INTRODUCTION

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Neisseria meningitidis infections continue to be a serious health problem worldwide. However, infection with one meningococcal strain in immunocompetent individuals seems to give life-long immunity to infection with homologous and heterologous serogroups, indicating that antibodies against subcapsular antigens may generate long-lasting and cross-protective immunity against meningococcal disease (3). Information regarding the immunogenicity of various outer membrane proteins during meningococcal infections is thus of interest in the complex process of determining the protective potential of future meningococcal vaccine components.

The relative contribution of bactericidal versus opsonic antibodies in the protection against meningococcal disease is not known. Whereas the majority of studies concerning the immune response following meningococcal disease and vaccination have focused on the role of human serum bactericidal activity against meningococci, some reports indicate that phagocytic elimination of meningococci is an important host defense mechanism (7, 26, 28, 30) which is stimulated by serum opsonins (complement and antibodies). Increasing serum opsonic activity against meningococci has been demonstrated during meningococcal disease (18, 19), and disease-induced opsonins have been shown to recognize meningococcal outer membrane components PorA and PorB (23).

There is considerable interest in the vaccine potential of N. meningitidis transferrin binding proteins (TbpA and TbpB, forming the TbpA+B complexes). Tbps are surface exposed on all meningococcal outer membranes in iron-restricted environments like mammalian tissue fluids. The TbpA+B complexes are involved in the uptake of iron from human transferrin (hTf) (16), which is necessary for bacterial survival and growth. Whereas TbpA is a highly conserved protein, two major families of TbpB molecules with high and low molecular masses have been identified (27). Antibodies to meningococcal TbpA+B complexes and to the isolated proteins have been detected in both patients and carriers (2, 12, 15, 20), and these antibodies have been shown to cross-react with Tbps isolated from different meningococcal strains (15). Tbps have also been shown to elicit bactericidal and protective antibodies in laboratory animals (4, 10, 24). No study has so far investigated the opsonic activity of anti-Tbp antibodies.

The aim of this study was to evaluate whether anti-Tbp serum opsonins are produced in response to meningococcal infections. Anti-TbpA+B opsonic activities were quantified in acute and convalescent sera from 40 patients infected by a variety of meningococcal strains, using TbpA+B from two different strains expressing high- and low-molecular-weight (MW) TbpBs in an antigen-specific opsonophagocytosis and oxidative burst assay (21, 22). The anti-TbpA+B opsonic activities were compared to anti-TbpA+B immunoglobulin G (IgG) responses, as evaluated by enzyme-linked immunosorbent assays (ELISAs).

	MATERIALS AND METHODS

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Patients and disease-causing strains. Serum samples were obtained from 40 survivors (22 females and 18 males; ages, 14 to 58 years; median age, 18 years) of meningococcal disease within the first day of admission to Haukeland University Hospital, Bergen, Norway, between admission and 6 weeks (intermediate samples, available between days 3 and 24; median day, 15; n = 39 [Table 1]) and 6 weeks after admission.

Fluorochromes and buffers. Polystyrene microspheres (Fluoresbrite Plain Microspheres PC Red; Polysciences Inc., Warrington, Pa.) and the oxidative burst indicators dihydrorhodamine 123 (DHR-123) and rhodamine 123 (R-123) (Molecular Probes, Eugene, Oreg.) were used (22, 29). Dulbecco's PBS (DPBS) was used with 5 × 10³ M glucose and 5 mg of bovine serum albumin (BSA; Boehringer Mannheim GmbH, Mannheim, Germany) per ml (DPBS-GA) as well as 9 × 10⁴ M CaCl₂ · 2H₂O and 5 × 10⁴ M MgSO₄ · H₂O (21).

TbpA+B complexes. TbpA+B complexes were prepared as described previously (2) from N. meningitidis K454 (B:15:P1.7,16 with TbpB of approximately 85 kDa) (15) and B16B6 (B:2a:P1.2 with TbpB of approximately 68 kDa) (27). These strains were chosen because the TbpBs are representative of the two families of high- and low-MW TbpB described by Rokbi et al. (27). The TbpA+B concentrations were determined by densitometry after SDS-PAGE, as evaluated against various concentrations of transferrin on the gels (6). The TbpA+B solution from strain K454 was concentrated from 535 to 800 µg/ml by a vacuum-operated filter unit (Immersible-CX units with agitator and low-binding ultrafilters with 10,000-MW cutoff; Millipore) to equal the concentration of the TbpA+B preparation from strain B16B6 prior to adsorption to polystyrene beads.

TbpA+B-coating of fluorescent beads. Fluorescent polystyrene beads were coated with TbpA+B complexes from N. meningitidis K454 and B16B6. Briefly, 300 µl of fluorescent beads (4.55 × 10¹⁰ beads/ml) were washed twice in borate buffer (0.1 M boric acid [pH 8.5]) and incubated with 480 µg of TbpA+B complexes (600 µl of the 800-µg/ml solutions) with end-over-end rotation at room temperature (RT) overnight. Remaining sites on the bead surfaces were blocked with 2% (wt/vol) BSA in 0.1 M boric acid before suspension in storage buffer (21) and kept protected from daylight in aliquots at 4°C until used. The TbpA+B complexes retained the ability to bind hTf after adsorption to beads, as evaluated by binding of peroxidase-conjugated hTf (Jackson ImmunoResearch Laboratories).

Leukocytes. As previously described, human leukocytes were separated and adjusted to 1.25 × 10⁷ nonlymphocytes (polymorphonuclear leukocytes and monocytes, i.e., the potentially phagocytosing cells) per ml in DPBS-GA (21).

Phagocytosis and oxidative burst assays. TbpA+B-beads and control beads coated with BSA were opsonized with patient and control sera and incubated with DHR-123 and leukocytes as previously described for other antigen-coated beads (21-23) except that the incubation time was extended to 15 min followed by flow cytometry (FCM) analysis (Coulter Epics XL-MCL flow cytometer; Coulter Corporation, Harpenden, England).

FCM parameters. Nonlymphocytes were analyzed for associated R-123 and bead fluorescence (22). The percentage of phagocytosing nonlymphocytes was defined as the percentage of nonlymphocytes with associated bead fluorescence, and the mean number of beads per phagocytosing cell was calculated by dividing the mean bead fluorescence associated with nonlymphocytes by the fluorescence of single beads (21, 22). The phagocytosis product (PP) was defined as the percentage of phagocytosing nonlymphocytes multiplied by the mean number of beads per phagocytosing cell (23). The PP values were designated with the strain from which the TbpA+B complexes were isolated (PP_K454 and PP_B16B6, respectively). Oxidative burst activity was reflected by the mean nonlymphocyte R-123 fluorescence (23).

ELISAs. Purified TbpA+B complexes (1 µg/ml in 0.05 M sodium carbonate buffer [pH 9.6], 100 µl per well) isolated from N. meningitidis K454 and B16B6 were coated onto 96-well plates (Maxisorb Immuno-plate; Nunc, Roskilde, Denmark) and incubated overnight at RT. Blocking was performed with 200 µl of PBS containing 0.01% (vol/vol) Tween 20 and 10% (vol/vol) newborn calf serum (blocking buffer) for 1 h at RT. Serial dilutions of test sera were prepared in blocking buffer in 96-well Serowell plates (Bibby Sterilin Ltd., Stone, Staffordshire, England), transferred to the coated plates, and incubated at RT for 2 h. After washing, 100 µl of an appropriate dilution of either biotin-conjugated anti-human pan-IgG (Stratech Scientific, Luton, England) or biotin-conjugated anti-human Ig subclass-specific antibodies (Sigma, Poole, Dorset, England) was added to each well and incubated for 1 h. The plates were washed, and 100 µl of an appropriate dilution of streptavidin-HRP conjugate (Pierce, Chester, Cheshire, England) was added to each well and incubated for 1 h at RT. Finally, after washing the plates, 100 µl of TMBlue substrate solution (Intergen, Milford, Mass.) was added to each well and incubated with shaking for 10 min at RT. The reaction was stopped with 50 µl of 2 M sulfuric acid per well, and absorbances were measured at 450 nm on a Titertek ELISA reader (MCC 340; Life Sciences International, Basingstoke, Hampshire, England). All plates contained duplicate rows of pooled 6-week-postinfection sera as a standard. ELISA titers were expressed as the reciprocal of serum dilutions required to obtain the midpoint of the standard dose-response curve, and titers on each plate were adjusted to the standard serum titer.

CLSM. Confocal laser scanning microscopy (CLSM) (MRC 1000; Bio-Rad, Hemel Hempstead, England) was performed immediately after incubation of opsonized TbpA+B- and BSA-coated beads with leukocytes as described for the FCM assay (21-23).

Statistical methods. The FCM results are presented as means of duplicate measurements. Nonparametric statistics (median and range) were employed. Wilcoxon's signed rank test was used to determine differences between data. A P value of <0.05 was considered statistically significant. Correlations were evaluated by Spearman's rank correlation coefficient.

	RESULTS

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Phagocytosis. The amount of opsonins that recognized TbpA+B complexes increased in patient sera during the course of meningococcal disease, as reflected by enhanced phagocytosis of opsonized antigen-coated beads by human leukocytes (Fig. 1). Increases in patient serum opsonic activities were most frequently reflected in both the percentage of phagocytosing nonlymphocytes and the mean number of beads per cell, as indicated in the summary of FCM parameters obtained with TbpA+B-beads opsonized with sera from patients in groups I to V (Table 2). However, since both of these parameters are required to describe the total opsonophagocytosis, the product of these two parameters (PP) was used to present the anti-TbpA+B serum opsonic activities (PP_K454 and PP_B16B6, using beads with TbpA+B from strains K454 and B16B6, respectively) (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Serum opsonic activities of individual patients in groups I to V (Table 1) against meningococcal TbpA+B complexes from meningococcal strains K454 and B16B6 during meningococcal disease as reflected by PPK454 and PPB16B6, respectively. Note different scales on the PPK454 and PPB16B6 ordinates. The antigen-specific opsonic activities were measured in sera obtained on admission to hospital (admission), between admission and 6 weeks later (intermediate), and 6 weeks after admission (6 weeks).

Oxidative burst. The phagocyte oxidative burst responses induced by antigen-specific opsonins were reflected by the intracellular oxidation of the nonfluorescent substrate DHR-123 by reactive oxygen metabolites to green-fluorescent R-123 (Table 2). The mean R-123 fluorescence after stimulation with opsonized TbpA+B-coated beads corresponded to the PP values obtained with admission sera (r = 0.41 and 0.92 [P < 0.01] for anti-K454 TbpA+B and anti-B16B6 TbpA+B activities, respectively), with intermediate sera (r = 0.90 and 0.99 [P < 0.01] for anti-K454 TbpA+B and anti-B16B6 TbpA+B activities, respectively), and with 6-week sera (r = 0.78 and 0.99 [P < 0.01] for anti-K454 TbpA+B and anti-B16B6 TbpA+B activities, respectively).

ELISA. The medians and ranges of serum titers obtained with each antigen in intermediate samples are given in Table 3; the highest titers were observed with TbpA+B from strain B16B6. Greater than fivefold rises in titer during meningococcal disease were found against K454 TbpA+B in 40% of intermediate and 42.5% of 6-week sera and against B16B6 TbpA+B in 60% of intermediate sera and 45% of 6-week sera. Furthermore, 40% of sera showed a greater than fivefold rise in titer to Tbps isolated from both strains. Sera were also judged to have a positive response to each antigen when a titer greater than three times the dilution cutoff was obtained. Using this analysis, 67.5% of intermediate and 70% of 6-week sera were positive for K454 TbpA+B, while 82.5% of intermediate and 70% of 6-week sera were positive for B16B6 TbpA+B.

CLSM. CLSM images confirmed that opsonized TbpA+B-beads were phagocytosed and that R-123 fluorescence was induced (not shown).

	DISCUSSION

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We have demonstrated for the first time that human serum opsonins produced during meningococcal disease recognize epitopes on meningococcal TbpA+B complexes. The antigen-specificity needed to perform this study was ensured by adsorption of isolated TbpA+B to polystyrene beads. Also, the antigens retained the ability to bind hTf after adsorption to the beads, indicating that their conformation was similar to that of native proteins. This provided a mimicry of bacteria with selected antigens surface-exposed and available for in vitro recognition and attachment of TbpA+B-specific serum opsonins.

Increased patient anti-TbpA+B serum opsonic activities were detected during disease. The overall magnitudes of the patient opsonic responses against TbpA+B from strain B16B6 were significantly higher than those observed against complexes from strain K454 (Fig. 1). This could in part be due to a 10% lower degree of adsorption of TbpA+B complexes from strain K454 to the beads than that of the B16B6 complexes. However, the IgG titers against B16B6 TbpA+B were higher than against K454 TbpA+B in the same intermediate sera, strongly indicating that the higher amount of antibodies detected against TbpA+B from strain B16B6 during disease were responsible for the higher opsonic activities against TbpA+B from strain B16B6 than from K454.

Even though considerable variations between individuals were observed in the anti-Tbp A+B serum opsonic activities, significant correlations were found between the PP_K454 and PP_B16B6 values, indicating human opsonic cross-reactivity against TbpA+B epitopes. Also, in spite of higher PP_B16B6 in patient group IV, the PP values were found to be statistically independent of the MW of the TbpB of the disease-causing strains. Accordingly, the cross-reactive opsonins may be directed against the more conserved TbpA molecule or against epitopes present on both high- and low-MW TbpBs or epitopes formed by the TbpA+B complex. A previous study with rabbit sera and Western blot techniques demonstrated little cross-reaction between the higher- and lower-MW TbpB isotypes (27). Also, antibodies raised in rabbits to Tbps from B16B6 have been shown to be bactericidal, but only against strains with the same TbpB MW isotype (24). The immune responses to TbpA and TbpB have, however, been shown to vary considerably depending on the route of antigen administration, the immunological method used, and the species-specific immune reactivity in various murine versus human host species (2). Thus, conclusions regarding the immunogenicity of TbpA and TbpB and, accordingly, the usefulness of Tbps as meningococcal vaccine candidates depend on data on the human immune responses to Tbps. Antibodies to meningococcal TbpA+B complexes and to the isolated proteins have been detected in both patients and carriers (2, 12, 15, 20), and these antibodies have been shown to cross-react with Tbps isolated from different meningococcal strains (15). To further clarify the cross-reactiveness of the anti-Tbp patient opsonins observed in the present study, studies are planned to evaluate patient opsonic responses against separate TbpA and TbpB molecules.

The anti-TbpA+B IgG responses were primarily of subclasses IgG1 and IgG3. These are the most effective to trigger complement activation and bind to Fc receptors (1, 8, 31), which implies that the anti-TbpA+B opsonic activities primarily are due to disease-induced antigen-specific IgG1 and IgG3 antibodies. Whereas IgG1 was detected against TbpA+B from both strains, IgG3 was found almost entirely against TbpA+B from strain K454. The reason for this is not known.

Phagocytic uptake of bacteria without initiation of intracellular killing mechanisms has been suggested as a means by which meningococci invade the host (3, 13). Since both ingestion and oxidative killing are probably needed to mount an effective phagocytic clearance of invading bacteria, the effects of human anti-TbpA+B opsonins on both of these steps were evaluated in the present study. With this in vitro approach to reflect the antigen specificity of the serum opsonic activities of surviving patients, the results indicate that anti-TbpA+B antibodies generate functional serum opsonic responses.

Meningococcal Tbps are essential for the acquisition of iron from hTf and are expressed by all strains during growth in iron-restricted environments such as human tissue fluids. Accordingly, it is thought that Tbps are vital for meningococcal pathogenicity. The importance of Tbps for gonococcal pathogenicity has been demonstrated in a human challenge experiment, as a mutant lacking TbpA+B failed to cause disease (9). Murine antibodies raised against the TbpA+B complex have been shown to block the bacterial acquisition of iron from hTf (25). TbpA+B complexes have also been shown to elicit bactericidal antibodies following immunization of mice (4, 10, 24) and after vaccination of adult volunteers (11), and the present study demonstrates that human opsonic antibodies produced in response to meningococcal disease are directed against TbpA+B epitopes. The multifunctional nature of anti-Tbp antibodies suggests that the meningococcal TbpA+B complex may be a multipotent meningococcal vaccine candidate.

We conclude that human serum opsonins produced during meningococcal disease recognize meningococcal TbpA+B complexes and that both phagocytosis and intracellular oxidative burst activities are initiated by anti-TbpA+B opsonins. TbpA+B from strain B16B6 was the most immunogenic in both ELISA and functional phagocytic assays, but extensive opsonic cross-reactivity was demonstrated against TbpA+B from the two strains. Functional studies with separate TbpA and TbpB molecules as target antigens are planned to clarify the relative importance of the TbpA and TbpB components of the complex as mediators of serum opsonic responses during meningococcal disease.