From the beginning of 2002 and 2012, severe respiratory syndrome coronavirus (SARS‐CoV) and Middle East respiratory syndrome coronavirus (MERS‐CoV) crossed the species barriers to infect humans, ...causing thousands of infections and hundreds of deaths, respectively. Currently, a novel coronavirus (SARS‐CoV‐2), which has become the cause of the outbreak of Coronavirus Disease 2019 (COVID‐19), was discovered. Until 18 February 2020, there were 72 533 confirmed COVID‐19 cases (including 10 644 severe cases) and 1872 deaths in China. SARS‐CoV‐2 is spreading among the public and causing substantial burden due to its human‐to‐human transmission. However, the intermediate host of SARS‐CoV‐2 is still unclear. Finding the possible intermediate host of SARS‐CoV‐2 is imperative to prevent further spread of the epidemic. In this study, we used systematic comparison and analysis to predict the interaction between the receptor‐binding domain (RBD) of coronavirus spike protein and the host receptor, angiotensin‐converting enzyme 2 (ACE2). The interaction between the key amino acids of S protein RBD and ACE2 indicated that, other than pangolins and snakes, as previously suggested, turtles (Chrysemys picta bellii, Chelonia mydas, and Pelodiscus sinensis) may act as the potential intermediate hosts transmitting SARS‐CoV‐2 to humans.
Highlights
The critical residues of S protein RBD binding with ACE2 indicated the potential intermediate hosts transmitting SARS‐CoV‐2 to humans.
The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that ...SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
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•SARS-CoV-2 uses ACE2 to enter target cells•SARS-CoV-2 and SARS-CoV bind with similar affinities to ACE2•Structures of SARS-CoV-2 spike glycoprotein in two conformations•SARS-CoV polyclonal antibodies inhibit SARS-CoV-2 spike-mediated entry into cells
SARS-CoV-2, a newly emerged pathogen spreading worldwide, binds with high affinity to human ACE2 and uses it as an entry receptor to invade target cells. Cryo-EM structures of the SARS-CoV-2 spike glycoprotein in two distinct conformations, along with inhibition of spike-mediated entry by SARS-CoV polyclonal antibodies, provide a blueprint for the design of vaccines and therapeutics.
The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we ...investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
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•Over 100 mutations were selected for analyses on their infectivity and antigenicity•The dominant D614G itself and combined with other mutations are more infectious•Ablation of both N331 and N343 glycosylation at RBD drastically reduced infectivity•Ten mutations such as N234Q, L452R, A475V, and V483A was markedly resistant to some mAbs
Eighty natural variants and 26 glycosylation spike mutants of SARS-CoV-2 were analyzed in terms of infectivity and antigenicity using high throughput pseudovirus assay in conjunction with neutralizing antibodies.
Coronavirus disease‐2019 (COVID‐19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection has come out as a major threat for mankind in recent ...times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS‐CoV‐2), its mode of interaction with the angiotensin‐converting enzyme‐2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll‐like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS‐CoV‐2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS‐CoV‐2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS‐CoV‐2 from a bat and also revealed that TLR4 may have a crucial role in the virus‐induced inflammatory consequences associated with COVID‐19. Therefore, selective targeting of TLR4‐spike protein interaction by designing competitive TLR4‐antagonists could pave a new way to treat COVID‐19. Finally, this study is expected to improve our understanding on the immunobiology of SARS‐CoV‐2 and could be useful in adopting spike protein, ACE2, or TLR‐guided intervention strategy against COVID‐19 shortly.
Highlights
Spike glycoprotein of SARS‐CoV‐2 is phylogenetically close to bat coronavirus.
SARS‐CoV‐2 spike glycoprotein strongly binds with ACE2 receptor protein from both human and bat origin.
Cell surface TLR4 is most likely to be involved in recognizing molecular patterns from SARS‐CoV‐2.
Although most mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome are expected to be either deleterious and swiftly purged or relatively neutral, a small proportion ...will affect functional properties and may alter infectivity, disease severity or interactions with host immunity. The emergence of SARS-CoV-2 in late 2019 was followed by a period of relative evolutionary stasis lasting about 11 months. Since late 2020, however, SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations, in the context of 'variants of concern', that impact virus characteristics, including transmissibility and antigenicity, probably in response to the changing immune profile of the human population. There is emerging evidence of reduced neutralization of some SARS-CoV-2 variants by postvaccination serum; however, a greater understanding of correlates of protection is required to evaluate how this may impact vaccine effectiveness. Nonetheless, manufacturers are preparing platforms for a possible update of vaccine sequences, and it is crucial that surveillance of genetic and antigenic changes in the global virus population is done alongside experiments to elucidate the phenotypic impacts of mutations. In this Review, we summarize the literature on mutations of the SARS-CoV-2 spike protein, the primary antigen, focusing on their impacts on antigenicity and contextualizing them in the protein structure, and discuss them in the context of observed mutation frequencies in global sequence datasets.
The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the ...effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody
. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab
, S2X259
and S2H97
. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.
The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute ...respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein
show promise therapeutically and are being evaluated clinically
. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies
in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs
. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.
SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive ...possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known ...about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant
, which has now been reported in ...94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b2
. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.
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