Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs—including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria—could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Coronaviruses are a group of positive-stranded, enveloped RNA viruses that circulate broadly among humans, other mammals, and birds, causing respiratory, enteric, or hepatic diseases (1). In the last two decades, coronaviruses have caused three major outbreaks: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the recent coronavirus disease 2019 (COVID-19) (2, 3). As of December 7, 2020, COVID-19 has already caused 50 million infections, leading to 1 million deaths globally. The pathogen responsible is a novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (4, 5). Phylogenetic and epidemiological analyses suggest that SARS-CoV, MERS-CoV, and SARS-CoV-2 likely originated from bats, with SARS-CoV spreading from bats to palm civets to humans, and MERS-CoV spreading from bats to camel to humans (6). However, the intermediate host of SARS-CoV-2, fueling spillover to humans, remains unknown.The SARS-CoV-2 genome encodes a spike (S) protein, the receptor-binding domain (RBD) of which binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral entry (5, 7). Following binding of ACE2, the S protein is subsequently cleaved by the host transmembrane serine protease 2 (TMPRSS2) to release the spike fusion peptide, promoting virus entry into target cells (7). It has been demonstrated that the interaction of a virus with species-specific receptors is a primary determinant of host tropism and therefore constitutes a major interspecies barrier at the level of viral entry (8). For example, murine ACE2 does not efficiently bind the SARS-CoV or SARS-CoV-2 S protein, hindering viral entry into murine cells; consequently, a human ACE2 transgenic mouse was developed as an in vivo model to study the infection and pathogenesis of these two viruses (9, 10).ACE2 is expressed in a diverse range of species throughout the subphylum Vertebrata. Several recent studies demonstrated that ferrets, cats, dogs, and some nonhuman primates are susceptible to SARS-CoV-2 (11–15). However, the exact host tropism of SARS-CoV-2 remains unknown and it is urgent to identify the putative zoonotic reservoirs to prevent future outbreaks. Numerous studies have predicted ACE2 orthologs/SARS-CoV-2 S binding affinity or energies but lack of support by virus infection experimentation (16–21). In this study, we experimentally assessed ACE2 orthologs from a broad range of species for their ability to support SARS-CoV-2 entry. Our data demonstrate that an evolutionarily diverse set of ACE2 species variants can mediate SARS-CoV-2 entry, suggesting that SARS-CoV-2 has a broad host range at the level of virus entry that may contribute to cross-species transmission and viral evolution.     

Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike

Binding of the spike protein of SARS-CoV-2 to the human angiotensin-converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds to a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.

Angiotensin-converting enzyme 2 (ACE2) is an enzyme that catalyzes the hydrolysis of angiotensin II into angiotensin (1–7) to counterbalance the ACE receptor in blood pressure control (1). A single transmembrane helix anchors ACE2 into the plasma membrane of cells in the lungs, arteries, heart, kidney, and intestines (2). The vasodilatory effect of ACE2 has made it a promising target for drugs treating cardiovascular diseases (3).ACE2 also serves as the entry point for several coronaviruses into cells, including SARS-CoV and SARS-CoV-2 (4–6). The binding of the spike protein of SARS-CoV and SARS-CoV-2 to the peptidase domain (PD) of ACE2 triggers endocytosis and translocation of both the virus and the ACE2 receptor into endosomes within cells (4). The human transmembrane serine protease 2, TMPRSS2, primes spike for efficient cell entry by cleaving its backbone at the boundary between the S1 and S2 subunits or within the S2 subunit (4). The structure of the ACE2 receptor in complex with the SARS-CoV-2 spike receptor binding domain (RBD) (7–9) reveals the major RBD interaction regions as helix H1 (Q24–Q42), a loop in a beta sheet (K353–R357), and the end of helix H2 (L79–Y83). With a 4-Å heavy-atom distance cutoff, 20 residues of ACE2 interact with 17 residues of the RBD, forming a buried interface of ∼1,700 Ų (7).The structure of full-length ACE2 has been resolved in complex with B⁰AT1 (also known as SLC6A19) (9). B⁰AT1 is a sodium-dependent neutral amino acid transporter (10). ACE2 functions as chaperone for B⁰AT1 and is responsible for its trafficking to the plasma membrane of kidney and intestine epithelial cells (11). Although it was speculated that B⁰AT1 prevents ACE2 cleavage by TMPRSS2 and thus could suppress SARS-CoV-2 infection (9, 12), other studies showed that SARS-CoV-2 can infect human small intestinal enterocytes where ACE2 is expected to be in complex with B⁰AT1 (13).Both the ACE2 receptor and the spike protein are heavily glycosylated. Several glycosylation sites are near the binding interface (7, 9, 14, 15). Whereas the focus has largely been on amino acid interactions in the ACE2–spike binding interface (16, 17), the role of glycosylation in binding has been recognized (7, 18–20). The extracellular domain of the ACE2 receptor has seven N-glycosylation sites (N53, N90, N103, N322, N432, N546, and N690) and several O-glycosylation sites (e.g., T730) (9, 14). Among ACE2 glycosylation sites, the only well-characterized position regarding the effect on the spike binding and viral infectivity is N90. It is known from earlier SARS-CoV studies that glycosylation at the N90 position might interfere with virus binding and infectivity (21). Also, recent genetic and biochemical studies showed that mutations of N90, which remove the glycosylation site directly, or of T92, which remove the glycosylation site indirectly by eliminating the glycosylation motif (NXT), increase the susceptibility to SARS-CoV-2 infection (22, 23).We use extensive molecular dynamics (MD) simulations to gain a detailed molecular-level understanding of how ACE2 glycosylation impacts the host–virus interactions. Glycosylation sites N90 and N322 of human ACE2 emerge as major determinants of its binding to SARS-CoV-2 spike. Remarkably, glycans at these sites have opposite effects, interfering with spike binding in one case, and strengthening binding in the other. Our findings provide direct guidance for the design of targeted antibodies and therapeutic inhibitors of viral entry.     

Cross-species recognition of SARS-CoV-2 to bat ACE2

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2–related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

The coronavirus disease 2019 (COVID-19), caused by infection with the novel coronavirus (CoV) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a major threat to global health with an increasing number of infected cases globally, and the end to this pandemic is still full of uncertainties (1–3). Seven CoVs have been reported to infect humans: SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), NL-63, OC43, 229E, HKU1, and the newly emerging SARS-CoV-2. Among the SARS-CoV-2 proteins, the spike (S) protein, which consists of an N-terminal S1 subunit and a C-terminal S2 subunit, is critical for the recognition of host cell receptors and serves as the key determinant of host specificity for CoVs (4). The C-terminal domain of the S1 subunit, also known as the receptor binding domain (RBD), binds to the human angiotensin-converting enzyme 2 (hACE2), the receptor for SARS-CoV and the human coronavirus NL-63 (5–9). Monoclonal antibodies (mAbs) that block the binding between SARS-CoV-2 RBD and ACE2 could efficiently inhibit virus infection in host cells expressing ACE2 (10, 11). Animal studies revealed that a single dose of these neutralizing mAbs showed promising therapeutic efficacy in reducing both viral load and pathological lung damage in hACE2 transgenic mice or rhesus macaques (10, 11). Recently, the structures of the complex between SARS-CoV-2 S protein (or RBD) with hACE2 have been determined, showing similar binding mode to that of SARS-CoV but with enhanced affinity (5–7).Bats are considered as the reservoir host animals of SARS-CoV-2, and several SARS-CoV-2-related CoVs have been identified from bats (12, 13). The binding assay showed that bat ACE2 (bACE2) from Rhinolophus macrotis (bACE2-Rm) bound to SARS-CoV-2 RBD efficiently (14). The entry capacity of SARS-CoV-2 through ACE2 orthologs from 46 bat species was evaluated by virus-host receptor binding and infection assays. The results indicated that although some bACE2 receptors could mediate SARS-CoV-2 entry, there are many bACE2 receptors that do not yet support SARS-CoV-2 entry (15). To date, the genome most closely related to SARS-CoV-2 is RaTG13, which was identified from a Rhinolophus affinis sampled from Yunnan Province in 2013 and had a 92.9% amino acid identity in the S gene (13). RmYN02 was also a coronavirus identified from bat with 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome (12). Yongyi Shen and colleagues (16) reported a coronavirus isolated from Malayan pangolin and shared 90.7% amino acid identity with SARS-CoV-2 in the S proteins. In addition, a coronavirus that showed 97.4% amino acid identity with SARS-CoV-2 in the RBD region was identified from another batch of Malayan pangolin (17). Bioinformatics analysis indicated that bats are the primary reservoir for the SARS-CoV-2 lineage (18). However, the origin of SARS-CoV-2 is full of uncertainties and whether or not there is an intermediate host is still unknown. Moreover, more than 1,400 species of bats have been identified and extensive species diversity among different species of bats has resulted in varied susceptibility for CoVs. Research into the mechanisms of viral entry and virus–host interaction would not only benefit our understanding of this virus but is also important for the design of antivirals and vaccines. However, the structure of bACE2 has not been determined, and the molecular basis of the binding between SARS-CoV-2 S protein and bACE2 has not been well studied.Here, we report that SARS-CoV-2 RBD can bind to bACE2-Rm with substantially lower affinity than that to hACE2, and infection of host cells carrying bACE2-Rm was also investigated with pseudotyped or wild SARS-CoV-2. Interaction mechanisms between SARS-CoV-2 RBD and bACE2-Rm were elucidated by determining the structure of the SARS-CoV-2 RBD and bACE2-Rm complex. The results of this study would broaden our understanding of the receptor binding mechanisms for SARS-CoV-2.     

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 10⁴-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Coronaviruses are enveloped RNA viruses that infect many mammalian and avian species. The current devastating COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has resulted in over 90 million infections and over 2 million deaths worldwide. The severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) are also highly pathogenic human pathogens. SARS-CoV resulted in the SARS epidemic in 2002 with over 8,000 infections and a 10% fatality rate, and MERS-CoV resulted in the MERS epidemic in 2012 with over 877 infections and a ∼36% fatality rate.Entry into a host cell is the critical first step in the viral life cycle. Of the four major structural proteins encoded by the coronavirus genome, the spike protein plays a crucial role in viral attachment, fusion, and entry (1, 2). The spike protein of SARS-CoV-2 is present on the virion surface as a trimer and mediates recognition of human angiotensin-converting enzyme 2 (ACE2) on the surface of host cells, subsequently triggering membrane fusion (3–6). The receptor-binding domain (RBD) localized on the N-terminal subunit (S1) mediates receptor binding and is a target of neutralizing antibodies (7–14). The SARS-CoV-2 RBD spans residues 319 to 541 with the receptor-binding motif (RBM) spanning residues 438 to 506, which contains most of the contacting residues of SARS-CoV-2 that bind to human ACE2 (6, 15). Recently, three SARS-CoV-2 variants of concern, B.1.1.7, B.1.351, and B.1.1.248, are known to carry several mutations within the spike protein. In particular, all three variants share one specific mutation called D614G, which replaced the initial SARS-CoV-2 strain to become the dominant form of the virus circulating globally and is thought to promote transmission of the virus but with no impact on pathogenesis (16). Within the RBD, B.1.1.7 carries N501Y mutation; B.1.351 carries K417N, E484K, and N501Y mutations; and B.1.1.248 carries K417N/T, E484K, and N501Y mutations (17, 18). E484K has recently been demonstrated to reduce virus neutralization for several monoclonal antibodies and polyclonal human sera (19–21). N501 is one of six key contact residues within the RBD, and the N501Y mutation increases binding affinity to human and mouse ACE2 (22, 23). The spike proteins from SARS-CoV-2 and SARS-CoV are ∼80% identical, and both RBDs bind ACE2 (6, 15, 24). Antiviral therapies against human coronaviruses that block receptor engagement and the ability to undergo membrane fusion will effectively inhibit the process of viral entry and block infection (25).Alpacas, llamas, and camels have evolved one of the smallest naturally occurring antigen recognition domains called nanobodies (26). Nanobodies are ∼12 to 15 kDa in size, highly stable across a wide range of pH and temperature, display strong binding affinities to target proteins, and can be expressed with high yields in bacterial, yeast, and mammalian expression systems (27, 28). In particular, due to their stability, nanobodies are highly suited for development as potential bio-inhaled therapies against respiratory diseases (29). Indeed, Ablynx developed an inhaled anti–respiratory syncytial virus nanobody, ALX-0171, which has robust antiviral effects and reduces symptoms of virus infection in animal models (30). Recently, neutralizing nanobodies against SARS-CoV-2 were identified using several approaches, including immunization of llamas, yeast surface display of synthetic nanobodies, and phage display of naïve llama nanobody library or humanized synthetic nanobody library (31–40). These nanobodies were readily engineered into different multivalent forms, fused to Fc domains, and affinity matured to increase neutralization potency (32, 33, 36, 37, 40). These studies also show that nanobodies and their derivatives retain function either upon lyophilization, heat treatment, or aerosolization, suggesting a potential avenue for development of inhaled therapeutics against COVID-19 (32, 33).While most nanobodies show potent neutralization against SARS-CoV-2 using in vitro assays, very few studies have examined the in vivo efficacy of nanobodies for the prevention and treatment for COVID-19 (41, 42). Due to their small size, nanobodies are rapidly cleared through renal elimination, which poses challenges for in vivo studies (43). However, the short half-life of nanobodies can be overcome via fusion to larger proteins such as albumin or to the Fc fragment of IgG (41–43). Furthermore, to the best of our knowledge, no study has examined the potential for nanobody mixtures in the prevention of COVID-19 in vivo, which may be advantageous for controlling more highly infectious variants and reducing the potential for virus escape mutations to develop (7). Due to their unique properties, it will be important to further understand the potential of nanobodies as therapeutics against SARS-CoV-2.By screening nanobody phage display libraries generated from two alpacas immunized with coronavirus spike and RBDs, we identified a collection of nanobodies that bound to RBD with low nanomolar (nM) affinities, inhibited RBD-ACE2 complex formation, and neutralized SARS-CoV-2. X-ray crystallography, cryogenic electron microscopy (cryo-EM), and epitope binning experiments identified several combinations of noncompeting nanobodies as potential antibody mixture combinations. Nanobody-Fc fusions effectively blocked ACE2 receptor engagement with naturally occurring RBD variants present in human populations, showed potent neutralization against wild-type (WT) SARS-CoV-2 and an N501Y D614G variant, and, when used prophylactically, protected mice infected with a SARS-CoV-2 N501Y D614G variant.     

From the Cover: Decoy nanoparticles protect against COVID-19 by concurrently adsorbing viruses and inflammatory cytokines

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need to rapidly develop therapeutic strategies for such emerging viruses without effective vaccines or drugs. Here, we report a decoy nanoparticle against COVID-19 through a powerful two-step neutralization approach: virus neutralization in the first step followed by cytokine neutralization in the second step. The nanodecoy, made by fusing cellular membrane nanovesicles derived from human monocytes and genetically engineered cells stably expressing angiotensin converting enzyme II (ACE2) receptors, possesses an antigenic exterior the same as source cells. By competing with host cells for virus binding, these nanodecoys effectively protect host cells from the infection of pseudoviruses and authentic SARS-CoV-2. Moreover, relying on abundant cytokine receptors on the surface, the nanodecoys efficiently bind and neutralize inflammatory cytokines including interleukin 6 (IL-6) and granulocyte−macrophage colony-stimulating factor (GM-CSF), and significantly suppress immune disorder and lung injury in an acute pneumonia mouse model. Our work presents a simple, safe, and robust antiviral nanotechnology for ongoing COVID-19 and future potential epidemics.

COVID-19 pandemic, caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; also known as 2019-nCoV) (1), has resulted in more than 25 million infections and 840,000 deaths worldwide as of August 31, 2020 (2). Over the past 240 years, there have been several global epidemics caused by emerging and reemerging viruses, such as SARS-CoV, influenza A (H1N1) pdm09 virus, Zika virus, Ebola virus, and, most recently, SARS-CoV-2 (3). Each time, the lack of available drug or vaccine has greatly hindered effective protection against such an emerging viral threat (4). Thus, it remains a grand challenge and is of paramount importance to rapidly develop therapeutic strategies for ongoing COVID-19 or future potential epidemics.Similar to SARS-CoV, the spike protein (S protein) of SARS-CoV-2 plays a vital role in viral infection. The S protein consists of S1 and S2 subunits: The S1 subunit engages human angiotensin converting enzyme II (ACE2) as the entry receptor, while the S2 subunit further facilitates viral fusion and entry (5–7). Responding to viral entry and infection, abundant inflammatory cytokines are up-regulated by macrophages/monocytes to eliminate pathogens and promote tissue repair (8). However, sustainably evaluated levels of inflammatory cytokines, characterized as cytokine release syndrome (CRS or “cytokine storm”), may in turn, exacerbate the inflammatory state and lead to immune dysfunction (9). Clinically, most patients with COVID-19 show mild symptoms, but ∼20% of patients progress to severe pneumonia, septic shock, and/or multiple organ failure owing to the CRS (10). Thus, in addition to vaccine development, approaches that block the viral entry involving ACE2 and treatments that suppress the aberrant inflammatory responses have become major focuses for COVID-19 (11).Several antiviral drugs, including remdesivir, are being actively tested and have shown encouraging effects on the early intervention in SARS-CoV-2 infection (12, 13). However, there are very few drug candidates targeting late-stage infection-associated CRS. Interleukin-6 (IL-6), a proinflammatory cytokine, plays a pivotal role in many immunological diseases (14), and granulocyte−macrophage colony-stimulating factor (GM-CSF) is a myelopoietic growth factor involved in immune regulation (15). Several preclinical and clinical studies have reported that monoclonal antibodies targeting IL-6 and GM-CSF may potentially curb immunopathology caused by SARS-CoV-2 (16, 17), while it remains challenging to suppress CRS owing to the multiplicity of cytokine targets and the complexity of cytokine interactions (18).Recent advances in nanotechnology and materials science, especially in lipid nanoparticles, offer many promising opportunities for infectious diseases (19–25). For instance, engineered liposomes, cell membrane nanosponges, and exosomes have been demonstrated to bind and neutralize bacterial toxin (19, 20, 26). Additionally, we have recently shown that biomimetic synthetic strategies involving synchronous synthesis and display of proteins on cell surface enable efficient development of cellular nanovesicles displaying proteins with native orientation, structure, and activity (22, 25, 27). Therefore, we hypothesize that we can genetically engineer ACE2 on cell surface and efficiently produce cellular nanovesicles displaying ACE2 to compete with host cells for SARS-CoV-2 binding (28–30). More importantly, recent reports involving cell membrane-coated nanoparticles for neutralization of broad-spectrum cytokines further promise the employment of engineered cellular nanovesicles for COVID-19 (18, 31).Here, we develop an engineered cell membrane nanodecoy for COVID-19. Briefly, the nanodecoys were established in three steps: 1) genetically engineering ACE2 on human embryonic kidney 293T cells, 2) collecting cell membrane nanovesicles from engineered 293T/ACE2 cells and human myeloid mononuclear THP-1 cells, and 3) fusing the resulting two nanovesicles (Fig. 1A). In this design, the nanodecoys inherit abundant ACE2 and cytokine receptors from the source cells, enabling effective intervention of COVID-19 by concurrently neutralizing viruses and inflammatory cytokines (Fig. 1 B and C).Open in a separate windowFig. 1.Schematic illustration of nanodecoys against COVID-19. (A) Preparation of nanodecoys by fusing cellular membrane nanovesicles derived from genetically edited 293T/ACE2 and THP-1 cells. The nanodecoys, displaying abundant ACE2 and cytokine receptors, compete with host cells and protect them from COVID-19 by neutralizing (B) SARS-CoV-2 and (C) inflammatory cytokines, such as IL-6 and GM-CSF.     

Host barriers to SARS-CoV-2 demonstrated by ferrets in a high-exposure domestic setting

Ferrets (Mustela putorius furo) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic disease, one of whom was confirmed positive for SARS-CoV-2. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on viral and antibody assays. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein Spike (S) mutations associated with mustelids. While we found evidence that angiotensin-converting enzyme II provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin cleavage efficiency. These data support the idea that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. We propose two mechanistically grounded hypotheses for mustelid host adaptation of SARS-CoV-2, with possible effects that require additional investigation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is a zoonotic member of Coronaviridae that emerged in 2019 as a major viral pandemic (1). As of February 2021, there have been ∼102 million confirmed COVID-19 cases globally and ∼2.2 million deaths (2). SARS-CoV-2 uses angiotensin I converting enzyme-2 (ACE2) as its primary cellular receptor for host entry and infection (3–5). In silico analyses of ACE2 genes in diverse mammalian species show that residues important to viral binding are moderately conserved between humans and several domestic animals, and a broad range of species have been demonstrated to be permissive to infection in vitro and in vivo (6–10).It is not yet known whether natural infection of animals plays a role in public health epidemiology or has the potential to establish endemic reservoirs and threaten wildlife. SARS-CoV-2 has been observed to be capable of natural human-to-animal reverse zoonoses, transmitting from infected individuals into mink (11), dogs (12), and felines (13–15). American mink (Neovison vison) are currently the only species observed to have natural human-to-animal spillover and onward transmission (11). To date, at least 27 mink farms in The Netherlands, Spain, Denmark, and United States have reported outbreaks, including at least one probable case of mink-to-human transmission (16, 17).SARS-CoV-2 has also been shown to productively infect several species, including ferrets and domestic cats, in vivo (9, 10, 18, 19). Ferrets (Mustela putorius furo) are of special relevance to laboratory studies of respiratory viruses like Influenza A virus and recapitulate clinical pathophysiological aspects of human disease. Given their susceptibility to experimental infection and onward transmission via direct and indirect contact, ferrets have been proposed as an animal model to study SARS-CoV-2 transmission. Based on in vivo data, we expect all naïve ferrets in direct contact with an infected ferret will 1) become infected, 2) have measurable viral shedding or RNA via oral swabs up to 19 d postinfection, and 3) seroconvert with measurable antibodies against SARS-CoV-2 receptor binding domain (RBD) (18, 19).In March 2020, during the first wave of the SARS-CoV-2/COVID-19 pandemic in the New England area, we developed a rapid response study to investigate the potential for human-to-animal spillover and onward transmission in domestic, farm, and wildlife species (CoVERS: Coronavirus Epidemiological Response and Surveillance). The goal of CoVERS is to understand whether and how SARS-CoV-2 transmission is occurring at these interfaces, to refine public health guidelines, investigate whether there are additional risks to animal or human health associated with spillover, and evaluate the potential for establishment of endemic reservoirs. In the CoVERS in-home study, participants are sent a “swab and send” kit, which provides materials and instructions to safely take longitudinal nasal and oral samples from their animals, store them in their freezers, and send them back for viral screening. This community science approach allows wide surveillance with no risk of human transmission, as kits are decontaminated and opened in biosafety cabinets. Here, we highlight one enrolled household that created an exceptional natural experiment with direct relevance to our understanding of SARS-CoV-2 reverse zoonosis and animal models of disease.     

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR^−/−mice, IFNAR^−/−-hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2–specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

In December 2019, a novel coronavirus disease (COVID-19) was first identified in Wuhan City, Hubei Province, People’s Republic of China. The causative agent was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On 11 March 2020 the World Health Organization (WHO) declared COVID-19 a global pandemic (1–3). It spread rapidly within China and swept into at least 200 countries within 3 mo. Symptoms are primarily pneumonia, as with two other important human coronaviruses (CoVs), SARS-CoV-1 and Middle East respiratory syndrome (MERS)-CoV (1–3). As of 1 February 2021, more than 102,399,513 cases had been reported worldwide, with 2,217,005 deaths (∼2.2% mortality). There is an urgent need to develop a safe and efficacious vaccine to protect the populace from this new virus. Globally, more than 300 SARS-CoV-2 vaccine candidates are in preclinical development (4–6) and at least 30 vaccine candidates have entered human clinical trials (4, 5, 7, 8). Among them, vaccines based on messenger RNA (mRNA), inactivated virus, and adenovirus vectors (Ad5-nCoV and ChAdOx1) are now in phase III clinical trials. Excitingly, preliminary results indicate that these vaccines are highly efficacious, reaching 90 to 95% effectiveness against SARS-CoV-2 infection in some cases. The durability of the protection conferred by these vaccine candidates is unknown. Although these vaccine candidates are highly promising, exploration of other vaccine platforms is needed.The CoV spike (S) protein is the main target for neutralizing antibodies that inhibit infection and prevent disease. As such, the S protein is the primary focus for CoV vaccine development (9, 10). The CoV S protein is a class I fusion protein trimer that is incorporated into virions as they bud into the endoplasmic reticulum–Golgi intermediate compartment. For SARS-CoV-2, S is cleaved into S1 and S2 subunits by furin before the virion is released. The S1 subunit contains the receptor-binding domain (RBD) that attaches to the hACE2 receptor on the surface of a target cell. The S2 subunit is further cleaved by TMPRSS2 (or cathepsin L/B) and possesses the membrane-fusing activity (9, 11, 12). Both S and its RBD have been shown to be immunogenic for many CoVs (13–15). The native S in the virion is in its “prefusion” form. Upon triggering, the prefusion S (preS) undergoes significant conformational changes to insert its fusion peptide into the target cell membrane and bring the virion and cell membranes together, arriving at its postfusion S form as it causes the membranes to fuse. For paramyxoviruses, pneumoviruses, and HIV, it has been shown that prefusion forms of glycoprotein are more potent in inducing neutralizing antibodies than their postfusion forms (16–20). Currently, whether the SARS-CoV-2 preS protein is more immunogenic than the postfusion S protein is unknown.Live attenuated measles virus (MeV) vaccine has been one of the safest and most efficient human vaccines and has been used in children since the 1960s (21, 22). Worldwide MeV vaccination campaigns have been very successful in controlling measles. MeV is an enveloped nonsegmented negative-sense RNA virus that belongs to the genus Morbillivirus within the Paramyxoviridae family. MeV is an excellent vector to deliver vaccines for human pathogens primarily because of its high safety, efficacy, and long-lived immunity (22, 23). MeV has previously been shown to be a highly efficacious vaccine vector for many viral diseases such as HIV (24, 25), SARS-CoV-1 (26, 27), MERS-CoV (28, 29), respiratory syncytial virus (30), hepatitis B and C viruses (31), influenza virus (30, 32), chikungunya virus (CHIKV) (33), and flaviviruses (Zika virus, dengue virus, West Nile virus, and yellow fever virus) (34–36). Recent human clinical trials have demonstrated that an recombinant MeV (rMeV)-based CHIKV vaccine is safe and highly immunogenic in healthy adults, even in the presence of preexisting anti-MeV vector immunity (33).In this study, we developed a series of rMeV-based vaccine candidates expressing different forms of the SARS-CoV-2 S protein and evaluated them in cotton rats, IFNAR^−/−mice, IFNAR^−/−-hCD46 mice, and golden Syrian hamsters. We found that all SARS-CoV-2 S antigens are highly expressed by the MeV vector. Among these vaccine candidates, rMeV expressing stabilized preS (rMeV-preS) and full-length S (rMeV-S) proteins were the most potent in triggering SARS-CoV-2–specific antibodies. Animals immunized with rMeV-preS induced the highest level of neutralizing antibodies that were higher than convalescent sera of patients recovered from COVID-19, and the highest Th1-biased T cell immune response. Furthermore, hamsters immunized with rMeV-preS provided complete protection against SARS-CoV-2 challenge and lung pathology.     

Just 2% of SARS-CoV-2−positive individuals carry 90% of the virus circulating in communities

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and possibly also superspreaders.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged into the human population in late 2019 (1), presumably from animal reservoirs (2, 3). During the ensuing world-wide pandemic, already more than 3 million lives have been lost due to the virus. Spread of SARS-CoV-2 has thus far been extremely difficult to contain. One key reason for this is that both presymptomatic and asymptomatic infected individuals can transmit the virus to others (4–13). Further, it is becoming clear that certain individuals play a key role in seeding superspreading events (14–17). Here, we analyzed data from a large university surveillance program. Viral loads were measured in saliva, which has proven to be an accessible and reliable biospecimen in which to identify carriers of this respiratory pathogen, and the most likely medium for SARS-CoV-2 transmission (18–20). Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We find that the distribution of SARS-CoV-2 viral loads on our campus is indistinguishable from what has previously been observed in symptomatic and hospitalized individuals. Strikingly, these datasets demonstrate dramatic differences in viral levels between individuals, with a very small minority of the infected individuals harboring the vast majority of the infectious virions.     

A data-driven approach to identify risk profiles and protective drugs in COVID-19

As the COVID-19 pandemic is spreading around the world, increasing evidence highlights the role of cardiometabolic risk factors in determining the susceptibility to the disease. The fragmented data collected during the initial emergency limited the possibility of investigating the effect of highly correlated covariates and of modeling the interplay between risk factors and medication. The present study is based on comprehensive monitoring of 576 COVID-19 patients. Different statistical approaches were applied to gain a comprehensive insight in terms of both the identification of risk factors and the analysis of dependency structure among clinical and demographic characteristics. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells by binding to the angiotensin-converting enzyme 2 (ACE2), but whether or not renin−angiotensin−aldosterone system inhibitors (RAASi) would be beneficial to COVID-19 cases remains controversial. The survival tree approach was applied to define a multilayer risk stratification and better profile patient survival with respect to drug regimens, showing a significant protective effect of RAASi with a reduced risk of in-hospital death. Bayesian networks were estimated, to uncover complex interrelationships and confounding effects. The results confirmed the role of RAASi in reducing the risk of death in COVID-19 patients. De novo treatment with RAASi in patients hospitalized with COVID-19 should be prospectively investigated in a randomized controlled trial to ascertain the extent of risk reduction for in-hospital death in COVID-19.

The need of discovering rapidly new findings on COVID-19 medications has been rushing publication, whereas the type of data collected in emergency needed a further degree of caution in data management and analysis with respect to the usual observational designed studies. Hence, in the recent literature, standard statistical approaches often failed to provide reliable and reproducible results and to control for the highly correlated structure among covariates, while accounting for potential confounders in risk prediction (1).COVID-19 is characterized by highly variable clinical manifestations and severity, ranging from asymptomatic to multiorgan failure (2, 3). Older age and cardiovascular comorbidities are among the most important risk factors influencing the virus−host interaction and the clinical outcome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (4–6). Understanding the relationship between cardiovascular disease (CVD), therapy, and COVID-19 outcomes is important to guide clinical and public health interventions. Several treatment approaches, also in light of previous comorbidities, have been adopted to reduce COVID-19 mortality in hospitalized patients. Among other medications, renin−angiotensin−aldosterone system inhibitors (RAASi) have been a major object of interest (7). There are two major arms of RAAS; one arm, the Angiotensin II (Ang II) type 1 receptor (AT1R) pathway, is proinflammatory and can cause acute lung injury (8). The other arm, the angiotensin-converting enzyme 2 (ACE2)−Ang-(1–7)−Mas receptor (MasR) pathway is anti-inflammatory because ACE2 metabolizes Ang II, thus reducing its levels and converting it to the anti-inflammatory peptide, Ang-(1–7) (9). ACE2 is the receptor for coronaviruses, including SARS-CoV-2 (10). When SARS-CoV-2 binds to ACE2, the enzyme is no longer functional, and therefore the proinflammatory Ang II-AT1R is no longer blocked by the ACE2−Ang-(1−7)−MasR pathway; this imbalance can cause acute lung injury (11). RAASi have been hypothesized to influence the clinical course of COVID-19 because of the role of ACE2 as a functional receptor for the virus entrance into the cells (12–14). Initially, some authors raised concerns regarding the potential harm of RAASi in COVID-19, but these were not confirmed, and, more recently, a potential protective role was postulated, but until now not unequivocally proven (11, 15–18).     

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2–infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Since the outbreak of COVID-19, the disease has been spreading worldwide rapidly (1–4). Although both COVID-19 and severe acute respiratory syndrome (SARS) cause severe respiratory illness, epidemiological and clinical data suggest that the disease spectrum of COVID-19 is markedly different from that of SARS. COVID-19 shows a longer incubation period (around 6.4 d, range: 0 to 24 d) than SARS; interpersonal transmission could occur from presymptomatic individuals (5, 6); asymptomatic infection has been widely reported and severely jeopardizes the prevention system in a community (5); a significant portion of recovered patients still shed genetic materials of the virus in the upper respiratory tract and digestive tract, leading to their hospitalization for a considerably longer time (7–9); and some recovered patients show redetectable viral RNA after being discharged from the hospital (7). The desynchronization of viral titer and clinical symptom development suggest that SARS coronavirus 2 (SARS-CoV-2) could have undergone extensive replication in infected host cells without being effectively detected by host antiviral immunity (10).Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infection by directly eradicating the virus-infected cells (11). In a virus-infected cell, major histocompability complex class Ι (MHC-Ι) molecules present peptides derived from various viral proteins. Once the T cell receptor on CD8⁺ T cells recognizes the special signal presented by MHC-Ι–peptide complex, CTLs release various toxic substances (i.e., perforins, granzyme, and FasL) that directly induce the death of viral-infected cells as well as cytokines such as interferon-γ, TNF-α, and IL-2 (11). Thus, the cells supporting viral replication will be eliminated, and the spread of viruses will be effectively prevented (12). Some viruses that cause chronic infection, such as HIV type 1 (HIV-1) and Kaposi’s sarcoma–associated herpes virus (KSHV), can disrupt antigen presentation for immune activation by down-regulating MHC-Ι expression on the surface of cells and evading immune surveillance (13–15). In the current study, we investigated whether the SARS-CoV-2 virus could affect the antigen presentation system and assist viruses in evading immune surveillance.In this study, we report that SARS-CoV-2 virus leads to MHC-Ι down-regulation in both infected human angiotensin–converting enzyme 2 (hACE2)-expressing HEK293T (HEK293T/Hace2) cells and infected lung epithelial cells of hACE2 transgene mice. We screened all SARS-CoV-2 structural proteins and unidentified open reading frames (ORFs) and found that ORF8, which shares the least homology with SARS-CoV among all viral proteins, can directly interact with MHC-Ι molecules and mediate their down-regulation through the autophagy pathway. In addition, we obtained healthy human donor–derived CTLs sensitized to the SARS-CoV-2 epitope SARS-CoV spike protein–derived peptide-1 (SSp-1, RLNEVAKNL) and CTLs isolated from a patient recovering from COVID-19 that responded to a mixture of SARS-CoV-2 peptides. ORF8-expressing cells and SARS-CoV-2–infected cells were found to be more resistant to CTL lysis. Knockdown of ORF8 protein expression in SARS-CoV-2–infected cells restored MHC-Ι expression and consequently cell sensitivity to CTL lysis. Collectively, our results strongly suggested that ORF8 induced MHC-Ι down-regulation and provided protection against CTLs in SARS-CoV-2–infected host cells.     

Rapid identification of a human antibody with high prophylactic and therapeutic efficacy in three animal models of SARS-CoV-2 infection

Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

The severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) (1) has spread worldwide thus requiring safe and effective prevention and therapy. Inactivated serum from convalescent patients inhibited SARS-CoV-2 replication and decreased symptom severity of newly infected patients (2), suggesting that monoclonal antibodies (mAbs) could be even more effective. Human mAbs are typically highly target specific and relatively nontoxic. By using phage display we have previously identified a number of potent fully human mAbs (m396, m336, and m102.4) against emerging viruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) (3), Middle East respiratory syndrome coronavirus (MERS-CoV) (4), and henipaviruses (5, 6), respectively, which are also highly effective in animal models of infection (7–10); one of them was administered on a compassionate basis to humans exposed to henipaviruses and successfully evaluated in a clinical trial (11).Size and diversity of phage-displayed libraries are critical for rapid selection of high-affinity antibodies without the need for additional affinity maturation. Our exceptionally potent antibody against the MERS-CoV, m336, was directly selected from a very large (size ∼10¹¹ clones) library from 50 individuals (4). However, another potent antibody, m102.4, against henipaviruses was additionally affinity matured from its predecessor selected from a smaller library (size ∼10¹⁰ clones) from 10 individuals (6). Thus, to generate high-affinity and safe mAbs we used very large (size ∼10¹¹ clones each) naive human antibody libraries in Fab, scFv, or VH format using peripheral blood mononuclear cells (PBMCs) from a total of 490 individuals obtained before the SARS-CoV-2 outbreak. The complementarity-determining regions (CDRs) of the human VH domains were grafted (except CDR1 which was mutagenized or grafted) from our other libraries as previously described (12).Another important factor to consider when selecting effective mAbs is the appropriate antigen. Similar to SARS-CoV, SARS-CoV-2 uses the spike (S) glycoprotein to enter into host cells. The S receptor binding domain (RBD) binds to its receptor, the human angiotensin-converting enzyme 2 (hACE2), thus initiating a series of events leading to virus entry into cells (13). We have previously characterized the function of the SARS-CoV S glycoprotein and identified its RBD which is stable in isolation (14). The RBD was then used as an antigen to pan phage-displayed antibody libraries; we identified potent antibodies (4, 7) more rapidly and the antibodies were more potent than when we used the whole S protein or S2 as panning antigens. In addition, the SARS-CoV RBD-based immunogens are highly immunogenic and elicit neutralizing antibodies which protect against SARS-CoV infections (15). Thus, to identify SARS-CoV-2 mAbs, we generated two variants of the SARS-CoV-2 RBD (amino acids [aa] 330 to 532) (SI Appendix, Fig. S1) and used them as antigens for panning of our libraries.     

The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2

The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.

The spike glycoproteins of coronaviruses are responsible for receptor binding and membrane fusion during the initial stages of virus infection (1). Viruses that have spike proteins containing the amino acid substitution D614G are currently predominant in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and it has recently been shown that G614 viruses have higher infectivity and produce higher viral loads than D614 viruses (2–4).We, and others, have shown that the D614 spike adopts several different conformations, including a “closed” conformation in which the receptor-binding domain (RBD) is partly buried and cannot bind to the human ACE2 receptor. We have also shown that both furin cleavage (5) and the presence of ACE2 (6) increase the proportion of the spikes that adopt open conformations and suggested that the D614G substitution could also promote the spike’s “opening” (6). To better understand the impact of the D614G substitution we have now solved the cryo-electron microscopy (cryo-EM) structure of the G614 spike and compared it to that of the D614 spike recently solved by us and others (5, 7, 8).     

Temperature and population density influence SARS-CoV-2 transmission in the absence of nonpharmaceutical interventions

As COVID-19 continues to spread across the world, it is increasingly important to understand the factors that influence its transmission. Seasonal variation driven by responses to changing environment has been shown to affect the transmission intensity of several coronaviruses. However, the impact of the environment on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains largely unknown, and thus seasonal variation remains a source of uncertainty in forecasts of SARS-CoV-2 transmission. Here we address this issue by assessing the association of temperature, humidity, ultraviolet radiation, and population density with estimates of transmission rate (R). Using data from the United States, we explore correlates of transmission across US states using comparative regression and integrative epidemiological modeling. We find that policy intervention (“lockdown”) and reductions in individuals’ mobility are the major predictors of SARS-CoV-2 transmission rates, but, in their absence, lower temperatures and higher population densities are correlated with increased SARS-CoV-2 transmission. Our results show that summer weather cannot be considered a substitute for mitigation policies, but that lower autumn and winter temperatures may lead to an increase in transmission intensity in the absence of policy interventions or behavioral changes. We outline how this information may improve the forecasting of COVID-19, reveal its future seasonal dynamics, and inform intervention policies.

In late 2019, a novel coronavirus originating in Wuhan City (Hubei, China) (1) began to rapidly spread through the human population. Since March 2020, this disease, COVID-19, has been recognized as a global pandemic by the World Health Organization. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a close relative of the 2003 SARS coronavirus (1), although it appears to have several differences, including a higher basic reproduction number (R0; the average number of people infected by a carrier at the onset of an epidemic) (2). Understanding the factors influencing SARS-CoV-2 transmission is key for understanding the current patterns of transmission and for refining predictions of the future spread of SARS-CoV-2. Other coronaviruses display seasonal cycles of transmission, and up to 30% of seasonal “colds” are caused by coronaviruses (3). Thus, as many countries alter and relax the nonpharmaceutical interventions initially imposed to control COVID-19, there is a pressing need to understand whether environmental factors will enhance or drive additional “waves” of COVID-19 outbreaks as places move through seasonal climate patterns (4).SARS-CoV-2 is an enveloped RNA virus which is structurally (if not phylogenetically) similar to other RNA viruses such as influenza, Middle East respiratory syndrome, and HcoV-NL63 (5) that are known to display seasonal dynamics due to their physical properties. For example, high temperatures and low humidity can have a negative effect on influenza transmission by reducing the efficiency of respiratory droplet transmission (6, 7). Similar effects are seen in transmission of coronaviruses (8–10), where high environmental temperatures break down viral lipid layers to inactivate virus particles that are in the air or deposited on surfaces (9, 11). However, assessing the role of environment during a disease outbreak is challenging (12) because human factors such as population density, herd immunity, and behavior are likely the main drivers of transmission (13–16). Moreover, the nonpharmaceutical control measures and behavioral changes in response to COVID-19 have been unprecedented in the modern era. These difficulties have hindered the quantification of the impact of environment on SARS-CoV-2 transmission, making it harder to generalize and synthesize observations across regions with their differing climates. Despite these caveats, various early studies have already reported effects of environmental variables such as temperature, humidity, ultraviolet (UV) levels, and wind speed on the transmission of SARS-CoV-2 (16–24). While, in general, most studies appear to support increased transmission rates under cool, dry conditions (18), conflicting results have been observed (21, 25), and, collectively, the environmental signal appears to be weak (4). Much of the variability in these early results is likely due to the use of inappropriate response variables (such as cases or fatalities) which fail to capture the intrinsic variations in transmission intensity driven by the effects of nonpharmaceutical intervention measures (4). Furthermore, COVID-19 has taken hold in many places with diverse climates, and there are obvious examples of high transmission rates under warmer conditions, such as in Brazil (26), India (27), and Iran (28).Accurate assessment of the role environmental factors have played so far in the spread of SARS-CoV-2 may provide insight into the future seasonality of the disease. This is because seasonal outbreaks of viruses are often driven by their responses to favorable (seasonal) changes in weather (29). Most epidemiological forecasts make use of some variant of the Susceptible–Infected–Recovered (SIR) framework and/or focus on the impacts of government-level mitigation (e.g., refs. 30 and 31). Few epidemiological models incorporate environmental impacts, and, when they do, they assume COVID-19 responds in a manner identical to related coronaviruses, because we lack data on SARS-CoV-2’s environmental (and thus seasonal) responses (e.g., ref. 18). This is despite theoretical demonstrations of the potential role of environment in driving future seasonality of SARS-CoV-2 (22, 32) and the empirical evidence in structurally similar viruses outlined above. Efforts to incorporate climate into COVID-19 forecasting have focused on regression-type models of cases and fatalities (e.g., ref. 17), which are unreliable when diseases are in the growth/expansion phase (33). Furthermore, such models conflate environmental controls on occurrence with other drivers such as public health interventions (e.g., the effects of lockdown measures to contain the pandemic) (33), as both are changing similarly through time. Such models are unlikely to yield useful insights and may be misleading to policy makers (12). To address this knowledge gap, there is a need for a true synthesis of environmental modeling with well-established epidemiological approaches.Here we investigate the role of environment in the transmission of SARS-CoV-2 by incorporating environmental factors into an existing epidemiological framework that has been applied globally (34–36), and to the United States in particular (37). The United States is a large country with great variation in climate across which case and policy intervention data are comparable, permitting us to disentangle the role of environmental drivers in SARS-CoV-2 transmission. We begin by exploring associations between the environment (temperature, humidity, UV radiation, and population density) and transmission intensity independently estimated before and during stay-at-home orders (henceforth termed “lockdown”). We used the basic reproduction number (R0) for our prelockdown transmission intensity estimates, and the time-varying reproduction number (Rt, the reproduction number, R, at a given time, t) averaged across an appropriate time window for our during-lockdown estimates. Our independent analysis of R0 focuses on a single snapshot (the beginning) of the virus’s outbreak in each state, and reveals whether differences in transmission across states are correlated with differences in environment across space at that snapshot in time. Critically, this independent analysis allows us to investigate the role of environment in the absence of any temporally correlated changes in climate and transmission rate. After confirming a potential role for the environment, we verify and more accurately quantify the relative roles of temperature and population density by integrating them into an existing semimechanistic epidemiological framework (37). While we find strong evidence that temperature and population density are associated with SARS-CoV-2 transmission, we emphasize that our findings also reconfirm that the major drivers of transmission rates are public policy and individual behavior. Through our use of existing, robust sources of forecasts and models, our findings can be easily incorporated into workflows already used by policy makers, as we detail here.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein

The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a ∼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2−specific sequence, ₇₃YIDI₇₆. Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.

The severity of the current COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to past outbreaks of Middle East respiratory syndrome, SARS, and other betacoronaviruses in humans begs the question as to its molecular basis. The accessory protein ORF8 is one of the most rapidly evolving betacoronavirus proteins (1–7). While ORF8 expression is not strictly essential for SARS-CoV and SARS-CoV-2 replication, a 29-nucleotide deletion (Δ29) that occurred early in human to human transmission of SARS-CoV, splitting ORF8 into ORF8a and ORF8b, is correlated with milder disease (8). A 382-nucleotide deletion (Δ382) in SARS-CoV-2 (9, 10) was also found to correlate with milder disease and a lower incidence of hypoxia (11).SARS-CoV-2 ORF8 is a 121-amino acid (aa) protein consisting of an N-terminal signal sequence followed by a predicted Ig-like fold (12). With <20% sequence identity to SARS-CoV ORF8, SARS-CoV-2 ORF8 is remarkably divergent. ORF8 proteins from both viruses possess a signal sequence for endoplasmic reticulum (ER) import. Within the lumen of the ER, SARS-CoV-2 ORF8 interacts with a variety of host proteins, including many factors involved in ER-associated degradation (13). Presumably, ORF8 is secreted, rather than retained in the ER, since ORF8 antibodies are one of the principal markers of SARS-CoV-2 infections (14). Several functions have been proposed for SARS-CoV-2 ORF8. ORF8 disrupts IFN-I signaling when exogenously overexpressed in cells (15). It has been shown that ORF8 of SARS-CoV-2, but not ORF8 or ORF8a/ORF8b of SARS-CoV, down-regulates MHC-I in cells (16).These observations suggest the relationship between ORF8 structure, function, and sequence variation may be pivotal for understanding the emergence of SARS-CoV-2 as a deadly human pathogen. Yet not only is there no three-dimensional structure of any ORF8 protein from any coronavirus, there are no homologs of known structure with sequence identity sufficient for a reliable alignment. SARS and SARS-CoV-2 ORF7a are the most closely related templates of known structure (17), yet their core is approximately half the size of ORF8, and their primary sequence identity is negligible. Therefore, we determined the crystal structure of SARS-CoV-2 ORF8. The structure confirms the expected Ig-like fold and overall similarity of the core fold to SARS-CoV-2 ORF7a. The structure reveals two novel dimer interfaces for SARS-CoV-2 ORF8 unique relative to all but its most recent ancestors in bats. Together, our results set the foundation for elucidating essential aspects of ORF8 biology to be leveraged for the development of novel therapeutics.     

Global analysis of more than 50,000 SARS-CoV-2 genomes reveals epistasis between eight viral genes

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.

The pandemic of the disease COVID-19 has so far led to the confirmed deaths of more than 852,000 people (1) and has hurt millions. As the health crisis has been met by nonpharmacological interventions (2, 3) there has been significant economic disruption in many countries. The search for vaccine or treatment against the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is therefore a worldwide priority. The Global Initiative on Sharing All Influenza Data (GISAID) repository (4) contains a rapidly increasing collection of SARS-CoV-2 whole-genome sequences, and has already been leveraged to identify mutational hotspots and potential drug targets (5). Coronaviruses, in general, exhibit a large amount of recombination (6–9). The distribution of genotypes in a viral population can therefore be expected to be in the state of quasi-linkage equilibrium (QLE) (10–12), and directly related to epistatic contributions to fitness (13, 14). We have determined a list of the largest such contributions from 51,676 SARS-CoV-2 genomes by a direct coupling analysis (DCA) (15, 16). This family of techniques has earlier been used to infer the fitness landscape of HIV-1 Gag (17, 18) to connect bacterial genotypes and phenotypes through coevolutionary landscapes (19) and to enhance models of amino acid sequence evolution (20). We apply a recent enhancement of this technique to eliminate predictions that can be attributed to phylogenetics (shared inheritance) (21). We find that eight predictions stand out between pairs of polymorphic sites located in genes nsp2 and ORF3a, in genes nsp4 and ORF8, and between genes nsp2, nsp6, nsp12, nsp13, nsp14 and ORF3a. Most of these sites have been documented in the literature when it comes to single-locus variations (22–27). The nsp4–ORF8 pair was additionally found to be strongly correlated, in an early study (28). It does not show prominent correlations today, but is ranked second in our global analysis. The epistasis analysis of this paper brings a different perspective than correlations, and highlights pair-wise associations that have remained stable as orders of more SARS-CoV-2 genomes have been sequenced.     

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic coronavirus disease (COVID-19) (1–3), resulting in more than 209 million infections worldwide since December 2019. SARS-CoV-2 is the seventh coronavirus (CoV) infecting humans beyond endemic human CoVs (HCoV-HKU1, -NL63, -OC43, and -229E), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV-2 preferentially infects the airway epithelial cells (1, 4), although viral infection has been detected in a variety of human organs, including the lungs, pharynx, heart, liver, brain, kidneys, and digestive system organs (5–7), causing upper respiratory diseases, fever, and severe pneumonia in humans.CoVs were not considered to be highly pathogenic to humans until the outbreak of SARS-CoV started in 2002 in Guangdong province, China (8–10). The primary determinant of CoV tropism is their spike (S) glycoproteins, which mediate the viral infection by binding to membrane receptors on the host cells and could be cleaved to the N-terminal S1 and C-terminal S2 subunit by the host proteases such as transmembrane protease serine 2 (TMPRSS2) and Furin (11). Studies have shown that ACE2, a cellular receptor for SARS-CoV, also binds SARS-CoV-2 S and serves as the entry point for SARS-CoV-2 (3, 12). However, ACE2 cannot fully explain the tissue tropism of SARS-CoV-2 because of the virus detection in tissues with little ACE2 expression, such as the liver, brain, and blood, and even in the lung, only a small subset of cells expresses ACE2 (SI Appendix, Fig. 1B) (13–16). Recently, neuropilin-1 (NRP1) (17, 18) and heparan sulfate (19, 20) have been identified as cofactors implicated in enhancing ACE2-dependent SARS-CoV-2 infection, while tyrosine-protein kinase receptor (AXL) (21) and CD147 (22) were identified as receptors involved in SARS-CoV-2 cell entry independently of ACE2. These highlight the multiple routes of SARS-CoV-2 infection, likely contributing to high infectivity and spread of COVID-19. Hence, SARS-CoV-2 is assumed to be dependent on other potential receptors or coreceptors to facilitate its infection in humans.Here, we applied engineered ascorbate peroxidase APEX2-based subcellular proteomics (23–25) to capture spatiotemporal membrane protein complexes interacting with SARS-CoV-2 S in living cells. We found that the nonmuscle myosin heavy chain IIA (MYH9) specifically interacts with SARS-CoV-2 S protein. The C-terminal domain (designated as PRA) of MYH9 interacts with the S2 subunit and the N-terminal domain (NTD) of the S1 subunit, and PRA overexpression facilitates a pan-CoV entry into host cells. In addition, depletion of MYH9 significantly reduced authentic SARS-CoV-2 infection in human lung cell lines. Mechanistically, a myosin inhibitor but not TMPRSS2 and CatB/L inhibitors blocked SARS-CoV-2 infection in PRA-A549 cells, and the presence of ACE2 is required for MYH9 mediated SARS-CoV-2 entry. Collectively, our results suggest that MYH9 is a coreceptor of ACE2 for SARS-CoV-2 infection in human pulmonary cells.