Science is compiled in full! Chinese scientists have structurally revealed a powerful therapeutic antibody and SARS-CoV-2 and SARS-CoV mechanisms.

!--:page title"--July 26, 2020 /--- According to the World Health Organization (WHO), the 2019 coronavirus pandemic has infected more than 7 million people in more than 200 countries and killed 400,000, according to the World Health Organization (WHO).
the pathogen that caused the pandemic is an emerging coronavirus: SARS-CoV-2, which is associated with the closely related SARS-CoV, which belongs to the Coronavirus family of beta coronavirus.
the stinging glycoprotein (S) located in the SARS-CoV-2 and SARS-CoV envelopes has approximately 80% amino acid sequence consistency, and both viruses use human angiotensin conversion enzyme 2 (hACE2) to enter the host cell.
cell entry is achieved through viral-receptor binding mediated by The S protein homologous tripolymer, followed by viral-host membrane fusion, where this binding is carried out through the receptor binding domain (RBD) of the S protein.
destroys this key role of S proteins in infection establishment as the main goal of neutralizing antibodies, as well as therapeutic interventions and vaccine design. several SARS-CoV neutralantibodies (NAbs) previously characterized by
have been shown to have very limited neutrality to SARS-CoV-2.
in these neutralizing antibodies, CR3022 is a weak neutral antibody against SARS-CoV, although it can be closely combined with SARS-CoV-2, but there is no neutral effect on the latter, which indicates that there may be conformational differences between neutralizing and epialdes.
recent studies have reported that two SARS-CoV neutral and antibodies 47D11 and S309 have also been found to neutralise SARS-CoV-2, suggesting that the beta coronavirus belongs to the B-spectrum memory in a wide range of cross-neutraland and tabalist.
recovery 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 epimostes of SARS-CoV-2, which hinders the development of effective immunotherapy for the virus.
THE RBD of SARS-CoV and SARS-CoV-2 has about 75% amino acid sequence consistency, so it is possible to find cross neutralizing antibodies targeting RBD. In a new study,
, researchers from the Chinese Academy of Sciences, the University of 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 Co., Ltd. used phage display technology to build an antibody bank that was produced from peripheral lymphocytes derived from mice immunized with recombinant SARS-CoVD.
use SARS-CoV-2 RBD as a target for screening the phage antibody pool to find potential targets.
will show antibodies bound to SARS-CoV-2 RBD as chimeric antibodies for further optimization and test their neutralactivity using a pseudo-viral system based on blister-based oral inflammatory virus (VSV).
research published online July 23, 2020 in the journal Science, entitled "R.R.S. neutral for-fieldization to SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody".
humanized antibody cloning 014, which has strong neutralization and activity of SARS-CoV-2 fake virus, and named it H014 in the antibodies tested.
to evaluate the real-time combination of H014 and SARS-CoV-2 RBD or SARS-CoV RBD using the OCTET system to assess the combination of affinity. both 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 at the subM level (Figure 1A).
Figure 1. H014 is a therapeutically valuable B-line coronavirus cross-neutralizing antibodies, pictured from Science, 2020, doi:10.1126/science.abc5881.
the neutralized and tested of the pseudovirus showed that H014 had strong neutrality: 50% neutralized concentration value (IC50) for SARS-CoV-2 and SARS-CoV pseudovirus, respectively, 3 nM and 1 nM (Figure 1B).
the spot reduction test of the real SARS-CoV-2 strain (Beta-CV/Beijing/AMMS01/2020) verified its neutralactivity: its IC50 was 38 nM, 10 times lower than observed in a fake virus system (Figure 1C).
the 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 infection.
in this mouse model, sars-CoV-2 enters the lungs and replicates like in humans, showing lung lesions five days after infection, due to the expression of hACE2 on the surface of the lung cells.
hACE2 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 and other treatments) in 5 x 105 PFU's SARS-CoV-2 (BetaCoV/Beijing/AMMS01/2020) nasal infection.
all mice challenged by the virus were killed on day 5.
Although the viral load on the lungs of the mice in the PBS group (control group) soared to about 107 RNA copies/g (Figure 1D) on the fifth day, it is worth noting that in the prevention group and the prevention plus treatment group, H014 treatment reduced the viral titness of their lungs by about 10 times and 100 times on the fifth day (Figure 1D).
lung pathology analysis showed that SARS-CoV-2 caused mild interstitial pneumonia after phosphate buffer (PBS) treatment, characterized by inflammatory cell immersion, alveolar interval thickening and significant vascular damage.
, in contrast, no significant alve osmoblast lesions or phenomenal haemorrhage were observed in the lung slices of mice treated with H014 (Figure 1E), indicating that H014 had a potential therapeutic effect in the treatment of COVID-19.
the overall structure of the SARS-CoV-2 S protein tripolymer is similar to that of SARS-CoV and other coronaviruses. Each monomer
S protein consists of two functional sub-bases.
The Fusion of the S1 subkey binding host cell receptor, and the S2 sub-mediated viral membrane and the host cell membrane.
the four domains within the S1 subkey are the N-end domain (NTD), the RBD, and two sub-domains (SD1 and SD2), which are located adjacent to the cutting point of S1/S2.
the 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 receptor's accessible state, which should be substable.
!--/ewebeditor:!---!--:pagetitle"--Cryoscope-EM structure table of a composite of the stable SARS-CoV-2 S protein and H014 Fab fragments It was found that the compound used three different conformation states, corresponding to one RBD open and two RBD close (state 1), two RBD opens plus one RBD off (state 2), and three RBD all open (state 3) (Figure 2A).
interesting, despite the presence of too many Fabs, the structure of the SARS-CoV-2 S protein tripolymer completely off (state 4) without any Fab binding was also observed in 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 the S proteins of the four states asymmetricly at resolutions of 3.4 to 3.6 amy.
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 for focus classification and fine processing, and further improved 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 using the binding interface structure
.
Figure 2. Cryo-EM structure of a complex of SARS-CoV-2 S protein tripolymers and H014, pictured from Science, 2020, doi:10.1126/science.abc5881.
The D014 identifies the conformation epitope located on the side of the open RBD and only involves protein/protein contact, which is different from the receptor binding base sequence (RBM) (Figure 2B).
H014 repertoire (paratope, i.e. antibody decision cluster) constitutes 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, resulting in approximately 1000 square sq.m. Amy burial area (Figure 2C).
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 contact.
this H014 epitope consists of 21 residuals, mainly located in the area of alpha 2-beta 2-2-386, s3 (residual s405-408, 411-413), alpha 4 (residual 439) and 4 (residual 503), thus forming a cavity on one side of the RBD (Figure 2, BD and BD).
a 12-amino acid residue CDRH3 inserted into the cavity, and CDRH3, which is rich in hydrophobic residue (YDPYVM), is in contact with the 3rd and edge contacts of the RBD's five-strand edifice beta-folding (beta 2) region (Figure 2D). the close ness of
RBD and H014 is mainly due to the extensive hydrophobic interactions contributed by two blocks ( patch ) , one of which is made by F54 from CDRH2 , Y101 from CDRH3 , and A411, P412 and Y from RBD The other block is composed of Y49 from CDRL2, P103, Y104, Y105 and V407, V503, and Y508 from RBD (Figure 2E).
in addition, hydrophilic 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 (Figure 2E).
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.
, 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.
carrying the above amino acid residual base mutations and other reported alternative mutations of the recombinant RBD structure and H014 binding affinity is difficult to distinguish, which suggests that H014 may be currently spread inglia to the global SARS-CoV-2 strain has a wide range of neutrality.
the 17 residuals (81%) between SARS-CoV-2 and SARS-CoV are the same in the 21 amino acid residues at this H014 table, which explains the cross-reactivity and similar affinity.
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 Immune Adsorption Test (ELISA) show that H014 is able to compete with recombinant hACE2 with the RBD of SARS-CoV-2 and SARS-CoV, with EC50 values of 0.7 nM and 5 nM (Figure 3A), respectively.
, H014 can effectively block the binding of SARS-CoV-2 RBD to 293T cells expressing ACE2, as well as recombinant ACE2 and 293T cells expressing SARS-CoV-2 S protein (Figure 3B).
Figure 3. H014's Neutral mechanism, pictured from Science, 2020, doi:10.1126/science.abc5881.
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.
again, hACE2 can be removed from the S protein tripolymer and replaced by H014 (Figure 3C).
in order to further validate these results in a cell-based virus infection model, they performed real-time RT-PCR analysis to quantify residual virus attachment at 4 degrees C.