Science: Chinese scientists have structurally revealed a powerful therapeutic antibody and SARS-CoV-2 and SARS-CoV mechanisms.

According to the World Health Organization (WHO), the 2019 coronavirus epidemic (COVID-19) pandemic has infected more than 7 million people in more than 200 countries and killed 400,000 people, according to the World Health Organization (WHO)!---- The pathogen that led to this pandemic is an emerging coronavirus: SARS-CoV-2, which is closely related to THE SARS-CoV, which belongs to the coronavirus family b lineageThe stingy glycoprotein (S) located in the SARS-CoV-2 and SARS-CoV envelopes has approximately 80% consistency of amino acid sequences, and both viruses use human angiotensin conversion enzyme 2 (hACE2) into host cellsCell entry is achieved through viral-receptor binding mediated by The S-protein homologous, followed by viral-host membrane fusion, where by the Receptor Binding Domain (RBD) of the S proteinDestroying this key role of S protein in infection establishment is the primary goal of neutralizing antibodies and is also the focus of therapeutic interventions and vaccine designSeveral sars-CoV neutralantibodies (NAbs) previously characterized have been shown to have very limited neutrality to SARS-CoV-2In these neutralizing antibodies, CR3022 is a weak neutral antibody against SARS-CoV, although it can be tightly bound to SARS-CoV-2, but there is no neutral effect on the latter, which indicates that there may be conformational differences in neutralizing the epitheleRecent studies have reported that two SARS-CoV neutralandizing antibodies 47D11 and S309 have also been found to neutralise SARS-CoV-2, suggesting that the beta coronavirus belongs to the B lineage in a wide range of cross-neutral and epitopeRecovery plasma containing SARS-CoV-2 neutralizing antibodies has been found to provide clear protection for PATIENTs with COVID-19, but there is still a gap in scientists' understanding of the immune progenitor characteristics and key epialities of SARS-CoV-2, which hinders the development of effective immunotherapy for the virusThe RBD of SARS-CoV and SARS-CoV-2 has approximately 75% amino acid sequence consistency, so it is possible to find cross neutralizing antibodies targeting RBDIn a new study, researchers from the Chinese Academy of Sciences, the University of Chinese Academy of Military Medicine, the Chinese Academy of Military Medicine, the Chinese Academy of Medicine and Drug Administration, the Chinese Academy of Medical Sciences, the Beijing Concord Medical College, Beijing Shenzhou Cell Biotechnology Company and Beijing Yiju Shenzhou Technology Company used phage display technology to build an antibody bank produced from RNA extracted from peripheral lymphocytes in mice immunized with recombined SARS-CoV RBDUse SARS-CoV-2 RBD as a target to screen the phage antibody bank to find potential targetsAntibodies closely bound to SARS-CoV-2 RBD will be displayed for further optimization as chimeric antibodies and their neutralizing activity is tested using a pseudovirus system based on the blister-based oral inflammatory virus (VSV)The findings were published online July 23, 2020 in the journal Science with the title "R.R.Sneutral for life-for-cyvs-2 and SARS-CoV by a potent therapeutic antibody"In the test of antibodies, the SARS-CoV-2 fake virus has a strong neutralized active antibody cloning 014 humanized, and named it H014In order to evaluate the combination of affinity, the OctET system is used to monitor the real-time combination of H014 and SARS-CoV-2 RBD or SARS-CoV RBDBoth The H014 IgG and its Fab fragments show a close bond with SARS-CoV-2 RBD and SARS-CoV RBD, which have a considerable affinity with the two RBDs at the subM levelThe neutralized and tested of the pseudovirus showed that H014 had strong neutrality: 50% neutralized and concentrated values (IC50) for SARS-CoV-2 and SARS-CoV pseudoviruses were 3 nM and 1 nM, respectivelyThe spot reduction test on the real SARS-CoV-2 strain (BetaCoV/Beijing/AMMS01/2020) verified its neutralization activity: its IC50 was 38 nM, 10 times lower than observed in a pseudo-virus systemThe authors then attempted to assess the protective effects of H014 in their previously established hACE2 humanized mouse model, which is sensitive to SARS-CoV-2 infectionIn this mouse model, because of the expression of hACE2 on the surface of the lung cells, SARS-CoV-2 enters the lungs and replicates like in humans, showing lung lesions five days after infectionHACE2 humanized mice were treated with 50mg/kg H014 after 4 hours of nasal infection (1 dose, treatment) or 12 hours before and 4 hours (2 doses, preventive plus therapy) in 5 x 105 PFU SARS-CoV-2 (BetaCoV/Beijing/AMMS01/2020) nasal infectionAll mice challenged by the virus were killed on day 5Although the viral load on the lungs of the pbS group (control group) of mice soared to about 107 copies/g on the fifth day, it is worth noting that in the prevention group and the prevention plus treatment group, H014 treatment reduced the viral droplets of their lungs by about 10 times and 100 times on the fifth day, respectivelyLung pathology analysis showed that SARS-CoV-2 caused mild interstitial pneumonia after phosphate buffer (PBS) treatment, characterized by inflammatory cell immersion, alveoscosine thickening and significant vascular damageIn contrast, no significant alveolar epithelial cell lesions or phenomotic bleeding were observed in the lung slices of mice treated with H014, indicating that H014 had a potential therapeutic effect in the treatment of COVID-19The overall structure of the SARS-CoV-2 S protein tripolymer is similar to that of SARS-CoV and other coronavirusesEach monomer of the S protein consists of two functional sub-basesS1 subkeys bind to host cell receptors, while S2 sub-based mediated viral membranes and host cell membranes mergeThe four domains within the S1 subki comprise the N-end domain (NTD), the RBD, and the two sub-domains (SD1 and SD2), which are located adjacent to the cutting sites of S1/S2The hinged motion of the RBD produces two different conformational states, called "off" and "open", where the close state corresponds to the receptor's inaccessible state, and the open state corresponds to the state in which the receptor is accessible, which should be substableThe low-temperature electroscope (Cryo-EM) structure representation of the transcellular domain of the stable SARS-CoV-2 S protein and the h014 Fab fragment snare found that the composite used three different conformation states, corresponding to one RBD open and two RBD close (state 1), two RBD open s and one RBD off (state 2) and three RBD all open states (state 3) Interestingly, despite the presence of too many Fabs, the completely off (state 4) OFB-CoV-2 S protein tripolymer structure without any Fab binding was also observed during the three-dimensional classification of Cryo-EM data, which suggested that the binding site of H014 was exposed by protein "breathing" and then performed random RBD movement (Figure 2A) The authors reconstructed S proteins in the four states asymmetricly at resolutions of 3.4 to 3.6 Ai However, due to the heterogeneity of conformation, the electronic potential map of the interface between RBD and H014 is relatively weak To solve this problem, they used a "block-based" reconstruction method to focus classification and fine-tuning, further improving the local resolution to 3.9 Ai, thus making the analysis of interaction patterns more reliable The interaction between H014 and S proteins was analyzed in detail by combining interface structure H014 identifies the conformation almost adhesion sitcom on the side of the open RBD and only involves protein/protein contact, unlike receptor binding base sequence (RBM) H014 complements (paratope, i.e antibody decision clusters) constitute all six complementary deterministic zones (CDR) rings (CDRL1-3 and CDRH1-3) and unusual heavy-chain frames (HF-R, residual 58-65), which interact closely with RBD to create approximately 1000 square sq.au burial areas The variable areas of The H014 light and heavy chains contribute approximately 32% and 68% of the buried surface area, respectively, through hydrophobic and hydrophilial exposure This H014 epitope consists of 21 residues, mainly located in the area of alpha 2-beta 2-2-386, s3 (residual s405-408, 411-413), alpha 4 (residual 439) and s4 (residual 503) region, thus forming a cavity on the side of the RBD A CDRH3, a 12-strong amino acid residue, is inserted into the cavity, and CDRH3, which is rich in hydrophobic residue (YDPYVM), comes into contact with the 3 and edge of the rbD's five-strand edifice (beta-2) region The close ness between RBD and H014 is mainly due to the extensive hydrophobic interactions contributed by two blocks ( patches ) , one of which is made up of F54 from CDRH2 , Y101 from CDRH3 , and A411, P412 from RBD Formed with Y508, another block consists of Y49 from CDRL2, P103, Y104, Y105 from CDRH3, and V407, V503, and Y508 from RBD In addition, hydrophilicity contact from CDRH1 and HF-R further enhances the interaction of RBD-H014, resulting in a very high binding affinity at the sub-nM level at temperatures of 25 degrees C or 37 degrees C The remains that make up the table are mostly conservative, with three unit-point mutations (R408I, N439K and V503F) present in the SARS-CoV-2 strain currently reported to be transmitted in the population In addition, some SARS-CoV-2 isolates carry a common mutation, V367F, in RBD, which is located near the main epitope patch (epitope patch) alpha2-beta 2-2 The binding affinity of recombinant RBD structures and H014, carrying the above amino acid residual base mutations and other reported alternative mutations, is difficult to distinguish, suggesting that H014 may have extensive neutrality and activity in the SARS-CoV-2 strain currently spreading globally Of the 21 amino acid residues at this H014 table, there are 17 residuals (81%) of SARS-CoV and SARS-CoV, which explains the cross-reactivity and similar affinity of the combination To study whether H014 interferes with the binding of RBD and hACE2 to SARS-CoV or SARS-CoV, the authors conducted competitive binding experiments at the protein and cellular levels The results of the Enzyme-Linked Immunosorption Test (ELISA) show that H014 is able to compete with recombinant hACE2 with the RBDof sars-CoV-2 and SARS-CoV, with EC50 values of 0.7 nM and 5 nM, respectively In addition, H014 can effectively block the binding of SARS-CoV-2 RBD and 293T cells expressing ACE2, as well as recombinant ACE2 and 293T cells expressing the SARS-CoV-2 S protein To verify the potential complete closure of H014 to the S-protein tripolymer, the authors conducted two sets of surface plasma resonance (SPR) tests, first exposing the S-protein tripolymer to H014 and then to hACE2, or vice versa As expected, the binding of H014 completely blocks the attachment of hACE2 to the S protein tripolymer Furthermore, hACE2 can be removed from the S protein tripolymer and replaced by H014 To further validate these results in a cell-based viral infection model, they performed real-time RT-PCR analysis to quantify the viral load on the surface of the host cell that remained at 4 degrees C and was exposed to the antibody before or after the virus was attached to the virus H014 effectively blocks SARS-CoV-2 from adhering to the cell surface in a dose-dependent manner, and virus particles that have been bound to the cell surface can be partially released by H014 By superimposing the structure of the H014-SARS-CoV-2 tripolymer S protein complex with the structure of the ACE2-SARS-CoV-2 RBD complex, it can be found that the cause of the conflict between hACE2 and H014 is the region of the junction of the junction of the top of the RBD (s)4 This observation is very different from that of most known SARS-CoV RBD-targeted antibody complexes, where these targeted antibodies directly identify RBM Thus, H014's ability to block SARS-CoV-2 attached to host cells can be attributed to a stereoscopic conflict with hACE2 Similar to RBM, this H014 epitope is only accessible in an open state, indicating that it has this EFFECT similar to RBM--- involves dynamic interference with the interaction with the host cell The structures observed by these authors, together with previously reported coronavirus S structures (excluding human coronavirus HKU1), have revealed the transition between S1 sub-based respiratory-mediated "off" and "open" conformations Unlike the hinged motion of the RBD observed in most structures, the conformational transition from "off" to "open" observed in these authors' structures mainly involves two rotation steps: 1) SD1 rotates counterclockwise about 25 degrees around the hinge point (at the amino acid residue 320); and 2) the RBD itself rotates counterclockwise by about 60 degrees The conformation rotation of SD1 and RBD in near-end positions causes the changes in the far end position to be amplified, thus opening up enough space for the combination of H014 or ACE2 What is not clear is whether the particular conformational changes observed in the composite structures of these authors are the result of H014 participation or the synergetic movement of SD1 and RBD Body fluid immunity is essential to prevent coronavirus infection, and passive immunity of restorative plasma has been shown to be effective in treating SARS and COVID-19 It has previously been reported that some SARS-CoV-specific neutralizing antibodies, including m396, 80R and F26G19, are mainly combined with RB under RBD.