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Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies.

ABSTRACT: Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.

SUBMITTER: Xu H

PROVIDER: S-EPMC9284966 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies.

Xu Hai H Li Ling L Deng Bihua B Hong Weiming W Li Ruiting R Guo Zijie Z Hou Jibo J Govinden Roshini R Chenia Hafizah Y HY

Scientific reports 20220715 1

Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 12 ...[more]

PMID: 35840654

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Project description:BackgroundThe emergence of the coronavirus disease 2019 (COVID-19) pandemic and the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) requires the continuous development of safe, effective, and affordable prevention and therapeutics. Nanobodies have demonstrated antiviral activity against a variety of viruses, providing a new candidate for the prevention and treatment of SARS-CoV-2 and its variants.MethodsSARS-CoV-2 glycoprotein spike 1 subunit (S1) was selected as the target antigen for nanobody screening of a naïve phage display library. We obtained a nanobody, named Nb-H6, and then determined its affinity, inhibition, and stability by ELISA, Competitive ELISA, and Biolayer Interferometry (BLI). Infection assays of authentic and pseudotyped SARS-CoV-2 were performed to evaluate the neutralization of Nb-H6. The structure and mechanism of action were investigated by AlphaFold, docking, and residue mutation assays.ResultsWe isolated and characterized a nanobody, Nb-H6, which exhibits a broad affinity for S1 and the receptor binding domain (RBD) of SARS-CoV-2, or Alpha (B.1.1.7), Delta (B.1.617.2), Lambda (C.37), and Omicron (BA.2 and BA.5), and blocks receptor angiotensin-converting enzyme 2 (ACE2) binding. Moreover, Nb-H6 can retain its binding capability after pH or thermal treatment and effectively neutralize both pseudotyped and authentic SARS-CoV-2, as well as VOC Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.2 and BA.5) pseudoviruses. We also confirmed that Nb-H6 binds two distinct amino acid residues of the RBD, preventing SARS-CoV-2 from interacting with the host receptor.ConclusionOur study highlights a novel nanobody, Nb-H6, that may be useful therapeutically in SARS-CoV-2 and VOC outbreaks and pandemics. These findings also provide a molecular foundation for further studies into how nanobodies neutralize SARS-CoV-2 and variants and imply potential therapeutic targets for the treatment of COVID-19.

Dataset Information

Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies.

Publications

Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies.

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