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Engineered Human Monoclonal scFv to Receptor Binding Domain of Ebolavirus.

ABSTRACT: (1) Background: Ebolavirus (EBOV) poses as a significant threat for human health by frequently causing epidemics of the highly contagious Ebola virus disease (EVD). EBOV glycoprotein (GP), as a sole surface glycoprotein, needs to be cleaved in endosomes to fully expose a receptor-binding domain (RBD) containing a receptor-binding site (RBS) for receptor binding and genome entry into cytoplasm for replication. RBDs are highly conserved among EBOV species, so they are an attractive target for broadly effective anti-EBOV drug development. (2) Methods: Phage display technology was used as a tool to isolate human single-chain antibodies (HuscFv) that bind to recombinant RBDs from a human scFv (HuscFv) phage display library. The RBD-bound HuscFvs were fused with cell-penetrating peptide (CPP), and cell-penetrating antibodies (transbodies) were made, produced from the phage-infected E. coli clones and characterized. (3) Results: Among the HuscFvs obtained from phage-infected E. coli clones, HuscFvs of three clones, HuscFv4, HuscFv11, and HuscFv14, the non-cell-penetrable or cell-penetrable HuscFv4 effectively neutralized cellular entry of EBOV-like particles (VLPs). While all HuscFvs were found to bind cleaved GP (GPcl), their presumptive binding sites were markedly different, as determined by molecular docking. (4) Conclusions: The HuscFv4 could be a promising therapeutic agent against EBOV infection.

SUBMITTER: Densumite J

PROVIDER: S-EPMC8147973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A human monoclonal antibody panel (PD4, PD5, PD7, SC23, and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 spike (S) protein, only PD5, PD7, and SC23 were able to bind to the receptor binding domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localized within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD-binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognized the uncleaved S protein, indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining were demonstrated for each isolate, SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. IMPORTANCE The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore, an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS- CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID-19 patients to monitor the RBD in cells infected with SARS-CoV-2 clinical isolates. These immunological reagents specifically recognize the correctly folded RBD and were used to monitor the appearance of the RBD in SARS-CoV-2-infected cells and identified the site where the RBD first appears.

Project description:BackgroundAntigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.Methods and findingsIn parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.ConclusionsThe C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.

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Engineered Human Monoclonal scFv to Receptor Binding Domain of Ebolavirus.

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