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

Abstract

Background: Mechanisms of acquired protection to malaria in asymptomatic Plasmodium falciparum carriers are only partially understood. Among them, the role plays by the self-reactive antibodies has not been clarified yet. In this study, the relationship between repertoires of circulating self-reactive and parasite-specific immunoglobulin G (IgG), their correlation with cytokine levels, and their association with protection against malaria was investigated in asymptomatic Plasmodium falciparum-infected Gabonese children.

Methods: The diversity of P. falciparum-specific antibody repertoire was analysed using a protein micro-array immunoassay, the total auto-antibody repertoire by quantitative immunoblotting and circulating cytokine levels were measured by ELISA in endemic controls (EC) and P. falciparum-infected children from Gabon with asymptomatic (AM) or mild malaria (MM). The association of self- and parasite-specific antibody repertoires with circulating cytokines was evaluated using single linkage hierarchical clustering, Kruskal-Wallis tests and Spearman's rank correlation.

Results: Children with AM exhibited an IgG response to merozoite surface protein 3 (MSP3) but not to MSP1-19, although their levels of total P. falciparum-specific IgG were similar to those in the MM group. Moreover, the asymptomatic children had increased levels of autoantibodies recognising brain antigens. In addition, a correlation between IL-10 levels and parasite load was found in AM and MM children. These two groups also exhibited significant correlations between plasma levels of IL-10 and IFN-γ with age and with total plasma IgG levels. IL-10 and IFN-γ levels were also associated with auto-antibody responses in AM.

Conclusions: Altogether, these results indicate that a self-reactive polyclonal response associated with increased IgG to MSP3 and high plasma levels of IL-10 and IFN-γ may contribute to protective immune mechanisms triggered in asymptomatic P. falciparum infection in Gabonese children.

Figure 1

Epidemiological and serological characteristics of the study population. (A) Parasite loads (percentage of P. falciparum-infected red blood cells) in the EC, AM and MM groups. (B) Correlation between total IgGs and age of the children in EC, AM and MM groups. (C) Levels of IgG recognizing total P. falciparum antigens in the EC, AM and MM groups. (D) Proportion of patients with >3 pg/ml of P. falciparum-specific IgG in the respective clinical groups.

Figure 2

Repertoire of Plasmodium falciparum antigenic diversity recognized by infected children in Gabon. (A) Percentage of individuals in the study population that recognized at least one P. falciparum antigen. (B) Antibody patterns and percentage of positive patients for each of the 20 P. falciparum-specific antigens tested in the total study population. (C) Kruskal-Wallis analysis of the antibody responses against the 20 parasite antigens in all groups. (D) Plasma levels (pg/mL) of anti-P. falciparum MSP3-3D7 antibodies in EC, AM and MM. (E) Single linkage hierarchical clustering of the antibody response against the 20 P. falciparum antigens tested in the different groups. (F) Heat map representing fold change of the antibody response and Mann–Whitney p-value comparing the different groups: AM vs MM, AM vs EC, and EC vs MM. Antigens: MSP1 block 2 PA repeats; MSP1 block 2 3D7 Wellcome repeats; MSP1 block 2 3D7 full length; MSP1 block 2 3D7 Wellcome full length; MSP1 block 2 MAD20 full length; MSP1 block 2 RO33 full length; MSP1 block 2 K1 super repeats; MSP1 block 2 K1 flanking; MSP1 block 2 MAD20 repeats; MSP1 block 2 3D7 repeats; MSP3 3D7; MBP; MSP3 K1; MSP2 3D7; MSP2 FC27; AMA-1; GST; MSP1-19 GST; PfEMP1; NANP.

Figure 3

Patterns of brain self-reactive IgG responses in EC, AM and MM groups. PCA-1 scores for (A) the different groups, (B) relation to age, and (C) relation to total plasma IgGs.

Figure 4

Relationship between auto-antibody response and Plasmodium falciparum-specific antibody response. (A) Relationship of the auto-antibody response to anti-P. falciparum IgG response in the EC, AM and MM groups, and division of response patterns into three sub-groups: α (anti-P. falciparum IgG <3 pg/ml; F1 > 1.5), β (anti-falciparum IgG >3 pg/ml; F1 < 1.5), δ anti-falciparum IgG <3 pg/ml; F1 < 1.5). (B) Frequencies of patients in α and β sub-groups in the EC, AM and MM groups.

Figure 5

Cytokines levels in respect to clinical status. Plasma levels of IFN-γ (A) TNF (B) and IL-10 (C) in the EC, AM and MM groups at the day of hospitalization and before treatment.

Figure 6

Interaction between cytokine response and auto-antibody patterns. Correlation between self-reactivity (PCA-1 score) and (A) IL-10 plasma levels or (B) IFN-g plasma levels in AM patients older than 18 months. (C) Correlation between IL-10 plasma concentrations and parasite loads at the day of hospitalization and before treatment.