INTRODUCTION

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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 strongly indicate that phagocytic killing of meningococci is an important host defense mechanism, particularly against serogroup B meningococci (6, 32, 36, 38). Effective uptake and ingestion of meningococci are dependent on the deposition of opsonins (complement and antibodies) on epitopes exposed on the bacterial surfaces (8, 35, 43). Increasing human serum opsonic activity has been demonstrated during the course of meningococcal disease and after vaccination with a complex serogroup B outer membrane vesicle (OMV) preparation, using whole meningococci as target antigens in functional assays (1, 13, 14, 18, 24, 27, 39). Furthermore, patient serum opsonic activity has been shown to correlate positively with levels of immunoglobulin G (IgG) antibody to meningococcal PorA and PorB proteins (17). However, functional assays for direct identification of bacterial antigens that are recognized by serum opsonins have not been available.

The aim of the present study was to evaluate whether functional opsonins produced in response to meningococcal disease are directed against meningococcal outer membrane components and, if so, to determine whether patient opsonins recognize PorA and PorB proteins. We employed a recently developed functional assay that quantifies antigen-specific serum opsonic activity, as reflected by induction of phagocytosis and oxidative burst mechanisms in human leukocytes (25, 26). Fluorescent polystyrene beads coated with OMVs and purified porins from mutants of the same meningococcal strain (44/76; B:15:P1.7,16) were opsonized with acute- and convalescent-phase sera from 40 surviving patients infected by a variety of meningococcal strains. The leukocyte phagocytosis and oxidative burst induced by opsonized antigen-coated beads were quantified by flow cytometry (FCM), visualized by confocal laser scanning microscopy (CLSM), and correlated to serum anti-OMV and antiporin Ig levels as measured by enzyme-linked immunosorbent assays (ELISAs).

	MATERIALS AND METHODS

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Patients. Serum samples were obtained from 40 survivors (22 females and 18 males; age, 14 to 58 years; median age, 18) of meningococcal disease on admission to Haukeland Hospital, University of Bergen, Bergen, Norway, between admission and 6 weeks later (intermediate samples, available between days 3 and 24; median day, 13; n = 38) (Table 1) and at 6 weeks after admission. The patients were allocated into five groups (groups I to V) according to the serogroups, serotypes, and serosubtypes of meningococcal strains isolated from cerebrospinal fluid or blood (Table 1). Four clinical disease categories were employed (Table 1) (18). Six patients had been injected twice with an OMV vaccine produced from strain 44/76 in a large-scale vaccine trial (5, 10), and patient 4 was immunized with the Meningococcal Polysaccharide A+C Vaccine (Pasteur-Merieux Serums and Vaccins, Lyon, France), 1 to 4 years prior to disease (Table 1).

Fluorochromes and buffers. Polystyrene microspheres with incorporated red fluorescent dye (Fluoresbrite Plain Microspheres PCRed; Polysciences Inc., Warrington, Pa.) and with a size similar to that of meningococci (1 µm in diameter) were used for the opsonophagocytosis studies. The oxidative burst substrate dihydrorhodamine 123 (DHR 123) (Molecular Probes, Eugene, Oreg.) was used to measure leukocyte oxidative burst activity. DHR 123 is converted intracellularly to green fluorescent rhodamine 123 (R-123) by reactive oxygen intermediates (37). Dulbecco's phosphate-buffered saline (DPBS) (pH 7.4) (25) was supplemented with 5 × 10³ M glucose and 5 mg of bovine serum albumin (BSA) (Boehringer GmbH, Mannheim, Germany) per ml (DPBS-GA), as well as with 9 × 10⁴ M CaCl₂ · 2H₂O and 5 × 10⁴ M MgSO₄ · H₂O (DPBS-GACM).

Antigens. OMVs from Neisseria meningitidis 44/76 (B:15:P1.7,16) were prepared as for vaccine production (10). Meningococcal PorA (class 1) and PorB (class 3) outer membrane proteins were purified by detergent extraction and column chromatography from mutant variants of strain 44/76 (44/7634 and 44/7614, lacking PorB and PorA, respectively, as well as RmpM [class 4 protein]) (15, 16). The mutant strains were employed to minimize contamination with nonporin proteins, and gel electrophoresis demonstrated negligible lipopolysaccharide contamination (data not shown). PorA and PorB proteosomes were prepared as described previously (47).

Antigen adsorption to beads. Meningococcal OMV and PorA and PorB proteosomes were adsorbed to fluorescent polystyrene beads as described previously (25). In brief, after two washes in borate buffer (0.1 M boric acid, pH 8.5), 500 µl of beads (4.55 × 10¹⁰ beads/ml) were incubated with an excess of each antigen (600 µg) with end-over-end rotation at room temperature (20°C) overnight (20 h). The degree of antigen adsorption to the bead surfaces was determined by using the bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill.) (25). Remaining active sites on the bead surfaces were blocked with 2% BSA in 0.1 M boric acid (pH 8.5) to avoid adsorption of nonspecific serum proteins during the subsequent incubation with human serum. The antigen-coated beads were suspended in storage buffer (25) and stored at 4°C until used.

Leukocytes. Leukocytes were separated from freshly drawn, heparinized venous blood from one healthy nonsmoker by an erythrocyte-lysing method, as described previously (25). The leukocyte suspension was adjusted to 1.25 × 10⁷ nonlymphocytes (monocytes and polymorphonuclear leukocytes, i.e., the potentially phagocytosing cells) per ml in DPBS-GA.

FCM analysis of phagocytosis and oxidative burst assay. Polystyrene beads (20 µl; 2.5 × 10⁸ beads/ml) coated with meningococcal OMVs or PorA or PorB proteosomes and control beads coated with BSA were opsonized with patient serum (5 µl; 5% of final incubation volume) in 96-well microtiter plates (96 WELL Polypropylene Cluster, U-bottom with polystyrene lid; Costar Corporation, Cambridge, Mass.) for 7.5 min with shaking at 37°C in the presence of 20 µl of DHR/123 (10 µg/ml) and 35 µl of DPBS-GACM per well. Donor leukocytes (20 µl; 1.25 × 10⁷ nonlymphocytes/ml, which provides a bead/nonlymphocyte ratio of 20:1) were added to each well, and the incubation was continued for 7.5 min. Phagocytosis was terminated by addition of 200 µl of ice-cold PBS with 0.02% EDTA to each well. The samples were kept briefly on ice and diluted 1/5 prior to a 30-s FCM analysis (Epics XL-MCL; Coulter Corporation, Harpenden, England) (25).

FCM parameters. Lymphocytes and nonlymphocytes were discriminated and quantified by combined measurements of forward-angle light scatter and side-angle light scatter, and nonlymphocytes were gated to separate forward-angle light scatter versus log fluorescence cytograms and analyzed for associated R-123 (FL1) and bead (FL2) fluorescence. The percentage of phagocytosing nonlymphocytes was defined as the percentage of nonlymphocytes with associated bead fluorescence. The mean number of beads per phagocytosing cell was calculated by dividing the mean bead fluorescence associated with gated nonlymphocytes by the fluorescence of single beads (25, 26). The phagocytosis product (PP) was defined as the percentage of phagocytosing nonlymphocytes multiplied by the mean number of beads per phagocytosing cell, and the PP values were denoted with the antigen employed (i.e., PP_OMV when OMVs were adsorbed to beads). Oxidative burst activity was reflected by the mean nonlymphocyte R-123 fluorescence.

ELISA. Serum IgG antibodies to OMVs were quantified as described previously (29, 30). In brief, twofold dilutions of sera were applied to OMV-coated microtiter plates (4 µg of protein/ml in 0.1 M Tris-HCl [pH 8.6] with 100 µl/well at 4°C for at least 24 h) and incubated for 2 h at 37°C. Immunosorbance-purified biotinylated sheep anti-human IgG antibody mixed with streptavidin and alkaline phosphatase-biotin conjugate at optimal dilutions were added, and 2-p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.) was used as substrate. After a 30-min incubation at room temperature, the absorbances were read at 405 nm (Emax microplate reader; Molecular Devices, Sunnyvale, Calif.). The levels of anti-OMV IgG antibodies were determined from the standard curve of a standard serum by using SOFTmax four-parameter analysis (Molecular Devices).

CLSM. Immediately after incubation of opsonized antigen-coated beads and leukocytes as described for FCM, CLSM (MRC 1000; Bio-Rad, Hemel Hempstead, United Kingdom) was performed to visualize phagocytosis and R-123 formation (25, 26).

Statistical methods. The FCM results are presented as means of triplicate measurements. 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. SPSS 7.5.1 for Windows and SigmaStat software were used.

	RESULTS

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Phagocytosis. The amount of patient opsonins that recognized the complex OMV antigen and the purified PorA and PorB proteins increased during meningococcal disease, as reflected by enhanced phagocytosis of opsonized antigen-coated beads by human leukocytes (Fig. 1). Serum opsonic responses were demonstrated by increases in both the percentage of phagocytosing nonlymphocytes and the mean number of beads per cell, as shown in the summary of FCM parameters obtained with the porin-coated 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 (the PP) was used to reflect the opsonic activities.

View larger version (43K): [in this window] [in a new window]   FIG. 1.   Serum opsonic activities of individual patients in groups I to V (Table 1) against meningococcal PorA and PorB and OMVs from 44/76 (B:15:P1.7,16) strains during meningococcal disease, as reflected by leukocyte PPs (the percentage of phagocytosing nonlymphocytes multiplied by the number of opsonized, antigen-coated beads per cell). The antigen-specific opsonic activities were measured in sera obtained on admission to hospital (admission), between admission to hospital and 6 weeks later (intermediate), and 6 weeks after admission (6 weeks).

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Patient serum opsonic activities against meningococcal OMVs, PorA, and PorB, using antigen-coated beads as targets for functional patient opsonins before exposure to human leukocytes and FCM to reflect the resulting leukocyte PPs (the percentage of phagocytosing nonlymphocytes multiplied by the number of beads per cell). Spearman rank correlation coefficients are given for the plots of PPPorA versus PPOMV (upper panel) and PPPorB versus PPOMV (lower panel) induced by patient sera obtained between admission to hospital with meningococcal disease and 6 weeks later (intermediate sera; n = 38). Simple regression lines with 99% confidence intervals are shown.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Box plots of leukocyte PPPorA values induced by sera from meningococcal disease patients infected with a meningococcal serosubtype homologous or heterologous to that of the strain from which the PorA target antigen was extracted (P1.7,16). The upper and lower levels of the boxes represent the 75th and 25th percentiles, and the error bars represent the 90th and 10th percentiles. The open circles denote outliers. The solid lines represent the median values, and the dotted lines represent the means.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Box plots of leukocyte PPPorB values induced by sera from meningococcal disease patients infected with a meningococcal serotype homologous or heterologous to that of the strain from which the PorB target antigen was extracted (serotype 15). The upper and lower levels of the boxes represent the 75th and 25th percentiles, and the error bars represent the 90th and 10th percentiles. The open circles denote outliers. The solid lines represent the median values, and the dotted lines represent the means.

Oxidative burst. The oxidative burst responses were reflected by the formation of R-123 (Table 2). The mean nonlymphocyte R-123 fluorescence after stimulation with opsonized OMV- and porin-coated beads corresponded to the PP values with admission sera (r = 0.88 [P < 0.01], r = 0.34 [P < 0.05], and r = 0.84 [P < 0.01] for anti-OMV, anti-PorA, and anti-PorB activities, respectively), intermediate sera (r = 0.83, 0.91, and 0.95 [P < 0.01] for anti-OMV, anti-PorA, and anti-PorB activities, respectively), and 6-week sera (r = 0.97, 0.80, and 0.99 [P < 0.01] for anti-OMV, anti-PorA, and anti-PorB activities, respectively).

ELISA. IgG antibodies recognizing OMV epitopes were detected in sera from all but one patient (no. 33) during meningococcal disease. The anti-OMV IgG levels in 38 available intermediate sera (median, 39 µg/ml; range, <2.5 to 681 µg/ml) were significantly higher than those in admission sera (median, 4 µg/ml; range <2.5 to 59 µg/ml) and 6-week sera (median, 22 µg/ml; range, <2.5 to 384 µg/ml) (P < 0.001 for both). The patient anti-OMV IgG levels corresponded to the anti-OMV opsonic activity, as reflected by PP_omv values (r = 0.68, 0.50, and 0.72 [P < 0.01] for all admission, intermediate, and 6-week sera, respectively) (results for intermediate sera for PP_omv versus anti-OMV IgG are shown in Fig. 5).

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Correlation plots between levels of IgG against meningococcal PorA, PorB, and OMVs and PP values induced by antigen-specific opsonins in patient sera obtained between admission to hospital with meningococcal disease and 6 weeks later (intermediate sera).

CLSM. Phagocyte internalization of opsonized OMV- and porin-coated beads and the intracellular R-123 oxidative responses were visualized by CLSM, as shown with PorA-coated beads opsonized with intermediate serum from patient no. 35 (Fig. 6). The cell nucleus appeared black against the green R-123 in the cytoplasm, in which the opsonized antigen-coated beads were located. Control beads coated with BSA and unopsonized antigen-coated beads were not phagocytosed and did not initiate R-123 formation (not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 6.   CLSM front- and side-view images of phagocytosed PorA-coated beads and green R-123 oxidative burst fluorescence.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have demonstrated that human serum opsonins produced in response to meningococcal disease are directed against meningococcal outer membrane components and, for the first time, have specifically identified opsonins that recognize meningococcal PorA and PorB displayed in a purified form on the surface of beads. Significant increases in both phagocytosis and oxidative burst activity were observed with PorA- and PorB-coated beads opsonized with patient sera obtained during meningococcal disease (Fig. 1; Table 2). The results indicate that opsonins directed against these structures are induced by natural infection.

Previously, increasing amounts of human serum opsonins during meningococcal disease and after OMV vaccination have been demonstrated by using ethanol-fixed bacteria in functional opsonophagocytosis assays (1, 13, 14, 18, 24, 39), and such serum opsonic activity in patients has been shown to correlate with levels of IgG antibody to purified meningococcal PorA and PorB (17). To directly characterize the antigen specificity of the opsonic activities, we recently replaced the bacteria with antigen-coated beads (25, 26). Phagocytosis and oxidative burst activity induced by opsonized OMV-coated beads correlated with results obtained with opsonized ethanol-fixed meningococci (25). The epitopes exposed on OMV, PorA, and PorB may be different from those exposed on live bacteria (1a, 1b). In the present study, the vesicle and proteosome formulations were employed to present the antigens in as close to the native conformation as presently possible. The antigen specificity of the antiporin opsonic activity was further ensured by the extraction of porins from mutant strains lacking potentially contaminating proteins. Accordingly, the assay should reflect serum opsonic activities to defined bacterial structures.

The patient anti-PorA and anti-PorB opsonic activities correlated with the anti-OMV opsonic activity (Fig. 2), which suggests that patient opsonins recognize porin epitopes on the vesicles. However, higher anti-PorB opsonic activities than anti-PorA opsonic activities were observed (Fig. 1 to 4), and these were further found to correlate with higher IgG levels against PorB than against PorA in intermediate sera (Fig. 5). Higher IgG antibody levels against class 3 (PorB) than against class 1 (PorA) during meningococcal infections have previously been reported (17). The results suggest that PorB epitopes are more immunogenic than PorA during meningococcal disease. A strong immune response to PorB was also observed after three doses of an OMV vaccine, in contrast to two doses (34, 45). Thus, viable bacteria seem to induce a more rapid immune response to PorB than the OMV vaccine.

The PorA and PorB proteins were recognized by serum opsonins produced in patients infected by meningococci with serogroups, serotypes, and serosubtypes both homologous and heterologous to the strain from which the target antigens were extracted (Fig. 1; Table 1). With convalescent-phase sera, the median PP_PorB values were markedly higher after infections with homologous serotypes (Fig. 4), whereas the median PP_PorA values were higher after infections with heterologous serosubtypes (Fig. 3). However, no statistically significant differences were found when all anti-PorA and anti-PorB opsonic activities induced by meningococci of homologous versus heterologous serosubtypes and serotypes were compared. This may be explained by considerable interindividual differences in the PP_PorA and PP_PorB values (Fig. 1, 3, and 4). However, the presence of cross-reactive patient opsonic activities concurs with the serotype- and serosubtype-independent IgG responses observed after meningococcal disease (15, 16, 20, 28). Thus, the production of antiporin opsonic antibodies during meningococcal disease may at least in part be initiated by non-serotype and non-serosubtype-specific epitopes shared by various meningococcal outer membrane components.

In accordance with previous findings, the disease-induced antiporin IgG antibodies were primarily of subclasses IgG1 and IgG3 (15, 16). These are known to trigger complement activation and Fc receptor binding (2, 7, 40), which implies that the observed antiporin opsonic responses were due primarily to antigen-specific IgG1 and IgG3 antibodies.

Modest amounts of serum IgA antibodies recognizing PorA and PorB proteins were detected. Previous studies have proposed that a high fraction of IgA recognizing meningococcal components increases the disease susceptibility and that circulatory IgA can block the serum bactericidal activity and possibly also the opsonic activity of antimeningococcal antibodies (12, 22, 23). However, Haneberg et al. recently found that vaccinee IgA antibodies did not reduce the serum bactericidal activities (19). In the present study, only four admission sera had detectable antiporin (PorB) IgA antibodies, and no association was found between the IgA levels during disease and the magnitude of the phagocytic responses. Accordingly, the antiporin IgA antibodies did not seem to affect the opsonophagocytosis.

Phagocyte internalization of opsonized OMV-coated beads and initiation of intracellular oxidative burst mechanisms have previously been demonstrated by CLSM (25, 26). CLSM generates serial sections through individual phagocytes and projects fluorescent images from separate sections into one, which visualizes the total number of attached and internalized beads and the locations of beads and R-123 within individual phagocytes. The CLSM image supports the FCM results by confirming that both phagocytosis and oxidative burst are initiated by patient opsonins against bead-associated meningococcal porins (Fig. 6).

The higher anti-OMV opsonic activity observed in admission sera from previously OMV-vaccinated patients may be due to prevailing serum opsonins prior to infection but is most probably due to a rapid secondary immune response. Previous studies have shown that even though both IgG antibodies to OMV and species-specific bactericidal and opsonic antibodies were induced after similar two-dose OMV vaccinations, the anti-OMV IgG levels were significantly decreased after one year (13, 21, 24, 34, 46). Low levels of anti-OMV opsonic antibodies prior to infection may accordingly explain the occurrence of vaccine failures among the included patients. The finding of a suboptimal protective efficacy of 57% during a 29-month observation period (5) has intensified investigations concerning the OMV immunization scheme and routes and the search for the protective antigens for inclusion in improved vaccines (3, 4, 11, 19, 29, 31, 34, 41, 42, 45, 48).

The mechanisms responsible for the development of protective immunity to meningococcal disease remain unclear (3, 4, 31). Ross et al. demonstrated that serogroup B meningococci are more resistant to complement-mediated bactericidal activity of normal serum than serogroup A and C meningococci but are susceptible to phagocytic killing by polymorphonuclear leukocytes after opsonization (36). Furthermore, since genetic variations in the phagocyte Fc receptors have been shown to influence the susceptibility to meningococcal disease and since low opsonic activity against meningococci in admission sera is associated with serious and fatal meningococcal disease (6, 18), opsonophagocytosis seems to be of importance in the defense against meningococcal disease. Accordingly, knowledge about the opsonic activity of antibodies to meningococcal structures in natural infections is of value in the complex process of selecting future meningococcal vaccine constituents.

Immunocompetent survivors of meningococcal disease appear to acquire immunity to infection with heterologous serogroups, an indication that subcapsular antigens can generate cross-protective immune responses (3, 9). Our functional in vitro model with purified meningococcal antigens adsorbed to beads seems to facilitate dissection of subcapsular components that are recognized by disease-induced serum opsonins and accordingly may contribute to the identification of opsonin-inducing antigens for inclusion in future vaccines. Serum opsonins produced in response to a variety of disease-causing strains recognized OMV components and in particular PorB epitopes and initiated phagocytosis and oxidative burst activity, which suggests that these antigens are attractive meningococcal vaccine candidates.