Vol. 58, No. 9

INFECTION AND IMMUNITY, Sept. 1990, p. 2828-2833 0019-9567/90/092828-06$02.00/0 Copyright C 1990, American Society for Microbiology

Murine Monoclonal Antibodies to Klebsiella pneumoniae Protect against Lethal Endotoxemia and Experimental Infection with Capsulated K. pneumoniae ELIANE MANDINE,' MARIE-FRANCE SALLES,' RENE ZALISZ,l MONCEF GUENOUNOU,2 AND PIERRE SMETS1* Centre de Recherches Immunologiques Roussel-Uclaf, Societe des Usines Chimiques des Laboratoires Frangais, Laboratoires Cassenne, Osny,l and Unite Associee et Centre National de la Recherche Scientifique

40622 et Departement de Microbiologie, Faculte de Medecine, Paris,2 France Received 6 March 1990/Accepted 21 June 1990

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopolysaccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 1OA5) were directed against the lipid A fraction. Only one (2A4) recognized the 0 antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.

The incidence of gram-negative bacteremias is increasing, and shock occurs in about 40% of cases, with 40 to 90% mortality (24, 33). Many difficulties remain in the treatment of these bacteremias, since gram-negative bacteria are often resistant to antibiotics and, furthermore, since this pathology occurs frequently in patients who are weakened by other underlying diseases. Escherichia coli is the most common etiologic agent, causing one-third of gram-negative bacteremias; Klebsiella pneumoniae is the second most prevalent, followed by Pseudomonas species (18). Because of the major role played by endotoxin in this pathological state, the idea of an immunological approach to therapy has long been envisaged. Encouraging results in terms of diminution of mortality and morbidity were obtained in humans (2, 19, 37) after the administration of plasma rich in antilipopolysaccharide (LPS) antibodies. Antibodies occurring in animals after immunization with smooth bacteria or LPSs are generally directed against the outermost 0-side-chain oligosaccharide of LPS, which is serotype or species specific. No cross-reactivity is observed with these antibodies. In contrast, it has been postulated that antibodies directed against less well-exposed but more highly conserved elements of the core and lipid A regions of LPS possess cross-reactive and protective properties (5, 9, 33, 35). Antibodies directed against these masked, highly conserved antigenic determinants may be obtained either by immunization with rough mutants, which lack the 0 antigen and thus partially reveal the core-lipid A regions (1, 6, 20, 36), or by inoculation of LPS-associated proteins plus LPS (LAP-LPS) (12). Indeed, protein-containing LPSs are known to be highly immunogenic (3, 12). By immunizing mice with the J5 mutant of E. coli 0111:B4 (killed bacteria plus LAP-LPS), we obtained monoclonal antibodies (MAbs) which were protective in endotoxemia

*

Corresponding author. 2828

and experimental infection models (28). This report describes the production of murine MAbs directed against the outer membrane determinants, particularly the core region, of K. pneumoniae (01:K2) LPS. Using immunization with LAP-LPS preparations, we tested the effectiveness of some of these antibodies in lethal endotoxemia and infection protection models. MATERIALS AND METHODS Bacterial stock and antigen preparation. K. pneumoniae 01:K2 (CIP 52145), 03:K25 (CIP 52230), 04:K15 (CIP 52219), and R:K18 (CIP 52222) and Klebsiella ozaenae 02:K4 (CIP 52211) strains were purchased from the Collection Nationale de Culture de Microorganismes (Institut Pasteur, Paris, France). The R:K18 strain is rough or has an 0 serotype not yet determined (23). LAP-LPS from K. pneumoniae was prepared as previously reported (R. Zalisz and M. F. Salles, European patent EP 49,182, April 1982). Briefly, bacteria were grown in tryptocasein soy broth. Harvested bacteria were lysed by being heated for 90 min at 70°C, followed by 8 days of incubation at 37°C in the presence of EDTA-sodium mercurothiolate-lysozyme. These bacteria were then treated with acetone and methanol. The organic supernatants were discarded, and the remaining material was suspended in distilled water (30 g/liter) and stirred for 16 h at 4°C. After centrifugation, the supernatants were purified by ultrafiltration on Hi P100 hollow fibers (Amicon S.A.R.L., Paris, France). The LPS from K. pneumoniae 01:K2 was either extracted by the hot phenol-water method of Westphal et al. (34) and then purified as previously described (22) or derived from the LAP-LPS by high-performance liquid chromatography as described by Kol et al. (16). The LPS subunits were kindly provided by B. Fournet (CNRS, Lille, France) (17). The LPS (Westphal preparation) from E. coli 0111:B4 was purchased from Difco Laboratories (Detroit, Mich.). The LPSs from K. pneumoniae 03:K25, 04:K15, and R:K18 and

VOL. 58, 1990

K. ozaenae 02:K4 were prepared by the Westphal method (34). Production of hybridomas. Two groups of female BALB/c mice (Iffa-Credo, l'Arbresle, France) were injected intraperitoneally with 20 ,ug of RU 41821 (LAP-LPS from K. pneumoniae). Booster doses were administered intravenously on days 21 (20 ,ug) and 35 (10 jig) for group 1 and on day 20 (10 ,ug) for group 2. Three days after the last injection, spleen cells were fused as described by Kohler and Milstein (15) with nonsecreting Sp2/0-Agl4 murine myeloma cells (kindly donated by C. Grosclaude, Institut National de la Recherche Agronomique, Grignon, France) in standard medium with polyethylene glycol 1000 (E. Merck AG, Darmstadt, Federal Republic of Germany). The fused cells were selected in hypoxanthine-azaserine medium. The supernatants were tested for the presence of antibodies against K. pneumoniae LAP-LPS antigen by a two-step enzyme-linked immunosorbent assay (ELISA). Antibodysecreting hybridomas were cloned by limiting dilution. Ascitic fluids were obtained from BALB/c mice primed with pristane (2,6,10,14-tetramethylpentadecane) 2 weeks before intraperitoneal injection of 5 x 105 hybridoma cells. Antibodies against E. coli J5 were obtained as previously reported (28). Detection and characterization of antibody production by hybridomas. The reactivity of the MAbs toward LAP-LPS was determined by a two-step ELISA. Ten micrograms of LAP-LPS per ml in 0.05 M carbonate buffer (pH 9.6) was coated on Immulon microplates (Dynatech, Marnes-LaCoquette, France) before any remaining sites were blocked with bovine serum albumin (1 mg/ml). Cell culture supernatants were diluted with 0.01 M phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20. Bound antibodies were detected with a rabbit antibody to mouse immunoglobulin (Zymed Laboratories Inc., San Francisco, Calif.), followed by an alkaline phosphatase-conjugated goat antibody to rabbit immunoglobulin (Zymed). p-Nitrophenylphosphate (Sigma Chemical Co., St. Louis, Mo.) was used as the substrate and, after 30 min of incubation, the optical densities were read at 405 nm with an MR 610 microplate reader (Dynatech). The specificity of antibodies for the LPS subunits was determined by a one-step ELISA. The following concentrations of coating antigens were used: 10 ,ug/ml (LPS) and 30 ,ug/ml (lipid A) in 0.05 M carbonate buffer (pH 9.6) and 100 jig/ml (core) and 30 ,ug/ml (O antigen) in 0.01 M PBS (pH 7.4). Bound antibodies were detected with alkaline phosphatase-conjugated rabbit antibody to mouse immunoglobulin (Zymed). The same ELISA technique was used to determine the reactivity towards the LPSs extracted from the other Klebsiella strains. Immunoglobulin isotypes were determined with the concentrated culture supematant by double immunodiffusion (Ouchterlony) with anti-isotype sera (Nordic Immunological Laboratories, Tilburg, The Netherlands). Antibody purification. MAbs were purified by two precipitations in 45% saturated ammonium sulfate. Following dialysis against pyrogen-free PBS, the MAb concentrations were determined by radial immunodiffusion with Diffu-Gen (Tago Inc., Burlingame, Calif.). Detection of endotoxin. The presence of endotoxin was determined by the chromogenic Limulus amebocyte lysate test (Microbiological Associates Bioproducts, Walkersville, Md.). LPS lethal toxicity assay. All materials (solutions, vials, etc.) used for the purification were pyrogen free. Ascitic

IMMUNOPROTECTIVE ANTI-K. PNEUMONIAE MAbs

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TABLE 1. MAb characterizationa Antibody

2A4 3A3 3C2 3C4 6C5 6D5 7D2 1OA5 11C3 12B6 Sp2/0

Iso-

Specificity of antibody (optical density at 405 nm) for K. pneumoniae O1:K2

type

IgG3 IgM IgM IgM IgGl IgG3 IgG2b IgG3 IgG2b IgG3

LAP-LPS

LPS

Lipid A

Core

0 antigen

0.595 >2 >2 >2 0.558 1.941 >2 0.267 >2 >2 0.025

0.041 >2 >2 >2 0.022 >2 >2 0.082 >2 >2 0.024

0.055 0.278 0.322 0.502 >2 0.083 0.287 >2 0.478 0.284 0.008

0.007 >2 >2 >2 0.017 0.034 1.277 0.008 >2 >2 0.008

0.906 0.038 0.016 0.096 0.196 0.020 0.041 0.044 0.118 0.037 0.006

a Antibody isotypes were determined by double immunodiffusion (Ouchterlony). The specificities of the antibodies for LAP-LPS, LPS, and LPS subunits were tested by a one-step ELISA with supematants at a 1:10 dilution. An optical density of