INFECTION AND IMMUNITY, May 1999, p. 2552–2560 Vol. 67, No.

5
0019-9567/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Human Opsonins Induced during Meningococcal Disease Recognize

Outer Membrane Proteins PorA and PorB
A. K. LEHMANN,1* A. HALSTENSEN,1 I. S. AABERGE,2 J. HOLST,2 T. E. MICHAELSEN,2,3
S. SØRNES,1 L. M. WETZLER,4 AND H.-K. GUTTORMSEN5
Medical Department B, University of Bergen, Bergen,1 and Department of Vaccinology, National Institute of Public Health,2
and Institute of Pharmacy, Department of Pharmacognosy, University of Oslo,3 Oslo, Norway, and Maxwell Finland
Laboratory for Infectious Diseases, Boston Medical Center, Boston University School of Medicine,4 and
Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital,
Harvard Medical School,5 Boston, Massachusetts
Received 13 October 1998/Returned for modification 24 November 1998/Accepted 20 January 1999

Human opsonins directed against specific meningococcal outer membrane structures in sera obtained
during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent
phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class
3) proteins purified from mutants of the same strain (44/76; B:15:P1.7.16) were adsorbed to fluorescent beads,
opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed
to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs)
demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both
porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV
values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians
[ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with
higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immu-
nosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities
between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens
were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an
indication that both of these important steps in the in vitro phagocytic elimination of meningococci are
initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human
patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by
this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in
future vaccines.

Whereas the majority of studies concerning the immune ease are directed against meningococcal outer membrane com-
response following meningococcal disease and vaccination ponents and, if so, to determine whether patient opsonins
have focused on the role of human serum bactericidal activity recognize PorA and PorB proteins. We employed a recently

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against meningococci, some reports strongly indicate that
phagocytic killing of meningococci is an important host de-
fense mechanism, particularly against serogroup B meningo-
cocci (6, 32, 36, 38). Effective uptake and ingestion of menin-
gococci are dependent on the deposition of opsonins
(complement and antibodies) on epitopes exposed on the bac-
terial surfaces (8, 35, 43). Increasing human serum opsonic
activity has been demonstrated during the course of meningo-
coccal 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 immunoglob-
ulin G (IgG) antibody to meningococcal PorA and PorB pro-
teins (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 func-
tional opsonins produced in response to meningococcal dis-
* Corresponding author. Mailing address: Medical Department B,
University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway.
Phone: 47 55 97 50 00. Fax: 47 55 97 29 50. E-mail: Anne.Lehmann
@medb.uib.no.
developed functional assay that quantifies antigen-specific se-
rum 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 oxida-
tive 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
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 admis-
sion 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 5 38) (Table 1) and at 6 weeks after admission. The
patients were allocated into five groups (groups I to V) according to the sero-
groups, serotypes, and serosubtypes of meningococcal strains isolated from ce-
rebrospinal fluid or blood (Table 1). Four clinical disease categories were em-
ployed (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 A1C Vaccine (Pasteur-

2552
VOL. 67, 1999 HUMAN OPSONINS RECOGNIZE MENINGOCOCCAL PorA AND PorB 2553

TABLE 1. Clinical characteristics of patients with diameter) were used for the opsonophagocytosis studies. The oxidative burst
meningococcal disease (n 5 40) substrate dihydrorhodamine 123 (DHR 123) (Molecular Probes, Eugene, Oreg.)
was used to measure leukocyte oxidative burst activity. DHR 123 is converted
Time of intracellularly to green fluorescent rhodamine 123 (R-123) by reactive oxygen
Intermediate intermediates (37). Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4) (25)
Group and Meningococcal Disease preadmission
sampling was supplemented with 5 3 1023 M glucose and 5 mg of bovine serum albumin
patient no.a strain categoryb symptoms
day
(h) (BSA) (Boehringer GmbH, Mannheim, Germany) per ml (DPBS-GA), as well as
with 9 3 1024 M CaCl2 z 2H2O and 5 3 1024 M MgSO4 z H2O (DPBS-GACM).
I (n 5 8) Antigens. OMVs from Neisseria meningitidis 44/76 (B:15:P1.7,16) were pre-
1 B:15:P1.7,16 2 12 15 pared as for vaccine production (10). Meningococcal PorA (class 1) and PorB
2 B:15:P1.7,16 1 12 10 (class 3) outer membrane proteins were purified by detergent extraction and
3 B:15:P1.7,16 1 18 16 column chromatography from mutant variants of strain 44/76 (44/76D3D4 and
44/76D1D4, lacking PorB and PorA, respectively, as well as RmpM [class 4
4 (V2A1C [1]) B:15:P1.7,16 3 20 12
protein]) (15, 16). The mutant strains were employed to minimize contamination
5 B:15:P1.7,16 4 11 16 with nonporin proteins, and gel electrophoresis demonstrated negligible lipo-
6 B:15:P1.7,16 1 25 10 polysaccharide contamination (data not shown). PorA and PorB proteosomes
7 B:15:P1.7,16 4 24 9 were prepared as described previously (47).
8 B:15:P1.7,16 2 10 17 Antigen adsorption to beads. Meningococcal OMV and PorA and PorB pro-
teosomes were adsorbed to fluorescent polystyrene beads as described previously
II (n 5 7) (25). In brief, after two washes in borate buffer (0.1 M boric acid, pH 8.5), 500
9 C:15:P1.7,16 4 20 ml of beads (4.55 3 1010 beads/ml) were incubated with an excess of each antigen
10 C:15:P1.7,16 3 20 24 (600 mg) 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
11 C:15:P1.7,16 4 48 17 using the bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill.) (25).
12 C:15:P1.7,16 1 16 8 Remaining active sites on the bead surfaces were blocked with 2% BSA in 0.1 M
13 C:15:P1.7,16 1 24 5 boric acid (pH 8.5) to avoid adsorption of nonspecific serum proteins during the
14 C:15:P1.7,16 2 16 7 subsequent incubation with human serum. The antigen-coated beads were sus-
15 C:15:P1.7,16 3 30 4 pended in storage buffer (25) and stored at 4°C until used.
Leukocytes. Leukocytes were separated from freshly drawn, heparinized ve-
III (n 5 10) nous blood from one healthy nonsmoker by an erythrocyte-lysing method, as
16 B:15:P1.2 3 23 13 described previously (25). The leukocyte suspension was adjusted to 1.25 3 107
nonlymphocytes (monocytes and polymorphonuclear leukocytes, i.e., the poten-
17 B:15:P1.2,5 4 36 5
tially phagocytosing cells) per ml in DPBS-GA.
18 B:15:P1.12,13 2 12 24 FCM analysis of phagocytosis and oxidative burst assay. Polystyrene beads
19 B:15:P1.12,13 2 11 15 (20 ml; 2.5 3 108 beads/ml) coated with meningococcal OMVs or PorA or PorB
20 B:15:P1.12 4 24 18 proteosomes and control beads coated with BSA were opsonized with patient
21 B:15:P1.12 4 18 5 serum (5 ml; 5% of final incubation volume) in 96-well microtiter plates (96
22 (V2B [3]) B:15:P1.12 1 14 13 WELL Polypropylene Cluster, U-bottom with polystyrene lid; Costar Corpora-
23 (V2B [3]) B:15:P1.12 3 18 8 tion, Cambridge, Mass.) for 7.5 min with shaking at 37°C in the presence of 20
24 (V2B [3]) B:15:P1.12 1 14 10 ml of DHR/123 (10 mg/ml) and 35 ml of DPBS-GACM per well. Donor leuko-
cytes (20 ml; 1.25 3 107 nonlymphocytes/ml, which provides a bead/nonlympho-
25 (V2B [1]) B:15:P1.12 1 26 3
cyte 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 ml of ice-cold PBS with
IV (n 5 6) 0.02% EDTA to each well. The samples were kept briefly on ice and diluted 1/5
26 C:2a:P1.2 4 20 6 prior to a 30-s FCM analysis (Epics XL-MCL; Coulter Corporation, Harpenden,
27 C:2a:P1.2 2 11 13 England) (25).
28 C:2a:P1.2 4 11 14 An argon laser operating at 488 nm produced excitation in the fluorochromes.
29 C:2a:P1.2 2 28 4 The green R-123 fluorescence was collected between 505 and 545 nm (FL1
30 (V2B [4]) C:2a:P1.2 2 20 15 channel), and the red bead fluorescence was collected between 560 and 590 nm
31 (V2B [4]) C:2a:P1.2 2 10 17 (FL2 channel). Electronic color compensations eliminated spectral overlaps be-
tween the fluorochromes. The FCM coincidence rate was repeatably 1 to 2% (25,

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26). The FCM was calibrated daily with fluorescent beads (DNA-Check, EPICS
V (n 5 9) Alignment Fluorospheres; Coulter Corporation, Hialeah, Fla.).
32 B:NT:P1.12 4 18 13 FCM parameters. Lymphocytes and nonlymphocytes were discriminated and
33 B:NT:P1.16 1 24 13 quantified by combined measurements of forward-angle light scatter and side-
34 B:NT:P1.3 4 20 8 angle light scatter, and nonlymphocytes were gated to separate forward-angle
35 B:NT:NT 3 30 14 light scatter versus log fluorescence cytograms and analyzed for associated R-123
36 B:4:NT 4 10 16 (FL1) and bead (FL2) fluorescence. The percentage of phagocytosing nonlym-
37 B:4:P1.12 2 25 9 phocytes was defined as the percentage of nonlymphocytes with associated bead
fluorescence. The mean number of beads per phagocytosing cell was calculated
38 B:8:P1.15 1 18
by dividing the mean bead fluorescence associated with gated nonlymphocytes by
39 B:19:P1.15 4 16 8 the fluorescence of single beads (25, 26). The phagocytosis product (PP) was
40 Microscopyc 3 11 14 defined as the percentage of phagocytosing nonlymphocytes multiplied by the
a mean number of beads per phagocytosing cell, and the PP values were denoted
V2A1C, immunized with meningococcal polysaccharide A1C vaccine; with the antigen employed (i.e., PPOMV when OMVs were adsorbed to beads).
V2B, immunized with meningococcal serogroup B OMV vaccine. Numbers in Oxidative burst activity was reflected by the mean nonlymphocyte R-123 fluo-
brackets indicate years since vaccination. rescence.
b
1, meningitis; 2, septicemia with shock; 3, meningitis and septicemia with ELISA. Serum IgG antibodies to OMVs were quantified as described previ-
shock; 4, septicemia without shock and with or without meningitis. ously (29, 30). In brief, twofold dilutions of sera were applied to OMV-coated
c
Gram-negative diplococci in cerebrospinal fluid. microtiter plates (4 mg of protein/ml in 0.1 M Tris-HCl [pH 8.6] with 100 ml/well
at 4°C for at least 24 h) and incubated for 2 h at 37°C. Immunosorbance-purified
Merieux Serums and Vaccins, Lyon, France), 1 to 4 years prior to disease (Table biotinylated sheep anti-human IgG antibody mixed with streptavidin and alkaline
1). phosphatase-biotin conjugate at optimal dilutions were added, and 2-p-nitrophe-
Sera from five persons without a history of meningococcal disease and reaction nyl phosphate (Sigma Chemical Co., St. Louis, Mo.) was used as substrate. After
mixtures without serum were included as controls. Control admission and con- a 30-min incubation at room temperature, the absorbances were read at 405 nm
valescent-phase sera were obtained from a patient with pneumococcal meningitis (Emax microplate reader; Molecular Devices, Sunnyvale, Calif.). The levels of
and a patient with varicella-zoster meningoencephalitis. All sera had normal anti-OMV IgG antibodies were determined from the standard curve of a stan-
complement activity (as determined by 50% hemolytic complement units) and dard serum by using SOFTmax four-parameter analysis (Molecular Devices).
were stored at 270°C until used. The amounts of serum IgA, IgM, and IgG main and subclass antibody-recog-
Fluorochromes and buffers. Polystyrene microspheres with incorporated red nizing epitopes on purified PorA and PorB were determined as described pre-
fluorescent dye (Fluoresbrite Plain Microspheres PCRed; Polysciences Inc., viously (15, 16, 47), with secondary antibodies and/or conjugates from Sigma
Warrington, Pa.) and with a size similar to that of meningococci (1 mm in Chemical Co. IgG was quantified in micrograms per milliliter against a separate
2554 LEHMANN ET AL. INFECT. IMMUN.

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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).

ELISA with wells coated with Fab-specific anti-human IgG and dilutions of an
IgG standard. IgG subclass titrations were performed with mouse anti-human
IgG1, IgG2, IgG3, and IgG4 as secondary antibodies and alkaline phosphatase-
conjugated goat anti-mouse IgG as the conjugate. Twofold serial dilutions of sera
started at 1:50 for IgG subclass analyses and at 1:100 for IgA and IgM analyses,
and the levels were calculated as the reciprocal serum dilutions that gave an A405
of 1.0 after 1 h of incubation.
CLSM. Immediately after incubation of opsonized antigen-coated beads and
leukocytes as described for FCM, CLSM (MRC 1000; Bio-Rad, Hemel Hemp-
stead, 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. Cor-
relations were evaluated by Spearman’s rank correlation coefficient. SPSS 7.5.1
for Windows and SigmaStat software were used.
RESULTS
Phagocytosis. The amount of patient opsonins that recog-
nized the complex OMV antigen and the purified PorA and
PorB proteins increased during meningococcal disease, as re-
flected 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
VOL. 67, 1999 HUMAN OPSONINS RECOGNIZE MENINGOCOCCAL PorA AND PorB 2555

TABLE 2. Phagocytosis and oxidative burst activities mediated by anti-PorA and anti-PorB serum opsonins in meningococcal disease patients
Median (range) for beads coated with:
Patient
Parameter PorA proteosomes PorB proteosomes
group
Admissiona Intermediateb 6 weeksc Admission Intermediate 6 weeks

% Phagocytosing I 8 (3–11) 23 (4–81) 10 (5–32) 12 (4–40) 95 (16–96) 88 (44–95)

nonlymphocytes II 11 (4–30) 44 (7–92) 15 (8–91) 22 (5–51) 91 (12–96) 94 (23–96)
III 10 (6–87) 58 (13–91) 15 (6–88) 19 (9–96) 95 (44–97) 88 (27–97)
IV 7 (5–13) 70 (15–94) 46 (9–93) 18 (7–81) 92 (77–95) 91 (27–94)
V 10 (7–34) 73 (11–95) 24 (10–92) 15 (7–89) 56 (14–97) 44 (10–97)

Beads/phagocytosing I 1.3 (1.2–1.4) 1.4 (1.3–2.7) 1.3 (1.2–1.5) 1.4 (1.3–1.5) 8.0 (1.3–10.8) 4.1 (1.7–9.8)
nonlymphocyte (mean no.) II 1.3 (1.3–1.4) 1.7 (1.4–4.5) 1.3 (1.2–3.9) 1.5 (1.4–2.0) 7.0 (1.4–2.0) 6.1 (1.5–9.9)
III 1.4 (1.3–3.6) 2.1 (1.3–4.0) 1.3 (1.3–3.9) 1.5 (1.3–8.6) 8.6 (1.6–10.5) 4.9 (1.4–9.9)
IV 1.3 (1.3–1.5) 2.2 (1.7–4.9) 1.6 (1.3–2.9) 1.5 (1.4–3.2) 5.5 (3.0–9.3) 5.2 (1.5–8.0)
V 1.3 (1.2–1.4) 2.7 (1.2–5.1) 1.4 (1.2–4.0) 1.4 (1.3–3.7) 4.5 (1.6–10.9) 1.5 (1.3–9.6)

PPd I 10 (4–14) 33 (5–219) 13 (6–45) 17 (5–60) 759 (21–1,026) 356 (75–931)

II 15 (5–42) 88 (11–414) 20 (10–355) 31 (7–102) 642 (17–1,008) 567 (35–950)
III 12 (8–313) 144 (17–360) 20 (8–343) 26 (12–826) 817 (70–1,019) 440 (38–950)
IV 9 (7–12) 152 (20–461) 76 (12–270) 26 (10–259) 503 (231–874) 473 (41–752)
V 12 (10–48) 199 (15–485) 34 (13–368) 21 (9–329) 169 (20–1,057) 79 (13–931)

Oxidative burste I 0.2 (0.2–0.2) 0.3 (0.2–3.9) 0.2 (0.2–0.4) 0.2 (0.2–0.3) 13.8 (0.2–18.6) 4.9 (0.4–17.1)
II 0.2 (0.2–2.2) 0.2 (0.2–9.2) 0.2 (0.2–7.7) 0.3 (0.2–2.8) 7.7 (0.2–18.3) 10.3 (0.2–18.3)
III 0.2 (0.2–6.6) 2.2 (0.2–7.2) 0.2 (0.2–6.3) 0.3 (0.2–11.0) 13.4 (0.4–18.8) 6.6 (0.3–19.1)
IV 0.2 (0.1–0.2) 1.4 (0.2–6.2) 0.8 (0.2–4.8) 0.2 (0.2–1.8) 6.7 (2.0–12.1) 6.5 (0.3–11.0)
V 0.2 (0.2–0.2) 1.8 (0.2–8.9) 0.3 (0.2–7.9) 0.2 (0.2–2.4) 5.9 (0.2–20.3) 0.3 (0.2–18.6)
a
Sera obtained on admission to hospital.
b
Sera obtained on day 4 to 24 (median, day 13) (Table 1).
c
Sera obtained 6 weeks after admission to hospital.
d
Percent phagocytosing nonlymphocytes multiplied by mean number of beads per phagocytosing cell.
e
Mean nonlymphocyte R-123 fluorescence.

per cell, as shown in the summary of FCM parameters ob-
tained 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 opsonoph-
agocytosis, the product of these two parameters (the PP) was
used to reflect the opsonic activities.
Increases in anti-OMV opsonic activity were detected in 38
rosubtype P1.7,16 strains induced 2.8-fold (intermediate sera)-
and 2-fold (6-week sera)-higher median PPPorA values than
sera from patients with P1.7,16 strains (Fig. 3). However, no
statistically significant differences were found between the
PPPorA values induced by serosubtype P1.7,16 versus non-
P1.7,16 convalescent-phase sera (P 5 0.27 and P 5 0.21 for
intermediate and 6-week sera, respectively).

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(95%) of the patients during the course of disease, and the
highest PPOMV values were registered with intermediate sera
(median PPOMV, 1,438; range, 72 to 1756; n 5 38) (Fig. 1;
Table 2). Increased PPPorB values were observed in 37 inter-
mediate sera (97%) with a median PPPorB of 754 (range, 17 to
1,057). In contrast, only 22 intermediate sera (58%) induced
increased PPPorA values, with a lower median PPPorA of 107
(range, 4 to 485). The difference in the magnitudes of the
PPPorA and the PPPorB values obtained with intermediate sera
was statistically significant (P , 0.0001). However, both the
PPPorA and the PPPorB values were positively correlated with
the PPOMV values (Fig. 2).
The patients were grouped according to the main sero-
groups, serotypes, and serosubtypes of the disease-causing
strains (Table 1). Patients in groups I and II were infected by
meningococcal strains with the serotype (serotype 15) and se-
rosubtype (P1.7,16) homologous to those of the strain from
which the target antigens were extracted, whereas strains with
heterologous serotypes and/or serosubtypes were isolated from
patients in groups III to V. No statistical differences in the
serum opsonic activity were found between the groups; how-
ever, considerable interindividual variations in both the anti-
OMV and the antiporin opsonic responses were demonstrated
in patient sera within each group (Fig. 1; Table 2).
Convalescent-phase sera from patients infected by non-se-
Convalescent-phase sera from patients infected by serotype
15 meningococci induced 1.8-fold (intermediate sera)- and 3.8-
fold (6-week sera)-higher median PPPorB values than sera from
patients infected by other strains (Fig. 4). However, no statis-
tically significant differences between the PPPorB values in-
duced by serotype 15 versus non-serotype 15 convalescent-
phase sera were found (P 5 0.20 and P 5 0.13 for intermediate
and 6-week sera, respectively).
Small amounts of serum opsonins against all employed an-
tigens were detected in most of the patients on admission to
hospital (Fig. 1). The highest opsonic activities were registered
in intermediate samples, and these were significantly higher
than those measured with admission sera (P , 0.001 for
PPOMV, PPPorA, and PPPorB values). Anti-OMV, anti-PorA,
and anti-PorB opsonic activities were still significantly in-
creased in 6-week sera (P , 0.0001 for PPOMV, PPPorA, and
PPPorB values, compared with admission values), whereas a
decline was registered between intermediate and 6-week sam-
plings (P , 0.0001 for all values).
Six patients had been vaccinated with a serogroup B menin-
gococcal OMV vaccine (strain 44/76; B:15:P1.7,16) 1 to 4 years
prior to infection caused by B:15:P1.12 (n 5 4) and C:2a:P1.2
(n 5 2) strains (Table 1). Whereas the vaccinee admission sera
induced higher PPOMV values (median PPOMV, 749; range, 603
to 1,306) than admission sera from nonvaccinated patients
2556 LEHMANN ET AL. INFECT. IMMUN.

FIG. 3. Box plots of leukocyte PPPorA values induced by sera from menin-
gococcal disease patients infected with a meningococcal serosubtype homolo-
gous 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.

Control admission and convalescent-phase sera induced low

PP and oxidative burst responses with all antigens, which were
similar to those observed with admission sera from nonvacci-
nated meningococcal disease patients (data not shown). Sera
from healthy controls induced highly variable PPOMV values
(median PPOMV, 55; range 6 to 1,167) and R-123 activity (me-
dian, 0.17; range, 0.14 to 7.40) and low PPPorA and PPPorB
values and R-123 activities (median PPPorA, 7 [range, 5 to 14],
median PPPorB, 6 [range, 4 to 10]; and mean R-123 fluores-

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

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of PPPorA versus PPOMV (upper panel) and PPPorB versus PPOMV (lower panel)
induced by patient sera obtained between admission to hospital with meningo-
coccal disease and 6 weeks later (intermediate sera; n 5 38). Simple regression
lines with 99% confidence intervals are shown.

(P 5 0.028), no differences in the PPOMV values induced by

convalescent-phase sera were found. No statistically significant
differences in the PPPorA and PPPorB values between vacci-
nated and nonvaccinated patients were found (data not
shown). As for the other patients, the PP values induced by
vaccinee intermediate sera were higher than those obtained
with their admission sera (P 5 0.028 for PPOMV, PPPorA, and
PPPorB values).
Oxidative burst. The oxidative burst responses were re-
flected by the formation of R-123 (Table 2). The mean non-
lymphocyte R-123 fluorescence after stimulation with opso-
nized OMV- and porin-coated beads corresponded to the PP
values with admission sera (r 5 0.88 [P , 0.01], r 5 0.34 [P ,
0.05], and r 5 0.84 [P , 0.01] for anti-OMV, anti-PorA, and FIG. 4. Box plots of leukocyte PPPorB values induced by sera from menin-
anti-PorB activities, respectively), intermediate sera (r 5 0.83, gococcal disease patients infected with a meningococcal serotype homologous or
0.91, and 0.95 [P , 0.01] for anti-OMV, anti-PorA, and anti- 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
PorB activities, respectively), and 6-week sera (r 5 0.97, 0.80, 75th and 25th percentiles, and the error bars represent the 90th and 10th
and 0.99 [P , 0.01] for anti-OMV, anti-PorA, and anti-PorB percentiles. The open circles denote outliers. The solid lines represent the me-
activities, respectively). dian values, and the dotted lines represent the means.
VOL. 67, 1999 HUMAN OPSONINS RECOGNIZE MENINGOCOCCAL PorA AND PorB 2557

cence, ,0.18 with all sera). Control beads coated with BSA
and unopsonized antigen-coated beads induced low PP values
and R-123 formation (PP values of ,10 and mean R-123
fluorescence of ,0.20).
ELISA. IgG antibodies recognizing OMV epitopes were de-
tected in sera from all but one patient (no. 33) during menin-
gococcal disease. The anti-OMV IgG levels in 38 available
intermediate sera (median, 39 mg/ml; range, ,2.5 to 681 mg/
ml) were significantly higher than those in admission sera (me-
dian, 4 mg/ml; range ,2.5 to 59 mg/ml) and 6-week sera (me-
dian, 22 mg/ml; range, ,2.5 to 384 mg/ml) (P , 0.001 for both).
The patient anti-OMV IgG levels corresponded to the anti-
OMV opsonic activity, as reflected by PPomv values (r 5 0.68,
0.50, and 0.72 [P , 0.01] for all admission, intermediate, and
6-week sera, respectively) (results for intermediate sera for
PPomv versus anti-OMV IgG are shown in Fig. 5).
IgG antibodies recognizing PorA and PorB proteins were
detected in sera from all patients during meningococcal dis-
ease, and the highest levels were found in intermediate serum
samples (Table 3). The anti-PorB IgG levels (median, 0.4
mg/ml [range, 0.1 to 47.4 mg/ml]; median, 22.7 mg/ml [range, 0.3
to 457.7 mg/ml]; and median, 6.8 mg/ml [range, 0.4 to 290.3
mg/ml] for all admission, intermediate, and 6-week sera, re-
spectively) were higher than the anti-PorA IgG levels (median,
0.2 mg/ml [range, 0.1 to 4.7 mg/ml]; median, 3.1 mg/ml [range,
0.1 to 90.9 mg/ml]; and median, 1.7 mg/ml [range, 0.1 to 53.5
mg/ml] for all admission, intermediate, and 6-week sera, re-
spectively) (P , 0.001 for all). The antiporin IgG levels were
correlated to the antiporin opsonic activity (r 5 0.25 [P , 0.12],
r 5 0.71 [P , 0.01], and r 5 0.75 [P , 0.01] for anti-PorA IgG
versus PPPorA values and r 5 0.70, 0.63, and 0.88 [P , 0.01] for
anti-PorB IgG versus PPPorB values, using admission, interme-
diate, and 6-week sera, respectively) (values for intermediate
sera are shown in Fig. 5).
The antiporin IgG responses were mainly of the IgG1 and/or
IgG3 subclass (Table 3). More intermediate sera exhibited
detectable IgG1 and IgG3 antibody levels against the PorB
protein than against PorA (Table 3), and the median IgG1 and
IgG3 levels against PorB were higher than those against PorA
(P , 0.05 for differences in IgG1 and IgG3 levels in admission,
intermediate, and 6-week sera). IgG2 antibodies recognizing

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PorA and PorB were observed in only four and five sera,
respectively (Table 3). Antiporin IgG4 was not found (Table
3).
Antiporin IgM responses were detected in sera from the
majority of patients during meningococcal disease, and the
anti-PorB IgM responses were more pronounced than the anti-
PorA responses (P , 0.001 for admission, intermediate, and
6-week samples). The highest antiporin IgM titers were found
in intermediate sera (Table 3), but the antiporin IgM levels did
not correlate with the antiporin opsonic activity (r , 0.35 [P .
0.05] for all sera).
Four admission sera had detectable levels of anti-PorB IgA
(data not shown), whereas anti-PorA and anti-PorB IgA were
found in 10 and 24 intermediate sera, respectively (Table 3). FIG. 5. Correlation plots between levels of IgG against meningococcal PorA,
No association between the IgA levels and the opsonic activi- PorB, and OMVs and PP values induced by antigen-specific opsonins in patient
ties was found. sera obtained between admission to hospital with meningococcal disease and 6
CLSM. Phagocyte internalization of opsonized OMV- and weeks later (intermediate sera).
porin-coated beads and the intracellular R-123 oxidative re-
sponses were visualized by CLSM, as shown with PorA-coated DISCUSSION
beads opsonized with intermediate serum from patient no. 35
(Fig. 6). The cell nucleus appeared black against the green We have demonstrated that human serum opsonins pro-
R-123 in the cytoplasm, in which the opsonized antigen-coated duced in response to meningococcal disease are directed
beads were located. Control beads coated with BSA and un- against meningococcal outer membrane components and, for
opsonized antigen-coated beads were not phagocytosed and the first time, have specifically identified opsonins that recog-
did not initiate R-123 formation (not shown). nize meningococcal PorA and PorB displayed in a purified
2558 LEHMANN ET AL. INFECT. IMMUN.

TABLE 3. Meningococcal disease patient intermediate sera with ificity of the opsonic activities, we recently replaced the bacte-
antiporin (PorA and PorB) IgG, IgA, and IgM antibodies, ria with antigen-coated beads (25, 26). Phagocytosis and oxi-
as evaluated by ELISA dative burst activity induced by opsonized OMV-coated beads
PorA PorB correlated with results obtained with opsonized ethanol-fixed
Ig meningococci (25). The epitopes exposed on OMV, PorA, and
n Median (range)a n Median (range)a
PorB may be different from those exposed on live bacteria (1a,
IgG 38 3.1 (0.1–90.9) 38 22.7 (0.9–457.7) 1b). In the present study, the vesicle and proteosome formu-
lations were employed to present the antigens in as close to the
IgG1 22 339 (58–5,200) 32 1,125 (74–31,500) native conformation as presently possible. The antigen speci-
IgG2 4 147 (87–370) 5 100 (59–100) ficity of the antiporin opsonic activity was further ensured by
IgG3 11 151 (50–501) 21 661 (60–12,300)
the extraction of porins from mutant strains lacking potentially
IgG4 0 0
contaminating proteins. Accordingly, the assay should reflect
IgA 10 344 (124–1,740) 24 449 (150–1,800) serum opsonic activities to defined bacterial structures.
IgM 36 430 (100–3,200) 37 1,600 (100–5,040) The patient anti-PorA and anti-PorB opsonic activities cor-
a
related with the anti-OMV opsonic activity (Fig. 2), which
Only for sera with detectable Ig levels. IgG is quantified as micrograms per
milliliter; IgA, IgM, and IgG subclass titers are presented as reciprocals of serum suggests that patient opsonins recognize porin epitopes on the
dilutions. vesicles. However, higher anti-PorB opsonic activities than an-
ti-PorA opsonic activities were observed (Fig. 1 to 4), and these
form on the surface of beads. Significant increases in both were further found to correlate with higher IgG levels against
phagocytosis and oxidative burst activity were observed with PorB than against PorA in intermediate sera (Fig. 5). Higher
PorA- and PorB-coated beads opsonized with patient sera IgG antibody levels against class 3 (PorB) than against class 1
obtained during meningococcal disease (Fig. 1; Table 2). The (PorA) during meningococcal infections have previously been
results indicate that opsonins directed against these structures reported (17). The results suggest that PorB epitopes are more
are induced by natural infection. immunogenic than PorA during meningococcal disease. A
Previously, increasing amounts of human serum opsonins strong immune response to PorB was also observed after three
during meningococcal disease and after OMV vaccination doses of an OMV vaccine, in contrast to two doses (34, 45).
have been demonstrated by using ethanol-fixed bacteria in Thus, viable bacteria seem to induce a more rapid immune
functional opsonophagocytosis assays (1, 13, 14, 18, 24, 39), response to PorB than the OMV vaccine.
and such serum opsonic activity in patients has been shown to The PorA and PorB proteins were recognized by serum
correlate with levels of IgG antibody to purified meningococcal opsonins produced in patients infected by meningococci with
PorA and PorB (17). To directly characterize the antigen spec- serogroups, serotypes, and serosubtypes both homologous and

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FIG. 6. CLSM front- and side-view images of phagocytosed PorA-coated beads and green R-123 oxidative burst fluorescence.
VOL. 67, 1999 HUMAN OPSONINS RECOGNIZE MENINGOCOCCAL PorA AND PorB
heterologous to the strain from which the target antigens were
extracted (Fig. 1; Table 1). With convalescent-phase sera, the
median PPPorB values were markedly higher after infections
with homologous serotypes (Fig. 4), whereas the median PP-
PorA values were higher after infections with heterologous se-
rosubtypes (Fig. 3). However, no statistically significant differ-
ences were found when all anti-PorA and anti-PorB opsonic
activities induced by meningococci of homologous versus het-
erologous serosubtypes and serotypes were compared. This
may be explained by considerable interindividual differences in
the PPPorA and PPPorB 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 menin-
gococcal disease may at least in part be initiated by non-
serotype and non-serosubtype-specific epitopes shared by var-
ious 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 activa-
tion 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 pro-
posed that a high fraction of IgA recognizing meningococcal
components increases the disease susceptibility and that circu-
latory IgA can block the serum bactericidal activity and possi-
bly 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 asso-
ciation 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 opsonoph-
agocytosis.
Phagocyte internalization of opsonized OMV-coated beads
and initiation of intracellular oxidative burst mechanisms have
previously been demonstrated by CLSM (25, 26). CLSM gen-
erates 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 ini-
tiated by patient opsonins against bead-associated meningo-
coccal porins (Fig. 6).
The higher anti-OMV opsonic activity observed in admis-
sion 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 oc-
currence 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 protec-
2559
tive immunity to meningococcal disease remain unclear (3, 4,
31). Ross et al. demonstrated that serogroup B meningococci
are more resistant to complement-mediated bactericidal activ-
ity of normal serum than serogroup A and C meningococci but
are susceptible to phagocytic killing by polymorphonuclear leu-
kocytes after opsonization (36). Furthermore, since genetic
variations in the phagocyte Fcg 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 ap-
pear to acquire immunity to infection with heterologous sero-
groups, 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 op-
sonins 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 meningococ-
cal vaccine candidates.
ACKNOWLEDGMENTS
The serotyping of N. meningitidis was performed at the National
Institutes of Public Health (NIPHs) in Oslo, Norway, and in Birming-
ham, United Kingdom. We thank Lisbeth Meyer Næss at NIPH, Oslo,
Norway, for advice concerning the anti-OMV ELISA. We thank Edu-
ardo Ramirez for assistance with the CLSM, which was provided by the
FFS-Medical Research Center, University of Bergen, Bergen, Norway.
The complement hemolytic activity was measured by the Department
of Microbiology and Immunology, Haukeland Hospital, Bergen, Nor-
way.
This study was supported by grants from Nina’s Memorial Fund,
Norway.
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