WHO/BS/2016.2302 WHO collaborative study to assess the suitability of a WHO International Reference Panel for Ebola virus VP40 antigen

Overview

Although the Ebola virus (EBOV) outbreak in West Africa has ended, WHO’s preparedness activities continue with the aim to ensure that all countries are operationally ready to effectively and safely detect, investigate, and report potential Ebola virus disease (EVD) cases, and to mount an effective response to any EVD outbreak. Thus, the need remains for the development of point-of-care (POC) rapid tests for specific and accurate diagnoses of EBOV without requirements for complex lab equipment or highly trained staff. This report describes the work undertaken for the development and evaluation of candidate EBOV recombinant protein preparations to serve as WHO International Reference Reagents (IRR) for EBOV VP40 for monitoring the performance of POC tests for antigen detection and presumptive diagnosis of EVD. Nine freeze-dried samples, derived from recombinant EBOV VP40, GP and/or NP protein and formulated in defibrinated plasma, were tested for EBOV antigens by 8 laboratories from 4 countries in a blinded manner. Assay methods used include POC rapid tests for detection of EBOV VP40, GP and/or NP as well as enzyme linked immunosorbent assays (ELISA) for VP40 and sequential detection of GP and VP40 by western blot analysis. The study assessed the ability of the participating laboratories to correctly identify samples as positive or negative for EBOV antigen(s). Where available, a semi-quantitative assessment of sample reactivity is also presented.

The study showed that some assays were not able to detect certain EBOV antigens. This may be due to low assay sensitivity, the absence of relevant antigen epitopes or conformations and/or differences in epitope recognition by antibodies used in the assays antigen capture or detection. The consequences of using recombinant EBOV antigens derived from bacterial and eukaryotic expression systems are discussed. The freeze-dried candidates containing VP40 only and showing weakly positive/trace reactivity (sample 5) and low but unequivocal reactivity (sample 3) along with the negative control (sample 8) may have utility as a WHO IRR panel for use in monitoring the performance of rapid POC tests for the detection of VP40 provided the potential limitations of the panel are understood.

Initial data from accelerated thermal degradation of the material obtained at 1 month is insufficient for predicting the long-term stability of the candidate IRR and it is recommended that they are shipped at -20°C. Further work is required for developing reference materials for EBOV GP and NP assays.