We describe the development of a simple and rapid micro neutralization assay for RSV based on a recombinant RSV construct expressing the EGFP protein. To this aim, we generated two recombinant RSV viruses: one harbouring EFGP at the 3’ proximal position (E1-rRSV-X) and one harbouring EGFP between the SH and G gene (E7-rRSV-X). Both recombinant RSV clones were constructed with an EGPF gene flanked by RSV gene start and stop signals, ensuring the expression of EGFP in virus infected cells. E7-rRSV-X was selected to further develop the neutralization assay since growth properties of this virus on Vero cells were indistinguishable from the parental rRSV-X virus, while E1-rRSV-X was shown to be attenuated. This suggests that positioning the EGFP gene at the 3’ end of the viral genome, i.e. before the first gene, most likely negatively affects transcription and, by extension, virus growth, a phenomenon that has been described previously
[17–19]. Using the current assay, virus neutralization titers can be determined within 2 days of incubation without requiring fixation, staining and manual counting of plaques. The results showed a strong correlation between automated EFGP and manual counting of immunostained plaques for the detection of RSV neutralizing antibody titers.
Serum neutralizing antibody titers to respiratory syncytial virus (RSV) are higher when assayed in the presence of guinea pig complement
[14, 15]. In the current assay, the addition of complement also resulted in increased virus neutralizing titers compared to incubation without complement. However, the observation that the amount of virus needed to obtain similar plaque counts in the presence of complement suggest a more complex, possibly confounding influence of complement on the outcome of the virus neutralization assay. While apparently improving the sensitivity of the assay, the opposite may be happening simultaneously through a direct inhibitory effect of complement, as suggested by the need for higher MOI levels in the test. By directly eliminating a portion of the input virus, thereby decreasing the number of targets for VN antibodies, complement may actually decrease the sensitivity of the assay. Therefore we chose to omit the use of complement in our neutralization assay. These findings are in agreement with Yoder et al.
, who reported that addition of complement did not enhance the ability to detect increased antibody titers after natural infection in humans and have not found evidence to support its use in the assay. As these authors, we believe that the addition of complement introduces an additional, possibly confounding and variable in the performance of the test and we would advocate that RSV neutralization assays be carried out without complement, in order to provide a more standardized and robust assay.
The main advantage of the currently developed method is that the most time consuming, labour intensive parts of the RSV neutralization assay (i.e. fixation, staining and plaque counting) are replaced by computerized image scanning and analysis. These features result in a faster and more efficient assay and ensure less subjective results through the exclusion of operator bias. Another advantage of the automated counting system is that it is capable of detecting and differentiating plaques of different morphology. This feature could be used to detect serum neutralizing antibodies against other plaque forming viruses, although this requires recombinant viruses harbouring EGFP. In addition, the fluorescence ELISPOT reader can also be used to detect virus infected cells by staining with fluorescently labelled antibodies as an alternative for the classical plaque forming unit (PFU) assay. In fact, we have developed such a fluorescent plaque forming assay for quantification of RSV that correlates well with the classical PFU assay (data not shown).
Using the fluorescence ELISPOT reader, a plate can be processed within 5 min, including reading, counting and analysis. Robotic automation of plate loading could be introduced for high throughput purposes. These assets would render the fluorescent neutralization assay a valuable tool for analysis of humoral immune responses in vaccine studies and for serological studies in naturally infected hosts. Taken together, our virus neutralization assay is a major improvement compared to other described micro neutralization assays
[9, 10] which still require either manual plaque counting or immunostaining. Recently, a flow cytometry-based assay to assess RSV-specific neutralizing has been described
. As our method, this method is based on the expression of EGPF by the viral genome and can be performed within two days. However this assay requires manual treatments prior to analysis while our fluorescence-based plaque reduction assay can be measured directly.