HLA-A2 supertype-restricted cell-mediated immunity by peripheral blood mononuclear cells derived from Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls

Abstract

Understanding HLA-restricted adaptive host immunity to defined epitopes of malarial antigens may be required for the development of successful malaria vaccines. Fourteen epitopes of preerythrocytic malarial antigens known to mediate cytotoxic T-lymphocyte responses against target cells expressing HLA-A2-restricted epitopes were synthesized and pooled based on antigen: thrombospondin-related anonymous protein (TRAP), circumsporozoite protein (CSP), and export protein 1 (Exp-1) peptides. HLA-A2 supertype (*0201, *0202, *0205, *6802) peripheral blood mononuclear cells collected from 774 Malian children, aged 3 months to 14 years, with severe Plasmodium falciparum malaria matched to uncomplicated malaria or healthy controls were stimulated with the HLA-A2-restricted peptide pools. Significant gamma interferon production, determined by enzyme-linked immunospot assay to at least one of the three peptide pools, was observed in 24/58 (41%) of the severe malaria cases, 24/57 (42%) of the uncomplicated malaria cases, and 34/51 (67%) of the healthy controls. Significant lymphoproliferation to these peptides was observed in 12/44 (27%) of the severe malaria cases, 13/55 (24%) of the uncomplicated malaria cases, and 18/50 (36%) of the healthy controls. Responses to individual peptide pools were limited. These studies confirm the presence of adaptive cell-mediated immunity to preerythrocytic malaria antigens in volunteers from Mali and demonstrate that suballeles of the HLA-A2 supertype can effectively present antigenic epitopes. However, whether these immune responses to TRAP, CSP, and Exp-1 malarial proteins play a substantial role in protection remains a matter of controversy.

FIG. 1.

Trial profile demonstrates enrollment numbers, HLA-A2 supertype determination, and quantity of specimens available for immunologic assays. A portion of sample PBMC did not survive the freeze process and were noted to be dead upon thaw. Unresponsive cells appeared intact but did not release IFN-γ in response to positive stimulant (anti-CD3/anti-CD28 beads). Reasons for not performing an immunologic assay included utilization of cells in alternate experiments, cell loss during transit, collection of insufficient cell numbers, and death or loss of follow-up of study participants.

FIG. 2.

Bar graph depiction of IFN-γ-secreting cells determined by ELISPOT as SFC per 10⁶ PBMC in HLA-A2 (*0201, *0202, *0205, *6802)-positive children with severe malaria and age- and residence-matched uncomplicated-malaria cases or healthy controls. Production of IFN-γ in response to pooled HLA-A2-restricted malaria peptide pools or media was measured (pool 1, thrombospondin-related anonymous protein; pool 2, circumsporozoite protein; pool 3, export protein 1) in both the wet and dry seasons. Standard errors are depicted.

FIG. 3.

Geometric mean stimulation indices of proliferating PBMC derived from children with severe or uncomplicated malaria and healthy controls matched by age and residence. Lymphoproliferation as determined by [H³]thymidine incorporation after stimulation with HLA-A2-restricted malaria peptide pools or media was measured (pool 1, thrombospondin-related anonymous protein; pool 2, circumsporozoite protein; pool 3, export protein 1) in both the wet (transmission) and dry seasons. Results are depicted as a combination of the responses to all three pools in each enrollment group. The asterisk depicts significant difference between wet and dry seasons (P < 0.05).