Technical note
A flow cytometry-based assay to assess RSV-specific neutralizing antibody is reproducible, efficient and accurate

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Abstract

Respiratory syncytial virus (RSV) is an important cause of respiratory infection in people of all ages, and is the leading cause of hospitalization in infants. Although commercially available monoclonal antibody is available for passive prophylaxis of neonates at risk of severe disease, there is no available vaccine to prevent RSV. Measurement of neutralizing activity will be a key endpoint for vaccine evaluation. Assessment of neutralizing antibody against RSV has been limited to traditional plaque reduction, which is time-consuming and inherently operator dependent and highly variable. Here, we describe a flow cytometry-based RSV-specific neutralization assay which is more rapid than traditional methods, highly sensitive and highly reproducible.

Introduction

Respiratory syncytial virus (RSV) is a pneumovirus in the family Paramyxoviridae, which also includes metapneumovirus. RSV is a major cause of infant respiratory infection, especially severe pneumonia and bronchiolitis (Glezen et al., 1981, Collins and Graham, 2008). There are three envelope proteins F, G, and SH. Both F and G are glycosylated and represent the targets of neutralizing antibodies. F-specific neutralizing antibody is known to be protective, and there is a licensed monoclonal antibody, Synagis® (Palivizumab) that is used to passively protect high risk infants from severe disease (Johnson et al., 1997). Assessment of neutralizing activity in preclinical or clinical samples has been primarily by traditional plaque reduction neutralization (PRNT) or microneutralization (Anderson et al., 1985). PRNT suffers from limited sensitivity and nonspecificity, and is prone to technician error, is tedious, labor-intensive, and is not as reproducible as newer reporter pseudovirus methods developed for other viral diseases (Mascola et al., 2002, Pierson et al., 2006, Martin et al., 2008). Additionally the PRNT assay is time-consuming and not easily adapted to high throughput technology. Here we describe an efficient, highly reproducible flow cytometry-based assay to detect RSV neutralization with high sensitivity and specificity.

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Material and methods

Virus: Viral stocks of RSV expressing Green Fluorescent Protein (GFP) and based on the A2 strain of RSV, were prepared and maintained as previously described (Graham et al., 1988). GFP-RSV was constructed and provided by Mark Peeples and Peter Collins, as previously reported (Hallak et al., 2000). The titer of the virus stocks used for the experiments was 2.5 × 107 pfu/ml.

Cell line: HEp-2 cells were maintained in Eagle's minimal essential medium containing 10% fetal bovine serum (10% EMEM) and

Results

The assay was optimized for consistency and sensitivity. Parameters assessed included cell culture, viral titer and infection duration. Sub confluent HEp-2 cells with a passage number between 1 and 20 were determined to be the optimal cell type. The optimized condition included a cell count of 5 × 104 per well in a 96 well culture plate, which was freshly seeded, with a control well viral infection rate of 6–12%. As the virus replication cycle and cell growth cycle affect virus infection rates,

Discussion

Flow cytometry-based measurement of neutralizing activity for RSV is reliable and reproducible when the analysis is preformed under the optimized conditions defined here. Data derived from assessment of monoclonal antibody (palivizumab), human plasma, mouse serum and rabbit serum indicate that when performed in optimal conditions as described, the RSV flow cytometry-based neutralization assay is reproducible, quantitative, and follows the “percentage law”.

The flow cytometry-based neutralization

Acknowledgements

The authors thank Dr. John Mascola for critical editing and advice and Dr. Theodore Pierson for expert opinion and education regarding neutralization and the importance of considering the Law of Mass Action in assay development.

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Published by Elsevier B.V.