Asymptomatic Plasmodium falciparum infection in children is associated with increased auto-antibody production, high IL-10 plasma levels and antibodies to merozoite surface protein 3

Venous blood was collected in EDTA on days 0 (before treatment), 7 and 30. Plasma was separated and stored at −80°C until use. Parasitaemia (expressed as the percentage of infected erythrocytes) was determined by microscopic examination of Giemsa-stained thin blood smears.

Plasmatic levels of IFN-γ, TNF and IL-10 were measured in EC, AM and MM at day 0, 7 and 30 using a sandwich-type ELISA (Kit OptEIA set, Pharmingen, BD Bioscience, France) according to the manufacturer’s recommendations.

The quantification of total IgG in children plasma from all groups was done by ELISA. At day 0, 7 and 30 after their inclusion in the study. Briefly, 96 microwell plates (reacti-bind 96 EIA Plate 100/PKG, Pierce) were coated with 5 μg/ml of purified sheep polyclonal anti-human IgG (Sigma-Aldrich, France) followed by overnight incubation at 4°C. After blockage with 1% gelatine and washing with 1% gelatin-PBS, serial dilutions of plasma samples were incubated overnight at 4°C. Bound IgG was detected using a peroxidase-conjugated polyclonal anti-human-IgG (The Binding Site, Birmingham UK). Binding was revealed using 0.5 mg/ml O-phenylenediamine (OPD) substrate (Sigma-Aldrich, France) and 0.03% H₂O₂ in 0.05 M phosphate-citrate buffer, pH5 and the product was quantified from the optical density (OD) at 450 nm. The mean of OD value was fitted into the sigmoidal standard curve using a specific ELISA programme running in Igor version 3.16 (Wavemetrics, Lake Oswego, OR).

Total P. falciparum specific plasmatic IgGs were quantified using ELISA. Briefly, ninety-six-well plates (Reacti-Bind 96 EIA Plat Corn Not, Pierce) were coated overnight at 4°C with 5 μg/ml of P. falciparum 3D7 schizont-stage protein extract. Plates were blocked with 1% gelatine. One-hundred ml of 1:100 dilution of patient plasma was added to each well in duplicate, incubated for one hour at 37°C, and developed using anti-human IgG-HRP and OPD (Sigma-Aldrich, France). Ten per cent sodium dodecyl sulphate was added after 30 min and the OD measured at 450 nm. Concentration of P. falciparum-specific IgG in the samples was calculated from a standard curve using 0.001 − 2 mg/ml of purified human IgG. For every sample tested a cut-off value was estimated using a pool of plasma from children living in Europe who had never been exposed to P. falciparum infection.

The plasma parasite-specific antibodies were profiled using a protein micro-array immuno-assay [14]. Each array contained 20 recombinant antigens derived from leading blood-stage vaccine candidates, including antigenic variants of MSP1, MSP2 and MSP3, apical membrane antigen 1 (AMA1), P. falciparum erythrocyte membrane protein 1 (PfEMP1), and circumsporozoite protein epitope NANP-4. Arrays were incubated with 100 μl of 1:100 dilution of plasma for 15 min at 37°C and washed for 5 min in PBS-Tween. Arrays were then incubated with Alexa Fluor 546-labelled anti-human IgG monoclonal antibody (1:1,000; Jackson Immunoresearch Europe, UK) for 20 min. Alexa 555 diluted 1:1 in PBS was used as signal control. Ten mg/ml bovine serum albumin was used as a negative control. Anti-human IgG (Sigma, France) was used as carry-over control. Arrays were washed twice in PBS-Tween, dried under nitrogen, and scanned with a GMS 428 scanner (Affymetrix, Santa Clara, CA, USA). Acquired images were analysed with the GenePix image analysis software (version 3.0.6.89, Axon Instruments, CA, USA). The mean intensity of duplicate spots was analysed using the linear range of the individual slide standard micro-array [14].

The PANAMA-Blot method was used to determine the antibody repertoire to brain antigens as previously described [15,16]. PANAMA-Blot is a powerful approach allowing the analysis of the self-reactive antibodies repertoire against a large panel of antigens. Briefly, proteins from normal human brain, from a volunteer donor after death, were separated on 10% SDS-PAGE at 25 mA, and were transferred onto nitrocellulose membranes (Schleicher & Schull, Dassel, Germany) by semi-dry electrotransfer (Pasteur Institute, Paris) for 1 h at 0.8 mA/cm2. Membranes were blocked by incubation overnight at room temperature with 0.2% Tween 20 in PBS (PBST), and were then incubated with plasma samples diluted 1:20 in PBST (non-adjusted assay), or at a total IgG concentration of 200 μg/ml (adjusted assay), in a Cassette Miniblot System (Immunetics, MA, USA). Membranes were incubated for 4 h at room temperature with gentle shaking. Bound IgG were detected with rabbit anti-human IgG–alkaline phosphatase (Sigma-Aldrich, France) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Promega, France) incubated for 90 minutes at room temperature. Immunoreactivity was detected with NBT/BCPI (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) and the quantitative signals were normalized using the IGOR 3.16 software (Wavemetrics, Lake Oswego, OR, USA). A standard plasma sample (a mixture of diverse plasma samples from Gabonese individuals diluted 1:20) was applied in duplicate for intensity adjustment.

Immunoblot data were analysed by multivariate statistical methods, using IGOR software (Wavemetrics, Lake Oswego, OR, USA). After baseline subtraction, the standard migration scale was divided into sections around individual peaks of immunoreactivity. Section-wise absorbance values were subjected to principal component analysis (PCA). The first component (PCA-1) was the one-dimensional fitting vector that accounted for the largest proportion of variance and factor 2 (PCA-2) was the second-best orthogonal one-dimensional vector fitting the data. Quantitative comparisons between groups were done either with the Mann–Whitney (between two groups) or Kruskal-Wallis tests (>two groups). Qualitative association was tested by Pearson’s c² test. The association between continuous quantitative parameters was assessed by Spearman’s rank correlation. The trend over time was tested by the paired t-test; p-values <0.05 were considered significant. Linear discriminant analysis was performed on log-transformed data, and samples with incomplete information were rejected. The significance of contributing parameters was calculated by backward elimination, using partial F tests on variances explained by models with or without the respective parameters. The significance criterion was a Bonferroni-corrected p-value <0.05. Single linkage hierarchical clustering of the antibody response against the 20 P. falciparum antigens tested in the different groups was performed using the hclust function in R software. For the heatmaps, fold change of the antibody response and was calculated using the formula Fc = μ1/μ2 for μ1 > μ2 and Fc = −μ2/μ1 for μ1 < μ2, where μ1 is the mean of the first group and μ2 the mean of the second group compared. The same methods were used for brain self-reactive IgG responses clustering and heatmaps.