Fifteen PfEMP1 sequences were downloaded from the sequence database maintained at the National Center for Biotechnology Information via PubMed Medline. Gene identification number and names of the sequences are as follows: g1297091, 3D7var1; g6601436, A4 tres var partial; g886375, var-1 Dd2; 1809295, FCR3varT11.1; g914919, MCvar1; g4760402, varCS2 (CSAP) Like; g3845343, PFB 1055c; g3540145, Var IT 4/25/5; g1517814, var ITG 2F6-P21; g914921, var-2 MC132; g886378, var PF 23AFCR3; g2944095, var prot PNG; g5163174, var C2 itG Exon 2; VAR7-like molecule of DD2, (accession number Y11909); and Var MC, (accession number U27338).

The sequences were then aligned using the Megalign program of DNA star computer software. A peptide similar to most peptides in a relatively conserved block was chosen and synthesised by 9-Fluorenylmethoxycarbonyl (Fmoc) chemistry using the multiple peptide synthesiser (Biotech Instrument Limited) as described 9. The degree of sequence homology of the chosen peptide to the others in a conserved block is shown in Table 1. Three pools of relatively conserved peptides were prepared as shown in Fig. 1 and Table 1. Since previous studies 101112 show that the peptides recovered from purified MHC class II molecules range from 9 to 30 amino acids or more, we have only chosen peptides from conserved blocks where the amino acid length is not less than 9. Longer peptides were included as they would be processed before presentation by the antigen-presenting cell. Although there are no algorithms or programmes available that can predict MHC class II-binding peptides with probabilities of greater than 50%, screening of the selected peptides using SYFPEITHI database epitope prediction programme 13 predicted that at least 13 out of the peptides used contain one or more epitopes theoretically able to bind one or more HLA-DR molecules.

The ages of the Benin malaria exposed donors ranged from 4–45 years (male to female ratio was 4:6). The ages of the UK unexposed control volunteers ranged from 20–45 years (male to female ratio was 3:7). Blood (5 mL from children to 20 mL from adults) was taken from donors living in and around Cotonou, Benin during the dry season of 1992. In this region, malaria is holoendemic with maximal transmission during the rainy season 14. 28% of the donors were parasitaemic at the time of blood collection, and parasitaemia ranged from 0.0004% to 0.1%. None of the UK volunteers had travelled to a malaria endemic area at any time. Peripheral blood mononuclear cells (PBMC) were separated over Ficoll/paque (Pharmacia Biotech.) using standard procedures. The cells were resuspended in foetal calf serum (FCS) containing 10% DMSO and frozen by controlled-gradient freezing at -70°C for 48h and finally in liquid Nitrogen until use. The Benin samples were transported from Benin to the UK to be analysed simultaneously with the UK samples that were frozen using a similar method. We have shown previously that the CD4 T cell response of PBMC frozen in this manner is as good as that of freshly isolated cells to antigens such as tetanus toxoid 15. Viability of recovered cells from the Benin samples ranged from 70 to 90% and above 95% for the UK sample. Ethical approval was given by the Vice Dean of Medical School of the University of Benin in accordance with the Helsinki declaration and by the Barnet Health Authority Ethics Committee, UK.

PBMC were thawed rapidly at 37°C, washed twice in RPMI 1640, centrifuged at 415 g for 5 min at room temperature and counted. The cells were resuspended at 5 × 10⁶ cells in 250 μL diluent for PKH26 (Sigma) labelling, and stained for 1 min by incubation with PKH26 dye at room temperature as described previously 1516. PKH26 dye incorporation into cell membrane was stopped by adding two times the volume of 100% human AB serum. Cells were then washed three times, and staining was assessed by flow cytometry. After labelling and washing the viabililty as assessed by Trypan Blue was greater than 75%. The labelled cells were re-suspended in fresh RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES and 10% heat-inactivated AB serum medium. The cultures were set up in triplicates at 2 × 10⁵ cells/well in a 96 well U-bottomed plate (Nunclon™, Gibco) in a final volume of 200 μL with or without antigen. Test antigens were used at a final concentration of 5 nM for each peptide in a pool. Phytohaemagglutinin (PHA) (Sigma) was used as a control for cell viability at a final concentration of 2.0 μg/mL. Plates were incubated at 37°C, 5% CO₂, in a humidified atmosphere for 7 days, after which time supernatants were removed for cytokine measurement and the cells were stained with monoclonal antibodies directed against CD3 and CD4. The relative numbers of dividing CD4⁺ lymphocytes were determined by flow cytometry.

The antibody used to determine the phenotype of the responding cells was allophycocyanin (APC) conjugated anti-CD4, 13B8.2 (mouse IgG1) (Pharmingen).

Cultured cells were washed in sterile PBS containing 0.5% w/v bovine serum albumin, 5.0 mM EDTA pH 8.0 and 0.01% w/v sodium azide (Sigma) and incubated with the antibody for 30 mins. Excess antibody was washed off in PBS and the cells fixed in 1% paraformaldehyde in PBS and transferred into FACS tubes for analysis.

Flow cytometry was performed on a FACscalibur machine using Cell Quest software (Becton Dickinson). Viable cell population was defined by using forward and 90° light scatter. A reduction in the fluorescence intensity of PKH26 labelling was used as an index of cell division 1516. The use of APC conjugated antibody allowed the measurement of dividing CD4⁺ T cells. The flow cytometer was set to acquire events for 2 min. This allows the relative numbers of recovered viable cells within each well (a measure of the magnitude of the response) to be determined. Any cell exhibiting a reduction in PKH26 fluorescence intensity was classified as having divided in response to the antigen. The background number of divided CD4 T cells in the absence of antigen varied widely between donors (30–8152), therefore, a donor was considered to have responded by CD4 T cell division if the mean number of dividing cells in triplicate wells containing an antigen was equal to or above 2 times the mean value of triplicate wells without antigen. The percentage of dividing CD4 population relative to the total population of CD4 positive cells ranged from 1.2% – 10%

The amount of IL-10 in the supernatants after 7 days of cultures was measured by ELISA using paired antibody kits (Pharmingen/ Beckton Dickinson, Oxford, UK) and following the manufacturers instructions. The response of a donor was defined as positive when the mean concentration of IL-10 produced in triplicate wells containing antigen was significantly greater (i.e. p < 0.05, Student T test) than the mean concentration of IL-10 production in triplicate wells without antigen of that donor.

The limit of detection for the ELISA assay for IL-10 was 4 pg/mL. The background level of IL-10 production differed widely between individual donors (range: 0 to 149 pg/mL). For this reason, the concentration of IL-10 produced by each donor in response to the peptide antigens was taken as the net concentration after subtraction of the background of that donor without antigen. Standard Errors of the means were usually in the range of 15–20% (not shown).

The results were analysed by the Two-tailed Student T test and the Fisher's exact Test.

We have determined the in vitro CD4 T cell division in response to three pools of relatively conserved peptides from three regions of PfEMP1, namely, DBLα (pool 1), CIDRα and CIDRβ + DBL β-δ (pool 2) and EXON 2 (pool 3) (Table 1 and Figure 1). All the three peptide pools elicited a CD4 T cell response in a proportion of malaria-exposed and non-exposed donors (Figure 2). The magnitude of the CD4 T cell division was significantly higher in the Benin malaria-exposed donors when compared to the UK malaria-unexposed donors for peptide pools 1 and 3 (Two tailed Student T test, p = 0.025 and 0.045 for peptide pools 1 and 3, and p = 0.062 for pool 2 peptides) (Table 2). Using a cut off of 2 stimulation indices to define a positive response, the prevalence of positive CD4 T cell response among the malaria-exposed Benin donors to any of the three peptide pools ranged from 21–37% (Figure 2). However, using the Fisher's Exact test, this was not statistically different from the prevalence of the responders among the UK malaria-unexposed donors (4–20%) (Figure 2). The absence of CD4 T cell division in response to the antigens by some donors was not due to loss of cellular viability, as cells from all donors divided in response to PHA, (data not shown).

IL-10 was measured in the supernatants after 7 days of culture in the presence of the three PfEMP-1 peptide pools. The prevalence of positive IL-10 responses to any of the peptide pools ranged from 16–21% among the malaria-exposed donors living in Benin and ranged from 0–7% among the malaria-unexposed UK. Because of the small sample size, the differences observed in the prevalence of positive IL-10 responders between the Benin malaria-exposed donors and the UK malaria-unexposed donors was not statistically significant, using the Fisher's Exact test (data not shown). The magnitude of IL-10 production was significantly higher only in the culture supernatants of the Benin malaria-exposed donors stimulated with pool 3 (EXON 2) peptides when compared with the magnitude of IL-10 production in the culture supernatants of the UK non-exposed donors (31.1 ± 10.4 pg/mL and 3.4 ± 1.2 pg/mL respectively; p = 0.03).

Comparison of IL-10 production and CD4 T cell division for each donor indicated an inverse correlation between the 2 responses (Figure 3). UK donors with positive IL-10 production to peptides pools 2 and 3 had no positive CD4 T cell response. Conversely, those UK donors whose CD4 T cells divided in response to the peptide pools did not produce IL-10 (Figure 3). Amongst the Benin donors, only those with the lowest CD4 T cell division produced IL-10 (27% of donors).