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Sterile protective immunity against malaria in humans is induced by immunization with irradiated Plasmodium falciparum sporozoites (1, 2, 7, 9, 19), is dependent on CD8⁺ T cells (5, 20, 27), and is presumed to be directed against antigens expressed by irradiated sporozoites in infected hepatocytes (10, 11). Accordingly, a major approach to developing a malaria vaccine that duplicates the excellent protection induced by the irradiated sporozoite vaccine is to identify CD8⁺ T-cell epitopes on parasite proteins expressed by irradiated sporozoites in hepatocytes (10, 17). To date, 32 P. falciparum CD8⁺ T-cell epitopes derived from five proteins known to be expressed in infected hepatocytes have been reported (6; Table 1).

One potential challenge to the development of epitope-based vaccines is the polymorphism of major histocompatibility complex (MHC) class I molecules. Many HLA-A molecules can be grouped into different HLA supertypes which are characterized by largely overlapping peptide-binding repertoires and are present in high frequencies, irrespective of the particular ethnicity considered. Focusing on the major HLA supertypes generally simplifies the process of development of epitope-based vaccines (21, 22, 24). HLA-A1 is one of five HLA antigens (A1, A2, A3, A11, and A24) expressed in a high proportion of different populations (12, 24) and is the second most common antigen expressed by Caucasians (26 to 36.5%), after HLA-A2 (42 to 64%) (6; Table 2). Recent data also suggest that HLA-A*01 might represent a prototype allele of an HLA-A1 supertype composed of several alleles with similar peptide-binding motifs (22). Thus, HLA-A*01-restricted epitopes should be considered for inclusion in multiepitope peptide-based vaccines.

Accordingly, we searched the P. falciparum circumsporozoite protein (PfCSP) 3D7 sequence for sequences containing potential HLA-A*01 binding motifs, specifically a threonine (T), serine (S), or methionine (M) at position 2 or an aspartic acid (D), glutamic acid (E), serine (S), or threonine (T) at position 3 and a tyrosine (Y) at the C terminus (3, 13-15). Two peptides, amino acids (aa) 31 to 40 (NTRVLNELNY) and aa 310 to 319 (EPSDKHIKEY), predicted to contain an HLA-A*01 peptide-binding motif were synthesized and analyzed for their affinity of binding (23, 25) to HLA-A*01. Peptides which bound to HLA-A*01 with high affinity (50% inhibitory concentration [IC₅₀], 500 nM) were then assessed for their capacity to induce peptide-specific recall cytotoxic T-lymphocyte (CTL) responses from peripheral blood mononuclear cells (PBMC) from three volunteers expressing the HLA-A*01 molecule (Table 3) (7) and immunized with P. falciparum irradiated sporozoites.

Generation of effector cells. PBMC from irradiated sporozoite-immunized volunteers were infected with recombinant vaccinia virus expressing the entire PfCSP 3D7 sequence at 10 PFU/cell (16) or stimulated with 10 µg of synthetic PfCSP peptide (aa 310 to 319 [EPSDKHIKEY]) per ml for 6 days as described previously (16). In some experiments effector cell populations were depleted of CD8⁺ T cells using anti-CD8⁺-coated Dynabeads.

CTL assay. CTL assays and transient transfection of autologous and HLA-mismatched lymphoblastoid B-cell lines (B-LCL) were performed as described previously (16) using plasmid DNA expressing the 3D7 PfCSP gene (VR2510) or the same plasmid without the PfCSP insert (VR1020) (8) or autologous and HLA-mismatched B-LCL pulsed at 10 µg/ml with peptide PfCSP 3D7 (aa 310 to 319) or control peptide from P. falciparum sporozoite surface protein 2 (PfSSP2 [RRHNWVNHA]) (28).

Antigen-specific CD8⁺ CTL against endogenously synthesized PfCSP are induced in HLA-A*01-irradiated sporozoite-immunized volunteers. To determine whether immunization with radiation-attenuated P. falciparum sporozoites could generate anti-PfCSP CTL responses in HLA-A*01 individuals (Table 3), and if so, whether such CTL would recognize endogenously synthesized antigen, immune PBMC from irradiated sporozoite-immunized volunteers (4, 16, 17) were stimulated in vitro with autologous PBMC infected with recombinant PfCSP-expressing vaccinia virus. Cytolytic activity was assessed against autologous or HLA-mismatched B-LCL transiently transfected with PfCSP-encoding DNA (VR2510) or control DNA (VR1020). Effector cells from all three volunteers lysed autologous B-LCL transiently transfected with PfCSP-encoding DNA (Fig. 1) but not autologous B-LCL transfected with control DNA or HLA-mismatched B-LCL targets transfected with PfCSP-encoding DNA. These data demonstrated that an antigen-specific, HLA-restricted CTL response against endogenously presented PfCSP was induced in all three HLA-A*01-positive irradiated sporozoite-immunized volunteers.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   CTL lysis of target cells expressing endogenously synthesized PfCSP. Immune PBMC from volunteer 16 (A), volunteer 4 (B), and volunteer 17 (C) were stimulated with autologous PBMC infected with recombinant vaccinia virus expressing the entire gene of PfCSP 3D7 (PfCSP vaccinia). Cytotoxicity was assessed in a CTL assay against autologous and HLA-mismatched B-LCL transiently transfected with PfCSP-encoding plasmid DNA (2510) or vector control DNA (1020) at an 80:1 effector/target ratio.

Identification of an HLA-A*01-restricted epitope on PfCSP 3D7. We next determined whether these effector cells could recognize either of two predicted HLA-A*01 epitopes from PfCSP (aa 31 to 40 [NTRVLNELNY] and aa 310 to 319 [EPSDKHIKEY]). Two peptides containing these epitopes were synthesized, and their HLA-A*01 binding capacity was estimated to be 2,604 and 147 nM, respectively, as described previously (14, 23). Only one peptide, PfCSP 310-319, bound with an IC₅₀ of less than 500 nM. Based on our previous work we consider a peptide that binds with an affinity between 50 and 500 nM to be a high binder (23), and studies have established a correlation between high binding affinity and immunogenicity (23). Accordingly, PBMC from the three HLA-A*01 volunteers were stimulated with peptide PfCSP 3D7 310-319. In parallel, PBMC cultures were also stimulated with PBMC infected with recombinant PfCSP-expressing vaccinia virus. Autologous B-LCL were pulsed with either the PfCSP 3D7 310-319 peptide or with a control peptide or were transiently transfected with PfCSP-encoding DNA (VR2510) or control DNA (VR1020). The CTL response elicited by peptide PfCSP 3D7 310-319 was peptide specific, since effectors stimulated with peptide PfCSP 3D7 310-319 lysed targets pulsed with peptide PfCSP 3D7 310-319 but only minimally lysed or failed to lyse targets pulsed with the control peptide (Fig. 2). Furthermore, the CTL response was genetically restricted, since no significant CTL activity was detected when HLA-mismatched targets were pulsed with peptide PfCSP 3D7 310-319 (Fig. 2A and 3). The CTL response was dependent on CD8⁺ T cells, since depletion of CD8⁺ T cells from the effector cells eliminated the CTL activity (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Antigen-specific, MHC-restricted, CD8+ T-cell-dependent CTL activity in HLA-A*01 volunteers. Immune PBMC from volunteer 4 (A) and volunteer 17 (B) were stimulated with peptide PfCSP 3D7 310-319 (EPSDKHIKEY) (PfCSP peptide) or PfCSP-expressing vaccinia virus (PfCSP vaccine). Cytotoxicity was assessed in a CTL assay at an 80:1 effector/target ratio. Autologous or HLA-mismatched B-LCL were pulsed with peptide PfCSP 3D7 310-319 or a control peptide from the PfSSP2 sequence. Where indicated, cells were depleted of CD8+ T cells using Dynabeads (PfCSP peptide-CD8).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Antigen-specific, MHC-restricted CTL response in volunteer 16 (HLA-A*01). Immune PBMC from volunteer 16 were stimulated with peptide PfCSP 3D7 310-319. Cytolytic activity was then assessed in a CTL assay at an 80:1 effector/target ratio using autologous and HLA-mismatched B-LCL transiently transfected targets with a plasmid expressing PfCSP-encoding DNA (2510) or vector control DNA (1020) as a negative control.

Recognition of endogenously synthesized PfCSP by peptide PfCSP 3D7 310-319 stimulated immune effectors. Next, we wanted to assess whether effector cells induced by the PfCSP 3D7 310-319 peptide could recognize target cells that endogenously synthesized the PfCSP antigen. Accordingly, PBMC from volunteer 16 were stimulated with the PfCSP 3D7 310-319 peptide, and autologous B-LCL and HLA-mismatched B-LCL were transiently transfected with PfCSPs-encoding DNA (VR2510) or control DNA (VR1020). Data presented in Fig. 3 establish that peptide-stimulated effectors could recognize endogenously processed PfCSP, since they lysed autologous B-LCL targets transfected with the PfCSP gene but not autologous B-LCL transfected with control vector VR1020. Furthermore, this CTL response was genetically restricted, since CTL activity could not be detected using HLA-mismatched B-LCL transfected with the PfCSP gene (Fig. 3).