Protection against Pneumococcal Pneumonia in Mice by Monoclonal Antibodies to Pneumolysin

Recombinant PLY and antibodies.The MAbs used in this study were PLY-4, PLY-5, and PLY-7 {mouse anti-PLY immunoglobulin G1 κ chain [IgG1(κ)]}; 1.4G8.66 [mouse anti-pepsinogen C IgG1(κ)] (16) was used as an isotype-matched indifferent MAb. Recombinant PLY and rabbit polyclonal IgG to PLY (anti-PLY IgG) were obtained as already described (13, 14). Purified nonimmune rabbit IgG (NI-IgG) was purchased from Sigma Chemical Co.

Bacteria.S. pneumoniae D39 type 2 NCTC 7466 was obtained from The Spanish Type Culture Collection (Valencia, Spain). Lyophilized cells were restored in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) and were subcultured twice on blood agar plates (Biomedics, Madrid, Spain). The virulence of the type 2 strain was ensured by intraperitoneal injection of MF-1 mice with 10⁵ CFU and recovery of the strain from the peritoneal cavity during necropsy. This virulent strain was inoculated into 10 ml of BHI broth supplemented with 10% fetal calf serum (Gibco Laboratories, Life Technologies Ltd., Paisley, Scotland) and incubated overnight at 37°C. The culture was diluted to 100 ml and incubated at 37°C until it reached an optical density at 600 nm of 0.15 (10⁸ CFU/ml). This strain was stored as 1-ml aliquots at −70°C in BHI broth. Virulence and CFU were periodically checked. For challenge in mice, frozen suspensions of S. pneumoniae type 2 were thawed, pelleted, and washed three times with 1 ml of Hanks balanced salts solution (HBSS) (Flow Laboratories, Irvine, Scotland) diluted with distilled water. Cells were suspended in 250 μl of HBSS and used immediately.

Hemolysis and in vitro neutralization assays.Hemolysis and in vitro neutralization assays were performed as already described (15). Briefly, serial dilutions of toxin were incubated with 50 μl of 1.6% sheep erythrocytes in phosphate-buffered saline (PBS) for 30 min at 37°C. The concentration of toxin that lysed 50% of erythrocytes was considered 1 hemolytic unit. For the neutralization test, serial twofold dilutions of MAb were incubated with 2 hemolytic units of PLY for 15 min at 37°C. Fifty microliters of 1.6% sheep erythrocytes in PBS was added, and the plates were incubated at 37°C for 30 min. The minimal concentration of MAb which completely inhibited hemolysis was considered 1 neutralizing unit (NU).

Mice.MF-1 mice (Oxon, Harland Olac Ltd., Bicester, England) were bred at the University of Oviedo animal house. All animal studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Oviedo. Mice used in this report were 6 to 8 weeks old and weighed 30 ± 3 g (mean and standard deviation).

Mouse toxin lethality model.The model used for the toxin lethality studies involved injection into the mouse tail vein of various amounts of PLY: 3, 1.5, 0.75, and 0.375 μg diluted in 100 μl of pyrogen-free 10 mM PBS. Groups of 10 mice were injected with each amount of toxin in sterile nonpyrogenic PBS. The same volume of pyrogen-free PBS was injected into control mice. Lethality then was monitored for the subsequent 24 h, and the 50% lethal dose (LD₅₀) was calculated as described by Reed and Muench (34) and Dougherty (17).

Murine infection.Groups of 20 mice were lightly anesthetized with 3% (vol/vol) halothane over oxygen (3 to 4 liters min⁻¹) by using a methacrylate box connected to Fluovac 240 (Anaesthetizing Systems, Cheshire, England). Mice were intranasally infected with a lethal dose of 5 × 10⁶ CFU of S. pneumoniae D39 type 2 in 50 μl of HBSS applied atraumatically to the tip of the nose and involuntarily inhaled. Deaths were recorded for 20 days.

Passive protection studies.Groups of 20 mice were injected in the tail vein with 100 μg of PLY-4, PLY-5, PLY-7, or anti-PLY IgG in 200 μl of sterile nonpyrogenic PBS 1 h before purified toxin challenge or intranasal infection with S. pneumoniae D39 type 2. Control mice were injected with 100 μg of indifferent MAb (1.4G8.66), 100 μg of NI-IgG, or 200 μl of PBS. Mice received a second dose of antibodies 36 h after infection, under the same conditions as the first dose. Combinations of 2 MAbs (PLY-4 and PLY-7 at 50 μg each) and 3 MAbs (PLY-4, PLY-7, and PLY-5 at 34 μg each) were administered to mice under the same conditions as single MAbs.

Organ histological analysis and CFU.Groups of six mice were randomly selected 12, 24, 48, and 72 h postinfection and were deeply anesthetized. Blood was obtained from heart puncture and collected in heparinized tubes. After that, mice were euthanatized by cervical dislocation. The lungs were removed, weighed, and homogenized in an Ultra-Turrax T25 (Janke & Kunkel, IKA-Labortechnik, Staufen, Germany). Quantitative culturing on blood agar of blood and lung homogenates was done to determine the number of CFU. At each time point, one animal per group was randomly chosen for histological analysis. Lungs were removed, fixed in 10% buffered formalin, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin and viewed by light microscopy. Four sections separated by at least 200 μm were studied per animal.

Statistics.Differences between the median survival times for groups of mice were analyzed by the Mann-Whitney U test. Differences between the overall survival rates for groups of mice were analyzed by the Fisher exact test. Differences between CFU were analyzed by the two-tailed unpaired t test. The limit of statistical significance was a P value of 0.05.

Neutralization of toxemia in mice by anti-PLY MAbs.The in vitro neutralizing capacity of anti-PLY MAbs was tested; 1 NU of PLY-4, PLY-5, and PLY-7 was estimated to be equivalent to 4, 125, and 68 ng, respectively.

To assess the potential protective efficacy of these MAbs in vivo, we first determined the intravenous LD₅₀ of purified PLY in mice; it was found to be 1.06 μg. Injection of 2 LD₅₀s of the toxin into the tail vein of mice was, as expected, uniformly fatal in 2 h.

In the first in vivo experiment, preincubation of PLY (2 LD₅₀s) with a neutralizing amount of MAb PLY-7 for 30 min at 37°C was highly protective (100% survival). However, all of the mice injected with the same amount of this MAb 1 h prior to toxin administration died a few minutes later. Therefore, twofold increasing amounts of anti-PLY MAbs were sequentially assayed in groups of 10 mice. Mice given indifferent MAb, anti-PLY IgG, and NI-IgG were used as controls. Table 1 shows the doses of antibodies which protected the mice against toxin lethality. Animals injected with PLY-4, PLY-7, and anti-PLY IgG were protected against death, while mice that received indifferent MAb and NI-IgG did not survive. All of the mice given 6.25 μg (1,500 NUs) of PLY-4 survived toxemia, while 50 μg (735 NUs) of PLY-7 was necessary to obtain a similar effect. No degree of protection was observed with PLY-5 at the tested amount (1,600 NUs).

Observation of a synergistic effect of anti-PLY MAbs in protection against pneumococcal pneumonia.In order to evaluate the potential protection afforded by passive immunization, anti-PLY MAbs were administered singly or in combination. Groups of mice were intranasally challenged with 5 × 10⁶ CFU of S. pneumoniae type 2. Morbidity and mortality were monitored every 12 h. The first signs of disease were observed between 24 and 36 h; mice exhibited an increased rate of breathing, low mobility, fever, and bristly hair. The median survival time was 60 h, and all of the animals died before 6 days postinfection.

Anti-PLY MAbs (PLY-4, PLY-5, and PLY-7) and indifferent MAb were assayed individually in four groups of 20 mice. Mice treated with PLY-4 lived significantly longer than either PBS-treated mice (P < 0.0001) or indifferent MAb-treated mice (P = 0.0048) (Fig. 1). However, neither PLY-5 nor PLY-7 alone conferred significant protection against infection. Three groups of 20 mice were treated with a combination of antibodies. PLY-4 and PLY-7 were mixed prior to injection into mice. A significant increase in the median survival time was obtained for mice treated with the MAb mixture compared with both indifferent MAb-treated mice (P = 0.028) and PBS-treated mice (P = 0.0002). However, this treatment did not provide better protection than PLY-4 alone (Table 2). Mice that received a combination of three MAbs survived significantly longer than those that received PLY-4, indifferent MAb, or PBS. However, there was no significant difference from the results obtained for mice that received PLY-4 and PLY-7 in combination. Similarly, mice given anti-PLY IgG survived longer than mice given PLY-4, NI-IgG, or PBS. The median survival time for these mice was not significantly different from that for mice given either two MAbs or three MAbs.

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FIG. 1.

Antibody-mediated protection of mice by intravenous administration of anti-PLY antibodies in pneumococcal infection. Groups of 20 mice were intranasally infected with 2 LD₅₀s of S. pneumoniae type 2 and treated with antibodies. Each dot represents one mouse. Horizontal lines denote median survival times. indiff., indifferent.

The highest protection rate was observed with anti-PLY IgG (15 of 20 mice survived), but this rate was not significantly different from that observed after treatment with a combination of either two or three MAbs. The survival rates for mice receiving single MAbs were not significantly different from that of PBS-treated mice (Table 2). Although the overall survival rate for mice that received two MAbs (8 of 20) was numerically higher than that for mice that received PLY-4 (2 of 20) or PLY-7 (3 of 20), the difference did not reach statistical significance. The survival rate for mice that received three MAbs (10 of 20) was significantly higher than that for mice that received single MAbs but was not significantly higher than that for mice that received a combination of two MAbs (8 of 20).

Protection of mice against bacteremia and lung tissue injury by passive immunization with anti-PLY MAbs.The effects on the number of bacteria in blood and lungs were examined in mice treated with a combination of three MAbs (PLY-4, PLY-5, and PLY-7), indifferent MAb, and PBS. Because the majority of the control mice succumbed to infection by day 3, collection of samples was carried out before 72 h postinfection. Determinations of CFU in lungs of mice intranasally challenged with S. pneumoniae type 2 are shown in Fig. 2A. All of the mice exhibited intrapulmonary colonization with pneumococci throughout the period of experimentation. After 48 h, a significantly lower pneumococcal count was observed in lung homogenates from anti-PLY MAb-treated mice than in those from either PBS-treated mice (P = 0.0106) or indifferent MAb-treated mice (P = 0.0480). After 72 h postinfection, mice that had been treated with anti-PLY MAbs showed lower CFU than either PBS-treated mice (P = 0.0004) or indifferent MAb-treated mice (P = 0.0070). In both indifferent MAb- and PBS-treated mice, after 12 h postinfection, increasing numbers of bacteria were detected in blood samples, and all mice became bacteremic at 48 h postinfection (Fig. 2B). Lower frequencies of bacteremia were seen in MAb-treated mice than in PBS-treated mice at 48 h postinfection (16.6 versus 100%; P = 0.0152) or in both groups of control mice at 72 h postinfection (0 versus 100%; P = 0.0022).

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FIG. 2.

(A) Passive immunization with anti-PLY MAbs significantly decreased pneumococcal colonization of lungs with respect to that in PBS-treated and indifferent MAb-treated groups (P = 0.0106 and 0.0480, respectively) at 48 and 72 h postinfection (P = 0.0004 and 0.0070). (B) Anti-PLY MAb treatment decreased the number of bacteremic mice during pneumococcal pneumonia with respect to that in the PBS-treated group at 48 h (P = 0.0152) and with respect to that in both control groups at 72 h (P = 0.0022) postinfection. Symbols: •, PBS-treated mice; ○, mice treated with indifferent MAb; ⧫, mice treated with PLY-4, PLY-5, and PLY-7.

When lung tissues from PBS-treated mice were examined by light microscopy (Fig. 3, A1 and A2), severe inflammation was observed in areas surrounding vessels, bronchi, and bronchioles starting at 24 h postinfection. Numerous polymorphonuclear leukocytes and occasional hemorrhages were detected in these areas. Some intra-alveolar leukocytes were also seen. The inflammatory process increased during the following days. In contrast, most of the lung tissues from mice treated with anti-PLY MAbs appeared histologically normal (Fig. 3, B1 and B2), and only a few inflammatory cells could be detected in the alveoli and perivascular areas.

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FIG. 3.

(A1 and A2) Histological appearance of lungs of mice infected with S. pneumoniae type 2. Perivascular and peribronchiolar inflammation (arrows) can be seen at 48 h postinfection (A1). Numerous leukocytes can be seen in this peribronchiolar area. The bronchiolar epithelium (asterisk) is altered, and the alveoli show slight distension (A2). (B1 and B2) Lung sections of mice treated with anti-PLY MAbs at 48 h postinfection. Vessels, bronchioles, and alveoli appear structurally normal, with no signs of acute inflammation (B1). The bronchiolar epithelium (asterisk) and the alveoli do not show any signs of pathological alterations (B2). a, alveolus; b, bronchiole; v, vessel. Calibration bars: A1 and B1, 100 μm; A2 and B2, 50 μm.