Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays

Yu, Jingyou; Li, Zhenfeng; He, Xuan; Gebre, Makda S.; Bondzie, Esther A.; Wan, Huahua; Jacob-Dolan, Catherine; Martinez, David R.; Nkolola, Joseph P.; Baric, Ralph S.; Barouch, Dan H.

Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL2 adapted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here, we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed SΔCT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells compared to that with full-length spike (S). We further compared S and SΔCT13 in terms of their sensitivity to vaccine sera, purified convalescent-phase IgG, human ACE2 (hACE2) murine IgG (mIgG), and the virus entry inhibitor bafilomycin A1 (BafA1). We developed an SΔCT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra-assay and intermediate assay precision. Our data demonstrate that the SΔCT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. The development of a high-throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This SΔCT13-based pseudovirus neutralization assay should be broadly useful for the field.