E. coli Nanobodies
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ABSTRACT:
PROVIDER: PRJEB78364 | ENA |
REPOSITORIES: ENA
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Project description:Escherichia coli is a major cause of blood stream and urinary tract infections. Owing to the spread of antimicrobial resistance, it is often treated with an inadequate antibiotic. With the aim to accelerate the diagnostics of this key pathogen, we used the flycode technology to generate nanobodies against the conserved and highly abundant outer membrane protein OmpA. Two nanobodies each recognizing a different isoform of OmpA were shown by flow cytometry to recognize > 91% of 85,680 E. coli OmpA sequences deposited in a large bacterial genome database. Crystal structures of these nanobodies in complex with the respective OmpA isoform revealed interactions with all four surface accessible loops of OmpA. Steric hindrance caused by dense O-antigen layers initially impeded reliable capture of clinical E. coli strains. By generating nanobody constructs with long linkers and by thinning the O-antigen layer through alterations to growth medium and buffers, we achieved to capture <50 CFU/mL. Our work provides a framework to generate nanobodies for the specific and sensitive detection and capture of clinically relevant pathogenic bacteria.
2025-06-11 | PXD054264 | Pride
Project description:Selection of Novel Nanobodies Targeting EGFR Using Phage Display Library
| PRJEB35883 | ENA
Project description:Nanobodies are emerging as ideal instruments for drug design and several have recently been created to block SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not efficiently target Spike during viral entry. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for replication of the viral genome. To find out if this strategy efficiently blocked viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells subjected to LNP-mRNA-2NSP23 with multiple SARS-CoV-2 variants. Analysis of total RNA isolated form infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We propose that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.
2023-10-10 | GSE244714 | GEO
Project description:The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.
2023-06-21 | GSE202972 | GEO
Project description:In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. We are developing developed INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties. microenvironment, which can be distinct from draining lymph nodes. To identify targets, we selected a clone enriched for dendritic cells that binds to PHB2. Using single cell RNA sequencing, we observe PHB2 signaling is associated with activation in cDC1’s. Immunofluorescence confirmed that the nanobody colocalizes with PHB2 in regions along the cell membrane. Structural modeling with AlphaFold2 and antibody docking using Rosetta assist binding site predictions, thus could be used to guide nanobody selection for future aims. This work shows that INSPIRE-seq can interrogate complex microenvironments and may assist in developing therapeutics.
2023-11-16 | GSE223428 | GEO
Project description:Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Many vaccines have been deployed to date; however, the continual evolution of the viral receptor binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in South Africa (B.1.351) and the U.K. (B.1.1.7) have reduced plasma neutralization activity and crippled antibody cocktails that received emergency use authorization1-3. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to overcome viral escape. One potential alternative are camelid VHHs (also known as nanobodies), which can access conserved epitopes often hidden to conventional antibodies4-6. We here isolate anti-RBD nanobodies from llamas and mice engineered to produce VHHs from alpacas, dromedaries and camels. Through neutralization assays and cryo-electron microscopy we identify two “nanomouse” VHHs that circumvent RBD antigenic drift by recognizing a domain conserved in coronaviruses, away from the ACE2 binding motif. Conversely, llama nanobodies recognize the RBD-ACE2 interphase and as monomers they are ineffective against E484K or N501Y substitutions. Notably, as homotrimers those same VHHs neutralize RBD variants with ultrahigh (pM) affinity, rivaling the most potent antibodies produced to date against SARS-CoV-2. We conclude that multivalent nanobodies can avert SARS-CoV-2 escape mutants and thus they represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
2021-05-18 | GSE167310 | GEO
Project description:In the rapidly advancing field of synthetic biology, there is a critical need for technology to discover targeting moieties for therapeutic biologics. We developed INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.
2024-01-26 | PXD046363 | Pride
Project description:Generation of synthetic Acinetobacter baumannii specific nanobodies
| PRJNA837907 | ENA
Project description:This Series involves two studies: 1) The gene expression of E. coli K-12 BW25113 ompA mutant strain vs. wild type strain glasswool biofilm cells and E. coli K-12 BW25113 ompA mutant vs. wild type polystyrene biofilm cells. 2) The gene expression of E. coli BW25113 ompA/pCA24N_ompA vs. ompA/pCA24N suspension cells. Strains: E. coli K-12 BW25113 wild type, ompA mutant Medium: LB Cell type: Biofilm cells grown on glasswool and polystyrene surfaces Time: 15 h Temperature: 37C Strains: BW25113 ompA/pCA24N_ompA and ompA/pCA24N Medium: LB Time: 7 h Temperature: 37C Cell type: suspension cells, induced by 0.1 mM IPTG
2010-11-03 | E-GEOD-14064 | biostudies-arrayexpress