Monoclonal Antibody against Klebsiella Capsular Polysaccharide ...

Vol. 60, No. 5

INFEcrION AND IMMUNITY, May 1992, p. 1771-1778 0019-9567/92/051771-08$02.00/0 Copyright © 1992, American Society for Microbiology

Monoclonal Antibody against Klebsiella Capsular Polysaccharide Reduces Severity and Hematogenic Spread of Experimental Klebsiella pneumoniae Pneumonia THOMAS K. HELD,lt* MATTHIAS TRAUTMANN,1t MARTIN E. A. MIELKE,2 HERMANN NEUDECK,3 STANLEY J. CRYZ, JR.,4 AND ALAN S. CROSS5 Department of Infectious Diseases, 1 Department of Microbiology,2 and Department of Pathology,3 Klinikum Rudolf

Virchow Charlottenburg, Free University, W-1000 Berlin, Germany; Swiss Serum and Vaccine Institute, CH-3001 Bern, Switzerland4; and Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D. C. 203075 Received 30 September 1991/Accepted 18 February 1992

KiebsieUla pneumoniae is an important nosocomial pathogen causing severe pulmonary infections. The majority of clinical KiebsieUla isolates produce a high-molecular-weight capsular polysaccharide (CPS) which is of the dominant virulence factors. In the present study, we examined the potency of a murine immunoglobulin M monoclonal antibody (MAb) with specificity to KiebsieUla type 2 CPS to protect rats against experimental KiebsieUla pneumonia. The MAb did not prevent the invasion of virulent bacteria into the interalveolar space. However, the resolution of infection was accelerated in MAb-treated animals. This was demonstrated by (i) less severe weight loss and (ii) markedly reduced inflammatory reactions in the lung. The elimination of bacteria was significantly increased not only in the lungs but also in the livers of antibody-treated rats. This was reflected by reduced levels of circulating, soluble CPS and MAb-bound CPS. A mixture of human MAbs with specificity to CPS of clinically important Kiebsiella serotypes may prove to be a useful tool for the prevention or supportive treatment of Kiebsiela pneumonia.

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Kiebsiella pneumoniae is one of the most frequently isolated gram-negative pathogens in severe nosocomial infections (1, 5, 9, 13, 16, 25). Pulmonary infections due to K pneumoniae are often complicated by lung abscesses and multilobular involvement and have a high mortality rate (6, 17). The rapidly progressive clinical course of nosocomial Klebsiella pneumonia often leaves little time to institute effective antibiotic treatment. In addition, an increasing proportion of nosocomial K pneumoniae isolates are resistant to various antibiotics commonly used in intensive care units (4, 19, 20, 27). Thus, alternative approaches to the prophylaxis and supportive treatment of Kebsiella respiratory infections are needed. The majority of clinical Klebsiella isolates produce a high-molecular-weight capsular polysaccharide (CPS). The virulence of Klebsiella strains in human and animal models is strongly correlated with the degree of encapsulation (11, 12). In a preceding study, it has been shown that active immunization with purified CPS protects rats against lethal, experimentally induced Klebsiella pneumonia (10). However, antibodies induced by active immunization may appear too late to protect a patient at imminent risk of acquiring Klebsiella pneumonia. Recently, we have shown that a CPS-specific murine monoclonal antibody (MAb) is able to protect mice against lethal intravenous Klebsiella infection (35). While this observation indicated a beneficial effect of passive immunoprophylaxis under certain conditions, it was still doubtful whether this type of treatment would be effective in the more complicated pathophysiologic process

of intratracheally induced pulmonary infection. Regarding the pathogenesis of Klebsiella pulmonary infections, it has been speculated that, in addition to the capsule, lipopolysaccharide (LPS) may play a critical role for the development of the necrotic lesions frequently found in such cases (34). Thus, an MAb with specificity for CPS may not by itself be sufficient for full protection in pneumonic infection. In the present study, we therefore used the previously characterized MAb to study the effectiveness of CPS-specific passive immunotherapy in a clinically relevant pneumonia model.

MATERIALS AND METHODS

Bacteria. K pneumoniae Caroli (O1:K2) was used throughout the study. This organism exists in two variants, a heavily encapsulated, highly virulent strain and a less encapsulated, less virulent derivative obtained from the original strain by sequential in vitro passages. Both strains have been used in previous studies (35, 37). To enhance virulence of the heavily encapsulated strain, bacteria were injected intraperitoneally into C56BL/6J mice and reisolated from spleen homogenate 24 h later. The strains were grown in Trypticase soy broth (TSB), and log-phase cultures were adjusted densitometrically to the desired bacterial concentration, which was confirmed by plating serial 10-fold dilutions of the suspension on blood agar plates for colony counts. For determination of 50% lethal doses (LD50s), various bacterial concentrations of the two strains were injected intraperitoneally into C56BL/6J mice and animals were observed daily as previously described (37). LD50 determinations were done by using standard methods (30). To determine capsular size, bacteria were stained with India ink and photographed, capsular diameters were measured, and the real diameters were calculated. Animals. Pathogen-free female Lewis rats (180 to 230 g; Institute of Medical Microbiology, Free University, Berlin,

Corresponding author. t Present address: Department of Hematology and Oncology, Klinikum Rudolf Virchow Charlottenburg, Spandauer Damm 130, W-1000 Berlin 19, Germany. t Present address: Department of Gastroenterology, Klinikum Rudolf Virchow Wedding, W-1000 Berlin 65, Germany. *

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Germany), 8 to 12 weeks of age, were used throughout the protection study. Antibody. A murine MAb specific for K pneumoniae type 2 CPS (K2-CPS), i.e., MAb III/5-1, was used to protect the animals. This antibody is of the immunoglobulin M (IgM) class and promotes the uptake and killing of K pneumoniae K2 strains by granulocytes and activates complement after contact with the bacteria (35). Protection study. Animals were pretreated with an intravenous injection of K2-specific MAb III/5-1 (35) or bovine serum albumin (BSA) (Merck, Darmstadt, Germany) at a dose of 10 mg/kg of body weight in phosphate-buffered saline (PBS). Both preparations had been previously passaged through a Detoxi-Gel column (Pierce Chemical Co., Rockford, Ill.), according to the directions of the manufacturer, to remove potentially contaminating LPS (7). Two hours later, lobar pneumonia was induced by intrabronchial instillation of the highly virulent Klebsiella strain by the method of Domenico et al. (11). In short, rats were anesthetized with 1.5 mg of xylacine (Bayer, Leverkusen, Germany) and 6 mg of ketamine hydrochloride (Parke, Davis & Co., Munich, Germany) given intraperitoneally. The trachea was exposed and incised, and 100 RI of PBS containing approximately 3 x 104 CFU of K pneumoniae Caroli was placed into the left or the right lobe of the lung via a bead-tipped, curved inoculation needle. This infectious dose was chosen on the basis of preliminary histologic and bacteriologic studies which showed that at least 3 x 104 CFU was needed to produce progressive pneumonia (data not shown). Previous experiments showed that it was not possible to intubate one side of the lung selectively. The incision of trachea and skin was closed by suturing. The animals were returned to their cages and observed four times daily for body weight and clinical abnormalities. The investigators were blinded to the therapy used. Because mortality studies are now prohibited by law in Germany, we were not able to observe for mortality. Previous experiments in which quantitative blood cultures were performed gave variable results; therefore, bacterial colony counts of K pneumoniae were not performed in the final experiments. Instead, we monitored these animals for differences in bacterial colony counts in lungs, livers, and spleens, differences in concentrations of CPS and MAb III/5-1 in plasma and organs, and histologic evidence of pneumonia. Animals were sacrificed at 48 and 72 h after challenge, when severe disease was obvious in the control group. Quantification of viable K. pneumoniae in infected tissues. At the times indicated, rats were sacrificed by inhalation of ether. Immediately thereafter, blood samples were obtained by cardiac puncture and placed into sterile tubes containing 0.52 ml of an 0.106-mol/liter concentration of sodium citrate (Sarstedt, Nurnbrecht, Germany). The lungs, livers, and spleens were aseptically excised, placed into sterile 50-ml glass tubes, and homogenized on ice with a tissue homogenizer (Elverham-Potter, Braun, Melsungen, Germany). The homogenates were serially diluted (10-fold) and plated in duplicate onto Endo agar plates (Becton Dickinson, Heidelberg, Germany). After incubation at 37°C for 24 h, colonies of K pneumoniae were counted and bacterial counts were expressed as log1o CFU per organ (limit of detection, 100 CFU per organ). Bacterial counts in livers and spleens were used as an indicator of bacterial dissemination. Determination of circulating and tissue CPS. For the quantitation of CPS, blood samples and tissue homogenates were centrifuged (30 mim, 3,000 U min-') and the supernatants were filter sterilized (pore size, 0.45 ,m; Renner, Dannstadt, Germany) and stored at -20°C until further use. The en-

INFECT. IMMUN.

zyme-linked immunosorbent assay (ELISA) for the detection of K2-CPS was performed as previously described (36). In brief, 96-well flat-bottom microtiter plates (Greiner, Nurtingen, Germany) were coated with 5 jig of purified MAb III/5-1 per ml (100 RI per well) at 4°C for 18 h. After removal of antigen, 200 ,ul of filling buffer (PBS [pH 7.4], containing 0.5% bovine serum albumin, 0.5% casein, and 0.1% sodium azide [wt/vol]), was added to each well to block nonspecific binding sites. After incubation for 1 h at 37°C, the plates were washed three times with PBS and stored at 4°C until use. Standards of K2-CPS and appropriately diluted samples of plasma and tissue homogenate supernatants were placed in duplicate onto each plate (100 RI per well). After overnight incubation at 4°C, the plates were washed three times with PBS and 100 RI of appropriately diluted polyclonal rabbitanti-K2 CPS IgG (ELISA titer, 1:16,000) was added to each well. After further incubation at 4°C for 4 h, the plates were washed again three times with PBS. Bound rabbit IgG was detected with appropriately diluted alkaline phosphataseconjugated, heavy-chain-specific anti-rabbit IgG (Sigma, Deisenhofen, Germany) (100 IA per well, 4 h at 4°C). Plates were washed again three times with PBS, and color was developed by adding 200 RI of substrate solution (1 mg of p-nitrophenyl phosphate [Sigma] per ml in 1 M diethanolamine buffer [pH 9.8]) to each well. The optical density was read at 405 nm in a Titertek Multiskan Plus MK II reader (Flow Laboratories, Helsinki, Finland). The ELISA for the detection of circulating MAb III/5-1 in plasma was performed in the same manner, except that plates were coated with 25 ,ug of K2-CPS per ml. Standards of purified MAb III/5-1 and appropriately diiuted samples of plasma were placed in duplicate onto each plate (100 ,u per well) and incubated at 4°C for 4 h. Bound MAb III/5-1 was traced with appropriately diluted alkaline-phosphatase-conjugated, heavy-chain-specific anti-mouse IgM (Sigma) (100 RI per well, 4 h at 4°C), and reactions were developed as described above. To liberate bound CPS from MAb-CPS complexes, plasma and tissue homogenates were treated with protease VIII (Sigma) by the method of Kahn and Jones (18). The enzyme was dissolved at 10 mg/ml in PBS and added to the samples at a 1:1 ratio. After incubation at 37°C for 60 min, the samples were boiled in a water bath for 10 min to destroy residual protease activity. Previous experiments with purified K2-CPS, incubated with MAb III/5-1, showed that this procedure quantitatively released bound, and therefore not detectable, CPS. Histological examination. From each experimental group, two rats were randomly selected for histological examination. Animals were sacrificed with ether, and their lungs, spleens, livers, thymuses, mediastinal lymph nodes, and kidneys were placed in PBS containing 5% formalin. After fixation for 1 day, the tissues were fixed with ethanol and embedded in paraffin. Five-micrometer sections were placed on glass slides and stained with hematoxylin and eosin. Statistical methods. Body weights, bacterial counts, and CPS concentrations were compared by using the MannWhitney U test. Capsular sizes were compared with the t test, and numbers of noninfected tissues were compared by using the chi-square test (29, 31).

RESULTS Characterization of the experimental bacterial strains. When comparing log-phase cultures of the two variants of K. pneumoniae Caroli, we found significant differences in cap-

CPS-SPECIFIC IMMUNOTHERAPY IN A PNEUMONIA MODEL

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TABLE 1. Comparison of two variants of K pneumoniae Caroli Production of

L50 (CFU/mouse)'

soluble CPSCaslriz (rm)c (ngIl1O CFU)b

Highly virulent variant

3 orders of magnitude at 72 h [P < 0.01] after inoculation) in the MAb-treated rats and decreased significantly from 48 to 72 h after infection (P < 0.05) (Fig. 2). In the spleen, the numbers of noninfected tissues were significantly lower in MAb-treated animals than those in control animals at 48 h after infection (two of eight infected spleens in the MAb group versus six of eight in the control group [P < 0.05]) but not at 72 h postinfection. In the liver, bacterial counts of the MAb group were diminished from 0.8 log1o CFU (48 h postinfection) to less than the level of detection at 72 h postinfection, while the counts of the BSA group increased between 48 and 72 h after challenge, although this trend was not significant (Fig. 2). There was a significant difference in the numbers of infected livers between the two groups at 72 h after infection (zero of eight infected livers in the MAb group versus eight of eight in the control group [P < 0.0001]). CPS in plasma. To further evaluate the potential of MAb III/5-1 to restrict bacterial multiplication and severity of pneumonia, we determined the CPS concentrations in plasma. Mean CPS concentrations in the animals pretreated with MAb were significantly lower in plasma at 48 h after infection (Fig. 3). In additional experiments, proteasetreated and untreated homogenate supernatants of organs (lungs, livers, and spleens) of MAb-treated and control animals were compared. Results indicate that, by day 3, significant differences in the amount of released CPS were observed (data not shown).

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+72h +24h +48h h after infection FIG. 1. Course of body weight in MAb III/5-1 pretreated (0) and BSA-pretreated control ([1) animals after induction of lobar pneumonia with 3 x 104 CFU of K pneumoniae Caroli. **, significant difference between the two groups (Mann-Whitney U test) at P of