|Title||Development of a novel serological assay for the detection of rabies virus neutralising antibodies using lentiviral pseudotypes|
|Authors||Armson, B.L., Wright, E., McElhinney, L.M., Banyard, A.C., Goddard, T., Healy, D.M., Voller, K., Weiss, R.A. and Fooks, A.R.|
Rabies virus (RABV) causes an acute encephalitis that is almost invariably fatal. Whilst effective vaccines and post exposure prophylaxis are available it is estimated that over 55,000 human fatalities occur worldwide each year. This figure is, however, believed to be a gross underestimation of the actual number of human deaths, as a disproportionate number of fatalities occur across the developing world where reporting systems are mainly absent. The detection of virus neutralising antibodies (VNA) is key to the evaluation of vaccination status following pre-immunisation. Current tests for RABV VNAs require expensive reagents and equipment, along with biosafety containment level 3 facilities, something not easily achieved in rabies endemic areas.
The use of lentiviral pseudotypes as a vector for gene therapy is well documented. These have proven to be highly sensitive yet flexible platforms which can be adapted to allow the evaluation of vaccines and antiviral drugs against highly pathogenic viruses without the need for high level containment facilities or great expense. We have adapted this technology to allow the determination of serum neutralising antibody titres against highly pathogenic rabies and related lyssaviruses. The pseudotype assay only requires a small amount of serum in comparison to the standard fluorescent virus neutralisation assay (FAVN), and the use of different reporter genes, such as green fluorescent protein, luciferase, or β-galactosidase, makes it possible for the assay to be undertaken at low cost in laboratories throughout the world.
G-protein sequences from viral isolates representing each lyssavirus genotype and the newly classified Eurasian strains were cloned and co-expressed with lentiviral gag-pol and different reporter genes. The pseudotypes infected a number of target cell lines and produced titres almost equivalent to VSV-G protein pseudotypes. To date, over 500 sera have been evaluated concurrently using genotype 1, CVS-11 live virus (FAVN) and pseudotype neutralisation assays. Comparison of antibody titres reveals a 96.4% sensitivity and 100% specificity (r=0.83, p<0.001).
Neutralisation assays using pseudotypes with glycoproteins from other lyssavirus genotypes suggest that they provide a greater sensitivity compared to the current virus neutralization tests and will therefore allow a more accurate determination of serological response to these highly pathogenic infections. Importantly for the use of this assay in countries where the cold chain cannot be maintained, CVS-11 pseudotypes were highly stable during freeze-thaw cycles and storage at room temperature. These results suggest that the proposed pseudotype assay is a practical, effective and robust solution for rapid lyssavirus serosurveillance wherever it is needed.
|Conference||4th Annual Meeting EPIZONE|