Vol. 45, No. 1
INFECTION AND IMMUNITY, JUlY 1984, P. 217-221
0019-9567/84/070217-05$02.00/0 Copyright © 1984, American Society for Microbiology
Preparation and Characterization of a Nontoxic PolysaccharideProtein Conjugate That Induces Active Immunity and Passively Protective Antibody Against Pseudomonas aeruginosa Immunotype 1 in Mice GRACE C. TSAY* AND MICHAEL S. COLLINS
Microbiology Research Department, Cutter Laboratories, Division of Miles Laboratories,
Inc.,
Berkeley, California 94710
Received 27 January 1984/Accepted 9 April 1984
Acid treatment of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide generated a low-molecularweight polysaccharide fraction that was detectable in agar gel immunodiffusion but did not induce antibodies or resistance tb infection in mice. The polysaccharide was treated with periodate to generate additional aldehyde groups. Oxidized polysaccharide was covalently coupled by reductive amiration to 1,4-diaminobutylderivatized bovine serum albumin. Physical properties of the conjugate were characterized by gel filtration and high-pressure liquid chromatography. The gelation activity of the conjugate in the Limilus amoebocyte lysate assay was 4,000-fold less than native lipopolysaccharide by weight. Mice immunized with the conjugate resisted challenge with P. aeruginosa immunotype 1 that killed 90% of nice immunized with saline. Immunization with the conjugate vaccine induced humoral immunoglobulin G that passively protected normal and burned mice. These results indicate that conjugation of nonimmunogenic polysaccharide antigen of P. aeruginosa restores immunogenicity similar to that of native lipopolysaccharide without restoring endotoxicity inherent in lipopolysaccharide.
maintained on brain heart infusion agar slants at 4°C. On the day of an animal challenge, overnight cultures on brain heart infusion agar slants were subcultured onto fresh agar slants. After 4 to 5 h of incubation at 37°C, cells were harvested by washing the slants with phosphate-buffered saline (pH 7.4). Cells were washed once and adjusted to the desired concentration with saline. Inocula levels were determined by correlating plate counts with a 660-nm spectrophotomnetric reading of cell suspensions. Mice. Female Swiss-Webster mice (weight, 18 to 26 g) were obtained from Simonsen (Gilroy, Calif.). For each study, mice were matched by age and weight. They were housed at 10 mice per cage and freely given water and mouse chow. Extraction and purification of LPS. P. aeruginosa was cultured to late log phase in 10 liters of chemically defined glucose-glutamate salts medium (16) in a 14-liter Virtis fermenter. Cells were killed by addition of formaldehyde to 0.37%, harvested by centrifugation, washed once with distilled water, and lyophilized. Yields of cells were 20 to 30 g (dry weight). LPS was extracted by the hot phenol-water method of Westphal et al. (33). After dialysis to remove phenol, crude LPS was treated with RNase and DNase (Sigma Chemical Co., St. Louis, Mo.) in 0.1 M acetate buffer (pH 5.0) for 24 h. The pH was adjusted to 7.0 with NaOH, and LPS was treated with pronase (Sigma) for 24 h. Lowmolecular-weight substances were removed by diafiltration through a hollow fiber ultra filter (HIX-50; Amicon Corp., Lexingtorn, Mass.). Preparation of conjugate vaccine. LPS was hydrolyzed in dilute acetic acid (8). Insoluble lipid A was removed after centrifugation at 10°C. The supernatant fluid was adjusted to pH 7.0 with NaOH, and the remaining lipid A was removed by three extractions with 2 volumes of CHC13-methanol (2:1; vol/vol). PS in the aqueous phase was concentrated by rotary evaporation under vacuum. PS was fractionated by water elution on a Bio-Gel A-5m column (100 by 2.6 cm),
Pseudomonas aeruginosa is often a virulent pathogen in hosts whose immune system is compromised by neoplastic disease (30) or major thermal injury (21). Despite therapy with appropriate antibiotics, the mortality rate from P. aeruginosa bacteremia is high in these debilitated patients (14). Numerous studies have indicated that immunoglobulin G (IgG) antibody to lipopolysaccharide (LPS) is protective in experimentally infected animals (6, 22). A heptavalent vaccine containing LPS of the seven immunotypes of P. aeruginosa (13) is effective in inducing antibodies in humans (19) that are protective in experimentally infected animals (12). Attempts to immunize patients at high risk of P. aeruginosa infection with this vaccine, however, have been only moderately successful due in part to the potent endotoxin activity of LPS. Local and systemic adverse reactions to endotoxin, including fever, malaise, and pain at the injection site, can limit vaccine dosage (25, 35). In this study, we describe the preparation of a nontoxic P. aeruginosa vaccine consisting of purified low-molecularweight polysaccharide (PS) derived from immunotype 1 LPS covalently coupled to bovine serum albumin (BSA) and characterize the protective activity of hyperimmune mouse serum in experimentally infected burned and normal mice. (This paper was presented in part at the 67th Annual Meeting of the Federation of American Societies for Experimental Biology, April, 1983.)
were
MATERIALS AND METHODS Bacteria and inocula preparation. P. aeruginosa 1369 (Fisher-Devlin-Gnabasik immunotype 1) was obtained from J. A. Bass, Shriners' Hospital for Crippled Children, Galveston, Tex. The strain was kept at -60°C in brain heart infusion broth containing 10% glycerol. Working cultures
*
Corresponding author. 217
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TSAY AND COLLINS
followed by fractionation with water elution on a Sephadex G-25 column (100 by 2.6 cm). PS in the Sephadex G-25 void volume was lyophilized. Dextran (Pharmacia Fine Chemicals, Piscataway, N.J.) molecular weight standards were used to estimate the molecular weight of eluted PS fractions. PS was oxidized with NaIO4 to generate aldehyde groups (29). Oxidized PS was purified by water elution on a Sephadex G-25 column (100 by 2.6 cm) and lyophilized. Essentially fatty acid-free BSA (Sigma) was coupled to 1,4-diaminobutane (Aldrich Chemical Co., Milwaukee, Wis.) to increase the number of BSA amino groups (24). Oxidized PS was coupled to aminobutyl-BSA by reductive amination with sodium cyanoborohydride (3). The reaction products were fractionated by water elution on a Sephadex G-100 column (100 by 1.0 cm). Fractions containing both carbohydrate and protein were collected. High-pressure liquid chromatography. The elution profiles of BSA, aminobutyl-BSA, and the aminobutyl-BSA-PS conjugate were examined on an Altex high-pressure liquid chromatograph fitted with a Spherogel TSK-3000 column (7.5 mm by 60 cm) and a UV detector. The eluting buffer was 0.2 M sodium phosphate (pH 6.8). Chemical assays. Assay methods for 2-keto-3-deoxyoctonate (32), protein (20), RNA (4), DNA (1), total carbohydrate (9), reducing sugar (28), and free amino groups (18) have been described previously. Limulus amoebocyte lysate. The activities of LPS, 2 lots of PS, BSA, and 2 lots of BSA-PS conjugate were quantitated with reagents from Associates of Cape Cod. The Limulus assay was sensitive to 0.015 ng of Escherichia coli 0113 LPS per ml. Active immunization. Mice (weight, 18 to 20 g) were injected once weekly four times by the subcutaneous route with 0.1-ml saline solutions of the following substances: 37 ,ug of aminobutyl-BSA, 5 ,ug of PS, a mixture of aminobutylBSA and PS, or the conjugate containing 5 ,ug of PS covalently linked to 37 ,ug of BSA. Sera were obtained before immunization and after injections two through four. Mice were challenged after injection four with 10 times the 50% lethal dose (LD50) of P. aeruginosa 1369 by the intra-
peritoneal route. Passive immunization of normal mice. Mice (weight, 18 to 20 g) were passively immunized intraperitoneally with 0.05 to 0.10 ml of sera. Two hours later, mice were challenged intraperitoneally with 10 to 20 times the LD50 of P. aeruginosa 1369. Passive immunization of burned mice. Dorsal fur of mice (weight, 26 g) was clipped from head to tail. A 0.05-ml volume of serum or immunoglobulin was administered intraperitoneally. Two hours later, mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (60 to 80 mg/kg of body weight). An asbestos cloth with a 5.8-cm2 oval hole was placed over the back, and mice were given approximately a 10% full thickness body surface burn for 5 s with a Fischer gas burner. Inoculum suspended in 0.5 ml of saline was injected subcutaneously into the burn site (5). Burned mice were observed for 15 days after challenge. Serum fractionation. A volume of 1.9 ml of pooled sera from mice immunized with conjugate vaccine was fractionated by phosphate-buffered saline (pH 7.4) elution on a Sephadex G-200 column (100 by 2.6 cm). Each fraction containing protein was tested by agar gel immunodiffusion against goat anti-mouse IgG, IgA, and IgM (Kirkegaard and Perry Laboratories). Only fractions containing a single immunoglobulin class were used for passive immunization. Immunoglobulin concentration was adjusted to the same concentration as
INFECT. IMMUN.
found in serum by ultrafiltration on a Amicon PM10 membrane. Enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay (ELISA) was essentially that described by Engvall and Perlmann (11), as adapted to microtiter plates by Voller et al. (31). Wells of polystyrene microtiter plates (Cooke Laboratories, Alexandria, Va.) were coated with 200 ,ul of carbonate buffer (pH 9.6) containing 10 ,ug of LPS per ml. Alkaline phosphataseconjugated rabbit anti-mouse IgG and IgM were obtained from Miles Laboratories, Inc., Elkhart, Ind. The substrate pnitrophenylphosphate was obtained from Sigma. The yellow color that developed was read at 405 nm on a Dynatech MR580 ELISA spectrophotometer. The titer was considered to be the highest dilution of pooled sera that gave an absorbance at 405 nm of 0.100 after 30 min of incubation at 23°C. All plasma samples were run in duplicate, with reference serum included on each microtiter plate. Statistics. The significance of protection conferred by active and passive immunization was determined by the Fisher exact test. RESULTS PS antigen. Fractionation of immunotype 1 PS on Bio-Gel A-Sm yielded well-separated, high-molecular-weight (1.5 x 105 to 5 x 105) and low-molecular-weight (0.2 x 104 to 4 x 104) peaks (Fig. 1). Low-molecular-weight PS was further purified from the void fraction of Sephadex G-25. The yield of immunotype 1 PS after acetic acid hydrolysis of LPS was 15%, with approximately 50% of the total PS in the lowmolecular-weight range. To exclude the possibility of contamination of PS with trace amounts of LPS, only the lowmolecular-weight fraction (
