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. Author manuscript; available in PMC: 2025 Aug 1.
Published in final edited form as: Immunol Lett. 2024 Jun 24;268:106887. doi: 10.1016/j.imlet.2024.106887

In vitro antibody-mediated SARS-CoV-2 infection suppression through human ACE2 receptor blockade

Priscilla S Redd 1,2,3,4,*, Alyssa D Merting 2,3,4, John D Klement 2,3,4, Dakota B Poschel 2,3,4, Dafeng Yang 2,3,4, Kebin Liu 1,2,3,4
PMCID: PMC11256821  NIHMSID: NIHMS2006940  PMID: 38925442

SUMMARY

Vaccines and antibodies that specifically target or neutralize components of the SARS-CoV-2 virus are effective in prevention and treatment of human patients with SARS-CoV-2 infection. However, vaccines and SARSCoV-2 neutralization antibodies target a subset of epitopes of viral proteins, and the fast evolution of the SARS-CoV-2 virus and the continuing emergence of SARS-CoV-2 variants confer SARS-CoV-2 immune escape from these therapies. ACE2 is the human cell receptor that serves as the entry point for SARS-CoV-2 into human cells and thus is the gatekeeper for SARS-CoV-2 infection of humans. We report here the development of 4G8C11, an anti-human ACE2 receptor monoclonal antibody that recognizes ACE2 on human cell surfaces. We determined that 4G8C11 blocks SARS-CoV-2 and variant infection of ACE2+ human cells. Furthermore, 4G8C11 has minimal effects on ACE2 receptor activity. 4G8C11 is therefore a monoclonal antibody for ACE2 receptor detection and potentially an effective immunotherapeutic agent for SARS-CoV-2 and variants.

Graphical Abstract

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INTRODUCTION

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-3. Vaccines have been the first line of defense for SARS-CoV-2 and are highly effective in the prevention of its infection4-7. However, SARS-CoV-2 is highly evolutional and a breakthrough infection often occurs8,9. To treat patients with SARS-CoV-2 infection, two types of SARS-CoV-2 neutralization antibodies have been developed10-13. The first type is convalescent sera and recombinant SARS-CoV-2 neutralization antibody based on antibody sequences from patients. Early studies of other coronaviruses such as SARS-CoV-1 and the Middle East respiratory syndrome (MERS) indicate that treatment of patients with convalescent sera reduces the spreading of infection and mortality14,15. Passive antibody immunotherapy with convalescent sera is thus considered as an effective and timely approach for suppressing SARS-CoV-216-22. Immunotherapy by transferring the convalescent sera from recovered patients of SARS-CoV-2 to patients with severe disease resulted in improvement in their clinical status18,20,23. The second type is the SARS-CoV-2 neutralization monoclonal antibodies that neutralize the viral SARS-CoV-224-28. This type of defined monoclonal antibodies has high efficacy in reducing viral load, especially in patients whose immune response had not yet been initiated or who had a high viral load26. However, a major challenge for both types of SARS-CoV-2 neutralization antibodies is the loss of efficacy due to evolution and emergence of the SARS-CoV-2 variants of concern (VOC)29-33. Currently, the status of all VOC have been downgraded to variants being monitored (VBM)(The Centers for Disease Control and Prevention, United States). There is still potential for future VOC to occur.

The SARS-CoV-2 genome ranges from 29.8 kb to 29.9 kb and contains 10 open reading frames with the 5′ two-thirds of the genome consisting of orf1a/b which encodes orf1ab polyproteins34. The 3′ one third of the genome encodes genes for structural proteins including surface spike (SARS-2-S or S), envelope (E), membrane (M), and nucleocapsid (N) proteins. In addition, there are 16 non-structural proteins (nsp1-16) that mediate virus metabolism and interaction with the host immune response35. SARS-CoV-2 is highly mutagenic with rapid accumulation of mutations in its genomes, resulting in numerous variants with distinct phenotypes36-39. Five SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (BA.1-5), emerged and were declared as VOC at that time40-42, although currently they are VBM. The Alpha variant harbors mutations in the S protein receptor-binding domain (RBD: N501Y, P681H), the S protein N-terminal domain (NTD 144), and several non-spike mutations43-45. The Beta variants contains at least 9 mutations in the S protein, including N501Y, E484K and K417N that are also present in Gamma variant43,46,47. The Delta variant contains at least 23 mutations, including mutation E484Q and L452R in the RBD and mutations on ORF3 and ORF748. The Omicron variant is highly mutated and has more than 50 mutations, including more than 35 mutations in the S protein. The Omicron subvariants are highly immune evasive and can escape from vaccine-elicited immunity, convalescent antibodies, and SARS-CoV-2 neutralization monoclonal antibodies49-54. VBM therefore constitute a major challenge in SARS-CoV-2 prevention and treatment33,53,55-58.

Structural and functional studies using pseudovirus and SARS-CoV-2 isolates from human patients determined that the SARS-CoV-2 S protein binds to the angiotensin II converting enzyme (ACE2) receptor on human cell surfaces to enter the host cells to infect humans. The S protein consists of a N-terminal subunit for mediating receptor binding and a C-terminal subunit for virus cell membrane fusion. During viral entry into cells, the RBD of the N-terminal subunit recognizes and binds to the ACE2 receptor27,28,59,60. The S protein and the interactions between S and ACE2 are thus key determinants of human infection and SARS-CoV-2 pathogenesis. These findings suggest that ACE2 blockade therapy with an ACE2-specific antibody is an effective approach to block SARS-CoV-2 entry into human target cells since ACE2 is the receptor for all SARS-CoV-2 variants and therefore the gatekeeper of SARS-CoV-2 infection of humans12,13,61. ACE2 blockade therapy therefore should block all SARS-CoV-2 VBM. Another advantage of ACE2 blockade over SARS-CoV-2 blockade is the ration of virus load vs ACE2 receptor level. It is likely that virus load can be high in patients whereas the ACE2 receptor is restricted in specific cell types with a relative constant expression level. We report here the development of the human ACE2 neutralization monoclonal antibody 4G8C11 for the detection and quantification of human cell surface ACE2 receptor and for intervention of SARS-CoV-2 infection of humans.

RESULTS

ACE2 expression profiles in humans.

To determine human ACE2 expression profiles in human tissues and cells, we retrieved ACE2 transcriptomics datasets from the Human Protein Atlas (HPA) and Genotype-Tissue Expression (GTEx) datasets62. The highest ACE2 expression level is in the small intestines (Fig. 1A). ACE2 expression level is also high in the kidney, gallbladder, testis, and heart (Fig. 1A). To determine ACE2 expression level in the single cell level, human scRNA-seq datasets were retrieved from HPA that contains datasets from the Single Cell Expression Atlas, the Human Cell Atlas, the Gene Expression Omnibus, the Allen Brain Map, European Genome-phenome Archive, and the Tabula Sapiens63-82. At the cellular level, the highest ACE2 expression level is in enterocytes (Fig. 1B). Low levels of ACE2 expression is detected in several cell types including monocytes (Fig. 1B).

Figure 1. ACE2 expression level in human tissues and cells.

Figure 1.

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A. The human ACE2 transcript level in the indicated human tissues. ACE2 expression datasets were retrieved from the Human Protein Atlas. The ACE2 expression level is calculated as the normalized transcripts per million (nTPM) for each tissue from the Human Protein Atlas and GTEx. B. The human ACE2 expression level in the indicated human cell types. The expression value is based on human scRNA-seq datasets retrieved from 31 datasets from the Single Cell Expression Atlas, the Human Cell Atlas, the Gene Expression Omnibus, the Allen Brain Map, European Genomephenome Archive, and the Tabula Sapiens.

Generation of human ACE2 neutralization antibodies that selectively blocks SARS-2-S binding to ACE2 protein without significant inhibition of ACE2 receptor activity.

ACE2 is the receptor on the surface of human cells that serves as the entry point for SARSCoV-2 into cells and thus is the gatekeeper for SARS-CoV-2 infection of humans28. To block ACE2 access for SARS-CoV-2 with minimal effects on ACE2 normal physiological functions, we analyzed human ACE2 protein structure. Human ACE2 protein structure data was extracted from the protein database and analyzed using a computer program. There are two SARS-2-binding sites on ACE2 proteins27,59 (Fig. 2A). The ACE2 enzymatic catalytic site is apparently located on a different domain of the ACE2 protein (Fig. 2A). This finding provides the rationale to develop a neutralization antibody that selectively blocks these two SARS-2-S-binding sites but not the ACE2 enzymatic catalytic activity site83. We used recombinant full length human ACE2 protein to immunize mice. Antisera from 5 mice were analyzed for affinity to ACE2 protein, efficacy in blocking SARS-2-S binding to ACE2, and effects on ACE2 enzyme activity. All 5 sera showed high affinity for binding to human ACE2 protein in a dose-dependent manner (Fig. 2B). The antibodies showed various efficacy in blocking SARS-2-S binding to ACE2 with the highest efficacy in #4194 and #4193 (Fig. 2C). Antisera from 4 mice showed >70% inhibition of ACE2 receptor activity, but #4194 showed about 30% ACE2 receptor activity inhibition as compared to the control serum (Fig. 2D). #4194 was therefore chosen for further development.

Figure 2. Design and generation of human ACE2 neutralization antibodies.

Figure 2.

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A. Human ACE2 receptor and SARS-2-S interaction complexes. The structures were adapted from the protein database and analyzed for SARS-2-S-binding sites27,59 (Green and purple sites) and ACE2 enzyme catalytic site (yellow circle). B. Antisera from five mice immunized with recombinant human ACE 2 protein were analyzed for their affinity for human ACE2 protein by indirect ELISA assay. C. The antisera were screened at 3 dilutions for their ability to block the binding of SARS-2-S protein to ACE2 protein using the indirect ACE2:Spike S1-ELISA kit (Cat #:79945, BPS Bioscience, San Diego, CA). Pre represents serum from the mice prior to immunization. Post represents serum from the mice after the third immunization. The data was normalized to a positive control with no inhibitor. Average of 3 replicates. * p<0.05. D. The sera from the 5 immunized mice were screened for inhibition of ACE2 enzyme activity using an ACE2 enzyme activity assay kit (Cat #: K310, BioVision, Milpitas, CA). Shown is the result of mouse # 4194 pre and post serums at the highest dose (1:100).

Development of human ACE2 blockade monoclonal antibodies

Spleen cells from #4194 mouse were fused with myeloma cells and twenty plates were screened using indirect ELISA assay. The top twenty hybridoma cell lines with binding affinity in the same level as the parent antibody #4194 were selected (Fig. 3A). Five of the twenty cell lines were further validated and selected for single cell subcloning (Fig. 3B, Fig. S1). The monoclonal antibody clones maintained the activity of the parent antibody (antisera) #4194 (Fig. 2B & 3B).

Figure 3. Generation of five human ACE2 neutralization monoclonal antibodies.

Figure 3.

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A. Screening and establishment of 20 human ACE2 neutralization antibody hybridoma cell lines. B. Generation of 5 human ACE2 monoclonal antibody clones by single cell subcloning. The hybridoma culture supernatants were analyzed for binding to human ACE2 by ELISA.

To determine the binding affinity of these clones to the human ACE2 receptor on human cell surface. We obtained the human ACE2-expressing lung epithelial tumor cell line A549.hACE2. We also generated human ACE2-expressing colon tumor cell line SW620.hACE2 and human pancreatic tumor cell line MIAPaCa2.hACE2 using the plasmid pUNO1-hACE2 stable transfection. The transfected cells were selected by blasticidin. We then determined the binding affinity of these 5 clones to the human ACE2 receptor using these three ACE2-expressing human cell lines by flow cytometry. 4G8C11 and 7E10F10 bind to the ACE2 receptor, whereas clones 14C11B8, 4G12E5, and 17B6D4 failed to bind to the ACE2 receptor on the cell surface.

Hybridoma cells of clones 4G8C11, 14C11B8, and 7E10F10 were deposited in Bio X cell Corp for large-scale purification (Fig. S1). The purified antibodies were then sequenced. 4G8C11 and 7E10F10 have identical sequences and are thus the same clone. 14C11B8 has multiple heavy chains. 4G8C11 is thus used as the lead agent (Fig. 4). The purified 4G8C11 was then tested for binding affinity to the ACE2 receptor. 4G8C11 stably binds to ACE2 receptor on the human cell surfaces as determined by flow cytometry analysis (Fig. 5).

Figure 4. The heavy and light chain protein sequences of 4G8C11 monoclonal antibody.

Figure 4.

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Figure 5. 4G8C11 binds to human ACE2 receptor on the surface of human cells.

Figure 5.

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The indicated human cell lines were incubated with 4G8C11 (blue line) and mouse IgG isotype control antibody (black line), respectively for 30 min, washed with PBS, and incubated with FITC-conjugated antimouse IgG 2nd antibody for 30 min. Cells were washed, fixed, and analyzed by flow cytometry. Shown are overlaps of isotype control and ACE2-specific staining of the indicated cells. A549 cell line is used as an ACE2-negative control cell line.

4G8C11 blocks SARS-CoV-2 entrance into ACE2-positive human cells in vitro.

We then used the SARS-CoV-2 pseudovirus and the SARS-CoV-2 Delta variant pseudovirus models to test the efficacy of 4G8C11 in blocking SAR-CoV-2 infection of human lung epithelial tumor A549.hACE2 cells. A549.hACE2 overexpress human ACE2 receptor on the cell surface (Fig. 5). 4G8C11 blocked SARS-CoV-2 pseudovirus and its Delta variant infection of A549 cells in a dose-dependent manner (Fig. 6).

Figure 6. 4G8C11 blocks SAR-CoV-2 entrance into ACE2+ human cells in vitro.

Figure 6.

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A. A549.hACE2 cells were seeded in 96-well plates overnight. 4G8C11 mAb was added to the culture medium at the indicated concentrations for 30 min. SARS-CoV-2 pseudovirus particles were then added to the culture for 48h. Cells were analyzed for luciferase activity using firefly luciferase assay kit. B. A549.hACE2 cells were seeded in 96-well plates overnight. 4G8C11 mAb was added to the culture medium at the indicated concentrations for 30 min. SARS-CoV-2 Delta variant pseudovirus particles were then added to the culture for 48h. Cells were analyzed for luciferase activity using the ONE-Step Luciferase Assay System. The luciferase activity of control samples was set to 100%.

4G8C11 has minimal effect on human ACE2 enzymatic activity.

To determine the effect of 4G8C11 on human ACE2 enzyme activity, we performed the same in vitro ACE2 enzyme activity assay in the presence of the purified 4G8C11 monoclonal antibody. As expected, the positive control ACE2 inhibitor completely inhibited human ACE2 enzyme activity in vitro (Fig. 7A). Under the same assay conditions, 4G8C11 monoclonal antibody showed a dose-dependent inhibition of human ACE2 protein enzyme activity, albeit at a degree of less than 15% at the highest dose of 10 μg/ml concentration (Fig. 7B).

Figure 7. Human ACE2 monoclonal antibody clone 4G8C11 has minimal effect on human ACE2 enzyme activity in vitro.

Figure 7.

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A. ACE2 protein was analyzed for its enzyme activity in the presence of the positive control inhibitor using the ACE2 enzyme activity assay kit. B. ACE2 enzyme activity was analyzed as in A in the presence of 4G8C11 at the indicated concentrations.

DISCUSSION

Vaccines and viral neutralization antibodies are currently the two major approaches for SARSCoV-2 prevention and intervention. SARS-CoV-2 vaccines are highly effective and offer protection of SARS-CoV-2 infection4-7. However, breakthrough infections often occur and the VOC/VBM are capable of escape from vaccine-induced immunity8,9,84. To treat patients with breakthrough infection and infection by VOC/VBM, major effort has been put on the development of SARS-CoV-2 neutralization antibodies. As of January 24, 2024, 218 SARS-CoV-2 monoclonal antibodies have been developed (Stanford University Coronavirus antiviral %resistance database: https://covdb.stanford.edu/page/mab-tables). Four SARS-CoV-2 neutralization monoclonal antibodies (bamlanivimab+etesevimab, casirivimab+imdevimab, sotrovimab, and bebtelovimab) have received the emergency use authorizations (EUA) from the Food and Drug Administration (FDA) for the treatment of SARS-CoV-2 patients. However, the recent SARS-CoV-2 VBM exhibit decreased sensitivity to these neutralization antibodies29-31,85.

SARS-CoV-2 infects humans through binding to the ACE2 receptor on human cell surface28,59,70. Compared to SARS-CoV-2 virus, which often has high load in the infected human patients, ACE2 expression is expressed in limited human cell and tissue types and its expression is lower in most expressing cell types (Fig. 1). Furthermore, unlike the SAR-CoV-2 S protein which exhibits high mutation rate and mutagenic dynamics, ACE2 mutations are rare and its relativly stable86. Therefore, blocking the SARS-CoV-2 binding sites on ACE2 likely blocks all SARS-CoV-2 VOC/VBM from binding to ACE2 and therefore is potentially a more effective approach to treatment of SARS-CoV-2 infection. So far, only 2 ACE2 blocking monoclonal antibodies have been developed61,87,88.

Here we describe the development and functional characterization of human ACE2 monoclonal antibody 4G8C11. 4G8C11 was generated by using mammalian cell-expressed recombinant human ACE2 whole protein and selectively binds to the ACE2 receptor on human ACE2+ cell surface. The selective and stable binding on human cell surface indicate that 4G8C11 is not internalized by the cells and thus likely generates stable blocking efficacy against the SARS-2-S binding. In addition, 4G8C11 can be used for staining and quantifying human ACE2 receptor on human cell surface using flow cytometry application, and it has been licensed to Biolegend for commercial distribution to the scientific research community.

The major advantage of ACE2 blocking antibody is its ability to block the entrance of SARCoV-2 and potentially all VOCs/VBMs into human cells61,87,88 since ACE2 is the gatekeeper of SAR-CoV-2 infection of human cells 28,59,60,70. In the proof of principle studies with SARS-CoV-2 pseudo virus models, we determined that 4G8C11 effectively blocks infection of ACE2-overexpressing human lung cells by SARS-CoV-2 and SARS-CoV-2 variant Delta. Our data thus indicate that 4G8C11 is an effective human ACE2 neutralization monoclonal antibody with minimal inhibition of ACE2 receptor activity. There are at least 218 SARS-CoV-2, primarily SARS-CoV-2-S, neutralization antibodies that have been reported. Considering there are only 2 reported human ACE2 antibodies61,87,88, 4G8C11 warrants further development.

LIMITATIONS OF THIS STUDY

The blocking efficacy studies were performed in in vitro pseudo virus models. The in vivo efficacy of 4G8C11 therefore needs to be determined. In addition, in vivo toxicity also remains to be determined.

METHODS

Analysis of human ACE2 expression in human cells and tissues.

The ACE2 expression datasets in human tissues were retrieved from the Human Protein Atlas (HPA) (https://v19.proteinatlas.org) that contains 50 tissues transcriptomics data from HPA and Genotype-Tissue Expression (GTEx) (https://gtexportal.org) project62. The ACE2 expression level is calculated as the normalized transcripts per million (nTPM) for each tissue. The ACE2 expression level in human cells is based on human scRNA-seq datasets retrieved from HPA that contains 81 cell types from 31 datasets from the Single Cell Expression Atlas, the Human Cell Atlas, the Gene Expression Omnibus, the Allen Brain Map, European Genome-phenome Archive, and the Tabula Sapiens63-82. The ACE2 expression level is presented as the consensus normalized transcripts per million (nTPM).

Analysis of human ACE2 protein interaction sites with SARS-CoV-2 S protein.

The human ACE2 protein were adapted from the protein database and analyzed for SARS-2-S-binding sites27,59 and ACE2 enzyme catalytic site.

Indirect ELISA.

Recombinant human ACE2 protein (Biolegend cat#792008) was added to the clear flat bottom 96-well plate at 0.2 μg/well in 100 μl PBS and incubated at 4°C overnight. The wells were washed 3 times with PBS. 1x assay diluent (Biolegend, cat#421203, 200 μl/well) was added to the washed wells and incubated at room temperature for 1 h to block non-specific blocking. Serum or antibody (100 μl/well) were then added to each well and incubated at room temperature for 2h with shaking. The plate was washed 4 times with PBS (200 μl/well). HRP-conjugated goat anti-mouse IgG (100 μl, 1:1000 dilution, Biolegend cat#405306) was added to each well and incubated at room temperature for 1h. The wells were washed with 200 μl PBS 5 times. Add 100 μl freshly mixed 1:1 TMP substrate (Biolegend cat#421101) to each well and incubate in the dark for up to 20 min. Add 100 ml 1M H3SO4 to stop the reaction and read the absorbance at 450 nm and 570 nm. Subtract absorbance at 570 nm from absorbance at 450 nm and calculate net antibody binding affinity.

Development of human ACE2 monoclonal antibodies.

Recombinant human ACE2 protein was obtained from Biolegend (Cat#792008) and used for immunization of mice. Five BALB/c mice were administered with the ACE2 protein (25 μg/mouse) and adjuvant (inactivated and dried M. tuberculosis in mineral oil) mix in 200μl volume via intraperitoneal injection. The mice were boosted with the same ACE2 and adjuvant mix every two weeks for two more injections. Peripheral blood was collected from mice one week after the last immunoization and analyzed for human ACE2 protein binding affinity and effect on ACE2 enzymatic activity. The mouse that produces the serum with the lowest ACE2 enzymatic activity inhibition and highest human ACE2-binding affinity was selected for fusion. The fusion partner used was SP2/0 myeloma cells, and the fusion was carried out using electroporation. The fused hybridoma cells were seeded onto a 96-well plate, and the cell supernatant was collected for detection and screening. Indirect ELISA was used to coat the antigen on the ELISA plate, followed by addition of the cell supernatant and secondary antibodies to promote the selection of specific clones. The subcloning was performed using a limited dilution method to obtain a stable proliferating monoclonal cell line. Monoclonal cell lines were used for sequencing. Total RNA was isolated from the hybridoma cells following the manufacturer’s instructions. Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase. Antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure of rapid amplification of cDNA ends of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes.

ACE2 monoclonal antibody purification.

ACE2 monoclonal antibody is deposited in Bio X Cell Corp. Low endotoxin 4G8C11 was purified in Bio X Cell Corp. Hybridoma cells are cultured in hybridoma medium [Hybridoma-SFM medium (Fisher Scientific Cat# 12-045-084), 10% fetal bovine serum (Hyclone, cat# SH30109.03, and 1% Penicillin-Streptomycin (Fisher Scientific cat# 15140122)] to 70-80% confluence. Actively growing hybridoma cells are passaged three times in antibiotic free media. The cell supernatant is tested for mycoplasma contamination using Lonza’s MycoAlert PLUS Mycoplasma Detection kit (Cat #: LT07-701). Cells are expanded from a T150 T-Flask to a small spinner flask at a final volume of 300ml. Cells are counted daily, and once cells reach peak cell density, viability percentages are calculated daily. When cultures drop below 40% viability, the antibody productivity is assessed daily using a quantitative assay. When daily titers have peaked, cell supernatant is harvested, filtered, and concentrated prior to purification. The protein A/G column is equilibrated with buffer at the appropriate pH conditions and then the supernatant is loaded at a similar pH. Following elution, the antibody is immediately neutralized and dialyzed into PBS at 4°C. Upon completion of two 1:40 dialysis exchanges, the antibody is filtered using a 0.22uM sterile filter into a sterile container. Finally, endotoxin is measured using Associates of Cape Cod LAL gelclot reagent. All antibody lots have an endotoxin level <0.5EU/mg.

ACE2 enzymatic activity assay.

The ACE2 inhibitor screening kit (BioVision cat#K310-100, now Abcam Cat# ab273373) was used to determine ACE2 enzymatic activity according to the manufacturer’s instructions. The ACE2 enzyme recombinant protein was reconstituted in the dilution buffer. The diluted ACE2 enzyme was mixed with ACE2 assay buffer (1:25 ratio) and added to each well (50 μL) of the assay 96-well plate. Serum or purified 4G8C11 ACE2 monoclonal antibody (10 μl) was added to the assay wells in series dilution and incubated in room temperature for 10 min. Forty μL of ACE2 substrate mix was added into control & sample wells. The plate was read in a plate reader to measure fluorescence at 320/420 nm in kinetic mode for 1h at room temperature. Two time points in the linear range were choose and the enzyme activity was analyzed using Graph Prism.

Antibody sequencing.

Total RNA was isolated from the hybridoma cells using TRIzol reagent (Ambion, Cat# 15596-026). cDNA was synthesized from the total RNA using either isotype-specific anti-sense primers or universal primers and the PrimeScript 1st Strand cDNA synthesis kit (TaKaRa, Cat# 6110A). Antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure of rapid amplification of cDNA ends of Genscript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. Five clones with inserts of correct sizes were sequenced for each fragment. The sequences of different clones were aligned and the consensus sequence of these clones were generated.

Generation of ACE2+ human cell lines.

Human ACE2-encoding plasmid pUNO1-hACE2 was obtained from Invivogen (Cat# puno1-hace2). Human tumor cell lines MiaPaCa2 and SW620 were transfected with pUNO1 -hACE2 using Lipofectamine 2000 (Invitrogen). Stable cell lines MiaPaCa2.hACE2 and SW620.hACE2 were established by selection with blasticidin, followed by FACS sorting. A549.hACE2 cell line was obtained from Invivogen (Cat# a549-hace2).

Flow cytometry analysis.

4G8C11 were cultured and purified in protein G column in Bio X Cell Corp. Cells were incubated with IgG isotype control or purified 4G8C11 in 100 μl PBS plus 1% BSA at 4°C for 30 min, washed with PBS once, resuspended in 100 μl PBS plus 1% BSA, and incubated with FITC-conjugated anti-mouse IgG1 antibody (Biolegend). The stained cells were washed with PBS, fixed in 2% paraformaldehyde, and analyzed in a BD FACS flow cytometer.

Pseudoviral infection analysis in vitro.

The MyBioScience SARS-Cov-2 pesudovirus was used to determine SARS-Cov-2 infection of ACE2+ human cells. A549.hACE2 cells (2x104 cells/well) were seeded in 96-well plates in 50 μl DMEM with 10% FBS (Hyclone cat#SH30068.03) overnight. 4G8C11 antibody was then added to the culture in a various concentration and cultured in 37°C for 1h. Add 50 μl SARS-CoV-2 pseudoviral particles into each well, spin at 700 rpm for 15 min at 4°C, incubate the plate at 37°C for 2h, add 50 μl culture medium and culture the plate at 37°C for 48h. Add 20 μl firefly luciferase assay working solution (My BioScience cat# MBS434279) to the culture wells and incubate for 5 min in room temperature. Luminescence intensity was read in a plate reader. The Spike (B.1.617.2; Delta Variant) Pseudotyped Lentivirus assay kit (BPS Bioscience cat# 78215) was used to determine SAR CoV-2 Delta variant infection of ACE2+ human cells. A549.hACE2 cells were seeded in 96-well plate at 1x104 cells/well in 90 μl DMEM medium plus 10% FBS. Add 5 μl 4G8C11 antibody per well and incubate for 30 min at 37°C, followed by addition of 5 μl of SARS-CoV-2 Spike pseudotyped lentivirus into each well. Forty eight hours later, ONE-Step Luciferase Assay reagent (BPS Bioscience, cat#60690-1, 100 μl/well) was added to the culture and incubated at room temperature for 30 min. The plates were measured for luminescence using a plate reader.

Supplementary Material

1

ACKNOWLEDGEMENT

Grant support from the National Institutes of Health (R43CA250780, to PSR; R01CA227433 and R01CA278852, to KL; F30CA236436, to JDK; and F31CA257212, to ADM), and US Department of Veterans Affairs (I01CX001364 to KL).

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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DATA AVAILABILITY

4G8C11 has been licensed to Biolegend (San Diego, CA) and will be available to the research community.

REFERENCES

  • 1.Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei YY, et al. (2020). A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269. 10.1038/s41586-020-2008-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, et al. (2020). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273. 10.1038/s41586-020-2012-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, et al. (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727–733. 10.1056/NEJMoa2001017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, Diemert D, Spector SA, Rouphael N, Creech CB, et al. (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med 384, 403–416. 10.1056/NEJMoa2035389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Beladiya J, Kumar A, Vasava Y, Parmar K, Patel D, Patel S, Dholakia S, Sheth D, Boddu SHS, and Patel C (2024). Safety and efficacy of COVID-19 vaccines: A systematic review and meta-analysis of controlled and randomized clinical trials. Rev Med Virol 34, e2507. 10.1002/rmv.2507. [DOI] [PubMed] [Google Scholar]
  • 6.Ivanova EN, Shwetar J, Devlin JC, Buus TB, Gray-Gaillard S, Koide A, Cornelius A, Samanovic MI, Herrera A, Mimitou EP, et al. (2023). mRNA COVID-19 vaccine elicits potent adaptive immune response without the acute inflammation of SARS-CoV-2 infection. iScience 26, 108572. 10.1016/j.isci.2023.108572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chenchula S, Chandra MB, Adusumilli MB, Ghanta SN, Bommasani A, Kuttiappan A, Padmavathi R, Amerneni KC, Chikatipalli R, Ghanta MK, et al. (2024). Immunogenicity, clinical efficacy and safety of additional second COVID-19 booster vaccines against Omicron and its subvariants: A systematic review. Rev Med Virol 34, e2515. 10.1002/rmv.2515. [DOI] [PubMed] [Google Scholar]
  • 8.Tan ST, Kwan AT, Rodriguez-Barraquer I, Singer BJ, Park HJ, Lewnard JA, Sears D, and Lo NC (2023). Infectiousness of SARS-CoV-2 breakthrough infections and reinfections during the Omicron wave. Nat Med 29, 358–365. 10.1038/s41591-022-02138-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Amanatidou E, Gkiouliava A, Pella E, Serafidi M, Tsilingiris D, Vallianou NG, Karampela I, and Dalamaga M (2022). Breakthrough infections after COVID-19 vaccination: Insights, perspectives and challenges. Metabol Open 14, 100180. 10.1016/j.metop.2022.100180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chen Y, Zhao X, Zhou H, Zhu H, Jiang S, and Wang P (2023). Broadly neutralizing antibodies to SARS-CoV-2 and other human coronaviruses. Nat Rev Immunol 23, 189–199. 10.1038/s41577-022-00784-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ambrose N, Amin A, Anderson B, Barrera-Oro J, Bertagnolli M, Campion F, Chow D, Danan R, D'Arinzo L, Drews A, et al. (2023). Neutralizing Monoclonal Antibody Use and COVID-19 Infection Outcomes. JAMA Netw Open 6, e239694. 10.1001/jamanetworkopen.2023.9694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ge J, Wang R, Ju B, Zhang Q, Sun J, Chen P, Zhang S, Tian Y, Shan S, Cheng L, et al. (2021). Antibody neutralization of SARS-CoV-2 through ACE2 receptor mimicry. Nat Commun 12, 250. 10.1038/s41467-020-20501-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Park YJ, De Marco A, Starr TN, Liu Z, Pinto D, Walls AC, Zatta F, Zepeda SK, Bowen JE, Sprouse KR, et al. (2022). Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry. Science 375, 449–454. 10.1126/science.abm8143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Arabi YM, Al-Enezi F, Longuere KS, Balkhy HH, Al-Sultan M, Al-Omari A, Al-Hameed FM, Carson G, Shindo N, and Fowler R (2016). Feasibility of a randomized controlled trial to assess treatment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Saudi Arabia: a survey of physicians. BMC Anesthesiol 16, 36. 10.1186/s12871-016-0198-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cheng Y, Wong R, Soo YO, Wong WS, Lee CK, Ng MH, Chan P, Wong KC, Leung CB, and Cheng G (2005). Use of convalescent plasma therapy in SARS patients in Hong Kong. Eur J Clin Microbiol Infect Dis 24, 44–46. 10.1007/s10096-004-1271-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kumar GV, Jeyanthi V, and Ramakrishnan S (2020). A short review on antibody therapy for COVID-19. New Microbes New Infect, 100682. 10.1016/j.nmni.2020.100682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Shanmugaraj B, Siriwattananon K, Wangkanont K, and Phoolcharoen W (2020). Perspectives on monoclonal antibody therapy as potential therapeutic intervention for Coronavirus disease-19 (COVID-19). Asian Pac J Allergy Immunol 38, 10–18. 10.12932/AP-200220-0773. [DOI] [PubMed] [Google Scholar]
  • 18.Shen C, Wang Z, Zhao F, Yang Y, Li J, Yuan J, Wang F, Li D, Yang M, Xing L, et al. (2020). Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma. JAMA. 10.1001/jama.2020.4783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Duan K, Liu B, Li C, Zhang H, Yu T, Qu J, Zhou M, Chen L, Meng S, Hu Y, et al. (2020). Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad Sci U S A. 10.1073/pnas.2004168117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lee WT, Girardin RC, Dupuis Ii AP, Kulas KE, Payne AF, Wong SJ, Arinsburg S, Nguyen FT, Mendu DR, Firpo-Betancourt A, et al. (2020). Neutralizing Antibody Responses in COVID-19 Convalescent Sera. J Infect Dis. 10.1093/infdis/jiaa673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Lee WT, Girardin RC, Dupuis AP, Kulas KE, Payne AF, Wong SJ, Arinsburg S, Nguyen FT, Mendu DR, Firpo-Betancourt A, et al. (2021). Neutralizing Antibody Responses in COVID-19 Convalescent Sera. J Infect Dis 223, 47–55. 10.1093/infdis/jiaa673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Casadevall A, and Pirofski LA (2020). The convalescent sera option for containing COVID-19. J Clin Invest 130, 1545–1548. 10.1172/JCI138003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Boonyaratanakornkit J, Morishima C, Selke S, Zamora D, McGuffin SA, Shapiro AE, Campbell VL, McClurkan CL, Jing L, Gross R, et al. (2020). Clinical, laboratory, and temporal predictors of neutralizing antibodies to SARS-CoV-2 among COVID-19 convalescent plasma donor candidates. J Clin Invest. 10.1172/JCI144930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Fagre AC, Manhard J, Adams R, Eckley M, Zhan S, Lewis J, Rocha SM, Woods C, Kuo K, Liao W, et al. (2020). A potent SARS-CoV-2 neutralizing human monoclonal antibody that reduces viral burden and disease severity in Syrian hamsters. bioRxiv. 10.1101/2020.09.25.313601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Xiaojie S, Yu L, Lei Y, Guang Y, and Min Q (2020). Neutralizing antibodies targeting SARS-CoV-2 spike protein. Stem Cell Res 50, 102125. 10.1016/j.scr.2020.102125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Weinreich DM, Sivapalasingam S, Norton T, Ali S, Gao H, Bhore R, Musser BJ, Soo Y, Rofail D, Im J, et al. (2020). REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19. N Engl J Med. 10.1056/NEJMoa2035002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, and Veesler D (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281–292 e286. 10.1016/j.cell.2020.02.058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, et al. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271–280 e278. 10.1016/j.cell.2020.02.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zhang L, Kempf A, Nehlmeier I, Cossmann A, Richter A, Bdeir N, Graichen L, Moldenhauer AS, Dopfer-Jablonka A, Stankov MV, et al. (2024). SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency. Cell. 10.1016/j.cell.2023.12.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Islam S, Islam T, and Islam MR (2022). New Coronavirus Variants are Creating More Challenges to Global Healthcare System: A Brief Report on the Current Knowledge. Clin Pathol 15, 2632010X221075584. 10.1177/2632010X221075584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Davies NG, Abbott S, Barnard RC, Jarvis CI, Kucharski AJ, Munday JD, Pearson CAB, Russell TW, Tully DC, Washburne AD, et al. (2021). Estimated transmissibility and impact of SARSCoV-2 lineage B.1.1.7 in England. Science 372. 10.1126/science.abg3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tegally H, Wilkinson E, Giovanetti M, Iranzadeh A, Fonseca V, Giandhari J, Doolabh D, Pillay S, San EJ, Msomi N, et al. (2021). Detection of a SARS-CoV-2 variant of concern in South Africa. Nature 592, 438–443. 10.1038/s41586-021-03402-9. [DOI] [PubMed] [Google Scholar]
  • 33.Planas D, Veyer D, Baidaliuk A, Staropoli I, Guivel-Benhassine F, Rajah MM, Planchais C, Porrot F, Robillard N, Puech J, et al. (2021). Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature 596, 276–280. 10.1038/s41586-021-03777-9. [DOI] [PubMed] [Google Scholar]
  • 34.Wu A, Peng Y, Huang B, Ding X, Wang X, Niu P, Meng J, Zhu Z, Zhang Z, Wang J, et al. (2020). Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China. Cell Host Microbe 27, 325–328. 10.1016/j.chom.2020.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Huang Y, Yang C, Xu XF, Xu W, and Liu SW (2020). Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 41, 1141–1149. 10.1038/s41401-020-0485-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Maxmen A (2021). One million coronavirus sequences: popular genome site hits mega milestone. Nature 593, 21. 10.1038/d41586-021-01069-w. [DOI] [PubMed] [Google Scholar]
  • 37.Andersen KG, Rambaut A, Lipkin WI, Holmes EC, and Garry RF (2020). The proximal origin of SARS-CoV-2. Nat Med 26, 450–452. 10.1038/s41591-020-0820-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Khailany RA, Safdar M, and Ozaslan M (2020). Genomic characterization of a novel SARS-CoV-2. Gene Rep, 100682. 10.1016/j.genrep.2020.100682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Girma A (2023). The Many Mutations of the COVID-19 Variant: Current Perspectives on EG.5/Eris. Environ Health Insights 17, 11786302231217805. 10.1177/11786302231217805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.He X, Hong W, Pan X, Lu G, and Wei X (2021). SARS-CoV-2 Omicron variant: Characteristics and prevention. MedComm (2020) 2, 838–845. 10.1002/mco2.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Jabraeilian H, and Jamali Y (2024). Omicron vs. the rest: Assessing the competitive dynamics and coinfection scenarios of COVID-19 strains on a social network. PLoS One 19, e0287623. 10.1371/journal.pone.0287623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Carabelli AM, Peacock TP, Thorne LG, Harvey WT, Hughes J, Consortium C-GU, Peacock SJ, Barclay WS, de Silva TI, Towers GJ, and Robertson DL (2023). SARS-CoV-2 variant biology: immune escape, transmission and fitness. Nat Rev Microbiol 21, 162–177. 10.1038/s41579-022-00841-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Tao K, Tzou PL, Nouhin J, Gupta RK, de Oliveira T, Kosakovsky Pond SL, Fera D, and Shafer RW (2021). The biological and clinical significance of emerging SARSCoV-2 variants. Nat Rev Genet 22, 757–773. 10.1038/s41576-021-00408-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Volz E, Mishra S, Chand M, Barrett JC, Johnson R, Geidelberg L, Hinsley WR, Laydon DJ, Dabrera G, O'Toole A, et al. (2021). Assessing transmissibility of SARSCoV-2 lineage B.1.1.7 in England. Nature 593, 266–269. 10.1038/s41586-021-03470-x. [DOI] [PubMed] [Google Scholar]
  • 45.Laffeber C, de Koning K, Kanaar R, and Lebbink JHG (2021). Experimental Evidence for Enhanced Receptor Binding by Rapidly Spreading SARS-CoV-2 Variants. J Mol Biol 433, 167058. 10.1016/j.jmb.2021.167058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ramesh S, Govindarajulu M, Parise RS, Neel L, Shankar T, Patel S, Lowery P, Smith F, Dhanasekaran M, and Moore T (2021). Emerging SARS-CoV-2 Variants: A Review of Its Mutations, Its Implications and Vaccine Efficacy. Vaccines (Basel) 9. 10.3390/vaccines9101195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Wang P, Casner RG, Nair MS, Wang M, Yu J, Cerutti G, Liu L, Kwong PD, Huang Y, Shapiro L, and Ho DD (2021). Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization. Cell Host Microbe 29, 747–751 e744. 10.1016/j.chom.2021.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Shiehzadegan S, Alaghemand N, Fox M, and Venketaraman V (2021). Analysis of the Delta Variant B.1.617.2 COVID-19. Clin Pract 11, 778–784. 10.3390/clinpract11040093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Cao Y, Wang J, Jian F, Xiao T, Song W, Yisimayi A, Huang W, Li Q, Wang P, An R, et al. (2022). Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature 602, 657–663. 10.1038/s41586-021-04385-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Callaway E (2021). Heavily mutated Omicron variant puts scientists on alert. Nature 600, 21. 10.1038/d41586-021-03552-w. [DOI] [PubMed] [Google Scholar]
  • 51.Bhattacharyya RP, and Hanage WP (2022). Challenges in Inferring Intrinsic Severity of the SARS-CoV-2 Omicron Variant. N Engl J Med 386, e14. 10.1056/NEJMp2119682. [DOI] [PubMed] [Google Scholar]
  • 52.Abbasi J (2021). Researchers Tie Severe Immunosuppression to Chronic COVID-19 and Virus Variants. JAMA 325, 2033–2035. 10.1001/jama.2021.7212. [DOI] [PubMed] [Google Scholar]
  • 53.Liu L, Iketani S, Guo Y, Chan JF, Wang M, Liu L, Luo Y, Chu H, Huang Y, Nair MS, et al. (2022). Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2. Nature 602, 676–681. 10.1038/s41586-021-04388-0. [DOI] [PubMed] [Google Scholar]
  • 54.Kannan SR, Spratt AN, Sharma K, Chand HS, Byrareddy SN, and Singh K (2022). Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing antibodies. J Autoimmun 126, 102779. 10.1016/j.jaut.2021.102779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Kupferschmidt K (2021). Where did 'weird' Omicron come from? Science 374, 1179. 10.1126/science.acx9738. [DOI] [PubMed] [Google Scholar]
  • 56.Levin MJ, Ustianowski A, De Wit S, Launay O, Avila M, Templeton A, Yuan Y, Seegobin S, Ellery A, Levinson DJ, et al. (2022). Intramuscular AZD7442 (Tixagevimab-Cilgavimab) for Prevention of Covid-19. N Engl J Med 386, 2188–2200. 10.1056/NEJMoa2116620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Dong J, Zost SJ, Greaney AJ, Starr TN, Dingens AS, Chen EC, Chen RE, Case JB, Sutton RE, Gilchuk P, et al. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nat Microbiol 6, 1233–1244. 10.1038/s41564-021-00972-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Shi G, Li T, Lai KK, Johnson RF, Yewdell JW, and Compton AA (2024). Omicron Spike confers enhanced infectivity and interferon resistance to SARS-CoV-2 in human nasal tissue. Nat Commun 15, 889. 10.1038/s41467-024-45075-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Wang Q, Zhang Y, Wu L, Niu S, Song C, Zhang Z, Lu G, Qiao C, Hu Y, Yuen KY, et al. (2020). Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell. 10.1016/j.cell.2020.03.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Niu S, Zhao Z, Liu Z, Rong X, Chai Y, Bai B, Han P, Shang G, Ren J, Wang Y, et al. (2024). Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs. J Virol, e0115723. 10.1128/jvi.01157-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Chaouat AE, Brizic I, Kucan Brlic P, Atari N, Kliker L, Alfi O, Mandelboim M, Wolf D, Tafish L, Kol I, et al. (2022). Anti-human ACE2 antibody neutralizes and inhibits virus production of SARS-CoV-2 variants of concern. iScience 25, 104935. 10.1016/j.isci.2022.104935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Hikmet F, Mear L, Edvinsson A, Micke P, Uhlen M, and Lindskog C (2020). The protein expression profile of ACE2 in human tissues. Mol Syst Biol 16, e9610. 10.15252/msb.20209610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Bhat-Nakshatri P, Gao H, Sheng L, McGuire PC, Xuei X, Wan J, Liu Y, Althouse SK, Colter A, Sandusky G, et al. (2021). A single-cell atlas of the healthy breast tissues reveals clinically relevant clusters of breast epithelial cells. Cell Rep Med 2, 100219. 10.1016/j.xcrm.2021.100219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Chen J, Cheung F, Shi R, Zhou H, Lu W, and Consortium, C.H.I. (2018). PBMC fixation and processing for Chromium single-cell RNA sequencing. J Transl Med 16, 198. 10.1186/s12967-018-1578-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.De Micheli AJ, Spector JA, Elemento O, and Cosgrove BD (2020). A reference single-cell transcriptomic atlas of human skeletal muscle tissue reveals bifurcated muscle stem cell populations. Skelet Muscle 10, 19. 10.1186/s13395-020-00236-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Guo J, Grow EJ, Mlcochova H, Maher GJ, Lindskog C, Nie X, Guo Y, Takei Y, Yun J, Cai L, et al. (2018). The adult human testis transcriptional cell atlas. Cell Res 28, 1141–1157. 10.1038/s41422-018-0099-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.He S, Wang LH, Liu Y, Li YQ, Chen HT, Xu JH, Peng W, Lin GW, Wei PP, Li B, et al. (2020). Single-cell transcriptome profiling of an adult human cell atlas of 15 major organs. Genome Biol 21, 294. 10.1186/s13059-020-02210-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Hildreth AD, Ma F, Wong YY, Sun R, Pellegrini M, and O'Sullivan TE (2021). Single-cell sequencing of human white adipose tissue identifies new cell states in health and obesity. Nat Immunol 22, 639–653. 10.1038/s41590-021-00922-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Liao J, Yu Z, Chen Y, Bao M, Zou C, Zhang H, Liu D, Li T, Zhang Q, Li J, et al. (2020). Single-cell RNA sequencing of human kidney. Sci Data 7, 4. 10.1038/s41597-019-0351-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, Winter H, Meister M, Veith C, Boots AW, et al. (2020). SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J 39, e105114. 10.15252/embj.20105114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.MacParland SA, Liu JC, Ma XZ, Innes BT, Bartczak AM, Gage BK, Manuel J, Khuu N, Echeverri J, Linares I, et al. (2018). Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations. Nat Commun 9, 4383. 10.1038/s41467-018-06318-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Menon M, Mohammadi S, Davila-Velderrain J, Goods BA, Cadwell TD, Xing Y, Stemmer-Rachamimov A, Shalek AK, Love JC, Kellis M, and Hafler BP (2019). Single-cell transcriptomic atlas of the human retina identifies cell types associated with agerelated macular degeneration. Nat Commun 10, 4902. 10.1038/s41467-019-12780-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Parikh K, Antanaviciute A, Fawkner-Corbett D, Jagielowicz M, Aulicino A, Lagerholm C, Davis S, Kinchen J, Chen HH, Alham NK, et al. (2019). Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 567, 49–55. 10.1038/s41586-019-0992-y. [DOI] [PubMed] [Google Scholar]
  • 74.Qadir MMF, Alvarez-Cubela S, Klein D, van Dijk J, Muniz-Anquela R, Moreno-Hernandez YB, Lanzoni G, Sadiq S, Navarro-Rubio B, Garcia MT, et al. (2020). Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche. Proc Natl Acad Sci U S A 117, 10876–10887. 10.1073/pnas.1918314117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Sole-Boldo L, Raddatz G, Schutz S, Mallm JP, Rippe K, Lonsdorf AS, Rodriguez-Paredes M, and Lyko F (2020). Single-cell transcriptomes of the human skin reveal agerelated loss of fibroblast priming. Commun Biol 3, 188. 10.1038/s42003-020-0922-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Tabula Sapiens C, Jones RC, Karkanias J, Krasnow MA, Pisco AO, Quake SR, Salzman J, Yosef N, Bulthaup B, Brown P, et al. (2022). The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science 376, eabl4896. 10.1126/science.abl4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Ulrich ND, Shen YC, Ma Q, Yang K, Hannum DF, Jones A, Machlin J, Randolph JF Jr., Smith YR, Schon SB, et al. (2022). Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq. Dev Cell 57, 914–929 e917. 10.1016/j.devcel.2022.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Vento-Tormo R, Efremova M, Botting RA, Turco MY, Vento-Tormo M, Meyer KB, Park JE, Stephenson E, Polanski K, Goncalves A, et al. (2018). Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 563, 347–353. 10.1038/s41586-018-0698-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.Wagner M, Yoshihara M, Douagi I, Damdimopoulos A, Panula S, Petropoulos S, Lu H, Pettersson K, Palm K, Katayama S, et al. (2020). Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. Nat Commun 11, 1147. 10.1038/s41467-020-14936-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 80.Wang L, Yu P, Zhou B, Song J, Li Z, Zhang M, Guo G, Wang Y, Chen X, Han L, and Hu S (2020). Single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function. Nat Cell Biol 22, 108–119. 10.1038/s41556-019-0446-7. [DOI] [PubMed] [Google Scholar]
  • 81.Wang W, Vilella F, Alama P, Moreno I, Mignardi M, Isakova A, Pan W, Simon C, and Quake SR (2020). Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle. Nat Med 26, 1644–1653. 10.1038/s41591-020-1040-z. [DOI] [PubMed] [Google Scholar]
  • 82.Wang Y, Song W, Wang J, Wang T, Xiong X, Qi Z, Fu W, Yang X, and Chen YG (2020). Single-cell transcriptome analysis reveals differential nutrient absorption functions in human intestine. J Exp Med 217. 10.1084/jem.20191130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Gurwitz D (2020). Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics. Drug Dev Res. 10.1002/ddr.21656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84.Tuekprakhon A, Nutalai R, Dijokaite-Guraliuc A, Zhou D, Ginn HM, Selvaraj M, Liu C, Mentzer AJ, Supasa P, Duyvesteyn HME, et al. (2022). Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell 185, 2422–2433 e2413. 10.1016/j.cell.2022.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Dejnirattisai W, Huo J, Zhou D, Zahradnik J, Supasa P, Liu C, Duyvesteyn HME, Ginn HM, Mentzer AJ, Tuekprakhon A, et al. (2022). SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Cell 185, 467–484 e415. 10.1016/j.cell.2021.12.046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Raghav PK, Raghav A, Lathwal A, Saxena A, Mann Z, Sengar M, and Rajalingam R (2023). Experimental and clinical data analysis for identification of COVID-19 resistant ACE2 mutations. Sci Rep 13, 2351. 10.1038/s41598-022-20773-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Chen Y, Zhang YN, Yan R, Wang G, Zhang Y, Zhang ZR, Li Y, Ou J, Chu W, Liang Z, et al. (2021). ACE2-targeting monoclonal antibody as potent and broad-spectrum coronavirus blocker. Signal Transduct Target Ther 6, 315. 10.1038/s41392-021-00740-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Ou J, Zhang Y, Wang Y, Zhang Z, Wei H, Yu J, Wang Q, Wang G, Zhang B, and Wang C (2022). ACE2-Targeting antibody suppresses SARS-CoV-2 Omicron and Delta variants. Signal Transduct Target Ther 7, 43. 10.1038/s41392-022-00913-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2006940-supplement-1.docx (328KB, docx)

Data Availability Statement

4G8C11 has been licensed to Biolegend (San Diego, CA) and will be available to the research community.

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