A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Protocol for High Throughput Screening of Antibody Phage Libraries.

Phage display is a proven and widely used technology for selecting specific antibodies against desired targets. However, an immense amount of effort is required to identify and screen the desired positive clones from large and diverse combinatorial libraries. On the other hand, the selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias toward clones with randomly produced amber stop codons, making it more difficult to identify desirable binding antibodies. To overcome the screening of desired clones with amber codons, we present a step-by-step approach for effective phage library screening to isolate useful antibodies. The procedure calls for creating a simple new vector system for soluble production of phage ELISA positive binding clones with one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences, which is otherwise difficult in standard screening. Graphical abstract.

Characterization of a broadly cross reactive tetravalent human monoclonal antibody, recognizing conformational epitopes in receptor binding domain of SARS-CoV-2.

We used human semi-synthetic phage antibody gene libraries to select anti-SARS-CoV-2 RBD scFv antibody fragment and subsequent characterization of this novel tetravalent monoclonal antibody targeting conformational epitopes in the receptor binding domain of SARS-CoV-2. Binding studies suggest that II62 tetravalent antibody cross-reacts with RBD protein of SARS-CoV2 and its different variants of concerns. The epitope mapping data reveals that II62 tetravalent antibody targets an epitope that does not directly interferes with RBD: ACE2 interaction. Neutralization studies with live authentic SARS-CoV2 virus suggests that increase in valency of II62 mAb from monovalent to tetravalent doesn't perturbate virus interactions with the ACE2 expressing host cells in cytopathic effect-based (CPE) assay. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03272-6.