Plasmodium falciparum immunodominant IgG epitopes in subclinical malaria

Abstract

Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stage Plasmodium falciparum rapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients' IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.

Figure 1

Epitope mapping in the five P. falciparum antigens. Data was recorded from 15-mer microarrays of overlapping peptides covering the whole protein sequence. Data shows average fluorescence m (blue) and standard error (s) subtracted average fluorescence m-s (red) along the sequence of the indicated antigens. Data is presented in a set of 4 plots to distinguish incubation with sera from ASC or ANP patients and for high IgG (>60 μg mL⁻¹) and low IgG sera. n values are as follow: 10 for ASC/High IgG; 8 for ANP/Low IgG; 11 for ASC/Low IgG; 9 for ANP/High IgG. The positions of the selected 20-mer peptides are shown as overlay green bars. Ratios of cumulative signal intensities to Pf-specific IgG were not significantly different between High and Low IgG groups: average values 7.6 × 10⁻³ ± 0.01 and 5.6 × 10⁻³ ± 0.01, respectively.

Figure 2

Comparison of IgG reactivity frequency between serum from adult patients with (blue: ASC) and without (red: ANP) parasitemia from Breman-Asikuma. X-axis shows amino acid sequence length of each antigen. Y-axis shows fluorescence signals above a cutoff stablished as the third quartile of the recorded data. Data from high IgG sera are shown. The positions of the selected 20-mer peptides are shown as overlay green bars.

Figure 3

IgG reactivity of adult serum samples from the endemic malaria region with eight selected 20-mer epitopes. Data shows specific IgG concentration present in sera against 20-mer peptides sequences of START (1.1, 1.3); HSP70-2 (2.2); putative protein kinase Uniprot Q8I2Q0 (3.3); HSP60 (4.1, 4.3); and PDI8 (5.1, 5.3). Three different sera groups were used: asymptomatic subclinical (ASC, n = 8), undetected malaria parasite (ANP, n = 8) and no malaria exposed controls (NC, n = 4). The 16 sera used from endemic malaria region were selected from the patients indicated with an asterisk in Table S2. Mean values are given by horizontal lines; boxes depict 25/75 percentiles and whiskers cover data excluding outliers at 1.5 coefficient. Mann-Whitney nonparametric tests were run: * P < 0.05; ** P < 0.01.

Figure 4

IgG reactivity of children serum samples from the endemic malaria region with four selected 20-mer epitopes. Data shows specific IgG concentration present in sera against 20-mer peptides sequences of START (1.1, 1.3) and PDI8 (5.1, 5.3). Three different sera groups were used: clinical malaria samples (CCM, parasitemia >0.1%); asymptomatic subclinical (CSC), and undetected malaria parasite (CNP). Mean values are given by horizontal lines; boxes depict 25/75 percentiles and whiskers cover data excluding outliers at 1.5 coefficient. Mann-Whitney nonparametric tests were run: *P < 0.05.