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Human CD4⁺ T Cells Induced by Synthetic Peptide Malaria Vaccine Are Comparable to Cells Elicited by Attenuated Plasmodium falciparum Sporozoites¹

Department of Medical Parasitology, New York University, School of Medicine, New York, NY

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4⁺ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4⁺ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN- as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4⁺ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4⁺ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.

	Introduction

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Despite the fact that 40% of the world’s population is at risk of malaria infection, there is currently no vaccine available to reduce the enormous burden of morbidity and mortality caused by the Plasmodium parasite. The hope of developing a vaccine to eliminate the pre-erythrocytic stages of the parasite, thus preventing the blood stage infections that cause clinical disease, is based on the ability of the attenuated sporozoite to elicit sterile immunity in experimental rodents, primates, and human volunteers (1). The logistics of developing an attenuated vaccine for humans are daunting, as sporozoites cannot be grown in vitro and protection against human species of malaria has thus far been elicited only by exposure of volunteers to bites of large numbers of irradiated infected mosquitoes (2, 3, 4, 5).

A synthetic Plasmodium falciparum peptide vaccine, termed (T1BT*)₄-N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl) (P3C),⁴ comprised of repetitive B cell epitopes and a universal T cell epitope from the circumsporozoite (CS) protein, was shown in a phase I trial to elicit anti-repeat Ab titers comparable in magnitude to those observed following sporozoite immunization (6). The CS repeat region is a target of protective Abs that inhibit sporozoite infectivity by immobilizing the parasite and blocking invasion of host cells (1, 7). The two T cell epitopes contained in the vaccine were defined by CD4⁺ T cell clones derived from volunteers immunized by multiple exposures to the bites of irradiated mosquitoes infected with P. falciparum (8, 9). One of these CS epitopes, termed T*, is a universal T cell epitope that is recognized by DR 1-, 4-, 7-, and 9-restricted T cell clones from sporozoite-immunized volunteers and that binds to multiple HLA class II molecules in vitro and elicits immune responses in mice of diverse genetic background (9, 10). Protection against P. falciparum sporozoite challenge in volunteers immunized with a recombinant CS protein vaccine, termed RTS,S, has been correlated with CD4⁺ T cells and CD8⁺ T cells that recognize epitopes overlapping the T* region (11). Although CD8⁺ T cells play an important role in immunity in murine studies, CS peptide-induced CD4⁺ T cells specific for the Plasmodium yoelii ortholog of T*, as well as CD4⁺ T cells induced by peptides from other pre-erythrocytic stage Ags, can protect against sporozoite challenge (12, 13, 14).

To analyze the fine specificity and potential effector function of human CD4⁺ T cells induced by peptide immunization, a panel of clones was isolated at various time points from volunteers immunized with (T1BT*)₄- P3C. The genetic restriction, core epitopes, strain specificity, and cytokine profiles of these clones were determined and compared with those we have previously defined using clones from sporozoite-immunized volunteers (9, 15, 16). These studies are the first to demonstrate that a synthetic peptide vaccine containing minimal epitopes of P. falciparum CS protein can elicit long-lived CD4⁺ T cell responses comparable to those elicited by the attenuated sporozoite.

	Materials and Methods

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Antigens

The synthetic peptide vaccine, (T1BT*)₄-P3C, was comprised of a 48-mer peptide containing T and B cell epitopes of the malaria CS protein ligated to a tetrabranched lysine core modified to contain the synthetic lipopeptide P3C as an endogenous adjuvant (Refs.6, 17 , and 18 and Fig. 1). The (DPNANPNV)₂ sequence represented the T1 epitope, located in the minor repeat region of the P. falciparum CS protein, and the (NANP)₃ sequence represented the B cell epitope located in the major repeat region. The T* sequence, EYLNKIQNSLSTEWSPCSVT, representing aa 326–345 of the P. falciparum CS NF54 isolate (19), was originally identified by clones from sporozoite-immunized volunteers (9, 15). This epitope was found to bind to multiple human and murine class II alleles in vivo and in vitro (9, 10), suggesting its potential as a universal T cell epitope for inclusion in vaccines.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Schematic of (T1BT*)4-P3C peptide vaccine showing the tandemly synthesized T1 [(DPNANPNV)2], B [(NANP)3], and T* (EYLNKIQNSLSTEWSPCSVT) epitopes of the P. falciparum (NF54) CS protein ligated to a tetrabranched lysine core modified with the P3C lipopeptide adjuvant (6 ).

Recombinant P. falciparum CS protein, containing the entire CS sequence of the NF54 isolate (19), exclusive of 26 aa at the N terminus and 13 aa at the C terminus, was expressed in Escherichia coli and purified by column chromatography (8). Similarly purified E. coli-expressed murine dihydrofolate reductase protein served as a negative control protein.

T cell clones

T cell clones were derived from PBMC obtained at various time points after s.c. immunization with 1 mg of (T1BT*)₄-P3C administered on days 0, 28, and 84. Briefly, 1 x 10⁶ PBMC/ml were cultured with T1BT* peptide (4 µM) for 3 days and expanded, without further Ag stimulation, by twice weekly addition of fresh complete RPMI 1640 medium containing 10% AB⁺ serum and rIL-2 (100U/ml). After 3–4 wk in vitro culture, the short-term T cell lines were screened for recognition of the T1BT* peptide and positive cell lines were immediately cloned by limiting dilutions in 96-round-bottom plates in medium with PHA-P (Difco), 100 U/ml rIL-2, and 10⁵ allogeneic irradiated (2500 rad) PBMC (16). Positive clones were expanded with mitogen and allogeneic cells in medium containing 100 U/ml rIL-2. To avoid skewing the T cell repertoire, clones were cryopreserved and thawed before experiments and were not maintained by long-term passage in vitro.

Phenotyping of the cells was carried out using commercial FITC-labeled mAb specific for CD4 or CD8, followed by PE-labeled mAb specific for markers for activation (CD69), memory cells (CD45RO, CD45RA), and chemokine receptor CCR7 (24) (BD Biosciences). The washed cells were fixed before analysis of 10,000 cells by FACS using CellQuest software (BD Biosciences).

Proliferation assays

The clones were assayed for Ag specificity in 3-day proliferation assay, as previously described (9). Triplicate wells of 2 x 10⁴ T cell clones in complete RPMI 1640 + 10% FCS were stimulated with 10–20 µg/ml peptide or E. coli expressed recombinant P. falciparum CS protein, using irradiated EBV-transformed B cell lines (EBV-B) (2 x 10⁴) or autologous PBMC (5 x 10⁴) as APCs. Wells were pulsed with 1µCi [³H]Tdr at 48 h and incubated for an additional 16–18 h before harvesting and scintillation counting. Results are expressed as stimulation indices calculated as cpm in wells with Ag/cpm in wells without Ag.

Genetic restriction of the T cell clones was determined using mAb specific for monomorphic determinants of DR (mAb L243 (ATCC HB-55; American Type Culture Collection), DQ (mAb SPV-L3), or DP (mAb B7/21) class II molecules (BD Biosciences) to inhibit peptide presentation. Donor-matched and mismatched peptide-pulsed EBV-B cells of known class II haplotypes were used to define the specific genetic restriction element used for Ag presentation (9).

Cytokine assays

Culture supernatants were collected at 24 and at 48 h for cytokine measurements. IL-2 in 24 h supernatants was measured by bioassay based on proliferation of an IL-2-dependent cell line, CTLL-2 (9). Results were expressed as stimulation index, calculated as cpm induced by supernatants from wells stimulated with Ag/cpm stimulated by supernatants from wells without Ag. Cytokines of Th1- (IL-2, TNF-, IFN-) and Th2- (IL-4, IL-6, IL-10) type subsets were determined using a Cytometric Bead Array (CBA) kit (BD Biosciences) and a FACSCalibur flow cytometer. CBA results were expressed as picograms per milliliter and clones producing >5 pg/ml were considered positive.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Fine specificity of CD4⁺ T cell clones

In the initial phase I study, low levels of PBMC proliferation and IL-2 responses to T1BT* peptide were detectable in 7 of 10 volunteers immunized with (T1BT*)4 peptide (6). PBMC analyses, however, are limited by the cell numbers available at each time point, the low frequency of Ag-specific cells in the peripheral circulation, and the inability to analyze genetic restriction and phenotype of the responding T cells. To evaluate the fine specificity and function of peptide-induced T cell responses, and to allow comparisons with CD4⁺ T cells derived from volunteers immunized with attenuated P. falciparum sporozoites (9, 15), clones were derived from the immunized volunteers at various time points following immunization with (T1BT*)₄-P3C. All of the clones were CD4⁺, CD8^–, and of memory phenotype (CD69⁺, CD45RO⁺, CD45RA^–, MHC class II⁺, CCR7^–) when analyzed by FACS (data not shown).

The fine specificity of the T cell clones was defined by stimulating cells with the individual T1, B, or T* epitopes contained in the triepitope vaccine (Table I). The majority of clones derived after the first boost (day 42) or second boost (day 98), proliferated and produced IL-2 when stimulated with the T1BT* or T* peptides, but not the repeat epitopes (NANP)₃ or T1. Importantly, T*-specific clones were also detected on day 373, 10 mo after the final immunization, indicating that long-lived malaria-specific memory CD4⁺ T cells were induced by peptide immunization.

Similar analyses were conducted for all 10 peptide-immunized volunteers and the fine specificity of these clones are summarized in Table II. CD4⁺ T cell clones specific for the T1BT* immunogen were detected in 7 of 10 volunteers, consistent with results obtained with PBMC (6). The presence of peptide-specific CD4⁺ T cell clones in each volunteer correlated with anti-CS Ab titers. A total of 213 T1BT*-specific clones were derived from 7 volunteers with high anti-CS Abs (GMT 2100), while peptide-specific cells were not detected in 3 volunteers (volunteers 03, 06, 07) with low Ab responses (GMT 254) (Table II).

Genetic restriction of T*-specific clones

T*-specific clones were derived from peptide-immunized volunteers expressing a broad range of HLA class II haplotypes (Table III). Using a combination of inhibition with class II mAb and Ag presentation by EBV-B typing cells, the T* clones were found to be restricted by multiple DR and DQ alleles. Clones from volunteer 04 used the broadest range of restriction elements, with T*-specific cells restricted by three class II alleles, DRB1*1103, DQB1*0301, and DQB1*0602 (Fig. 2A, Table III).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Genetic restriction of T*-specific clones from peptide-immunized volunteers 04, 05, 09, and 10 (A–D). HLA class II alleles used in Ag presentation were defined using peptide-pulsed EBV-B cell lines sharing one or more DR or DQ alleles. Results shown as percentage of IL-2 response elicited by T* peptide-pulsed autologous EBV-B cell as APC.

The majority of clones from volunteer 10, derived on day 42 (n = 6) or day 373 (n = 6), demonstrated an identical pattern of DRB1*0403-restricted T*-specific responses as shown for clone 10-16 (Fig. 2D, Table III). APC expressing shared DRB1*0701 (EBV-B ox03) or DRB4*01 (EBV-B ox03 and ox15) molecules, or the closely related DRB1*04 subtypes DRB1*0401 (EBV-B 9032) or DRB1*0404 (Ox 15), did not present the T* peptide to volunteer 10 clones.

Although most of the peptide-induced clones were monogamous, clones 9-2E10 and 10-36 from volunteers 09 and 10 were promiscuous and recognized T* in the context of more than one DR 4 molecule. Clone 9-2E10 recognized T* when presented by DRB1*0401 and DRB1*0404 APCs (Fig. 2C,

In addition to the six class II alleles defined for clones from volunteers 04, 05, 09, and 10 (DRB1*0401, 0403, 0404, and 1103; DQB1*0301 and 0602), additional DR and DQ restriction elements function in presentation of T* peptide. Anti-DR mAb inhibited T* clones from volunteer 08 (DRB1*1401,1602), while anti-DQ MAB inhibited volunteer 15 clones (DQB1*02,0302) (Table III). The broad range of genetic restriction found in the T*-specific clones from the peptide-immunized volunteers is consistent with T* epitope functioning as a universal T cell epitope in vivo, as predicted by the clones from sporozoite-immunized volunteers (9) and the ability of T* peptide to bind to multiple DR and DQ molecules in vitro (10).

Core epitope mapping

Universal Th epitopes from parasite, viral, and bacterial proteins contain multiple core epitopes that can bind in more than one frame to class II MHC molecules and thus elicit responses in individuals of diverse genetic backgrounds (25, 26). The minimal core epitopes recognized by DRB1*0403-restricted clones from volunteer 10 are shown as an example of the diversity of core epitopes within the T* sequence that are recognized in the context of a single class II molecule (Table IV). One clone (10-47, day 373) recognized only the 20-mer peptide and was not stimulated by truncated T* peptides containing N- or C-terminal aa deletions. Other clones (day 42 clones 10-7, -16, -21, and -27) recognized core sequences as small as 10 aa, LNKIQNSLST, tolerating truncations of four N-terminal and eight C-terminal residues. A second 10-aa core KIQNSLSTEW was recognized by clones from both day 42 (10-38 and -20) and day 373 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29), as was a 12-mer core, EYLNKIQNSLST (day 42 clones 10-6, -17, and day 373 clones 10-36, -17, -40). Thus, a total of five distinct cores within the T* sequence were used by DRB1*0403-restricted T cell clones from volunteer 10.

The N terminus of T* overlaps a polymorphic region of the CS protein while the C terminus overlaps the highly conserved region II of CS that functions in targeting sporozoites to the liver (reviewed in Ref.27). Studies in peptide-immunized mice and naturally infected individuals have suggested that the CS polymorphisms function in immune evasion (21, 28, 29). Nevertheless, protection in sporozoite-immunized individuals is not strain specific (2) and CD4⁺ T cell clones derived from protected sporozoite-immunized volunteers cross-reacted with a large number of P. falciparum strain variant peptides (9).

When strain specificity of the peptide-induced T*-specific cells was tested, a broad range of cross-reactivity for variant peptides representing P. falciparum isolates from Africa, South America, and Asia was found (Table VI). Highly strain cross-reactive clones from volunteer 04 recognized all peptides with two amino acid substitutions and several with three (Q - - KR peptide and K - - Q - - K peptide) or four (Q - - KT - K) amino acid substitutions (Table VI, Volunteer 04 section). In volunteer 09 clones, clones from an early time point (day 42) displayed a diverse pattern, ranging from strain-specific (clone 09-10) to broadly cross-reactive (clone 09-11) (Table VI, Volunteer 09 section). In contrast, volunteer 09 clones from a later time point (day 373) were all highly cross-reactive, suggesting an increase in cross-reactivity with time postimmunization. The day 373 clones recognized variants with two or three amino acid substitutions and the majority (seven of eight clones) recognized the 7G8 peptide that differs from NF54 strain at four amino acid positions (Q - - K - - K - - I). The broad range of strain cross-reactivity was not due to expansion of a single clone, as TCR of clones from day 373 expressed different V families, including V2, 3, 9, 14, and 18 (J. M. Calvo-Calle, unpublished observations).

Several of the variant peptides elicited heterocyclic responses, i.e., higher levels of proliferation/IL-2 production than that elicited by the homologous NF54 strain peptide. Variants containing two amino acid changes (E₃₂₆Q, K and N₃₂₉T, K, Q) frequently stimulated IL-2 production >2-fold higher that that elicited by the NF54 sequence (Table VI). The heterocyclic responses were unique to each clone TCR, as the same variant peptides did not elicit increased proliferation from other clones (day 373, clones 10-40 vs clone 10-17) nor from clones from different volunteers. Clone-specific heterocyclic responses were also noted in sporozoite-immunized volunteers (9) and the biological relevance of these responses remain to be defined.

Correlation of core epitope and strain cross-reactivity

The recognition of multiple core epitopes within the T* sequence ensures a diverse T cell repertoire. Clones from different volunteers tended to skew toward recognition of core epitopes from either the polymorphic N terminus or the conserved C terminus of the T* sequence (Fig. 3). Volunteer 04 clones recognized mostly C-terminal cores (nos. 2–6), while volunteer 10 clones recognized core epitopes that overlapped more frequently with the N-terminal cores (nos. 9 and 10). Volunteer 09 cores were unique in that clones from early time points frequently recognized N-terminal cores (core nos. 9 and 10), while all clones from day 373 recognized a single C terminus core, SLSTEWSP (core no. 8). Molecular analysis of TCR used by the day 373 clones from volunteer 09 indicate that this core was recognized in the context of different V families, including V2, 3, 9, 14, and 18 (J. M. Calvo-Calle, unpublished observations).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Correlation of core epitope specificity and strain cross-reactivity. The number of variant peptides and minimal core epitope recognized by clones derived on day 42 (open symbols) or day 373 (closed symbols) are summarized, based on data from Tables V and VI. The cores were assigned arbitrary numbers 1–10, with the amino acids overlapping the conserved region II of the CS protein shown in bold in the C-terminal minimal cores (nos. 2–8). Polymorphic residues in the N terminus are underlined.

Th cell subtypes T*-specific clones

The T*-specific clones derived from sporozoite-immunized volunteers were Th0 and produced IL-2,-4, -5, IFN-, TNF-, with variable expression of IL-10 and IL-6 depending on the clone, as measured by RT-PCR and ELISA (Ref.16 ; E. H. Nardin, unpublished observations). Similarly, the majority of the clones from the peptide-immunized volunteers were Th0 type, with a mixture of Th1- (IL-2, TNF-, IFN-) and Th2- type (IL-4, IL-6, IL10) cytokines detectable in supernatant of T*-stimulated cell cultures (Table VII). The ratio of Th1/Th2-type cytokines produced by the clones varied in the different volunteers and with time postimmunization. The majority of the clones produced IFN-, with a trend toward higher levels produced by day 373 clones (range 1679–3963 pg/ml) vs day 42 clones (range 1221–2232 pg/ml). In contrast, the number of clones secreting Th2-type cytokine (IL-4) varied, depending on the donor. In volunteer 09, 60% of the day 42 clones, but only 29% of day 373 clones, produced IL-4, suggesting skewing toward Th1-type responses in long-lived memory cells. Similarly, 100% of volunteer 10 day 42 clones produced IL-4 while only 67% of the clones were IL-4 positive on day 373.

Recent murine and human studies indicate that the cytokine profile of Th0-type CD4⁺ memory T cell clones can remain plastic and amendable to modulation by Ag stimulation in different environments (30, 31). CD4⁺ T cell clones derived from a malaria-exposed European donor were shown to switch from IFN- to IL-10 production when costimulated with CS variant peptides, suggesting that CS polymorphisms can function as altered peptide ligands in parasite immune evasion (32).

To determine whether the cytokine pattern of the peptide-induced T*-specific clones changed following stimulation with CS variant peptides, culture supernatants from strain-specificity experiments (Table VII) were assayed for Th1/Th2 cytokines. Although altered peptide ligands can dissociate proliferative and cytokine responses (30, 33), the cytokine responses to the variant peptides were found to be consistent with proliferation and IL-2 bioassays (Fig. 4). Th1/Th2 cytokines were not detected in the supernatant of cells that did not proliferate in response to peptide stimulation, indicating that the variant peptides were not partial agonists. For clones that made both IL-10 and IFN- in response to T* (e.g., clones from volunteers 04 and 10), stimulation with variant peptides did not shift the cytokine pattern toward a more Th2-type response, i.e., increased IL-10/IL-4 and decreased IFN- production. Conversely, clones that did not produce IL-10 when stimulated with the homologous NF54 T* peptide (e.g., volunteer 09 clones) did not produce IL-10 when stimulated with variant peptides.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Cytokines produced by T*-specific clones in response to stimulation with homologous NF54 T* peptide or variant peptides. Th1-type (IFN-) and Th2-type (IL-10, IL-4) cytokines in culture supernatants were measured by CBA and IL-2 responses were measured by bioassay. Response to variant peptides is shown as percentage of response obtained with NF54 T* peptide. Two clones obtained on day 373 are shown for each volunteer. Similar results were obtained with day 42 clones (data not shown).

	Discussion

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Analysis of the genetic restriction of the peptide-induced clones demonstrated that the DRB1*0401 molecule was used as restriction element by T*-specific clones from both sporozoite-immunized (9) and peptide-immunized volunteers (Table III). In addition, five new restriction elements for the T* epitope were identified by the peptide-induced clones, three DR molecules (DRB1*0403, 0404, and 1103), and two DQ molecules (DQB1*0301 and 0602) (Table III). As found in the sporozoite-immunized volunteers (9), multiple class II alleles could function as restriction elements in the same volunteer, e.g., clones from volunteer 04 were restricted by three different class II alleles, DRB1*1103, DQB1*0301, and DQB1*0602.

Expression of class II molecules known to bind the T* epitope in vitro or in vivo did not predict genetic restriction of the peptide-induced clones. Although DRB1* 0701-restricted clones were isolated from a sporozoite-immunized volunteer (9) and soluble DRB5*0101 (DR51) can bind T* peptide in vitro (10), these DR molecules were not used as restriction elements by T*-specific clones from donors expressing these alleles (volunteers 04, 09, and 10) (Table III) (6). Overall there was a pattern of allelic dominance of DR 4 molecules in the presentation of the T* peptide. In volunteer 10, only DRB1*0403-restricted clones were detected despite coexpression of DRB1*0701, a genetic restriction element used by sporozoite-induced clones. Similarly, only DRB1*0401-restricted clones were detected in volunteer 09, who was heterozygous for DRB5*0101 molecules (6) that can bind T*in vitro (10), and DQB1*0602, a restriction element used by volunteer 04 clones. Peptide competition for HLA binding in vivo may have contributed to allele dominance as T* is predicted by computer algorithms to bind with higher affinity to DRB1*04 alleles than other class II molecules (35, 36). In our peptide competition assays, T* peptide bound with high affinity to soluble DRB1*0401 (IC₅₀ 0.7 µM), but with lower affinity to soluble DQB1*0301 (IC₅₀ 100 µM) or DRB5*0101 (IC₅₀ 80 µM) molecules (10).

Differences in peptide-binding affinity for class II alleles or, alternatively, expression of lower levels of DQ on APC (37), may have contributed to predominance of DRB1*0401-restricted T* clones in volunteer 09. However, other factors potentially play a role in allelic dominance. DR7-restricted T*-specific clones were not detected in volunteer 10 despite the fact that DR 7 and DR 4 have a common peptide-binding motif and similar binding affinity for T* (10, 38). Moreover, two of the three (T1BT*)₄-P3C immunized volunteers of DR 7 haplotype (volunteers 03 and 07) developed only low Ab titers, and T*-specific T cells were not detected in these volunteers (Table III) (6). The DRB1*0701 haplotype is associated with low/nonresponders to both the (T1B)₄ malaria peptide vaccine (34) and hepatitis B virus surface Ag vaccine (39), suggesting that genetic factors linked to the DR7 haplotype may play a role in modulating CD4⁺ T cell responses.

The presence of multiple core epitopes within universal T cell epitopes ensures recognition by a large number of HLA molecules (25, 26). A total of 10 overlapping core epitopes were defined within the T* sequence that functioned in the context of DR and/or DQ molecules (Fig. 3). As found with sporozoite-induced clones (9), the core epitopes recognized by the peptide-induced clones varied in length, ranging from 8 to 20 aa. The same core epitope core no. 5 (KIQNSLSTEW) was recognized by DR7-restricted clones from a sporozoite-immunized volunteer (9) and by both DR- and DQ-restricted peptide-induced clones (volunteers 04 and 10), despite the different motifs that function in binding of peptides to these class II alleles (40). Other cores (e.g., core nos. 9 and 10) were recognized by closely related DR subtypes (DRB1*0401 and 0403) which differ in the peptide-binding cleft at aa residues 71, 86, and 74, suggesting that the peptide binds in a similar register to these DR 4 subtypes. Consistent with similarity of the peptide-binding cleft, promiscuous DRB1*0401/0404-restricted clones were identified in volunteer 09 and promiscuous DRB1*0403/0401-restricted clones in volunteer 10 (Fig. 2, C and D). Promiscuous clones expressing TCR that recognize the same peptide in the context of different class II molecules have also been noted in clones specific for other universal T cell epitopes (41).

The core epitopes recognized by the sporozoite-induced clones, as well as the majority of the peptide-induced clones, overlapped amino acid residues from the highly conserved region II of the P. falciparum CS protein (Fig. 3). In contrast, the N terminus of the T* sequence is polymorphic, with 28 variants reported thus far, containing a limited repertoire of amino acid substitutions at defined positions (E₃₂₆Q, K, D; N₃₂₉T, K,Q; K₃₃₀R, I, T, E) (22, 23, 42, 43). At the present time, it is not known what structural or functional requirements limit variability in position and in amino acid usage in this region of the CS protein. The fact that all substitutions maintain an amphipathic helix secondary structure suggests that this region is critical for protein function, potentially maintaining conformation needed for CS ligand interaction with cellular receptors of the mammalian host or mosquito vector (27, 44).

Early studies based on mice immunized with peptides containing only the CS polymorphic region (Th2R) suggested that amino acid changes abrogated immune recognition and thus potentially functioned in immune evasion (21, 29). However, in volunteers immunized with irradiated P. falciparum sporozoites or a recombinant CS protein vaccine, protection against sporozoite challenge was not strain-specific (2, 45). Consistent with strain cross-reactivity of protective immunity, T*-specific T cell clones from sporozoite- (9) and peptide-immunized volunteers (Table VI) recognized multiple T* variants found in geographically diverse P. falciparum isolates. Many of the clones were highly cross-reactive, recognizing variants with three or four amino acid substitutions (e.g., volunteer 09 day 373 clones). Importantly, as found in the sporozoite-immunized volunteers (9), strain cross-reactive clones were isolated from all of the peptide-immunized volunteers. The fact that clones derived from peptide-immunized volunteers on day 373, 10 mo after the last immunization, were frequently more cross-reactive than day 42 clones (Table VI, Fig. 3) is also important for vaccine efficacy. These clones represent long-lived memory T cells providing the potential for anamnestic responses to diverse P. falciparum isolates.

Core specificity plays a role in strain cross-reactivity, as recognition of C terminus cores containing the region II conserved amino acid residues of the CS protein was associated with higher levels of strain cross-reactivity (Fig. 3). This correlation was particularly striking in the volunteer 09 day 373 clones that recognized the conserved C terminus SLSTEWSP (core no. 8) which were highly cross-reactive (Table VI, Volunteer 09 section). Although variant peptides have been hypothesized to function as altered peptide ligands that can induce a shift from a Th1-type to a Th2-type cytokine response (32), no skewing of the cytokine profiles following stimulation with variant peptides was noted in the T*-specific clones (Fig. 4).

The fact that clones could be isolated from all of the peptide-immunized volunteers who developed high Ab titers after immunization (Table II) supports the conclusion that the T*-specific CD4⁺ T cells functioned as Th cells in vivo. IL-6 and IFN- are helper factors for differentiation of B cells, and IL-4 derived from CD4⁺ T cells is required for development of murine CS-specific CD8⁺ memory T cells (46). As found for the sporozoite-induced clones (16), the majority of the peptide-induced clones were Th0 type that secreted high levels of IL-2 and IFN- (>1500 pg/ml), as well as variable levels of Th2-type cytokines IL-4 and IL-6, depending on the donor (Table VII). The Th0 clones from volunteers 09 and 10 had a lower percentage of IL-4-positive clones at day 373 when compared with day 42, while clones derived from volunteer 04 produced IL-4, as well as high levels IL-10, at both days 42 and 373. Importantly, IFN- a critical cytokine for humoral and cellular immune responses, was produced by the majority of clones from all of the volunteers at all time points.

The high levels of IFN- produced by the peptide-induced clones correlated with a predominantly IgG1 and IgG3 Ab response in the (T1BT*)₄-P3C-immunized volunteers (6). In addition to functioning as a differentiation factor for B cells producing opsonizing Abs, IFN- also activates macrophages thus enhancing phagocytosis and clearance of opsonized sporozoites (47). The presence of opsonizing Abs specific for CS has been correlated with protection in volunteers immunized with the recombinant CS protein vaccine RTS,S (48).

In addition, IFN- functions directly in immune resistance and is a potent inhibitor of hepatic intracellular exoerythrocytic forms (EEF) development in the rodent malaria model (49, 50, 51). Murine CD4⁺ T cells induced by malaria peptide immunization can mediate IFN--dependent protective immunity against sporozoite challenge (13, 14). In recent studies, protection of naturally immunized African adults and RTS,S-immunized volunteers has been correlated with the presence of CS-specific IFN--secreting CD4⁺ T cells (11, 52).

Within the liver, IFN- and other proinflammatory cytokines can up-regulate MHC class II on Kupffer cells and induce expression of MHC class II on hepatocytes and sinusoidal endothelial cells (53). Therefore, professional as well as nonprofessional hepatic APC have the potential to present CS and stimulate IFN- production by CD4⁺ T cells to inhibit EEFs. Moreover, the potential for direct lysis of EEF-infected liver cells also exists, as murine cytotoxic CD4⁺ T cells from sporozoite-immunized mice can passively protect naive recipients from sporozoite challenge (54). The human peptide-induced T*-specific clones were cytolytic for peptide-pulsed target cells (J. M. Calvo-Calle, manuscript in preparation) as were T*-specific CD4⁺ CTL from sporozoite-immunized volunteers (15).

In volunteers immunized with the RTS,S malaria vaccine, protection correlated with the presence of IFN--producing CD4⁺ and CD8⁺ T cells that recognized epitopes within the C-terminal region of the CS protein that overlaps the T* epitope (11, 55). Protection in the RTS,S-immunized volunteers, however, was short-lived, lasting 2 mo (56, 57). In the current study, long-lived T*-specific CD4⁺ T cells that secreted high levels of IFN- were detectable 10 mo following the final immunization with peptide vaccine. T*-specific clones were also isolated from the sporozoite-immunized volunteer 1 year after immunization, at a time when protection against a second P. falciparum sporozoite challenge was demonstrated (9, 58).

In summary, the fine specificity, broad genetic restriction, and cytokine profiles of T*-specific CD4⁺ T cells elicited by peptide vaccination were comparable to those elicited by the attenuated P. falciparum sporozoite. These results encourage the hope that simple, well-defined malaria peptide vaccines containing T and B cell epitopes of pre-erythrocytic stage Ags can be developed for effective malaria immunoprophylaxis.

	Acknowledgments

 
We gratefully acknowledge the expert technical assistance provided by Rita Altszuler and Diana Barrios Rodrigues. We thank Victor Nussenzweig for critical review of the manuscript. Human rIL-2 was provided by M. Gately through the National Institutes of Health AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI 45138 and AI25085.

² Current address: Regeneron Pharmaceuticals, Old Saw Mill River Road, Tarrytown, NY 10591.

³ Address correspondence and reprint requests to Dr. Elizabeth H. Nardin, Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010. E-mail address: nardie01{at}med.nyu.edu

⁴ Abbreviations used in this paper: P3C, N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl); CS, circumsporozoite; EEF, exoerythrocytic form.

Received for publication May 3, 2005. Accepted for publication September 28, 2005.

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