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

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.

Isolation and Characterizations of A Novel Recombinant scFv Antibody Against Exotoxin-A of Pseudomonas Aeruginosa

“…In a 2019 study by Sirijan Santajit et al, they produced human antibodies against subdomain Ia exotoxin A of Pseudomonas using phage display technology. However, in the present study, domain 1 subdomains (domains Ia and Ib) were targeted for antibody production, which had a greater neutralizing power (18). An older study also produced a mouse antibody against exotoxin A, but due to adverse immune responses we decided to produce a completely human antibody (19).…”

“…Totally, six rounds of biopanning were carried out to select domain I -speci c phage clones. [18,19]. After adding 10 9 helper phages to the wells and incubation for 1hr at 37 °C, the plate was centrifuged at 3000…”

Human Single-chain Variable Fragments Neutralize Pseudomonas aeruginosa Quorum Sensing Molecule, 3O-C12-HSL, and Prevent Cells From the HSL-mediated Apoptosis

“…Tapryal et al, 2013). A repertoire of E. coli clones carrying recombinant huscfv-phagemids was previously retrieved from a HuscFv phage display library (Kulkeaw et al, 2009) by panning with P. aeruginosa exotoxin A (Santajit et al, 2019). Moreover, because the previously produced murine mAb, RS2-1G9, has been known as the P. aeruginosa quorum quencher, we used computerized antibody structure superimposition to select the bacterial derived-HuscFvs that shared structural homology with the murine mAb RS2-1G9 antigen-binding site.…”

“…Nowadays, any engineered fully human antibody format can be generated in vitro using phage display technology, invented by Nobel laureate, George Pearson Smith ( Smith, 1985 ) as a biological tool ( Santajit et al, 2019 ). The target antigens, such as proteins or peptides, attached to a carrier surface, e.g., fixed cell, plastic bead, or well of an ELISA plate, can be used as bait to fish out phage clones that display recombinant antibodies binding to the antigen from an antibody phage display library ( Kulkeaw et al, 2009 ).…”

“…The human single-chain variable fragments (HuscFvs) to the 3O-C12-HSL were generated based on the principles of the polyspecific property of an antibody, i.e., one antibody can bind different antigens by paratope adaptation to accommodate distinct antigens, such as through differential engagements of the complementarity determining regions (CDRs), and the molecular mimicry of the antigens (different antigens can share surface topologies in terms of shape or chemical nature) (Tapryal et al, 2013). In this study, HB2151 Escherichia coli clones carrying phagemids with inserted HuscFv genes (huscfvs) were previously selected from a HuscFv phage display library (Kulkeaw et al, 2009) using Pseudomonas exotoxin A (ETA) as antigen in the phage-biopanning process (Santajit et al, 2019). Genes coding for HuscFvs of individual E. coli clones were sequenced and deduced, and the canonical CDRs and framework regions (FRs) of both VH and VL domains were determined based on the numbering scheme of Chotia and Kobat (Abhinandan and Martin, 2008).…”