Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2.

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets-the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity-and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2-specific T-cells.

BACKGROUND: Rapid tests to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. METHODS: Using a rapid whole blood assay requiring a minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T-cell responses in 31 healthcare workers using flow cytometry. RESULTS: 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T-cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR-negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce interferon (IFN)-γ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, coexpressing IFN-γ and tumour necrosis factor-α and also Granzyme B. CONCLUSIONS: This proof-of-concept study presents a scalable alternative to peripheral blood mononuclear cell-based assays to enumerate and phenotype SARS-CoV-2-responding T-cells, thus representing a practical tool to monitor adaptive immunity due to natural infection or vaccine trials.

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

Conditional Regulation of Gene Expression by Ligand-Induced Occlusion of a MicroRNA Target Sequence.

RNA switches that modulate gene expression with small molecules have a number of scientific and clinical applications. Here, we describe a novel class of small regulatory on switches based on the ability of a ligand-bound aptamer to promote stem formation between a microRNA target sequence (miR-T) and a complementary competing strand. Two on switch architectures employing this basic concept were evaluated, differing in the location of a tetracycline aptamer and the region of a miR-21 target sequence (miR-21-T) masked by its competing strand. Further optimizations of miR-21-T and its competing strand resulted in tetracycline-regulated on switches that induced luciferase expression by 19-fold in HeLa cells. A similar switch design based on miR-122-T afforded 7-fold regulation when placed in tandem, indicating that this approach can be extended to additional miR-T. Optimized on switches introduced into adeno-associated virus (AAV) vectors afforded 10-fold regulation of two antiviral proteins in AAV-transduced cells. Our data demonstrate that small-molecule-induced occlusion of a miR-T can be used to conditionally regulate gene expression in mammalian cells and suggest that regulatory switches built on this principle can be used to dose expression of an AAV transgene.

Inhibition of Middle East respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody.

We identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also significantly inhibited infection. These findings indicate that both 2F9 and YS110 are potential therapeutic agents for MERS-CoV infection. YS110, in particular, is a good candidate for immediate testing as a therapeutic modality for MERS.

The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies.

The spike (S) protein of the recently emerged human Middle East respiratory syndrome coronavirus (MERS-CoV) mediates infection by binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). Here we mapped the receptor binding domain in the S protein to a 231-amino-acid fragment (residues 358 to 588) by evaluating the interaction of spike truncation variants with receptor-expressing cells and soluble DPP4. Antibodies to this domain--much less so those to the preceding N-terminal region--efficiently neutralize MERS-CoV infection.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

Predicting the efficacy of COVID-19 convalescent plasma donor units with the Lumit Dx anti-receptor binding domain assay.

BACKGROUND: The novel coronavirus SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.5 million people, but no cure exists. Although passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable therapeutic option, the selection of optimal units for therapy in a timely fashion remains a barrier. STUDY DESIGN AND METHODS: Since virus neutralization is a necessary characteristic of plasma that can benefit recipients, the neutralizing titers of plasma samples were measured using a retroviral-pseudotype assay. Binding antibody titers to the spike (S) protein were also determined by a clinically available serological assay (Ortho-Vitros total IG), and an in-house ELISA. The results of these assays were compared to a measurement of antibodies directed to the receptor binding domain (RBD) of the SARS-CoV2 S protein (Promega Lumit Dx). RESULTS: All measures of antibodies were highly variable, but correlated, to different degrees, with each other. However, the anti-RBD antibodies correlated with viral neutralizing titers to a greater extent than the other antibody assays. DISCUSSION: Our observations support the use of an anti-RBD assay such as the Lumit Dx assay, as an optimal predictor of the neutralization capability of CCP.

Donor Anti-Spike Immunity is Related to Recipient Recovery and Can Predict the Efficacy of Convalescent Plasma Units.

BACKGROUND: The novel coronavirus, SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.31 million people within the last year and yet no cure exists. Whereas passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable option, selection of optimal units for therapy and lack of clear therapeutic benefit from transfusion remain as barriers to the use of CCP. STUDY DESIGN AND METHODS: To identify plasma that is expected to benefit recipients, we measured anti-SARS-CoV2 antibody levels using clinically available serological assays and correlated with the neutralizing activity of CCP from donors. Neutralizing titer of plasma samples was measured by assaying infectivity of SARS-CoV-2 spike protein pseudotyped retrovirus particles in the presence of dilutions of plasma samples. We also used this assay to identify evidence of passive transfusion of neutralizing activity in CCP recipients. RESULTS: Viral neutralization and anti-spike protein antibodies in 109 samples from 87 plasma donors were highly varied but modestly correlated with each other. Recipients who died of COVID-19 were found to have been transfused with units with lower anti-spike antibody levels and neutralizing activity. Passive transfer of neutralization activity was documented in 62% of antibody naive plasma recipients. CONCLUSIONS: Since viral neutralization is the goal of CCP transfusion, our observations not only support the use of anti-spike SARS-CoV2 serology tests to identify beneficial CCP units, but also support the therapeutic value of convalescent plasma with high titers of anti-spike antibodies.

An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020.

CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome.

The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity.

SARS coronavirus 2 (SARS-CoV-2) isolates encoding a D614G mutation in the viral spike (S) protein predominate over time in locales where it is found, implying that this change enhances viral transmission. We therefore compared the functional properties of the S proteins with aspartic acid (S D614 ) and glycine (S G614 ) at residue 614. We observed that retroviruses pseudotyped with S G614 infected ACE2-expressing cells markedly more efficiently than those with S D614 . This greater infectivity was correlated with less S1 shedding and greater incorporation of the S protein into the pseudovirion. Similar results were obtained using the virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, S G614 did not bind ACE2 more efficiently than S D614 , and the pseudoviruses containing these S proteins were neutralized with comparable efficiencies by convalescent plasma. These results show S G614 is more stable than S D614 , consistent with epidemiological data suggesting that viruses with S G614 transmit more efficiently.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells.

Rapid tests to evaluate SARS-CoV-2-specific T cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. Using a rapid whole blood assay requiring minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T cell responses in 31 healthcare workers, using flow cytometry. 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce IFNγ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, co-expressing IFNγ and TNFα and also Granzyme B. This proof of concept study presents a scalable alternative to PBMC-based assays to enumerate and phenotype SARS-CoV-2-responding T cells, thus representing a practical tool to monitor adaptive immunity in vaccine trials. SUMMARY: In this proof of concept study, we show that SARS-CoV-2 T cell responses are easily detectable using a rapid whole blood assay requiring minimal blood volume. Such assay could represent a suitable tool to monitor adaptive immunity in vaccine trials.

An engineered receptor-binding domain improves the immunogenicity of multivalent SARS-CoV-2 vaccines.

The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.

A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo.

Widespread use of gene therapy technologies is limited in part by the lack of small genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components1-5. In this study, we engineered a class of type III hammerhead ribozymes to develop RNA switches that are highly efficient at cis-cleaving mammalian mRNAs and showed that they can be tightly regulated by a steric-blocking antisense oligonucleotide. Our variant ribozymes enabled in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent and up to 223-fold regulation of protein expression over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease6, we demonstrated regulated expression of physiological levels of erythropoietin with a well-tolerated dose of the inducer oligonucleotide. These small, modular and efficient RNA switches may improve the safety and efficacy of gene therapies and broaden their use.

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity.

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus ) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat ( R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.