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

Abstract

A nontoxic Pseudomonas aeruginosa exotoxin A (PE), which has the carboxyl-terminal 38 amino acid residues of native PE deleted, was used as an antigen to immunize BALB/c mice, which were then challenged with native PE in order to raise monoclonal antibodies (MAbs) that can neutralize PE cytotoxicity. A murine MAb against PE, designated MAb B7, was established. MAb B7 was characterized in terms of its ability to neutralize PE cytotoxicity, epitope mapping, inhibition of PE receptor binding, and influence on cellular processing of PE and ADP-ribosylation activities. We found that MAb B7 could neutralize PE cytotoxicity in cell culture and in BALB/c mice. The epitope recognized by MAb B7 was mapped to the carboxyl-terminal amino acid residues 575 to 595 of PE. Consistent with the results of epitope mapping, MAb B7 did not block PE receptor-binding activity or the cellular processing of PE but strongly inhibited the ADP-ribosylating activity of PE. In addition, MAb B7 retained strong binding to PE even at pH 4.0, indicating that the complex of MAb B7 and PE is stable in the phagolysosome. On the basis of these observations, the neutralization of PE cytotoxicity by MAb B7 could be due to its binding to the carboxyl terminus of PE. As a result, MAb B7 may interfere with the interaction of the carboxyl-end amino acid residues REDLK of PE with cellular factors. However, we could not rule out the possibility that MAb B7 directly blocks the ADP-ribosylation activity of PE in the cytosol.

Protection against Exotoxin A (ETA) and Pseudomonas aeruginosa Infection in Mice with ETA-Specific Antipeptide Antibodies

“…Domain III, aa 396 to 613, constitutes the enzymatic portion of ETA (9,11). To date, several studies have been conducted in order to understand the immunochemistry of ETA and to identify the immunodominant neutralizing epitopes of this molecule (4,15,16,17,18,24,25). Such studies are essential for the development of immunotherapeutic approaches for treating infections caused by toxin-producing strains of P. aeruginosa and for elucidating the structure-function relationship of ETA.…”

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

“…D461, Q485, E546, E547 and E553 formed the edge of the ETA-NAD + binding cavity and participated in hydrogen bonding with two ribose moieties of the NAD + molecule 40 . Moreover, a previous study indicated that the ETA epitopes recognized by murine monoclonal antibody clone B7 (MAbB7), which displayed strong neutralizing capacity on ETA cytotoxicity, were mapped to the C-terminal residues 575–595 of the toxin 41 . Although the results of ETA-HuscFv interaction by computerized simulation needs experimental validation, it is plausible that the effectiveness of HuscFv-C41 in neutralizing the apoptotic activity of ETA might be through interfering with the catalytic activity and inhibition of ADP-ribose transfer from NAD + to eEF-2.

Figure 10Computerized interaction of modeled-ETA and HuscFvs and residues that were predicted to form contact interfaces between them.

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

“…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). S. NATHAN (20) used phage Display technology for isolation of the antibody against Burkholderia pseudomallei by several biopaning rounds.…”

“…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…”