A new study of novel coronavirus-neutralizing antibodies led by Gao Fu, director of the Chinese Center for Disease Control and Prevention and a member of the Chinese Academy of Sciences, and other scholars shows that B38 and H4 antibodies block the binding of the novel coronavirus S protein receptor-binding region RBD to the human cell receptor ACE2.
Mouse model tests showed that both antibodies reduced viral load in the lungs of mice after infection.
The study also provides a structural basis for vaccine design.
The above study was published on May 7, local time on the bioprinting website medRxiv ("A non-competing pair of human neutralizing antibodies blocks COVID-19 virus binding to its receptor ACE2").
The team isolated four human-derived monoclonal antibodies from a recovering Chinese patient, all of which showed neutralizing ability in vitro, with B38 and H4 antibodies being particularly promising.
The work was carried out jointly by research teams from Capital Medical University, Beijing Institute of Life Sciences, Chinese Academy of Sciences, Key Laboratory of Pathogenic Microorganisms and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Chinese University of Science, Shenzhen Third People's Hospital, China Agricultural University, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and China Center for Disease Prevention and Control.
Corresponding authors of the paper are Gao Fu, Director of the China Center for Disease Control and Prevention and a member of the Chinese Academy of Sciences; Feng Feng, Associate Researcher, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences; and Liu Lei, President of Shenzhen Third People's Hospital.
To date, there are no specific drugs or vaccines for the new coronavirus.
Currently, most therapeutic developments are targeting the "key" to human invasion by novel coronaviruses, the S-protein (prickly glycoprotein), which mediates viral and host cell binding and invasion and consists of two structural domains, S1 and S2, mediating receptor binding and membrane fusion, respectively. The receptor-binding domain (RBD) is located in S1.
The research team believes that screening for neutralizing antibodies against novel coronaviruses using RBD proteins is a priority strategy that can be adopted today.
They first obtained four different antibodies (B5, B38, H2, and H4) from patients recovering from novel coronavirus pneumonia whose supernatants showed the ability to bind to the novel coronavirus RBD but failed to bind to the RBD of SARS-CoV, suggesting that the RBD epitopes of SARS-CoV and novel coronavirus are immunologically distinct.
These four antibodies showed different binding affinities to the novel coronavirus RBD, with Kd ranging from minus 7 orders of magnitude 10 to minus 9 orders of magnitude M. Among them, H4 showed a relatively high binding affinity, with Kd of 4.48nM, while B5 showed a relatively weak binding affinity, with Kd of 305nM. B38 and H2 bound to RBD with Kd of 70.1nM and 14.3nM, respectively.
All four antibodies showed neutralizing activity, with IC50 (semi-inhibitory concentration) values ranging from 0.177 μg/ml to 1.375 μg/ml.B38 was the most active antibody, followed by H4, H2, and B5.
The team next evaluated the ability of each antibody to block the competition of viral RBD to bind to ACE2, and the results showed that B38 and H4 were able to fully compete with ACE2 and bind to RBD. In contrast, B5 showed partial competition, while H2 showed a combination with RBD, but not with ACE2.
It was also shown that B38 and H4 recognize different epitopes on the RBD and partially overlap.
To explore the protective efficacy of B38 and H4 against novel coronavirus attacks in vivo, the research team conducted treatment experiments in mice. Twelve hours after the attack, the team injected hACE2 transgenic mice with a single 25 mg/kg dose of B38 or H4.
The results showed that mice in the B38 group, which had a significant post-attack weight loss compared to the control and H4 groups, recovered at 3 days (dpi) after infection.
The team also examined viral RNA copies in mouse lung tissues at 3 dpi, and the relative RNA copy changes in both the B38 and H4 groups were significantly lower than those in the control group, with 32.8% and 26% lower than those in the control group, respectively.
To further elucidate the structural basis of the antibody neutralization mechanism, the team prepared RBD-B38 and RBD-H4 Fab complexes by in vitro incubation and purification.
Three complementary decision regions (CDRs) on the heavy chain and two complementary decision regions on the light chain are involved and the viral RBD acts. There are 36 residues in the viral RBD that interact with B38, 21 of which interact with the heavy chain and 15 of which interact with the light chain.
Sequence comparison showed that only 15 of the 36 residues in the epitope were conserved between the novel coronavirus and SARS-CoV. This explains the specific responsiveness of B38.
Further analysis of the binding interface interactions revealed that the 470 ring of the viral RBD plays an important role in binding to the B38 HCDR1 ring.
The team also investigated the structural basis of the B38 blockade of COVID-19 virus RBD interaction with ACE2. The results showed that both VH and VL of B38 caused the spatial dislocation of RBD binding to ACE2 (Figure 4C).
Notably, RBD did not exhibit significant conformational changes in the two forms that bound to B38 and in the two forms that bound to hACE2.
Further analysis revealed that 18 of the 21 amino acids on the RBD were identical when bound to both, which clearly explains why B38 completely eliminated the binding of the novel coronavirus RBD to the receptor.
Overall, the novel coronavirus and SARS-CoV RBDs have high homology and both binds to the ACE2 receptor to infect their hosts.
However, the binding affinity is different, with the novel coronavirus RBD exhibiting more atomic interaction with hACE2 than the SARS-CoV RBD and therefore exhibiting a higher binding affinity for receptor binding.
To date, no studies have reported antibodies that cross-neutralize SARS-CoV2 and SARS-CoV by competing for ACE2 binding sites.CR3022 from recovering SARS patients can bind to RBDs from both viruses.
However, while it can neutralize SARS-CoV, it cannot neutralize the novel coronavirus.
The study emphasizes that a comprehensive understanding of the humoral immune response to novel coronaviruses needs to be carried out in a larger number of patients.
In addition, cocktail antibodies should be considered as an alternative treatment strategy to avoid potential escape mutations.
"As the novel coronavirus epidemic continues to spread, the identification of the viral RBD protein epitope is critical and will provide valuable information for the development of a vaccine, In addition, the molecular characterization of neutralizing antibodies against epitopes facilitates the development of small molecule or peptide drugs/inhibitors."
The neutralizing antibody identified in this study is expected to be a candidate antibody for the prevention and treatment of novel coronaviruses.
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