Abstract
ABSTRACTNeutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log2; linear over a range of 4.27 to 9.65 log2 50% inhibitory concentration (IC50); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.
Highlights
Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants
To determine the optimal concentration of an RSV strain A2 recombinant virus expressing green fluorescent protein (RSVA-GFP) to add to the assay, the RSVA-GFP stock virus was diluted to cover a range of concentrations that would be quantifiable in a 96-well plate by use of an imaging system
Virus input concentrations ranging from 100 to 1,400 fluorescent focus-forming units (FFU)/well were used in the neutralization assay to test human serum that had been pooled into high, medium, and low-neutralizingtiter samples
Summary
Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. The plaque reduction neutralization test (PRNT) has been the gold standard for measurement of RSV-neutralizing activity, several more efficient RSV microneutralization (MN) assays have been developed [5,6,7,8,9] While some of these methods have short incubation times, most require one of the following: a virus inoculum medium exchange, postinfection processing, or multiple days of incubation to perform an assay. The qualification of the assay and control trending data demonstrated that this RSVA FFA-MN assay has the sensitivity, linearity, precision, reproducibility, and robustness that are required to detect RSV-specific neutralizing antibodies elicited following natural infection or vaccination
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