CD4+ T cell responses to the Plasmodium falciparum erythrocyte membrane protein 1 in children with mild malaria

Abstract

The immune response against the variant surface Ag Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against malaria. We have investigated the development and maintenance of CD4(+) T cell responses to a small semiconserved area of the Duffy binding-like domain (DBL)α-domain of PfEMP1, the DBLα-tag. Young children were followed up longitudinally, and parasites and PBMCs were isolated from 35 patients presenting with an acute case of uncomplicated malaria. The DBLα-tag from the PfEMP1 dominantly expressed by the homologous parasite isolate was cloned and expressed as recombinant protein. The recombinant DBLα-tag was used to activate PBMCs collected from each acute episode and from an annual cross-sectional survey performed after the acute malaria episode. In this article, we report that CD4(+) T cell responses to the homologous DBLα-tag were induced in 75% of the children at the time of the acute episode and in 62% of the children at the following cross-sectional survey on average 235 d later. Furthermore, children who had induced DBLα-tag-specific CD4(+)IL-4(+) T cells at the acute episode remained episode free for longer than children who induced other types of CD4(+) T cell responses. These results suggest that a wide range of DBLα-tag-specific CD4(+) T cell responses were induced in children with mild malaria and, in the case of CD4(+)IL-4(+) T cell responses, were associated with protection from clinical episodes.

FIGURE 1.

Schematic presentation of the study design. From each child, blood samples were collected at the acute malaria episode or during the cross-sectional survey (postacute). PBMCs and plasma were separated and stored. RBCs were separated from the acute blood sample and total parasite RNA isolated using Trizol. Total RNA was reversed transcribed and DBLα-tag sequences amplified by PCR. DBLα-tag PCR products were cloned and 10–20 clones sequenced. The dominant DBLα-tag sequence was cloned into an expression vector and the recombinant DBLα-tag purified. The recombinant DBLα-tag originating from PfEMP1 expressed on the clinical parasite isolate a child was infected with (the homologous DBLα-tag) was used to stimulate stored PBMCs from that child and CD4⁺ T cell responses were analyzed by intracellular cytokine staining and flow cytometry.

FIGURE 2.

Cumulative percentage of cytokine producing CD4⁺ T cells after stimulation with homologous DBLα-tag. Shown are cumulative bar-graphs of the percentage of DBLα-tag specific CD4⁺ T cells producing cytokines as indicated for each child at the acute episode (A) and at the cross-sectional survey (B). Children with asymptomatic parasitaemia at the cross-sectional survey are indicted (*). Children with missing data at the cross-sectional survey (n = 9) are also indicated (§).

FIGURE 3.

Phenotype of CD4⁺ T cells stimulated with the homologous DBLα-tag. The phenotype of CD4⁺ T cell in response to the homologous DBLα-tag was classified for each child as follows: Th1: IFN-γ– but no IL-4–secreting CD4⁺ T cells; Th2: IL-4– but no IFN-γ–secreting CD4 T cells; Other: All other responses including mixed Th1/Th2 responses; No responses: children who did not induce any Ag-specific CD4⁺ T cells. (A) Shown are pie charts of the proportion of all children who induced a given CD4⁺ T cell phenotype in response to the DBLα-tag at the acute episode (n = 35) and at the cross-sectional survey in May 2009 (n = 26). (B) Number of episodes before the acute event in children with different CD4⁺ T cell phenotypes. Shown are median (horizontal bar) and 25th and 75th percentile (vertical bars). The differences in the number of episodes in children with a dominant Th2 response compared with the number of episodes in children with other responses were not significant.

FIGURE 4.

Relationship between CD4⁺ T cell responses to cys2 and cys4 PfEMP1. (A) Pie chart representing the proportion of recombinant DBLα-tags falling into cys2 (PoLV group1-3), cys4 (PoLV group 4 and 5) or none (PoLV group 6) of these groups (16). (B) Odds ratios and 95% confidence intervals are plotted for CD4⁺ T cells secreting a cytokine as indicated when children were infected parasites dominantly expressing either cys2 PfEMP1 (circle) or cys4 PfEMP1 (triangle). Children infected with cys2 PfEMP1-expressing iRBCs were more likely to induce CD4⁺IL10⁺ responses whereas children infected with cys4 PfEMP1-expressing iRBC were more likely to induce CD4⁺IFN-γ⁺ T cell responses. *p < 0.05.

FIGURE 5.

Survival plots with time to first episode of clinical malaria over a 12 mo period. Survival plots for children who induced DBLα-tag specific CD4⁺ T cells secreting a given cytokine. Children who made a CD4⁺IL-4⁺ T cell response (n = 14) remained episode free longer than children who did not (HR = 0.31, CI 0.12–0.79, p = 0.014, adjusted for age). Hazard ratios for all other types of T cell responses were not significant. *Log rank test for equality.

FIGURE 6.

Ab responses to the homologous DBLα-tag at the acute episode and the cross-sectional survey. Shown are corrected OD values of DBLα-tag specific Ab responses for each child at the time of acute disease and at the cross-sectional survey for IgG, Ig4, and IgE.