Qualification of a reporter virus microneutralization assay for evaluation of influenza specific antibodies in human clinical trials

Abstract

Neutralizing antibodies can block the entry of viruses into host cells. Next-generation influenza vaccines should stimulate the production of high levels of neutralizing antibodies capable of preventing influenza infection or reducing the severity of disease. Currently, multiple assays and protocols are used to measure influenza neutralization and there is a critical need to compare antibody responses elicited by different candidate vaccines tested in different populations to advance the most promising vaccine candidates to efficacy studies and licensure. A standardized approach for safely testing human sera against influenza viruses is to employ replication restricted reporter influenza viruses in which an essential viral gene (i.e., polymerase basic 1, PB1, gene) is replaced with a reporter gene. The replication restricted reporter viruses can be propagated in specific cell lines expressing the deleted viral gene, which make them suitable for biosafety level 2 (BSL-2) laboratory. Qualification of this method is needed for standardized testing in human clinical trials.

In compliance with Good Clinical Laboratory Practice (GCLP) guidelines, we qualified a reporter virus microneutralization method by evaluating linearity, precision, accuracy, range, limits of detection and quantitation, specificity and robustness for A/New Caledonia/20/1999 (H1N1). We utilized unique sets of human clinical trial samples positive for influenza specific antibodies to enable development and testing. We compared the reporter virus microneutralization assay to the standard microneutralization assay with three additional viruses: A/California/07/2009 (H1N1), A/Singapore/INFIMH-16-0019/2016 (H3N2) and A/Texas/71/2017 (H3N2). The qualified reporter virus microneutralization assay was linear (R2 = 0.99, p-value <0.0001), precise (0–8 % GCV repeatability), accurate (%RE −18 to −12 %) with LLOQ of 19.4, robust for laboratory testing conditions (i.e. incubation times, temperature) and correlated with the traditional microneutralization assay (Spearman's r = 0.84–0.98, p < 0.001 for four virus strains). This qualified method will enable defining correlates of protection and contribute to meta-analyses of future human clinical trials.