An ACE2 Microbody Containing a Single Immunoglobulin Fc Domain Is a Potent Inhibitor of SARS-CoV-2

Takuya Tada,Chen Fan,Jennifer S Chen,Ramanjit Kaur,Kenneth A Stapleford,Harry Gristick,Belinda M Dcosta,Craig B Wilen,Crina M Nimigean,Nathaniel R Landau

doi:10.1016/j.celrep.2020.108528

Abstract

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a “microbody,” in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of β coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.

Highlights

As the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) continues to spread worldwide, there is an urgent need for preventative vaccine and improved therapeutics for treatment of COVID-19
SARS-CoV-2 D19 Spike Protein Was Incorporated in Pseudotyped Virion and Had Higher Infectivity As a means to study SARS-CoV-2 entry, we developed an assay based on SARS-CoV-2 spike protein pseudotyped lentiviral reporter viruses
Pseudotyped viruses were produced in 293T cells cotransfected with the dual nanoluciferase/GFP reporter lentiviral vector pLenti.GFP.NLuc, Gag/Pol expression vector pMDL and full-length spike protein, D19 spike protein, vesicular stomatitis virus G protein (VSV-G) expression vector, or without an envelope glycoprotein expression vector

Summary

Introduction

As the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) continues to spread worldwide, there is an urgent need for preventative vaccine and improved therapeutics for treatment of COVID-19. The development of therapeutic agents that block specific steps of the coronavirus replication cycle will be highly valuable both for treatment and prophylaxis. Virus entry is advantageous because as the first step in virus replication, it spares target cells from becoming infected and because drugs that block entry do not need to be cell permeable as the targets are externally exposed. In SARS-CoV-2 entry, the virus attaches to the target cell through the interaction of the spike glycoprotein with its receptor, the angiotensin-converting enzyme 2 (ACE2) (Li, 2015; Li et al, 2003, 2005), a plasma membrane protein carboxypeptidase that degrades angiotensin II to angiotensin-(1–7) (Ang-(1–7)), a vasodilator that promotes sodium transport in the regulation of cardiac function and blood pressure (Kuba et al, 2010; Riordan, 2003; Tikellis and Thomas, 2012). ACE2 binding triggers spike protein-mediated fusion of the viral envelope with the cell plasma membrane or intracellular endosomal membranes. These include cells in the lungs, arteries, heart, kidney, and intestines (Harmer et al, 2002; Ksiazek et al, 2003; Leung et al, 2003)

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