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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.

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).

YycFG, one of the two-component systems involved in the regulation of biofilm formation, has attracted increasing interest as a potential target of antibacterial and antibiofilm agents. YycG inhibitors for Staphylococcus aureus and Staphylococcus epidermidis have been developed, but Enterococcus faecalis remains underexplored. Herein, we selected and identified novel candidate molecules against E. faecalis targeting histidine kinase YycG using high-throughput virtual screening; six molecules (compound-16, -30, -42, -46, -59, and -62) with low cytotoxicity toward mammalian cells were verified as potential YycG inhibitors through an autophosphorylation test and binding kinetics. Compound-16 inhibited planktonic cells of E. faecalis, including the vancomycin- or linezolid-resistant strains. In contrast, compound-62 did not affect planktonic growth but significantly inhibited biofilm formation in static and dynamic conditions. Compound-62 combined with ampicillin could synergistically eradicate the biofilm-embedded viable bacteria. The study demonstrates that YycG inhibitors may be valuable approaches for the development of novel antimicrobial agents for difficult-to-treat bacterial infections.

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional “down” conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD “up”. Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern. Supplementary Information The online version contains supplementary material available at 10.1007/s13238-021-00871-6.

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.

DNA transfection is routinely used for delivering expression of gene of interest to target cells. Transfected DNA has been known to activate cellular DNA sensor(s) and innate immune responses, but the effects of such responses on transfected DNA are not fully understood. STING (stimulator of interferon genes) is an important adaptor protein in cellular innate immune response to various DNA and RNA stimuli and upon activation induces significant type I interferon responses. In this work, we characterized the effects of STING on gene expression driven by transfected double-stranded DNA. We observed that gene expression from transfected DNA was repressed in the presence of overexpressed STING, but increased if endogenous STING was knocked down through RNA interference. Endogenous chromosomal genes and chromosome-integrated exogenous genes were not affected by such STING-mediated restriction, which did not depend on DNA circularity or linearity, promoter used, or bacterial sequences in transfected DNA. Mechanistically, STING-mediated repression of transfected DNA correlates with reduced mRNA levels, and partially involves the induction of interferon β production by STING. Collectively, these data indicate that episomal double-stranded DNA is targeted by STING-mediated cell defense.

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.

SAMHD1 (Sterile Alpha Motif and Histidine-aspartate Domain containing protein 1) has been documented as a host factor that restricts HIV-1 and some DNA viruses. In this work, we attempted to explore possible effects of SAMHD1 on exogenous DNA and show that SAMHD1 exerts a general inhibition on the expression of exogenous DNA in vitro and in mice. This inhibition is achieved through repressing transcription of exogenous DNA. Intriguingly, unlike SAMHD1’s restriction of HIV-1, such restriction does not require the dNTPase or RNase activities, or T592 phosphorylation of SAMHD1. Mechanistically, SAMHD1 enhances the expression of interferon regulatory factor-1 (IRF1), while IRF1 upregulation was demonstrated to inhibit exogenous DNA expression in a similar fashion as SAMHD1. IFNλ1, whose induction has been associated with IRF1 activation, is dispensable for SAMHD1/IRF1-mediated restriction of exogenous DNA, and neither type I nor II interferons appear to be involved. We also demonstrate that SAMHD1/IRF1-mediated restriction can effectively inhibit hepatitis B virus (HBV) antigen expression and progeny virus production in mouse models. In conclusion, these data support restriction of exogenous DNA as a novel function of SAMHD1.

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. In mouse models of HBV persistence, interleukin 21 (IL-21) has been identified as a potent inducer of viral clearance. Strict hepatotropism makes recombinant HBV (rHBV) vectors ideal for liver-targeting gene delivery. Previously, we established an rHBV vector termed 5c3c, which is highly replicative by itself, but requires HBV envelope proteins provided in trans to produce virions. 5c3c-based rHBV virions are capable of delivering cargo gene expression driven by HBV Sp1 promoter into infected hepatocytes. In this work, we explore the feasibility of using 5c3c-derived rHBV for liver-specific delivery of IL-21 as treatment of chronic HBV infection. Methods: 5c3c-derived rHBV replicons harboring mouse or human IL-21 genes (termed 5c3c-mIL-21 and 5c3c-hIL-21 respectively) were constructed and then tested for the production of rHBV virions in vitro and in vivo. 5c3c-mIL-21's anti-HBV effects were determined in chronic HBV mouse model. Furthermore, superinfection by rHBV virions was analysed using HBV-infected HepG2/NTCP cells and human liver chimeric mice. Results: 5c3c-mIL-21 and 5c3c-hIL-21 were efficiently replicative and produced enveloped virions when provided with envelope proteins, both in vitro and in vivo. In mouse model of HBV persistence, IL-21 expressed from injected 5c3c-mIL-21 replicon induced complete viral clearance. 5c3c-mIL-21 and 5c3c-hIL-21 virions could infect HepG2/NTCP cells and engender sustained IL-21 expression. Most importantly, IL-21-expressing rHBV virions could superinfect HBV-infected HepG2/NTCP cells and human hepatocytes in human liver chimeric mice, and engender sustained IL-21 expression and rHBV production. Conclusion: These data suggest the high potential of 5c3c-derived IL-21-expressing rHBV as a novel therapeutic against chronic HBV infection.

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470–495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450–458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic., Neutralizing antibodies are currently one versatile strategy to treat SARS-CoV-2 infection. Here, Li et al. characterize three monoclonal antibodies neutralizing authentic virus infection in vitro and in vivo by targeting the receptor binding domain as evidenced by Cryo-EM.

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.

The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus causing acute respiratory tract infection in humans. The virus has the characteristics of rapid transmission, long incubation period and strong pathogenicity, and has spread all over the world. Therefore, it is of great significance to select appropriate animal models for antiviral drug development and therapeutic effect evaluation. Here, we review and compare the current animal models of SARS-CoV-2.

The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases., Graphical abstract The dePEGylated lipopeptides were designed, synthesized, and screened for the most effective one, EKL1C, which was shown to have broad-spectrum anti-coronavirus activity, potent protection of human angiotensin-converting enzyme 2 (hACE2) transgenic mice against authentic coronavirus infection, and high stability.Image 1

Background The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed. Methods Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses. Results Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect. Conclusions Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19.

Chronic hepatitis B virus (HBV) infection causes hepatitis, liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA) is the sole viral transcription template and not affected by current treatment options, constituting a key determinant of HBV persistence. Novel therapeutics with demonstrable effectiveness against cccDNA are required. Methods: Previously, we established an HBV persistence mouse model using replicon plasmid derived from a clinical isolate (termed BPS) and identified IL-21 as a potent clearance-inducer. We also described another persistence mouse model based on cccDNA mimics produced in vivo termed recombinant cccDNA (rcccDNA). In this work, effectiveness of IL-21-based gene and cellular therapies was tested using these models. Results: In both models of HBV persistence, single injections with adeno-associated virus (AAV) expressing murine IL-21 highly efficiently induced clearance of both HBV markers from serum, and more importantly, BPS DNA and rcccDNA from mouse liver. Mechanistically, IL-21-induced clearance was associated with activation and liver infiltration of CD8+ T cells, and CD8 antibody injections negatively affected AAV-IL-21 effectiveness. More notably, adoptive transfer of CD8+ T cells from AAV-IL-21-cured mice engendered clearance in acceptor HBV persistence mice. Furthermore, cured mice were protected against re-challenge with long-lived memory. Most significantly, infusion of splenocytes from treatment-naive mice stimulated ex vivo with IL-21 protein and HBV antigen could also induce clearance in treatment-naive mice. Conclusion: These data demonstrate IL-21-based gene and cellular therapies as valid candidates for treating chronic HBV infections, with potential in removing cccDNA-harboring hepatocytes via activated CD8+ T cells accompanied by long-term protective memory.

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

Significance COVID-19, caused by SARS-CoV-2, is a major global health threat. The host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. We found that SARS-CoV-2 has the potential to infect a broad range of mammalian hosts, including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria. Those species may be at risk for human-to-animal or animal-to-animal transmissions of SARS-CoV-2. Our study highlights the importance of banning illegal wildlife trade and consumption, and enforcing the importance of surveilling animals in close contact with humans as potential zoonotic reservoirs to prevent outbreaks in the future., 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.

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., The SARS-CoV-2 spike protein contains a multi-basic cleavage site. Here, the authors show how this multi-basic cleavage site affects entry of SARS-CoV-2 into cells and transmission in the hamster model and identify host factors affecting entry of SARS-CoV-2 in a genome-wide CRISPR screen.

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes., Graphical Abstract, Highlights • Antibodies against SARS-CoV-2 S protein are isolated from an elite neutralizer • Receptor-binding domain (RBD) antibodies target four groups of non-overlapping epitopes • Group IV antibodies induce antibody-dependent enhancement (ADE) of entry in Raji cells • Group II/III antibodies neutralize SARS-CoV-2 without mediating ADE of entry in vitro, Zhou et al. clone human antibodies against the SARS-CoV-2 S protein from an elite neutralizer and reveal the association of antibody-dependent enhancement (ADE)/neutralizing activities in vitro with four distinct groups of non-overlapping epitopes on the receptor-binding domain (RBD).

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen–antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections., Here, the authors identify and characterize two mouse-derived monoclonal antibodies against SARS-CoV-2 spike protein that target different epitopes in RBD and block the interaction S/ACE2 and show that a formulated humanized version cocktail exhibits prophylaxis and therapeutic antiviral effects in an hACE2-adenovector expressed mouse model.

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.

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., Author summary SARS-CoV-2 spike protein could bind cellular receptor ACE2 for cell entry in a species-specific manner. A diverse of mammalian ACE2 proteins could be used by SARS-CoV-2 for entry, but ACE2 proteins of koala or mouse cannot bind with viral spike protein. We compared the koala or mouse ACE2 with human ACE2, and found Thr at 31 position of koala ACE2 or His at 353 position of mouse ACE2 as the restrictive residue which limits its function as the viral receptor, respectively. Interestingly, koala or mouse ACE2 could gain the receptor function once the restrictive reside was replaced by human counterpart by genetic engineering. This study could facilitate our understanding of the genetic basis of ACE2 as the functional receptor of SARS-CoV-2, which could inform the animal model development.

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious threats to global health and economy, thus calling for the development of safe and effective vaccines. The receptor-binding domain (RBD) in the spike protein of SARS-CoV-2 is responsible for its binding to angiotensin-converting enzyme 2 (ACE2) receptor. It contains multiple dominant neutralizing epitopes and serves as an important antigen for the development of COVID-19 vaccines. Here, we showed that immunization of mice with a candidate subunit vaccine consisting of SARS-CoV-2 RBD and Fc fragment of human IgG, as an immunopotentiator, elicited high titer of RBD-specific antibodies with robust neutralizing activity against both pseudotyped and live SARS-CoV-2 infections. The mouse antisera could also effectively neutralize infection by pseudotyped SARS-CoV-2 with several natural mutations in RBD and the IgG extracted from the mouse antisera could also show neutralization against pseudotyped SARS-CoV and SARS-related coronavirus (SARSr-CoV). Vaccination of human ACE2 transgenic mice with RBD-Fc could effectively protect mice from the SARS-CoV-2 challenge. These results suggest that SARS-CoV-2 RBD-Fc has good potential to be further developed as an effective and broad-spectrum vaccine to prevent infection of the current SARS-CoV-2 and its mutants, as well as future emerging SARSr-CoVs and re-emerging SARS-CoV.

Significance The COVID-19 pandemic caused by SARS-CoV-2 infection has led to more than 840,000 deaths worldwide as of August 31, 2020. Unfortunately, no licensed vaccine or specific treatment is available right now. Herein, we report a decoy nanoparticle against COVID-19. The decoy nanoparticles were constructed by fusing cell membrane nanovesicles derived from genetically engineered cells, which stably express SARS-CoV-2 receptor ACE2, and human monocytes, which display abundant cytokine receptors. By competing with host cells, these nanodecoys efficiently adsorb viruses and inflammatory cytokines such as IL-6 and GM-CSF. These two functionalities allow effective intervention of viral infection and its associated immune disorder, presenting a promising therapeutic strategy for COVID-19 and other potential epidemics., 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.

Coronavirus interaction with viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter the host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkeys, 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 of susceptibility of ACE2 orthologs to viral entry, we compared koala and mouse ACE2 sequences with human ortholog, and identified the key residues in koala or mouse ACE2 that restrict its viral receptor activity. Humanization of these critical residues could render the capabilities of koala and mouse ACE2 to bind viral spike protein and facilitate the viral entry. Our work identifies the genetic determinant of ACE2 for SARS-CoV-2 susceptibility, and a single mutation could restore the mouse ACE2 receptor activity, providing a potential avenue for the development of mouse model of SARS-CoV-2.

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1–3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients’ plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

SUMMARYHost cellular receptors are key determinants of virus tropism and pathogenesis. Virus utilizes multiple receptors for attachment, entry, or specific host responses. However, other than ACE2, little is known about SARS-CoV-2 receptors. Furthermore, ACE2 cannot easily interpret the multi-organ tropisms of SARS-CoV-2 nor the clinical differences between SARS-CoV-2 and SARS-CoV. To identify host cell receptors involved in SARS-CoV-2 interactions, we performed genomic receptor profiling to screen almost all human membrane proteins, with SARS-CoV-2 capsid spike (S) protein as the target. Twelve receptors were identified, including ACE2. Most receptors bind at least two domains on S protein, the receptor-binding-domain (RBD) and the N-terminal-domain (NTD), suggesting both are critical for virus-host interaction. Ectopic expression of ASGR1 or KREMEN1 is sufficient to enable entry of SARS-CoV-2, but not SARS-CoV and MERS-CoV. Analyzing single-cell transcriptome profiles from COVID-19 patients revealed that virus susceptibility in airway epithelial ciliated and secretory cells and immune macrophages highly correlates with expression of ACE2, KREMEN1 and ASGR1 respectively, and ACE2/ASGR1/KREMEN1 (ASK) together displayed a much better correlation than any individual receptor. Based on modeling of systemic SARS-CoV-2 host interactions through S receptors, we revealed ASK correlation with SARS-CoV-2 multi-organ tropism and provided potential explanations for various COVID-19 symptoms. Our study identified a panel of SARS-CoV-2 receptors with diverse binding properties, biological functions, and clinical correlations or implications, including ASGR1 and KREMEN1 as the alternative entry receptors, providing insights into critical interactions of SARS-CoV-2 with host, as well as a useful resource and potential drug targets for COVID-19 investigation.

SUMMARYThe global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found 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 instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

The pandemic of COVID-19 caused by SARS-CoV-2 has posed serious threats to global health and economy, thus calling for the development of safe and effective vaccines. The receptor-binding domain (RBD) in the spike protein of SARS-CoV-2 is responsible for its binding to ACE2 receptor. It contains multiple dominant neutralizing epitopes and serves as an important antigen for the development of COVID-19 vaccines. Here, we showed that immunization of mice with a candidate subunit vaccine consisting of SARS-CoV-2 RBD and Fc fragment of human IgG, as an immunopotentiator, elicited high titer of RBD-specific antibodies with robust neutralizing activity against both pseudotyped and live SARS-CoV-2 infections. The mouse antisera could also effectively neutralize infection by pseudotyped SARS-CoV-2 with several natural mutations in RBD and the IgG extracted from the mouse antisera could also show neutralization against pseudotyped SARS-CoV and SARS-related coronavirus (SARSr-CoV). Vaccination of human ACE2 transgenic mice with RBD-Fc could effectively protect mice from the SARS-CoV-2 challenge. These results suggest that SARS-CoV-2 RBD-Fc has good potential to be further developed as an effective and broad-spectrum vaccine to prevent infection of the current SARS-CoV-2 and its mutants, as well as future emerging SARSr-CoVs and re-emerging SARS-CoV.

In this study, we tested the in vitro inhibitory activity of griffithsin (GRFT) against infection of pseudotyped and live SARS-CoV-2 infection, in order to repurpose the application of GRFT as a potential prophylactic or therapeutic to prevent or treat COVID-19.

Summary COVID-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired heavy and light chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find 3 main epitopes in RBD recognized by these antibodies, and epitope B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross-neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates., Graphical Abstract, Highlights 11 neutralizing antibodies against SARS-CoV-2 targeting 3 main epitopes on RBD Epitope A antibody 414-1 shows neutralizing IC50 at 1.75 nM Epitope B antibody 553-15 can enhance the neutralizing abilities of other antibodies One neutralizing antibody, 515-5, can cross-neutralize SARS-CoV pseudovirus, Wan et al. identify 11 potent neutralizing antibodies against COVID-19 from 11 convalescent patients. These human neutralizing antibodies target 3 separate epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and epitope B antibody (553-15) can enhance the neutralizing abilities of most other antibodies.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. At present, drug options for systemic treatment of HCC are very limited. There is an urgent need to develop additional effective drugs for HCC treatment. In the present study, we found that proscillaridin A (ProA), a cardiac glycoside, exerted a strong anticancer effect on multiple HCC cell lines. ProA significantly inhibited the cell proliferation, migration, and invasion of HCC cells. ProA also had a marked inhibitory effect on the progression of HCC in the MHCC97H xenograft nude mouse model. ProA-mediated suppression of HCC was closely related to cell apoptosis. ProA-treated HCC cells displayed significant mitochondrial damage and elevated reactive oxygen species production, resulting in profound cell apoptosis. Meanwhile, ProA also played a role in autophagy induction in HCC cells. Defects in autophagy partially relieved ProA's anticancer effect in HCC cells. Our findings demonstrate that ProA can effectively inhibit HCC progression and may serve as a potential therapeutic agent for HCC treatment.

Objective Human astroviruses (HAstVs) are single-stranded RNA viruses of the Astroviridae family. Infection of classic HAstVs is one of the most common causes of acute viral gastroenteritis (infectious viral diarrhea). There is a lack of data on the prevalence and genetic characterization of classic HAstVs in acute viral gastroenteritis in the whole population. This study aimed to investigate the epidemiological trend, genotypes, viral co-infections, and viral loads of classic HAstVs in Shanghai, China, from January 2015 to December 2016. Methods A total of 6,051 non-redundant stool samples were collected in outpatients with acute diarrhea in Shanghai from January 2015 to December 2016. One-step real-time RT-PCR was used for screening viral diarrhea, including rotavirus A, rotavirus B, rotavirus C, norovirus genotype I and II, classic human astroviruses, and sapovirus. Real-time PCR was used for screening human enteric adenoviruses. Conventional RT-PCR was used for the amplification of viral fragments for genotyping. PCR products were sequenced and used for the construction of phylogenetic trees. Results The detection rate of classic HAstVs was 1.55% (94/6,051). The prevalence of HAstV infection displayed a typical winter/spring (December to March) seasonality and was highest in the 5-14 age group. Eighty-six samples were genotyped, which revealed HAstV-1 as the most prevalent genotype, followed by HAstV-5, HAstV-4, HAstV- 2, HAstV-8, and HAstV-3. There was a dramatic rise in the prevalence of HAstV-4 from December 2015 to March 2016, and the viral loads of HAstV-4 were significantly higher than those of other genotypes. Among the mixed infection samples, noroviruses were found to be the most frequently co-infected enteric viruses with HAstV. Conclusion Multiple genotypes of classic HAstVs circulated in Shanghai from January 2015 to December 2016. For the first time, HAstV-3\4\5\8 were detected in Shanghai.

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.

The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats 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. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 representative ACE2 orthologs among eighty orthologs for functional analysis and it showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind 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. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. 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.

The COVID-19 pandemic is spreading rapidly, highlighting the urgent need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We describe here the development of a phage-displayed single-domain antibody library by grafting naïve CDRs into framework regions of an identified human germline IGHV allele. This enabled the isolation of high-affinity single-domain antibodies of fully human origin. The panning using SARS-CoV-2 RBD and S1 as antigens resulted in the identification of antibodies targeting five types of neutralizing or non-neutralizing epitopes on SARS-CoV-2 RBD. These fully human single-domain antibodies bound specifically to SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of them were found to potently neutralize pseudotyped and live virus, and therefore may represent promising candidates for prophylaxis and therapy of COVID-19. This study also reports unique immunogenic profile of SARS-CoV-2 RBD compared to that of SARS-CoV and MERS-CoV, which may have important implications for the development of effective vaccines against SARS-CoV-2.

The newly emerged pandemic coronavirus, SARS-CoV-2, has posed a significant public health threat worldwide. However, the mode of virus transmission and tissue tropism is not well established yet. Recent findings of substantial liver damage in patients and ACE2+ cholangiocytes in healthy liver tissues prompted us to hypothesize that human liver ductal organoids could serve as a model to determine the susceptibility and mechanisms underlining the liver damage upon SARS-CoV-2 infection. By single-cell RNA sequencing, we found that long-term liver ductal organoid culture preserved the human specific ACE2+ population of cholangiocytes. Moreover, human liver ductal organoids were permissive to SARS-CoV-2 infection and support robust replication. Notably, virus infection impaired the barrier and bile acid transporting functions of cholangiocytes through dysregulation of genes involved in tight junction formation and bile acid transportation, which could explain the bile acid accumulation and consequent liver damage in patients. These results indicate that control of liver damage caused directly by viral infection should be valued in treating COVID-19 patients. Our findings also provide an application of human organoids in investigating the tropism and pathogenesis of SARS-CoV-2, which would facilitate novel drug discovery.

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca2+ as a secondary messenger in all examined species to date. However, the downstream signal of Ca2+ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca2+ , was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca2+ inhibitor, Lacl3 , attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference-mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)-11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca2+ and plays an important role in PBAN-induced male sex pheromone biosynthesis and female mating acceptance.

Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L89PKVLHKR96 on HBx, a segment that is highly conserved across genotypes and coincidentally overlaps with the DDB1-interacting segment. HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.

Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (LncRNA) with unclear mechanism in Alzheimer’s disease (AD) progression. Here, we found that NEAT1 down-regulates in the early stage of AD patients and APPswe/PS1dE9 mouse. Moreover, knockdown of NEAT1 induced de-polymerization of microtubule (MT) and axonal retraction of nerve cells by dysregulation of the FZD3/GSK3β/p-tau signaling pathway. Histone acetylation analysis at the Frizzled Class Receptor 3 (FZD3) promoter shows a marked decreased in the levels of the H3K27 acetylation (H3K27Ac) after NEAT1 knockdown. Our data demonstrates that P300/CBP recruited by NEAT1 to the FZD3 promoter and induced its transcription via histone acetylation. In recent years a growing number of evidences have shown an abnormal brain glucose homeostasis in AD. In the present study we also observed an abnormal brain glucose homeostasis and enhanced sirtuin1 (SIRT1) activity after knockdown of NEAT similarly as in AD. Our results provided insight into the role of NEAT1 in the maintenance of MT stability and its effect on glucose metabolism during early stages of AD.

With widespread abuse of antibiotics, bacterial resistance has increasingly become a serious threat. Acinetobacter baumannii has emerged as one of the most important hospital-acquired pathogens worldwide. Bacteriophages (also called “phages”) could be used as a potential alternative therapy to meet the challenges posed by such pathogens. Endolysins from phages have also been attracting increasing interest as potential antimicrobial agents. Here, we isolated 14 phages against A. baumannii, determined the lytic spectrum of each phage, and selected one with a relatively broad host range, named vB_AbaP_PD-6A3 (PD-6A3 for short), for its biological characteristics. We over-expressed and purified the endolysin (Ply6A3) from this phage and tested its biological characteristics. The PD-6A3 is a novel phage, which can kill 32.4% (179/552) of clinical multidrug resistant A. baumannii (MDRAB) isolates. Interestingly, in vitro, this endolysin could not only inhibit A. baumannii, but also that of other strains, such as Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). We found that lethal A. baumannii sepsis mice could be effectively rescued in vivo by phage PD-6A3 and endolysin Ply6A3 intraperitoneal injection. These characteristics reveal the promising potential of phage PD-6A3 and endolysin Ply6A3 as attractive candidates for the control of A. baumannii-associated nosocomial infections.

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

At least twelve percent of human cancers are caused by virus infection. To understand whether other viruses are associated with human cancers, a viral metagenomics approach was used to analyze the composition of the viral communities of the serum of the patients with Hodgkin’s lymphoma and Non-Hodgkin lymphoma. In this report, a human anellovirus TTMV named TTMV-SH was discovered from three patients with Hodgkin’s Lymphoma. The complete genome of TTMV-SH is 2812nt in length. Phylogenetic analysis based on ORF1 indicated that TTMV-SH of the 11 isolates cluster with TTMV strain TLMV-CBD231 sharing only 60.3%-62% sequence similarity, and the sequences divergence is 41.5%-43.1%, which indicates that TTMV-SH is a novel species. The TTMV-SH prevalence in HL group, especially in nodular sclerosing Hodgkin's lymphomas (NCHL), was significantly higher than in the healthy group implicated that the TTMV-SH may be associated with Hodgkin’s lymphoma, especially NCHL.

Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (KD = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.