Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo

Abstract

Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. In vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin Gl [IgGl]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgGl), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but onl weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgGl), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.

Protective Properties of Nontoxic Recombinant Exotoxin A (Domain I-II) Against Pseudomonas aeruginosa Infection

“…Exotoxin A is extremely lethal, causes histopathological changes within the liver, induces apoptosis of the hepatocytes and enhances the expression of different pro-inflammatory cytokines (12). Research has shown that these damaging effects are neutralized in the presence of anti-exotoxin A antibodies that are essential for protection against P. aeruginosa infections (12,16,17). Native exotoxin A is toxic and its modification into toxoid form by different methods and its immunologic properties have been reported in several in vitro and in vivo studies (7,12,16,17).…”

Characterization of monoclonal antibody B7, which neutralizes the cytotoxicity of Pseudomonas aeruginosa exotoxin A

“…Therefore, it is worthwhile to screen murine MAbs for strong neutralizing activity prior to their selection for treatment of infection. Recently, some anti-PE MAbs have been described (4,11,22,23). Most of them recognize the binding domain of PE; thus, they can neutralize PE cytotoxicity by inhibiting PE binding to target cells (4,23).…”

“…MAbs against different domains of PE would be needed to neutralize residual PE cytotoxicity. Chia et al (4) and Ohtsuka et al (22) described two anti-PE MAbs which inhibited the ADP-ribosylation activity of PE but failed to neutralize PE cytotoxicity in cell culture and in vivo. Galloway et al (11), on the other hand, cloned a MAb whose epitope resides at amino acid residues near His-426 of domain III.…”

“…The substrate solution contained 0.54 mg of 2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) per ml and 0.03% H 2 O 2 in 0.1 M citric acid. ELISA was also used to detect the pH-dependent binding of MAbs to PE as previously described (22). In brief, MAbs were prepared at a concentration of 1 g/ml by dissolving the MAbs in 50 mM 3,3-dimethylglutaric acid-NaOH buffer containing 0.01% bovine serum albumin (BSA), to give pHs of 3.4, 4.0, 5.0, 6.0, and 7.0.…”

“…ADP-ribosylation activity assay. Purified MAbs were assayed for their potential to inhibit the ADP-ribosylation activity of PE as previously described (22). Wheat germ extracts enriched for EF-2 were used as a source of EF-2.…”