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The Journal of Immunology, 2002, 169: 3200-3207.
Copyright © 2002 by The American Association of Immunologists

Age-Dependent Cellular Immune Responses to Plasmodium vivax Duffy Binding Protein in Humans1

Jia Xainli*, Moses Baisor{ddagger}, Will Kastens*, Moses Bockarie{ddagger}, John H. Adams§ and Christopher L. King2,*,{dagger}

* Division of Geographic Medicine, Case Western Reserve University and {dagger} Veterans Affairs Medical Center, Cleveland, OH 44106; {ddagger} Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea; and § University of Notre Dame, South Bend, IN 46556


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Plasmodium vivax merozoite Duffy binding protein (DBP) contains a cysteine-rich region II (DBPII) that binds to the Duffy Ag receptor for chemokines on erythrocytes, which is essential for parasite invasion. Cellular immune responses to DBPII have not been reported in P. vivax endemic populations, although they may contribute to partial acquired immunity. To examine host cellular immunity to DBPII and identify major T cell epitopes, PBMCs from 107 individuals (2–68 years old) were examined for cytokine production by ELISPOT and/or ELISA to rDBP and overlapping peptides (displaced by 2 aa spanning a 170-aa region of DBPII corresponding to the critical binding motif to the Duffy Ag receptor for chemokines). In P. vivax-exposed subjects, 60 and 71% generated significant rDBP-induced IFN-{gamma} and IL-10 production, respectively, 11% stimulated IL-2, and IL-5 and IL-13 were not detected. Children <5 years of age had reduced levels and frequency of rDBP-induced IL-10 and IFN-{gamma} production compared with partially immune older children and adults (p < 0.01). Five major T cell epitopes were identified. Three of these T cell epitopes contained polymorphic residues present in the population. Peptides synthesized corresponding to these variants induced IFN-{gamma} and IL-10 production to one variant and little response to the other variant in the same individual. These results demonstrate age-dependent and variant-specific cellular immune responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in endemic populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The species Plasmodium vivax is the most widely distributed malaria species and is estimated to cause 70–90 million clinical cases of malaria annually; although rarely fatal, it can cause a very debilitating illness (1). Its prevalence is likely to increase because of growing drug resistance (1). P. vivax, like other species of human Plasmodium, initiates erythrocyte invasion through expression of several surface and apical organelles on the merozoite that bind to erythrocyte surface proteins (2, 3). One well-characterized ligand-receptor interaction involves the Duffy binding protein (DBP)3 expressed on the P. vivax merozoite and its corresponding receptor on erythrocytes, the Duffy Ag receptor for chemokines (DARC) (3, 4, 5). When mutation in the promoter of the DARC gene in erythroid precursor cells prevents its expression on erythrocytes (6), complete resistance to P. vivax infection occurs (7, 8). Alternative erythrocyte invasion pathways have not been identified so far (9). Over 90% of sub-Saharan Africans lack DARC expression, and as a result P. vivax malaria has a limited distribution in Africa. Thus, the P. vivax DBP represents one of the most promising vaccine candidate Ags against malaria, yet little is known about the host immune response against this molecule.

The P. vivax DBP is a 140-kDa protein that belongs to a family of erythrocyte binding proteins characterized by a functionally conserved cysteine-rich region (3, 10). This cysteine-rich region occurs in region II (DBPII), which has been shown to contain the binding motifs necessary for adherence to DARC on the erythrocyte (11, 12). Critical binding motifs in DBPII have been mapped to a region between amino acids 291 and 460 (13). Although most residues are conserved within this binding motif, ~20% of the residues are highly polymorphic (14, 15, 16).

It is has been hypothesized that these polymorphisms in region II of P. vivax DBP arose from immune selection (14, 16). This presumably occurred because these polymorphic regions represent B and/or T cell epitopes recognized by the host immune response. Ab responses to rDBP have been demonstrated in endemic populations of P. vivax infection in Papua New Guinea (PNG) and Colombia that increase in prevalence and titer with age, suggesting that they may contribute to acquired immunity in these populations (17, 18). However, no studies have examined T cell immune responses to DBP. The present study investigates the hypothesis that host cellular immunity to DBP contributes to age-acquired immunity in a P. vivax endemic population by examining whether rDBP-induced T cell responses increase in frequency and intensity with age coincident with a decline in P. vivax infection. We have also identified major T cell epitopes in DBPII, some of which contained polymorphic residues. We investigate whether these variants alter T cell reactivity in residents of a highly endemic population for P. vivax in PNG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study site and population

Study subjects resided in three adjacent villages collectively referred to as Liksul, located 50 km north of Madang, PNG, directly across from Kar Kar Island. The villages extend 1–2 km inland from the ocean along rugged, coastal hills. Residents belong to the Bargam ethnic and language group (http://www.sil.org/ethnologue/countries/Papu.html). Subsistence farming and copra production are the principal occupations. Inhabitants receive malaria treatment at a nearby health center. In June and July 1999, a demographic survey of the total population of Liksul was undertaken, followed by collection of peripheral venous blood in February 2000 and blood smear analysis for malaria parasites as previously described (19). Although malaria is transmitted throughout the year, February is wetter than June and is associated with increased malaria transmission. The study was approved by the Institutional Review Boards at Case Western Reserve University and the Papua New Guinea Institute of Medical Research.

DNA preparation and PCR amplification of genes encoding P. vivax dbpII

DNA was extracted from 200 µl of whole blood samples individually by spin blood kits (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The final extract was eluted with 200 µl of deionized distilled water and stored at -20°C. Region II (aa 285–521) of the P. vivax DBP was amplified with primers complementary to conserved regions of this gene. Nest I forward and reverse primers are as follows: 5'-GATAAAACTGGGGAGGAAAAAGAT and 5'-CTTATCGGATTTGAATTGGTGGC, respectively. The nest I reaction (25-µl reaction volume) was conducted using 1 µl of template, 1.5 mM MgCl2, 100 nM of each deoxynucleotide triphosphate, 5 pmol of each primer, and 1.25 U of Platinum Taq polymerase (Life Technologies, Rockville, MD) in the supplied buffer. The nest I cycling conditions were as follows: initial denaturation of 2 min at 94°C; five cycles of 1 min at 94°C, 2 min at 59°C, and 2 min at 72°C; thirty cycles of 1 min at 94°C, 1 min at 54°C, and 2 min at 72°C; and a final extension of 10 min at 72°C. One microliter of the nest I reaction was used in the nest II reaction as template. Nest II forward and reverse primers are as follows: 5'-GATCGAAGATATCAATTATGTA and 5'-TATCATAAGGAGTTACGATAC, respectively. The reaction and cycling conditions for the nest II reaction (50-µl reaction volume) were similar to the nest I conditions except that 53°C was used as the annealing temperature in the first 5 cycles and 48°C was used as the annealing temperature in the last 25 cycles. Three microliters of the nest II 712-bp amplicons were visualized by electrophoresis on a 1% agarose gel in 1x TAE buffer with 0.5 µg/µl ethidium bromide.

Recombinant Ags and peptides

Expression and purification of rDBP were described previously (17). Briefly, a portion of DBP (Sal-I, isolate GenBank accession no. M37514) (20) from aa 177 to 815 that includes regions II to IV was inserted in frame with glutathione S-transferase (GST) in the plasmid expression vector pGEX-2T (17). Fig. 1Go shows the structure of P. vivax DBP and the binding motif to DARC (aa 291–460), the region mapped for T cell epitopes. To isolate the DBP from GST component, the expressed protein was cleaved with thrombin. The GST was then removed from the mixture with reduced glutathione agarose CL-4B bead (Fluka, Buchs, Switzerland). Polyclonal rabbit sera raised to rDBP bound to P. vivax schizonts and could inhibit binding of COS7 cells transfected with rDBP protein to DARC plus erythrocytes (21), indicating that the expressed molecule is conformationally correct. Recombinant DBP was mixed with polymyxin-coated beads (Pierce, Rockford, IL) to remove endotoxin according to the manufacturer’s protocol. Some endotoxin remained in the stock solution (0.13 ng/ml), which was then used at a 1/200 final dilution in culture. This is well below levels required for monocyte activation.



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  		FIGURE 1. The molecular structure of the DBP shows regions II and VI (shaded areas), which are cysteine-rich regions of the molecule. There are 10 cysteines in region II. The region between cysteines 4 and 7 (aa 291–460) contains the critical binding motif to DARC.

 
Peptide synthesis was performed using a pin method of peptide synthesis (Chiron Mimotopes, Victoria, Australia; www.mimotopes.com) of 15-mer peptides displaced by 2 aa for a total of 79 peptides spanning the 170-aa critical binding region of DBPII (13). Peptide purity was 65–75%, as determined by HPLC. Synthetic peptides of 15 aa in length were chosen because they are comparable to naturally processed peptides by MHC class II molecules (9–14 residues) (22). Peptides overlapping by 2 aa allowed fine mapping of epitopes. The sequences were based on the most common alleles identified in the population (42) (GenBank accession nos. AF469515–602). In addition, the peptide GSEPLFEGVFCSSSSFLSLS, corresponding to the epidermal growth factor-like motif at the C terminus of P. vivax MSP119 (GenBank accession no. M75674), was identified as a T cell epitope (our unpublished observations) and used to stimulate lymphocyte responses simultaneously with that for DBP peptides. Recombinant MSP19 was obtained as described previously (19).

Preparation of PBMC and culture conditions

Blood samples were collected in Vacutainers with EDTA (K3; BD Biosciences, San Jose, CA) and PBMC were isolated with Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) by centrifugation as previously described (23). Culture medium contained RPMI 1640 supplemented with 10% heat-inactivated (40 min at 56°C) autologous plasma, 4 mM L-{gamma}-glutamine, 25 mM HEPES, and 40 µg/ml gentamicin (C-RPMI; BioWhittaker, Walkersville, MD). PBMC were cultured at 1.5 x 106/ml in C-RPMI in 0.2-ml cultures with medium alone, Mycobacterium tuberculosis purified protein derivative (PPD; 5 µg/ml; Stats Serum Institute, Copenhagen, Denmark), rDBP (10 µg/ml), PMA (50 ng/ml; Calbiochem, La Jolla, CA) ionomycin (1 µg/ml; Calbiochem), or each one of the 79 peptides (17 µM). The peptide concentration used to stimulate PBMC was selected to account for differences in the predicted solubilities of the peptides used.

ELISPOT

PBMCs were cultured on multiscreen plates (MultiScreen-IP ELISPOT plates; Millipore, Bedford, MA) coated with 4 µg/ml anti-human IFN-{gamma} (M-700; Endogen, Woburn, MA) at 4°C overnight. After a 3-day incubation at 37°C with 5% CO2, the supernatant was saved for determination of other cytokines by ELISA. Biotinylated secondary Ab M-701 was added (Endogen) at 2 µg/ml, followed by avidin-peroxidase (DAKO, Glostrup, Denmark) at 1/2,000 at room temperature for 2 h. The spot color was developed by adding 3-amino-9-ethylcarbozole dissolved in N,N-dimethylformide and diluted 1/30 in 0.1 M acetate buffer (pH 5) containing a dilution of 30% H2O2. The plates were observed for spot development for a maximum of 1 h at room temperature and then were washed three times with dH2O (200 µl/well) to stop the reaction. Plates were dried overnight at room temperature, images of wells were acquired and saved on compact disc using an automated ImmunoSpot Series 1 (Cellular Technology, Cleveland, OH), and spots were enumerated on an ImmunoSpot Satellite analyzer (Cellular Technology) using software especially designed for the ELISPOT assay.

Measurement of cytokines by ELISA

Cytokines were measured by ELISA and expressed in picograms per milliliter by interpolation from standard curves based on recombinant lymphokines (24). Ab pairs for capture and detection, respectively, were as follows: IL-5, clones TRFK5 and 5D10 (BD PharMingen, San Diego, CA); IL-13 (polyclonal goat anti-human Ab; Endogen) and detecting IL-13 mAb (Endogen); IFN-{gamma}, M-700 and M-701 (Endogen); IL-10, 18551D and 18652D (BD PharMingen). All detection Abs were biotin labeled. The limits of detection were as follows: IL-5, 18 pg/ml; IL-13, 16 pg/ml; IFN-{gamma}, 10 pg/ml; IL-10, 16 pg/ml.

Statistical analysis

Analysis of spot number or cytokine levels were analyzed by using the Wilcoxon rank-sum test comparing responses between controls and subjects living in a P. vivax endemic areas. A Student t test was used to compare log-transformed data between different age groups. The {chi}2 test examined differences in proportions between experimental groups. A p < 0.05 was considered significant using two-sided tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Infection status of the study subjects

Ninety-one of 1025 (9%) residents in the study village were blood smear positive for P. vivax and 358 of 1025 (35%) were positive using a nested PCR for dbpII. Cellular immune responses were examined in 10% of the population selected to represent all age groups and for the presence of P. vivax infection (Table IGo). In this subset of study subjects, the median age for smear-positive individuals was lower (7 years) compared with smear-negative individuals (29 years). There was 93% concordance between P. vivax blood smear and PCR positivity.


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  		Table I. Characteristics of the study subjects (n = 107) examined for T cell responses

 
T cell immune response to rDBP

The frequency of IFN-{gamma}-secreting cells was evaluated by ELISPOT in 35 study subjects and 13 nonexposed North American subjects of Asian decent (ages, 18–63 years; six male and seven female) (Fig. 2Go). Recombinant DBP-induced IFN-{gamma} in controls subjects was not significantly higher than in cultures containing medium alone (Fig. 2Go; p = 0.2). A positive response in PNG donors was considered if it exceeded the mean + 2 SD of control subjects in response to rDBP. This cut-off was >66 spots per 106 PBMC of IFN-{gamma}-secreting cells for the ELISPOT analysis and >172 pg/ml rDBP-induced IFN-{gamma} for ELISA. Twenty-six of 34 (74%) subjects had significantly increased numbers of rDBP-induced IFN-{gamma}-secreting lymphocytes compared with controls. The frequency of DBP-specific lymphocytes was similar to that of PPD-specific lymphocytes in many individuals. A similar analysis was performed for 70 additional subjects for whom only DBP-induced IFN-{gamma} was measured in culture supernatants. ELISPOT analysis (which is more labor intensive and required more cells) was not performed on these additional individuals because the ELISA and ELISPOT analysis generated almost identical rDBP- and peptide-induced IFN-{gamma} responses (data not shown). Fifty-nine percent (41 of 70) produced significantly more IFN-{gamma} than controls. All subjects generated significant levels of PPD-driven IFN-{gamma} production assessed by ELISPOT (Fig. 2Go) and ELISA (data not shown).



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  		FIGURE 2. Recombinant DBP induced IFN-{gamma}-secreting cells in residents of P. vivax endemic area of PNG (•) and unexposed individuals residing in North American ({circ}). The number of IFN-{gamma}-secreting cells was determined by ELISPOT analysis during a 72-h culture in medium with 10% heat-inactivated autologous plasma at a concentration of 3 x 105 PBMC per well in 96-well ELISPOT plates. Bars represent geometric means. The number of IFN-{gamma}-secreting cells was determined in parallel cultures stimulated with PPD of M. tuberculosis. **, p < 0.01; ***, p < 0.001 compared with nonexposed individuals.

 
Although most PNG residents demonstrated little or no spontaneous IL-10 release, some levels exceeded that observed in control subjects (Fig. 3Go). Recombinant DBP-induced IL-10 in controls subjects was not significantly higher than in cultures containing medium alone (p = 0.11). A response in PNG donors was considered positive if it exceeded the mean + 2 SD of control subjects in response to rDBP (e.g., >314 pg/ml rDBP-induced IFN-{gamma}). Based on this criterion, rDBP induced IL-10 production in 76 of 107 (71%) subjects. Mycobacterial Ag PPD also stimulated IL-10 in some subjects, but the levels were markedly lower than that observed in responses to rDBP (Fig. 3Go). Recombinant DBP stimulated IL-2 production in 8 of 75 subjects examined (11%) and failed to induce any detectable IL-5 or IL-13 release.



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  		FIGURE 3. Recombinant DBP-induced IL-10 production in residents of P. vivax endemic area of PNG ({blacktriangleup}) and unexposed individuals residing in North American ({triangleup}). The IL-10 was measured in the same culture supernatants from cells that were used for ELISPOT analysis described in Fig. 1Go or from identical culture conditions without ELISPOT analysis. Bars represent geometric means. The dashed line indicates the lower limits of sensitivity for the ELISA. ***, p < 0.001 compared with nonexposed individuals.

 
Recombinant DBP-induced IFN-{gamma} and IL-10 production is greater in semi-immune children and adults compared with children <5 years old

The prevalence (and levels) of P. vivax parasitemia peaked at 3–4 years of age and then declined in the community (Fig. 4Go) based on peripheral blood smears, consistent with development of partial immunity among children 5 years of age and older. This decline in parasitemia correlated with significantly greater rDBP-induced IL-10 in children >=5 years of age compared with children <=4 years of age (Table IIGo). A greater proportion of children >=5 years also had rDBP-induced IFN-{gamma} production when the percentages of responders were combined for both the ELISPOT and ELISA (5 of 18 (28%) in children <5 years compared with 55 of 86 (64%) in those 5 years and older; p = 0.005, {chi}2 test; Table IIIGo). Subjects aged 5 years and older had equivalent levels of rDBP-induced IL-10 (Table IIGo) and IFN-{gamma} production (data not shown).



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  		FIGURE 4. The prevalence of P. vivax blood smear-positive individuals in the overall study population (n = 1025).

 

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  		Table II. Young children have a diminished rDBP-induced IL-10 production

 

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  		Table III. Relationship of age and rDBP-induced IFN-{gamma} production

 
Active malaria infection may either inhibit rDBP-induced T cell responses in some individuals and/or enhance T responses in others. Neither of these possibilities seemed likely because the proportion of P. vivax smear-positive individuals with rDBP-induced IL-10 (35 of 52, 67%) and/or IFN-{gamma} (28 of 53, 53%) was equivalent to smear-negative individuals (IL-10, 36 of 50, 72%; and IFN-{gamma}, 32 of 51, 63%) or between individuals that were PCR positive or negative for P. vivax (data not shown).

Major T cell epitopes of DBPII binding motif

To determine the principal T cell epitopes in the 170-aa critical binding region of DBP a total of 79 synthetic 15-mer peptides displaced by 2 aa spanning the 170-aa binding motif to DARC (13) were used to activate PBMC in separate cultures for each study subject. No peptide-driven IFN-{gamma} or IL-10 production by PBMC exceeded that observed with medium alone in any control subjects from North American residents of Asian descent (n = 13) or Papua New Guineans living in Madang town who were not exposed to P. vivax (n = 3). Therefore, a positive response was any peptide-driven IFN-{gamma} and IL-10 production that exceeded the mean + 3 SD of triplicate cultures containing medium for each individual studied. Five dominant T cell epitopes were identified corresponding to peptides 5, 13, 16, 20, and 66 (Fig. 5Go and Table IVGo). Peptide 13 induced the greatest proportion and frequency of IFN-{gamma}-secreting cells (Figs. 5A and 6A) and proportion of individuals that produced IFN-{gamma} in culture supernatants (for peptide 13, 55%; peptide 5, 46%; peptide 20, 45%; peptide 16, 30%; peptide 66, 15%). By contrast, peptide 5 stimulated the greatest amount of IL-10 (Fig. 6GoB). The geometric mean levels of peptide 5-driven IL-10 (Fig. 6GoB; 401 pg/ml) was statistically equivalent to that observed with rDBP (Fig. 3Go; 515 pg/ml).



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  		FIGURE 5. T cell epitopes for DBPII binding motif to DARC as determined by the proportion of individuals that generated significant levels of IFN-{gamma} and IL-10 in response to 15-mer synthetic peptides displaced by 2 aa. A, A portion of an ELISPOT plate showing the number of IFN-{gamma}-secreting cells in 4 x 105 PBMC from a representative individual in response to peptides 12–17. B and C, The proportion of positive responders. For IFN-{gamma} (B), both ELISPOT and cytokine data were combined to determine the portion of positive responders. Culture conditions were identical to those described in Figs. 1Go and 2Go. Peptide-driven IFN-{gamma} and IL-10 production that exceeded the mean + 3 SD of triplicate cultures in the same individual without peptide were scored as a positive response. An asterisk indicates peptides that met the criteria for positivity.

 

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  		Table IV. Major T cell epitopes in the 170-aa binding motif of DBPII

 


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  		FIGURE 6. The number of IFN-{gamma}-secreting spots in response to peptides corresponding to the major T cell epitopes of the 170-aa binding motif of DBP. A, Culture conditions are identical to that described in Fig. 2Go; however, the absolute number of IFN-{gamma}-secreting spots are indicated per well rather than per 106 PBMC. B, IL-10 levels in response to peptides corresponding to the major T cell epitopes of the 170-aa binding motif of DBP (A). Culture conditions are identical to that described in Fig. 2Go. Bars represent geometric means ± SEM.

 
The amount of IL-10 produced by PBMC to each individual peptide corresponding to the five major T cell epitopes accounted for 9–15% (r2 = 0.09–0.15; all p < 0.0001) of the overall variability of rDBP-induced IL-10 production. When the cumulative amount of IL-10 production was calculated for each of the 5 peptides added together (e.g., the amount of cytokine produced in five cultures containing peptides corresponding to T cell epitopes), this accounted for 41% (r2 = 0.41, p < 0.0001) of the variability of rDBP-induced IL-10. In contrast to IL-10 production, only peptide 13-induced IFN-{gamma} correlated with rDBP-induced IFN-{gamma} (e.g., r2 = 0.46, p < 0.0001).

The cumulative levels of peptide-induced IL-10 (using just the five peptides associated with dominant T cell epitopes) was significantly higher in children >=5 years (geometric mean = 343 ± 91 pg/ml) compared with children <5 years (geometric mean = 97 ± 29 pg/ml, p < 0.01). Similarly, peptide 13-induced IFN-{gamma} was higher in children >=5 years (geometric mean = 108 ± 37 pg/ml) compared with younger children (geometric mean = 41 ± 9 pg/ml, p < 0.05, Student’s t test). By contrast, a peptide corresponding to a portion of the immunostimulatory epidermal growth factor-like motif of the C-terminal region of P. vivax MSP119 failed to show an age-associated increase in IL-10 and/or IFN-{gamma} production. Twelve of 17 (71%) children <5 years of age responded to the MSP119 peptide (IFN-{gamma} and/or IL-10 response greater than mean + 3SD of MSP119 stimulated PBMC from unexposed control subjects) compared with 54 of 68 (79%) in children >=5 years of age. The levels of recombinant MSP119-induced IL-10 and IFN-{gamma} in children <5 years were also statistically equivalent to that for individuals >=5 years of age (children <5 years: IL-10, 245 ± 102 pg/ml; IFN-{gamma}, 73 ± 22 pg/ml; older children and adults: IL-10, 359 ± 89 pg/ml; IFN-{gamma}, 108 ± 43 pg/ml; p > 0.05 for all comparisons).

Polymorphisms in T cell epitopes affect lymphocyte reactivity

The high degree of polymorphisms in DBPII may have arisen from immune selection by altering the host immune response to these variants (14, 16). To examine this hypothesis we first examined whether polymorphisms occurred in the dbpII gene encoding identified dominant T cell epitopes (42). Of the five peptides corresponding to major T cell epitopes, three contained polymorphic residues (Table IVGo). One polymorphism is at codon 308, corresponding to position 10 in peptide 5, an arginine (R) to serine (S) mutation. The other polymorphism occurred at codon 333, a leucine (L) to phenylalanine (F) at position 13 in peptide 16 and at position 5 in peptide 20.

To evaluate whether these polymorphic residues affected T cell reactivity, seven additional adult subjects from the same population were examined for IFN-{gamma} production in response to peptides 5 and 16 that contained polymorphisms present in the study population. Peptides were synthesized that differed by a single amino acid corresponding to each identified allele (Table IVGo). Four subjects tested demonstrated T cell responses to rDBP and the peptides (Fig. 7Go). Two subjects demonstrated 3- to >18-fold reduction in the number of IFN-{gamma}-secreting cells to the 308R (peptide 5) and 333F (peptide 16) compared with 308S and 308L alleles (Fig. 7Go). Allele frequencies of 303R and 333F were significantly lower in the study population (frequency, <0.27) compared with the 303S and 333L alleles (>0.77) (42).



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  		FIGURE 7. Alteration of a single amino acid corresponding to different alleles identified in parasites in the population present in two T cell epitopes affects levels of peptide-driven cytokine responses. Culture conditions are identical to those shown in Fig. 2Go. Bars represent means ± SD of triplicate cultures. No spots were observed in cultures with medium alone for subjects 1, 2, and 4. For subject 2 an average of two spots was observed in cultures with medium alone. Peptide 5 (sequence shown in Table IVGo) has two alleles corresponding to codon 308 with either serine (S) or arginine (R) in position 10 as indicated by 5-S or 5-R. Similarly, peptides corresponding to two alleles for codon 333 also correspond to position 13 in peptide 16 (see Table IVGo for sequence) with either a leucine (L) or phenylalanine (F) designated at 16-L and 16-F. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with parallel cultures with same peptide that differed by only a single amino acid, as indicated above.

 
In subjects 1, 3, and 4, the frequency of peptide-induced IFN-{gamma} responses was higher than that observed to rDBP. This may have been a result of cross-regulation by IL-10, because rDBP induced much higher levels of IL-10 (1130 and 560 pg/ml for subjects 1 and 3, respectively) compared with peptide-induced IL-10 (126 and 368 pg/ml for subjects 1 and 3, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that most individuals in P. vivax endemic populations develop cellular immune responses to rDBP and to several dominant T cell epitopes of DBP located in the binding motif to DARC. The proportion of individuals developing T cell responses to DBP increased rapidly within the first 4 years of life such that by 5–9 years of age 80% of children responded. The prevalence and intensity of P. vivax infection markedly declined in this age group, suggesting that host cellular immune responses to this molecule may participate in development of acquired immunity. This delay in T cell reactivity with age is unlikely to result from a lack of prior malaria infection, because most children have been repeatedly infected by 5 years of age, as demonstrated by the high proportion of children <4 years of age who developed T cell responses to P. vivax MSP119, a much more abundant blood stage molecule than DBP. The delay in T cell reactivity to DBP compared with MSP119 may have occurred for several reasons. First, DBP are contained in micronemes, organelles within the merozoite (25), and their release to the merozoite surface is thought to occur only after the merozoite has begun erythrocyte invasion, which limits the amount of Ag released and exposure to the host immune response to this essential invasion ligand. Second, regions of DBPII are highly polymorphic and may generate variant, non-cross-reactive T cell epitopes such that T cell responses and immunity develop only after the cumulative responses to multiple parasite strains.

To investigate this latter hypothesis, T cell epitopes were determined by screening with multiple peptides (79 in all) in one assay for IFN-{gamma} and IL-10 for each subject. Five dominant T cell epitopes emerged, three of which contained polymorphic residues that did not cross-react in some individuals. Some subjects responded to some dominant T cell epitopes and others did not (e.g., Fig. 7Go, subject 2), suggesting genetic restriction. Other subjects reacted to just one allele and not the other (Fig. 7Go, subjects 3 and 4), suggesting that these two individuals experienced repeated infection to one strain and not to the other. Alternatively, genetic restriction may also account for this difference if the polymorphism occurred at an anchor residue for peptide binding to MHC class I/II. This is unlikely for the mutations observed because neither amino acid substitution at residue 308 is negatively charged (negatively charged or hydrophobic residues are usual anchor residues for MHC class II binding) (22). In either case this lack of cross-reactive alleles corresponding to T cell epitopes suggests that they arose by host immune pressure. It also supports the hypothesis that the slow acquisition in levels of T cell responses with age occurs, in part, because of parasite variation in DBPII. These are tentative conclusions considering the small number of donors examined showing variant-specific T cell responses. However, one of the conserved T cell epitopes (peptide 13) generated the strongest IFN-{gamma} responses relative to other peptides that may be particularly attractive for incorporation into a vaccine. This peptide may be functionally conserved because it could contain residues necessary for binding to DARC. It is possible that additional polymorphic residues in this T cell epitope may exist in other populations with a different distribution of MHC class I/II alleles. However, so far this has not been reported from parasite isolates sequenced for DBPII from Colombia, PNG, or Korea (15, 16, 26).

Natural single amino acid polymorphisms of T cell epitopes of Plasmodium falciparum circumsporozoite protein have also been shown to affect levels of T cell reactivity (27). It has been suggested that CD8+ T cells could select parasite-infected hepatocytes expressing variants of the circumsporozoite protein (28). CD4+ cells may also select for parasite variants of DBP because effector mechanisms such as Ab-dependent cellular inhibition, along with humoral immunity, have been postulated to protect against blood stage infection (29, 30).

In children >4 years of age, >80% responded to one or more peptides corresponding to the major T cell epitopes, similar to that observed to rDBP. This suggests that most of the T cell responses to rDBP can be reproduced with one or several peptides. Overall, peptide-induced T cell responses as measured by IFN-{gamma} and/or IL-10 production correlated with that observed with rDBP. A failure to see a closer correlation may have occurred because the rDBP Ag used included a larger portion of the DBP (e.g., regions II-IV) (3, 20) and may contain additional T cell epitopes other than the region of molecule represented by the synthetic peptides. The peptide that induced the strongest IFN-{gamma} responses differed from that for IL-10 (Fig. 6Go) and varied in their correlation with levels of cytokine stimulated by rDBP. This indicates that peptide-specific lymphocytes probably differ in their relative amount of IFN-{gamma} or IL-10 secreted. Although the cellular origin of the cytokine production was not evaluated in the present study, the predominant cell source is likely to be T cells, because IL-2 is exclusively and IFN-{gamma} is predominantly secreted by T cells in PBMC (31, 32). It is unlikely that non-T cells produced much IL-10 to peptides because no peptide-induced IL-10 was observed in control subjects or for most peptides examined in the study subjects. The reason different T cell epitopes in the same molecule induce different cytokine patterns is unclear. It may be related to differences in MHC class II binding, its immunodominance relative to other T cell epitopes, whether it cross-reacts with T cells expanded in response to closely related Ags, or immune interference by one variant with another (33). This difference in the type of peptide-induced cytokine response permits selection of T cell epitopes that favor IFN-{gamma} relative to IL-10 responses.

The greater production of DBP-induced IFN-{gamma} and IL-10 with increasing age suggests that these cytokines may help to mediate protective immunity. IFN-{gamma} and IL-10 production in response to pre-erythrocytic and/or blood-stage Ags has been shown to correlate with decreased levels of parasitemia or clinical disease with P. falciparum infection (34, 35, 36, 37, 38). The studies of pre-erythrocyte stage immunity may be relevant to DBP because its homolog in P. falciparum, the erythrocyte binding Ag 175, has been recently reported to be expressed in pre-erythrocytic stages of parasites to which the host can induce an immune response without blood stage infection (39). It is unknown whether P. vivax DBP is also expressed in the pre-erythrocytic stages of the parasite.

Exactly how IFN-{gamma} and/or IL-10 mediate blood stage immunity is unclear. IFN may act indirectly by boosting monocyte function to increase clearance of infected erythrocytes or enhance Ab-dependent cellular-mediated destruction of parasites (40). Although IL-10 has been widely characterized as an immunosuppressive cytokine, it has also been shown to mediate proinflammatory responses such as IL-10-dependent, CD4+ cell-mediated tumor rejection (41). The strong IL-10 response may also participate in enhancing Ab production and therefore its correlation with protection.

In some individuals, a single peptide stimulated greater IFN-{gamma} and/or IL-10 production compared with rDBP. Recombinant DBP may have contained a small amount of LPS or other contaminants that may induce IL-10. These could, in turn, modulate rDBP-induced IFN-{gamma} responses in vitro. Alternatively, each epitope may stimulate a different pattern of cytokine response that may cross-modulate their respective cytokine production in vitro. It is possible that construction of a subunit vaccine with selected immunodominant T cell epitopes may generate a stronger protective immune response than full recombinant Ag.

In conclusion, these studies describe for the first time naturally acquired cellular immunity to DBP in endemic populations. These observations, along with the essential role of DBP for P. vivax infection, make this an ideal malaria vaccine. The selection of an Ag for vaccination requires a detailed understanding of natural immune responses elicited by the protein as described in the current study for successful control or clearance of parasites. It still remains to be established whether these responses are indeed protective. This will require studies first to track the epidemiology of specific variants over space and time and to correlate these responses with cellular and humoral Ab responses to defined regions of DBPII. Next, longitudinal studies will be needed to determine whether strong immune responses to DBP predicts partial protection from infection and disease to the homologous strains in semi-immune individuals.


   Acknowledgments
 
We thank the residents of Liksul for their cooperation, Kerry Lorry for reading blood smears, and Jennifer Cole-Tobian and Sigifredo Pedraza for reviewing the manuscript.


   Footnotes
 
1 This work was supported by the Department of Veterans Affairs Medical Research Service. Back

2 Address correspondence and reprint requests to Dr. Christopher L. King, Department of Medicine, Division of Geographic Medicine, Room W137, 10900 Euclid Avenue, Cleveland, OH 44106-4983. E-mail address: cxk21{at}po.cwru.edu Back

3 Abbreviations used in this paper: DBP, Duffy binding protein; GST, glutathione S-transferase; PPD, purified protein derivative; DARC, Duffy Ag receptor for chemokines; PNG, Papua New Guinea. Back

Received for publication April 5, 2002. Accepted for publication July 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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