A new study of neutralizing antibodies in a new coronavirus led by Gao Fu, director of the Chinese Center for Disease Control and Research and a member of the Chinese Academy of Sciences, shows that B38 and H4 antibodies can prevent the binding region of the new coronavirus S protein receptor RBD to the binding region of the human cell receptor ACE2. Mouse model tests showed that the two antibodies reduced the viral load in the lungs after infection in mice. The study also provides a structural basis for vaccine design.
The above study was published on May 7 local time on the bio-preprinted website medRxiv (“A non-non-competition of human neutrality of human neutrality antibody block COVID-19 virus to its body ACE2” “A neutral antibody for non-competitive humans can block the binding of COVID-19 virus with its receptor ACE2”). The team isolated four human-derived monoclonal antibodies from a Chinese rehabilitation patient, which were shown in vitro, with B38 and H4 antibodies in particular having potential.
The work was carried out by the scientific research team of Capital Medical University, Beijing Institute of Life Sciences of the Chinese Academy of Sciences, Cas for Microbiology and Research Institute of Chinese Academy of Sciences, Chinese Academy of Sciences, Third People’s Hospital of Shenzhen, China Agricultural University, Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and the China Center for Disease Control and Prevention. The paper is written by Gao Fu, director of the Chinese Center for Disease Control and Prevention, a member of the Chinese Academy of Sciences, Gao Lei, associate researcher of the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and Liu Lei, director of the Third People’s Hospital of Shenzhen.
To date, there are no specific drugs or vaccines for the new coronavirus. Currently, most treatment development programs are aimed at the “key” of new coronaviruses invading humans, the S protein (protoglytin). S-protein-mediated virus and host cell binding and invasion, it is composed of S1 domain and S2 domain two parts, respectively, mediated receptor binding and membrane fusion. The receptor binding domain (RBD) is located in S1. The team believes that screening anti-neoviral neutralizing antibodies through the use of RBD proteins is a priority strategy available today.
They first obtained four different antibodies (B5, B38, H2 and H4) from patients with new coronary recovery period, and their upper-clear sepsis showed that they could bind to the new coronavirus RBD, but failed to bind to THE RBD of SARS-CoV, indicating that the RBD epitopeof of SARS-CoV and the new coronavirus was different in immunology.
These 4 antibodies show different binding capacity to the new coronavirus RBD, with Kd ranging from minus 7 to 10 minus 9 times M. Where H4 showed relatively high binding affinity, Kd was 4.48nM, while B5 showed a relatively weak combined affinity, and Kd was 305nM. B38 and H2 are combined with RBD, with Kd at 70.1nM and 14.3nM, respectively.
All 4 antibodies show neutral activity, with IC50 (semi-inhibited concentration) values ranging from 0.177 to 1.375 ?g/ml. B38 is the most effective antibody, followed by H4, H2 and B5.
The team then assessed the competitiveness of each antibody-blocking virus, RBD, in combination with ACE2, and found that B38 and H4 could compete fully with ACE2 and bind to RBD. Instead, B5 shows partial competition, while H2 shows a combination with RBD, but not ace2.
Studies have also shown that B38 and H4 can identify different epitopes on RBD and partially overlap.
To explore the protective effects of B38 and H4 against neo-coronavirus attacks in the body, the team conducted mouse therapy experiments. After 12 hours of poisoning, the team injected 25 mg/kg of b38 or H4 in a single dose of B38 or H4 in hACE2 genetically modified mice. The results showed that mice in the B38 group whohad a significant weight loss after attacking the drug recovered within 3 days (dpi) after infection, compared with the control group and the H4 group.
The team also tested copies of viral RNA in mouse lung tissue at 3 dpi. The relative RNA copy changes in both the B38 group and the H4 group were significantly lower than those in the control group, and were reduced by 32.8% and 26%, respectively, compared to the control group.
To further clarify the structural basis of the antibody neutralization mechanism, the team prepared the RBD-B38 and RBD-H4 Fab complexes through in vitro incubation and purification. The three complementary decision zones (CDRs) on the heavy chain and the two complementary decision zones on the light chain participate and the role of the virus RBD. There are 36 residual bases in the virus RBD interacting with B38, of which 21 are interacting with heavy chains and 15 are interacting with light chains.
The sequence comparison showed that of the 36 residues in the epitope, only 15 were conservative between the neo-coronavirus and SARS-CoV. This explains the specific reactive nature of B38. Further analysis of the interaction of the combined interface reveals that the 470 ring of virus RBD plays an important role in the binding with the B38 HCDR1 ring.
The team also studied the structural basis of the interaction between b38 blocking COVID-19 virus RBD and ACE2. The results show that both the VH and VL of B38 cause the spatial bit resistance (Figure 4C) combined with RBD and ACE2. It is worth noting that RBD did not show significant conformation altogether in the two forms of binding with B38 and with hACE2. Further analysis found that 18 of the 21 amino acids on RBD were exactly the same when combined, which clearly explains why B38 completely eliminates the binding of the new coronavirus RBD to the receptor.
In general, the RBDs of the neo-coronavirus and SARS-CoV have a high homologous nature and are both associated with ACE2 receptors to infect the host. However, binding affinity is different, the new coronavirus RBD interacts more with the atoms of SARS-CoV RBD and hACE2, and therefore exhibits higher binding affinity for receptor binding.
So far, no studies have reported antibodies through competitive ACE2 binding site intersections with SARS-CoV2 and SARS-CoV. CR3022 from patients with SARS during recovery can be combined with RBDs from both viruses. However, although it can be moderated to SARS-CoV, it cannot be medium to new coronavirus.
The study stressed that a comprehensive understanding of the immune response of the new coronavirus’s body fluids needs to be done in more patients. In addition, cocktail antibodies should be considered as an alternative treatment strategy to avoid potential escape mutations. “As the new coronal outbreak continues to spread, the identification of the viral RBD protein epitope is critical, which will provide valuable information for the development of vaccines. In addition, the molecular characteristics of neutralizing antibodies for epitopes contribute to the development of small molecules or peptide drugs/inhibitors. “
In summary, the study concluded that neutralizing antibodies identified in this study are expected to become candidate antibodies for the prevention and treatment of new coronaviruses.