Assessing the binding of four Plasmodium falciparum T helper cell epitopes to HLA-DQ and induction of T-cell responses in HLA-DQ transgenic mice

Abstract

A subunit vaccine for Plasmodium falciparum malaria will need to contain well-defined T helper cell epitopes that induce protective immune responses to the parasite. One major barrier to the use of subunit vaccines is the requirement for T helper cell epitopes to be presented by the HLA class II molecules that are present in the population being vaccinated. Since the majority of malaria studies have focused on HLA-DR, little information on the role of HLA-DQ in the binding and immune response to malarial epitopes is available. This study used an in vitro peptide-binding assay to predict the extent of HLA-DQ binding of four conserved T helper cell epitopes identified from asexual-stage malaria vaccine candidate antigens. Epstein-Barr virus (EBV)-transformed human B-cell lines expressing 14 different DQ molecules (DQ2.1, -2.2, -4.1, -4.2, -5.1 to -5.3, -6.1, -6.2, -6.4, -7.1, -7.3, -8, and -9) representing all broad serological specificities, including common DQ molecules present in populations in areas where malaria is endemic, were used in the binding assay. Moreover, an HLA-DQ transgenic mouse model was employed to evaluate the correlation between the in vitro DQ binding of the peptides and the generation of in vivo immune responses following peptide immunization. This study identified two broad DQ-binding peptides, ABRA#14 and SERA#9. ABRA#14 also induced T-cell proliferation and Th1-associated cytokine production in DQ8(+) transgenic mice. The combination of peptide binding to EBV-transformed cell lines and DQ transgenic mice provides a method for identifying additional T-cell epitopes for inclusion in a vaccine.

FIG. 1

Binding specificities of malarial blood stage T-cell epitopes ABRA#14 (10 μM), SERA#9 (10 μM), MSP-1#2 (50 μM), and MSP-1#3 (50 μM) to DQ allelic products expressed on B-LCLs including class II-negative control line BLS-1. Binding was performed at pH 4.4, 5.6, and 7.0. The solid horizontal line indicates a high mean fluorescence, and the dashed line indicates an intermediate mean fluorescence (5,000 fluorescence units). The DQA1/DQB1 alleles encoding each DQ molecule are shown in Table 2. Data represent the means ± standard deviations of three independent experiments.

FIG. 2

Responses to malaria peptides in HLA-DQ8 transgenic mice correlate with the DQ8 binding result. In vitro proliferative responses of LNC and SPC from HLA-DQ8⁺/H-2 A^b0 mice immunized with ABRA#14, SERA#9, MSP-1#2, and MSP-1#3 as described in Materials and Methods and cultured with the same peptide at 1 or 10 μM compared to those of cells incubated with medium alone. Data are means ± standard deviations from six replicate well cultures from one out of two representative experiments.

FIG. 3

Cytokine production by LNC and SPC of HLA-DQ8 transgenic mice and HLA-DQ8-negative littermates in response to immunization with the ABRA#14 peptide. Cells were cultured in vitro with medium alone or with ABRA#14 (10 μM) and harvested at 24, 48, and 72 h of culture. Cytokine profiles were measured by enzyme-linked immunosorbent assay. Data represent the means of duplicate wells from one out of two representative experiments.

FIG. 4

The response to ABRA#14 peptide in HLA-DQ8 transgenic mice is mediated by CD4⁺ cells and the HLA-DQ molecule, with a background response by CD8⁺ cells in HLA-DQ8-negative littermates. LNC and SPC from HLA-DQ8⁺/H-2 Ab⁰ mice (A) and HLA-DQ8⁻/H-2 Ab⁰ mice (B) were challenged with ABRA#14 (10 μM) in the presence of the indicated MAbs. Results of responses in the absence of MAb (none) and unstimulated cultures are also indicated. Only results for MAbs at 1 μg/ml are shown here. The inhibition level was not altered by using a higher concentration of each MAb. The data are means ± standard deviations of six replicate well cultures from one out of two representative experiments.

FIG. 5

Cell cultures depleted of either CD4 or CD8 T cells from lymph nodes (A) and spleens (B) of immunized HLA-DQ8 transgenic mice were used in a cell proliferation assay. Proliferation levels of unseparated cells cultured without magnetic bead treatment (CD4⁺/CD8⁺), CD4-depleted cells (CD8⁺), and CD8-depleted cells (CD4⁺) are shown. Bars represent incubation with the ABRA#14 peptide at 1 and 10 μM and incubation with medium alone. Data are means ± standard deviations of six replicate well cultures from one out of two representative experiments.