Study design
The study was conducted in the district of Tori Bossito in southwest Benin between July 2007 and January 2010, where 567 children were followed-up from birth to 18 months of age. This follow-up consisted in an epidemiological, parasitological and immunological follow-up detailed elsewhere [26,27,28]. Health workers recruited by the program visited the infants at home every week in order to check their health status. In case of fever (axillary temperature > 37.5 °C), a rapid diagnostic test (RDT) and/or thick blood smear examination (TBS) were performed. In case of symptomatic malaria (fever and positive RDT/TBS), an anti-malarial treatment (artemether/lumefantrine) was administered according to the national guidelines that were applied at the time of the study.
TBS were also performed monthly to detect asymptomatic malaria infections. In addition, mothers were invited to bring their infants to the health centre, at any time, in case of fever (suspected by the mother) or clinical signs, whether or not related to malaria, and the same protocol was applied. Venous blood samples were collected quarterly for hematological and immunological measurements. Venous blood was centrifuged for plasma isolation and genomic DNA extraction. IgG1 and IgG3 responses against P. falciparum vaccine antigen candidates AMA1, MSP1, MSP3, MSP2-3D7, MSP2-FC27, GLURP-R0 and GLURP-R2 were quantified at 6, 9, 12, 15 and 18 months by ELISA [6]. IgG purification was performed for the 106 plasma samples with sufficient volumes at 15th or 18th month time-points. Among them, 27 samples belonged to the group of infants able to control asymptomatic malaria infections over time (CAIG).
The control of asymptomatic malaria infection was defined as follow: the clearance of blood P. falciparum parasites or at least maintain P. falciparum at very low parasitaemia, below the detection threshold of thick blood smear [6]. CAIG group was compared with two other groups: a group of 53 children infected by P. falciparum but not able to control asymptomatic malaria infection (IG group) and a group of 23 children for which P. falciparum has not been detected by TBS examination during the survey (NPfDG group). These two groups were composed of all other infants for whom sufficient plasma volume was available.
IgG purification and IgG controls
Ab SpinTrap™ columns containing protein G coupled to Sepharose were used to purify IgG from 60 μl of plasma diluted 1/10 in phosphate buffered saline (PBS), as per manufacturer's instructions (Sigma-Aldrich). IgG concentration was measured using a NanoDrop®ND-1000 spectrophotometer, and samples were stored at −80 °C. Purified IgG samples were diluted in PBS to obtain a final concentration of 50 ng/mL before performing OP assays. Pooled IgG from malaria-exposed hyperimmune Liberian adults or malaria-non immune Danish blood donors served as internal controls [29].
Plasmodium falciparum culture and preparation of merozoites
P. falciparum line NF54 was cultured and merozoite isolation was performed as described previously [30]. Briefly, parasites were maintained in O + human erythrocytes at 3–4% haematocrit in parasite growth medium (RPMI 1640 Gibco™ supplemented with 25 mM HEPES, 0.5% AlbuMAX, 4 mM l-glutamine, 0.02 g/l hypoxanthine, and 25 μg/ml gentamicin) at 37 °C in a humidified 5% CO2, 2% O2 and 93% N2 atmosphere. The parasitaemia was monitored by examination of Giemsa-stained thin blood smears, and parasite cultures were synchronized with 5% sorbitol treatment for 10 min. Synchronized trophozoite-stage parasites were harvested using a magnetic separation column (Vario MACS) and subsequently treated with 10 μM of epoxysuccinyl-l-leucylamido (4-guanidino) butane (E64) for 6–8 h to allow schizonts to mature without rupture. Segmented schizonts were centrifuged, resuspended in 4–6 ml RPMI 1640 medium, and then filtered through a 1.2 μm/32 mm syringe filter to obtain merozoites. The filtrate was passed over an LS MACS Column twice to remove the haemozoin. Free merozoites were stained with 10 μg/ml of ethidium bromide (EtBr) for 30 min before used in OP assay.
Human neutrophil purification and culture of THP-1 cells
Primary neutrophils were purified from freshly drawn blood from a healthy Danish donor as described previously [17]. Briefly, blood was layered over a Percoll solution (1.077 g/ml) and centrifuged at 800 g for 25 min. The granulocyte-containing band was transferred to ten volumes of RBC lysis solution (155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA) and incubated for 10 min.
The cells were centrifuged and further purification of neutrophils was carried out with the EasySep Human Neutrophil Isolation Kit (StemCell Technologies) according to the manufacturer’s protocol. For OP assay, neutrophils were resuspended in cell medium (RPMI 1640, 10% fetal bovine serum (FBS), 4 nM l-glutamine and 25 μg/ml gentamicin) and distributed in 96-well, U-bottom plates (1 × 105 cells/150 µl/well). The human monocytic cell line THP-1 was maintained as described previously [31]. Likewise, neutrophils, viable THP-1 cells were distributed in 96-well, U-bottom plates (1 × 105 cells/150 µl/well).
Opsonic phagocytosis assay measured by flow cytometry
The OP assay was performed as described in detail [16]. Briefly, freshly isolated EtBr-stained merozoites were opsonized with 0.05 mg/ml IgG (controls and test samples) for 20 min. Aliquots of 50 μl of opsonized merozoites were co-incubated with pre-seeded primary neutrophils or THP-1 cells. The plates were incubated for 35 min at 37 °C in a 5% CO2 humidified incubator. To stop phagocytosis, plates were centrifuged for 5 min at 400 g at 4 °C, followed by 2 washes with cold fluorescence-activated cell sorting (FACS) buffer (PBS + 0.5% BSA + 2 mM EDTA). Then, cells were fixed with 200 μl of FACS fixative (FACS buffer + 2% paraformaldehyde) and kept at 4 °C until analysed in a CytoFLEX S flow cytometer (Beckman Coulter). Viable neutrophils or THP-1 cells were gated by forward scatter and side scatter properties, and EtBr-positive events were enumerated using primary neutrophils or THP-1 cells alone [16]. The percentage of primary neutrophils or THP-1 cells containing EtBr-stained merozoites determined the level of phagocytosis, which was expressed as the phagocytosis index (PI). Data analyses were performed with Kaluza Analysis Software (version 2.1).
IgG Gm phenotypes
The determination of Gm allotypes was performed before our study and included in our statistical analyses. Detailed description on the determination of IgG Gm allotypes of the Tori Bossito cohort is available elsewhere [26]. Briefly, the Gm allotype determination was performed for infants for whom a sufficient quantity of plasma (200 µl) was available at 15 months of age. The Gm allotype diversity of infants was confirmed by means of its consistency (inheritance) with this of both biological parents. G1m [1,2,3, 17] and G3m [5, 6, 10, 11, 13,14,15,16, 21, 24, 28] allotypes were determined in plasma samples by a standard haemagglutination inhibition method which allowed to consider resulting Gm phenotypes [31]. Due to the constant presence of the G1m1,17 phenotype in the population group under study, only the G3m phenotype diversity was considered.
Considering G3m allotypes, no child presented G3m16 or G3m21, both located on the CH2 domain of the IgG heavy chain, and absent in populations from sub-Saharan Africa. Similarly, due to the uncertain location of the Gm28 allotype on either IgG1 or IgG3 sub-classes among sub-Saharan Africans [17], this allotype was discarded from the analysis. Therefore, the differentiation of infants was based on the remaining G3m allotypes combined into four G3m alleles mostly encountered in Africa, that are G3m5,10,11,13,14, G3m5,6,11,24, G3m5,6,10,11,14 and G3m10,11,13,15 [17]. The homozygous or heterozygous carriage of these alleles led to the ten-following possible G3m phenotypes: G3m5,10,11,13,14; G3m5,6,10,11,14; G3m5,6,10,11,13,14; G3m5,6,11,24; G3m5,6,10,11,13,14,24; G3m5,6,10,11,14,24; G3m10,11,13,15; G3m5,10,11,13,14,15; G3m5,6,10,11,13,14,15 and G3m5,6,10,11,13,15,24.
IgG quantification
IgG quantification was performed before our study and included in the statistical analyses.
The Enzyme Linked Immunosorbent Assay (ELISA) was used to assess antibody levels against seven merozoite antigens. Details about the recombinant proteins and the procedure are described elsewhere [13, 29]. Briefly, recombinant proteins (0.1 μg/well) diluted in PBS were coated on MaxiSorp Nunc plates and blocked with 3% powdered-milk 0.1% PBS-Tween 20. For total IgG quantification, plasma samples were tested at 1:500 (for AMA1) and 1:100 (for MSP2-3D7, MSP2-FC27, MSP1, MSP3, GLUR-R0 and GLURP-R2). To quantify cytophilic IgG subclasses (IgG1 and IgG3), samples were used at 1:50 dilution against all the antigens. Horseradish peroxidase-conjugated anti-human IgG1 (NL16 clone) at 1:2000 dilution and anti-human IgG3 (ZG4 clone) at 1:5000 dilution (Skybio, France) were used as detection antibodies. The plates were washed thrice with PBS Tween-20 (0.1%) NaCl (0.5 M) after blocking, primary and detection antibodies. ADAMSEL software (Auditable Data Analysis and Management System for ELISA) was used to transform optical density (OD) values into antibody concentrations [32].
Statistical analyses
The statistical analysis was carried out using the Stata software version 13. Comparisons of demographic and clinical characteristics were investigated between the CAIG, IG and NPfDG groups using Chi-square test for categorical variables and Mann–Whitney U-test and Kruskal–Wallis test for quantitative variables. Kruskal–Wallis test was used to determine if the distribution of OP values was different between groups while the Chi-square test compared the proportion of infants with low/high OP (based on the median OP values) between groups.
Finally, the correlation between OP values, groups (CAIG, IG and NPfDG), antibody levels against P. falciparum merozoite antigens and G3m phenotypes carriages were studied by multivariate regressions. Multivariate regressions studied the correlation between OP values, groups, antibody levels and G3m phenotypes using control variables. In this study, only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. The correlation between OP values and groups (CAIG, IG and NPfDG) was studied using a multivariate ordered logit regression model; NPfDG, IG, and CAIG was coded 0, 1 and 2, respectively, according to OP values observation. If the coefficient and P value was significant and positive, the OP values increased with groups in the order 0, 1 and 2 otherwise the OP values did not increase with groups. The association between OP values, IgG levels and G3m phenotypes were studied using a generalized linear multivariate regression model where only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. In these models, validity assumptions (normality, heteroscedasticity) have been checked and also the deviance and Pearson were shown as regards the model fitting.
Ethics
The Faculté des Sciences de la Santé institutional review board and the IRD’s Consultative Ethics Committee approved the study protocol. All mothers in this study signed an informed consent before enrollment (which also included their infants) with the possibility to withdraw at any time.