Long-Term Multiepitopic Cytotoxic-T-Lymphocyte Responses Induced in Chimpanzees by Combinations of Plasmodium falciparum Liver-Stage Peptides and Lipopeptides

Animals.A total of six malaria-negative adult chimpanzees (Pan troglodytes) (Table 1) were enrolled in immunization experiments. The animals were randomly selected and housed in the Biomedical Primate Research Centre, Rijswijk, The Netherlands. Prior to the initiation of the study, all of the animals went through a detailed investigation, which included (i) assessment of current or past Plasmodium infection; (ii) determination of antibodies with specificity for sporozoites and erythrocytic stages of P. falciparum; (iii) determination of antibodies directed against the four pre-erythrocytic-stage Ags used for immunization; and, finally, (iv) determination of T-cell responses to LS peptides in preimmune lymphocyte cultures. Blood smears were prepared on several occasions and examined for the presence of parasites. None of these tests showed any evidence of current or past infection in any animal. Two weeks prior to the start of the study, no significant T-cell response to any LS peptides used in the present study was observed in preimmunization lymphocyte samples from the studied chimpanzees. Experimental procedures were performed under standard conditions in compliance with the institutional animal care and use committee and according to the relevant laws related to the conduct of animal experiments.

Peptides and lipopeptides.The sequences, localizations, and numbers of the amino acids of the 12 peptides and lipopeptides derived from four P. falciparum pre-erythrocytic Ags (strain T.9.96) are shown in Table 1. All peptides were synthesized by the solid-phase method on a benzhydrylamine resin (Applied Biosystems, Foster City, Calif.) by using a standard t-butyloxycarbonyl (Boc)-benzyl strategy and systematically acetylated at the end of the synthesis (7, 9, 11). A Boc-L-Lys (Fmoc [9-fluorenylmethoxy carbonyl]) group was introduced in the C-terminal end of the peptide and coupled to a palmitic acid moiety as previously described (7, 9-13, 19, 49). The crude peptides and lipopeptides were purified by reversed-phase high-pressure liquid chromatography. Peptides and lipopeptides were checked for homogeneity by analytical reversed-phase high-pressure liquid chromatography and for identity by amino acid analysis and molecular mass determination on a Bio Ion 20 plasma desorption mass spectrometer (Bio Ion AB, Uppsala, Sweden). Peptides and lipopeptides were 95 and 90% pure, respectively, with the exception of the STARP lipopeptide, which was insoluble in water. To prevent aggregation, the crude STARP lipopeptide (100 μg) was solubilized with trifluoroacetic acid in the presence of sodium dodecyl sulfate (600 μg), precipitated with ether, and then solubilized with water. Excess sodium dodecyl sulfate was eliminated by extensive dialysis. The synthesis of the STARP-derived mixotope was performed as described previously (45).

Immunization of chimpanzees.In view of ethical considerations and bearing in mind the requirements of a prospective vaccine in individuals with different HLA backgrounds, we designed a strategy in which reliable CTL responses could be generated in a limited number of outbred animals by vaccinating the animals with combinations of LS peptides and lipopeptides and taking into consideration previous findings that the subcutaneous route of immunization elicits strong and stable CTL responses (3, 7, 15, 48). These peptides and lipopeptides were injected subcutaneously and separately in different sites on the left or right shoulder areas. A mixture of nonlipidated peptides (100 μg of each) was injected in a single site by using Montanide ISA51 (SEPPIC, Quai d'Orsay, France). The Montanide ISA51 (100 μl) was emulsified with equal volumes of phosphate-buffered saline (pH 7.4) containing peptides. On the other hand, the mixtures of lipopeptides (100 μg of each) in phosphate-buffered saline (pH 7.4) were injected without adjuvant at a different site. The toxicity of the peptides and lipopeptides preparations was evaluated by observations of local and systemic side effect reactions after immunization.

The immunization regimen for each animal is shown in Table 2. The LSA3 in association with LSA1, SALSA, and STARP peptides and lipopeptides were injected subcutaneously in Demi, Karlien, and Iris at weeks 0, 3, and 6. The LSA1 and LSA3 peptides and lipopeptides were injected subcutaneously in Dirk at weeks 0, 12, and 15. Chimpanzees Bram and Fuad served as nonimmunized and adjuvant injected controls.

Cytotoxic T-cell assays. (i) Generation of CTL lines.CTL lines were generated as previously described (9, 11, 15). Briefly, heparinized venous blood was collected from the immunized and control animals at various time points, and peripheral blood mononuclear cells (PBMC) were collected after centrifugation on a Ficoll-Hypaque density gradient (Pharmacia LKB, Uppsala, Sweden). Purified cells were then resuspended in complete medium consisting of RPMI 1640 (Gibco Laboratories, Grand Island, N.Y.) supplemented with streptomycin (100 μg/ml), penicillin (100 IU/ml), l-glutamine (50 mM), and 10% of heat-inactivated normal human AB⁺ serum. The human serum was previously tested for its ability to support normal chimpanzee T-cell growth. Cells were grown at 2 × 10⁶/ml of complete medium in 25-cm² Falcon flasks and restimulated with immunizing peptides (10 μg/ml of each). Cells were incubated in a humidified incubator at 37°C and 5% CO₂. Then, 10 U of human recombinant interleukin-2 (Goenzyme, San Diego, Calif.)/ml was added to the cultures after 72 h, and cytotoxic assays were performed at days 7 to 10.

(ii) Target cells.Autologous phytohemagglutinin (PHA) blasts were used as target cells. PHA blasts were generated from each animal by in vitro stimulation of 2 × 10⁶ to 3 × 10⁶ PBMC with 0.5 μg of L-PHA (Sigma, St. Louis, Mo.)/ml. Blasts were maintained by biweekly medium changes. Target cells were incubated overnight at 37°C in the presence or absence of 20 μg of test or control individual peptide/ml and 3 μg of human β₂-microglobulin (Sigma)/ml in RPMI medium (Gibco, San Diego, Calif.) supplemented with 20% human AB serum. Target cells were then labeled with 150 μCi of ⁵¹Cr (sodium chromate; ICN Biomedical, Inc., Irvine, Calif.) for 1 h at 37°C, washed three times, and then plated in triplicate (at 5 × 10³ cells per well) in a 96-well round-bottom microtiter plate (Corning, New York, N.Y.). ⁵¹Cr-labeled target cells mock peptide pulsed were set up as controls. In order to minimize the nonspecific cytolytic activity caused by natural killer cells during the ⁵¹Cr release assay, nonlabeled human erythroleukemia natural killer-sensitive cells (line K562) were added at 20-fold excess in each well.

(iii) Chromium release assay.CTL assays were performed as described previously (9, 11, 15). Briefly, 5 × 10³⁵¹Cr-labeled target cells, pulsed or nonpulsed with a same concentration of peptide were incubated with CTL lines in triplicate wells, either with RPMI 1640 complete medium alone (for spontaneous release) or with 5% Triton X-100 or 1 N HCl (for maximum release), in round-bottom 96-well tissue culture plates. Target and effector cells were then incubated for 5 h at 37°C in 5% CO₂-air mixture, and the ⁵¹Cr activity from each well was then measured in 100 μl of supernatant by using a beta-plate scintillation counter (Wallac LKB 1205, San Jose, Calif.). Unless specified, assays were performed at a standardized effector/target ratio of 40:1. In all assays, the spontaneous ⁵¹Cr release in the absence of CTLs was <20% of maximum release by 5% Triton X-100 or 1 N HCl. The classical percent lysis was calculated as follows: 100 × [(experimental release − spontaneous release)/(total release − spontaneous release)]. The percent specific lysis was calculated as the percent lysis with peptide subtracted by the percent lysis without peptide. As we previously described (7, 9-11, 13), a positive result was defined as at least 10% higher specific lysis of peptide-pulsed cells than either control peptide-pulsed or nonpulsed target cells.

In some experiments, a fraction of effector cells was treated with anti-human CD4 or CD8 mouse monoclonal antibodies and thereafter with sheep anti-mouse monoclonal antibodies conjugated to magnetic beads (Dynal, Inc., Great Neck, N.Y.) to enrich CD8 or CD4 cell populations. Beads were added to T-cell lines at a bead/cell ratio of 50:1. The mixture was gently rotated at room temperature for 20 min before the beads were separated by using a magnetic particle concentrator (Dynal). Negatively selected cells were washed twice, resuspended in appropriate volume of RPMI 1640 complete medium, and used directly in CTL assays as described above. More than 95% of the cell populations were effectively depleted, as evaluated by fluorescence microscopy and flow cytometry analysis.

Statistical analysis.Figures represent data from at least two independent experiments. The data are expressed as the mean ± the standard deviation of means and analyzed by using the Student t test by using the Statview II statistical program (Abacus Concepts, Berkeley, Calif.).

Selection of potential T-cell epitopes within the LS P. falciparum Ags.We selected potential T-cell epitopes within the deduced primary amino acid sequence of LS protein Ags based on the fact that T-cell and B-cell epitopes have been frequently observed to cluster within limited regions of Ags (9, 38). To search for such regions, we focused on possible hinge regions between the repetitive sequences, which frequently contain B-cell epitopes, and the nonrepetitive sequences of the molecules. These hinge regions are less constrained than other parts of the molecules and therefore more readily accessible to proteolysis, an event that precedes T-cell epitope presentation in association with MHC molecules (24). We then searched for potential T-cell epitopes in the immediate vicinity of the repeat regions by using the “epitope cluster analysis” and the Garnier-Robson algorithms (29). Sequences from the repetitive areas that show putative conserved α-helical conformations, which are accessible to an acceptable mimicry by peptide synthesis, were also included. To search for potential epitopes within SALSA sequences, which in contrast to most malarial Ags does not contain repetitive regions, we selected sequences from two nonoverlapping areas that show a high tendency to adopt α-helical conformation. In contrast to the remaining Ags, the STARP structure includes a complex repeat central domain consisting of a mosaic of degenerated 10-amino-acid repeats. There is limited size variation in this domain, resulting from highly localized duplication events. The STARP-R peptide represents the nondegenerated units present as tandem copies in STARP. The STARP-M peptide is a mixotope peptide containing all of the observed degenerated sequences and, in addition, those potentially occurring but as yet nondescribed, provided they keep the same secondary structure (45). We selected 12 regions altogether within the LSA1, LSA3, SALSA, and STARP sequences, and the corresponding peptides were synthesized (Table 1). The sizes of these peptides were chosen to be between 20 and 41 residues since multiple T-cell epitopes corresponding to separate MHC class I restriction elements have been frequently described to segregate and overlap in relatively limited regions within many Ags (25, 31).

Induction of multiepitopic CTL responses in chimpanzees by combinations of LS peptides and LS lipopeptides.To determine whether P. falciparum LSAs induce CTL responses in chimpanzees and to obtain maximal information from limited numbers of animals, we immunized four animals with a combination of peptides and lipopeptides derived from the LSA3 molecule, since the latter appears to be the one with greatest potential (2, 11, 12, 19), together with those derived either from LSA1 (Dirk and Demi), SALSA (Karlien), or STARP (Iris) (Table 2). After each immunization, T-cell lines were derived from each animal, and their cytolytic activities against target cells were determined upon exposure to recall individual peptides. As shown in Fig. 1, three of the six unmodified peptides (two derived from LSA3 and one from SALSA Ags) and four of the six lipopeptides (one derived from LSA1, two from LSA3, and one from SALSA Ags) elicited significant CTL responses in chimpanzees. These CTLs were perceptibly heterogeneous and multiepitopic with various intensities to a given Ag in each animal (Fig. 1). Of the four peptides and lipopeptides selected from LSA1, LSA1-J lipopeptide elicited significant CTL responses in both Dirk and Demi (Fig. 1A and B). Lower but significant levels of CTLs were generated against the LSA1-REP in Dirk, but no CTL activity was detected against the remaining LSA1 peptides (i.e., LSA1-NR and LSA1-TER) (Fig. 1A and B). T-cell lines derived from the chimpanzee Karlien showed cytolytic activity against SALSA2 peptide and low but consistent CTLs against SALSA1 peptide (Fig. 1C). None of the STARP lipopeptides elicited significant levels of CTL activities in chimpanzee Iris (Fig. 1D). The T cells lines derived from chimpanzees Dirk, Demi, Karlien, and Iris showed cytolytic activity against LSA3-NRII, LSA3-RE, and/or LSA3-CT1 peptides (Fig. 1G to J). Interestingly, all of the animals had developed responses against the various LSA3 peptides. Three animals showed good responses to LSA3-NRII (Fig. 1G, H, and J), three showed good responses to LSA3-RE (Fig. 1G, I, and J), and two showed good responses to LSA3-CT1 (Fig. 1H and I). A similar profile of LSA3-specific CTL responses was recorded from Dirk immunized with LSA3, together with LSA1 peptides and lipopeptides (Fig. 1G). Collectively, although CTL activity had been detected against each of the four LSAs, the consistency and the level of CTL activity induced by LSA3 peptide epitopes points to the strong T-cell immunogenicity of LSA3.

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FIG. 1.

CTL response induced in chimpanzees by combinations of peptides and lipopeptides derived from P. falciparum LSA1, LSA3, and SALSA Ags. Animals were injected subcutaneously with peptides in Montanide ISA51 and lipopeptides in saline from the indicated Ag (top of each histogram). CTL lines were derived upon in vitro stimulation of PBMC with pools of recall peptides as described in Materials and Methods. CTL activities were assayed against individual recall peptides. MSP3-C peptide (from an erythrocytic Ags) was used as the control. The CTL responses elicited by LSA1 (A and B), SALSA (C) STARP (D), and LSA3 (G to J) peptides and lipopeptide are shown at an effector/target ratio of 40:1. CTL responses recorded in control animals are shown in histograms E, F, K, and L. Assays were performed in triplicates and bars represent standard error of the mean. Asterisks in front of peptides in the x axis indicate that a lipopeptide form was used for in vivo immunization. The results are representative of three independent experiments.

Antigen specificity and CD8⁺-T-cell dependence of CTL activities induced in chimpanzees by LS peptides and lipopeptides.Effector CTLs from Karlien were reactive to target cells sensitized with either SALSA1 or SALSA2 but not to target cells sensitized with heterologous control peptides from LSA1 Ag (Fig. 1C). In similar experiments with Demi, CTL lines reactive to LSA3-NRII and LSA3-CT1 did not lyse target cells sensitized with SALSA peptides (data not shown). These results indicate a lack of cross-reactive CTL epitopes within the four pre-erythrocytic Ags. The specificity of these responses was further confirmed by the lack of cytolytic activity to the control MSP3-C peptide from an erythrocytic Ags (Fig. 1) and by the lack of cytolytic activity of PBMC from both adjuvant-injected (Bram) (Fig. 1E and K) and nonimmunized control animals (Fuad) (Fig. 1F and L). The lack of cytolytic activity in PBMC sampled before immunization demonstrated that in vivo priming by LS peptides or lipopeptides was required in order to induce a CTL response (data not shown).

To address the question of MHC restriction, heterologous target cells sensitized with immunizing peptides were introduced. SALSA-specific CTL lines derived from Karlien that were able to lyse autologous target cells sensitized with either SALSA1 or SALSA2 were unable to lyse heterologous target cells from Dirk sensitized with the same peptides (i.e., SALSA1 and SALSA2) (Fig. 2A), suggesting MHC restriction of the CTL responses.

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FIG. 2.

Genetic restriction (A) and CD8⁺ T-cell dependence (B) of the P. falciparum LSA-specific CTLs induced in chimpanzees. (A) CTLs induced in chimpanzee Karlien by SALSA peptide and lipopeptide were assayed against either autologous target cells (black histogram) or heterologous target cells (gray histogram). (B) CTL lines were derived from chimpanzee Dirk, immunized with LSA3 peptides and lipopeptides, and their cytolytic activities were assayed against LSA3-NRII or LSA3-RE peptides either as bulk CTLs (○) or after depletion of CD4⁺ (•) or of CD8⁺ (▪) T cells. Assays were performed in triplicate, and bars represent the standard error of the mean. The results are representative of two independent experiments. E:T ratio, effector/target ratio.

The CTL responses were CD8⁺ T cell dependent since the cytolytic activities were abolished after in vitro depletion of CD8⁺ effector cells, but were not affected upon depletion of CD4⁺ cells (Fig. 2B). The cytolytic activities specific to both LSA3-NRII and LSA3-RE peptides, recorded from Dirk, were abolished by elimination of CD8⁺ cells but were unaffected when CD4⁺ cells were eliminated from the T-cell culture (Fig. 2B). Collectively, these results point to new regions within LSA1, LSA3, and SALSA Ags each bearing at least one CD8⁺-CTL epitope. These epitopes are distinct from those previously identified in humans living in malaria-endemic areas (1, 2, 32, 51) and in primate models (9, 11, 12, 14, 19, 46, 47, 57). In addition, these results strongly suggest that multiepitopic and MHC-restricted CD8⁺ CTLs can be simultaneously primed by combinations of LS peptides in Montanide ISA51 and lipopeptides in saline.

Long-lasting memory of CTL responses induced in chimpanzees by LS peptide or lipopeptide immunization.To determine whether memory CTLs had been induced, chimpanzees were monitored for up to 10 months after immunization. Nine months after Dirk's last immunization with LSA3 peptides and lipopeptides, significant CTL responses were still detectable. The long-term CTL responses were particularly stable for two of LSA3 peptides (LSA3-RE and LSA3-NRII), which were detected even 9 months after the final immunization. Indeed, the percent specific lysis had only decreased from 22.3 to 18.7% for the LSA3-RE peptide and from 28.3 to 18.3% for the LSA3-NRII peptide. In a similar experiment with chimpanzee Iris, the LSA3-NRII-specific CTLs that were at 15.3% specific lysis 4 weeks after the third final immunization remained at a significant level 5 months postimmunization (i.e., 12.9% specific lysis). However, not all LS peptides are equally efficient at inducing a long-lasting CTL memory. For instance, although LSA3-CT1 peptide initially elicited significant CTL response in both Demi and Karlien, the response declined to a nonsignificant level within 2 months after the final immunization (i.e., from 20.7 to 5.2% in Demi and from 14.7 to 6.3% in Karlien). The specific lysis of 24.3% generated by LSA1-J peptide in chimpanzee Dirk within the first months after the final immunization declined to a nonsignificant level of 6.8% 4 months later. The CTL responses to SALSA2 peptide recorded in chimpanzee Karlien remained detectable at a significant level up to 6 months after the third immunization (i.e., from 21.9 to 18.7% specific lysis). The percentage of specific lysis of 14.3% induced by SALSA1 peptide detected in Karlien 4 weeks after the final immunization declined 6 months later to a nonsignificant level of 7.3% lysis. Finally, no CTL activity was detected in Iris, when tested 1, 2, and 4 months after STARP immunization. Together, these results showed that long-lasting CTL responses were generated in outbred chimpanzees by combinations of LS peptides or lipopeptides, particularly those derived from the LSA3 Ag.

Consistency of CTL responses induced by LS peptides or lipopeptides in outbred chimpanzees.To evaluate the consistency of the CTL induced by each LS antigen during the 10-month follow-up period for the chimpanzees, up to six PBMC samples were assayed from each animal, and the number of animals that responded persistently to a given Ag was determined. It is important to remember that CTL activity cannot be demonstrated in each assay, and thus an animal is considered a responder if CTL responses were positive at least in two of three consecutive samples. Using such criteria, both Dirk and Demi immunized with LSA1 peptides and lipopeptides developed persistent cytolytic activity to LSA1-J peptide, whereas weaker CTL responses were induced by the remaining three peptides (i.e., LSA1-REP, LSA1-NR, and LSA1-TER) (Fig. 1A and B and Table 3). Both SALSA1 and SALSA2 peptides induced persistent CTLs in Karlien (Fig. 1C); in addition to those previously recorded in two additional chimpanzees (15). In contrast, the PBMC from STARP-immunized Iris were never shown to have a CTL activity. Interestingly, the LSA3 peptides were consistently immunogenic in five of six (83% of responders) immunized animals (Fig. 1G to J). The LSA3-NRII lipopeptide generated higher and consistent CTL responses in four of six animals (Fig. 1G to J). The LSA3-RE peptide generated persistent CTLs in three of six animals (Fig. 1G to J) (A. Luty et al., unpublished data) and the LSA3-CT1 in two of six animals (Demi and Karlien). Only one in six animals immunized with LSA3 peptides and lipopeptides (Demi) developed borderline CTL response to LSA3-NRI peptide (Table 3). The magnitude and persistence of CTL responses, induced by LSA3 in five of six outbred animals strongly suggest that this Ag is immunodominant (Table 3). The CD8⁺-CTL responses generated by the LSA3-NRII peptide showed in the present study, together with the CD4⁺-T-cell responses previously demonstrated with the same peptide epitope (7, 9-12, 14, 19), point to LSA3-NRII sequence as bearing one or several CTL and T-helper epitopes and therefore stand as an immunologically critical region of the LSA3 Ag.