Development of a pseudotype-based neutralization test for SARS-COV-2 antibody measurement

Abstract

The gold standard for measuring anti-SARS-CoV-2 neutralizing antibody is a live-virus neutralization test (VNT). However, VNT is rather complex and must be performed in a biosafety level 3 facility. Therefore, a gene-encoding spike protein of the original SARS-CoV-2 variant was designed to develop a lentivirus-based SARS-CoV-2 pseudotype in this study to replace the live virus, which can be conducted in a biosafety level 2 facility. The 2nd generation lentiviral pseudotype with green fluorescent protein (GFP) and the 3rd generation lentiviral pseudotype with luciferase as a reporter system, respectively, provided a sufficient titer (TCID50/mL) for a neutralization test. However, the 3rd generation lentivirus with luciferase reporter system was inconsistent, with high variability. Therefore, the 2nd generation lentiviral pseudotype with GFP was selected and developed further into a pseudotype neutralization test (PVNT). When tested against 128 serum samples from the ChulaCoV-19 trial, the diagnostic sensitivity and specificity of this test were 98.91% and 100%, respectively, in comparison with the VNT. Standard serum samples with known titers were tested to convert the PVNT50 titer into international units (IU/mL) and compared with the standard VNT method. A moderate level of agreement was observed between the 2 tests, with a correlation coefficient of 0.740 (p-value