Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors.

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism. Phage mimotopes of the SARS-CoV-2 Spike S1 protein have been developed using a combination of phage display and molecular mimicry concepts, termed here "phage mimicry", supported by bioinformatics methods. The key elements of the phage mimicry concept include: (1) preparation of a collection of p8-type (landscape) phages, which interact with authentic active receptors of live human cells, presumably mimicking the binding interactions of human coronaviruses such as SARS-CoV-2 and its variants; (2) discovery of closely related amino acid clusters with similar 3D structural motifs on the surface of natural ligands (FGF1 and NRP1), of the model receptor of interest FGFR and the S1 spike protein; and (3) an ELISA analysis of the interaction between candidate phage mimotopes with FGFR3 (a potential alternative receptor) in comparison with ACE2 (the authentic receptor).

Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein.

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the "pairing potential" of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the "inside" or "outside" of the infected cell. Their therapeutic potential will be discussed.

Protocol for constructing large size human antibody heavy chain variable domain (VH) library and selection of SARS-CoV-2 neutralizing antibody domains.

This protocol is a comprehensive guide to phage display-based selection of virus neutralizing VH antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 1011) naïve human antibody heavy chain domain library, (2) SARS-CoV-2 antigen expression and stable cell line construction, and (3) library panning for selection of SARS-CoV-2-specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

Cross-Reactive SARS-CoV-2 Neutralizing Antibodies From Deep Mining of Early Patient Responses.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

Discovery of Antivirals Using Phage Display.

The latest coronavirus disease outbreak, COVID-19, has brought attention to viral infections which have posed serious health threats to humankind throughout history. The rapid global spread of COVID-19 is attributed to the increased human mobility of today's world, yet the threat of viral infections to global public health is expected to increase continuously in part due to increasing human-animal interface. Development of antiviral agents is crucial to combat both existing and novel viral infections. Recently, there is a growing interest in peptide/protein-based drug molecules. Antibodies are becoming especially predominant in the drug market. Indeed, in a remarkably short period, four antibody therapeutics were authorized for emergency use in COVID-19 treatment in the US, Russia, and India as of November 2020. Phage display has been one of the most widely used screening methods for peptide/antibody drug discovery. Several phage display-derived biologics are already in the market, and the expiration of intellectual property rights of phage-display antibody discovery platforms suggests an increment in antibody drugs in the near future. This review summarizes the most common phage display libraries used in antiviral discovery, highlights the approaches employed to enhance the antiviral potency of selected peptides/antibody fragments, and finally provides a discussion about the present status of the developed antivirals in clinic.

Phage Display Technique as a Tool for Diagnosis and Antibody Selection for Coronaviruses.

Phage display is one of the important and effective molecular biology techniques and has remained indispensable for research community since its discovery in the year 1985. As a large number of nucleotide fragments may be cloned into the phage genome, a phage library may harbour millions or sometimes billions of unique and distinctive displayed peptide ligands. The ligand-receptor interactions forming the basis of phage display have been well utilized in epitope mapping and antigen presentation on the surface of bacteriophages for screening novel vaccine candidates by using affinity selection-based strategy called biopanning. This versatile technique has been modified tremendously over last three decades, leading to generation of different platforms for combinatorial peptide display. The translation of new diagnostic tools thus developed has been used in situations arising due to pathogenic microbes, including bacteria and deadly viruses, such as Zika, Ebola, Hendra, Nipah, Hanta, MERS and SARS. In the current situation of pandemic of Coronavirus disease (COVID-19), a search for neutralizing antibodies is motivating the researchers to find therapeutic candidates against novel SARS-CoV-2. As phage display is an important technique for antibody selection, this review presents a concise summary of the very recent applications of phage display technique with a special reference to progress in diagnostics and therapeutics for coronavirus diseases. Hopefully, this technique can complement studies on host-pathogen interactions and assist novel strategies of drug discovery for coronaviruses.

Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.