Functionally active monoclonal antibody that recognizes an epitope on the O side chain of Pseudomonas aeruginosa immunotype-1 lipopolysaccharide

Abstract

A murine monoclonal antibody (MAb) was prepared against Pseudomonas aeruginosa immunotype-1 (It-1) lipopolysaccharide (LPS). The MAb bound It-1 LPS in the enzyme-linked immunosorbent assay and in the immunodiffusion and immunoblotting assays, agglutinated and opsonized It-1 bacteria, and protected against challenge with live It-1 organisms in a murine burn infection model. All of these activities were immunotype specific. Correlation of the opsonic and protective properties of the MAb with its recognition site on the LPS O side chain confirmed that such immunotype-specific determinants are important targets for protective antibodies in Pseudomonas disease. The functional equivalence of this MAb and polyclonal antibodies from hyperimmune plasma underscores the therapeutic potential of single MAbs which recognize critical determinants in the LPS 0 side chain. 656 single MAbs directed toward appropriate epitopes on the exposed 0 side chain of Pselidomonas LPS and perhaps other LPSs.MATERIALS AND METHODS LPSs. P. aeruginosa It-1 LPS (Fisher-Devlin-Gnabasik system [12]), purified by hot phenol-water extraction (38) and gel filtration chromatography, was obtained from List Biological Laboratories, Campbell, Calif. Trichloroacetic acid-extracted LPSs from Fisher immunotypes 2 through 7 (15) were obtained from M. Fisher, Parke, Davis & Co., Detroit, Mich.Preparation of MAbs. Female BALB/c mice (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) were immunized with four weekly intraperitoneal (i.p.) injections of 108 heat-killed P. aeruiginosa It-I organisms (obtained from M. Fisher, Parke, Davis). Four days after the final immunization, the spleens were removed aseptically and dissociated into a single-cell suspension. Approximately 5 x 107 spleen cells were fused with 5 x 107 non-immunoglobulin-producing Sp 2/0-Ag 14 myeloma cells (33) (American Type Culture Collection, Rockville, Md.; CRL 1581) by using 4,000-molecular-weight polyethylene glycol (50% [wt/vol] in water) as previously described (8). After fusion, the cells were washed and suspended in 80 ml of medium containing hypoxanthine, aminopterin, and thymidine. This mixture was dispensed in 100-[il portions into 96-well tissue culture plates (Costar, Cambridge, Mass.) previously seeded with 3 x 105 BALB/c spleen cells. The cultures were incubated in a 10% CO2 atmosphere at 37°C and fed with hypoxanthineand thymidine-containing medium on day 7. The supernatants from viable cultures were screened by enzyme-linked immunosorbent assay (ELISA) for antibody (see below) on days 14 and 21. Hybridomas from positive wells (i.e., optical density > 0.8) were cloned by limiting dilution in 96-well culture plates containing 3 x 105 splenic feeder cells per well. Positive clones were recloned three times, grown in large-scale culture in serum-free medium,

Generation and characterization of murine antiflagellum monoclonal antibodies that are protective against lethal challenge with Pseudomonas aeruginosa

“…Preliminary experiments in our laboratory indicated that opsonophagocytosis by the antiflagellum MAbs did occur, but the efficiency of opsonization varied according to the bacterial isolate used in the assay. Furthermore, opsonophagocytic activity of the antiflagellum MAbs was far inferior to that of specific anti-LPS MAbs (unpublished observation), which are very efficient opsonins (48,49). The fact that antiflagellum MAbs were less effective opsonins than anti-LPS MAbs does not preclude the possibility that opsonization of bacteria by antiflagellum antibodies is involved in prevention of lethal infection.…”

A protective human monoclonal antibody directed to the outer core region of Pseudomonas aeruginosa lipopolysaccharide

“…Antisera and MAbs reactive with the 0 polysaccharide of P. aeruginosa LPS have been reported to act as opsonins and effectively protect mice from death caused by experimental bacteremia (11,21,22,25,29). Almost all of the antibodies so far reported were directed to serotype-specific epitopes on LPS and were only active against a specific serotype out of 17 serotypes.…”

“…Lipopolysaccharide (LPS), mucoid exopolysaccharide, flagella, and outer membrane protein F of P. aeruginosa have been extensively studied for their effectiveness as protective antigens (1,6,9,14). Recently, monoclonal antibodies (MAbs) specific for LPS have been obtained and shown to be highly protective (2, 18, 21, 22, 25,26,29).…”

“…LPS consists of three regions, 0 polysaccharide, core oligosaccharide, and lipid A, which are covalently bound to each other. Antibodies to 0 polysaccharide have been demonstrated to be more effective than antibodies to the other components of LPS in experimental animal infection models (13,18,21,22,25). However, the former antibodies have narrow efficacy spectra, because each of them is only effective against strains belonging to the specific 0 serotype that is recognized by the antibody.…”

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

“…While some strains of P. aeruginosa are killed by complement-dependent bacteriolysis, most strains associated with noscomial infections are killed only following opsonization and phagocytosis [1]. Passive protection studies using sera of animals immunized with fipopolysaccharide (LPS) [2], murine monoclonal antibodies (MAbs) [3,4] and human MAbs [5,6] reactive with LPS provide evidence that antibodies to serotype-specific antigens are highly protective in experimental P. aeruginosa infectious. This report describes the opsonic activity of five human IgM MAbs against serotypespecific LPS antisens of P. aeruginosa and examines their protective activity in a lethal model of Pseudomonas burn wound sepsis.…”