, Anna-Lena Hammarin, Britta Wahren, Frank D Goebel, Volker Erfle, Gerd Sutter
Affiliations

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus

Antonio Cosma et al. Clin Diagn Lab Immunol. 2004 Mar.

Abstract

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

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Figures

FIG. 1.
FIG. 1.
Set-up of the MVA-gfp neutralization assay. (a) Representative side scatter-forward scatter plot with a gate (R1) used to discriminate intact cells from cellular debris. (b and c) Representative histogram plots showing, respectively, a neutralizing sample and an untreated control sample. The percentages of Gfp-expressing cells are shown on each histogram plot. (d) B-LCL were infected with different numbers of PFU of MVA-gfp/cell, and the Gfp expression was measured after 14 h. (e and f) Different amounts of MVA-gfp were used in the neutralization assay (⧫ 0.5 PFU/cell; ▪, 1 PFU/cell; ◊ 2 PFU/cell; □, 6 PFU/cell), and the percentages of neutralization are shown for six serial dilutions. The same experiment was carried out with human plasma collected before (f) and 2 weeks after (e) the vaccination with MVA-nef.
FIG. 2.
FIG. 2.
Neutralizing antibodies in mouse and rabbit specimens. (a) Sera from two MVA-vaccinated mice (⧫, ▪) and two naive mice (◊, □) were tested in ELISA for the presence of MVA-binding antibodies. (b) The same mouse sera were tested using the MVA-gfp neutralization assay. (c) An anti-vaccinia virus rabbit serum used for plaque-based titration assay was tested using the MVA-gfp neutralization assay (▪), together with an unrelated rabbit control serum (□). All three panels show the results of one representative experiment out of two.
FIG. 3.
FIG. 3.
A preparation of VIGs was tested in three parallel assays. (a) Neutralization activity of the VIG measured using the standard vaccinia virus Elstree PRNT. The average from six independent experiments is shown. (b) Neutralization activity of the VIG measured using an MVA PRNT. The average from two experiments is shown. (c) Neutralization activity of the VIG measured using the MVA-gfp neutralization assay. The average from three independent experiments is shown.
FIG. 4.
FIG. 4.
The neutralizing activity detected with the MVA-gfp assay is antibody mediated. A serum, from an individual vaccinated three times with MVA-nef was IgG depleted (◊) or mock depleted (▪) and then tested in the MVA-gfp neutralization assay. The results of one representative experiment out of three are shown.
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