Detection of EXP1-Specific CD4+ T Cell Responses Directed Against a Broad Range of Epitopes Including Two Promiscuous MHC Class II Binders During Acute Plasmodium falciparum Malaria

Abstract

Background: T cells are thought to play a major role in conferring immunity against malaria. This study aimed to comprehensively define the breadth and specificity of the Plasmodium falciparum (P. falciparum)-specific CD4+ T cell response directed against the exported protein 1 (EXP1) in a cohort of patients diagnosed with acute malaria. Methods: Peripheral blood mononuclear cells of 44 patients acutely infected with P. falciparum, and of one patient infected with P. vivax, were stimulated and cultured in vitro with an overlapping set of 31 P. falciparum-specific 13-17-mer peptides covering the entire EXP1 sequence. EXP1-specific T cell responses were analyzed by ELISPOT and intracellular cytokine staining for interferon-γ production after re-stimulation with individual peptides. For further characterization of the epitopes, in silico and in vitro human leukocyte antigen (HLA) binding studies and fine mapping assays were performed. Results: We detected one or more EXP1-specific CD4+ T cell responses (mean: 1.09, range 0-5) in 47% (21/45) of our patients. Responses were directed against 15 of the 31 EXP1 peptides. Peptides EXP1-P13 (aa60-74) and P15 (aa70-85) were detected by 18% (n = 8) and 27% (n = 12) of the 45 patients screened. The optimal length, as well as the corresponding most likely HLA-restriction, of each of these two peptides was assessed. Interestingly, we also identified one CD4+ T cell response against peptide EXP1-P15 in a patient who was infected with P. vivax but not falciparum. Conclusions: This first detailed characterization of novel EXP1-specific T cell epitopes provides important information for future analysis with major histocompatibility complex-multimer technology as well as for immunomonitoring and vaccine design.

Keywords: CD4+; CD8+; HLA binding; HLA class II; Plasmodium falciparum; Plasmodium vivax; T cell epitope; malaria.

Figure 1

Intracellular cytokine staining (ICS) for IFNγ production of CD4+ T cells after re-stimulation with single EXP1 peptides. (A) Exemplary ICS dot blot of a P. falciparum-specific T cell response. Every positive response in the ELISPOT assay was confirmed by re-stimulation with a single peptide intracellular cytokine staining (ICS) assay for IFNγ production. CD4+ T cells were gated (an exemplary gating strategy is shown in Supplementary Figure 2), (left) negative control, (right) positive peptide response against EXP1-P15 by HH-03. DMSO and R10-medium were added to the negative control. (B) Overview of the relative location of the P. falciparum-specific T cell responses detected by one or more patients of the cohort against one or more of the 31 single EXP1 peptides. Response frequency (RF) is the number of patients that had a specific T cell response against a single peptide divided by the number of patients that were tested (n = 45). The center of EXP1, EXP1-P13 to EXP1-P20 (aa60-109), showed a high immunogenicity.

Figure 2

(A–D) Breadth of the EXP1-specific T cell response in comparison with relevant clinical parameters. Clinical parameters, including parasitaemia, CRP, hemoglobin and thrombocytes were examined but no significant (ns) correlation with the CD4+ T cell response could be shown. (A) Response or no response compared to parasitaemia [%]. (B) Response or no response compared to C-reactive protein (CRP) [mg/dl]. (C) Response or no response compared to hemoglobin [g/dl]. (D) Response or no response compared to thrombocytes [1,000/μl].

Figure 3

(A) Fine-mapping of malaria epitope EXP1-P13 (HH-09). (B) Fine-mapping of EXP1-P15 (HH-03). (C) Testing of variants for EXP1-P20 (HH-03). Experiments were performed by ICS for IFNγ production after stimulation with peptides of different length and with varying sequences. (A) For EXP1-P13 we used 15-, 13-, and 11-mer N- as well as C- terminal truncations with PBMCs of patient HH-09 (DRB1*08:04, *11:01P). Truncation 1 showed the highest CD4+ T cell response. The core epitope of EXP1-P13 is marked in black. (B) 16-, 14-, and 12-mer N- as well as C- terminal truncations were tested for EXP1-P15. PBMCs of patient HH-03 (DRB1*04:05P,*15:02P) were used for this experiment. EXP1-P15 Truncation 1 showed the highest CD4+ T cell response as well as EXP1-P15 Variant 2 which contains Serine (S) instead of Alanine (A). For EXP1-P15 and P20 variants were tested. Variant 1 of EXP1-P20 contained the amino acid Arginine (R) instead of Histidine (H) and Variant 2 Arginine (R) instead of Lysine (K).