Cellular responses to Plasmodium falciparum erythrocyte membrane protein-1: use of relatively conserved synthetic peptide pools to determine CD4 T cell responses in malaria-exposed individuals in Benin, West Africa
© Sanni et al; licensee BioMed Central Ltd. 2002
Received: 27 March 2002
Accepted: 26 April 2002
Published: 26 April 2002
Plasmodium falciparum erythrocyte membrane protein-1, a variant antigen of the malaria parasite, is potentially a target for the immune response. It would be important to determine whether there are CD4 T cells that recognise conserved regions. However, within the relatively conserved region, there is variation. It is not possible to test T cell responses from small field samples with all possible peptides.
We have aligned sequences that are relatively conserved between several PfEMP1 molecules, and chosen a representative sequence similar to most of the PfEMP1 variants. Using these peptides as pools representing CIDRα, CIDRβ and DBLβ-δ domains, DBLα domain, and EXON 2 domain of PfEMP1, we measured the CD4 T cell responses of malaria-exposed donors from Benin, West Africa by a FACS based assay.
All the three peptide pools elicited a CD4 T cell response in a proportion of malaria-exposed and non-exposed donors. CD4 T cell proliferation occurs at a relatively higher magnitude to peptide pools from the DBLα and EXON 2 in the malaria-exposed donors living in Benin than in the UK malaria-unexposed donors.
These findings suggest that an immunological recall response to conserved peptides of a variant antigen can be measured. Further testing of individual peptides in a positive pool will allow us to determine those conserved sequences recognised by many individuals. These types of assays may provide information on conserved peptides of PfEMP1 which could be useful for stimulating T cells to provide help to P. falciparum specific B cells.
Plasmodium falciparum infection is a major source of morbidity and mortality in malaria-endemic areas . The manifestation of severe malaria is more common in non-immune children living in, and non-immune visitors to, endemic regions, suggesting that the acquisition of specific immunity is an important factor in preventing morbidity and mortality from malaria infection . However, the immunity to malaria infection is partial, as adults living in malaria hyper-endemic zone can be parasitaemic or indeed develop malarial disease. The immunological mechanisms and the antigens to which acquired immunity is directed, are not fully known, although the erythrocytic stage of the parasite has been reported to be the primary target . The variant antigens, expressed on the surface of P. falciparum infected red blood cells and believed to be a mechanism of immune evasion by the malaria parasite, are important antigens worthy of consideration as targets for malaria immunity. One such antigen used in this study is P. falciparum erythrocyte membrane protein-1 (PfEMP1).
Hence, we have aligned the sequences of published PfEMP-1 molecules, synthesised a peptide similar to most peptides in a relatively conserved block and pooled the peptides into three groups which correspond to regions of the whole PfEMP1 molecule (Figure 1). The CD4 T cell responses of malaria-exposed donors (from Benin, West Africa) was determined by a flow cytometry method, and compared to the CD4 T cell responses of malaria-unexposed UK donors. Using this method, we have been able to show that CD4 T cells responses to conserved regions of a variant antigen can measured. As the T cell responses to malarial antigens are often low  and it has been suggested that cytokines such as IL-10 may inhibit T cell proliferation , we have also measured IL-10 production after stimulation with the different peptide pools.
Materials and methods
PfEMP1 sequences, alignment and synthesis
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 degree of sequence homology between chosen peptide and variants within relatively conserved blocks of PfEMP1.
Peptides in each pool
Length of peptide
Number of variants compared
Identity with variants
Similarity with variants
Pool 1 Peptides from DBLα region
1aa:11], 2aa:2, 5aa:4, 7aa:1, 8aa:1
2aa:4, 3aa:2, 4aa:4, 1aa:1
1aa:2, 3aa:3, 1aa:2, 6 aa:2, 10aa:1
1aa:2, 2aa:4, 3aa:1
2aa:3, 3aa:1, 4aa:1, 6aa:2, 7aa:3
2aa:4, 3aa:3, 4aa:2, 5aa:1, 6aa:1
2aa:2, 3aa:2, 4aa:1, 5aa:3, 6aa:1, 7aa:2
Pool 2 peptides from CIDRα and β and DBLβ-δ regions
5aa:2, 6aa:3, 7aa:1, 8aa:3, 9aa:1
1aa:1, 2aa:3, 4aa:1
1aa:1, 2aa:4, 3aa:1, 4aa:2, 6aa:1, 8aa:1
1aa:3, 2aa:3, 4aa:1, 8aa:1
1aa:1, 2aa:1, 3aa:4, 4aa:2, 5aa:1
3aa:5, 4aa:1, 5aa:1, 7aa:1, 10aa:1
1aa:2, 2aa:1, 3aa:1, 5aa:1, 7aa:1
1aa:2, 5aa:1, 6aa:1 8aa:2, 9aa:1, 10aa:1,
1aa:1, 9aa:2, 22aa:1, 23aa:2
1aa:3, 6aa:2, 9aa:1
Pool 3 peptides from Exon 2 region
2aa:4, 3aa:2, 4aa:1, 12 aa:2
1aa:2, 2aa:1, 6aa:1, 7aa:2
1aa:3, 3aa:1, 7aa:1, 10aa:1
1aa:6, 2aa:2, 14aa:1
1aa:2, 3aa:2, 5aa:1, 6aa:1
1aa:2, 2aa:1, 4aa:1, 5aa:1
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 . 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 . 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.
In vitro assay for cell division
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 [15, 16]. 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.
Antibodies for 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.
Measurement of cell division by flow cytometry
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 [15, 16]. 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%
Detection of cytokines by ELISA
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.
Prevalence and magnitude of the CD4 T cell proliferation
CD4 T cell proliferation to three pools of relatively conserved peptides of PfEMP-1
Pools of peptides
Mean CD4 T cell proliferation (Log10)
DBLα (pool 1)
2.47 ± 0.15 1] (24)2]
1.94 ± 0.18 (24)
P = 0.025
CIDRα + DBLβ-δ (pool 2)
2.47 ± 0.14 (23)
2.02 ± 0.20 (21)
P = 0.062
Exon 2 (pool 3)
2.44 ± 0.15 (24)
1.94 ± 0.20 (22)
P = 0.045
Prevalence and magnitude of cytokine production
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).
In this study we have shown that the magnitude of the CD4 T cell response to peptide pools representing parts of DBLα and Exon 2 domains of PfEMP1 (Table 2) was significantly higher in the Benin malaria-exposed donors when compared to the UK malaria-unexposed donors. These results suggest that, despite the extensive variation of PfEMP1, exposure to P. falciparum may induce a CD4 T cell response to conserved or shared regions of DBLα and EXON 2 of this molecule. A more detailed study with a larger number of PBMC from immune adults would allow us to determine whether individual peptides within these pools were responsible for the enhanced responses, and thus whether conserved peptides might be useful for eliciting CD4 T cells that are able to help variant specific B cell responses.
All CD4 T cell divisions to the peptides were relatively small; only rarely were divisions of greater than 5 stimulation indices observed (Figure 2). These results agree with several earlier studies showing low T cell responses to a variety of malaria antigens [7, 16–18] which may in part be caused by selective sequestration of specific cells in lymphoid organs [19, 20]. It is also possible in our case that either the peptides selected did not bind the different MHC Class II molecules, or they were not representative of the repertoire of PfEMP1 molecules in the parasite population of this endemic area.
The repertoire of PfEMP1 molecules expressed on parasites in this or any area of P. falciparum transmission is not known. Since the conservation between the fifteen variants sequenced from a variety of parasites from different areas was high, we would expect some of these peptides to be representative of those in the sample population. As tools become available that enable characterisation of the PfEMP1 molecules in different parasites, it will become possible to test T cell responses against peptides from PfEMP1 that are known to be expressed in the parasites infecting the individuals under study.
CD4 T cell responses can be inhibited in vitro and in vivo by cytokines such as TGFβ and IL-10 [21, 22]. Therefore we determined whether IL-10 was produced in the culture supernatants of PBMC from Benin and UK donors in response to the three peptide pools. Here we show that greater amounts of IL-10 were produced in the cultures of the Benin malaria-exposed donors stimulated with pool 3 (EXON 2) peptides. Exposed or non-exposed donors, whose cultures produced high levels of IL-10 in response to any of the peptide pools generally had no or low CD4 T cell responses and vice versa. These data, although from a small sample, suggest that strong IL-10 responses are not compatible with CD4 T cell proliferation in vitro. Proliferation assays carried out in the presence of anti-IL-10 antibodies or anti-IL-10 receptor antibodies would clarify the anti-proliferative role of IL-10 in these responses; such studies are underway. The fact that a small fraction of malaria-unexposed UK donors also respond to these peptide pools could indicate that there are cross-reactive or shared T cell epitopes with other antigens among the three peptides pools used in the in vitro stimulation as has been described earlier for some malaria antigen specific T cell clones .
Our data suggest that the use of synthetic peptides representing conserved sequences of PfEMP1 might be of value in determining regions of the molecule recognised by T cells of a wide variety of donors exposed to different variants of PfEMP1. Further dissection of the responses to individual peptides may allow us to select regions of PfEMP1 that could be useful for a peptide-based vaccine. It would be very encouraging for vaccine design if CD4 T cell helper responses for variant specific B cells could be directed to common or conserved regions of PfEMP1.
In summary, we have shown that CD4 T cell division to conserved peptides of some regions of PfEMP1 occurs at a relatively higher magnitude in donors living in a malaria endemic zone when compared with malaria-unexposed donors. These findings indicate a secondary response to these peptides and, hence, have important implications in malaria vaccine development, as the relatively conserved peptides inducing the T cell division might be useful for stimulating variant specific B cell help in a multi-component vaccine. At present, we have used peptide mixtures from different regions of PfEMP1 because of limitations in the number of PBMC available from each donor. An important extension of this work is to map out the specific peptides responsible for the induction of the observed CD4 T cell division in the responders and determine whether other variants of the peptides produce a similar response or act as altered peptide ligands. The response of individuals with different HLA class II haplotype to these peptides would also determine their usefulness in vaccination.
We would like to thank Frank Albano and Ching Li for the critical reading of the manuscript, and the Medical Research Council for funding this study. Part of this work was supported by the EU INCO-DC programme. Latifu Sanni was the recipient of a Wellcome Trust Travelling Research Fellowship (053488).
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