Human monoclonal antibody S309 – Cross-neutralization of SARS-CoV and SARS-CoV2


An antibody first identified in a blood sample from a patient who recovered from Severe Acute Respiratory Syndrome in 2003 inhibits related coronaviruses, including the cause of COVID-19.

The antibody, called S309, is now on a fast-track development and testing path at Vir Biotechnology in the next step toward possible clinical trials.

Laboratory research findings on the S309 antibody are reported in the May 18 edition of Nature. The title of the paper, available here, is: “Cross-neutralization of SARS-CoV and SARS-CoV2 by a human monoclonal antibody.”

The senior authors on the paper are David Veesler, assistant professor of biochemistry at the University of Washington School of Medicine, and Davide Corti of Humabs Biomed SA, a subsidiary of Vir.

The lead authors are Dora Pinto and Martina Beltramello of Humabs, as well as Young-Jun Park and Lexi Walls, research scientists in the Veesler lab, which for several years has been studying the structure and function of the infection mechanisms on a variety of coronaviruses

“We still need to show that this antibody is protective in living systems, which has not yet been done,” Veesler said.

“Right now there are no approved tools or licensed therapeutics proven to fight against the coronavirus that causes COVID-19,” he added. If the antibody is shown to work against the novel coronavirus in people, it could become part of the pandemic armamentarium.

Veesler said that his lab is not the only one seeking neutralizing antibodies for COVID 19 treatment. What makes this antibody different is that its search did not take place in people who had COVID-19, but in someone who had been infected 17 years ago during a SARS epidemic.

“This is what allowed us to move so fast compared to other groups,” Veesler said.

The scientists identified several monoclonal antibodies of interest from memory B cells of the SARS survivor.

Memory B cells form following an infectious illness. Their lineage can last, sometimes for life. They usually remember a pathogen, or one similar to it, that the body has ousted in the past, and launch an antibody defense against a re-infection.

Several of the antibodies from the SARS survivor’s memory B cells are directed at a protein structure on coronaviruses. This structure is critical to the coronaviruses’ ability to recognize a receptor on a cell, fuse to it, and inject their genetic material into the cell. This infectivity machinery is located in the spikes that crown the coronavirus.

The S309 antibody is particularly potent at targeting and disabling the spike protein that promotes the coronavirus entry into cells.

It was able to neutralize SARS CoV-2 by engaging with a section of the spike protein nearby the attachment site to the host cell.

Through their cryo-electronmicroscopy studies and binding assays, the researchers learned that the S309 antibody recognizes a binding site on the coronavirus that is conserved across many sarbocoviruses, not just the SARS and COVID-19 viruses.

That is probably why this antibody, instead of being single-minded, is able to act against related coronaviruses.

Credit: UW Medicine.

Combining the S309 antibody with other, though weaker, antibodies identified in the recovered SARS patient enhanced the neutralization of the COVID-19 coronavirus.

This multiple antibody cocktail approach might help limit the coronavirus’ ability to form mutants capable of escaping a single-ingredient antibody treatment, according to the researchers.

The scientists noted that they hope these initial results pave the way for using the S309 antibody, alone or in a mixture, as a preventive measure for people at high-risk of exposure to the COVID-19 coronavirus or as post-exposure therapy to limit or treat severe illness.

Other research institutions participating in this research include Institut Pasteur in France, the Università della Svizzera Italiana in Switzerland, and Washington University in St. Louis, Missouri.

SARS-CoV-2 biology and replication machinery: genome organisation and viral proteins expressed during the infection cycle

SARS-CoV-2 is a positive-sense, single-stranded RNA beta-coronavirus with a 30 kilobase genome that encodes viral proteins in up to 14 open reading frames (Orfs).1,2 At the 5’ end of the genome, a single

Orf encodes a polyprotein that auto-proteolytically cleaves into 16 non-structural proteins (Nsp1-16) that form the replicase-transcriptase complex.2 The 16 protein replicase-transcriptase consists of multiple enzymes essential to viral genome replication, including the viral RNA-dependent RNA polymerase and other enzymes such as endo- and exonucleases essential to nucleic acid metabolism.2

It is suspected that as many as 13 Orfs are expressed from the 3’ end of the viral genome, including four major viral structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N). Structural proteins of SARS- CoV-2 form the viral capsid that encapsulates the genome, while also facilitating entry to human cells through the human angiotensin converting enzyme 2 (ACE2) receptor.2

Lessons from SARS-CoV-1 and MERS-CoV: conserved viral replication machinery and identification of host pathways commonly utilised in coronavirus infection

Despite our limited knowledge of SARS-CoV-2, pathogenic coronaviruses have been widely studied since the SARS coronavirus outbreak of 2003 and the MERS (Middle East Respiratory Syndrome) coronavirus outbreak which began in 2012.

While there are differences in both infectivity and mortality rates between SARS/MERS and the current SARS-CoV-2 virus, the genome size (30 kb) and organisation of replicase-transcriptase and structural protein Orfs used in all three viruses is highly conserved.3 

Therefore, SARS and MERS research has already helped in identifying potential viral and host drug targets to block coronavirus replication.  

Notably, research on MERS-CoV infection has identified host autophagy pathways, which includes mTOR-PI3K signalling, as essential host components in coronavirus replication.5 

The host ubiquitin system also plays an important role in MERS-CoV utilisation of autophagy pathways during infection and research has shown that blocking specific kinases (SKP2) important in autophagy decreases MERS- CoV replication 28,000 fold in vitro.4

Other host pathways determined to be highly important in SARS and MERS-CoV replication include vesicle trafficking within the endoplasmic reticulum of host cells. It has been determined that the replicase-transcriptase machinery of the coronavirus assembles at the host endoplasmic reticulum (ER) and viral structural proteins assemble within the host ER, making it an essential cellular component and potential drug target to block both viral genome replication and capsid assembly in the formation of new virus particles during infection.3

Proteomic-chemoinformatic approach to COVID-19 host-pathogen drug target identification

Scientists in the Krogan lab used an affinity purification mass spectrometry proteomics approach to first screen all SARS-CoV-2 proteins against human cell lines to determine high confidence viral-human protein interactions.2 

With the genome sequence of SARS-CoV-2 available, the Krogan Lab cloned, tagged and expressed 26 of 29 viral proteins in HEK293 cell lines.2 Viral proteins were then affinity purified using streptavidin magnetic beads and then after overnight digestion the purified proteins were sequenced using protein mass spectrometry.2 

In total, 332 high confidence SARS-CoV-2 human protein-protein interactions were identified. Upon completion of the SARS-CoV-2 human protein interactome, chemoinformatics database searches were completed to identify all FDA-approved compounds or compounds in clinical and pre-clinical investigation that target host proteins identified in the screen.2 

Host pathway in proteome interaction screenCorresponding SARS-CoV-2 proteins interacting with host pathway
DNA replicationNsp1
Epigenetic and gene expression regulatorsNsp5, Nsp8, Nsp13, E
Vesicle traffickingNsp6, Nsp7, Nsp10, Nsp13, Nsp15, Orf3a, E, Orf8
Lipid modificationSpike
RNA processing and regulationNsp8, N
Ubiquitin ligasesOrf10
Host signalingNsp8, Nsp13, N, Orf9b
Nuclear transport machineryNsp9, Nsp15, Orf6
CytoskeletonNsp1, Nsp13
MitochondriaNsp4, Nsp8, Orf9c
Extracellular matrixNsp9
Source: Gordon, D et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug Repurposing bioRxiv preprint doi:

COVID-19 targets and pathways identified with existing FDA-approved drugs or compounds under pre-clinical investigation

The study’s authors identified 66 human proteins in host pathways targeted by 69 existing FDA-approved drugs or compounds currently being investigated.

Notable host pathways shown to interact with SARS-CoV-2 during its replication include antiviral pathways in the innate immune response such as the stress granule protein G3BP1, a known antiviral protein that induces the innate immune antiviral response.

This led the authors to hypothesise SARS-CoV-2 may have mechanisms to inhibit host innate antiviral immunity.2 Another important pathway the authors noted was the host ubiquitin system and the novel Orf10 of SARS-CoV-2 interacting with multiple members of the Culin 2 E3 Ligase E3 ligase complex.2 

Mammalian viruses often utilise the host ubiquitin system in viral replication, leading the authors to suggest targeting the ubiquitin system with compounds such as the small molecule Pevonedistat in future pre-clinical investigation.2

With the 66 putative drug targets identified, the authors are now testing all 69 compounds in pre-clinical SARS-CoV-2 infection assays to determine their efficacy in blocking viral replication,2 aiding the global race to identify effective therapies to combat the current COVID-19 pandemic.

Existing drug classes targeting identified COVID19 host pathways
Bromodomain (BRD) inhibitors
CK2 inhibitors
HDAC1/2 inhibitors
mTOR inhibitors
Nuclear export inhibitors
Sigma factor inhibitors
NEK9 inhibitors
CEP250 inhibitor
PPIAIMPDH2 modulator
Translation inhibitors
Viral transcription inhibitors
ACE inhibitors
Serine protease I inhibitors
Inhibitors of mitochondrial translation
Source: Gordon, D et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug Repurposing bioRxiv preprint doi:

Mechanism of S309-mediated neutralization of SARS-CoV-2 and SARS-CoV

The cryoEM structure of S309 bound to SARS-CoV-2 S presented here combined with the structures of SARS-CoV-2 SB and SARS-CoV SB in complex with ACE216-18,36 indicate that the Fab engages an epitope distinct from the receptor-binding motif and would not clash with ACE2 upon binding to S (Figure 3a-b). Biolayer interferometry analysis of S309 Fab or IgG binding to the SARS-CoV-2 SB domain or the S ectodomain trimer confirmed the absence of competition between the mAb and ACE2 for binding to SARS-CoV-2 S (Figure 3c and Extended Data Fig. 8).

Figure 3:
Mechanism of neutralization of S309 mAb.
a-b, Ribbon diagrams of S309 and ACE2 bound to SARS-CoV-2 SB. This composite model was generated using the SARS-CoV-2 S/S309 cryoEM structure reported here and a crystal structure of SARS-CoV-2 S bound to ACE216. c, Competition of S309 or S230 mAbs with ACE2 to bind to SARS-CoV SB (left panel) and SARS-CoV-2 SB (right panel). ACE2 was immobilized at the surface of biosensors before incubation with SB domain alone or SB precomplexed with mAbs. The vertical dashed line indicates the start of the association of mAb-complexed or free SB to solid-phase ACE2. d, Neutralization of SARS-CoV-MLV by S309 IgG1 or S309 Fab, plotted in nM (means ±SD is shown, one out of two experiments is shown). e, mAb-mediated ADCC using primary NK effector cells and SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of 3-4 donors genotyped for their FcγRIIIa (mean±SD, from two independent experiments). f, Activation of high affinity (V158) or low affinity (F158) FcγRIIIa was measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb). g, mAb-mediated ADCP using Cell Trace Violet-labelled PBMCs as phagocytic cells and PKF67-labelled SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of four donor (mean±SD, from two independent experiments). h, Activation of FcγRIIa measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb).

To further investigate the mechanism of S309-mediated neutralization, we compared side-by-side transduction of SARS-CoV-2-MLV in the presence of either S309 Fab or S309 IgG. Both experiments yielded comparable IC50 values (3.8 and 3.5 nM, respectively), indicating similar potencies for IgG and Fab (Fig. 3d).

However, The S309 IgG reached 100% neutralization, whereas the S309 Fab plateaued at ∼80% neutralization (Fig. 3d). This result indicates that one or more IgG-specific bivalent mechanisms, such as S trimer cross-linking, steric hindrance or aggregation of virions37, may contribute to the ability to fully neutralize pseudovirions.

Fc-dependent effector mechanisms, such as NK-mediated antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) can contribute to viral control in infected individuals.

We observed efficient S309- and S306-mediated ADCC of SARS-CoV-2 S-transfected cells, whereas the other mAbs tested showed limited or no activity (Fig. 3e and Extended Data Fig. 9a).

These findings might be related to distinct binding orientations and/or positioning of the mAb Fc fragment relative to the FcγRIIIa receptors. ADCC was observed only using NK (effector) cells expressing the high-affinity FcγRIIIa variant (V158) but not the low-affinity variant (F158) (Fig. 3e).

These results, which we confirmed using a FcγRIIIa cell reporter assay (Fig. 3f), suggest that S309 Fc engineering could potentially enhance activation of NK cells with the low-affinity FcγRIIIa variant (F158)38.

Macrophage or dendritic cell-mediated ADCP can contribute to viral control by clearing virus and infected cells and by stimulating T cell response via presentation of viral antigens39,40. Similar to the ADCC results, mAbs S309 and S306 showed the strongest ADCP response (Fig. 3g and Extended Data Fig. 8b).

FcγRIIa signaling, however, was only observed for S309 (Fig. 3h). These findings suggest that ADCP by monocytes was dependent on both FcγRIIIa and FcγRIIa engagement. Collectively, these results demonstrate that in addition to potent in vitro neutralization, S309 may leverage additional protective mechanisms in vivo, as previously shown for other antiviral antibodies41,42.

Funding: This study was supported by the National Institute of General Medical Sciences {R01GM120553), National Institute of Allergy and Infectious Diseases (HHSN272201700059C), Pew Biomedical Scholars Award, Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, University of Washington Arnold and Mabel Beckman cryoEM Center, the Pasteur Institute, and the beamline at the Advanced Light Source at Lawrence Berkley National Laboratory. The researchers obtained viral genomic sequences from GISAID’s EpiFlu Database, hosted by the German government.

University of Washington


1 Lu, R et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.  VOLUME 395, ISSUE 10224, P565-574, FEBRUARY 22, 2020

2 Gordon, D et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug Repurposing bioRxiv preprint doi:

3 Kindrachuk, J et al. Coronaviruses: An Overview of Their Replication and Pathogenesis Methods Mol Biol. Author manuscript; available in PMC 2016 Jan 1.

4 Nils C. Gassen. SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection Nat Commun. 2019 Dec 18;10(1):5770. doi: 10.1038/s41467-019-13659-4.

5 Kindrachuk, J et al. Antiviral Potential of ERK/MAPK and PI3K/AKT/mTOR Signaling Modulation for Middle East Respiratory Syndrome Coronavirus Infection as Identified by Temporal Kinome Analysis Antimicrob Agents Chemother. 2015 Feb;59(2):1088-99. doi: 10.1128/AAC.03659-14. Epub 2014 Dec 8.

16 Yan, R. et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444–1448, doi: 10.1126/science.abb2762 (2020)

18 Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature, doi: 10.1038/s41586-020-2180-5 (2020)

36 Li, F., Li, W., Farzan, M. & Harrison, S. C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864–1868, doi: 10.1126/science.1116480 (2005)

37 Klasse, P. J. & Sattentau, Q. J. Occupancy and mechanism in antibody-mediated neutralization of animal viruses. J Gen Virol 83, 2091–2108, doi: 10.1099/0022-1317-83-9-2091 (2002)

38 Wang, X., Mathieu, M. & Brezski, R. J. IgG Fc engineering to modulate antibody effector functions. Protein Cell 9, 63–73, doi: 10.1007/s13238-017-0473-8 (2018).CrossRefGoogle Scholar
39 He, W. et al. Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice. Nat Commun 8, 846, doi: 10.1038/s41467-017-00928-3 (2017).CrossRefGoogle Scholar
40 DiLillo, D. J. & Ravetch, J. V. Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect. Cell 161, 1035–1045, doi: 10.1016/j.cell.2015.04.016 (2015).CrossRefPubMedGoogle Scholar
41 Corti, D. et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333, 850–856, doi: 10.1126/science.1205669 (2011).Abstract/FREE Full TextGoogle Scholar
42 Hessell, A. J. et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature 449, 101–104, doi: 10.1038/nature06106 (2007).

50 Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 14, 290–296, doi: 10.1038/nmeth.4169 (2017)


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