Human cytotoxic T lymphocytes against the Plasmodium falciparum ...

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3300-3304, April 1991 Medical Sciences

Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein (malaria/sporozoite/transfection/protective immunity/vaccine)

ANITA MALIK*, JAMES E. EGANt, RICHARD A. HOUGHTENt, JERALD C. SADOFFt, AND STEPHEN L. HOFFMAN*§ *Malaria Program, Naval Medical Research Institute, Bethesda, MD 20889; tDivision of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307; and tTorrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121

Communicated by Louis H. Miller, December 27, 1990 (received for review November 7, 1990)

W. Weiss, personal communication) epitopes transfer protection. Accordingly, there are efforts to produce vaccines for humans that induce CTL against the P. falciparum CS protein (13). However, since humans have never been shown to have CTL against any malaria protein, there is no human data to support development of such vaccines and no assay for assessing them. We describe such an assay and report that humans immunized with irradiated sporozoites produce CTL against the P. falciparum CS protein.

Cytotoxic T lymphocytes (CTL) against the ABSTRACT circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodiumfalk4iarm CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falkiparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P.fakiparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8 T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B1O.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria.

MATERIALS AND METHODS Mouse CTL Assay. The assay was performed as described (9). Briefly, 5 x 106 spleen cells from B10.BR-H-2k mice immunized with recombinant vaccinia virus were incubated with 2 x 10W L cells (H-2k) transfected with the CS proteinencoding gene (CS gene) of the 7G8 clone of P. falciparum (9). Six days later effector cells were incubated with 51Crlabeled L cells in the presence or absence of peptide, and the 5tCr release was measured. Immunization. After approval by an institutional review board, four human volunteers gave informed consent and were immunized by the bites of irradiated (1.5 x 104 rads) Anopheles stephensi mosquitoes infected with sporozoites of either the NF54 strain (17) or the 3D7 clone (16) of the NF54 strain of P. falciparum. Approximately 2 weeks after the last immunization, volunteers were challenged by exposure to mosquitoes infected with 3D7 (homologous) or 7G8 (heterologous) sporozoites. The details of these experiments are being reported elsewhere. Recombinant Vaccinia Virus V71. A recombinant vaccinia virus, V71, containing the CS gene of the 7G8 clone of P. falciparum has been described (14). Control vaccinia virus (vSC-8) contains the Escherichia coli IacZ gene. Synthetic Peptides. Synthetic peptides (see Table 1) reflecting amino acid residues 311-400 of clone 7G8 (15) P. falciparum CS protein and 368-390 of the identical 3D7 and NF54 sequences (16, 17) were synthesized as described (18). Effector Cells. Peripheral blood mononuclear cells (PBMC) were separated from heparinized venous blood or from leukophoresis runs by density gradient centrifugation. Recombinant Vaccinia Virus-Stimulated Effector Cells. PBMC from immunized donors were stimulated in vitro for 6 days with V71. One million PBMC per ml were infected for 1 hr at 370C at a multiplicity of infection of 10:1 with V71. The V71-infected PBMC were washed twice and then cultured with uninfected PBMC from the same volunteer. The expression of CS protein in V71-infected cells was confirmed by

After inoculation by mosquitoes, mature sporozoites pass through the blood to the liver where each uninucleate sporozoite can develop within a hepatocyte during -1 week to a schizont containing 1-5 x 104 uninucleate merozoites. When this schizont ruptures, each merozoite can invade an erythrocyte initiating the stage responsible for human disease. Immunization of mice (1) and humans (2, 3) with irradiated sporozoites protects against sporozoite challenge but not against challenge with infected erythrocytes. The protective immunity is against the sporozoite in circulation or the parasite within hepatocytes. In some mice protection after vaccination with irradiated rodent malaria sporozoites is eliminated by in vivo depletion of CD8' T cells (4, 5), and T cells from immune mice eliminate malaria-infected hepatocytes from in vitro culture (6), suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens on infected hepatocytes contribute to this protection. The circumsporozoite (CS) protein is the major surface protein of malaria sporozoites (7, 8). Mice immunized with irradiated Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii sporozoites develop CTL against a single epitope in the carboxyl-terminal region of their respective CS proteins (9-11). CTL against the P. yoelii epitope eliminate infected hepatocytes from culture (11), and CTL clones against the P. berghei (10) and P. yoelii (ref. 12;

Abbreviations: CTL, cytotoxic T lymphocytes; CS, circumsporozoite; PBMC, peripheral blood mononuclear cells; EBV, Epstein-Barr virus; V71, recombinant vaccinia virus containing the P. falciparum gene encoding CS protein; mAb, monoclonal antibody. §To whom reprint requests should be addressed.

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immunofluorescence assay (IFA) with an anti-P. falciparum CS protein monoclonal antibody (mAb), NFS2. Peptide-Stimulated Effector Cells. Immune PBMC (3 x 106) were stimulated in 24-well plates in a final volume of 2 ml of RPMI 1640 medium containing 10% (vol/vol) heatinactivated pooled human serum in the presence of peptide. Two days after initiation of the culture, human recombinant interleukin 2 (rIL-2; Cetus) was added (50 units/ml) with anti-CD3 mAb G19.4 (0.05 ,ug/ml) to stimulate T cells (19). Plates were incubated at 37°C (5% C02/95% air) for 6 days. Target Cells. Lymphoblastoid B cells from volunteers were transformed with Epstein-Barr virus (EBV) and maintained as long-term lines (20). Transfection of Target Cells. Forty-eight hours before an assay, EBV-transformed B cells (target cells) were transfected with the gene encoding CS. In a final volume of 100 pu, 10 ,ug of plasmid DNA (the 7G8 CS gene containing the entire coding sequence for the P. falciparum CS protein cloned into the EcoRI site of the expression vector pcEXV-3) (9), and 30-50 ,ug of Lipofectin reagent {1:1 (wt/wt) liposome formulation of cationic lipid N-[1-(2,3-dioleyloxy)-propyl]-N,NNtrimethylammonium chloride and dioleoyl phosphatidylethanolamine in membrane-filtered water; Bethesda Research Laboratories} were incubated at room temperature for 15 min. This mixture of DNA and Lipofectin reagent was incubated with target cells (1 x 106 cells per 60-mm Petri dish) in serum-free medium (Optimum Medium, GIBCO) for 5-10 hr at 37°C, and then fresh medium [RPMI 1640 medium with 20%o (vol/vol) fetal bovine serum] was added. Expression of CS protein was confirmed by indirect immunofluorescence assay with mAb NFS2. Peptide Labeling of Target Cells. Cells were incubated overnight with synthetic peptides in RPMI 1640 medium with 10% fetal bovine serum. CTL Assay. Effector cells were washed twice with RPMI 1640 medium/10% fetal bovine serum and added to roundbottom 96-well plates. Target cells were labeled with 51Cr for 1 hr at 37°C and washed twice with medium; after reexposure to the peptide, 5 x 103 cells were added to wells containing effector cells. After 6 hr at 37°C, 51Cr in supernatants collected by using a Skatron SCS System (Skatron, Sterling, VA) was assayed (Clinigamma-1272, LKB). The percent specific 51Cr release was: 100 x (experimental release spontaneous release)/(maximum release - spontaneous release). Maximum release was determined from supernatants of 51Cr-labeled cells lysed by 5% Triton X-100, and spontaneous release was determined from labeled cells incubated without effector cells. Depletion of CD8+ and CD4+ Effector T Cells. After 6 days of peptide stimulation, CD8+ and CD4+ T cells were depleted from effector cells by using Dynabeads (Dynal, Great Neck,

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NY) coated with anti-CD8 (M-450 CD8) and anti-CD4 (M-450 CD4) mAbs. Briefly, 1 x 107 effector cells were incubated with 2 x 108 magnetic beads coated with anti-CD4 or -CD8 mAbs for 1 hr in an ice bath, and then the beads were removed with a magnet (MPC-1). Cells were washed and used in the assay as described above. Fluorescence-activated cell sorter (EPICS Profile, Coulter) analysis with anti-CD4 and -CD8 mAbs indicated depletion of 98% of CD4+ and 99% of CD8+ T cells.

RESULTS Cross Reactivity in Mice of 3D7 and 7G8 CTL Epitope. Development of an assay required reagents to stimulate effector cells and to label target cells. We had peptides based on the sequence of the 7G8 CS protein, a recombinant vaccinia virus containing the 7G8 CS protein gene (V71), and a construct of the 7G8 CS gene for transfection of target cells. However, volunteers were immunized with the NF54 strain of P. falciparum and the 3D7 clone of the NF54 strain. The NF54 strain (17) and the 3D7 clone (16) are reported to encode identical CS protein sequences. However, the 7G8 protein sequence differs from these at several sites (Table 1). In particular, there is a single amino acid difference within the CTL epitope that had been identified for the 7G8 CS protein in B10.BR mice (9). Therefore, we studied mice to determine if this change would affect CTL recognition. Mice were immunized with V71, and spleen cells were stimulated in vitro for a week with L cells transfected with the P. falciparum CS gene (7G8) (9). L cells labeled with either peptide were lysed by these effector cells (Fig. 1); the single amino acid change did not alter CTL recognition. Antigen-Specific, Genetically Restricted, CD8+ T CellDependent CTL. In initial experiments we used V71 to stimulate effector cells and to infect EBV-transformed B cells as target cells. The results suggested CTL activity but were not definitive because there was activity against target cells infected with the control vaccinia, VSc-8. We then transiently transfected autologous EBV-transformed B cells with the 7G8 CS gene. When PBMC from volunteer 1H (Tables 2 and 3) were infected with V71 and used to stimulate his PBMC for 6 days in vitro, the effector cells lysed autologous target cells transfected with the 7G8 CS gene but did not lyse nontransfected transformed B cells or HLA-mismatched transformed MG cells (Table 2) transfected with the CS gene (Fig. 2a). Activity was antigen specific and genetically restricted. At an effector-to-target cell ratio of 60:1, specific lysis was >60%, indicating that transfection was efficient. Using vaccinia virus to stimulate effector cells required large numbers of PBMC. To conserve cells and to define the specificity of response, we used peptides to stimulate effector T cells. We next asked if the murine CTL epitope, CS-(368390) tricosapeptide, could stimulate effector cells to lyse

Table 1. Amino acid sequences of the peptides used in this study and the residues at which the NFS4/3D7 and 7G8 CS protein sequences differ Residues Clone Segment Sequence 1 151-170 7G8 P N A N P N A N P N A N P N A N P N A N 2 311-330 7G8 N A N A N N A V K N N N N E E P S D K H 3D7 S 3 321-340 7G8 N N N E E P S D K H I E Q Y L K K I K N 3D7 K E N Q 4 331-350 7G8 I E Q Y L K K I K N S I S T E W S P C S 3D7 K E N L Q 5 351-370 7G8 V T C G N G I Q V R I K P G S A N K P K 6 368-390 7G8 K P K D E L D Y E N D I E K K I C K M E K C S 7 3D7 A 8 381-400 7G8 K K I C K M E K C S S V F N V V N S S I In addition to the differences shown at residues 316, 332, 333, 336, 339, 342, and 376, sequences differ at residue 117 (G -* A), and amino acids 78-96 of the 7G8 sequence are deleted from the NF54/3D7 sequence (16, 17).

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Table 3. Outcome of challenge with sporozoites and CTL assays Sporozoite challenge Homologous Heterologous Performed Positive (3D7) (7G8) Volunteer 6 9 1H Unprotected Not done 3W Not done 11 7 Protected 2 12 4R Protected Protected 0 5 5H Protected Protected

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Effector/target ratio FIG. 1. A single amino acid change in the CS-(368-390) peptide does not alter CTL recognition in mice. B10.BR-H-2k mice were immunized by i.v. injection of 107 plaque-forming units of vaccinia virus that contained the 7G8 CS protein gene (V71). Eight weeks later, spleen cells were stimulated in culture for 6 days with a clone of L cells transfected with the 7G8 CS protein gene (9). They were then studied for cytolytic activity by using three targets: L cells pulsed with 100 ,g of 7G8 CS-(368-390) peptide (A) or of 3D7 CS-(368-390) peptide (e) per ml or L cells not exposed to peptide (o).

target cells. Peptide-stimulated effector cells lysed peptidepulsed and transiently transfected target cells (Fig. 2b). In a third experiment with volunteer 1H, we showed that the cytolytic activity was dependent on CD8+ T cells, was antigen specific, and was genetically restricted (Fig. 2c). Using PBMC from volunteer 4R (Tables 2 and 3), we showed that activity was dependent on CD8+ T cells and was antigen specific (Fig. 3) but was not eliminated by depletion of CD4+ T cells. In similar experiments with volunteer 3W (Tables 2 and 3), PBMC stimulated in vitro with CS-(368-390) lysed peptide-coated autologous target cells but not mismatched target cells or autologous target cells pulsed with a control peptide (Fig. 4a). The effector cells lysed transfected target cells, and the activity was dependent on CD8+ T cells (Fig. 4b). Thirteen weeks after volunteer 3W's last immunization and 11 weeks after challenge with live sporozoites, we studied six peptides reflecting amino acids 311-400 ofthe 7G8 P. falciparum CS sequence for their capacity to stimulate volunteer 3W's effector cells and label his target cells. Only 7G8 CS-(368-390) peptide was associated with activity (Fig. 4c). In the same experiment the CS-(368-390) peptide from the 3D7 sequence had similar activity (Fig. 4d). PBMC from volunteer 5H (Table 3) were never shown to contain CTL. The least experiments were conducted with 5H because his blood yielded low numbers of PBMC. It is important to note that CTL activity could not be demonstrated among the three responders each time an assay was conducted (Table 3). Table 2. HLA phenotypes of the four volunteers immunized with irradiated sporozoites and the individuals from whom control target cells were obtained HLAHLAHLA- HLA- HLA- HLAVolunCw B DR DRw A teer DQw * * 1 1I 35,4,11,24 3W 1,7 1,2 53,8,35 4,1,3 4R 7,w11 3,7 52,53 44,w57 6,1,28 5H 7,w12 2,7 52,53 13,w22 3,6 24,30 MG ND w6,9 1,2 2,7 23,w74 7,w72 ND ND ND CG 8,17 6,7 1,2 Asterisks indicate samples not typeable by conventional methods. ND, not done.

There has been speculation regarding the role of CTL in protective immunity after immunization with irradiated malaria sporozoites. The requirement for CD8+ T cells (4, 5), the presence of CTL after immunization with irradiated sporozoites (9-11) and recombinant vaccines (refs. 21 and 22; M. Sedegah, personal communication), and the protective capacity of CTL (10, 12) have been established in mice. However, humans had never been shown to produce CTL to any malaria or other parasite protein. We have demonstrated that three of four volunteers immunized with irradiated P. falciparum sporozoites developed T cells that lysed autologous target cells and that this activity has the characteristics of CTL; it is antigen specific, dependent on CD8+ T cells, and genetically restricted. Construction of targets for human CTL assays has often been difficult. Transfection of autologous EBV-transformed lymphoblastoid cells and phytohemagglutinin-stimulated blasts with recombinant viruses, particularly vaccinia virus, has been used (23). We were unable to acceptably standardize our assay with the CS protein-encoding vaccinia recombinant. We attempted stable transfection of EBV-transformed cells with the CS gene, using calcium phosphate precipitation, electroporation, and Lipofectin reagent but were unsuccessful. However, in two volunteers, 1H and 3W, we were consistently able to use Lipofectin reagent to transiently transfect the CS gene into EBV-transformed cells and express the CS protein on the cell surface. Although genes encoding class I HLA proteins have been similarly transfected (24), we are unaware of successful gene transfection and use of expressed CTL target cell antigens. A striking finding in malaria preerythrocytic vaccine development has been the usefulness of rodents for developing approaches to study human immunity. After demonstrating that immunization of volunteers with irradiated sporozoites elicited CTL against the CS protein (Fig. 2a), we showed that at least one human CTL epitope is included within the same 22 amino acid residues (368-390) shown to be a CTL epitope in H-2k mice (9). A shared mouse and human CTL epitope was recently described in human immunodeficiency virus 1 reverse transcriptase (23) but otherwise has not been reported. Furthermore, as in mice (Fig. 1), a single amino acid difference between the immunogen and antigen on the target cells did not affect CTL activity. Additional experiments will determine if, as suggested in Fig. 4c, this is the only CTL epitope in this region ofthe CS protein. However, NF54/3D7 sporozoites were used for immunization, and the peptides studied in Fig. 4c reflected the 7G8 sequence. Since the NF54/3D7 and 7G8 sequences vary by five amino acids between residues 331 and 350 (Table 1), use of homologous peptides may reveal CTL epitopes in this region. Studies in mice have demonstrated that T-cell proliferative and helper responses to the CS protein are often genetically restricted (25). Yet, three of the four individuals produced CTL against an epitope included within the CS-(368-390) peptide. Study of more individuals and a panel of targets may more clearly define a pattern of restriction or multiple CTL epitopes within residues 368-390. The frequency of positive donors may be due to promiscuity of the peptide for different

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FIG. 2. CTL activity in volunteer 1H. (a) Genetically restricted CTL against the CS protein. After 369 bites of irradiated infected mosquitoes during 9 months, and 2 weeks after the last exposure, PBMC were stimulated with autologous PBMC infected with the CS protein-encoding vaccinia virus construct V71. These effector cells lysed 1H targets transfected with the CS protein gene (o) but had little activity against nontransfected 1H target cells (A) or against target cells from donor MG (Table 2) transfected with the CS gene (A). (b) Antigen-specific CTL against the CS-(368-390) peptide of clone 7G8. After 462 bites of irradiated infected mosquitoes during 11 months, and 2 weeks after the last exposure, PBMC were stimulated with 25 ,.g of CS-(368-390) peptide per ml. These effector cells lysed 1H target cells transfected with the 7G8 CS gene (r) or pulsed overnight with 25 jig of CS-(368-390) peptide per ml (o) but had little activity against 1H targets pulsed overnight with 25 ,ug of CS-(151-170) peptide per ml (Table 1) (A) or 1H targets not exposed to peptide (v). (c) CD8' T-cell dependence of CTL activity against CS-(368-390) peptide. After 607 bites of irradiated infected mosquitoes during 12 months, and 2 weeks after the last exposure, PBMC were stimulated with 25 ,ug of clone 7G8 CS-(368-390) peptide per ml. The effector cells lysed 1H target cells transfected with clone 7G8 CS gene (l) but had little activity against 1H nontransfected targets (A) or against targets from donor MG cells transfected with the CS gene (A). The activity against 1H targets transfected with the CS gene was reversed by depletion of CD8' T cells (o).

HLA molecules, as has been seen for a CS peptide and class II HLA molecule (26), or to a sharing of a few HLA class I molecules among the three CTL-positive donors in the study. Donors 1H and 3W share HLA-B35 and HLA-Cw4, and donors 3W and 4R share HLA-A1. However, the studies in which CTL from donor 3W (HLA-A1) did not lyse the CG target cells (HLA-A1) (Fig. 4d) argue against the presentation of the peptide by HLA-A1 to the 3W CTL. One of our goals was to develop an assay for predicting protective immunity. Here again findings in mice predicted the findings in humans. Transfer of CTL clones against an epitope on the P. yoelii CS protein protects against sporozoite challenge; CTL against the CS protein are adequate alone to confer protection (refs. 10 and 12; W. Weiss, personal communication). However, BALB/c mice immunized with recombinant Salmonella (22) or pseudo-rabiesvirus (M. Sedegah, personal communication) and B1O.D2 mice immunized with irradiated P. yoelii sporozoites (11) produced CTL against the same epitope as the protective CTL clones, but were not protected. CTL against the CS protein may contribute to protection, but the presence of such CTL does not indicate protection. Volunteer 1H had 25

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irradiated infected mosquitoes during 7 months and 2 weeks after the last exposure, PBMC were stimulated with 25 A.g of clone 7G8 CS-(368-390) peptide per ml. Effector cells lysed tar-


) and control (*) TC. (d) CTL activity against the CS-(368-390) fragment of clone 3D7. Thirteen weeks after the last immunization, PBMC were stimulated with 50 jug of 3D7 CS-(368-390) peptide per ml. These effector cells lysed 3W target cells pulsed with the same peptide (*) but did not lyse 3W target cells not exposed to peptide (c) or CG target cells (Table 2) pulsed with the 3D7 CS-(368-390) peptide (m).

study. We thank Dr. S. Kumar of the National Institutes of Health for the CS protein plasmid DNA for transfections, for advice, and for manuscript review; Drs. J. D. Haynes and D. Gordon of the Walter Reed Army Institute of Research for support; Dr. B. Moss and Dr. W. Biddison of the National Institutes of Health for recombinant vaccinia and manuscript review, respectively; Dr. F. Ennis of the University of Massachusetts Medical Center for the CG cell line; Cetus for rIL-2; and the following scientists of the Naval Medical Research Institute: Mrs. N. Hensel and Dr. R. Hartzman for HLA serotyping and EBV transformation; Dr. C. June for the anti-CD3 mAb; Drs. M. Sedegah and K. Jackson for EBV transformation; and Dr. Y. Charoenvit for the anti-P. falciparum mAb. The staffs of the leukophoresis laboratories at the National Naval Medical Center and National Institutes of Health are thanked for support. This work was supported by Naval Medical Research Development and Command Work Unit 3M473750D808AQ133 and 3M162770A870AN121 and Office of Naval Research Contract N00014-83-C-0355. 1. Nussenzweig, R. S., Vanderberg, J. P., Most, H. & Orton, C. (1969) Nature (London) 222, 488-489. 2. Clyde, D., Most, H., McCarthy, V. C. & Vanderberg, J. P. (1973) Am. J. Med. Sci. 266, 169-177. 3. Rieckmannn, K. H., Beaudoin, R. L., Cassells, J. S. & Sell, K. W. (1979) Bu!l. W.H.O. 57, Suppl. 1, 261-265. 4. Schofield, L., Villaquiran, J., Ferreira, A., Schellekens, H., Nussenzweig, R. S. & Nussenzweig, V. (1987) Nature (London) 330, 664-666. 5. Weiss, W. R., Sedegah, M., Beaudoin, R. L., Miller, L. H. & Good, M. F. (1988) Proc. Nat!. Acad. Sci. USA 85, 573-576. 6. Hoffman, S. L., Isenberg, D., Long, G. W., Sedagah, M., Szarfman, A., Waters, L., Hollingdale, M. R., Meide, V. D., Finbloom, D. & Ballou, W. R. (1989) Science 244, 1078-1081. 7. Yoshida, N., Nussenzweig, R. S., Potocnjak, P., Nussenzweig, V. & Aikawa, M. (1980) Science 207, 71-73. 8. Miller, L. H., Howard, R. J., Carter, R., Good, M. F., Nussenzweig, V. & Nussenzweig, R. S. (1986) Science 234, 1349-1356. 9. Kumar, S., Miller, L. H., Quakyi, I. A., Keister, D. B., Houghten, R. A., Maloy, W. L., Moss, B., Berzofsky, J. A. & Good, M. F. (1988) Nature (London) 334, 258-260.

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