key: cord-282795-kje7rn57
authors: Zheng, Yue; Larragoite, Erin T.; Lama, Juan; Cisneros, Isabel; Delgado, Julio C.; Slev, Patricia; Rychert, Jenna; Innis, Emily A.; Williams, Elizabeth S.C.P.; Coiras, Mayte; Rondina, Matthew T.; Spivak, Adam M.; Planelles, Vicente
title: Neutralization Assay with SARS-CoV-1 and SARS-CoV-2 Spike Pseudotyped Murine Leukemia Virions
date: 2020-09-21
journal: bioRxiv
DOI: 10.1101/2020.07.17.207563
sha: 
doc_id: 282795
cord_uid: kje7rn57

Antibody neutralization is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal MLV genome encoding firefly luciferase. These pseudovirions provide a practical means of assessing immune responses under laboratory conditions consistent with biocontainment level 2.

Coronaviruses are a group of enveloped RNA viruses with a positive-sense single-stranded 24 RNA genome ranging from 26-32 kilobases, which can cause respiratory tract infections. In December 25 2019, a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 26 was identified in China and has caused a global ongoing pandemic of coronavirus disease (COVID-27 19). To date, SARS-CoV-2 has spread to 188 countries (https://coronavirus.jhu.edu/). More than 29 28 million cases and 900,000 deaths have been reported at the time of this writing.

Enveloped viruses are known to efficiently package their core elements with heterologous 30 envelope glycoproteins, giving rise to the so called 'pseudotypes' or 'pseudoviruses ' 

Neutralization titers NT50 and NT80 were calculated using Prism 8 (GraphPad, US).

To generate pseudovirion particles, three plasmids were co-transfected into HEK293FT cells. The 

HIV-1 LAI gp160-pseudotyped virus is used as a negative control as it utilizes CD4 as a primary 89 receptor, which is present in SupT1 cells but absent in HEK293T.

Pseudotyped MLV viruses were tested on HEK293FT, HEK293T-ACE2, Huh7 and SupT1 cells.

HEK293FT cells were used as a control cell line, which is known to lack of susceptibility of 

To test for specificity of neutralization, we asked whether neutralizing antibodies from SARS-

CoV-2 patients would exhibit cross-reactivity against a pseudotype expressing SARS-CoV-1 ( Figure   112 4). We tested samples #1, 2 and 3, which had the highest NT50 and NT80. None of these sera had 

Characterization of 162 spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with 163 SARS-CoV

Veesler D: Structure, Function, and 165 Antigenicity of the SARS-CoV-2 Spike Glycoprotein

The 167 D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases 168 infectivity

High-efficiency gene 170 transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral 171 vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G

Fate of 174 the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr

Pseudotyping Viral Vectors With Emerging Virus Envelope 177 Proteins

The coronavirus spike protein is a class I