Team:Shanghai HS/Engineering
Engineering
Introduction
Coronavirus disease 2019 (COVID-19) is caused by a new type of coronavirus (SARS-CoV-2). The virus infects the human body through the binding of the S protein on viral surface to the angiotensin-converting enzyme 2 (ACE2) receptor on human cells. To date, more than 236 million COVID-19 confirmed cases and more than 4.8 million deaths have been reported worldwide. The S protein of SARS-CoV-2 is prone to mutation. Mutations in the receptor binding domain (RBD) in the S protein can lead to changes in the ability of the virus to infect and may lead to immune escape. At present, a number of SARS-CoV-2 neutralizing antibodies targeting RBD have been confirmed to have weakened or even lost inhibitory effects on mutant strains. By contrast, the receptor ACE2 is highly conserved. The antibodies targeting ACE2 has been proven to strongly inhibit the replication of SARS-CoV-2 in vivo and in vitro without affecting the activity of ACE2. It is of great significance to detect whether the ACE2 antibody inhibits the new coronavirus epidemic mutant B.1.1.7.
Design
ELISA and infection assays by HIV pseudovirus with SARS-CoV-2 S protein were designed to test the inhibitory effect of ACE2 antibodies.
ELISA (Enzyme linked immunosorbent assay) refers to a qualitative and quantitative detection method that binds soluble antigens or antibodies to solid-phase carriers such as polystyrene, and uses the specific binding of antigens and antibodies to carry out immune reactions. The solid carries in the design are recombinant SARS-CoV-2 B.1.1.7 S1 domain or RBD region only. The figure 1 is the maps of construct used to produce recombinant B.1.1.7 S1 and RBD.
Figure 1 Map of constructs used to produce recombinant B.1.1.7 S1 and RBD. Composite parts containing functional sequence element to transcript and translate the coding region of S1 or RBD.
HIV backbone plasmids labeled with firefly luciferase are co-transfected with the plasmid of S protein. The S protein coated pseudovirus enters the cell through ACE2, and the activity of Luciferase is detected to determine the infection efficiency of the pseudovirus.
Build
Two recombinant plasmids PTT5-S1-his-B.1.1.7 and PTT5-RBD-his-B.1.1.7 were constructed by using homologous recombination. The constructs were confirmed by DNA sequencing.
Test
Pseudo-typed coronaviruses with full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63 could infect HEK293F cells that ectopically express human ACE2, while SARS-CoV-2-D614G showed significantly enhanced infectivity when compared to the original SARS-CoV-2 (table 1). Incubation with monoclonal antibody 3E8 targeting ACE2 fully abolished the infectivity of all pseudoviruses.
Table 1 The IC50 values of 3E8 in blocking pseudo-typed coronaviruses.
Learn
Our results highlighted again the importance of epitope outside or on the verge of RBD/ACE2 interface and would facilitate future endeavors searching for broad-spectrum anti-coronavirus approaches. Overall, we presented evidence that 3E8 is a promising therapeutic candidate for the coronavirus pandemic and believe that it represents a significant conceptual advance in fighting COVID-19, which keeps evolving and may open the door for more ACE2-targeting drug discovery and development.
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