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Antibody-Dependent Cellular Inhibition Is Associated With Reduced Risk
Against Febrile Malaria in a Longitudinal Cohort Study Involving Ghanaian
Children
Article  in  Open Forum Infectious Diseases · March 2015
DOI: 10.1093/ofid/ofv044
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B R I E F R E P O R T
Antibody-Dependent Cellular however, the possible mechanisms by which these antibodies
function have not been adequately explained. The antibody-de-
Inhibition Is Associated With pendent cellular inhibition (ADCI) assay measures the overall
Reduced Risk Against Febrile functional effect of antibodies and monocyte (MN) collabora-
Malaria in a Longitudinal Cohort tion on in vitro parasite growth and has aided the discovery
and development of malaria vaccine candidates such as mero-
Study Involving Ghanaian Children zoite surface protein 3 and the glutamate rich protein [1, 2]. Sev-
eral studies have assessed ADCI activity in relation to malaria
Regis W. Tiendrebeogo,1,2 Bright Adu,1,2,a Susheel K. Singh,1,2 immunity [1–5]; however, the importance of this mechanism
Morten H. Dziegiel,3 Issa Nébié,4 Sodiomon B. Sirima,4
1 in protection against malaria in longitudinal cohort studiesMichael Christiansen, Daniel Dodoo,5 and Michael Theisen1,2
(LCS) is yet to be evaluated. In this study, we successfully as-
1Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum
Institut, Copenhagen, 2Centre for Medical Parasitology at Department of sessed the relationship between ADCI activity of IgG before
International Health, Immunology, Microbiology, and Department of Infectious malaria season and P. falciparum infection outcome for the
Diseases, Rigshospitalet, University of Copenhagen, and 3Blood Bank KI 2034, first time in well characterized LCS of Ghanaian children.
Copenhagen University Hospital, Denmark; 4Centre National de Recherche et de
Formation sur le Paludisme, Ouagadougou, Burkina Faso; and 5Noguchi Memorial
Institute for Medical Research, University of Ghana, Legon MATERIALS AND METHODS
The antibody-dependent respiratory burst and opsonic Ethics Statement
phagocytosis assays have been associated with protection The LCS was approved by the Institutional Review Board of
against malaria; however, other mechanisms may also be in- Noguchi Memorial Institute for Medical Research of University
volved. The antibody-dependent cellular inhibition (ADCI) of Ghana, Accra, Ghana. Written informed consent was ob-
assay is yet to be correlated with protection in longitudinal tained from parents or guardians of children before enrollment
cohort studies (LCS). We investigated the relationship be- into the study. Use of Danish blood donor samples was ap-
tween ADCI activity of immunoglobulin G before malaria proved by the Scientific Ethics Committee of Copenhagen
season and risk of malaria in a LCS involving Ghanaian chil- and Frederiksberg, Denmark.
dren. High ADCI activity was significantly associated with re-
duced risk against malaria. Findings here suggest a potential Study Design
usefulness of the ADCI assay as a correlate of protection to From May 2008 to February 2009, a 42-week malaria LCS,
guide malaria vaccine studies. detailed elsewhere [6], was conducted in Asutsuare, Ghana. In
Keywords. antibody-dependent cellular inhibition; chil- brief, 797 children (aged 1–12 years) were enrolled and ob-
dren; longitudinal cohort study; malaria; monocytes; Plasmodi- served both actively and passively for malaria case detection.
um falciparum. Baseline venous blood plasma was stored at −80°C until use,
and thick and thin film blood slides (TTBS) were obtained.
Sickle cell status and ABO blood group were determined by
The importance of immunoglobulin (Ig) G antibodies in immu- the sodium metabisulphite test and a commercial blood group-
nity against Plasmodium falciparummalaria remains undisputed; ing kit (Biotec Laboratories Limited, UK), respectively. Once
every month, TTBS was obtained from each child for asymp-
tomatic parasitemia assessment. Febrile malaria was defined
Received 4 December 2014; accepted 30 March 2015.
aPresent affiliation: Noguchi Memorial Institute for Medical Research, University of Ghana, as fever (axillary temperature ≥37.5°C, measured or reported)
Legon, Ghana. with slide positive for any asexual P. falciparum parasitemia
Correspondence: Michael Theisen, DMSc, PhD, Department of Clinical Biochemistry and Im-
munology, Statens Serum Institute, 5 Artillerivej, 2300 Copenhagen S, Denmark (mth@ssi.dk). and at least 1 other sign of malaria such as vomiting, diarrhoea,
Open Forum Infectious Diseases or malaise. At the end of the study, children in whom parasite-
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases mia was associated with febrile malaria were considered sus-
Society of America. This is an Open Access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ ceptible, whereas those who did not experience any febrile
by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any malaria despite parasitemia were considered protected. Others
medium, provided the original work is not altered or transformed in any way, and that the work
is properly cited. For commercial re-use, please contact journals.permissions@oup.com. had no detectable parasitemia by microscopy and no malaria
DOI: 10.1093/ofid/ofv044 symptoms [6].
BRIEF REPORT • OFID • 1
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Antibodies To facilitate interplate comparisons, sample ADCI activity
Test IgG was purified from approximately 300–500 µL of plasma (ie, SGI) values were normalized to the PHIG as follows: (Sam-
from Ghanaian children and Danish controls using protein G ple-SGI/Plate PHIG-SGI) × Mean PHIG-SGI all plates. The
coupled to Sepharose (GE Healthcare) as described previously PHIG-SGI coefficient of variation was 29.85%. The association
[7]. Purity and concentration were checked with sodium dodecyl between SGI and age (continuous scale) was assessed by multi-
sulfate polyacrylamide gel electrophoresis and NanoDrop, respec- variate linear regression analysis adjusting for covariates. To
tively, and IgG was stored at −20°C until use. Pooled IgG from evaluate whether the ADCI activity influences time-to-first ma-
hyperimmune African adults (PHIG) or malaria-naive Danes laria episode, SGI was categorized into tertiles. Kaplan–Meier
(PNIG) were the positive and negative controls, respectively [8]. survival curves and log-rank test were used to compare the ter-
tile categories, and hazard ratios were calculated by Cox propor-
Antibody-Dependent Cellular Inhibition Assay tional hazards regression analysis. Children were either high
The NF54 strain of P. falciparum was cultured and ADCI assay ADCI responders (with SGI values in the top tertile) or low
performed as described [8, 9]. In brief, Danish blood donor pe- ADCI responders (with SGI values in the bottom and middle
ripheral blood mononuclear cells were isolated by LymphoPrep tertile). Receiver-operating characteristic (ROC) analysis was
(Lonza), and approximately 2 × 105 MNs/well were selected used to determine sensitivity and specificity associated with
by adherence in a flat-bottom 96-well culture plate (Nunc, the SGI top tertile as a cutoff for classifying protected and sus-
Denmark). Highly synchronized schizont stage P. falciparum at ceptible children. Antibody titers were compared between high
0.5% parasitemia and 2.5% hematocrit were added at 100 µL/ and low ADCI responders (Welch’s t test). Raw P values and
well followed by test and control IgG at 0.5 mg/mL and 1.0 confidence limits with Bonferroni-corrected P values are
mg/mL final concentrations, respectively, to designated duplicate shown for antibody data analyses. Significance level was at 5%.
wells. Volume was adjusted to 200 µL with parasite growth me-
dium (PGM) (RPMI 1640 [Lonza] + 0.5% Albumax) [9]. An ad- RESULTS
ditional 50 μL PGMwas added per well at 48 hours and 72 hours,
and the assay was stopped after 96 hours. Final parasitemia was Study Demographics and Malaria Incidence
determined as described previously [8], and specific growth in- Febrile malaria incidence was low (15.1%), and 168 children were
hibitory index (SGI) was calculated: SGI = 100 × (1 – [%parasite- considered “definitively” exposed by recording at least 1 febrile
mia with MN and test antibodies/% parasitemia test antibodies]/ malaria episode (susceptible group) or 1 monthly blood slide
[%parasitemia with MN and PNIG/% parasitemia PNIG]). positive for asexual stage P. falciparum without any clinical
symptoms (protected group) throughout the study period [12].
Schizont Extract Enzyme-Linked Immunosorbent Assay Data presented here are for 98 (protected = 35; susceptible = 63)
Flat-bottom 96-well microtiter plates (Nunc) were coated with children with definitive exposure who had sufficient volumes of
100 µL/well of 5 μg/mL crude schizont antigen, obtained as de- baseline plasma available for IgG purification. These 98 children
scribed previously [10], in 0.05 M carbonate buffer and incubated were similar in age (P = .27, Welch’s t test) to the 168 exposed
overnight at 4°C. The remaining enzyme-linked immunosorbent children [12] but older (P = .025, Welch’s t test) than the entire
assay procedure was as described previously [11], with modifica- 669 children who successfully completed follow-up [6], because
tions. Samples were tested at 65 μg/mL and 130 μg/mL for IgG exposed but younger children were excluded from the current
and subclasses quantification, respectively. The detection anti- study due to insufficient plasma for IgG purification. Older chil-
bodies were as follows: horseradish peroxidase (HRP)-conjugated dren (6–12 year old) were protected from febrile malaria (odds
polyclonal rabbit anti-human IgG (Dako, Denmark) at 1:5000 di- ratio [OR] = 0.34; 95% CI, .13–.84; P = .021) compared with
lution; HRP-conjugated sheep anti-human IgG1 (AP006), IgG2 younger (1–5 year old) children. Similarly to previous reports
(AP007), or IgG3 (AP008) (The Binding Site, UK) at 1:4000, on the larger cohort [12], sex, sickle cell status, and ABO blood
1:2000, 1:3000 dilutions, respectively. group also had no influence on the outcome of P. falciparum in-
fection in this subpopulation (PLRT > .05) (Supplementary
Statistical Analysis Table 1). Baseline parasitemia levels showed no association
Data analyses were performed with R, version 3.1.2 (http://www.R- (P = .29) with febrile malaria in a multivariate logistic regression
project.org/). Age was categorized (1–5 and 6–12 years), and asso- adjusting for covariates.
ciations between febrile malaria and covariates (age group, sex,
sickle cell status, and ABO blood group) were assessed by multi- Antibody-Dependent Cellular Inhibition Activity Increases
variate logistic regression models and likelihood ratio tests (LRTs). With Age
The parasitemia after the 96-hour ADCI assay were mean = 3.67%, The distribution of ADCI activity (SGI) ranged from −30.39% to
95% confidence interval [CI], 3.12–4.22 and mean = 5.89%, 95% 103.10% with a median of 46.18%. Multivariate linear regression
CI, 5.21–6.57, for wells with and without MNs, respectively. found a significant increase (OR = 5.21; 95% CI, 1.22–22.27;
2 • OFID • BRIEF REPORT
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≥2500 did not change (OR = 0.93; 95% CI, .88–.97; P = .0026;
n = 64) the association between SGI and malaria. There was a
significant difference (log-rank test P = .00085) in the time-to-
first malaria episode for children in the different SGI tertiles
(Figure 1). Using children in the SGI bottom tertile as reference
in a Cox regressionmodel adjusted for age group, we noted a sig-
nificantly reduced risk of febrile malaria for children in the top
tertile (hazard ratio [HR] = 0.30; 95% CI, .15–.59; P = .00044)
but not those in the middle tertile (HR = 0.64; 95% CI, .37–
1.14; P = .13). Thus, it appears a rather high threshold of ADCI
activity was necessary to achieve a significant reduction in the
risk of febrile malaria. The ROC analysis showed that a cutoff
at the top SGI tertile (SGI≥ 52.5%) had a sensitivity and specif-
icity of 79.4% and 57.1%, respectively, in classifying protected
and susceptible children in this cohort.
Relationship Between Anti-schizont Extract Immunoglobulin
G Levels, Risk of Malaria, and Specific Growth Inhibitory Index
Total IgG (P = .0035), IgG1 (P = .031), and IgG3 (P = .001) but
Figure 1. High threshold of antibody-dependent cellular inhibition not IgG2 levels against crude schizont antigens were signifi-
(ADCI) activity is associated with reduced risk of febrile malaria. Children cantly associated with reduced risk against febrile malaria in a
were categorized based on ADCI activity (ie, specific growth inhibition multivariate logistic regression adjusting for age groups (Sup-
index [SGI]) into tertiles: top tertile (blue line), middle tertile (green line),
and bottom tertile (red line). There was a statistically signi cant difference plementary Table 2). To determine whether ADCI was associ-fi
in the risk of malaria (log-rank test P = .00085) among children in the dif- ated with IgG responses, children were categorized as either
ferent SGI categories. The Kaplan–Meier estimates of malaria-free proba- high (SGI ≥ 52.5%) or low (SGI < 52.5%) ADCI responders.
bility showed that children in the top SGI tertile category seemed to have a Only IgG3 levels was significantly different (t = 2.66; 95% CI,
much reduced risk compared with those in the middle and bottom tertiles. .59–4.06; P = .037) between high and low responders (Table 1).
Red crosses denote censored observations.
DISCUSSION
P = .026) in SGI with age of children but not with the other co- Using a well characterized LCS, we show for the first time that
variates (P > .05). ADCI activity of IgG is significantly associated with reduced risk
against febrile malaria. Although ADCI activity increased with age,
Association Between Antibody-Dependent Cellular Inhibition its association with malaria remained significant in multivariate
and Febrile Malaria models that were adjusted for age. Children with ADCI activity
Multivariate logistic regression analysis adjusting for the covar- within the top tertile of the distribution had a significantly delayed
iates showed increasing SGI was significantly (OR = 0.94; 95% time-to-first malaria episode in the cohort, and this threshold clas-
CI, .90–.97; P = .00056) associated with reduced risk against fe- sified protected and susceptible children with sensitivity and spe-
brile malaria. Redefining febrile malaria with parasitemia cificity of 79.4% and 57.1%, respectively. The lower specificity was
Table 1. Association Between Antibody-Dependent Cellular Inhibition and Antibody Responses Against Crude Schizont Antigensa
Anti-schizont High Responders Low Responders t Statistic Adjusted
Extract (n = 33) Mean (n = 65) Mean (95% CI) P Value P Value
IgG −0.08 −1.93 2.19 (.16–3.55) .032 .12
IgG1 1.33 0.067 1.23 (−.68–3.20) .20 .80
IgG2 −0.68 −1.80 1.40 (−.48–2.72) .17 .68
IgG3 −0.90 −3.23 2.66 (.59–4.06) .0093 .037
Abbreviations: CI, confidence interval; IgG, immunoglobulin G; SGI, specific growth inhibition index.
a Children were divided on the basis of SGI into high responder (ie, SGI values in the top tertile, SGI ≥ 52.5%) and low responder (ie, SGI values in the bottom and
middle tertile, SGI < 52.5%) groups, and antibody levels were compared between the groups by the Welch’s t test. Antibody titers were log to base 2 transformed,
and P values are shown without adjustment and after Bonferroni adjustment for multiple testing. Some antibody titers were negative after log base 2
transformation, which ultimately resulted in some negative mean values. Bold text indicate significant P value after multiple testing correction.
BRIEF REPORT • OFID • 3
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due to the overlapping distribution of ADCI activity among pro- malaria, suggesting its potential usefulness as a correlate of pro-
tected and susceptible children, suggesting that other mechanisms tection in malaria vaccine studies.
may contribute to protection in this cohort.
Several studies have evaluated the ADCI assay as a potential Supplementary Material
correlate of malaria immunity [3, 4], but until now its impor-
tance in assessing immunity in LCS had not been evaluated. Supplementary material is available online at Open Forum Infectious Diseases
The ADCI mechanism is thought to be triggered by synergistic (http://OpenForumInfectiousDiseases.oxfordjournals.org/).
activation of FcγRIIA and FcγRIIIA on MNs when cross-linked
with cytophilic antibodies bound to target antigens [4, 5]. This Acknowledgments
interaction signals the release of largely uncharacterized soluble We thank the children and their parents and guardians fromAsutsuare and
factors, including tumor necrosis factor (TNF)-α, which act in its environs who volunteered to participate in the study, and without whose
concert to block the division of surrounding intraerythrocytic cooperation this study would have been impossible. The Afro Immuno Assay
2 Field and laboratory personnel are duly acknowledged for their support.
uninucleate stage parasites [4]. The unspecific nature of the ac- Financial support. This study was supported by grants from the Danish
tive antiparasite mediators makes the ADCI mechanism para- Council for Strategic Research (grant 13127), the European and Developing
site strain independent [3], which suggests that it may play a Countries Clinical Trials Partnership (grants IP.2007.31100.001 and
TA.2007.40200.012), and the African Malaria Network Trust (grant 008/
vital role in premunition, a nonsterile form of immunity in 2008AIA).
which low levels of parasites are tolerated in the absence of clin- Potential conflicts of interest. All authors: No reported conflicts.
ical symptoms [13]. In malaria-endemic populations, protective All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
immunity develops gradually in relation to exposure to the par- tent of the manuscript have been disclosed.
asite and age. This is consistent with the significant association
of age with both protection against febrile malaria and increas- References
ing ADCI activity here. The apparent lack of protection with
sickle cell may be due to its low prevalence in the cohort. The 1. Oeuvray C, Bouharoun-Tayoun H, Gras-Masse H, et al. Merozoite sur-
face protein-3: a malaria protein inducing antibodies that promote Plas-
rather high ADCI activity necessary for protection against ma- modium falciparum killing by cooperation with blood monocytes.
laria may reflect the time-dependent nature of a mechanism Blood 1994; 84:1594–602.
that mirrors cumulative exposure and premunition. 2. Theisen M, Soe S, Oeuvray C, et al. The glutamate-rich protein
Malaria immunity may be a composite of several mechanisms. (GLURP) of Plasmodium falciparum is a target for antibody-dependent
monocyte-mediated inhibition of parasite growth in vitro. Infect
In the current cohort, we previously identified polymorphisms in Immun 1998; 66:11–7.
FcγRIIIB that modify antibody functionality to be associated with 3. Bouharoun-Tayoun H, Attanath P, Sabchareon A, et al. Antibodies that
risk of malaria [12], thus implicating neutrophil-mediated mech- protect humans against Plasmodium falciparum blood stages do not on
their own inhibit parasite growth and invasion in vitro, but act in coop-
anisms. In this study, cytophilic IgG antibodies were associated eration with monocytes. J Exp Med 1990; 172:1633–41.
with reduced risk [14], and the importance of IgG3 in the 4. Bouharoun-Tayoun H, Oeuvray C, Lunel F, et al. Mechanisms un-
ADCI mechanism was substantiated [5]. Although the ADCI derlying the monocyte-mediated antibody-dependent killing of Plas-
modium falciparum asexual blood stages. J Exp Med 1995; 182:
assay used here and the opsonic phagocytosis assay recently as- 409–18.
sociated with protection against malaria [15] were different, they 5. Jafarshad A, Dziegiel MH, Lundquist R, et al. A novel antibody-
share fundamental similarities. In essence, both assays are medi- dependent cellular cytotoxicity mechanism involved in defense against
ated by cytophilic IgG and involve merozoites opsonization and malaria requires costimulation of monocytes FcgammaRII and Fcgam-
maRIII. J Immunol 2007; 178:3099–106.
MN activation to release soluble factors, including TNF-α [4, 15], 6. Adu B, Dodoo D, Adukpo S, et al. Fc gamma receptor IIIB (FcgammaR-
which facilitate parasite killing [4]. Thus, it is plausible that these IIIB) polymorphisms are associated with clinical malaria in Ghanaian
2 mechanisms are not mutually exclusive; however, the extent of children. PLoS One 2012; 7:e46197.
7. Dahlback M, Jorgensen LM, Nielsen MA, et al. The chondroitin sulfate
any such potential overlap is not clear [4]. A-binding site of the VAR2CSA protein involves multiple N-terminal
A limitation of this study is the small sample size involved, domains. J Biol Chem 2011; 286:15908–17.
potentially restricting a wider generalization of our conclusions. 8. Tiendrebeogo RW, Adu B, Singh SK, et al. High-throughput tri-colour
flow cytometry technique to assess Plasmodium falciparum parasitae-
Nonetheless, we have clearly shown that the ADCI mechanism mia in bioassays. Malar J 2014; 13:412.
is associated with reduced risk against malaria in this LCS. Fur- 9. Jogdand PS, Singh SK, Christiansen M, et al. Flow cytometric readout
ther studies in larger cohorts would be crucial in improving our based on Mitotracker Red CMXRos staining of live asexual blood stage
understanding of ADCI in malaria immunity. malarial parasites reliably assesses antibody dependent cellular inhibi-
tion. Malar J 2012; 11:235.
10. Wahlgren M, Berzins K, Perlmann P, et al. Characterization of the
CONCLUSIONS humoral immune response in Plasmodium falciparum malaria. I.
Estimation of antibodies to P. falciparum or human erythrocytes by
means of microELISA. Clin Exp Immunol 1983; 54:127–34.
In conclusion, we have shown for the first time in a malaria LCS 11. Nebie I, Diarra A, Ouedraogo A, et al. Humoral and cell-mediated
that the ADCI assay is associated with reduced risk against immunity to MSP3 peptides in adults immunized with MSP3 in
4 • OFID • BRIEF REPORT
Downloaded from http://ofid.oxfordjournals.org/ at Danmarks NaturOG on May 13, 2015
malaria endemic area, Burkina Faso. Parasite Immunol 2009; 31: 14. Perraut R, Marrama L, Diouf B, et al. Distinct surrogate markers for
474–80. protection against Plasmodium falciparum infection and clinical malar-
12. Adu B, Jepsen MP, Gerds TA, et al. Fc gamma receptor 3B (FCGR3B- ia identified in a Senegalese community after radical drug cure. J Infect
c.233C>A-rs5030738) polymorphism modifies the protective effect of ma- Dis 2003; 188:1940–50.
laria specific antibodies in Ghanaian children. J Infect Dis 2014; 209:285–9. 15. Osier FH, Feng G, Boyle MJ, et al. Opsonic phagocytosis of Plasmodium
13. Sergent E. [Definition of immunity & premunition]. Ann Inst Pasteur falciparummerozoites: mechanism in human immunity and a correlate
(Paris) 1950; 79:786–97. of protection against malaria. BMC Med 2014; 12:108.
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