Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 < 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of COVID-19 Spike pseudotype virus

BackgroundCOVID-19 has been affecting global health since the end of 2019 and so far, no any sign shows the relief of the pandemic. The major issue for controlling the infection disease is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) has been reported to treat the disease, but underlying mechanism still keeps controversial. Purpose: The objective of this study was to investigate whether CQ and HCQ could be an ACE2 blocker to inhibit COVID-19 infection.MethodsIn our study, we used CCK-8 stain, flow cytometry and immunofluorescent stain to evaluated the toxicity and autophagy of CQ and HCQ respectively on ACE2 high expressed HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking, surface plasmon resonance (SPR) assays and molecule docking, and COVID-19 spike pseudotype virus was also used to observe the viropexis effect of CQ and HCQ in ACEh cells.ResultsResults showed that HCQ is slightly more toxic to ACE2h cells than CQ, both CQ and HCQ could bind to ACE2 with KD (7.31±0.62)e-7 and (4.82±0.87)e-7, respectively. They also exhibit equivalent suppression effect for the entrance of COVID-19 spike pseudotype virus into ACE2h cells.ConclusionsCQ and HCQ both inhibite the entrance COVID-19 virus into cell by blocking the binding of the virus with ACE2. Our finding provides a novel insight into the molecular mechanism of CQ and HCQ treatment effect on the virus infection.

Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drug Library

ABSTRACT Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design. IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.