Generation of Neutralizing Antipeptide Antibodies to the Enzymatic Domain of Pseudomonas aeruginosa Exotoxin A

Abstract

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.on July 3, 2020 by guest http://iai.asm.org/ Downloaded from MATERIALS AND METHODS Synthetic peptides. Specific amino acid sequences within ETA were selected for production of antibodies. Amino acid sequence selection for synthetic peptide synthesis was based on the analysis of hydrophilicity (Kyte-Doolittle), antigenic index (Jameson-Wolf), and surface probability (Emini) ( Fig. 1 and Table 1). Peptides were synthesized by the Synthetic Antigen Laboratory at the University of Texas, M. D. Anderson Cancer Center, Houston. Individual peptides were 12 to 45 aa long. Peptides, supplied as lyophilized powder, were reconstituted with distilled water to a stock solution of 10 mg/ml. A dilute solution of each peptide was conjugated to keyhole limpet hemocyanin (KLH) (Pierce, Rockford, Ill.) according to the manufacturer's recommendations. Because of the relatively large size of peptides 9 (45 aa) and 11 (29 aa), and based on our stud...

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

“…Antibodies to the 13 aa within the carboxyl half of peptide 11 were more efficient than antibodies to peptide 11 itself in neutralizing the cytotoxic and enzymatic activities of ETA. In the same study, we identified another synthetic peptide encompassing a region within the translocation domain of ETA (aa 289 to 333), which induced antibodies with moderate ability to neutralize the cytotoxic activity of ETA in vitro (5). Four synthetic peptides encompassing regions within the binding domain of ETA failed to induce ETAneutralizing antibodies (5).…”

“…In the same study, we identified another synthetic peptide encompassing a region within the translocation domain of ETA (aa 289 to 333), which induced antibodies with moderate ability to neutralize the cytotoxic activity of ETA in vitro (5). Four synthetic peptides encompassing regions within the binding domain of ETA failed to induce ETAneutralizing antibodies (5). In the present study, we examined the potential of neutralizing antipeptide antibodies to confer protection against ETA or infection with an ETA-producing strain of P. aeruginosa in mice.…”

“…Previously, we reported successful induction of neutralizing antipeptide antibodies to a short amino acid sequence representing a portion of the enzymatic domain of ETA (aa 596 to 625, designated peptide 11) (5). These antibodies provided in vitro protection to monolayers of 3T3 fibroblasts against ETA-induced inhibition of protein synthesis by specifically blocking ADP-ribosyltransferase activity (5). Antibodies to the 13 aa within the carboxyl half of peptide 11 were more efficient than antibodies to peptide 11 itself in neutralizing the cytotoxic and enzymatic activities of ETA.…”

“…Affinity-purified antibodies to selected synthetic peptides (3, 6, 9, and 11) encompassing regions within the translocation and enzymatic domains of ETA ( Fig. 1) were used in these studies (5). The 50% lethal dose (LD 50 ) (23) of ETA in Swiss Webster outbred mice was determined to be approximately 300 ng when it was injected intraperitoneally (i.p.).…”

“…Mouse serum from group IV (immunized with peptide 9, translocation domain) or group V (immunized with KLH) had no effect on ETA enzymatic activity in vitro. All of the different groups of BALB/c mice were challenged with a lethal a Four-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, Maine) (five per group) were repeatedly immunized for 7 months with immunogen (20 ng of ETA, 50 g of synthetic peptide, or 50 g of KLH along with Freund's adjuvant), according to the protocol described previously (5). After the antibody immune response reached a plateau level, as determined by enzyme-linked immunosorbent assay (5), all mice were challenged with a lethal dose of ETA (1 g/mouse) i.p.…”