Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

被引：553

作者：

Crawford, Katharine H. D. ^{[1
,2
,3
,4
]}

Eguia, Rachel ^{[1
,2
]}

Dingens, Adam S. ^{[1
,2
]}

Loes, Andrea N. ^{[1
,2
]}

Malone, Keara D. ^{[1
,2
]}

Wolf, Caitlin R. ^{[5
]}

Chu, Helen Y. ^{[5
]}

Tortorici, M. Alejandra ^{[6
,7
,8
]}

Veesler, David ^{[6
]}

Murphy, Michael ^{[9
]}

Pettie, Deleah ^{[9
]}

King, Neil P. ^{[6
,9
]}

Balazs, Alejandro B. ^{[10
,11
]}

Bloom, Jesse D. ^{[1
,2
,3
,12
]}

机构：

[1] Fred Hutchinson Canc Res Ctr, Div Basic Sci, Seattle, WA 98109 USA

[2] Fred Hutchinson Canc Res Ctr, Computat Biol Program, Seattle, WA 98109 USA

[3] Univ Washington, Dept Genome Sci, Seattle, WA 98195 USA

[4] Univ Washington, Med Scientist Training Program, Seattle, WA 98195 USA

[5] Univ Washington, Div Allergy & Infect Dis, Seattle, WA 98195 USA

[6] Univ Washington, Dept Biochem, Seattle, WA 98109 USA

[7] Inst Pasteur, F-75015 Paris, France

[8] CNRS, UMR 3569, Unite Virol Struct, F-75015 Paris, France

[9] Univ Washington, Inst Prot Design, Seattle, WA 98195 USA

[10] MIT, Ragon Inst, Massachusetts Gen Hosp, 77 Massachusetts Ave, Cambridge, MA 02139 USA

[11] Harvard Univ, Cambridge, MA 02138 USA

[12] Howard Hughes Med Inst, Seattle, WA 98103 USA

来源：

VIRUSES-BASEL | 2020年 / 12卷 / 05期

关键词：

SARS-CoV-2; COVID-19; coronavirus; neutralization assay; lentiviral pseudotype; Spike; cytoplasmic tail; ACE2; 293T-ACE2; luciferase; ALAYT; RESPIRATORY-SYNDROME CORONAVIRUS; HUMAN MONOCLONAL-ANTIBODY; SARS CORONAVIRUS; CONVALESCENT PLASMA; CYTOPLASMIC TAIL; PROTECTION; INFLUENZA; INFECTION; ESTABLISHMENT; VOLUNTEERS;

D O I：

10.3390/v12050513

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

引用

页数：15

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