Generation of synthetic Acinetobacter baumannii specific nanobodies
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE202972 | GEO | 2023/06/21
REPOSITORIES: GEO
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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: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:Kim2009 - Genome-scale metabolic network of
Acinetobacter baumannii (AbyMBEL891)
This model is described in the article:
Genome-scale metabolic
network analysis and drug targeting of multi-drug resistant
pathogen Acinetobacter baumannii AYE.
Kim HU, Kim TY, Lee SY.
Mol Biosyst 2010 Feb; 6(2):
339-348
Abstract:
Acinetobacter baumannii has emerged as a new clinical threat
to human health, particularly to ill patients in the hospital
environment. Current lack of effective clinical solutions to
treat this pathogen urges us to carry out systems-level studies
that could contribute to the development of an effective
therapy. Here we report the development of a strategy for
identifying drug targets by combined genome-scale metabolic
network and essentiality analyses. First, a genome-scale
metabolic network of A. baumannii AYE, a drug-resistant strain,
was reconstructed based on its genome annotation data, and
biochemical knowledge from literatures and databases. In order
to evaluate the performance of the in silico model,
constraints-based flux analysis was carried out with
appropriate constraints. Simulations were performed from both
reaction (gene)- and metabolite-centric perspectives, each of
which identifies essential genes/reactions and metabolites
critical to the cell growth. The gene/reaction essentiality
enables validation of the model and its comparative study with
other known organisms' models. The metabolite essentiality
approach was undertaken to predict essential metabolites that
are critical to the cell growth. The EMFilter, a framework that
filters initially predicted essential metabolites to find the
most effective ones as drug targets, was also developed.
EMFilter considers metabolite types, number of total and
consuming reaction linkage with essential metabolites, and
presence of essential metabolites and their relevant enzymes in
human metabolism. Final drug target candidates obtained by this
system framework are presented along with implications of this
approach.
This model is hosted on
BioModels Database
and identified by:
MODEL1507180029.
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To the extent possible under law, all copyright and related or
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the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
2015-07-30 | MODEL1507180029 | BioModels
Project description:A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.
2021-09-21 | GSE158906 | GEO
Project description:Background: Acinetobacter baumannii is one of the most dangerous multidrug-resistant pathogens worldwide. Currently, 50-70% of clinical isolates of A. baumannii are extensively drug-resistant (XDR) and available antibiotic options against A. baumannii infections are limited. There are still needs to discover specific de facto bacterial antigenic proteins that could be effective vaccine candidates in human infection. With the growth of research in recent years, several candidate molecules have been identified for vaccine development. So far, there is no public health authorities approved vaccine against A. baumannii. Methods: The purpose of this study was to identify immunodominant vaccine candidate proteins that can be immunoprecipitated specifically with patients’ IgGs. Relaying on hypothesis that IgGs of infected person have capacity to capture immunodominant bacterial proteins. Herein, outer membrane and secreted proteins of sensitive and drug resistant A. baumannii were captured by using IgGs obtained from patient and healthy control sera and were identified by LC-MS/MS analysis. Results: By using subtractive proteomic approach, we determined 34 unique proteins which were captured only in drug-resistant A. baumannii strain via patient sera. After extensive evaluation of predicted epitope regions, solubility, membrane transverse characteristics, and structural properties, we selected several notable vaccine candidates. Conclusion: We identified vaccine candidate proteins that triggered de facto response of human immune system against the antibiotic-resistant A. baumannii. Precipitation of bacterial proteins via patient immunoglobulins was a novel approach to identify the proteins which have potential to trigger to response in patient immune system.
2022-11-02 | PXD037507 | Pride
Project description:To identify cell adhesion molecules (CAMs) targeting bacterial membrane proteins within a synthetic bacteria-displayed nanobody library, we present a comprehensive whole-cell screening platform. This involves targeted amplicon sequencing to discover nanobodies targeting the natural adhesin, TraN. Furthermore, we employ deep mutational engineering to enhance the binding affinity of these nanobodies toward TraN.
2023-12-18 | GSE249103 | GEO
Project description:Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients and antibiotic treatment is compromised in multi-drug resistant strains resistant to beta-lactams, carbapenems, cephalosporins, polymyxins and tetracyclines. Among COVID-19 patients receiving ventilator support, multi-drug resistant A. baumannii secondary infection is associated with a two-fold increase in mortality. Here we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break resistance of A. baumannii to tetracycline class antibiotics.
2021-12-17 | GSE175535 | GEO
Project description:Comparative Genomic and Transcriptomic Characterization of Colistin-Resistant Acinetobacter baumannii
2019-01-23 | GSE109951 | 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:Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, our studies demonstrate that SFTSV surface glycoprotein (Glycoprotein N, Gn) is a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies play a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages including small molecular weight, high affinity, low immunogenicity and convenient production by gene engineering, etc. In this study, we combined next generation sequencing (NGS) with proteomics technology and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, and selected 6 of them for recombinant protein expression and purification. Among these 6 anti-Gn nanobodies, nanobody 57493 was validated to be highly specific for Gn.
2024-01-26 | PXD032731 | Pride