Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study

Abstract

Background: The factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM.

Methods: To identify immune responses associated with SM, a sex- and age-matched case-control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-α rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1β, TNF, TNF-β and TGF-β1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar's and Signtest.

Results: Compared to UM, matched children with SM had reduced levels of IgG against DBLα (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-β1 (P < 0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLα IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026).

Conclusions: The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.

Figure 1

IgG antibody responses to merozoite antigens (A) andP. falciparum(Pf) lysate (B). Seroprevalence is represented by bars as the % of responders; plasma levels are represented by dot plots, with horizontal lines indicating median values. Data from children with SM is shown as shaded bars/dots; data from children with UM is shown as open bars/dots. Results of matched comparisons are reported in the x axis as the number (%) of divergent pairs for IgG seroprevalence, and the median difference (inter-quartile range) of IgG levels between SM and UM. P-values were calculated using McNemar’s or Sign test. All comparisons were corrected by the Monte Carlo permutation test (1000 random permutations). Thresholds for seroprevalence (OD): MSP-1₁₉ = 0.560, EBA-175 = 0.628, AMA-1 = 0.578, DBLα = 0.489, P. falciparum lysate = 0.034. Median difference (inter-quartile range) of IgM levels against uninfected erythrocytes lysate between SM and UM = −0.01 [−0.03, 0.01], P = 0.175.

Figure 2

IgM antibody responses to merozoite antigens (A) andP. falciparum(Pf) lysate (B). Seroprevalence is represented by bars as the % of responders; plasma levels are represented by dot plots, with horizontal lines indicating median values. Data from children with SM is shown as shaded bars/dots; data from children with UM is shown as open bars/dots. Results of matched comparisons are reported in the x axis as the number (%) of divergent pairs for IgM seroprevalence, and the median difference (inter-quartile range) of IgM levels between SM and UM. P-values were calculated using McNemar’s or Sign test. All comparisons were corrected by the Monte Carlo permutation test (1000 random permutations). Thresholds for seroprevalence (OD): MSP-1₁₉ = 0.605, EBA-175 = 0.514, AMA-1 = 0.554, DBLα = 0.444, P. falciparum lysate = 0.057. Median difference (inter-quartile range) of IgG levels against uninfected erythrocytes lysate between SM and UM = −0.02 [−0.2, 0.1], P = 1.000.

Figure 3

Plasma concentrations of cytokines and chemokines.(A) Pro-inflammatory cytokines, (B) immuno-regulatory/anti-inflammatory cytokines, (C) Th1 cytokines, (D) pro-inflammatory chemokine and (E) Th2 cytokine. Percentage of detectable cytokines/chemokines is represented by bars; concentrations are represented by dot plots, with horizontal lines indicating median values. Data from children with SM is shown as shaded bars/dots; data from children with UM is shown as open bars/dots. Results of matched comparisons are reported in the x axis as the number (%) of divergent pairs for cytokine/chemokine prevalence, and the median difference (inter-quartile range) of cytokine/chemokine concentrations between SM and UM (in ng/mL for TGF-β1). P-values were calculated using McNemar’s or Sign test. All comparisons were corrected by the Monte Carlo permutation test (1000 random permutations).