Description: Publication date: 25 July 2018 Source: Vaccine, Volume 36, Issue 31 Author(s): M.S. Boukhvalova, A. Mbaye, S. Kovtun, K.C. Yim, T. Konstantinova, T. Getachew, S. Khurana, A.R. Falsey, J.C.G. Blanco Respiratory syncytial virus (RSV) is a significant cause of bronchiolitis and pneumonia. Protection against RSV is associated with neutralizing antibodies against the fusion (F) and attachment (G) glycoproteins. Several RSV vaccine candidates are in development, but their immunogenicity is hard to compare due to the little-understood differences between multiple RSV neutralizing antibody assays used. Existing assays utilize primarily Vero or HEp-2 cells, but their ability to detect G-neutralizing antibodies or antibodies against specific RSV strains is unclear. In this work, we developed an RSV microneutralization assay (MNA) using unmodified RSV and immortalized cell line derived from human airway epithelial cells (A549). Performance of A549-, HEp-2- and Vero-based MNA was compared under the same assay conditions (fixed amount of virus and cells) with regards to detection of neutralizing antibodies against RSV A or B viruses, G-reactive neutralizing antibodies, and effect of complement. Our results indicate that A549 cells yield the highest MNA titers, particularly in the RSV A/A2 MNA, are least susceptible to complement-enhancing effect of neutralizing titer readout and are superior to Vero or HEp-2 MNA at recognizing G-reactive neutralizing antibodies when no complement is used. Vero cells, however, can be more consistent at recognizing neutralizing antibodies against multiple RSV strains. The choice of substrate cells thus affects the outcome of MNA, as some immortalized cells better support detection of broader range of neutralizing antibodies, while others facilitate detection of G-targeting neutralizing antibodies, a long-thought prerogative of primary airway epithelial cells.