Identification of an anti–SARS–CoV-2 receptor-binding domain–directed human monoclonal antibody from a naïve semisynthetic library

Abstract

There is a desperate need for safe and effective vaccines, therapies and diagnostics for SARS-CoV-2, the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Here in, we use this technology to search for antibodies targeting the receptor binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semi-synthetic phage library against RBD, leading to the identification of a high-affinity single chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All the three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Based Novel Epitopes Induce Potent Immune Responses in vivo and Inhibit Viral Replication in vitro

“…Similarly, pooled sera from RBD-pep2 immunized sera detected soluble RBD protein at 1:1000 dilutions ( Figure 4F ). The sera from the pooled peptides were able to detect the soluble spike protein as a clear single band at an approximate molecular weight of ~175kDa, whereas, the pooled peptide sera from RBD-pep2 recognized the soluble RBD as a single band at an approximate molecular weight of ~29 kDa ( 27 , 28 ). To study the ability of the sera from immunized groups to detect expressed spike protein in viruses, Vero-E6 cells were infected with wild type SARS-CoV-2, Isolate USA-WA1/2020 virus using an MOI of 10 and 24 h p.i., the binding of the pooled sera from each immunized group to viruses was determined by indirect immunofluorescence ( Figure 4G ).…”

Immunological and cardio-vascular pathologies associated with SARS-CoV-2 infection in golden syrian hamster

“…The antibody response was measured against Spike, RBD and N protein post virus challenge at different time points. Soluble spike prefusion trimeric protein (S2P) (Wrapp et al, 2020), in house RBD (Ha et al, 2020) and N proteins were coated on Maxisorp plates (Nunc) at different protein concentrations (Spike S2P trimer; 1 μg/ml, RBD; 2μg/ml and N protein at 0.5ug/ml) in 1X carbonate/bicarbonate buffer, pH 9.6 overnight at 4 °C. The following day, the plates were blocked using 250 μl of PBS containing 5 % skimmed milk (blocking buffer).…”

“…In the absence of crystal structure of hamster ACE2 structures, the crystal structure of human ACE2 and RBD, pdb-ids 6m17, 6VW1 were considered as templates to generate the robust model of hamster through homology modeling (Srivastava et al, 2018). The robustness of each model was evaluated through ERRAT, PROSA and Ramachandran plot (allowed and disallowed %), followed by structure optimization through energy minimization to allow conformational relaxation of the protein structure (Ha et al, 2020). The same was carried out for neurolipin-1 protein of hamster as human neurolipin crystal structure (pdb-id 6JJQ) is reported.…”

Antibodies and Vaccines Target RBD of SARS-CoV-2

“…S309 and S310 recognize post-fusion conformation of SARS-CoV-2 S protein, and CR3022 exhibits non-neutralizing behavior against SARS-CoV-2. Non-NAb plays an essential role in the blockage of cell surface infection in vivo without perturbing virus entry, which could be useful for improving antibody-based therapeutics and diagnostics for COVID-19 ( Parray et al, 2020 ).…”