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HLA-DR-Promiscuous T Cell Epitopes from Plasmodium falciparum Pre-Erythrocytic-Stage Antigens Restricted by Multiple HLA Class II Alleles^{1 ,2}

Denise L. Doolan^3,*, Scott Southwood, Robert Chesnut, Ettore Appella, Eduardo Gomez, Allen Richards, Yuichiro I. Higashimoto, Ajesh Maewal, John Sidney, Robert A. Gramzinski, Carl Mason^§, Davy Koech^¶, Stephen L. Hoffman^* and Alessandro Sette

^* Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910; Epimmune, San Diego, CA 92121; Naval Medical Research Unit 2, Jakarta, Indonesia; ^§ U.S. Army Medical Research Unit-Kenya, Nairobi, Kenya; and ^¶ Kenya Medical Research Institute, Nairobi, Kenya

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously, we identified and established the antigenicity of 17 CD8⁺ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN- responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Malaria is the most important parasitic disease affecting the human species, exacting an estimated toll of five deaths and 950 clinical cases per minute (1). Development of a vaccine against malaria presents formidable obstacles in terms of parasite biology and host immune responses. Different Ags that may be targeted by protective immune responses are expressed during the different stages of the parasite’s complex life cycle, and only a limited number of epitopes are contained within a relatively small number of Ags. Furthermore, because of HLA polymorphism, immune responses directed against these epitopes are genetically restricted and a given epitope may be recognized by one individual but not by another. Accordingly, an efficacious vaccine against malaria will need to have the potential to induce responses against a number of epitopes that are recognized in the context of many different HLA alleles.

CD8⁺ T cells have been implicated as critical effector cells in protective immunity against pre-erythrocytic-stage malaria (reviewed in Refs. 2 and 3), the developmental stage preceding the manifestation of clinical symptoms and the subsequent transmission of disease. Active immunization and adoptive transfer experiments also provide strong evidence for a critical role for CD4⁺ T cells (4, 5), as well as for Abs (6, 7) and cytokines such as IFN- (reviewed in Ref. 2). These CD4⁺ T cells could either directly act as immune effector cells per se or indirectly provide help for the development of CD8⁺ T cell-mediated or Ab-mediated protective immunity. Accordingly, using a combination of immunochemical and cellular immunologic analyses based on specific HLA peptide binding motifs, we are designing a subunit vaccine against malaria comprising a number of CD8⁺ and CD4⁺ T cell epitopes from multiple Plasmodium falciparum pre-erythrocytic-stage Ags that are restricted by common HLA class I and HLA class II alleles.

However, a potential obstacle to the development of a vaccine designed to induce T cell-mediated protective immunity is the large degree of polymorphism of human HLA molecules. Therefore, we have focused on HLA alleles that would allow coverage of all racial and ethnic populations, and preference has been given to those peptides that bind to more than one MHC. In particular, we have capitalized on the fact that different class I HLA molecules overlap in their peptide binding specificities such that a given epitope may bind with high affinity to multiple HLA alleles. A large majority of all human HLA-A and -B molecules can be classified in one of nine different supertypes (8). HLA-A2, -A3, and -B7 supertypes have been studied in detail (reviewed in Ref. 9), and CD8⁺ T cell epitopes that belong to these supertypes have now been identified and validated (8, 10, 11, 12, 13).

However, class II epitope predictions on the basis of MHC binding motifs have been generally less accurate than those for class I epitopes. Lower accuracy is presumably due to the fact that the peptide binding groove of MHC class II molecules is less conformationally restricted as compared with that of MHC class I molecules (14). However, recent studies have revealed that a majority of HLA-DR alleles are associated with largely overlapping peptide-binding specificities (HLA-DR supertype) (15). Because the corresponding Ags are represented with a high frequency in different ethnic populations, these data suggest that effective population coverage could be achieved, also in the case of class II, by the use of peptides binding multiple HLA-DR molecules.

We have previously reported the identification of 17 CD8⁺ T cell epitopes on P. falciparum Ags that are recognized in the context of multiple HLA-A2, HLA-A3, and HLA-B7 class I supertypes (10). Here, we extend our earlier studies and exploit the recently defined HLA-DR supertype to identify 11 peptides from the same circumsporozoite protein (CSP),⁴ sprozoite surface protein 2 (SSP2), exported protein-1 (EXP-1), and liver-stage Ag-1 (LSA-1) Ags of P. falciparum that are capable of binding to at least five and up to 11 of 14 tested DR molecules commonly expressed in different populations. We further establish that all of the identified peptides are antigenic, as assessed by their capacity to recall specific lymphocyte proliferative and cytokine responses from PBMC of individuals experimentally immunized with radiation-attenuated P. falciparum sporozoites and naturally exposed to malaria.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence analysis

Using a customized computer algorithm analysis program (16), complete sequences of the Ags CSP (17), SSP2 (18), LSA-1 (19), and EXP-1 (20) from the 3D7 strain of P. falciparum were searched for the presence of 15 amino acid sequences conforming to the previously described HLA-DR binding motifs. Specifically, 15-mer sequences were selected that contained a 9-residue core bearing the DR supermotif described by O’Sullivan et al. (21) and Southwood et al. (15) with 3-residue N- and C-terminal flanking regions. Additional peptides were identified on the basis of an "extended" HLA-DR peptide binding motif that takes into consideration the effects of secondary anchor residues (15). In cases where two peptides that overlapped >90% were identified, a single longer peptide incorporating both sequences was synthesized. Multiple isolates were available for the CSP and SSP2 Ags, and only motif-containing peptides that are totally conserved in at least 80% or more of all P. falciparum isolates sequenced to date were considered for synthesis. Only one sequence was available each for the LSA-1 and EXP-1 Ags at the time of screening.

Peptide synthesis

Peptides were synthesized at Epimmune (San Diego, CA), as previously described (22), or purchased as crude material from Chiron Mimotopes (Chiron Corporation, Clayton, Victoria, Australia). Peptides synthesized at Epimmune were purified to >95% homogeneity by reverse-phase HPLC. Purity was determined on an analytical reverse-phase column, and their composition was ascertained by amino acid analysis and/or mass spectrometry analysis.

Cells

The EBV-transformed homozygous B lymphoblastoid cell lines LG2 (DRB1*0101 (DR1)), GM3107 (DRB5*0101 (DR2w2a)), MAT (DRB1*0301 (DR3)), PREISS (DRB1*0401 (DR4w4)), SWEIG (DRB1*1101 (DR5w11)), PITOUT (DRB1*0701 (DR7)), KT3 (DRB1*0405 (DR4w15)), Herluf (DRB1*1201 (DR5w12)), HO301 (DRB1*1302 (DR6w19)), OLL (DRB1*0802 (DR8w2)), and HID (DRB1*0901 (DR9)), or the transfected fibroblasts L466.1 (DRB1*1501 (DR2w2b)), TR81.19 (DRB3*0101 (DR52a)), and L257.6 (DRB4*0101 (DRw53)) were used as sources of human HLA class II molecules. Cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY), 50 µM 2-ME, and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA). Cells were also supplemented with 100 µg/ml of streptomycin and 100 U/ml of penicillin (Irvine Scientific, Santa Ana, CA). Large quantities of cells were grown in spinner cultures.

Cells were lysed for 30 min at 4°C with a lysis buffer of 50 mM Tris-HCl, pH 8.5, 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, and 2 mM PMSF (Calbiochem, San Diego, CA). Lysates were cleared of debris and nuclei by centrifugation at 15,000 x g for 30 min.

Affinity purification of HLA-DR molecules

Class II molecules were purified by affinity chromatography, essentially as described previously (23), using the mAb LB3.1 coupled to Sepharose CL-4B beads. Lysates were filtered twice through two precolumns of inactivated Sepharose CL4-B and protein A-Sepharose, and then passed over the anti-DR column. The anti-DR column was then washed with 10-column volumes of 10 mM Tris-HCl, pH 8.0, in 1% Nonidet P-40, PBS, 2-column volumes of PBS, and 2-column volumes of PBS containing 0.4% n-octylglucoside. Finally, DR molecules were eluted with 50 mM diethylamine in 0.15 M NaCl containing 0.4% n-octylglucoside, pH 11.5. A 1/25 volume of 2.0 M Tris, pH 6.8, was added to the eluate to reduce the pH to 8.0. The eluate was then concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, MA).

Class II peptide binding assays

Peptide binding assays were performed as described previously (15, 23). Specifically, purified human HLA class II molecules (5–500 nM) were incubated with various unlabeled peptide inhibitors and 1–10 nM ¹²⁵I-radiolabeled probe peptide for 48 h in PBS containing 0.05% Nonidet P-40 in the presence of a protease inhibitor cocktail. The final concentrations of protease inhibitors (each from Calbiochem) were: 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 µM pepstatin A, 8 mM EDTA, 6 mM N-ethylmaleimide, and 200 µM N--p-tosyl-L-lysine chloromethyl ketone. Final detergent concentration in the incubation mixture was 0.05% Nonidet P-40. Assays were performed at pH 7.0, except for DRB1*0301 and DRB4*0101, which were performed at pH 4.5 and 5.0, respectively (23). Class II peptide complexes were separated from free peptide by gel filtration on TSK200 columns (model 16215, TosoHaas, Montgomeryville, PA), and the fraction of bound peptide was calculated as previously described (23).

Radiolabeled peptides were iodinated using the chloramine-T method (23). The radiolabeled probe peptides used were hemagglutinin Y307–319 (sequence YPKYVKQNTLKLAT; DRB1*0101), tetanus toxoid 830–843 (sequence QYIKANSKFIGITE; DRB5*0101, DRB1*1101, DRB1*0701, DRB1*0802, DRB1*0901), myelin basic protein Y85–100 (sequence PVVHFFKNIVTPRTPPY; DRB1*1501), MT 65 kDa Y3–13 with Y7 substituted with F (sequence YKTIAFDEEARR; DRB1*0301), a nonnatural peptide (peptide 717.01, sequence YARFQSQTTLKQKT; DRB1*0401, DRB1*0405), a nonnatural peptide (peptide 717.10, sequence YARFQRQTTLKAAA; DRB4*0101), a naturally processed peptide (sequence EALIHQLKINPYVLS) (15, 24) of unknown origin eluted from a DRB1*1201 plus C1R cell line, integrin ß₃ Y24–37 (sequence YAWASDEALPLGSPR; DRB3*0101) (25), and an S836A analogue of tetanus toxoid 830–843 (sequence QYIKANAKFIGITE) for DRB1*1302.

In preliminary experiments, each DR preparation was titered in the presence of a fixed amount of the appropriate radiolabeled peptide to determine the concentration of class II molecules necessary to bind 10–20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these class II concentrations.

Inhibitor peptides were typically tested at concentrations ranging from 120 µg/ml to 1.2 ng/ml in two to four completely independent experiments. Under conditions where [label] < [MHC] and IC₅₀ [MHC], the measured IC₅₀ values are reasonable approximations of true K_d values. Peptides were classified as binders for each DR molecule for which its binding capacity was 1000 nM.

Study populations

Four populations were studied. Subjects were aged between 17 and 65 years, and informed consent was obtained in all cases. Parasitemia at the time of blood collection was assessed by examination of Giemsa-stained thick blood films. Smears were considered negative after examination of 200 oil-immersion fields (x1000).

Population 1. Irradiated sporozoite immunized volunteers (n = 7) were exposed to the bites of between 1008 to 1576 gamma-irradiated (1.5 x 10⁴ rad) Anopheles stephensi mosquitoes infected by membrane feeding with either the NF54 strain (26) or the corresponding 3D7 clone (27) of P. falciparum, as previously described (28). Exposure was during 9–14 sessions over a period of 23 mo. All subjects were Caucasian, the mean age was 42 years, and all were male. Immunological studies with these volunteers have been reported previously (10, 28, 29, 30, 31). Preimmunization samples collected before immunization with radiation-attenuated sporozoites and postimmunization samples were assayed simultaneously.

Population 2. Malaria-exposed Kenyan subjects (n = 13) were life-long residents of Saradidi, Siaya District, Nyanza Province, in the Asembo Bay area on the north and east shores of Lake Victoria, Western Kenya. In this area, transmission of malaria is very intense; the year-round prevalence of P. falciparum infection among children 6 mo to 6 years of age has been documented as 94.4–97.8% (32, 33, 34), the average daily entomological inoculation rate (number of bites by infectious Anopheline mosquitoes with P. falciparum sporozoites in their salivary glands per individual per night) has been estimated as 0.75 ± 0.97 infectious bites per person (32), and the average number of episodes of clinical malaria per year has been estimated as 2.1–2.5 episodes. The average number of infectious bites per person per year is estimated as 100–200, most bites occurring during the peak transmission periods (April to August and November to January), which coincide with seasonal rains. Samples studied here were collected from subjects in April 1987, just before the peak transmission season. Study subjects were estimated to have been bitten by >100 P. falciparum-infected mosquitoes during a 126-day period immediately following sample collection (32, 35). Subjects were almost exclusively of the Luo ethnic group. The mean age was 30 years, and all were male. Immunological studies with these volunteers have been reported previously (35, 36).

Population 3. Malaria-exposed Javanese transmigrants were residents of Arso Pir IV (Wonorejo), a lowland village located in the Arso District of northeastern Irian Jaya, the eastern-most province of Indonesia, 60 km south of the Pacific coast and 25 km west of the Papua New Guinea international border. This site has been described previously (37, 38). In this area, malaria transmission is moderately intense, the incidence rate of P. falciparum infections has been documented (38, 39) as 3.0 cases per year, and the cross-sectional prevalence of P. falciparum parasitemia ranges from 30% to 70%. The village is populated by native Irianese (20%) and by Javanese transmigrants (80%) who had moved voluntarily from malaria-free areas of West Java 5 years before the study. Study subjects were recruited from the transmigrant population and all were of the Javanese ethnicity. A total of 188 Javanese transmigrants were HLA typed, and a representative subset of 121 was selected for further study. Subjects reported no history of exposure to malaria before transmigration and were documented as experiencing an average of 1.00 clinical episodes per lifetime (range, 1–6 episodes) (44.6% of the population had no clinical episodes, 28.1% had 1 clinical episode, 17.4% had 2 clinical episodes, and 9.9% had 3 or more clinical episodes), with an average parasitemia of 181.61 parasites/µl. The mean age of study subjects was 33.5 years (range, 19–59), 78 were male (64.5%), and 43 were female (35.5%).

Population 4. Malaria-naive Javanese individuals were residents of Blitar, west central Java, and had no history of exposure to malaria. Blitar is one of several villages in West Java where residents of Arso Pir IV lived before transmigration. A total of 188 residents of Blitar were HLA typed, and a subset of 110 was selected for further study. Subjects were of the same Javanese ethnicity as the malaria-exposed Arso transmigrant subjects and were further matched where possible for socioeconomic status, age, and sex. The mean age was 32.4 years (range 17–65 years), 94 were male (52.2%), and 86 were female (47.8%).

HLA typing: frequency and projected population coverage analysis

Phenotypic frequencies of HLA class II alleles were established from peripheral blood samples using standard site-specific oligonucleotide PCR typing, in the case of the Javanese study population, or compiled from the literature (40) in the case of the major ethnic groups. The gene frequency of each B1, B3, B4, or B5 allele was then either directly tabulated or calculated from the Ag frequency using the binomial distribution formulae gf = 1 - (SQRT(1 - af)) (41), in which SQRT = square root.

To obtain overall population coverage estimates for each peptide, cumulative gene frequencies (gf) of DR Ags that bound the peptide were calculated. The cumulative Ag frequency (i.e., the fraction of individuals capable of binding the particular epitope) was then derived by the use of the inverse formula (af = 1 (1 - gf)2) (15), in which af = allelic frequency. The impact of the two DR5 subtypes DR11 and DR12 were considered separately because DRB1*1101 and DRB1*1201, representative of DR11 and DR12, respectively, were known to have different binding specificities. In the case of DR6, which is divided between DR13 and DR14, subtype frequencies were also used as the binding specificity of DR14 was not known.

The contribution of B3, B4, and B5 gene products to overall population coverage was also considered, based on the known strict linkage disequilibrium of the B3 gene product DR52a with DR3, of the B4 product DR53 with DR4, DR7, and DR9, and of the B5 product DR51 with DR2. A residual fraction (about 15%) of the genes in an average population are unspecified, using currently available HLA typing data (40). Therefore, to arrive at 100% accounting of genes, a fraction of the residual was added for each hit population cluster in proportion to the relative frequency of the cluster within the HLA-specified population. No adjustment was made for DRX.

The redundancy of coverage by the panel of epitopes is defined herein as the total number of different DR/peptide combinations potentially presented by a given individual and thus yielding a potentially immunogenic signal. Hence, because in the present study 11 peptides are considered, and each individual can express up to 4 different DR molecules, the theoretical maximum number of different peptide/DR combinations presented is 11 x 4 = 44.

The percentages of individuals yielding any given number of peptide/DR combinations known to bind with IC₅₀ of 1000 nM or less was then calculated using Monte Carlo analyses (42). For these analyses, model populations for each ethnic group identified in Table I were first constructed using the gene frequencies of DR Ags in the corresponding ethnic population. These model populations were constructed without considering linkage disequilibrium, and DR Ags were represented in direct proportion to their corresponding gene frequencies. The number of DR epitopes presented by each individual in the model population was then determined by tabulating, for each model individual, the number of DR epitopes/combinations associated with binding with an IC₅₀ 1000 nM. Finally, a histogram was generated to summarize the fraction of individuals in the population as a function of the number of DR-epitope combinations presented. A cumulative plot was also generated to determine the minimal number of DR-epitope combinations presented by 85% of the individuals in a given population.

PBMC collection and cultures

Peripheral blood was collected by venipuncture into heparinized vacutainers, and PBMC were isolated by standard Ficoll-Hypaque density gradient centrifugation. Cell concentration was adjusted as appropriate, and lymphoproliferative and cytokine assays were set up simultaneously. Cell culture medium consisted of RPMI 1640 containing 10 mM HEPES (Life Technologies) supplemented with 2 mM L-glutamine (Irvine Scientific or Life Technologies), 0.5 mM sodium pyruvate (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin (Irvine Scientific), and 10% heat-inactivated pooled human AB serum (Gemini Bioproducts, Calabasas, CA, or ICN Biomedical, Costa Mesa, CA).

Induction and assay of peptide-specific lymphoproliferative responses

In vitro induction of recall peptide-specific lymphoproliferative responses was achieved by culturing fresh (Javanese) or frozen (irradiated sporozoites and Kenyan) PBMC at a concentration of 2 x 10⁵ cells/well in quadruplicate in a volume of 0.2 ml complete medium in a round-bottom 96-well tissue culture plate in the presence of 200 and 20 µg/ml (Javanese) or 30, 10, and 3 µg/ml (irradiated sporozoites and Kenyan) of each peptide, without peptide (medium control; 8–12 wells/subject), or with mitogen (PHA at 10 µg/ml and Mycobacterium tuberculosis purified protein derivative at 5.0 µg/ml) for 6 days at 37°C in an atmosphere of 5% CO₂. Peptide concentrations were selected based on limitations imposed by cell numbers and peptide quantity in the different populations and preliminary studies with other peptides in the same populations. There was no association between peptide concentration and peptide-specific T cell responsiveness for any assay in any of the populations studied here (data not shown). Wells were pulsed with 1.0 µCi [³H]methyl thymidine (DuPont NEN, Boston, MA) for 16–18 h, and uptake was assessed by liquid scintillation spectroscopy (model LS6800, Beckman Coulter, Fullerton, CA). Results were expressed as a stimulation index (SI; cpm test sample/cpm medium control without peptide). Average medium control values were 263.1, 209.1, and 405.2 for irradiated sporozoites, Kenyan, and Javanese samples, respectively. As defined in previous studies (43, 44, 45), the response to a peptide was considered positive if the SI (ratio of stimulated to unstimulated cells) was >2.0.

Induction and assay of peptide-specific cytokine responses

Fresh (Javanese) or frozen (irradiated sporozoites and Kenyan) PBMC were cultured at a concentration of 1 x 10⁶ cells/ml per well in a volume of 1.0 ml complete medium in 24-well tissue culture plates (Costar, Cambridge, MA) in the presence of each peptide (10 µg/ml), without peptide (medium control), or with mitogen (PHA at 10 µg/ml and Mycobacterium tuberculosis purified protein derivative at 5.0 µg/ml) at 37°C in an atmosphere of 5% CO₂. Cell-free supernatants were collected at 5 days and stored at -70°C before analysis.

IFN-. The IFN- bioassay was based on a previously described protocol (46) using the WISH cell line and EMC virus. WISH cells were cultured in complete RPMI 1640 medium and were maintained by splitting twice weekly. The recombinant human IFN- standard was obtained commercially (Genzyme,Cambridge, MA; sp. act. = 1.0–4.75 x 10⁷ U/mg at 10 µg/ml, aliquoted into working stocks of 1 x 10⁵ U/ml that were stored at -135°C before use, working aliquots being stored at 4°C and reused for up to 4–6 wk). For the bioassay, WISH cells were washed three times, resuspended at a concentration of 1.0 x 10⁶ cells/ml, and aliquoted in 50-µl volumes (5 x 10⁴ cells/well) in flat-bottom 96-well plates. Fifty microliters of test samples (diluted 1:3, 1:9, and 1:27) or IFN- standard dilutions (100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 U/ml) were added in triplicate. Fifty microliters of medium was added to 12 wells to serve as the medium control. Cells were cultured at 37°C in an atmosphere of 5% CO₂ for 24 h or until the monolayer was confluent, and the medium was then aspirated. One hundred microliters of EMC virus (multiplicity of infection 1.0) was added to all wells excluding the three wells representing the cell control (without virus). Cells were cultured for an additional 24 h at 37°C, washed three times, fixed with 100 µl of 5% formaldehyde for 10 min at room temperature, stained with 100 µl of 0.05% crystal violet in 20% ethanol for 10 min at room temperature, washed with 100 µl of 100% methanol to dislodge dye from fixed cells, and then assessed for cytopathic effect relative to the virus control wells (100% cytopathic effect) and cell control wells (0% cytopathic effect) by reading the OD at 540 nm. The IFN- concentration of test samples was calculated by reference to the standard curve. Values were corrected for the appropriate dilution factor and the average of all three corrected values recorded for each subject.

IL-5, IL-10, and IFN-. IL-5, IL-10, and IFN- responses of irradiated sporozoite-immunized and Kenyan volunteers were assayed using commercially available kits (Endogen, Woburn, MA), according to manufacturer’s specifications. Samples were considered positive if the OD reading exceeded 1.5 times that of the background, and concentrations were calculated by interpolation from standard curves based on recombinant cytokine dilutions run in parallel on the same plate. Any background level of cytokine production in cultures not stimulated with peptide was subtracted from peptide-induced responses.

Statistical analysis

Nonparametric continuous outcome variables (mean peptide-specific cytokine response) were compared using the Mann-Whitney U rank sum test (two-tailed). Distributions of cytokine concentrations (log-transformed) did not deviate significantly from normality (data not shown). The prevalence of dichotomous outcome variables (frequency of peptide-specific proliferative T cell responses or cytokine responses) was assessed using the Pearson ² test (two-tailed) and the Fischer’s exact test (two-tailed). Analysis was conducted using the SPSS statistical program (SPSS version 8.0, SPSS, Chicago, IL). The level of significance was a p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selection of a panel of representative DR molecules

In the series of experiments described herein, we sought to identify a set of HLA class II-restricted epitopes that would allow redundant coverage of the world population, irrespective of ethnicity. Accordingly, we focused our attention on a set of the 10 different HLA-DR Ags most prevalent throughout the world. The phenotypic frequencies of these alleles in various common ethnic groups are detailed in Table I. A similar table has been published elsewhere (15) and is shown here for reference purposes only.

For each Ag, the most common HLA-DRB1 allelic form was studied, as representative of that particular Ag. However, in the case of DR4, both the DRB1*0401 and DRB1*0405 were studied, as one form is prevalent in Caucasians while the other is prevalent among Oriental ethnicities. Similarly, in the case of DR5, both DRB1*1101 and DRB1*1201 were studied, as the motif recognized by these molecules appears to be different. Also, DRB1*1201 is prevalent in Oriental ethnicities, while DRB1*1101 is most common in Caucasians. DR10 was not studied herein, but the frequency of this allele is relatively low in most ethnicities.

At the DR locus of humans, in addition to the product of the B1 gene that is expressed in conjunction with a monomorphic chain, the products of other genes (B3, B4, and B5) are also expressed, in conjunction with the same -chain. These DR molecules correspond to the serologic DR51, 52, and 53 Ags. Because of linkage disequilibrium, DR2-positive individuals usually express DR51, individuals expressing DR3, 11, 12, and 13 Ags usually express DR52, and individuals expressing DR4, 7, and 9 Ags usually express DR53. In the present study, DRB5*0101, DRB3*0101 and DRB4*0101 have also been studied as representative of the DR51, 52, and 53 Ags, respectively (Table I).

Identification of conserved high-affinity P. falciparum peptides containing specific HLA-DR binding motifs

Next, the sequences of the P. falciparum CSP, SSP2, LSA-1, and EXP-1 Ags were screened for the presence of specific HLA-DR binding motifs. Specifically, 15-mer sequences were selected that contained a 9-residue core bearing the DR supermotif described by O’Sullivan et al. (21) and Southwood et al. (15) with 3-residue N- and C- terminal flanking regions. In cases where two peptides that overlapped >90% were identified, a single longer peptide incorporating both sequences was synthesized.

Peptides were also selected on the basis of conservancy. For CSP and SSP2, where sequences of multiple variants of the same Ag were known at the time of analysis, peptides were considered for further study only if conservancy was >80%. However, only one sequence was available for LSA-1 and EXP-1 at the time of screening, so conservation could not be considered for these Ags.

Eighty-five P. falciparum-derived, HLA-DR supermotif-containing peptides were initially tested for binding to the DR molecules in a primary panel of assays, encompassing DRB1*0101, DRB1*0401, and DRB1*0701. Peptides binding at least two of these three DR molecules were then tested for binding to a secondary panel comprised of DRB*1501, DRB5*0101, DRB1*1302, and DRB1*0901 molecules. Peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, were screened for binding to the DRB1*0405, DRB1*1101, and DRB1*0802, molecules comprising a tertiary assay panel. Finally, peptides binding at least seven of the 10 DR molecules comprising the primary, secondary, and tertiary screening assays were further tested for binding to DRB3*0101, DRB4*0101, DRB1*0301, and DRB1*1201.

Accordingly, eight different HLA-DR cross-reactive peptides were identified that bound at least seven of the 14 HLA-DR molecules tested. In the course of these studies, three other peptides were also identified that bound five or six of the DR molecules tested. Together, these 11 peptides (Table II) include at least one from each of the four P. falciparum Ags considered: three were derived from CSP, five from SSP2, two from EXP-1 and one from LSA-1. To further address the issue of conservancy, P. falciparum DNA regions coding for the 11 selected peptides were also retrospectively sequenced in 10 to 13 P. falciparum parasite isolates from Arso (E. Gomez et al., manuscript in preparation) and analyzed in more recent deposits to the GenBank database. As shown in Table II, all of the peptides studied were found to be highly conserved in both cases, with conservancy values in the 83–100% range; in fact, seven of the 11 peptides were totally conserved. All peptides in which polymorphisms have been reported derive from the SSP2 Ag.

Projected population coverage by the 11 epitopes in various different ethnicities

Also shown in Table II is the projected potential population coverage of each peptide, based on its capacity to bind HLA-DR molecules with IC₅₀ 1000 nM. Calculations of population coverage were based on the assumption that the DR molecules tested are representative of all subtypes of the same Ag and on the basis of known linkage disequilibriums, with the exception of DR6w13 and DR52a, as described in more detail in Material and Methods. As shown, all peptides were predicted to be bound by at least 63% and up to 93% of individuals in an average population.

Using population frequencies of the various different DR Ags in different ethnic populations, we also calculated and tabulated projected population coverages, defined as the total number of different DR/peptide combinations potentially presented in a given individual and thus yielding a potentially immunogenic signal. Hence, because in the present study 11 peptides are considered, and each individual can express up to four different DR molecules; the theoretical maximum number of different peptide/DR combinations presented is 11 x 4 = 44.

The percentage of individuals yielding any given number of peptide/DR combinations known to bind with an IC₅₀ of 1000 nM or less is shown in Fig. 1a. It can be seen that only 3% of individuals are not predicted to bind any peptide. The average number of DR-peptide combinations presented is 14.1. On Fig. 1b cumulative population coverages are also shown, as a function of ethnicity. It can be seen that regardless of ethnicities, >85% of the individuals are predicted to be capable of recognizing four or more epitope/DR combinations.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Projected population coverage in model populations by the panel of 11 epitopes. a, Percent of individuals projected to present the indicated number of HLA-DR/P. falciparum epitope combinations bound in a model average population, generated by averaging the gene frequencies in Caucasian, North American Black, Japanese, Chinese, and Hispanic populations (). Also shown is the cumulative plot of percent projected population coverage (). b, Cumulative plot of population coverage in five different major ethnicities (Caucasian, North American Black, Japanese, Chinese, and Hispanic).

In conclusion, the analysis presented herein supports the notion that the selected set of peptides should allow redundant coverage of a large fraction of the human population, regardless of ethnicity.

Antigenicity of HLA-DR-supertype degenerate binding peptides for irradiated sporozoite-immunized individuals: lymphoproliferative responses

In the next series of experiments, the 11 selected HLA-DR degenerate binding peptides (Table II) were tested for their ability to elicit in vitro recall lymphoproliferative responses from frozen PBMC of seven Caucasian volunteers immunized with irradiated sporozoites. HLA allelic frequencies in this population have been established previously using serological typing and oligonucleotide-specific PCR typing. Of the five individuals typed, three expressed HLA-DRB1*0701, two expressed DRB1*0101, and one expressed each of DRB1*0301, DRB1*1101, DRB1*1201, DRB1*1501, and DRB1*1701. All of these molecules are represented in the assay panel described in Tables I and II.

As defined in Materials and Methods, a lymphoproliferative response was considered positive if the SI was >2.0. According to this criteria, all 11 HLA-DR degenerate, high-binding peptides tested were recognized as helper T lymphocyte (HTL) epitopes by PBMC derived from HLA-matched sporozoite-immunized volunteers (Table III).

Representative data, derived from samples collected pre- and postimmunization with irradiated sporozoites from one volunteer who expressed the alleles DRB1*0301 and DRB1*1701 is shown in Fig. 2.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Recall peptide-specific, lymphoproliferative responses in an individual experimentally immunized with sporozoites to HLA-DR-supertype peptides. PBMC from an immune volunteer who expressed the HLA-DRB1*0301 and HLA-DRB1*1701 alleles, collected pre- or postimmunization with irradiated sporozoites, were stimulated in vitro with each of the peptides (at 30, 10, and 3 µg/ml each), or without peptide, as described in Materials and Methods. Data are expressed as a SI, and the response to a peptide was considered positive if the SI was >2.0.

Antigenicity of HLA-DR-supertype degenerate binding peptides for irradiated sporozoite-immunized individuals: cytokine responses

In parallel, we also assessed the ability of the HLA-DR degenerate high-binding peptides to induce the production of cytokines (IFN-, IL-5, and IL-10) by frozen PBMC of the seven volunteers immunized with irradiated sporozoites. IFN- was studied because it has been implicated previously in pre-erythrocytic-stage protective immunity (2) and because it may be considered a marker of Th1-type immune responses. IL-5 was studied as a marker of Th2-type immune responses, and IL-10 was studied because it is known to be involved in the regulation of immune responses. Recently, other studies (47, 48) have implicated a role for IL-10 in protective immunity against malaria. An overall summary of the frequency and magnitude of the peptide-specific induction of IFN-, IL-5, and IL-10 from irradiated sporozoite-immunized volunteers expressing class II molecules of the HLA-DR supertype for each of the 11 peptides tested is shown in Table III.

All 11 of the peptides were able to induce a recall peptide-specific IFN- response from PBMC of volunteers immunized with irradiated sporozoites (mean response, 128.2 pg/ml; range, 2.2–708.2 pg/ml). Specifically, peptide-specific IFN- responses were detected in a total of 66.2% (51/77) of assays. The frequency of response ranged between 28.6% (two of seven donors tested for reactivity to peptide SSP2–62) and 85.7% (six of seven donors tested for reactivity to peptides SSP2–223, EXP-82, SSP2–527, and SSP2–509).

In contrast, peptide-specific IL-5 responses were detected in only 11.7% (9/77) of assays (mean response, 13.1 pg/ml; range, 2.0–61.1 pg/ml). Of the 11 peptides, four peptides were not able to induce an IL-5 response in any of the volunteers tested, five peptides were able to induce a response in only one of the seven volunteers (the same volunteer for all peptides), and the other two peptides (SSP2–223 and EXP-71) were able to induce a response in only two of the seven volunteers.

Likewise, peptide-specific IL-10 responses were detected in only 15.6% (12/77) of assays (mean response, 74.2 pg/ml; range, 26.3–115.7 pg/ml). Five of the 11 peptides were not able to induce IL-10 production from PBMC of any of the seven volunteers, two peptides were able to induce IL-10 production from only one of the seven volunteers, three of the peptides were able to induce IL-10 from only two of the seven volunteers, and one peptide (CSP-2) was able to induce IL-10 production from four of the seven volunteers.

A general correlation existed between the proliferation and IFN- release data, with the EXP-82, EXP-71, and SSP2–527 peptides being most active in both assays.

These data are consistent with a cytokine profile mostly of the Th1 type for the HLA-DR degenerate binding peptides. Furthermore, the responses appeared to be induced by exposure to P. falciparum because peptide-specific cytokine responses could not be generated from prebleeds of the same volunteers collected before sporozoite immunization (data not shown).

In summary, the data presented above demonstrate that all of the epitopes predicted on the basis of the in vitro analysis are indeed naturally processed and presented in vivo because all peptides induced a peptide-specific lymphoproliferative response and peptide-specific cytokine responses in volunteers immunized with irradiated sporozoites. These data also establish that a T cell repertoire capable of being expanded as a result of deliberate immunization exists for each of them.

Antigenicity of degenerate peptides for recall HTL responses from individuals naturally exposed to hyperendemic malaria

In the next series of experiments, we examined whether peptide-specific lymphoproliferative and cytokine responses could also be recalled from frozen PBMC of semiimmune individuals with life-long natural exposure to hyperendemic malaria, with an estimated average daily entomological inoculation rate of 0.75 ± 0.97 infectious bites per person and an average number of 2.1–2.5 episodes of clinical malaria per year.

A total of 13 HLA-typed Kenyan individuals were studied, all of whom expressed DR alleles (including DR1, 3, 6, 9, 11, and 13) predicted to bind the 11 selected peptides. These peptides were tested for their ability to elicit in vitro recall lymphoproliferative responses under the same conditions and according to the same criteria established for the irradiated sporozoite-immunized volunteers above. Results are summarized in Table IV.

Overall, recall lymphoproliferative responses were detected in 22.4% (32/143) of assays. The fraction of individuals responding to each peptide varied between 7.7% (one of 13 individuals tested for reactivity to peptides CSP-53 and SSP2–509) and 46.2% (seven of 13 individuals tested for reactivity to peptide LSA-13). The magnitude of peptide-specific lymphoproliferative responses also varied, with an overall mean SI for responders of 3.0 (range, 2.0–6.7). Representative data for one individual (4Z) who expressed the alleles B1*0901, B1*1302, B3*0301, and B4*0101 is presented in Fig. 3.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Recall peptide-specific, lymphoproliferative responses in an individual naturally exposed to hyperendemic malaria to HLA-DR-supertype peptides. PBMC from a semiimmune individual with lifelong exposure to hyperendemic malaria who expressed the HLA-DRB1*0901, B1*1302, B3*0301, and B4*0101 alleles were stimulated in vitro with each of the peptides (at 30, 10, and 3 µg/ml each), or without peptide, as described in Materials and Methods. Data are expressed as a SI, and the response to a peptide was considered positive if the SI was >2.0.

Expression of HLA-DR Ags in a Javanese population naturally exposed to mesoendemic malaria

Next, we planned to examine whether peptide-specific lymphoproliferative and cytokine responses could also be recalled from PBMC of semiimmune Javanese individuals with a more limited exposure (5 years) to a lower intensity of malaria transmission, with an estimated average daily entomological inoculation rate of 0.28 infectious bites per person and an average of 1.00 episodes of clinical malaria per life time.

Individuals in the Javanese study population were HLA typed using oligonucleotide-specific PCR methods. It was noted (Table V) that the most prevalent alleles in this population were HLA-DRB1*1202, DRB1*1500, DRB1*1502, and DRB1*0701, which were expressed in 65.2, 17.0, 25.9, and 19.6% individuals, respectively. As expected, the most prevalent HLA-DR alleles studied (Table I) afforded an actual coverage of the Javanese population of 100%.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Projected population coverage in the Javanese study population by the panel of 11 epitopes. Percent of individuals in the Javanese projected to present the indicated number of HLA-DR/epitope combinations. Genetype values were derived as described in Materials and Methods. Also shown is the cumulative plot of percent population coverage ()

The ability of the 11 HLA-DR degenerate binding peptides to elicit in vitro recall lymphoproliferative responses from fresh PBMC of naturally exposed Javanese subjects was assessed next. As noted for the immune irradiated sporozoite-immunized volunteers and semiimmune naturally exposed Kenyan subjects, all 11 peptides were recognized as HTL epitopes by PBMC from the Javanese subjects (Table VI). A representative profile for one individual (4031) who expressed the alleles DRB1*1202, DRB1*1500, DRB3*0301, DRB5*0101, DQB1*0301, and DQB1*0600 is presented in Fig. 5. The frequency of the recall lymphoproliferative responses was markedly reduced as compared with the responses of the irradiated sporozoite-immunized and Kenyan populations. Specifically, recall lymphoproliferative responses were detected in only 5.8% (77/1320) of assays, the fraction of individuals responding to each peptide varying between 2.5% (three of 121 individuals tested for reactivity to the peptide CSP-53, SSP2–527, and SSP2–509) and 10.8% (13 of 121 individuals tested for reactivity to the peptide CSP-375). With regard to the magnitude of peptide-specific lymphoproliferative responses, the overall mean SI of responders was 2.9 (range, 2.0–8.9).

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Recall peptide-specific, lymphoproliferative responses in an individual naturally exposed to mesoendemic malaria to HLA-DR-supertype peptides. PBMC from a semiimmune individual with 5 years exposure to mesoendemic malaria who expressed the HLA-DRB1*1202, DRB1*1500, DRB3*0301, DRB5*0101, DQB1*0301, and DQB1*0600 alleles were stimulated in vitro with each of the peptides (at 200 and 20 µg/ml each), or without peptide, as described in Materials and Methods. Data are expressed as a SI, and the response to a peptide was considered positive if the SI was >2.0.

In summary, the data presented above demonstrate that significant recall lymphoproliferative and IFN- responses were detected for all 11 peptides in each of the three populations tested. Therefore, the T cell repertoire for each of the epitopes predicted on the basis of the in vitro peptide binding studies and demonstrated to be antigenic in a population experimentally immunized with high-dose sporozoite inoculum can be primed by natural exposure to both hyperendemic and mesoendemic malaria.

Frequency and magnitude of HTL responses as a factor of transmission intensity

The data presented above (Tables III, IV, and VI) suggest that the frequency and magnitude of peptide-specific lymphoproliferative and cytokine responses induced by experimental exposure to the bites of hundreds of infected mosquitoes are significantly greater than those induced by natural exposed to malaria and that the responses induced by natural exposure similarly reflect the level of transmission intensity. A comparison of the frequencies of lymphoproliferative and IFN- responses on a population basis are presented in Figs. 6 and 7, respectively.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Population comparison of HLA-DR-supertype peptide-specific lymphoproliferative responses. PBMC from immune volunteers experimentally immunized with irradiated sporozoites (Irr.Spz.; n = 7), from semiimmune individuals with lifelong exposure to high transmission hyperendemic malaria (Kenyan; n = 13), or from semiimmune individuals with 5 years exposure to lower transmission mesoendemic malaria (Javanese; n = 121) were stimulated in vitro with each of the peptides, or without peptide, and assayed for peptide-specific lymphoproliferative responses as described in Materials and Methods. For each population, the percentage responding to each of the peptides (SI > 2.0) is presented. Mean frequencies of lymphoproliferative responses were 53.2% (Irr.Spz.) vs 22.4% (Kenyan) vs 5.8% (Javanese).

The frequency of peptide-specific IFN- response ranged from 28.6 to 85.7% (51/77, 66.2% overall) in irradiated sporozoite-immunized volunteers, from 7.7 to 61.5% (39/143, 27.3% overall) in Kenyans, and from 4.5 to 16.9% (84/962, 8.7% overall) in the Javanese population. As with the lymphoproliferative response, the effect of population on frequency of IFN- response was highly significant: irradiated sporozoite vs Kenyan, p < 1.0 x 10^-8; irradiated sporozoite vs Arso, p < 1.0 x 10^-8; Kenyan vs Arso, p < 1.0 x 10^-8.

We have previously reported a similar effect of sporozoite exposure on frequency and magnitude of CD8⁺ CTL and cytokine responses (10). The data in the present study as well as that reported previously are consistent with observations that all volunteers immunized with radiation-attenuated P. falciparum sporozoites are protected against experimental challenge, while individuals naturally exposed to malaria acquire a degree of protective immunity that reflects in part the degree of exposure to malaria. These data are consistent with studies indicating that the induction of protective immunity following immunization with irradiated sporozoites is affected by the number of immunizing bites (with an apparent threshold of exposure to 900–1000 bites being required to confer protection), the interval between immunization and challenge, and the number of bites used in the challenge (L. Goh, S. L. Hoffman, et al., unpublished data).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have used a combination of immunochemical and cellular analyses based on HLA-specific peptide binding motifs and in vitro binding assays with purified HLA class II molecules to identify 11 degenerate HTL epitopes from four P. falciparum pre-erythrocytic-stage Ags that are restricted by multiple HLA-DR alleles. The sequences of the P. falciparum Ags CSP, SSP2, LSA-1, and EXP-1 were screened for the presence of the HLA-DR-supertype binding motif. A total of 85 peptides were identified. Fifty-two of these peptides contained the HLA-DR motif described by O’Sullivan et al. (21) (3x CSP, 26x SSP2, 11x LSA-1, 12x EXP-1) and an additional 33 peptides were identified on the basis of an "extended" HLA-DR peptide binding motif (15) (6x CSP, 7x SSP2, 15x LSA-1, 4x EXP-1). Twenty-one of the 85 peptides (24.7%) bound with high affinity to at least one HLA-DR molecule in vitro. Eleven of the 21 exhibited degenerate binding to between five and 11 of 14 common HLA-DR molecules, illustrating that degenerate HLA-DR binding capacity appears to be relatively frequent.

These 11 HLA-DR binding peptides were selected for further study and tested for their capacity to induce recall lymphoproliferative and cytokine responses from the PBMC of seven volunteers immunized with irradiated sporozoites, 13 volunteers exposed to hyperendemic malaria in western Kenya, and 121 individuals exposed to mesoendemic malaria in Irian Jaya. Each of the 11 peptides was shown to be recognized by T cells from individuals experimentally immunized with radiation-attenuated sporozoites or naturally exposed to malaria. The finding that each of the 11 peptides identified was antigenic for both recall lymphoproliferative and IFN- responses in each of three distinct populations is remarkable. This high frequency of success is likely attributed to the fact that all peptides were preselected on the basis of their ability to bind with high affinity to multiple HLA molecules in vitro and parallels previous results that identified 17 HLA class I degenerate CD8⁺ T cell epitopes (10). In the case of the class I (10), only seven of 49 peptides tested for binding to HLA-A*0201, eight of 203 peptides tested for binding to HLA-A3/A11 and two of 24 peptides tested for binding to HLA-B7 were selected for antigenicity studies and all were shown to be recognized as epitopes for both CTL and cytokine responses. Here, 11 of 85 peptides cross-reactive for binding to HLA-DR molecules in vitro were assessed for their ability to be recognized by peptide-specific lymphoproliferative and IFN- responses, and all were found to be antigenic.

Because each individual can express up to four different HLA-DR molecules, and the peptides studied here were selected on the basis of their ability to bind multiple DR molecules in vitro, the present data do not address the degeneracy of the recall immune responses to each of the peptides at the level of individual HLA-DR alleles. Nonetheless, the data described above establish that each peptide epitope is capable of inducing a peptide-specific recall immune response in the context of multiple HLA-DR molecules. Likewise, studies of the biological relevance of the MHC binding affinity of peptides indicate that there is a correlation between the affinity of binding of peptides to MHC molecules and their ability to be recognized by specific T cells (15, 49). This aspect was not addressed in the present study, because nonbinding peptides were not tested. In fact, all of the peptides studied here (Table II) bound multiple HLA-DR molecules in vitro with an affinity <1000 nM, which is the threshold previously associated with immunogenicity for HLA class II epitopes (15).

Previously, Sinigaglia and colleagues (50) reported a conserved CSP epitope (CSP 378–398, sequence DIEKKIAKMEKASSVFNVVNS) that is recognized by HLA-DR1-, 2-, 4-, 5-, and 7-specific human T cell clones. More recently, another conserved CSP epitope that is presented by multiple HLA class II DR molecules (CSP 326–345, sequence EYLNKIQNSLSTEWSPCSVT) has been identified (51).

Interestingly, a high degree of overlap was noted (Table VII) between the regions of the P. falciparum Ags from which the 11 HLA-DR degenerate HTL epitopes peptides were identified with those of the 17 HLA class I degenerate CD8⁺ T cell epitopes identified previously. Other studies have demonstrated a similar overlap of CD4⁺ T cell and CD8⁺ T cell epitopes (52, 53, 54) or have reported that mouse and human T cell epitopes map to similar regions for malaria (55) or other systems (56, 57).

Together with the HLA class I degenerate CD8⁺ T cell epitopes identified previously (10), the class II epitopes identified herein may be incorporated in a vaccine designed to protect humans against P. falciparum malaria via T cell-mediated immune responses. Such a multivalent vaccine incorporating a wide repertoire of specificities at the epitope level would be expected to circumvent the problem of genetic restriction of the host immune response that has thus far been a major obstacle for malaria vaccine (60). On the basis of the frequencies of HLA-DR Ags in the general population, it was expected that 85% of the study population could be covered by at least one epitope. In the present study, 100% coverage was demonstrated in a real setting. Indeed, 100% of the Javanese study population was determined to have the capacity to present four or more DR-peptide epitopes. These results further validate the DR supermotif approach as a method to identify vaccine candidate epitopes.

In conclusion, herein we have analyzed the CSP, SSP2, EXP-1, and LSA-1 Ags of P. falciparum and identified 11 peptide epitopes capable of binding with high affinity (IC₅₀ < 10,000 nM) to between five and 11 of the 14 common HLA-DR alleles expressed by a high proportion of different ethnicities. From our data, it is inferred that all epitopes are naturally processed and an active T cell repertoire exists for each one of them, because all peptides recall specific lymphoproliferative and IFN- responses in individuals experimentally immunized with radiation-attenuated sporozoites. Further, this T cell repertoire could be primed by natural exposure because all peptides induced specific recall lymphoproliferative and IFN- responses in individuals naturally exposed to malaria. Finally, the frequency and magnitude of the recall immune response was influenced by the intensity of exposure to P. falciparum sporozoites.

These data provide considerable experimental support for the development of a subunit malaria vaccine comprising a small number peptide binding specificities that would be predicted to be broadly efficacious in the majority of all racial and ethnic populations.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Population comparison of HLA-DR-supertype peptide-specific IFN- responses. PBMC from immune volunteers experimentally immunized with irradiated sporozoites (Irr.Spz.; n = 7), from semiimmune individuals with lifelong exposure to high transmission hyperendemic malaria (Kenyan; n = 13), or from semiimmune individuals with 5 years exposure to lower transmission mesoendemic malaria (Javanese; n = 121) were stimulated in vitro with each of the peptides, or without peptide, and assayed for in vitro induction of peptide-specific IFN- production as described in Materials and Methods. For each population, the percentage responding to each of the peptides is presented. Mean frequencies of IFN- responses were 66.3% (Irr.Spz) vs 27.3% (Kenyan) vs 8.7% (Javanese).

	Acknowledgments

	Footnotes

 
¹ This work was supported by funds awarded from the U.S. Department of Defense to an Independent Research Grant (No. 30811), in part by funds allocated to the Naval Medical Research and Development Command Work Unit Nos. 61102A. 00101BFX.1431 and 6287A00101EFX.1432, and in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health Contracts N01-AI-45241 and N01-AI-95362.

² The studies reported herein were conducted in accordance with U.S. Navy, Kenyan Ministry of Health, and Republic of Indonesia regulations governing the protection of human subjects in medical research. The research protocols employing human subjects in this study were reviewed and approved by the Naval Medical Research Institute’s Committee for the Protection of Human Subjects, the Walter Reed Army Institute of Research Human Use Committee, the Kenya Medical Research Institute/National Ethical Review Committee, and the Indonesian Communicable Diseases Research Center Committee for the Protection of Human Subjects. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy or naval services at large, the Department of Defense, or the Indonesian Ministry of Public Health.

³ Address correspondence and reprint requests to Dr. Denise L. Doolan, Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue (Room 3W41/3W16), Silver Spring, MD 20910-7500.

⁴ Abbreviations used in this paper: CSP, circumsporozoite protein; SSP2, sporozoite surface protein 2; EXP-1, exported protein-1; LSA, liver-stage Ag-1; SI, stimulation index; HTL, helper T lymphocyte.

Received for publication October 1, 1999. Accepted for publication April 26, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. World Health Organization. 1996. Malaria Fact Sheet No. 94. World Health Organization, Geneva, Switzerland.
2. Hoffman, S. L., E. D. Franke, M. R. Hollingdale, and P. Druilhe. 1996. Attacking the infected hepatocyte. In Malaria Vaccine Development, Ch. 3. S. L. Hoffman, ed. ASM Press, Washington, D.C., p. 35.
3. Doolan, D. L., B. Wizel, S. L. Hoffman. 1996. Class I restricted cytotoxic T lymphocyte responses against malaria-elucidation on the basis of HLA peptide binding motifs. Immunol. Res. 15:280.[Medline]
4. Wang, R., Y. Charoenvit, G. Corradin, P. de la Vega, E. D. Franke, S. L. Hoffman. 1996. Protection against malaria by Plasmodium yoelii sporozoite surface protein 2 linear peptide induction of CD4⁺ T cell- and IFN- dependent elimination of infected hepatocytes. J. Immunol. 157:4061.[Abstract]
5. Charoenvit, Y., V. F. Majam, G. Corradin, J. B. Sacci, R. Wang, D. L. Doolan, T. R. Jones, E. Abot, M. E. Patarroyo, F. Guzman, S. L. Hoffman. 1999. CD4⁺ T-cell- and interferon-dependent protection against murine malaria by immunization with linear synthetic peptides from a Plasmodium yoelii 17-kilodalton hepatocyte erythrocyte protein. Infect. Immun. 67:5604.[Abstract/Free Full Text]
6. Charoenvit, Y., S. Mellouk, C. Cole, R. Bechara, M. F. Leef, M. Sedegah, L. F. Yuan, F. A. Robey, R. L. Beaudoin, S. L. Hoffman. 1991. Monoclonal, but not polyclonal antibodies protect against Plasmodium yoelli sporozoites. J. Immunol. 146:1020.[Abstract]
7. del Guercio, M. F., J. Alexander, R. T. Kubo, T. Arrhenius, A. Maewal, E. Appella, S. L. Hoffman, T. Jones, D. Valmori, K. Sakaguchi, H. M. Grey, A. Sette. 1997. Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE) for antibody responses in vivo. Vaccine 15:441.[Medline]
8. Sette, A., J. Sidney. 1999. Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50:201.[Medline]
9. Sidney, J., H. M. Grey, R. T. Kubo, A. Sette. 1996. Practical, biochemical and evolutionary implications of the discovery of HLA class I supertypes. Immunol. Today 17:261.[Medline]
10. Doolan, D. L., S. L. Hoffman, S. Southwood, P. A. Wentworth, J. Sidney, R. W. Chesnut, E. Keogh, E. Appella, T. B. Nutman, A. A. Lal, et al 1997. Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7:97.[Medline]
11. Bertoni, R., J. Sidney, P. Fowler, R. W. Chesnut, F. V. Chisari, A. Sette. 1997. Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J. Clin. Invest. 100:503.[Medline]
12. Chang, K. M., N. H. Gruener, S. Southwood, J. Sidney, G. R. Pape, F. V. Chisari, A. Sette. 1999. Identification of HLA-A3 and -B7-restricted CTL response to hepatitis C virus in patients with acute and chronic hepatitis C. J. Immunol. 162:1156.[Abstract/Free Full Text]
13. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G. Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, A. Sette, V. Barnaba. 1999. Presence of effector CD8⁺ T cells in hepatitis C virus-exposed healthy seronegative donors. J. Immunol. 162:6681.[Abstract/Free Full Text]
14. Madden, D. R.. 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13:587.[Medline]
15. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chesnut, H. M. Grey, A. Sette. 1998. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160:3363.[Abstract/Free Full Text]
16. Gulukota, K., J. Sidney, A. Sette, C. DeLisi. 1997. Two complementary methods for predicting peptides binding major histocompatibility complex molecules. J. Mol. Biol. 267:1258.[Medline]
17. Campbell, J. R.. 1989. DNA sequence of the gene encoding a Plasmodium falciparum malaria candidate vaccine antigen. Nucleic Acids Res. 17:5854.[Free Full Text]
18. Rogers, W. O., A. Malik, S. Mellouk, K. Nakamura, M. D. Rogers, A. Szarfman, D. M. Gordon, A. K. Nussler, M. Aikawa, S. L. Hoffman. 1992. Characterization of Plasmodium falciparum sporozoite surface protein 2. Proc. Natl. Acad. Sci. USA 89:9176.[Abstract/Free Full Text]
19. Zhu, J., M. R. Hollingdale. 1991. Structure of Plasmodium falciparum liver stage antigen-1. Mol. Biochem. Parasitol. 48:223.[Medline]
20. Sanchez, G. I., W. O. Rogers, S. Mellouk, S. L. Hoffman. 1994. Plasmodium falciparum: exported protein-1, a blood stage antigen, is expressed in liver-stage parasites. Exp. Parasitol. 79:59.[Medline]
21. O’Sullivan, D., T. Arrhenius, J. Sidney, M. F. Del Guercio, M. Albertson, M. Wall, C. Oseroff, S. Southwood, S. M. Colon, F. C. Gaeta, A. Sette. 1991. On the interaction of promiscuous antigenic peptides with different DR alleles: identification of common structural motifs. J. Immunol. 147:2663.[Abstract/Free Full Text]
22. Ruppert, J., J. Sidney, E. Celis, R. T. Kubo, H. M. Grey, A. Sette. 1993. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74:929.[Medline]
23. Sidney, J., S. Southwood, C. Oseroff, M. F. del Guercio, A. Sette, H. M. Grey. 1998. The measurement of MHC/peptide interactions by gel infiltration. Curr. Protocols Immunol. 18:3.1.
24. Falk, K., O. Rotzschke, S. Stevanovic, G. Jung, H. G. Rammensee. 1994. Pool sequencing of natural HLA-DR, DQ, DP ligands reveals detailed peptide motifs, constraints of processing and general rules. Immunogenetics 39:230.[Medline]
25. Wu, S., K. Maslanka, J. Gorski. 1997. An integrin polymorphism that defines reactivity with alloantibodies generates an anchor for MHC class II peptide binding: a model for unidirectional alloimmune responses. J. Immunol. 158:3221.[Abstract]
26. Ponnudurai, T., A. D. E. M. Leeuwenberg, J. H. Meuwissen. 1981. Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. Trop. Geog. Med. 33:50.[Medline]
27. Walliker, D., I. A. Quakyi, T. E. Wellems, T. F. McCutchan, A. Szarfman, W. T. London, L. M. Corcoran, T. R. Burkot, R. Carter. 1987. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236:1661.[Abstract/Free Full Text]
28. Egan, J. E., S. L. Hoffman, J. D. Haynes, J. C. Sadoff, I. Schneider, G. E. Grau, M. R. Hollingdale, W. R. Ballou, D. M. Gordon. 1993. Humoral immune response in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am. J. Trop. Med. Hyg. 49:166.
29. Malik, A., J. E. Egan, R. A. Houghton, J. C. Sadoff, S. L. Hoffman. 1991. Human cytotoxic T lymphocytes against Plasmodium falciparum circumsporozoite protein. Proc. Natl. Acad. Sci. USA 88:3300.[Abstract/Free Full Text]
30. Wizel, B., R. Houghten, P. Church, J. A. Tine, D. E. Lanar, D. M. Gordon, W. R. Ballou, A. Sette, S. L. Hoffman. 1995. HLA-A2 restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite protein 2 epitopes in sporozoite-immunized volunteers. J. Immunol. 155:766.[Abstract]
31. Wizel, B., R. A. Houghten, K. Parker, J. E. Coligan, P. Church, D. M. Gordon, W. R. Ballou, S. L. Hoffman. 1995. Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum surface sporozoite protein 2. J. Exp. Med. 182:1435.[Abstract/Free Full Text]
32. Beier, J. C., C. N. Oster, F. K. Onyango, J. D. Bales, J. A. Sherwood, P. V. Perkins, D. K. Chumo, D. V. Koech, R. E. Whitmire, C. R. Roberts, C. L. Diggs, S. L. Hoffman. 1994. Plasmodium falciparum incidence relative to entomologic inoculation rates at a site proposed for testing malaria vaccines in western Kenya. Am. J. Trop. Med. Hyg. 50:529.
33. McElroy, P. D., J. C. Beier, C. N. Oster, C. Beadle, J. A. Sherwood, A. J. Oloo, S. L. Hoffman. 1994. Predicting outcome in malaria: correlation between rate of exposure to infected mosquitoes and level of Plasmodium falciparum parasitemia. Am. J. Trop. Med. Hyg. 51:523.
34. Beadle, C., P. D. McElroy, C. N. Oster, J. C. Beier, A. J. Oloo, F. K. Onyango, D. K. Chumo, J. D. Bales, J. A. Sherwood, S. L. Hoffman. 1995. Impact of transmission intensity and age on Plasmodium falciparum density and associated fever: implications for malaria vaccine design. J. Infect. Dis. 172:1047.[Medline]
35. Hoffman, S. L., C. N. Oster, C. Mason, J. C. Beier, J. A. Sherwood, W. R. Ballou, M. Mugambi, J. D. Chulay. 1989. Human lymphoctye proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria. J. Immunol. 142:1299.[Abstract]
36. Hoffman, S. L., C. N. Oster, C. V. Plowe, G. R. Woollett, J. C. Beier, J. D. Chulay, R. A. Wirtz, M. R. Hollingdale, M. Mugambi. 1987. Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science 237:639.[Abstract/Free Full Text]
37. Baird, J. K., T. R. Jones, E. W. Danudirgo, B. A. Annis, M. J. Bangs, H. Basri, Purnomo, S. Masbar. 1991. Age-dependent acquired protection against Plasmodium falciparum in people having two years exposure to hyperendemic malaria. Am. J. Trop. Med. Hyg. 45:65.
38. Jones, T. R., J. K. Baird, M. J. Bangs, B. A. Annis, H. Purnomo, S. Basri, S. Gunawan, P. D. McElroy Harjosuwarno, S. L. Hoffman. 1994. Malaria vaccine study site in Irian Jaya, Indonesia: Plasmodium falciparum incidence measurements and epidemiologic considerations in sample size estimation. Am. J. Trop. Med. Hyg. 50:210.
39. Ohrt, C., T. L. Richie, H. Widjaja, G. D. Shanks, J. Fitriadi, D. J. Fryauff, J. Handschin, D. Tang, B. Sandjaja, E. Tjitra, et al 1997. Mefloquine compared with doxycycline for the prophylaxis of malaria in Indonesian soldiers: a randomized, double-blind, placebo-controlled trial. Ann. Intern. Med. 126:963.[Abstract/Free Full Text]
40. Imanishi, T., T. Akaza, A. Kimura, K. Tokunaga, T. Gojobori. 1992. Allele and Haplotype Frequencies for HLA and Complement Loci in Various Ethnic Groups Oxford University Press, Tokyo.
41. Tiwari, J. L., and P. I. Terasaki. 1985. The HLA complex. In HLA and Disease Associations. New York, Springer-Verlag.
42. Osborne, M. J., A. Rubinstein. 1994. A Course in Game Theory MIT Press, Cambridge, MA.
43. Good, M. F., D. Pombo, I. A. Quakyi, E. M. Riley, R. A. Houghten, A. Menon, D. W. Alling, J. A. Berzofsky, L. H. Miller. 1987. Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the polymorphic regions of the molecule. Proc. Natl. Acad. Sci. USA 85:1199.
44. Zevering, Y., R. A. Houghten, I. H. Frazer, M. F. Good. 1990. Major population differences in T cell response to a malaria sporozoite vaccine candidate. Int. Immunol. 2:945.[Abstract/Free Full Text]
45. Fidock, D. A., H. Gras Masse, J. P. Lepers, K. Brahimi, L. Benmohamed, S. Mellouk, C. Guerin Marchand, A. Londono, L. Raharimalala, J. F. Meis, et al 1994. Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants. J. Immunol. 153:190.[Abstract]
46. Vogel, S. N., R. M. Friedman. 1991. Measurement of antiviral activity induced by inerferons , ß, and . J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Straober, eds. Current Protocols in Immunology, Ch. 6.9.1 Wiley Interscience, National Institutes of Health, Bethesda, MD.
47. Plebanski, M., K. L. Flanagan, E. A. Lee, W. H. Reece, K. Hart, C. Gelder, G. Gillespie, M. Pinder, A. V. Hill. 1999. Interleukin 10-mediated immunosuppression by a variant CD4⁺ T cell epitope of Plasmodium falciparum. Immunity 10:651.[Medline]
48. Kurtis, J. D., D. E. Lanar, M. Opollo, P. E. Duffy. 1999. Interleukin-10 responses to liver-stage antigen 1 predict human resistance to Plasmodium falciparum. Infect. Immun. 67:3424.[Abstract/Free Full Text]
49. Sette, A., A. Vitiello, B. Reherman, P. Fowler, R. Nayersina, W. M. Kast, C. J. M. Melief, C. Oseroff, L. Yuan, J. Ruppert, et al. 1994. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol. 153.
50. Sinigaglia, F., M. Guttinger, J. Kilgus, D. M. Doran, H. Matile, H. Etlinger, A. Trzeciak, D. Gillessen, J. R. Pink. 1988. A malaria T-cell epitope recognized in association with most mouse and human MHC class II molecules. Nature 336:778.[Medline]
51. Moreno, A., P. Clavijo, R. Edelman, J. Davis, M. Sztein, D. Herrington, E. Nardin. 1991. Cytotoxic CD4⁺ T cells from a sporozoite-immunized volunteer recognize the Plasmodium falciparum CS protein. Int. Immunol. 3:997.5586.
52. Ou, D., L. A. Jonsen, D. L. Metzger, A. J. Tingle. 1999. CD4⁺ and CD8⁺ T-cell clones from congenital rubella syndrome patients with IDDM recognize overlapping GAD65 protein epitopes: implications for HLA class I and II allelic linkage to disease susceptibility. Hum. Immunol. 60:652.[Medline]
53. Gjertsen, M. K., J. Bjorheim, I. Saeterdal, J. Myklebust, G. Gaudernack. 1997. Cytotoxic CD4⁺ and CD8⁺ T lymphocytes, generated by mutant p21^ras (12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation. Int. J. Cancer 72:784.[Medline]
54. Lehtinen, M., M. H. Hibma, G. Stellato, T. Kuoppala, J. Paavonen. 1995. Human T helper cell epitopes overlap B cell and putative cytotoxic T cell epitopes in the E2 protein of human papillomavirus type 16. Biochem. Biophys. Res. Commun. 209:541.[Medline]
55. Dontfraid, F., M. A. Cochran, D. Pombo, J. D. Knell, I. A. Quakyi, S. Kumar, R. A. Houghten, J. A. Berzofsky, L. H. Miller, M. F. Good. 1988. Human and murine CD4⁺ T cell epitopes map to the same region of the malaria circumsporozoite protein: limited immunogenicity of sporozoites and circumsporozoite protein. Mol. Biol. Med. 5:185.[Medline]
56. Shirai, M., H. Okada, M. Nishioka, T. Akatsuka, C. Wychowski, R. Houghten, C. D. Pendleton, S. M. Feinstone, J. A. Berzofsky. 1994. An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans. J. Virol. 68:3334.[Abstract/Free Full Text]
57. Nehete, P. N., S. J. Schapiro, P. C. Johnson, K. K. Murthy, W. C. Satterfield, K. J. Sastry. 1998. A synthetic peptide from the first conserved region in the envelope protein gp160 is a strong T-cell epitope in HIV-infected chimpanzees and humans. Viral Immunol. 11:147.[Medline]
58. Connelly, M., C. L. King, K. Bucci, S. Walters, B. Genton, M. P. Alpers, M. Hollingdale, J. W. Kazura. 1997. T-cell immunity to peptide epitopes of liver-stage antigen 1 in an area of Papua New Guinea in which malaria is holoendemic. Infect. Immun. 65:5082.[Abstract]
59. Flanagan, K. L., M. Plebanski, P. Akinwunmi, E. A. Lee, W. H. Reece, K. J. Robson, A. V. Hill, M. Pinder. 1999. Broadly distributed T cell reactivity, with no immunodominant loci, to the pre-erythrocytic antigen thrombospondin-related adhesive protein of Plasmodium falciparum in West Africans. Eur. J. Immunol. 29:1943.[Medline]
60. Doolan, D. L., S. L. Hoffman. 1997. Multi-gene vaccination against malaria: a multi-stage, multi-immune response approach. Parasitol. Today 13:171.[Medline]

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