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    Figure 1.

    IFN-γ ELISpot responses to variants of 10 strains of Pf TRAP in Kenyan adults. Twenty-five healthy Kenyan adults (denoted K) were tested by IFN-γ ELISpot to a panel of 27 variant Pf TRAP peptides. Variants of overlapping peptides tp4, 5, and 6 were combined into three pools. A positive response is indicated by a filled in square. ND, not determined.

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    Figure 2.

    Cytokine reactivity to the TRAP peptide epitope variants Kenyan adults (denoted K) were tested in parallel ELISpot assays to a panel of 27 variant and 4 conserved Pf TRAP peptides for IFN-γ (N = 26), IL-4 (N = 15), and IL-10 (N = 11) reactivity. The first panel shows results for the 11 donors tested for all 3 cytokines, and the second panel shows results for those tested for IFN-γ and IL-4 reactivity only. A positive response is indicated by a filled-in square according to the key. ND, not determined.

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CELLULAR REACTIVITY TO THE P. FALCIPARUM PROTEIN TRAP IN ADULT KENYANS: NOVEL EPITOPES, COMPLEX CYTOKINE PATTERNS, AND THE IMPACT OF NATURAL ANTIGENIC VARIATION

KATIE L. FLANAGANWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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MAGDALENA PLEBANSKIWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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KENNEDY ODHIAMBOWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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ERIC SHEUWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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TABITHA MWANGIWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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COLIN GELDERWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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KEITH HARTWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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MOSES KORTOKWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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BRETT LOWEWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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KATHRYN J. ROBSONWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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KEVIN MARSHWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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ADRIAN V. S. HILLWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; KEMRI Centre for Geographic Medicine—Coast, Kilifi, Kenya; Infection & Immunity, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom

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Malaria vaccines based on thrombospondin-related adhesive protein of Plasmodium falciparum (Pf TRAP) are currently undergoing clinical trials in humans. This study was designed to investigate naturally acquired cellular immunity to Pf TRAP in adults from a target population for future trials of TRAP-based vaccines in Kilifi, Kenya. We first tested reactivity to a panel of 53 peptides spanning Pf TRAP and identified 26 novel T-cell epitopes. A panel of naturally occurring polymorphic variant epitope peptides were made to the most commonly recognized epitope regions and tested for ability to elicit IFN-γ, IL-4, and IL-10 production. These data provide for the first time a complex cytokine matrix mapping naturally induced T-cell responses to TRAP and suggest that T-cell responses boosted by vaccination with Pf TRAP could stimulate the release of competing pro- and anti-inflammatory cytokines. They further define polymorphic variants able to boost specific Th1, Th2, and possibly Tr1 reactivity.

INTRODUCTION

Three hundred million to 500 million cases of clinical Plasmodium falciparum malaria occur worldwide annually, with 1–2 million deaths, or an estimated death every 30 seconds.1 P. falciparum malaria poses an enormous economic burden on endemic countries, and despite worldwide efforts at control, malaria is on the increase. The development of an effective malaria vaccine would have a huge impact on this alarming situation. The observation that animals and humans immunized with irradiated sporozoites are protected against heterologous P. falciparum challenge provides the rationale for the development of pre-erythrocytic stage vaccines.2,3 Studies suggest that T-cell responses against the pre-erythrocytic stage antigens circumsporozoite (CS) protein, thrombospondin-related adhesive protein (TRAP) (also known as SSP24), and liver stage antigen-1 (LSA-1) may all have a protective role.513

Adults living in regions of the world holoendemic for P. falciparum rarely develop the severe manifestations of P. falciparum infection, although the mechanism of this immunity is poorly understood. Human studies of naturally exposed populations thus play an important role in our search for correlates of protection against P. falciparum and provide essential data on immune reactivity in populations prior to the testing of novel vaccines. TRAP-based malaria vaccines are currently undergoing clinical trials in humans in the United Kingdom,14 in The Gambia, West Africa,15 and more recently in Kilifi, Kenya, in East Africa. We have previously studied cellular reactivity to Pf TRAP in Gambian adults,16 but there are many reasons why reactivity might differ between different endemic populations including local strain variation, HLA variability, P. falciparum seasonality, bed net usage, and local disease patterns. A Th1 type response with IFN-γ production is thought to be the major protective cellular response at the pre-erythrocytic stage of infection, acting via the production of reactive nitrogen intermediates.1721 However cellular reactivity to Pf TRAP is likely to involve the activation of other cellular subsets with different cytokine profiles. We therefore tested IFN-γ, IL-4, and IL-10 ELISpot reactivity to peptides spanning the whole of Pf TRAP strain NF54 in semi-immune adults from Kilifi, Kenya, to determine the pattern of reactivity in this naturally exposed population. TRAP is less polymorphic than other vaccine candidate antigens such as CS protein. Despite this, numerous nonsynonymous mutations occur along the length of the antigen,2223 suggesting that antigenic variation is driven by immune selection pressure and may thus confer a survival advantage to the parasite. The strains of Pf TRAP present in Kenya was unknown, and thus we analyzed the infecting parasites in 105 Kenyan donors for the distribution and frequency of described polymorphisms and the presence of novel TRAP polymorphisms. We constructed a panel of peptide variants of the 14 most commonly recognized NF54 strain peptide epitopes to encompass 10 naturally occurring strains of Pf TRAP throughout the world. We tested for IFN-γ, IL-4, and IL-10 reactivity in healthy adult Kenyans to gain a comprehensive picture of the pattern of cytokine production to Pf TRAP in naturally exposed adults.

MATERIALS AND METHODS

Donors.

The study was approved by The Kenya Medical Research Institute national ethical and scientific review committees, and informed consent was obtained from all participants in the study. Sixty-five Kenyan adults (D1–65) from Kilifi district were recruited in June/July at the start of the high-transmission season (June to August), and a further 25 healthy adults (K1–25) 1 year later in September at the end of the high-transmission period. The majority of these 90 donors (76%, 68 of 90) were HIV negative blood transfusion donors, and the remaining 24% were not tested for HIV but were all healthy and not known to be infected. A further 105 healthy Kenyan adults and children that were recruited for a separate study were typed for TRAP polymorphisms in the local population. Fifteen malaria naïve adult employees at the John Radcliffe Hospital in Oxford were recruited and tested in the United Kingdom under similar conditions to the Kenyan study.

PBMC preparation.

Twenty to 50 mL of blood was taken from each adult donor, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) using standard methods. PBMCs were resuspended in complete medium RN5 (RPMI medium supplemented with 5% heat inactivated human AB serum, 2 mM glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin) for use in cellular assays. CD4+ and CD8+ cell depletions were performed using magnetic beads (Dynal, Bromborough, UK) according to the manufacturer’s instructions. Depletions were confirmed to be > 95% effective by flow cytometry.

TRAP polymorphism analysis.

DNA was extracted from a 200-μL sample of heparinized whole blood using Qiagen QIAmp Blood Kits (Qiagen, Crawley, UK) according to manufacturer’s instructions. The eluted DNA was frozen and transported to the United Kingdom for parasite typing. Parasite DNA was amplified using 2 rounds of PCR as published previously.24 PCR products were then incubated with the restriction enzymes Bgl II, Ssp I, Taq I, and Afl II (New England Biolabs, Hitchin, UK) and analyzed using 1.7% agarose gels, stained for 30 minutes with ethidium bromide and photographed under UV light.

Peptides.

A panel of fifty 20mer peptides (tp1–tp50; aa 1–510), overlapping by ten amino acids, derived from Pf TRAP clone NF54 were synthesized commercially (Research Genetics, Huntsville, AL). Three Pf TRAP 20mers covering the carboxy-terminus region (tp51, tp52, and tp53; aa 526–545, 541–560, and 555–574) were synthesized using a standard Fmoc/t-butyl solid-phase Zinsser Analytical synthesizer (Zinsser Analytic, Maidenhead, UK). Eleven TRAP pools were made by combining nonoverlapping TRAP peptides as detailed in our previous studies16 and were used in selected donors to narrow down the positive response before testing the individual peptides. The variant TRAP peptides derived from 10 naturally occurring strains of Pf TRAP were synthesised commercially (Research Genetics) (Table 1). All peptides were used at a final concentration of 25 μg/mL following preliminary titrations (not shown). Purified protein derivative (PPD) (Statens Seruminstitut, Copenhagen, Denmark) at 25 μg/mL was utilized as a positive control for the IFN-γ assays, and phytohaemagglutinin (PHA) at 10 μg/mL for the IL-4 and IL-10 assays.

ELISpot assays.

ELISpot assays were carried out using 96-well MAIP ELISpot plates (Millipore, Watford, UK) and human ELISpot kits (Mabtech, Nacka, Sweden) as described in detail elsewhere.16 Briefly, plates were coated with monoclonal anti-human antibody (IFN-γ mAb: 1-D1K; IL-4: IL-4-I; IL-10: 9-D7, all from Mabtech), washed, and blocked with R10 (RPMI with 10% fetal calf serum). Freshly isolated PB-MCs were resuspended at 4 × 106/mL in RN5, applied at 100 μL/well (4 × 105 cells/well) and tested against the TRAP peptide conditions, with PBS as the negative control and PPD (IFN-γ assays)/PHA (IL-4 and IL-10 assays) as positive controls. Plates were incubated for 16 hours at 37° C in 5% CO2, washed, and the appropriate biotinylated mAb (IFN-γ, IL-4, or IL-10, Mabtech) was applied at 1 μg/mL, followed by 1 μg/mL streptavidin alkaline phosphatase (Mabtech). Plates were then washed and developed with ALP conjugate substrate kit (Bio-Rad Laboratories, Hercules, CA) and the reaction stopped after 10–20 minutes by flicking out the contents and running under tap water. Each IFN-γ producing cell leaves a single spot or “footprint” in the ELISpot well. Wells were scored visually, using a dissection microscope, for the number of purple spots or spot-forming units (SFUs) per well, and the results were expressed as SFUs/106 PBMC.

HLA class II typing.

HLA class II phototyping was performed as described previously.25 Genomic DNA was amplified in 96-well thermowell V plates (Costar, High Wycombe, UK) using the Phoenix thermocycler (Techen, Cambridge, UK). PCR reaction mixtures consisted of 67 mM Tris Base pH 8.8, 16.6 mM ammonium sulfate, 2 mM magnesium chloride, 0.01% Tween 20, 200 μM of dNTP, 1–4 μM of each allele-specific primer, 0.1 μM of DRB1 control primers, 0.1–0.01 μg DNA, and 0.1875 units Taq Polymerase (Advanced Biotechnology, London, UK). The final volume for PCR reactions was 13 μL, consisting of 8 μL of buffer, DNA and enzyme mixture and 5 μL of primer mix (containing allele and control primers in distilled water). Ten microliters of mineral oil was overlayed and trays were sealed with thermowell sealer (Costar). PCR products were electrophoresed on 2% agarose gels containing 0.5 μg/mL ethidium bromide after the addition of 10 μL of loading buffer consisting of 0.05% Orange G, 30% v/v glycerol, and 0.5× TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0). Gels were run for 30–50 minutes at 15 V/cm in 0.5× TBE buffer and visualized using UV illumination.

Statistical methods.

The number of responder cells in the single test well were compared with the background negative well. ELISpot wells were scored positive using a statistical significance table, which assumes a Poisson distribution for the number of spots in each well, and gives a P value for the likelihood that the result is a true positive rather than a chance event, as described in previous studies.26 All responses with a 95% or more confidence (P < 0.05) that the result did not occur by chance were recorded as positive. Proportions of donors responding to different cytokines were compared used standard χ2 analysis, having first established that the background levels were comparable. Where the numbers in each group for comparison were very small, Fisher’s value for two-tailed analysis is given for the χ2 P value. The precursor frequency values (SFU/106 PBMC) were compared using t tests to look for evidence of a significant difference between groups.

RESULTS

IFN-γ reactivity to > 90% of 20mer peptides spanning the whole of TRAP.

IFN-γ ELISpot reactivity was tested to a panel of 53 overlapping 20mer TRAP peptides (tp1–tp53) encompassing almost the entire sequence of Pf TRAP strain NF54, using the same methodology and peptides as in our previous study of 50 adult Gambians.16 Freshly isolated PBMC from 65 adult Kenyans were either tested to the 11 peptide pools to screen for reactivity and then tested to the individual peptides using cryopreserved PBMC (N = 22); or tested immediately to all 53 individual TRAP peptides (N = 43). Sixty percent (39 of 65) of the Kenyan donors responded to one or more peptide (Table 2), which is almost double the reactivity rate of 32% (16 of 50) seen in the Gambian study (χ2 = 8.13, P = 0.004).16 Eighty-six percent (56 of 65) gave a positive PPD response. The magnitude of positive T-cell responses (specific responder cells/106 PBMC) was 33.6 SFU ± 2.1 SE/106 PBMC (arithmetic mean), which was comparable to our previous findings in Gambian adults (28.2 ± 1.2 SE/106 PBMC, P = 0.25).

Forty-eight of the 53 TRAP peptides (91%) were immunogenic in the 65 Kenyans, 26 of which are novel Pf TRAP clone NF54 epitopes (Table 2). CD4 and CD8 depletion studies were performed when cell numbers permitted, and all 5 epitopes tested were confirmed to elicit CD4+ responses (data not shown). We analyzed the HLA class II types for 29 of the 39 Kenyan responders, to assess for an association between HLA type and individual TRAP epitope responses (Table 3). Almost every responder had the haplotype DRB3*52 making it difficult to assess the role of this haplotype, otherwise we found only one TRAP peptide for which a consistent HLA type was found in all responders in that all 5 responders to tp6 were DQB1*05 (Table 3). This apparent lack of HLA restriction for individual epitopes suggests that the TRAP epitopes are recognised through multiple HLA types, supporting previous data demonstrating HLA-DR-promiscuous T cell epitopes in TRAP.27

Malaria naïve individuals do not respond to TRAP 20mer peptides.

Fifteen malaria naïve donors were tested in Oxford for IFN-γ ELISpot responses to the 10 pools of nonoverlapping 20mer TRAP peptides tp1–tp50 (aa 1–510) using freshly isolated PBMC and similar conditions to those used in the Kenyan study. None of the 15 naïve donors gave a positive response to the TRAP pools, while 73% (11 of 15) gave a positive response to PPD (not shown). This lack of reactivity in naïve adults is highly significant when compared with adult Kenyans (χ2 = 17.56, P = 0.00003), and suggests that none of the malaria naïve donors have circulating IFN-γ producing cells that are cross-reactive with Pf TRAP epitopes.

Reactivity to naturally occurring variants of PfTRAP epitopes.

Pf TRAP exhibits less polymorphism than other malaria antigens such as CS protein, however up to 5 naturally occurring variant residues arise at particular sites of the antigen.23 We selected 9 of the most frequently recognised Pf TRAP NF54 20mer peptides that contained polymorphic residues and synthesised the polymorphic variants from 10 common naturally occurring isolates of Pf TRAP from around the world including East Africa (7901), West Africa (3D7, NF54), Latin America (HB3, 7G8), and Southeast Asia (DD2, K1, ITO4, T996)23 (Table 1). The common Pf TRAP isolates in West Africa are well-known23 but have never been analyzed for Kenyan samples. We therefore analyzed the parasite isolates infecting 105 Kenyan donors at the end of a high-transmission season using restriction fragment length polymorphism. Those common in West Africa were found to predominate in Kilifi, with haplotype 42 being the most frequent (63% of donors) and complete absence of the 3′ Bgl II site which is thought to be due to an exclusively Southeast Asian polymorphism. Sixty-three percent of donors were infected with more than one Pf TRAP isolate (mean number of infecting parasite isolates 2.3 ± 1.5 SD), with up to 8 haplotypes in 1 donor. One might therefore expect the local population to exhibit broad ranging reactivity to the variant peptides of Pf TRAP. A new haplotype was also found in the Kilifi donors due to a novel Ssp1 site resulting from an asparagine to isoleucine substitution at position 403.

Twenty-five Kenyan adults were tested by IFN-γ ELISpot to the variant peptides. While 36% (9 of 25) of donors responded to NF54 strain peptides, 72% (18 of 25) of donors responded to at least one of the variant peptide panel (Figure 1). Thus testing with naturally occurring variant peptides rather than to a single local isolate doubled the number of positive responders. Forty-four percent (11 of 25) of the donors showed a degree of cross-recognition of variants of a particular epitope region, although generally cross-recognition was limited to 2 variants only (Figure 1). Four donors responded to 3 or 4 variants of a particular epitope, and in 3 of these donors reactivity was to variants of tp31, although 5 of the 9 donors who gave a tp31 region response did so to one variant only. The tp31 epitope region contains the proposed cell recognition sequence, RGD, which is likely to be of functional significance, although this has yet to be confirmed. Thirty-nine percent (16 of 41) of positive responses to any particular epitope region exhibited a degree of cross-recognition, and there was only one instance in which a donor responded to all variants of a particular epitope (donor K3 to all 4 tp31 variants) (Figure 1). Therefore although many Pf TRAP epitopes may be able to induce and be the target of cross-reactive immunity, specific regions did show strain specificity. If the latter are important for protection, this could explain the lack of association of TRAP strain NF54 specific IFN-γ ELISpot reactivity with protection observed in our longitudinal study in Kenya.28

Complex cytokine patterns in response to TRAP peptides suggest activation of multiple cellular subsets.

Twenty-three of the original 65 donors were assessed in parallel assays for reactivity to the 11 Pf TRAP peptide pools: 22 by IFN-γ , 21 by IL-4, and 8 by IL-10 ELISpot assays (Table 4). Fifty-nine percent (13 of 22) of donors responded to at least one pool by IFN-γ production, 29% (6 of 21) produced IL-4, and 63% (5 of 8) of donors tested produced IL-10 (Table 4). Thus, IFN-γ and IL-10 reactivity was comparable, with lower levels of IL-4 reactivity in these donors. Where IFN-γ and IL-4 responses were detected in the same donor, they were to different peptide pools, for example, D37 had an IFN-γ response to pools 4 and 9, and an IL-4 response to pools 2 and 7 (Table 4). Similarly the IL-4 and IL-10 responses were to different peptide pools in individual donors (D46, D47, D49). Two of the 5 responders (D44 and D46) produced IFN-γ and IL-10 to the same peptide pools, although we do not know to which individual peptides.

We speculated that variant TRAP peptides may selectively induce either Th1 (IFN-γ ), Th2 (IL-4 ± IL-10), or Tr1 (IL-10 alone) type cytokines. Fifteen of the 25 donors assayed for IFN-γ reactivity to the variant TRAP peptides were tested in parallel ELISpot assays for IL-4, 11 of whom were also tested for IL-10 reactivity. Fifty-five percent (6 of 11) of donors tested for IL-10 responses, and 47% (7 of 15) of donors tested for IL-4, gave positive T-cell responses to one or more TRAP peptides (Figure 2), indicating no significant difference in the proportion of donors who produced IL-4 or IL-10 (χ2 = 0.16, P = 0.69). The total number of IL-4 responses (20 positive responses in 15 donors) and IL-10 responses (12 positive responses in 11 donors) in this variant analysis were also comparable (P = 0.22). This contrasts with the higher IL-10 reactivity compared with IL-4 reactivity when testing with the peptide pools spanning Pf TRAP strain NF54 (Table 4). However, more donors responded to NF54 derived peptides by IL-10 production (5 of 11, 46%) than IL-4 production (2 of 15, 13%) in the variant study, suggesting that the NF54 strain selectively induces higher levels of IL-10 than IL-4, although numbers were too small to test for significance. This may be because NF54 is a common African strain, and hence may be more immunogenic in this African population. It would be interesting to test this hypothesis in the field.

Two donors (K8 and K12) gave entirely Th2 biased responses to TRAP variants, and 3 of the 11 donors for whom all 3 cytokines were tested were Th1 biased (donors K13, K21, K22) (Figure 2). For three of the donors IFN-γ was produced to certain variants, and IL-4 to other variants of the same epitope (donors K9, K10, K14). For example, donor K10 produced IFN-γ to tp37b and tp37d, and IL-4 to tp37c. Similarly, donor K14 produced IFN-γ to tp43b and IL-4 to tp43c. There were no cases of IL-10 production to one variant and IFN-γ to another, but the conserved peptide tp2 induced an IFN-γ and IL-10 response in the same donor (K7). Dual induction of opposing cytokines by the same conserved CD4 T cell epitope illustrates the complexity of T-cell immunity to P. falciparum in naturally exposed donors.

DISCUSSION

Animal studies suggest that T-cell reactivity to TRAP can provide protection against malaria challenge,79 and TRAP-based vaccines have reached phase II trials in humans.15 To test vaccine immunogenicity in malaria-exposed populations it is useful to understand the naturally acquired responses that are likely to be boosted by vaccination. This includes the identification of novel peptide epitopes, and we describe 26 novel 20mer Pf TRAP (strain NF54) peptide epitopes, which induce IFN-γ in naturally exposed Kenyans. Recognition through multiple HLA types has been described for class I and class II restricted responses to TRAP in humans,27,29 and our results further support this phenomenon. Such HLA promiscuity suggests that a limited number of epitopes could cover a wide range of common HLA types throughout the world, a feature advantageous for those designing poly-epitope vaccines against P. falciparum.

Reactivity occurred to peptides along the whole length of the antigen, with no clear areas of immunodominance, in agreement with our findings in adult Gambians.16 This is unlike reactivity to many other malaria antigens such as CS protein, Pf155/RESA or merozoite surface protein-1 (MSP-1) where reactivity tends to localise to certain immunodominant regions.3032 However, multiple T-cell epitopes have been described along the length of another pre-erythrocytic antigen, liver stage antigen-3 (LSA-3).33 Such widespread IFN-γ reactivity might suggest that Pf TRAP is a frequent target for antiparasite immunity, however it may also represent a parasite immune evasion strategy diverting host immune reactivity away from potentially protective epitope responses. Epitope “spreading” is a phenomenon whereby epitopes distinct from, and non-cross reactive with, an inducing epitope become major targets of an ongoing immune response. Although more commonly described in models of autoimmunity, it is also described in chronic persistent infections.3435 It is possible that initial exposure to TRAP stimulates an immunodominant response, but with repeated and prolonged exposure the response progresses to encompass lesser epitopes.

The TRAP vaccines undergoing clinical trials are based on a single parasite strain, and the significance of parasite polymorphism is not known. The majority of Kilifi donors were infected with multiple parasite strains (up to 8 per donor), so one would predict that donors are exposed to most of the naturally occurring variants within a short time, and would thus react to the variant peptides. However, if immune selection pressure has driven the antigenic variation by conferring a survival advantage for the parasite, then peptide sequence variation should cause loss of reactivity. Indeed, almost 60% of positive responders to the index NF54 strain showed no cross-reactivity with variants of the same epitope, supporting the latter hypothesis. A similar lack of cross-reactivity has been observed to variant epitope regions of CS protein.3638 Lack of reactivity could not be explained by HIV seropositivity, as the majority of donors in this study were known to be HIV negative. Another explanation is that ex vivo ELISpot responses are short lived, and indeed our previous studies in Kenya showed a lack of stability of these responses over 1 year.28 In only one instance did a donor respond to all the variants tested for a particular TRAP T cell epitope. Cross-recognition of variant T-cell epitopes by individual donors may be mediated either by different T cell clones, or by truly cross-reactive T-cell clones that recognize multiple variants through the same T-cell receptor. Certain of the variants tested have never been identified in Africans such as the tp43b RNI polymorphism (aa 426–428) (Table 1), which is thought to be exclusively Southeast Asian, and was not present in any of the 105 Kenyan samples tested. Reactivity to tp43b in 3 Kenyan donors (Figure 1) is therefore likely to be mediated by truly cross-reactive T cells that were primed by a variant of tp43 to which the donors had been exposed.

Many studies of vaccine immunogenicity test for IFN-γ reactivity, but overlook other cytokines. We therefore tested for IFN-γ , IL-4, and IL-10 reactivity in selected donors to both the NF54 strain and variant peptides. Isolated IL-10 production, in the absence of IL-4 production, was observed in many instances in this study suggesting a source of IL-10 other than Th2 cells. We speculate that some of the IL-10 production during acute P. falciparum may be derived from IL-10 producing Tr1 regulatory T cells,36,39 however the small number of donors tested precludes the drawing of any definitive conclusions regarding patterns of cytokine reactivity. Analysis of the cytokine responses to variant peptides demonstrated the extreme complexity of natural immunity to P. falciparum including the simultaneous production of IFN-γ and IL-4 to variants of the same epitope, and IFN-γ and IL-10 release to the same conserved epitope. For no variant region was there a differential induction of IFN-γ and IL-10 by variant epitopes, and thus no evidence that TRAP polymorphic variants use the same altered peptide ligand immune evasion strategy described for CS protein variants.36

We therefore describe the first comprehensive analysis of reactivity to Pf TRAP in Kenyans, and the first study of the effect of TRAP specific antigenic polymorphism in a naturally exposed population. This data provides fundamental baseline data for those testing TRAP-based malaria vaccines in malaria endemic regions and provides useful information for those attempting to interpret field studies of P. falciparum–specific cellular immunity in naturally exposed populations. Given the complexity of natural reactivity to Pf TRAP that we demonstrate here, it is hardly surprising that, despite intensive efforts, convincing T-cell correlates of protection have yet to be demonstrated in naturally exposed populations.

Table 1

Details of variant TRAP peptides

Peptide Sequence Strain
Naturally occurring variants were manufactured for the most frequently recognized NF54 epitopes in the 65 adults from Kilifi. The variants were derived from previous studies of naturally occurring Pf TRAP polymorphisms, and 10 natural isolates worldwide are represented by this panel of peptides as follows: East Africa (7901), West Africa (3D7, NF54), Latin America (HB3, 7G8), and Southeast Asia (DD2, K1, ITO4, T996).23 The index NF54 strain is denoted with the suffix “a” and the variants are suffixed with other letters. Overlapping peptides tp2, 3, and 4 were made up into 3 pools as follows: Pool 4–6a: tp4a + 5a + 6a; Pool 4–6b: tp4b + 5b + 6a; Pool 4–6c: tp4a + 5a + 6b. Polymorphic residues are indicated in bold type.
tp4a NIVDEIKYREEVCNDEVDLY NF54/FCR3/K1/DD2/HB3/7901/7G8/3D7/ITO4
tp4b NIVDEIKYSEEVCNDQVDLY T996
tp5a EVCNDEVDLYLLMDCSGSIR NF54/FCR3/K1/DD2/HB3/7901/7G8/3D7/ITO4
tp5b EVCNDQVDLYLLMDCSGSIR T996
tp6a LLMDCSGSIRRHNWVNHAVP 7G8/T996/ITO4/FCR3/K1/7901/3D7/ITO4
tp6b LLMDCSGSIRRHNWVKHAVP DD2/HB3
tp14a TNLTDALLQVRKHLNDRINR NF54/HB3/T996/3D7
tp14b TNLSDALLQVRKHLNDRINR ITO4/FCR3/K1/DD2/7G8
tp14c TNLTDALLEVRKHLNDRINR 7901
tp30a EPLDVPDEPEDDQPRPRGDN NF54/T996/3D7
tp30b EPLDVPHEPEDDQPRPRGDN ITO4/K1/FCR3
tp30c EPLDVPQEPEDDQPRPRGDN 7901/7G8/DD2/HB3
tp31a DDQPRPRGDNFAVEKPNENI NF54
tp31b DDQPRPRGDNSSVQKPEENI T996/7G8
tp31c DDQPRPRGDNFAVEKPKENI ITO4/3D7/FCR3/7901
tp31d DDQPRPRGDNFAVEKPEENI K1/DD2/HB3
tp37a PPNPPNPPNPDIPEQEPNIP ITO4/3D7/HB3/DD2/FCR3
tp37b PPNPPNPPNPDIPEQKPNIP T996/NF54
tp37c PPNPPNPPNPDIPERKPNIP 7901
tp37d PPNPPNPPNPDIPQQEPNIP 7G8/K1
tp38a DIPEQEPNIPEDSEKEVPSD ITO4/3D7/HB3/DD2/FCR3
tp38b DIPEQKPNIPEDSEKEVPSD T996/NF54
tp38c DIPERKPNIPEDSEKEVPSD 7901
tp38d DIPQQEPNIPEDSEKEVPSD 7G8/K1
tp43a NDKSDRYIPYSPLSPKVLDN NF54
tp43b NDKSDRSIPYSPLPPKVLDN ITO4/K1/FCR3
tp43c NDKSDRYIPYSPLPPNVLDN 7G8
tp43d NDKSDRNIPYSPLPPKVLDN T996
tp43e NDKSDRYIPYSPLAPKVLDN 3D7
tp43f NDKSDRYIPYSPLPPKVLDN 7901/HB3/DD2
Table 2

Summary of all positive IFN-γ ELISpot responses in 65 Kenyan adults

TRAP peptide Residues Sequence Number (%) responders Novel CD4/8
Summary of all positive responses to the 53 overlapping Pf TRAP peptides detected by IFN-γ ELISpot analysis of 65 adult Kenyans. The results for those peptides for which CD4 and CD8 depletion studies have been performed are shown. All peptides tested elicited CD4 responses, and 2 peptides induced CD4 and/or CD8 T-cell responses. Variant residues are indicated in bold type.
tp1 1–20 MNHLGNVKYLVIVFLIFFDL 1 (2%) Yes
tp2 11–30 VIVFLIFFDLFLVNGRDVQN 4 (9%)
tp3 21–40 FLVNGRDVQNNIVDEIKYRE 2 (5%) Yes
tp4 31–50 NIVDEIKYREEVCNDEVDLY 5 (12%)
tp5 41–60 EVCNDEVDLYLLMDCSGSIR 4 (9%) CD4/8
tp6 51–70 LLMDCSGSIRRHNWVNHAVP 5 (12%) CD4/8
tp7 61–80 RHNWVNHAVPLAMKLIQQLN 3 (7%) Yes
tp8 71–90 LAMKLIQQLNLNDNAIHLYA 2 (5%) Yes
tp9 81–100 LNDNAIHLYASVFSNNAREI 1 (2%) Yes CD4
tp12 111–130 KEKALIIIKSLLSTNLPYGK 2 (5%) Yes
tp13 121–140 LLSTNLPYGKTNLTDALLQV 1 (2%)
tp14 131–150 TNLTDALLQVRKHLNDRINR 4 (9%) CD4
tp15 141–160 RKHLNDRINRENANQLVVIL 2 (5%) Yes CD4
tp16 151–170 ENANQLVVILTDGIPDSIQD 2 (5%) Yes
tp17 161–180 TDGIPDSIQDSLKESRKLSD 1 (2%) Yes
tp18 171–190 SLKESRKLSDRGVKIAVFGI 2 (5%) Yes
tp19 181–200 RGVKIAVFGIGQGINVAFNR 3 (7%) Yes CD4
tp20 191–210 GQGINVAFNRFLVGCHPSDG 2 (5%) Yes
tp21 201–220 FLVGCHPSDGKCNLYADSAW 1 (2%) Yes
tp22 211–230 KCNLYADSAWENVKNVIGPF 2 (5%)
tp23 221–240 ENVKNVIGPFMKAVCVEVEK 3 (7%) CD4
tp24 231–250 MKAVCVEVEKTASCGVWDEW 1 (2%)
tp25 241–260 TASCGVWDEWSPCSVTCGKG 2 (5%)
tp26 251–270 SPCSVTCGKGTRSRKREILH 2 (5%) Yes CD4
tp27 261–280 TRSRKREILHEGCTSELQEQ 3 (7%) CD4
tp28 271–290 EGCTSELQEQCEEERCLPKR 3 (7%) Yes
tp29 281–300 CEEERCLPKREPLDVPDEPE 2 (5%) Yes
tp30 291–310 EPLDVPDEPEDDQPRPRGDN 3 (7%)
tp31 301–320 DDQPRPRGDNFAVEKPNENI 4 (9%) CD4
tp32 311–330 FAVEKPNENIIDNNPQEPSP 1 (2%) Yes CD4
tp34 331–350 NPEEGKGENPNGFDLDENPE 1 (2%) Yes
tp35 341–360 NGFDLDENPENPPNPPNPPN 1 (2%) Yes
tp36 351–370 NPPNPPNPPNPPNPPNPPNP 2 (5%) Yes
tp37 361–380 PPNPPNPPNPDIPEQEPNIP 4 (9%)
tp38 371–390 DIPEQEPNIPEDSEKEVPSD 4 (9%) CD4
tp39 381–400 EDSEKEVPSDVPKNPEDDRE 2 (5%)
tp40 391–410 VPKNPEDDREENFDIPKKPE 4 (9%)
tp42 411–430 NKHDNQNNLPNDKSDRYIPY 2 (5%) Yes
tp43 421–440 NDKSDRYIPYSPLSPKVLDN 2 (5%) CD4
tp44 431–450 SPLSPKVLDNERKQSDPQSQ 1 (2%) Yes
tp45 441–460 ERKQSDPQSQDNNGNRHVPN 1 (2%) Yes
tp47 461–480 SEDRETRPHGRNNENRSYNR 4 (9%) CD4
tp48 471–490 RNNENRSYNRKHNNTPKHPE 2 (5%)
tp49 481–500 KHNNTPKHPEREEHEKPDNN 2 (5%) Yes
tp50 491–510 REEHEKPDNNKKKAGSDNKY 1 (2%)
tp51 526–545 AGLAYKFVVPGAATPYAGEP 6 (14%) CD4
tp52 540–559 YAGEPAPFDETLGEEDKDLD 3 (7%) Yes
tp53 555–574 EDKDLDEPEQFRLPEENEWN 2 (5%) Yes
Table 3

HLA class II types of Kenyan epitope responders

Donor DRB1 DRB1 DRB5 DRB3 DRB4 DQB1 DQB1 tp peptide responses
Twenty-nine of the 39 Kenyan responders were HLA class II typed to see if epitope responders had common HLA class II haplotypes. The table clearly demonstrates that this was not the case, suggesting that each peptide could be recognized through more than one HLA class II type.
D11 10 11 52 5 tp1,2,7,12,14–17,23,25–26,35–36,38,42,50
D12 7 13 52 53 7 tp4,51–53
D13 1 11 52 5 7 tp15,16,19,27,28
D14 17 52 2 5 tp14,23,24,40,51
D15 11 12 52 5 7 tp51
D16 11 15 51 52 5 6 tp3,6,14,36
D17 11 51 52 tp14
D19 11 52 5 7 tp5,23,51
D21 1 13 52 53 2 5 tp6,30,39
D23 7 13 52 53 2 7 tp2,5,7,8,13,22,31,37,38
D24 11 17 52 5 tp5,47
D25 11 12 52 5 tp47,49,51
D27 18 52 5 tp6,8,22,25,26,30
D28 11 52 6 tp12,18
D30 11 13 52 5 6 tp5
D34 7 12 52 53 2 5 tp2,4,6,28,30,31,37–40,48,52
D35 13 17 52 2 6 tp9
D36 10 11 52 5 tp6
D37 13 17 52 ?53 2 tp4,29
D39 18 52 4 5 tp4,31
D40 13 52 5 tp18,20,34,37,40,51
D42 13 52 6 tp19
D44 12 51 5 6 tp43,52
D49 18 52 4 tp47
D51 7 13 52 53 2 5 tp43
D52 11 52 4 tp21
D55 11 13 52 5 7 tp27,42
D56 17 52 5 2 tp2–4,7,19,20,27–29,31–32,37–38,40,44,47–49,53
D57 13 52 6 7 tp45
Table 4

IFN-γ, IL-4, and IL-10 reactivity of Kenyan adults to NF54 strain TRAP peptide pools

Donor IFN-γ IL-4 IL-10
Twenty-three Kenyan adults were tested for reactivity by IFN-γ , IL-4, and IL-10 ELISpot assays to the 11 pools of overlapping Pf TRAP peptides derived from clone NF54. Positive responses to peptide pools are indicated for each donor. ND, not determined.
D9 nil nil ND
D10 Pool 9 nil ND
D17 Pool 4 nil ND
D19 Pool 3,5,11 nil ND
D20 nil nil ND
D21 Pool 6,9,10 nil ND
D22 nil ND nil
D31 nil nil ND
D32 nil nil ND
D34 Pool 1,2,4,6–11 nil ND
D37 Pool 4,9 Pool 2,7 ND
D43 nil ND Pool 1,6
D44 Pool 3,11 nil Pool 11
D45 nil nil nil
D46 Pool 5,6 Pool 11 Pool 1,5
D47 ND Pool 8 Pool 1,5
D48 Pool 2,3 nil nil
D49 Pool 7 Pool 1,11 Pool 5
D50 nil nil ND
D51 Pool 3 nil ND
D52 Pool 1 Pool 9 ND
D53 Pool 4 nil ND
D54 nil Pool 7 ND
13 of 22 (59%) 6 of 21 (29%) 5 of 8 (63%)
Figure 1.
Figure 1.

IFN-γ ELISpot responses to variants of 10 strains of Pf TRAP in Kenyan adults. Twenty-five healthy Kenyan adults (denoted K) were tested by IFN-γ ELISpot to a panel of 27 variant Pf TRAP peptides. Variants of overlapping peptides tp4, 5, and 6 were combined into three pools. A positive response is indicated by a filled in square. ND, not determined.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 3; 10.4269/ajtmh.2006.74.367

Figure 2.
Figure 2.

Cytokine reactivity to the TRAP peptide epitope variants Kenyan adults (denoted K) were tested in parallel ELISpot assays to a panel of 27 variant and 4 conserved Pf TRAP peptides for IFN-γ (N = 26), IL-4 (N = 15), and IL-10 (N = 11) reactivity. The first panel shows results for the 11 donors tested for all 3 cytokines, and the second panel shows results for those tested for IFN-γ and IL-4 reactivity only. A positive response is indicated by a filled-in square according to the key. ND, not determined.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 3; 10.4269/ajtmh.2006.74.367

*

Address correspondence to Katie L. Flanagan, MRC Laboratories, P.O. Box 273, Fajara, The Gambia. E-mail: kflanagan@mrc.gm

Authors’ addresses: Katie L. Flanagan, MRC Laboratories, P.O. Box 273, Fajara, The Gambia, Telephone: +220-4495442, Fax: +220-4495919, E-mail: kflanagan@mrc.gm. Magdalena Plebanski, Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, Australia, Telehone: +61-3-92870643, Fax: +61-3-92870601. Kennedy Odhiambo, Tabitha Mwangi, Moses Kortok, Brett Lowe, and Kevin Marsh, KEMRI Centre for Geographic Medicine—Coast, P.O. Box 230, Kilifi, Kenya, Telephone: +254-1255-22390, Fax: +254-1255-22063. Eric Sheu, Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, 260 Longwood Avenue, Boston, MA 02115, Telephone: +1-617-432-1738, Fax: +1-617-432-0232. Colin Gelder and Keith Hart, Tenovus Building, University of Wales College of Medicine, Heath Park, Cardiff, UK, Telephone: +44(0)-29 20, Fax: +44(0)-29 20. Kathryn J. Robson, MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford, UK, Telephone: +44-(0)1865-222379, Fax: +44(0)-1865-222500. Adrian V. S. Hill, University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK, Telephone: +44-(0)1865-287759, Fax: +44-(0)1865-287686.

Acknowledgments: We would like to thank all the donors and field workers in Kilifi. The study was approved by The Kenya Medical Research Institute national ethical and scientific review committees and is published with the permission of the director of KEMRI. We would like to thank Felicity May and Martin Williams for technical assistance with TRAP typing and HLA typing, respectively.

Financial support: K.L.F. was funded by a Wellcome Trust Tropical Training Fellowship, K.J.R. is funded by the Medical Research Council, K.M. is funded by a Wellcome Trust Career Post in Tropical Medicine, and A.V.S.H. is a Wellcome Trust Principal Fellow.

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Author Notes

Reprint requests: Katie L. Flanagan, MRC Laboratories, P.O. Box 273, Fajara, The Gambia, Telephone: +220-4495442, Fax: +220-4495919, E-mail: kflanagan@mrc.gm.
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