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

Abstract

Soluble forms of ACE2 have recently been shown to inhibit 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 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 COVID-19 that is active against viral variants and future coronaviruses.

Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

“…pLenti.GFP.NLuc dual GFP/nanoluciferase lentiviral vector, pcCOV2.Δ19S codon-optimized SARS-CoV-2 spike gene expression vector based on the Wuhan-Hu-1/2019 amino acid sequence with a termination codon at position 1255, HIV-1 Gag/Pol expression vector pMDL and HIV-1 Rev expression vector pRSV.Rev have been previously described 33 . Point mutations in pcCOV2.Δ19S open reading frame were introduced by overlap extension PCR.…”

“…293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37°C in 5% CO 2 . ACE2.293T cells are clonal cell-line that expresses high levels of human ACE2 and have been previously described 29,33 .…”

“…SARS-CoV-2 spike protein pseudotyped lentiviral stocks were produced by cotransfection of 293T cells with pMDL, pLenti.GFP-NLuc, pcCoV2.S-Δ19 (or variants thereof) and pRSV.Rev as previously described 33 . Virus stocks were normalized for reverse transcriptase activity 34 .…”

“…Virus stocks were normalized for reverse transcriptase activity 34 . Pseudotyped virus infections were done with 1 × 10 4 cells/well in 96 well tissue culture dishes at an MOI=0.2 33 . Luciferase activity was measured after 2 days using Nano-Glo luciferase substrate (Promega) and plates were read in an Envision 2103 microplate luminometer (PerkinElmer).…”

SARS CoV-2 nucleoprotein enhances the infectivity of lentiviral spike particles

“…In this regard, capitalizing on the interaction of the S protein with cellular ACE2, strategies have been reported which utilize chimeric ACE2 fused to the Fc region of human IgG as a potent neutralizing agent against spike-pseudotyped viruses 10 . This arsenal now includes variations such as mutations to the catalytic region of ACE2 (thereby preventing undesirable side-effects during in vivo administration), and modified Fc domains 10,11 . However, a common feature of these studies is that they invariably adopt pseudoviruses which are enveloped by spike, but do not incorporate contributions from any other SARS CoV-2 genes.…”