Specific Immune Responses and Enhancement of Murine Pulmonary Clearance of Moraxella catarrhalis by Intranasal Immunization with a Detoxified Lipooligosaccharide Conjugate Vaccine

Mice.Female BALB/c mice (6 to 8 weeks of age) were obtained from Taconic Farms Inc. (Germantown, N.Y.). The mice were housed in an animal facility in accordance with National Institutes of Health guidelines under animal study protocol 1009-01.

Bacterial strains and culture conditions.Strain 25238 (type A) was purchased from the American Type Culture Collection (Manassas, Va.). Strains 26404 and 26397 (types C and B) were obtained from the Culture Collection of the University of Göteborg, Department of Clinical Bacteriology, Göteborg, Sweden. Strain O35E was provided by E. J. Hansen (42). These strains were grown on chocolate agar at 37°C with 5% CO₂ for 16 h. Three to five isolated colonies from each strain were then transferred onto new plates and incubated for 3.5 to 4 h (mid-logarithmic phase). The strains were adjusted in phosphate-buffered saline (PBS) (pH 7.2) containing 0.1% gelatin, 0.15 mM CaCl₂, and 0.5 mM MgCl₂ to the required CFU as determined from a plot of optical density (or 65% transmission) at 540 nm against viable counts.

Conjugate vaccine.Purification of LOS from strain 25238, detoxification of the LOS, and conjugation of dLOS to a carrier, cross-reactive mutant (CRM9) of diphtheria toxin (31, 34), were performed (10). The composition of dLOS-CRM was 419 μg of dLOS and 222 μg of CRM per ml, with a molar ratio of dLOS to CRM of 40:1, based on an estimated M_r of 3,000 for dLOS, and M_r of 63,000 for CRM.

Immunizations.(i) i.n. immunization. Mice were anesthetized (intraperitoneally) with 0.1 ml of 2% ketamine (Fort Dodge Laboratories, Inc., Fort Dodge, Iowa) and 0.2% xylazine (Miles Inc., Shawnee Mission, Kans.) in PBS. The animals were then allowed to inhale 10 μl of vaccine or saline that was slowly instilled by micropipette into both nares. The amount of vaccine preparations inhaled was formulated to contain 5 μg of dLOS-CRM (carbohydrate content) mixed with 1 μg of cholera toxoid (CT) (List Biological Laboratories, Inc, Campbell, Calif.) as an adjuvant. The control mice were also immunized i.n. with a mixture of 5 μg of dLOS, 2 μg of CRM, and 1 μg of CT or with 1 μg of CT in PBS. All mice were given a total of four doses at 1-week intervals, with the last immunization 1 week before bacterial challenge or sample collection.

(ii). s.c. immunization. Mice were immunized subcutaneously (s.c.) with 5 μg of conjugate vaccine in 0.2 ml of normal saline mixed with Ribi-700 adjuvant (containing 50 μg of monophosphoryl lipid A and 50 μg of synthetic trehalose dicorynomycolate; Corixa Corporation, Mamilton, Mont.). All mice were given a total of three injections at 2-week intervals, with the last injection 1 week before bacterial challenge or sample collection.

Collection of secretions and sera.Saliva (∼100 μl) was collected first, from mice anesthetized with a mixture of ketamine and xylazine and then intraperitoneally. administered with 100 μl of 1-mg/ml pilocarpine (Sigma Chemical Co., St. Louis, Mo.) in PBS to stimulate salivation, using a transfer pipette (Becton Dickinson Labware, Franklin Lakes, N.J.). Then, the mice were euthanized with an overdose of methoxyflurane (Metophane; Mallinckrodt Veterinary Inc., Mundelein, Ill.), the nasal wash was obtained by flushing the nasal cavity from the pharynx through the nostrils 10 times with 200 μl of PBS (44). For the collection of the lung lavage fluid (also called bronchoalveolar lavage fluid), the trachea was exposed through the neck, a cannula was inserted in the trachea, and the lung lavage fluid was collected by instilling and recovering three times via the cannula with 1 ml of PBS (30). Fresh feces from the small intestines was suspended at a ratio of approximately 1:10 in PBS, supplemented with 0.05% NaN₃ and protease inhibitor (10 μg/ml; Sigma). After being vigorously vortexed for 15 min and subjected to centrifugation, the extract supernatant was removed and aliquoted. Peripheral blood was obtained via the axillary artery. Blood samples were allowed to clot at 4°C and then centrifuged in a refrigerated centrifuge. Sera were removed and divided into aliquots. All samples were stored at −70°C until assayed.

Preparation of lymphocytes from different organs.Lymphocytes were prepared from the spleens, the NALT, cervical lymph nodes (CLN), PPs, nasal passages (NP), and lungs (2, 12, 44). Mice were anesthetized and then bled. After exsanguination, the mouse palate with NALT was peeled from the rest of the nasal tissue and pooled into an ice-cold incomplete medium: RPMI 1640 containing l-glutamine, supplemented with 5% fetal bovine serum, 0.5% (vol/vol) antibiotic-antimycotic, gentamicin (20 μg/ml; Life Technologies Inc., Rockville, Md.), 5 × 10⁻⁵ M 2-mercaptoethanol, and 0.01 M HEPES (Sigma). Single cells were obtained from the spleens, PPs, CLN, lungs, and NP by pressing the organs through stainless-steel mesh or the frosted ends of two microscope slides in a 24-mm-diameter petri dish containing 5 ml of cold incomplete medium. Cell suspensions from the NALT, spleens, PPs, and CLN were washed twice with incomplete medium by centrifugation (200 × g for 8 min at 4°C). The lung and NP cell suspensions were layered on a Percoll (Sigma) density gradient after lysing the red blood cells. Viable lymphoid cells were recovered from the interface between the 40 and 75% Percoll solutions. Total cell yields were enumerated in a hemacytometer using 0.4% trypan blue to assess viability and kept in complete medium (10% fetal bovine serum).

ELISA.The titers of LOS-specific antibodies in the nasal washes, saliva, lung lavage fluid, fecal extracts, and sera were determined by enzyme-linked immunosorbent assay (ELISA) (17) with 96-well Nunc-Immuno plates (MaxiSorp surface; Nalge Nunc International, Roskilde, Denmark) coated with strain 25238 LOS (10 μg/ml) in PBS (pH 7.4) containing 10 mM MgCl₂ overnight at 4°C. A mouse antiserum against strain 25238 whole cells was used as a reference (10). Negative controls included presera and external secretions from naïve mice, giving optical density readings of less than 0.1. The antibody endpoint titer was defined as the highest dilution of sample giving an A₄₀₅ twofold greater than that of presera or external secretions from naïve mice.

ELISPOT.Enumeration of antibody-forming cells (AFCs) was performed by enzyme-linked immunospot assay (ELISPOT) (3, 45). A 96-well membrane-based plate (MultiScreen-HA plate, Millipore Corporation, Bedford, Mass.) was coated with 25238 LOS as described in ELISA. The plate was blocked with complete medium, and lymphocytes from spleens, NALT, CLN, PPs, NP, and lungs at appropriate concentrations were incubated at 37°C in 5% CO₂ for 6 h. Isotype-specific biotinylated anti-mouse IgA, -G, or -M reagents (Sigma) were added at 4°C for an overnight incubation, followed by incubation with avidin-peroxidase conjugate (5 μg/ml) diluted in PBS containing 0.05% Tween 20 for 1 h at room temperature. After 4-chloro-1-naphthol-H₂O₂ (Sigma) was added, the plates were incubated for 5 to 8 min at room temperature, and the reaction was stopped by thoroughly washing plates with tap water. Spots were enumerated using a stereomicroscope equipped with a vertical white light (Zeiss, Jena, Germany).

Bacterial challenge.Bacterial aerosol challenges were carried out one week after the last immunization in an inhalation exposure system (Glas-col, Terre Haute, Ind.) (18). Mice were challenged with 10-ml aliquots of live bacteria, 10⁸ to 10⁹ CFU/ml, in a nebulizer. At 6 h postchallenge, mouse lungs were removed and homogenized in 5 ml of PBS for 1 min at low speed in a tissue homogenizer (stomacher lab system model 80; Seward, London, United Kingdom). Each homogenate was diluted serially in PBS, and 50-μl aliquots of the homogenate and diluted samples were cultured on chocolate agar plates (Remel, Richmond, Va.). The bacterial colonies were counted after an overnight incubation at 37°C with 5% CO₂.

Statistical analysis.The viable bacteria were expressed as the geometric mean CFU of n independent observations ± the standard deviation (SD). Geometric means of reciprocal antibody titers were determined. Significance was determined by the two-tailed Student's t test.

Antibody responses against LOS of M. catarrhalis.Significant LOS-specific antibody responses were elicited by i.n. immunization with dLOS-CRM (Table 1). LOS-specific IgA titers in external secretions and in serum were increased in animals immunized with dLOS-CRM. An increase of 56-fold in the nasal wash or 48-fold in the lung lavage fluid compared with PBS controls was detected. LOS-specific IgA titers in the nasal wash were also elevated in animals immunized with dLOS plus CRM (sevenfold), but they were significantly lower than those of animals immunized with dLOS-CRM (P < 0.01). A significant increase in LOS-specific IgG titers was detected in mice immunized with dLOS-CRM compared with the PBS control groups: serum (fivefold), nasal wash (fivefold), and lung lavage fluid (ninefold). LOS-specific IgM titers were significantly increased only in serum from mice immunized with dLOS-CRM (fivefold) or with dLOS plus CRM (threefold).

LOS-specific AFCs in different lymphoid or nonlymphoid tissues.The dominant isotype of LOS-specific AFCs was IgA, followed by small numbers of IgG and IgM. i.n. immunization with dLOS-CRM resulted in detection of LOS-specific IgA AFCs in all tissues tested, including distant organs such as PPs and spleen (Table 2). The majority of LOS-specific IgA AFCs were located in the NP (203 per 10⁶ cells), followed by a small amount in other tested tissues, including lungs (52 per 10⁶ cells). i.n. immunization with dLOS plus CRM elicited a few LOS-specific IgA AFCs in NP but not in other tissues. LOS-specific IgM AFCs were detected in the spleen and CLN. No LOS-specific AFCs were found in any tissues from mice immunized with PBS.

Enhancement of bacterial clearance from murine lungs.One week after the final immunization, mice were challenged with the homologous strain 25238. The number of bacteria recovered from lungs 6 h postchallenge was significantly reduced in the dLOS-CRM group (87%; P < 0.01) compared with the PBS group (Table 1). Immunization with dLOS plus CRM did not significantly enhance bacterial clearance compared with the PBS group.

The relationship between LOS-specific antibody titers and bacterial counts from mouse lungs was further analyzed in the nasal wash, lung lavage fluid, saliva, fecal extract, and serum from dLOS-CRM-immunized and PBS-immunized mice. A negative correlation was found in the nasal wash IgA (r = −0.85; P < 0.01) or IgG (r = −0.76; P < 0.05), lung lavage fluid IgA (r = −0.912; P < 0.001) or IgG (r = −0.68; P < 0.05), or serum IgA (r = −0.68; P < 0.05) or IgG (r = −0.80; P < 0.01).

To define the kinetics of bacterial reduction in the immunized mice, the time course of bacterial clearance in the lungs was observed after challenging with strain 25238 (Fig. 1). Immunization with dLOS-CRM enhanced bacterial clearance in the lungs during the whole period and also reduced bacterial duration compared with natural clearance in the lungs of naïve mice (P < 0.01). Bacterial recovery became undetectable within 24 h postchallenge in the immunized mice, whereas naïve mice still carried a small amount of bacteria. The highest bacterial reduction occurred at 6 h (90%) and 12 h (92%) postchallenge, suggesting that 6 h postchallenge in mice retaining more bacteria is an optimal time point to observe the efficacy of immunization.

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FIG. 1.

Time course of bacterial recovery from mouse lungs challenged with M. catarrhalis strain 25238 after i.n. immunization with dLOS-CRM. Mice received i.n.-administered 10-μl aliquots of PBS containing a mixture of 5 μg of dLOS-CRM and 1 μg of CT four times at 1-week intervals or were kept as naïve mouse controls. One week after the last immunization, all the mice were challenged (via aerosol) with 10 ml of M. catarrhalis strain 25238 (5 × 10⁸ CFU/ml) in a nebulizer. Lungs were collected, homogenized, and cultured for bacterial counts at 0, 3, 6, 12, 24, and 48 h postchallenge. Bacterial recoveries from lungs are expressed as geometric means ± SD (error bars) from eight mice. **, P < 0.01 (for comparison with naïve mouse controls).

Effect of i.n. immunization with different doses of dLOS-CRM.To determine what doses would further enhance pulmonary clearance, mice were i.n. immunized with one to five doses of dLOS-CRM plus CT and then challenged with the strain 25238 (Fig. 2). The bacterial counts in the lungs were reduced following the increase of doses, and significant bacterial clearance was found at each dose compared to that observed in naïve mice (P < 0.05 or P < 0.01). There was no significant difference on the bacterial clearance in mice given three, four, or five doses. The number of bacteria in the lungs decreased from 75 to 66% compared with the naïve mice and showed the highest variation of bacterial counts after five doses. In terms of LOS antibody responses, four doses elicited the highest LOS-specific IgA and IgG titers, especially in the nasal wash, lung lavage fluid, and serum (Table 3). Five doses did not result in further enhancement of either antibody responses or pulmonary clearance.

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FIG. 2.

Comparison of protective effect induced by different doses of dLOS-CRM conjugate in mouse lungs challenged with the M. catarrhalis strain 25238. Mice were i.n. immunized at 1-week intervals with one to five doses consisting of 10 μl of PBS containing a mixture of 5 μg of dLOS-CRM and 1 μg of CT or were kept as naïve mouse controls. One week after last immunization, all the mice were challenged (via aerosol) with 10 ml of M. catarrhalis strain 25238 (5 × 10⁸ CFU/ml) in a nebulizer, and their lungs were collected 6 h postchallenge. Bacterial recoveries from the lungs are expressed as geometric means + SD (error bars) from eight mice. Statistical significance for comparison with naïve mouse controls: **, P < 0.01; *, P < 0.05.

Comparison of the effects of i.n. immunization and systemic immunization.Previous work showed that systemic immunization with dLOS-protein conjugates could enhance pulmonary clearance of M. catarrhalis (17). Therefore, groups of mice were immunized with dLOS-CRM by either the s.c. or i.n. route. Ribi was used as an optimal adjuvant for the s.c. route, while CT was used for the i.n. route. As shown in Table 4, i.n. immunization achieved an 80% bacterial clearance in the lungs compared to that observed in naïve mice (P < 0.01) following an aerosol challenge with strain 25238, while s.c. application showed a 58% bacterial reduction (P < 0.05). The i.n. route showed a higher level of bacterial clearance compared to s.c. application, although there was no statistical difference between the two groups. However, when the same adjuvant CT was used for both routes, i.n. immunization showed significantly higher levels of bacterial clearance compared to s.c. injection (80 versus 40%; P < 0.05, data not shown). In terms of LOS antibody responses, different patterns of AFC responses were elicited in tissues by the two routes of application (Table 5). LOS-specific IgA AFCs were detected in all tissues from the i.n. immunization group, but not in the s.c. injection group. s.c. injections with dLOS-CRM plus Ribi adjuvant elicited higher levels of LOS-specific IgG AFCs in the spleen and CLN compared to that elicited by i.n. immunization with dLOS-CRM plus CT. Significant differences between the two groups were observed (P < 0.05 or P < 0.01) in IgA titers of the nasal wash, lung lavage, and serum (higher with i.n.); in IgG titers of the lung lavage fluid and serum; and in IgM titers of serum (higher with s.c.).

Heterologous bacterial clearance from the lungs.To evaluate the heterologous protection of i.n. immunization on pulmonary clearance of M. catarrhalis, strain O35E (type A), 26397 (type B), or 26404 (type C) was used to challenge the animals (Tables 6 to 8). The protective effect of i.n. immunization was significant against strains O35E and 26404, with bacterial reduction of the strains in the lungs at 81 and 71%, respectively, compared to the PBS group (P < 0.01), but it was not significant against type B strain 26397, with bacterial reduction in the lungs at 38% (P > 0.05). The cross-reactivity of antibodies elicited by the dLOS-CRM from strain 25238 against the LOS of strain O35E (Table 6) or 26404 (Table 7) in the lung lavage fluid and serum was also analyzed. There was no significant difference between the titers of antibody against LOS of the serotype A or C strains. However, the titer of the cross-reactive antibody against LOS of the type B strain 26397 was significantly lower (Table 8).