Full text compilation! Chinese scientists published a Science paper that found that two non-competitive human neutralizing antibodies can block the SARS-CoV-2 virus binding to human ACE2 receptors

May 17, 2020 /
PRNewswire
BIOON/--- Coronary Virus disease (COVID-19) 2019 caused by the new coronavirus SARS-CoV-2 has become a pandemicThe virus has spread globally, causing fever, severe respiratory diseases and pneumoniaSystematic developmental analysis shows that the virus is closely related to severe acute respiratory syndrome (SARS-CoV), but appears to be more easily transmitted from person to personTo date, there have been no specific drugs or vaccinesThe SARS-CoV-2 virus belongs to the beta-coronavirus genus, which includes five pathogens capable of infecting humansOf the five pathogens, SARS-CoV and MERS-CoV are two highly pathogenic coronavirusesLike other coronaviruses, protoglycins (S proteins) homologous triolins located on the surface of SARS-CoV-2 virusplay play a vital role in receptor binding and viral entryThe S protein is a Class I fusion protein--- each S protein progenitor consists of an S1 and S2 domain, and the receptor binding domain (RBD) is located on the S1 domainPrevious studies have shown that, like SARS-CoV, SARS-CoV-2 also uses human ACE2 (hACE2) receptors to enter cellsScientists have found many neutralizing antibodies targeting SARS-CoV or MERS-CoVRBDTherefore, it is imperative to screen out neutralizing antibodies targeting SARS-CoV-2RBDin a new study,researchers from the Chinese Academy of Sciences, the University of Chinese Science and Technology, Capital Medical University, the China University of Science and Technology, shenzhen Third People's Hospital, china Agricultural University, Shanxi Institute of Advanced Innovation, the China Center for Disease Control and Prevention and the China Food and Drug Research Institute expressed SARS-CoV-2RBD protein as bait to isolate specific memory B cells from chowtortic blood mononucleosis (PBMC) in COVID-19 patientsThe variable regions of the coded anti-weight and light chains are amplified from different B cells and then cloned with the constant region of the antibody into the pCAGGS plasmidvector to produce IgG1 antibodies The findings were published online May 13, 2020 in the journal Science under the title "Anoncompeting a humanbodybodybodybodyblockCOVID-19virusbindingtoitsitetACE2." they amplification of 17 paired B-cell clones, three of which were the same (B5, B59, and H1) To determine the ability of antibody binding, plasmids containing pairs of heavy and light chains are transferred together into HEK293T cells to produce monoclonal antibodies (MAb) The ability to bind the liquid to RBD is then screened with a biolayer interferometer (bio-layerinterferometry, BLI) Use unrelated anti-SFTSVGn antibodies and SARS-CoV-specific antibodies as controls The up-liquidiation of four different antibodies (B5, B38, H2 and H4) is combined with SARS-CoV-2RBD, but not with SARS-CoVRBD, indicating that the epitopes of the two coronavirus RBDs are different in immunological Figure 1 Characterization of virus-specific neutralizing antibodies SARS-CoV-2 the four antibodies measured using surface plasma resonance (SPR) technology combined with SARS-CoV-2RBD solution constant (Kd) between 10
-7
and 10
-9
M (Figure 1, A-D) Next, they studied the neutralizing activity of the four antibodies to the SARS-CoV-2 virus (BetaCoV/Shenzhen/SZTH-003/2020) All four antibodies show neutralised activity, with ic
50
values ranging from 0.177?g/ml to 1.375?g/ml (Figure2, A to D) The mixtures B38 and H4 exhibit synergies and capabilities, even in the case of high viral titer (Figure 2E) Figure 2 Four antibodies can effectively neutralise SARS-CoV-2 viruses, two of which have a complementary inhibitory effect to assess the binding power of each antibody to inhibit RBD and ACE2, they conducted competitive tests using BLI and blocked tests using fluorescence-activated cell sorting (FACS) For BLI assays, streptomycin biosensors labeled with biotin-based RBD are saturated with antibodies and then, in the presence of soluble ACE2, allow the test antibodies to flow through The combination of B38 and H4 and ACE2 is fully competitive with RBD Instead, B5 shows partial competition, while H2 does not compete with ACE2 and RBD (Figures 1, E to H) The blocking test using FACS showed the same result (Figure 1I) In order to determine whether B38 and H4 are targetbound combined with the same epitope, they used BLI for epitope competition determination The NI-NTA sensor marked with RBD is saturated with the B38IgG, and then the H4IgG is allowed to flow through, or the Ni-NTA sensor is first combined with the H4IgG saturation, allowing the B38IgG to flow through Although RBD is saturated by the first antibody binding, but the second antibody can still bind to RBD, but there is a certain inhibitory effect This indicates that B38 and H4 identify different table holders on the RBD and have partial overlaps (Figures 1, J and K) to explore the protective efficacy of B38 and H4 in the body against the SARS-CoV-2 virus challenge, inject edg2 25 mg/kg B38 or H4 in hACE2 genetically modified mice after 12 hours of viral challenge Compared to the PBS control group and the H4 group, the B38 group lost weight slowly and recovered after 3 days of infection (Figure 3A) The number of copies of viral RNA in the lungs of mice was also measured 3 days after infection The number of RNA copies in both the B38 and H4 groups was significantly lower than that of the PBS group, with a decrease of 32.8% and 26% respectively (Figure 3B) These results show the same trend as in which Histopathological examination showed that severe bronchial pneumonia and interstitial pneumonia were observed in mice in the PBS control group, as well as edema, bronchial epithelial cell shedding, and lymphocytes leaching in the alveoli cavity (Figures 3, C and F) Mild bronchial pneumonia was observed in the H4 group (Figures 3, E and H), while lung lesions were not observed in the B38 group (Figures 3, D and G) the protective effect of monoclonal antibodies after infection with SARS-CoV-2 virus in a genetically modified mouse model in Figure 3.hACE2 consistent with the binding affinity between RBD and B38 or H4, stable compounds are obtained in both RBD-B38 and RBD-H4 mixtures The crystal structure of the RBD-B38Fab complex is parsed at 1.9 E resolution The three complementary determining regions (CDRs) on the B38 anti-weight chain and two CDRs on the light chain were involved in interaction with RBD (Figures 4, A, B and G to K) The buried surface area of B38 anti-weight chain and light chain on the table was 713.9 E and 497.7 E, respectively There are 36 residual bases in the RBD that are involved in interaction with B38, of which 21 are and 15 are involved in interactions with heavy and light chains, respectively (Figure 4B) The sequence comparison shows that only 15 of the 36 residual bases in the RBD table (defined as the residue buried by B38) are conservative between SARS-CoV-2 and SARS-CoV (Figures 4, D to F and Figure S3) It is worth noting that most of the contact between the interface between B38 and RBD is hydrophilic interaction Water molecules play an important role in the combination of SARS-CoV-2RBD and B38 (Figures 4, G and I to K) These differences explain the specific binding between B38 and SARS-CoV-2RBD instead of SARS-CoVRBD Figure 4 The structural analysis of B38 and SARS-CoV-2RBD complexes and the RBD binding epitope of B38 and hACE2 were compared to explore the structural basis for B38 to block the interaction between SARS-CoV-2RBD and hACE2, the structure of RBD/B38-Fab and RBD/hACE2 complex was superimposed The heavy-chain variable area of B38 and the light-chain variable zone both block the binding of hACE2 in the stereo structure (Figure 4C) It is worth noting that the RBD, which is combined with B38 or hACE2, has no significant conformation already, with a average square deviation of 0.489 E (194 atoms) Further analysis showed that 18 of the 21 amino acids on RBD were involved in the combination of RBD and B38 and hACE2 (Figure 4D), which also explains the ability of B38 to disrupt the binding between SARS-CoV-2RBD and hACE2 receptors as the COVID-19 outbreak continues to spread, the characterization of epitope suprapers on SARS-CoV-2RBD will provide valuable information for vaccine development In addition, the molecular characteristics of neutralizing antibodies targeting RBD epitopes contribute to the development of small molecules or peptide drugs/inhibitors These neutralizing antibodies themselves are promising candidates for the prevention and treatment of SARS-CoV-2 virus (biovalleybioon.com) Reference: Yan Wuetal.
Anonloninglystos block COVID-199virustoitsitititettace 2 Science, 2020, doi:10.1126/science.abc2241.