Serum Antibody Responses in Ethiopian Meningitis Patients Infectedwith Neisseria meningitidis Serogroup A Sequence Type 7�
Gunnstein Norheim,1 Abraham Aseffa,2 Mohammed Ahmed Yassin,3,4 Getahun Mengistu,5,6
Afework Kassu,5 Dereje Fikremariam,7 Wegene Tamire,7 Yared Merid,4 E. Arne Høiby,1Dominique A. Caugant,1,8 Elisabeth Fritzsønn,1 Torill Tangen,1 Tsegaye Alebel,9
Degu Berhanu,2 Morten Harboe,2,10 and Einar Rosenqvist1*Division of Infectious Disease Control, Norwegian Institute of Public Health (NIPH),1 Department of Oral Biology,
University of Oslo,8 and Institute of Immunology, University of Oslo, and Rikshospitalet-Radiumhospitalet Medical Center,10
Oslo, Norway; Armauer Hansen Research Institute (AHRI)2 and Department of Internal Medicine, Faculty of Medicine,Addis Ababa University,6 Addis Ababa, Southern Nations, Nationalities’ and Peoples’ Region (SNNPR) Health Bureau,
Awassa,4 Department of Microbiology and Parasitology, College of Medicine and Health Sciences, The University ofGondar,5 and North Gondar Zone Health Bureau, Gondar,9 and Yirgalem Hospital, Yirgalem,7
Ethiopia; and Liverpool School of Tropical Medicine, Liverpool, United Kingdom3
Received 20 October 2006/Returned for modification 9 January 2007/Accepted 5 February 2007
To elucidate critical components of protective immune responses induced during the natural course ofserogroup A meningococcal disease, we studied acute-, early-convalescent-, and late-convalescent-phase serafrom Ethiopian patients during outbreaks in 2002 to 2003. Sera were obtained from laboratory-confirmedpatients positive for serogroup A sequence type 7 (ST-7) meningococci (A:4/21:P1.20,9) (n � 71) and fromEthiopian controls (n � 113). The sera were analyzed using an enzyme-linked immunosorbent assay tomeasure levels of immunoglobulin G (IgG) against serogroup A polysaccharide (APS) and outer membranevesicles (OMVs) and for serum bactericidal activity (SBA) using both rabbit and human complement sources.Despite relatively high SBA titers and high levels of IgG against APS and OMVs in acute-phase patient sera,significant increases were seen in the early convalescent phase. Antibody concentrations returned to acute-phase levels in the late convalescent phase. Considering all patients’ sera, a significant but low correlation (r �0.46) was observed between SBA with rabbit complement (rSBA) using an ST-5 reference strain and SBA withhuman complement (hSBA) using an ST-7 strain from Ethiopia. While rSBA demonstrated a significant linearrelation with IgG against APS, hSBA demonstrated significant linear relationships with IgG against both APSand OMV. This study indicates that antibodies against both outer membrane proteins and APS may beimportant in providing the protection induced during disease, as measured by hSBA. Therefore, outer mem-brane proteins could also have a role as components of future meningococcal vaccines for the Africanmeningitis belt.
Neisseria meningitidis is responsible for recurring epidemicsof bacterial meningitis in the region of sub-Saharan Africadesignated the meningitis belt (36). Most of the cases in thisregion are caused by serogroup A meningococci (MenA), al-though serogroups W135, C, and X are also involved (42).Molecular epidemiological studies of meningococcal strainshave shown that a few complexes of related hypervirulentclones are responsible for the major part of the cases in themeningitis belt (42). Most recent serogroup A epidemics havebeen caused by a clonal group introduced to the meningitis beltfrom Mecca, Saudi Arabia, in 1987: the subgroup III/sequencetype 5 (ST-5) clonal complex (67). Strains belonging to thiscomplex have been very homogenous; nearly all expressed thesame PorA (serosubtype P1.20,9) and PorB (serotype 4/21)(59, 62). Between 1988 and 1999, ST-5 reached all the coun-tries of the meningitis belt, where it was responsible for severe
epidemics (42). In the mid-1990s, bacteria of the closely re-lated ST-7 emerged in Africa and progressively replaced theST-5 strains (42). This replacement reflects a significant ge-netic and epidemiological change (41, 45).
Ethiopia is part of the meningitis belt, and many epidemicshave been reported in the country since 1901 (45). In Ethiopia,the replacement of ST-5 by ST-7 strains occurred between1995 and 2000. We recently showed that Ethiopian ST-5 andST-7 strains differed in several loci associated with outer mem-brane antigens (45). These changes could be relevant for ex-plaining the clonal replacement.
An effective polysaccharide (PS) vaccine that could preventMenA disease in Africa has been available for nearly 3 decades(18, 46). However, the WHO does not recommend this vaccinefor routine immunization, and mass vaccination is performedonly in response to outbreaks, although there has been con-siderable dispute regarding this decision (1, 52). The newMenA conjugate vaccines (27) will hopefully provide long-term protection even when given to children below the age of2 years. However, the immunoglobulin G (IgG) response andserum bactericidal activity (SBA) following immunization withMenA conjugate do not differ much from those observed with
* Corresponding author. Mailing address: Department of Bacteriol-ogy and Immunology, Division of Infectious Disease Control, Norwe-gian Institute of Public Health, P.O. Box 4404, Nydalen, NO-0403,Oslo, Norway. Phone: 47 22 04 26 19. Fax: 47 22 04 25 18. E-mail:[email protected].
� Published ahead of print on 14 February 2007.
451
the MenA PS (APS) vaccine (14). Dissecting the humoralimmune response following disease may contribute to a betterunderstanding of the parameters that are important in relationto the development of improved vaccines.
A number of studies have determined the antibody responseinduced by MenA disease (2, 9, 10, 13, 17, 23, 31, 35, 50, 56,58). In general, high levels of antibody against APS are nor-mally observed in the population (40), but such levels may beobserved in acute-phase sera from MenA patients as well (31,58). Whereas anti-APS IgG antibodies can confer protectionagainst MenA disease, high levels of anti-APS IgA antibodiesin predisease or acute-phase sera may have a blocking effect onSBA and may be related to increased risk of MenA infection(20, 31). Despite preexistent high levels, IgG and IgM againstAPS increase significantly during early convalescent phase in alarge proportion of MenA patients to levels of the same mag-nitude as those obtained following vaccination with APS vac-cine (9, 10, 31, 56, 58). The antibodies induced by disease canbe directed against APS, outer membrane proteins, lipooligo-saccharide (LOS), and secreted proteins such as, e.g., IgA1protease (9, 10, 13, 58), but these are not necessarily bacteri-cidal (2, 23). Very few studies have characterized the noncap-sular antibody response and the SBA mounted during disease,and the number of patients and the duration of follow-up wereusually limited.
The objective of this study was to characterize the humoralresponse following MenA disease caused by subgroup III ST-7meningococci in Ethiopia in 2002 and 2003. The antibodyresponses against APS and outer membrane vesicles (OMVs)were quantified by an enzyme-linked immunosorbent assay(ELISA), and the functional activity was measured by an SBAassay using two different complement sources (human andrabbit serum) with two different serogroup A strains.
(Part of this work was presented at the 15th InternationalPathogenic Neisseria Conference in Cairns, Australia, Septem-ber 2006.)
MATERIALS AND METHODS
Patients. Ninety-five meningitis patients presenting at two study areas in NorthGondar Zone, Amhara Region, and Sidama and Gedio Zones, Southern Na-tions, Nationalities, and Peoples’ Region (SNNPR), Ethiopia, were included inthe study spanning April 2002 to June 2003, as described previously (45). Patientswere recruited consecutively in the first meningitis season, until at least eightpatients were included in each of the following intended age groups: infants (�6months to �2 years), young children (�2 to �6 years), older children andteenagers (�6 to �15 years), and adults (�15 years). During the second season,recruitment focused on including children below the age of 2 years, because toofew had been included in the first season. Of 95 patients, 71 were laboratoryconfirmed with meningococcal meningitis (45) (Table 1). An attempt was madeto obtain blood samples in the acute phase (0 to 7 days after the onset of disease),
the early convalescent phase (8 days to 6 weeks), and the late convalescent phase(�72 days after the onset of disease). The date of onset of disease was definedas the date of the first severe symptom related to the meningitis disease episodereported by the patient. Late-convalescent-phase blood samples (range, 70 to 610days after the onset of disease) were obtained through house visits by study teamsseeking the patients’ dwellings. Unless otherwise stated, the patients’ sera de-scribed are those from patients confirmed positive for N. meningitidis by cultureor PCR (Table 1). Eleven confirmed meningococcal disease patients were re-ported to have been vaccinated with APS vaccine previously; of these, tworeported that they had been vaccinated 3 months prior to admission, sevenreported that they had been vaccinated 8 to 12 months prior to admission, andtwo did not report the time of vaccination (45). Sera from patients not confirmedwith N. meningitidis by culture or PCR (Table 1) (45) were included in theanalysis for the purpose of later serological diagnostic confirmation.
Controls. Control blood samples were obtained from apparently healthy indi-viduals and from patients with nonmeningococcal disease in Ethiopia. Controlswere recruited from hospitals or health centers in Gondar in 2003 (n � 35;median age, 5.6 years; range, 0.8 to 55 years) and in SNNPR in 2002 (n � 39;median age, 28 years; range, 10 to 68 years) and 2003 (n � 39; median age, 12years; range, 1 to 25 years) (Table 1). Twenty-one individuals from SNNPR hadpreviously received the MenA/C PS vaccine. Of these, 4 had been vaccinated 5to 11 months prior to sampling, 16 had been vaccinated 1 or more years previ-ously, and the time of vaccination was not known for 1 individual. Forty-four serafrom Norwegian high school students (median age, 13.5 years; range, 12.7 to 13.9years) were also included; these were prevaccination sera from a clinical trial inNorway in 1992 (15). Unless otherwise stated, the control sera described arethose from Ethiopia, for which data are given in Table 1.
Preparation of APS and methylated human serum albumin. APS was pre-pared from strain Mk 83/94 (A:4/21:P1.20,9) (44) by phenol extraction as de-scribed elsewhere (19, 65). The purification yielded a product with negligibleamounts of protein, LOS, and DNA, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue and silver staining andby high-performance liquid chromatography analysis (Perkin-Elmer, Wellesley,MA) using a TSK-GEL G5000 PWXL column with UV detection at a � of 200nm (Biotech Support Group, East Brunswick, NJ). Human serum albumin wasmethylated (37), and 1:1 complexes of APS and methylated HSA were preparedas previously described (11).
APS ELISA. The APS ELISA was performed as described elsewhere (11).Binding of IgG and IgA antibodies to APS was detected using alkaline phos-phatase-conjugated goat anti-human IgG (Sigma-Aldrich, St. Louis, MO) (11).The CDC1992 serum (National Institute of Biologicals and Standards, PottersBar, United Kingdom) was used as an internal reference (24). In addition, twoother internal control sera were included.
Preparation of OMVs. OMVs were prepared by extraction with deoxycholate(dOMVs) from the MenA ST-7 strain Mk 686/02 and have been characterizedpreviously (43). The outer membrane proteins PorA, PorB, RmpM, Opa, OpcA,NspA, NadA, Omp85, and TdfH were present, along with approximately 5%LOS L11 relative to protein (43).
OMV ELISA. Binding of IgG to dOMVs was determined as described previ-ously (43). An internal serum standard for anti-dOMV IgG response, defined as1,000 arbitrary units (AU)/ml, was made by pooling early-convalescent-phasesera from five Ethiopian patients with confirmed MenA disease.
SBA assays. SBA assays were performed using the “tilt method” as previouslydescribed (7, 43), but with some modifications. To avoid possible interferencefrom antibiotics, sera were pretreated by mixing them with similar amounts ofpenicillinase at 100 international units/ml (Sigma-Aldrich) in Hanks buffered saltsolution. Sera were incubated at room temperature for 10 min and heat inacti-vated at 56°C for 30 min. Twofold dilutions of control sera, penicillinase-treated
TABLE 1. Number of individuals, sera available for analysis, and demographic characteristics for Ethiopian patients with systemic MenAdisease, nonconfirmed meningococcal disease patients, and controls
Patient category No. ofpatients
Total no.of sera
No. of sera from the following age group (yr): Median age(yr) (range)
a Values reflect the numbers of patients for whom data were available.b MD, meningococcal disease.
452 NORHEIM ET AL. CLIN. VACCINE IMMUNOL.
sera, or monoclonal antibodies were tested with 10 �l of the inoculum per wellin the presence of 25% complement in U-well polystyrene microplates (GreinerBio-One, Frickenhausen, Germany). The complement used was either a singlelot of serum from baby rabbits (PelFreez Biologicals, Clinical Systems, BrownDeer, WI) or plasma from a single human donor with no intrinsic bactericidalactivity. The ST-5 strain F8238, isolated from a patient in Kenya in 1989, wasused as a target strain (38) with rabbit complement (rSBA assay). The ST-7 strainMk 686/02 (43, 45), isolated from an Ethiopian patient in 2002, was used as atarget strain with human complement (hSBA assay). Both strains were serolog-ically characterized as A:4/21:P1.20,9:L11 OpcA� and had similar expression ofPorA and NadA but different Opa protein repertoires (43, 45). The reactionmixture was incubated for 60 min at 37°C in air.
SBA titers were expressed as the reciprocal of the final serum dilution givinga �50% reduction in CFU at 60 min. Sera with titers below the SBA assaydetection limit, �32 for the rSBA assay and �4 for the hSBA assay, wereassigned a titer of 2. An rSBA titer of �128 was defined here as putativelyprotective against MenA disease, in order not to overestimate protection (5, 64).With the hSBA assay, a titer of �4 was defined as putatively protective (16). Aseroresponder was defined as an individual demonstrating a �4-fold rise in theSBA titer. The seroconversion rate was defined as the proportion of individualsdemonstrating an SBA titer below the detection level in the acute phase whosubsequently achieved a �4-fold increase in the SBA titer in the early convales-cent phase. The CDC1992 serum (24), two positive-control sera (sera fromNorwegian teenagers after vaccination with APS plus serogroup C PS [15]), onenegative-control serum (Norwegian prevaccination serum), and the murine anti-P1.9 PorA monoclonal antibody (MN5-A10F) (49) were used as internal assayreferences.
Data analysis. Statistical analyses were performed using SPSS, version 13.0.1for Windows (SPSS Inc., Chicago, IL). The data in the serological assays wereconsidered to follow a nonnormal distribution and were therefore comparedusing nonparametric tests. For nonpaired data, group-to-group comparisonsusing Mann-Whitney t tests were first performed after an analysis-of-variancetest (Kruskal-Wallis) had verified a significant overall difference between allgroups. Paired data, such as groups of sera collected at different time points fromthe same patients, were compared using the Wilcoxon signed-rank sum test.Differences in proportions of responders above the predefined threshold valueswere compared using the chi-square test. Following log10 transformation of data,the relation between the rSBA and hSBA assay results was analyzed using thePearson correlation test, and the relation between antibody specificity and SBAresults was determined using univariate linear regression.
Ethical clearance. The investigators obtained ethical clearance from theAHRI/All Africa Leprosy TB & Rehabilitation Training Center (ALERT) Eth-ical Clearance Committee, the National Ethical Review Committee (EthiopianScience and Technology Agency), and the Norwegian Regional Committee forMedical Research Ethics in Western Norway (REK III). Written informedconsent was obtained from study participants or their parents/guardians (forthose below the age of 18 years or those with disturbed consciousness) beforeenrollment.
RESULTS
Demographic characteristics of patients and controls. Char-acteristics of the study participants are given in Table 1, to-gether with the numbers of sera available for analysis. TheEthiopian meningococcal meningitis patients and controls hadsimilar age and gender distributions (Table 1). However, insome assays, only selected sera were analyzed, and these werenot always matched for age and gender.
IgG response against APS. In sera from patients with me-ningococcal disease, the geometric mean concentrations(GMCs) of IgG against APS were 10.8 �g/ml in the acutephase, increasing to 33.4 �g/ml in the early convalescent phaseand declining to 13.7 �g/ml in the late convalescent phase(Table 2; Fig. 1a). The proportions of patient sera with a GMCof IgG against APS of �2 �g/ml were 98% in the acute phaseand 100% in the early and late convalescent phases. Limitingthe statistical analysis to paired sera only, we found a sig-nificantly higher IgG level in the early-convalescent-phasesera than in the acute-phase sera (P � 0.001), while thedifference in IgG levels between acute- and late-convales-cent-phase sera proved not to be significant (P � 0.26)(Tables 2 and 3). The proportion of patients with �4-foldincreases in anti-APS IgG levels from the acute to the earlyconvalescent phase was 35%, while the proportion with �2-fold increases was 59% (Table 3; Fig. 2a). The anti-APS IgGlevels in the acute- and late-convalescent-phase patient serawere not significantly different from those observed for con-trols, compared within each of the age groups. In contrast,anti-APS IgG levels in early-convalescent-phase sera weresignificantly higher than those for controls in all age groups,except for infants, where few sera were available. In acute-phase sera, we found no statistically significant difference inanti-APS IgG levels between age groups (P � 0.77) (Table2). On the other hand, a significant difference in anti-APSIgG levels between age groups was seen among controls(P � 0.003), and the proportions of sera with anti-APS IgGlevels above “thresholds” increased with age (Fig. 3a). Theproportions of sera from patients with anti-APS IgG levels
TABLE 2. GMCs of anti-APS IgG and anti-OMV IgG measured by ELISA in sera from Ethiopian patients withsystemic MenA disease and controls
IgG Age group(yr)
Analysis for:
PatientsControls
Acute phase Early convalescent phase Late convalescent phase
GMC (range) na GMC (range) n GMC (range) n GMC (range) n
VOL. 14, 2007 ANTIBODY RESPONSES TO SEROGROUP A MENINGOCOCCI 453
equal to or above the GMC for Ethiopian controls (9.4�g/ml) were 49%, 93%, and 72% for acute-, early-convales-cent-, and late-convalescent-phase sera, respectively. How-ever, the proportion of early-convalescent-phase sera frompatients with anti-APS IgG levels above the 99th percentile
for the control sera was only 7.1%. Anti-APS IgG levelswere significantly higher for teenage Ethiopian controls(�13 to �15 years) than for teenage Norwegian controls(GMC, 1.9 �g anti-APS IgG/ml; range, 0.3 to 11.4 �g IgG/ml; n � 44) (P � 0.001).
FIG. 1. Development of antibody responses against MenA bacteria in sera from MenA disease patients over time after the onset of disease,as measured by ELISAs for IgG against APS (a) and IgG against OMVs (b). In the same sera, rSBA against strain F8238 (c) and hSBA againststrain Mk 686/02 (d) were measured. conv., convalescent.
TABLE 3. GM fold increases in antibody levels in paired sera from Ethiopian patients with systemic MenA disease
Antibody
Acute vs early conv.a phase Acute vs late conv. phase
a Conv., convalescent.b Calculated by dividing the value for the individual serum sample from the early or late convalescent phase by the value for the corresponding acute-phase serum
sample.c n, number of sera analyzed.
454 NORHEIM ET AL. CLIN. VACCINE IMMUNOL.
IgA response against APS. In the patients’ acute-phase sera,the GMC of IgA against APS was 1.1 �g/ml (n � 25). Thislevel increased significantly, to 6.2 �g/ml, in early-convales-cent-phase sera, while in late-convalescent-phase sera it de-creased to 3.2 �g/ml, a level not significantly different from the
acute-phase level (Table 3). No differences between agegroups in the GMC of IgA against APS were observedamong the acute-phase patient sera (P � 0.77). A higherlevel of anti-APS IgA was observed in control sera than inacute-phase patient sera for the �6- to 15-year age group
FIG. 2. Antibody levels in paired acute- versus early-convalescent-phase sera from MenA disease patients. (a) Anti-APS IgG; (b) anti-OMVIgG; (c) rSBA; (d) hSBA.
FIG. 3. Age-related differences in antibody levels among Ethiopian control sera, expressed as percentages of sera above thresholds (eitherGMCs of these control sera or 50% of the GM). (a) rSBA and anti-APS IgG; (b) IgA against APS; (c) anti-OMV IgG.
VOL. 14, 2007 ANTIBODY RESPONSES TO SEROGROUP A MENINGOCOCCI 455
(P � 0.006). As was the case for IgG, a significant differencein anti-APS IgA levels (P � 0.003) between age groups wasobserved for controls (Fig. 3b).
IgG response against OMVs. The GMC of IgG againstdOMVs of strain Mk 686/02 increased significantly from acute-to early-convalescent-phase sera, while it declined in late-con-valescent-phase sera to the levels seen in acute-phase sera(Tables 2 and 3; Fig. 1b). The proportion of patients with�4-fold increases in anti-OMV IgG levels from acute to earlyconvalescent phase was 32%, while the proportion with �2-fold increases was 61% (Table 3; Fig. 2b). We found no sig-nificant differences in IgG levels between the various agegroups (P � 0.88). Higher levels of anti-OMV IgG were ob-served in acute-phase patient sera than in control sera forchildren �6 to �15 years old (P � 0.020) and for adults (P �0.017) (Table 2). In early-convalescent-phase sera, anti-OMVIgG levels were significantly higher than in control sera for allage groups except infants. In late-convalescent-phase sera,anti-OMV IgG levels were still significantly higher than thosein control sera among adults (P � 0.008). The proportions ofcontrol sera with anti-OMV IgG levels above two differentthresholds (GMC and 50% of GMC for controls, respectively)showed only a slight tendency to increase with age (Fig. 3c). Incontrol sera from Ethiopian individuals aged �13 to �15years, the anti-OMV IgG GMC was almost twice as high (P �
0.001) as that in control sera from Norwegian teenagers(GMC, 29 U/ml; range, 21 to 40 U/ml, n � 10).
SBA. The geometric mean titer (GMT) in acute-phase pa-tient sera was 47.3 by the rSBA method, increasing to 3,104 inearly-convalescent-phase sera and decreasing to 110 in late-convalescent-phase sera; the latter level was not significantlydifferent from that found in acute-phase sera (Tables 3 and 4;Fig. 1c). Correspondingly, the proportions of patient sera withprotective rSBA titers was 44% in the acute phase, 95% in theearly convalescent phase, and 56% in the late convalescentphase (Table 4). The proportion of patients who were rSBAseroresponders (i.e., who demonstrated a �4-fold increase)was 57% (20/35) (Table 3), while the rSBA seroconversion ratewas 100% (15/15). The rSBA GMT was significantly lower inacute-phase patient sera than in controls among adults (Table4) (P � 0.001). This was also the case when acute-phase serasampled at days 5 to 7 after onset were excluded from analyses.
In acute-phase patient sera, rSBA titers for children aged�2 to �6 years were significantly higher than those for indi-viduals aged �6 to 15 years (P � 0.045) or �15 years (P �0.001). rSBA titers were not significantly different between thelatter two groups (P � 0.088). In control sera, rSBA titers forthe �2-year age group were significantly lower than those forthe �2- to 6-year, �6- to 15-year, and �15-year age groups(P � 0.036 for all), while GMTs for the three latter age groups
TABLE 4. rSBA against target strain F8238 and hSBA against Mk 686/02 in sera from Ethiopian systemicMenA disease patients and controls
Group
rSBA hSBA
GMT (range) na
No. (%) of patientswith titer of: GMT (range) na
No. (%) of patientswith titer of:
�32 �128 �4 �16
PatientsAcute phase
All 47.3 (2–65,536) 62b 29 (47) 27 (44) 11.7 (2–4,096) 47 25 (53) 19 (40)0.5 to �2 yr 64 (2–2,048) 2 64 1�2 to �6 yr 1,024 (2–65,536) 10 58.7 (2–4,096) 8�6 to �15 yr 58.9 (2–32,768) 25 11.3 (2–512) 22�15 yr 11.7 (2–4,096) 24 5.0 (2–128) 16
Early convalescent phaseAll 3,104 (2–65,536) 40 38 (95) 38 (95) 81.2 (2–8,192) 35 27 (77) 24 (69)0.5 to �2 yr 0 0�2 to �6 yr 4,096 (128–65,536) 8 173 (2–2,048) 6�6 to �15 yr 2,724 (2–32,768) 17 32 (2–2,048) 15�15 yr 3,104 (2–65,536) 15 156 (2–8,192) 14
Late convalescent phaseAll 110 (2–32,768) 32 21 (66) 18 (56) 8.0 (2–1,024) 28 13 (46) 2 (29)0.5 to �2 yr 2 (2–2) 2 256 1�2 to �6 yr 45.3 (2–2,048) 4 2.8 (2–8) 4�6 to �15 yr 232 (2–32,768) 14 9.9 (2–1,024) 13�15 yr 121 (2–4,096) 12 6.5 (2–256) 10
ControlsAll 166 (2–131,072) 70 54 (77) 44 (64) 3.3 (2–256) 16 3 (19) 2 (13)0.5 to �2 yr 23.1 (2–2,048) 17 3.1 (2–16) 8�2 to �6 yr 192 (2–131,072) 17 6.7 (2–256) 4�6 to �15 yr 192 (2–4,096) 17 0�15 yr 737 (2–16,384) 19 2 (2–2) 4
a n, number of sera analyzed.b Includes one patient for whom age was not reported.
456 NORHEIM ET AL. CLIN. VACCINE IMMUNOL.
were not significantly different. However, the proportion ofsera with putative protective rSBA titers increased with age,from 40% for those aged �2 years to 82% for those aged �15years (Fig. 3a). rSBA titers in control sera from Ethiopianteenagers were significantly higher than those in control serafrom Norwegian teenagers (P � 0.001); the GMT of the Nor-wegian control sera was below the assay cutoff, and only 3/15(20%) had putative protective rSBA titers.
By the hSBA assay, the GMT was 11.7 in acute-phase patientsera; it increased significantly, to 81.2, in early-convalescent-phase sera and then decreased to 8.0 in late-convalescent-phase sera (Tables 3 and 4). The hSBA titers in the late-convalescent-phase patient sera were not significantly differentfrom those in the acute-phase sera (Tables 3 and 4). Corre-spondingly, among patients, the proportion of sera with puta-tive protective hSBA titers increased significantly, from 53% inthe acute phase to 77% in the early convalescent phase, beforedropping to 46% in the late convalescent phase (Table 4). Theproportion of patients who were hSBA seroresponders was52%, while the hSBA seroconversion rate was 53% (8/15).Among adults, the hSBA GMT was higher in early-convales-cent-phase patient sera than in control sera (P � 0.009). Thiswas the case also among patients �2 to �6 years of age,although it was not statistically significant. No other significantdifferences in hSBA GMTs were observed between control
sera and patient sera from any phase within the age groups(Table 4).
Vaccination status and serological results. Self-reporteddata on whether the patients or controls enrolled in this studywere vaccinated with APS in the last 3 years prior to enroll-ment were available from case record forms for 39/113 controlsand 60/71 patients with meningococcal meningitis. Among the60 patients reporting vaccination data, we observed no signif-icant difference in anti-APS IgG levels between individualswho reported that they had been vaccinated (n � 11) and thosewho reported that they had not (n � 49). Likewise, for the 39controls reporting vaccination data, we found no significantdifference in anti-APS IgG levels between those who said theyhad been vaccinated (n � 21) and those who said they had not(n � 18).
Correlations between assays. SBA is the primary surrogateassay for protection against meningococcal disease (7), but thecomplement source in the assay may be important for evalua-tion of the results. We therefore analyzed whether results fromour two different SBA methods (rSBA and hSBA) showedcovariation. Only a moderate but significant correlation be-tween rSBA and hSBA titers was observed for acute-phasesera (r � 0.52; n � 46) and late-convalescent-phase sera (r �0.41; n � 46) from patients and for controls (r � 0.51; n � 16),while no significant correlation was detected for early-conva-lescent-phase sera. Combining data from analyses of sera fromall patient phases (acute, early convalescent, and late conva-lescent), we observed a moderate but significant correlationbetween titers obtained by the rSBA and hSBA assays (r �0.46) (Fig. 4). The proportions with putatively protective titersby both the rSBA and the hSBA assay ranged from 13% to74% (Table 5). While as much as 15% of sera (acute phase)showed rSBA titers of �128 and hSBA titers of �4, as much as50% of sera (Ethiopian controls) showed hSBA titers of �4and rSBA titers of �128 (Table 5). The proportions of serawithout putative protective rSBA or hSBA titers were aboutone-third for acute- and late-convalescent-phase patient seraand for controls (Table 5). Some of the patients demonstrateda significant fall in anti-APS or anti-OMV IgG levels from theacute phase to the early convalescent phase (Fig. 2), but noneof these were among those showing SBA seroconversion (i.e.,having an SBA titer below detection level in the acute phaseand subsequently demonstrating a �4-fold titer rise).
However, analysis of whether those patients achieving a pu-tative protective hSBA titer were the same as those classifiedas seroresponders by the rSBA assay (i.e., a �4-fold rise from
FIG. 4. Correlation between rSBA titers and hSBA titers in all serafrom Ethiopian MenA patients (r � 0.46; P � 0.001; n � 109). Dottedlines indicate suggested MenA SBA titer thresholds for protection(�1:128 for rSBA and �1:4 for hSBA).
TABLE 5. Proportions of sera with SBA titers above putative protective thresholdsa for the subset of sera for whichboth rSBA and hSBA were analyzed
a Putative protective SBA titer thresholds were �1:128 for rSBA and �1:4 for hSBA.b n, number of sera analyzed.
VOL. 14, 2007 ANTIBODY RESPONSES TO SEROGROUP A MENINGOCOCCI 457
acute to early convalescent phase) showed no significant cor-relation between the results (P � 0.82; n � 33). Considering allpatient sera, both the rSBA and hSBA titers correlated signif-icantly with both anti-APS and anti-OMV IgG levels; however,there was low correlation between anti-OMV IgG levels andrSBA titers (Fig. 5). This pattern was confirmed by linearregression analysis, where we detected a significant linear re-lation between anti-APS IgG levels and rSBA titers (Fig. 5a)but not between anti-OMV IgG levels and rSBA titers (Fig.5b). In contrast, significant linear relationships were detectedboth between anti-APS IgG levels and hSBA titers (Fig. 5c)and between anti-OMV IgG levels and hSBA titers (Fig. 5d).For Ethiopian controls, rSBA titers correlated significantlywith levels of IgG against APS (r � 0.43).
rSBA titers correlated significantly with anti-APS IgA levelswhen all patient sera or all controls were analyzed (r � 0.50 forboth). In contrast, hSBA titers did not correlate with anti-APSIgA levels when either all patient sera or all controls wereanalyzed. We further analyzed anti-APS IgA levels in a subsetof control sera with high anti-APS IgG levels (above 4.7 �g/ml,
the concentration equivalent to 50% of the GMC of anti-APSIgG in control sera) from individuals aged �2 to �15 years inorder to obtain similar age distributions when comparing serawith and without protective rSBA titers. For this subset ofcontrol sera, we then observed that anti-APS IgA levels werehigher in sera with rSBA titers of �128 (GMC, 4.2 �g/ml; n �6) than in sera with rSBA titers of �1:128 (GMC, 2.6 �g/ml; n� 15) (P � 0.036). In contrast, anti-APS IgA levels for acute-phase patient sera with and without protective rSBA titerswere not significantly different.
Serological evaluation of patients not confirmed to havesuffered meningococcal disease. Following confirmation byculture and/or PCR of 71/95 patients having N. meningitidis intheir cerebrospinal fluid, we serologically evaluated whetherany of the remaining 24 meningitis patients could have sufferedmeningococcal meningitis as well (Table 1). Pairs of acute- andearly-convalescent-phase sera were available from 10 of thesepatients. Based on the results observed in our study for pa-tients with confirmed meningococcal meningitis (Table 3), asignificant antibody increase indicating that the patient had
FIG. 5. Scatter plots showing correlation between data on hSBA, rSBA, anti-OMV IgG, and anti-APS IgG in all sera from Ethiopian MenAdisease patients, along with the Pearson correlation coefficient r (P � 0.001 for all) and the least-squares linear regression equation, wheresignificant. (a) Anti-APS IgG versus rSBA; (b) anti-OMV IgG versus rSBA; (c) anti-APS IgG versus hSBA; (d) anti-OMV IgG versus hSBA.
458 NORHEIM ET AL. CLIN. VACCINE IMMUNOL.
gone through an episode of meningococcal meningitis wasdefined as a twofold rise in either the anti-APS or the anti-OMV IgG level. Significant rises in anti-APS IgG levels wereobserved for 6 of the 10 previously nonconfirmed patients.However, one of these had been immunized with APS vaccine1 day after the onset of the disease; thus, the anti-APS IgGresponse could have been due to vaccination. Two of the fivepatients with significant anti-APS IgG increases also showedsignificant increases in anti-OMV IgG levels. Of the remainingfour patients not confirmed with meningococcal disease, oneshowed a significant increase in anti-OMV IgG levels whiledemonstrating a significant decrease in anti-APS IgG levels. Insummary, there were serological indications of MenA infectionfor at least 6 of the 10 patients, thus increasing the number ofconfirmed and probable meningococcal disease patients in thisstudy to 77/95 (81.1%).
DISCUSSION
In this study we describe the antibody responses in patientsfrom Ethiopia with meningococcal disease caused by N. men-ingitidis serogroup A ST-7 organisms. Our main findings are asfollows: (i) most acute-phase patient sera and control serashow levels of IgG antibodies against APS substantially higherthan those previously assumed to be sufficient for protection(31); (ii) MenA infections stimulate significant increases inlevels of IgG against APS and OMVs and increase SBA levelsin these patients during the first month after the onset ofdisease; (iii) approximately 8 months after the onset of MenAdisease, the mean antibody levels decrease to levels foundduring the acute phase of the disease; (iv) the observed rSBAtiter variation can apparently be explained by variations inanti-APS IgG levels but only to a minor degree by variations inanti-OMV IgG levels. In contrast, a major part of the observedvariation in hSBA titers apparently can be explained by varia-tions in levels of IgG against both APS and OMVs.
Sera and data collection for the study relied on the existinghealth infrastructure and the doctors’ motivation for contrib-uting to research amid a resource-challenged practice. Thevaccination status was self-reported, since meningococcal vac-cination usually happens as mass vaccination in response toepidemics and is seldom recorded in a personal vaccinationcard.
Results from comparisons of sera from acute-phase patientswith sera from controls are very much dependent on the re-ported onset-to-admission time and the time it takes to mounta significant immune response after infection. The date of thefirst symptoms reported by patients may be an important un-certainty factor because of recall bias and possible differencesin the understanding of what may constitute a “serious symp-tom.” We chose to include sera collected up to 7 days after theonset of the first symptoms as acute-phase sera. Thus, in someinstances an immune response could already have been initi-ated in the period defined as acute. The anti-PS IgG responsefollowing PS vaccination usually starts to increase between 4 to10 days after exposure to the antigen (8), and 12/62 acute-phase patient sera were collected �5 days after the reportedonset of disease in our study. However, the antibody levels inthe acute-phase sera collected at different days after the onsetof disease did not show any increasing trend according to the
number of days after the onset of disease (Fig. 1). We com-pared acute-phase sera obtained 0 to 4 days after the onset ofdisease with acute-phase sera obtained 5 to 7 days after onsetbut found no significant differences between groups in GMantibody levels or SBA titers (data not shown). Furthermore,excluding acute-phase sera collected at days 5 to 7 gave resultssimilar to those obtained when all acute-phase sera were in-cluded. The anti-APS IgG levels in acute-phase sera weresimilar to those in control sera as well; thus, they were likelyrepresentative of the levels prior to response to the MenAinfection. However, further studies should be performed com-paring MenA SBA in acute-phase sera with that in control seraand also aiming at obtaining serial acute-phase sera closer tothe onset of infection.
Antibody response against APS. The anti-APS IgG levels inacute-phase patient sera were relatively high (�10 �g/ml) butnot significantly different from those in control sera (Table 2).This is in line with the findings of previous studies of patientsin the meningitis belt (9, 20), and levels were similar to thoseamong the adult population in Burkina Faso (40). In the men-ingitis belt, high levels of anti-APS antibodies are commonlyfound in healthy individuals (4, 9, 20, 21, 40). However, thelevels we observed were higher than those reported for acute-phase sera from Finnish and Egyptian MenA disease patientsas measured by a total-Ig radioimmunoassay (31, 58) and foracute-phase sera from Gambian MenA patients (9). Higheranti-APS IgG levels were seen in Ethiopian teenage controlsthan in Norwegian teenagers, confirming previous studies (4,54). Apart from previous vaccination with APS vaccine orprevious episodes of MenA disease, the main reason for thehigh levels is probably natural immunization, induced either bycarriage of meningococci in the nasopharynx (9) or by entericstimulation of cross-reactive PS structures (21, 51). Elevatedlevels of anti-APS IgG may also be due to nonspecific stimu-lation of APS-specific plasma cells, as observed for Finnishnon-MenA bacterial meningitis patients (30). Since popula-tions in developing countries are prone to undergo multipleinfections, e.g., by bacteria or protozoa, this may be an impor-tant cause of the high anti-APS IgG levels.
The antibody level suggested to confer protection againstMenA disease is �1 to 2 �g/ml of total Ig against APS (31, 47).One would therefore consider nearly all of our Ethiopian pa-tients and controls to be protected. However, the suggestedputative protective level was assigned from a phase III protec-tion trial with APS vaccine in Finland in the 1970s using anradioimmunoassay and a single-donor reference serum (47). Inusing results from the standardized APS ELISA (11), oneought to apply this criterion carefully: for example, the well-characterized human reference serum CDC1992, used as acontrol in our study, was obtained by pooling sera from 14 U.S.individuals vaccinated with APS vaccine (24). It is well suitedfor analyzing APS vaccine-induced antibodies, but the aviditiesof these antibodies may differ from those of the antibodiesinduced by MenA disease or carriage in the African popula-tion. A Finnish study suggested that the cross-reactive anti-APS antibodies in normal sera were of lower avidity than thoseobtained by immunization with APS vaccine (30). Borrow andcolleagues (28, 29) found that the avidity of anti-APS IgGcorrelated positively with MenA SBA in sera from MenA con-jugate vaccinees and that an APS vaccine induced anti-APS
VOL. 14, 2007 ANTIBODY RESPONSES TO SEROGROUP A MENINGOCOCCI 459
IgG of lower avidity than the MenA conjugate vaccine. Thelevel of anti-APS correlating with protection against MenAdisease may need to be specifically established for the menin-gitis belt area. However, the protective level of antibodies isdetermined not only by the quantity but also by the quality ofthese antibodies; thus, the Ig isotype, IgG subclass, and aviditytoward APS are probably all of importance, since these affectthe SBA.
From the acute to the early convalescent phase of disease,we found that the IgG against APS increased significantly(Table 3), to levels equivalent to or higher than those observedfollowing vaccination with APS vaccine (3, 25, 33). However,only 35% of the patients experienced a �4-fold increase inanti-APS IgG levels, while in Finnish MenA patients the cor-responding proportion was 62% (31). The already high acute-phase levels for the Ethiopian patients compared to the Finn-ish patients may explain the difference observed. The highantibody levels, as well as the effect of MenA carriage acqui-sition (9), also complicate the use of a specific anti-APS IgGn-fold increase for serological confirmation of MenA disease inthe meningitis belt.
In the late convalescent phase, the mean anti-APS IgG levelshad returned to levels similar to those found in acute-phasesera (Table 2). As far as we know, this is the first study onlong-term antibody levels following MenA disease. Similar re-ductions to prevaccination levels after �8 months have beenobserved in clinical trials with APS and conjugate-PS vaccines(14, 60), although others have seen elevated anti-APS IgGlevels for a longer period after APS vaccination (66). In Ethi-opian control sera, as well as in acute-phase patient sera, wefound an age-related increase in the proportions of sera withanti-APS IgG levels above 4.7 �g/ml, the concentration equiv-alent to 50% of the GMC in controls (Fig. 3a). Such an age-related increase is in line with the findings of previous studies(31, 40).
Several studies have suggested that IgA antibodies againstAPS may render the population susceptible to meningococcaldisease (4, 20, 21). Anti-PS IgA is reported not to activatecomplement via the classical pathway and is suggested to blockthe bactericidal effect of anti-PS IgG antibodies by competitivebinding (21, 26). Provided that there is a constant IgG concen-tration, there should thus be an inverse relation between theIgA concentration and the SBA titer (21). In our study, anti-APS IgA levels correlated well with rSBA titers. Also, amongEthiopian controls aged �2 to �15 years who had high anti-APS IgG levels, the anti-APS IgA levels were significantlyhigher for those with protective rSBA titers than for thosewithout. This contradicts the findings of a previous study ofsera of the Sudanese population (4). The possible role ofserum IgA in mediating the lysis of meningococci via the al-ternative complement activation pathway seems minor (61).The complement source may, however, be relevant for thepotential ability of IgA to mediate SBA, but further studies arerequired to test this hypothesis.
Antibody responses against OMVs. In line with the findingsof a previous study (9), IgG levels against outer membraneantigens were significantly higher in acute-phase patient serathan in control sera when individuals above the age of 6 yearswere compared (Table 2). This contrasts with a similar com-parison of anti-APS levels that showed no significant differ-
ences between acute-phase patient sera and control sera, andwith a similar comparison of rSBA titers, which were lower inacute-phase patient sera than in control sera. This disparitymay be explained by the exposure of a high proportion ofpatients to MenA carriage prior to disease, since carriage ofMenA organisms has been shown to induce IgG against bothcapsular and noncapsular antigens (10). Significantly lowerlevels of anti-OMV IgG were observed for Norwegian teenag-ers, a finding that also may be explained by different exposureto MenA carriage. Acute-phase sera from patients with sys-temic serogroup B and C meningococcal disease in Norwaydemonstrated anti-OMV IgG levels similar to those for healthypopulation controls, while for septicemia patients, significantlylower anti-OMV IgG levels were demonstrated at admission(22). Our findings for Ethiopian patients contrast with theseresults, but further studies of septicemia patients from themeningitis belt would be interesting.
Meningococcal meningitis was also found to induce signifi-cant rises in levels of IgG antibodies against OMVs, in linewith previous findings for MenA (9, 58) and MenB (22, 48, 53,57) patients. However, only 32% showed a �4-fold increase inlevels of IgG against OMVs, which can be explained by thealready high levels in acute-phase sera. Considering that a�2-fold increase in anti-OMV IgG levels was demonstratedfor 61% of confirmed MenA disease patients, approximatelythe same as the proportion demonstrating a �4-fold increasein SBA titers (Table 3), a �2-fold increase may be indicative ofMenA disease in the meningitis belt.
Functional activity of antibodies as analyzed by SBA. Wefound a significantly lower rSBA GMT in acute-phase patientsera than in control sera among adults, but not in the youngerage groups. However, few sera were available for comparisonin these age groups. A subcritical level of SBA was demon-strated to correlate with an increased risk of serogroup Cmeningococcal infection for U.S. military recruits, as measuredwith human complement against the prevailing serogroup Cstrain (16). However, this could not be confirmed by Green-wood et al. in a study of Gambian MenA patients, as measuredby using baby rabbit complement and a local MenA epidemicstrain (20). For both acute-phase patient sera and control sera,we observed high proportions considered protected by therSBA assay. This is in agreement with results from studies ofprevaccination sera in clinical vaccine trials (12). While theproportion of controls with protective rSBA titers increasedwith age, as was observed previously (3, 40), in acute-phasepatient sera the titers peaked in children aged �2 to �6 years.Among Ethiopian controls, the proportion with hSBA titers of�4 was 19%. Using a different assay, Goldschneider et al.found a similar proportion with hSBA titers of �4 against aMenA strain among baseline sera (predisease sera collected atenrollment) from meningococcal patients, while the propor-tion was 72% for baseline sera from individuals who did notcontract meningococcal disease (16). Achtman et al. have sug-gested that “disease might reflect the inability of the colonizedindividual to quickly mount a protective antibody response”(2); protection may thus consist of both a minimum level ofcirculating functional antibodies and a sufficient pool of mem-ory B cells specific for functional antibodies (32). EstablishingSBA assay conditions correlating with protection and assigning
460 NORHEIM ET AL. CLIN. VACCINE IMMUNOL.
a protective MenA SBA titer seem pivotal for the evaluation ofvaccines against MenA disease for the meningitis belt.
Significant titer rises were demonstrated for MenA patientsfrom the acute phase to the early convalescent phase, and theproportion of sera with protective hSBA titers increased from53% to 77%. This is higher than that reported for MenAmeningitis patients infected with clone IV-1 or subgroup IIIstrains from Gambia and Finland, respectively (2); in this studyvery few of the patients demonstrated the development ofbactericidal antibodies following disease. The high percentagesfound in our study may be due to differences in the SBA assays,although in sera from MenB patients aged 10 to 17 years, 75 to100% demonstrated the achievement of hSBA titers of �4during the convalescent phase (48). The proportion of menin-gococcal disease patient sera attaining protective rSBA titersincreased from 40% in the acute phase to 95% in the earlyconvalescent phase, a proportion similar to that achieved dur-ing vaccination with MenA vaccines (14). Since the rSBA titerswere already high in the acute-phase sera, it seems reasonablethat seroresponse rates were only 57% for rSBA (Fig. 2c). Theseroconversion rates for rSBA and hSBA were 100% and 53%,respectively. In comparison, vaccination of individuals 11 to 18years old with APS conjugate vaccines resulted in seroresponserates of 88% (rSBA) and 94% (hSBA) and seroconversionrates of 100% (rSBA) and 92% (hSBA) (34). Thus, in the shortterm, suffering from MenA disease seems to provide immuno-logical protection in a similar proportion of individuals asimmunization with APS and MenA conjugate vaccines.
A central question emerging from our findings is whethersurvivors of MenA disease in the meningitis belt are protectedagainst reinfection with a similar strain, i.e., what the durationof the achieved protection is. In this study, the general picturewas that although the proportion of individuals considered notprotected by rSBA or hSBA was only 3% in early convalescentphase, this decreased in the late convalescent phase to a pro-portion similar to that in the acute phase (Table 5). The timebetween the collection of early- and late-convalescent-phasesera varied greatly; thus, reporting the median response alonehides the gradual decay in IgG levels and SBA titers.
The returning waves of MenA disease in the meningitis beltcould be explained by the introduction of new clones in apopulation rendered susceptible due to waning immunity tothe prevalent clone and lack of immunity to the newly intro-duced clone (39). The introduction of new serogroup A N.meningitidis clones into the meningitis belt has been well doc-umented (2, 41, 42, 67), while the probable waning protectiveimmunity following disease has been the subject of only a fewstudies (2, 9). Vaccination with APS vaccine has been shown toinduce protection suggested to last as long as 3 years for adults(46). However, the dosing and presentation of antigen are verydifferent in vaccination and infection, and thus, the magnitudeof the response and the duration of protection conferred byinfection are not necessarily indicative of an antigen’s ability toinduce long-term protection when used as a vaccine (63).
Correlations. To evaluate the level of protection againstMenA disease, we analyzed sera by two different SBA assays(rSBA and hSBA). A major part of the patients apparentlyachieved immunological protection following MenA disease,since 74 to 94% of the patients mounted a protective antibodyresponse, by having putative protective titers of both rSBA and
hSBA in early convalescent phase (Table 5). However, theindividual results of our rSBA and hSBA assays correlated onlymodestly, and the individuals classified as protected by the twoassays were not the same. This is in agreement with previousobservations (34) and raises the questions of whether one ofthe methods may incorrectly categorize individuals as pro-tected and whether the SBA test can be used only for evalua-tion of susceptibility to disease at a population level and not onan individual basis.
Our study showed that the choice of method (parameters) iscritical in the evaluation of the ability of antibodies to conferSBA. In general, the titers obtained by the hSBA assay wereconsiderably lower than those obtained by the rSBA assay (Fig.4). This could be due to either the complement source or thetarget strain used. Using a limited number of sera, we com-pared the SBA titers against either strain F8238 or strain Mk686/02 obtained with human complement with those obtainedwith rabbit complement with the same strain. We then foundvery low hSBA activity by using the F8238 target strain (datanot shown), as also observed previously (64). On the otherhand, the ST-7 strain Mk 686/02 was more sensitive to rabbitcomplement (data not shown). The different titers obtained bythe rSBA and hSBA assays are primarily due to differences inthe complement source and target strains. Human anti-menin-gococcal IgG is known to mediate higher MenA SBA titers inthe presence of rabbit complement than in the presence ofhuman complement (64). Also, IgM against meningococcal PSis suggested to have a higher ability to mediate SBA with rabbitcomplement than with human complement (55). Part of theMenA rSBA response observed in the acute phase in our studymay thus have been caused by anti-MenA IgM. Although bothtarget strains in the SBA assays were characterized as A:4/21:P1.20,9:FetA 3-1:L11 (43, 45), subtle differences in outer mem-brane antigens between the ST-5 and ST-7 target strains usedin the SBA assay (6, 45), as well as in possibly variable amountsof capsule expressed, may have had an impact on the lowcorrelation observed between hSBA and rSBA.
By analyzing the specificity of the SBA response mountedduring disease, we showed that the variability in anti-APS IgGlevels explained a substantial proportion of the observed vari-ation in rSBA titers, whereas IgG against OMV apparently wasless important for the rSBA variation. This was also reflectedin the low correlation between rSBA titers and anti-OMV IgGlevels. Again, minor outer membrane antigen differences be-tween the rSBA target strain and the strain from which thedOMVs used in the ELISA were derived may have contributedto the observed correlations. In addition, some proportion ofthe rSBA variability may just as well be attributed to antibodiesdirected against, e.g., LOS or minor proteins not present in thedOMVs but expressed on live bacteria. In contrast to rSBA,the variability in hSBA could well be explained by both IgGagainst APS and IgG against OMVs, as reflected by correlationanalyses (Fig. 5). It seems from the sera studied here that bothIgG against APS and IgG against OMVs are important ininducing protection when the hSBA assay is used to assess theimmune response. SBA assays performed with human comple-ment may more closely reflect in vivo protection against me-ningococcal disease than SBA assays performed with rabbitcomplement. Thus, induction of antibodies toward noncapsu-lar antigens may represent a possibility for the improvement of
VOL. 14, 2007 ANTIBODY RESPONSES TO SEROGROUP A MENINGOCOCCI 461
current vaccines against MenA disease in the meningitis belt(43).
Further studies are needed to determine the specificities ofthe noncapsular antibody response during MenA disease andtheir relative importance for providing protection againstMenA disease. A notable proportion of sera had high concen-trations of IgG against APS and OMVs without conferringbactericidal activity (Fig. 5), irrespective of the SBA method.Any protection possibly conferred by antibody-mediated op-sonic activity following disease should thus be investigated withthese MenA disease patient sera. However, a first priorityshould be to standardize an SBA assay using a recent repre-sentative serogroup A strain with human complement and thento establish whether the initial Goldschneider studies, showinga correlation between SBA and protection (16), can be paral-leled in the meningitis belt.
ACKNOWLEDGMENTS
Berhanu Melak of the North Gondar Zonal Health Bureau isthanked for participating in the field study. Ulla Heggelund, KirstenKonsmo, and Berit Nyland are thanked for excellent technical assis-tance. Abdi Ali Gele is thanked for performing the OMV ELISA.Milada Smaastuen is thanked for statistical advice. The institutionsYirgalem Hospital, University of Gondar, North Gondar Zone HealthBureau, SNNPR Health Bureau, AHRI, and ALERT are thanked forexcellent support in this project.
This work was in part supported by grant 146185/730 from theResearch Council of Norway.
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