scholarly journals Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sirijan Santajit ◽  
Watee Seesuay ◽  
Kodchakorn Mahasongkram ◽  
Nitat Sookrung ◽  
Sumate Ampawong ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phage Display ◽  
Virulence Factors ◽  
Catalytic Domain ◽  
Exotoxin A ◽  
Single Chain ◽  
New Paradigm ◽  
Phage Display Technology ◽  
Single Chain Antibodies ◽  
Therapeutic Regime

Abstract Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.

scholarly journals Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic  - or  -Cells In Vivo

Diabetes ◽  
2009 ◽  
Vol 58 (10) ◽  
pp. 2324-2334 ◽  
Author(s):  
S. Ueberberg ◽  
J. J. Meier ◽  
C. Waengler ◽  
W. Schechinger ◽  
J. W. Dietrich ◽  
...  
Keyword(s):  
Phage Display ◽  
Imaging Agents ◽  
Single Chain ◽  
Direct Imaging ◽  
Phage Display Technology ◽  
Single Chain Antibodies ◽  
Display Technology

scholarly journals Human Single-Chain Antibodies That Neutralize Elastolytic Activity of Pseudomonas aeruginosa LasB

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 765
Author(s):  
Sirijan Santajit ◽  
Thida Kong-ngoen ◽  
Manas Chongsa-Nguan ◽  
Usa Boonyuen ◽  
Pornpan Pumirat ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune Modulation ◽  
Large Scale ◽  
Scale Production ◽  
Single Chain ◽  
Phage Display Technology ◽  
Single Chain Antibodies ◽  
Successful Infection ◽  
Elastolytic Activity

LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting Pseudomonas aeruginosa. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the P. aeruginosa. This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (huscfvs) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the huscfv-plasmid transformed Escherichia coli. HuscFvs of two transformed E. coli clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities in vitro. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by P. aeruginosa.

scholarly journals Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zahra Shadman ◽  
Safar Farajnia ◽  
Mohammad Pazhang ◽  
Mohammadreza Tohidkia ◽  
Leila Rahbarnia ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
High Reactivity ◽  
Scfv Antibody ◽  
Phage Library ◽  
Human Antibody ◽  
Exotoxin A ◽  
Phage Elisa ◽  
Human Scfv ◽  
Domain I

Abstract Background Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. Methods The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. Results Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. Conclusions The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

scholarly journals Identification of single chain antibodies to breast cancer stem cells using phage display

2009 ◽  
pp. NA-NA ◽  
Author(s):  
Deniz Gur ◽  
Suling Liu ◽  
Anurag Shukla ◽  
Stephanie C Pero ◽  
Max S Wicha ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Phage Display ◽  
Breast Cancer Stem Cells ◽  
Single Chain ◽  
Single Chain Antibodies

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

scholarly journals Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology

2017 ◽  
Vol 38 (6) ◽  
pp. 3915
Author(s):  
Greice Japolla ◽  
Ana Flávia Batista Penido ◽  
Greyciele Rodrigues Almeida ◽  
Luiz Artur Mendes Bataus ◽  
Jair Pereira Cunha Junior ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Bovine Herpesvirus ◽  
Single Chain ◽  
Phage Display Technology ◽  
Herpesvirus Type ◽  
Wide Range ◽  
Display Technology ◽  
Bovine Herpesvirus Type 1

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

scholarly journals Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa

2005 ◽  
Vol 385 (3) ◽  
pp. 667-675 ◽  
Author(s):  
Susan P. YATES ◽  
Patricia L. TAYLOR ◽  
René JØRGENSEN ◽  
Dana FERRARIS ◽  
Jie ZHANG ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Competitive Inhibition ◽  
Function Analysis ◽  
Binding Pocket ◽  
Ring Structure ◽  
Water Soluble ◽  
Exotoxin A ◽  
Ic50 Values

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant Ki of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å (1 Å=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

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