GENERATION OF NEUTRALIZING ANTIPEPTIDE ANTIBODIES TO THE ENZYMATIC DOMAIN OF PSEUDOMONAS-AERUGINOSA EXOTOXIN-A

Burn patients suffer a break in the physical barrier (skin), which, wh en combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas a eruginosa, 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 tre atment with antiexotoxin A (ETA) antibodies significantly increases su rvival in rats infected with toxin-producing strains of P. aeruginosa, we examined II synthetic peptides encompassing 12 to 45 amino acid (a a) 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 ET A. Animals immunized with various peptide combinations produced peptid e-specific antibodies that exhibited cross-reactivity to ETA. Two majo r epitopes were identified on the ETA molecule by experiments with pep tide-specific antibodies in enzyme-linked immunosorbent assay and immu noprecipitation. One of these epitopes was located in the translocatio n 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 (II I) (aa 626 to 638), Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that ab rogated the cytoxic activity of ETA in vitro. Antibodies to this epito pe blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enz ymatic active site of the ETA molecule. We conclude that polyclonal, a s well as monoclonal, antibodies to short peptides, representing smalt regions of ETA, may have therapeutic potential in passive immunizatio n or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.