Neutralization mechanism of a human antibody with pan-coronavirus reactivity including SARS-CoV-2.

Frequent outbreaks of coronaviruses underscore the need for antivirals and vaccines that can counter a broad range of coronavirus types. We isolated a human antibody named 76E1 from a COVID-19 convalescent patient, and report that it has broad-range neutralizing activity against multiple α- and ß-coronaviruses, including the SARS-CoV-2 variants. 76E1 also binds its epitope in peptides from γ- and δ-coronaviruses. 76E1 cross-protects against SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and therapeutic murine animal models. Structural and functional studies revealed that 76E1 targets a unique epitope within the spike protein that comprises the highly conserved S2' site and the fusion peptide. The epitope that 76E1 binds is partially buried in the structure of the SARS-CoV-2 spike trimer in the prefusion state, but is exposed when the spike protein binds to ACE2. This observation suggests that 76E1 binds to the epitope at an intermediate state of the spike trimer during the transition from the prefusion to the postfusion state, thereby blocking membrane fusion and viral entry. We hope that the identification of this crucial epitope, which can be recognized by 76E1, will guide epitope-based design of next-generation pan-coronavirus vaccines and antivirals.

Survival of SARS-CoV-2 in artificial seawater and on the surface of inanimate materials.

There is a potential risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread through human contact with seafood and the inanimate materials contaminated by the virus. In this study, we examined the stability of the virus in artificial seawater (ASW) and on the surface of selected materials. SARS-CoV-2 (3.75 log10 TCID50 ) in ASW at 22℃ maintained infectious about 3 days and at 4℃ the virus survived more than 7 days. It should be noticed that viable virus at high titer (5.50 log10 TCID50 ) may survive more than 20 days in ASW at 4℃ and for 7 days at 22℃. SARS-CoV-2 on stainless steel and plastic bag maintained infectious for 3 days, and on nonwoven fabric for 1 day at 22℃. In addition, the virus remained infectious for 9 days on stainless steel and non-woven fabric, and on plastic bag for 12 days at 4℃. It is important to highlight the role of inanimate material surfaces as a source of infection and the necessity for surface decontamination and disinfection.

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Delayed Antiviral Immune Responses in Severe Acute Respiratory Syndrome Coronavirus Infected Pregnant Mice.

Sex differences in immune responses had been reported to correlate with different symptoms and mortality in the disease course of coronavirus disease 2019 (COVID-19). However, whether severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection interferes with females' fertility and causes different symptoms among pregnant and non-pregnant females remains unknown. Here, we examined the differences in viral loads, SARS-CoV-2-specific antibody titers, proinflammatory cytokines, and levels of T cell activation after SARS-CoV-2 sub-lethal infection between pregnant and non-pregnant human Angiotensin-Converting Enzyme II (ACE2) transgenic mouse models. Both mice showed elevated levels of viral loads in the lung at 4 days post-infection (dpi). However, viral loads in the pregnant group remained elevated at 7 dpi while decreased in the non-pregnant group. Consistent with viral loads, increased production of proinflammatory cytokines was detected from the pregnant group, and the IgM or SARS-CoV-2-specific IgG antibody in serum of pregnant mice featured delayed elevation compared with non-pregnant mice. Moreover, by accessing kinetics of activation marker expression of peripheral T cells after infection, a lower level of CD8+ T cell activation was observed in pregnant mice, further demonstrating the difference of immune-response between pregnant and non-pregnant mice. Although vertical transmission did not occur as SARS-CoV-2 RNA was absent in the uterus and fetus from the infected pregnant mice, a lower pregnancy rate was observed when the mice were infected before embryo implantation after mating, indicating that SARS-CoV-2 infection may interfere with mice's fertility at a specific time window. In summary, pregnant mice bear a weaker ability to eliminate the SARS-CoV-2 virus than non-pregnant mice, which was correlated with lower levels of antibody production and T cell activation.

Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2.

Host cellular receptors play key roles in the determination of virus tropism and pathogenesis. However, little is known about SARS-CoV-2 host receptors with the exception of ACE2. Furthermore, ACE2 alone cannot explain the multi-organ tropism of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV, suggesting the involvement of other receptor(s). Here, we performed genomic receptor profiling to screen 5054 human membrane proteins individually for interaction with the SARS-CoV-2 capsid spike (S) protein. Twelve proteins, including ACE2, ASGR1, and KREMEN1, were identified with diverse S-binding affinities and patterns. ASGR1 or KREMEN1 is sufficient for the entry of SARS-CoV-2 but not SARS-CoV in vitro and in vivo. SARS-CoV-2 utilizes distinct ACE2/ASGR1/KREMEN1 (ASK) receptor combinations to enter different cell types, and the expression of ASK together displays a markedly stronger correlation with virus susceptibility than that of any individual receptor at both the cell and tissue levels. The cocktail of ASK-related neutralizing antibodies provides the most substantial blockage of SARS-CoV-2 infection in human lung organoids when compared to individual antibodies. Our study revealed an interacting host receptome of SARS-CoV-2, and identified ASGR1 and KREMEN1 as alternative functional receptors that play essential roles in ACE2-independent virus entry, providing insight into SARS-CoV-2 tropism and pathogenesis, as well as a community resource and potential therapeutic strategies for further COVID-19 investigations.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants.

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

Retraction Note to: SARS-CoV-2 infects T lymphocytes through its spike protein-mediated membrane fusion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection.

Massive production of efficacious SARS-CoV-2 vaccines is essential for controlling the ongoing COVID-19 pandemic. We report here the preclinical development of yeast-produced receptor-binding domain (RBD)-based recombinant protein SARS-CoV-2 vaccines. We found that monomeric RBD of SARS-CoV-2 could be efficiently produced as a secreted protein from transformed Pichia pastoris (P. pastoris) yeast. Yeast-derived RBD-monomer possessed functional conformation and was able to elicit protective level of neutralizing antibodies in mice. We further designed and expressed a genetically linked dimeric RBD protein in yeast. The engineered dimeric RBD was more potent than the monomeric RBD in inducing long-lasting neutralizing antibodies. Mice immunized with either monomeric RBD or dimeric RBD were effectively protected from live SARS-CoV-2 virus challenge even at 18 weeks after the last vaccine dose. Importantly, we found that the antisera raised against the RBD of a single SARS-CoV-2 prototype strain could effectively neutralize the two predominant circulating variants B.1.1.7 and B.1.351, implying broad-spectrum protective potential of the RBD-based vaccines. Our data demonstrate that yeast-derived RBD-based recombinant SARS-CoV-2 vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of SARS-CoV-2 vaccines to achieve global immunization.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

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.

A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry.

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Photo-catalyzed TiO2 inactivates pathogenic viruses by attacking viral genome.

Previous observations have been reported that viruses were inactivated using strong irradiation. Here, new evidence was disclosed by studying the effects of nanosized TiO2 on viral pathogens under a low irradiation condition (0.4 mW/cm2 at UVA band) that mimics the field setting. We showed that photo-activated TiO2 efficiently inhibits hepatitis C virus infection, and weak indoor light with intensity of 0.6 mW/cm2 at broad-spectrum wavelength and around 0.15 mW/cm2 of UVA band also lead to partial inhibition. Mechanistic studies demonstrated that hydroxyl radicals produced by photo-activated TiO2 do not destroy virion structure and contents, but attack viral RNA genome, thus inactivating the virus. Furthermore, we showed that photo-activated TiO2 inactivates a broad range of human viral pathogens, including SARS-CoV-2, a novel coronavirus responsible for the ongoing COVID-19 pandemic. In conclusion, we showed that photo-catalyzed nanosized TiO2 inactivates pathogenic viruses, paving a way to its field application in control of viral infectious diseases.