INFECTION AND IMMUNITY, Feb. 1990, p. 575-578
Vol. 58, No. 2
0019-9567/90/020575-04$02.00/0 Copyright C 1990, American Society for Microbiology
Identification of a T-Cell Epitope on the Circumsporozoite Protein of Plasmodium vivax F. W. GEORGE
IV,'* JUDY L.
LAW,2 KATHRYN A. RICH,'
AND
W. JOHN MARTIN'
Departments of Pathology' and Anatomy,2 University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, California 90033 Received 26 April 1989/Accepted 26 October 1989
A series of overlapping peptides, representing sequences in the vicinity of region II on the Plasmodium vivax circumsporozoite protein, was synthesized. One of the peptides (PV-23), a 20-mer containing the 6 C-terminal amino acids of region II, was found to evoke an in vitro T-celi proliferative response in spleen cells from C3Hf (H-2k12) mice immunized with the peptide. These results demonstrate that PV-23 contains a T-cell epitope. To our knowledge, this is the first report of a T-cell epitope on the circumsporozoite protein of P. vivax.
The last few years have seen a major expansion in worldwide efforts to develop an effective vaccine for human malaria. The variable results obtained with the in vivo testing of synthetic malaria vaccines containing portions of the circumsporozoite (CS)-protein repeat region (3, 5, 8, 10, 15) have intensified efforts to locate T-cell epitopes on malarial antigens. T-cell epitopes, those regions of a protein molecule which are specifically recognized by T lymphocytes, have been shown to significantly augment the antibody titers generated against the repeat region of Plasmodium falciparum CS protein (6, 13). Furthermore, a positive correlation between an in vitro proliferative response to such epitopes and resistance to malaria in individuals living in malaria-endemic regions has recently been reported (9). Although recent reports have described T-cell epitopes on the CS protein of P. falciparum (6, 7, 13), similar sequences have not yet been identified on the CS protein of Plasmodium vivax. We have screened the P. vivax CS protein for T-cell epitopes by using a murine model system. A recombinant DNA-derived CS protein of P. vivax has been shown to immunize mice of the H-2k haplotype (12). In an initial experiment, we sought to confirm that an in vitro proliferative response could be obtained with lymphoid cells derived from C3HfB/HeN (C3Hf) strain mice inoculated with P. vivax CS protein. C3Hf, a substrain of C3H/HeN, possesses a variant haplotype designated H-2k"? resulting from a mutation in the gene coding for the K molecule (14). C3Hf mice were inoculated with a highly purified, recombinant DNA-derived CS protein from the Belem strain of P. vivax, designated vivax-2 (4), encompassing amino acids (aa) 77 to 340 of the CS protein. Mice were inoculated subcutaneously at the base of the tail with 50 ,ug of CS protein or RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) emulsified in complete Freund adjuvant (CFA) and intraperitoneally with 100 jig of aqueous antigen or plain medium. On day 28, the spleens were removed and dissociated into medium. The lymphoid cells were isolated from erythrocytes by centrifugation on a Ficoll-Hypaque density gradient. The cells at the interface were washed and added at 2 x 105 cells per well to flat-bottom 96-well plates (Costar, Van Nuys, Calif.) in 0.2 ml of medium containing 10% fetal calf serum (GIBCO), 5 x 10' M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.), and gentamicin (50 jig/ml; Sigma) in the presence or absence of 100 jig of vivax-2 CS *
protein per ml. Cultures were incubated for 6 days. Proliferation was measured by adding 1.0 ,uCi of [3H]thymidine ([3H]TdR) (20 Ci/mmol; ICN Radiochemicals, Irvine, Calif.) to each microdilution plate well for the final 16 h of culture. At the end of the incubation period, the cells were harvested onto glass fiber filters and the levels of [3H]TdR incorporation were determined with a scintillation counter. A response to CS protein was observed in lymphoid cells from the CS protein-inoculated mouse (mean of triplicate wells the standard error of the mean, 28,582 322 cpm) but not in cells from the mouse receiving CFA alone (2,064 147 cpm). The responses of lymphoid cells cultured in the absence of CS protein were 2,706 490 and 1,629 298 cpm, respectively. Additional experiments (described below) with either spleen or lymph node cells from mice immunized in the same manner have confirmed this observation. To investigate the location of potential T-cell epitopes on the P. vivax CS protein, a series of overlapping synthetic peptides of 15 to 20 amino acids was constructed. Rather than synthesize peptides spanning the entire CS protein, we focused on sequences in the vicinity of region II, an area shown to contain several T-cell epitopes in the CS protein of P. falciparum (7). Peptides were synthesized by modified Merrifield chemistry on a model 430A automated peptide synthesizer (Applied Biosystems, Inc., Foster City, Calif.). They were chromatographed on Sephadex G-10 with 30% acetic acid, lyophilized, and stored at -20°C. Peptide solutions were prepared just before their use for inoculations and proliferative assays. The positions of these peptides on the P. vivax CS protein are diagrammed in Fig. 1. The peptide designations, positions, and sequences are as follows: PV-20 (aa 293 to 312), PNAKSVKEYLDKLETTVGTE; PV-21 (aa 298 to 317), VKEYLDKLETTVGTEWTPCS; PV-22 (aa 312 to 329), EWTPCSVTCGVGVRVRRR; PV-23 (aa 318 to 337), VTCGVGVRVRSRVNAANKKP; and PV-24 (aa 328 to 347), SRVNAANKKPEDLTLDDLET. To assess their immunogenicity, the peptides, in addition to CS protein, were used to immunize C3Hf mice as described above. After 17 to 36 days, the spleens or lymph nodes were removed and ±
±
±
±
dissociated into medium. Cultures were established in the presence or absence of 100 ,ug of either CS protein or peptide PV-22, PV-23, or PV-24 per ml, and proliferation was determined as described above. Pooled results from four experiments are summarized in Table 1. Lymphoid cells from uninoculated mice and from mice receiving only me-
Corresponding author. 575
576
INFECT. IMMUN.
NOTES
+NH 3
r-Region
295 (A)
r-Central Repeats-I
I-I
(LET)
325 310 r-Region II(S)
340
UVRVRRRVNAANKKPEDLTLDDLET -PNEKSVKEYLDKVRATVGTEWTPCSVTCGVGI I
-PV-20-
PV-21 PV-2 2 i
PV-2 3
I -PV-24FIG. 1. The location and amino acid sequence of synthetic peptides on P. vivax CS protein. The sequence presented is based on that published by Arnot et al. (1) and corresponds to the sequence for the vivax-2 CS protein (Chiron Corp.). Vivax-2 terminates at amino acid 340. Letters in parentheses denote amino acids which are different for the sequences of the designated synthetic peptides, whose sequences were based on findings published by Arnot et al. (2).
dium plus CFA were not stimulated by either CS protein or any of the peptides. In contrast, cells from the group of mice immunized with CS protein produced a significant proliferative response to CS protein (P < 0.05). The group of mice inoculated with PV-23 responded significantly to PV-23 (P < 0.006). Although CS protein-immunized mice as a group did not show a significant response to PV-23, the responses of some individual CS protein-immunized mice (two of four mice tested) were significant (t test for comparison of sample means, P < 0.01). Likewise, some PV-23-immunized mice gave individually significant responses to CS protein (two of five mice tested, P < 0.01). Mice inoculated with PV-24 did not respond to PV-24; one of four mice in this group did, however, give a significant response to PV-23 (P < 0.05). PV-22-inoculated mice produced no statistically significant responses to CS protein, PV-23, and PV-24. In other experiments, neither PV-20 nor PV-21 was found to immunize mice or to evoke a response in lymphoid cells from CS protein-immunized mice (data not shown). In some experiments, spleen cell responses were also analyzed by an alternative assay which detects cell proliferation and activation by monitoring the cleavage of the tetrazolium salt substrate MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] by mitochondrial enzymes. The assay used was a modification of that described by Mossman (11). In this assay, the amount of MTT solution converted from yellow to purple is proportional to the level of mitochondrial enzymes in a culture. Enzyme levels rise as a result of cellular activation or an increase in cell number. Mice were immunized, and in vitro cultures were initiated as detailed above. Two hours prior to the end of the 6-day incubation period, 0.1 ml of the medium from each well was removed and replaced with 0.1 ml of MTT solution (1 mg/ml TABLE 1. Proliferative Immunizing antigen
None CFA only CFA + CS protein CFA + PV-22 CFA + PV-23 CFA + PV-24
responses
in medium). After 2 h at 37°C, 0.1 ml of supernatant was removed from each well and 0.2 ml of dimethyl sulfoxide0.06 M HCl was added to each well to dissolve the blue formazan crystals which form as a product of MTT cleavage. The dimethyl sulfoxide was acidified to convert the phenol red in the medium to a yellow form not detected at the wavelength used to determine the MTT color change. The A540 in each well was then measured on an enzyme-linked immunosorbent assay plate reader (Biotek EL310). The results of the spleen cell assays as measured by this technique (Table 2) supported the findings obtained by [3H]TdR incorporation. For instance, the response of PV-23-inoculated mice to both PV-23 and CS protein was significant (P < 0.035). The responses of CS protein-immunized mice to CS protein and PV-23 were not significant by t test, but these values were more than three standard deviations above the mean response of mice inoculated with CFA alone. The responses of PV-22- and PV-24-inoculated mice to PV-23 were also more than three standard deviations above the mean response of CFA-treated mice under the same conditions. The data presented here demonstrate that the region of the P. vivax CS protein encompassed by peptide PV-23 is capable of immunizing mice of the H-2km2 haplotype and thus provide strong evidence that this sequence contains a T-cell epitope. To our knowledge, this is the first T-cell epitope described for the P. vivax CS protein. The crossreactivity in the responses to lymphoid cells from mice immunized with CS protein and from those receiving PV-23, as measured by the MTT assay (Table 2), suggests that this is a biologically relevant epitope recognizable by T cells after degradation of the CS protein by antigen-presenting cells. The positive responses of some PV-22 (Table 2)- and
of lymphoid cells from immunized mice to P. vivax synthetic peptides Mean of delta cpma + SEM in assays with:
CS protein
312 31 6,866 706 1,044 581
±
± ±
156 552 2,231 310 486 446
PV-22
-263 -543 -103 309 -349 -309
±
± ± ±
± ±
PV-23
180 161 108 146 41 255
-6 548 717 1,252 5,579 810
± ±
± ±
47 286 407 391 1,038 702
PV-24
-154 -115 135 -13 979 84
±
±
± ± ± ±
225 249 167 65 853 296
a Response of each mouse in a group. Values were calculated for each animal by taking the median value obtained from replicate (two to four) lymphoid cell cultures in the presence of antigen and subtracting from this the median value obtained for cultures without antigen. Each immunization group contained three to five mice. Values in boldface were found to be statistically significant compared with medium control values in the same group by using a two-tailed paired t test (P < 0.05 and P < 0.006, respectively). These values were also more than three standard deviations above the mean of responses to the same antigen by mice inoculated with CFA alone.
VOL. 58, 1990
NOTES
TABLE 2. MTT responses of lymphoid cells from immunized mice to P. vivax synthetic peptides Immunizing antigen
None CFA only CFA + CS protein CFA + PV-22 CFA + PV-23 CFA + PV-24
Mean delta A5401 (103) ± SEM in assays with: CS protein PV-22 PV-23 PV-24
7 0 134 -1 20 9
± 1 ± 3
+ 58b ± 1
±6 ± 5
-1 ± 2 4±2 -2 ± 4 -2 ± 4b -9 ± 2 23 ± 9 3 ± 7 23 ± 14b -11 ± 5 92 ± 25 -22 ± 6 31 ± job
-2 ± -2 ± 3± -4 ± 1± -3 ±
4 4 2 4 3 10
a MTT conversion. The response of each mouse in a group was determined by measuring color development due to MTT conversion in each lymphoid cell culture. Values were calculated for each animal by taking the median A540 obtained for replicate (two to four) cultures in the presence of antigen and subtracting from this the median value obtained for cultures without antigen. Each immunization group contained two to four mice. Values in boldface print were found to be statistically significant compared with the medium control values in the same group by using a two-tailed paired t test (P < 0.035) and were more than three standard deviations above the responses to the same antigen by mice inoculated with CFA alone. b Not significant by t test because of variance caused by the disparate response of one animal within each of the immunization groups. The mean values of these responses were, however, more than three standard deviations above the mean responses of animals inoculated with CFA alone.
PV-24 (Tables 1 and 2)-immunized mice to PV-23 indicate that the 10-amino-acid overlap regions between these peptides (aa 318 to 329 and 328 to 337) may contain portions of the epitope. The data also indicate that PV-23 was more effective than either PV-22 or PV-24 at eliciting an in vitro proliferative response and thus suggest that PV-22 and PV-24 may lack the amino acids within the epitope which are important for an optimal T-ceil response. Alternatively, this effect may be related to the ability of PV-23 to adhere to lymphoid cells, to the inability of PV-24 to do so (K. A. Rich et al., manuscript in preparation), and to our findings which suggest that the N-terminal portion of PV-22 may be immunosuppressive (data not shown). Further experimentation, utilizing a series of closely nested synthetic peptides, is necessary to identify the specific sequence required for maximal T-cell recognition and to determine any immunomodulatory effects of adjoining regions. The close correlation between the proliferation results presented in Table 1 and the data generated with the MTT assay (Table 2) is encouraging for two reasons. Firstly, since the MTT assay detects cellular responses by measuring parameters different from those analyzed in the [3H]TdR incorporation assay, the findings obtained with the MTT assays reinforce the [3H]TdR incorporation results. Secondly, these results suggest that the MTT assay could be utilized as a substitute for [3H]TdR in detecting lymphoid cell responses in malaria antigen assays when the use of a nonisotopic assay is preferable. Thus, the MTT assay could prove useful for conducting cellular-proliferation assays on malarial vaccine subjects in the field, where access to large equipment, such as scintillation counters, may be limited. That PV-23 may describe an important T-cell epitope is suggested by a recent report by Nardin et al. (12). They found that the antibody response to vivax-1 recombinant CS protein (aa 77 to 315) is haplotype restricted. In their discussion, however, they state that the response to vivax-2 is not restricted. While no data were presented in support of this statement, it is nevertheless interesting that 20 of the 25 C-terminal amino acids which make up the sequence difference between vivax-1 and vivax-2 are contained within PV-23. Thus, it is tempting to speculate that the epitope
577
contained within this peptide may play a role in minimizing Ir gene restriction. It is interesting that the epitope we have identified is similar in location to one of the human T-cell epitopes on the P. falciparum CS protein which Good et al. have described (7). This finding indicates that the area around region II may well contain T-cell epitopes in other malarial species. It also suggests that human T cells may recognize the epitope on PV-23. It will be important to determine directly the responsiveness of human lymphoid cells to the T-cell epitope contained within PV-23. Accordingly, studies are under way in our laboratory to test the in vitro response of human T cells, derived from donors previously infected with P. vivax malaria, to the various synthetic peptides. We thank Ian Bathurst of Chiron Corp. (Emeryville, Calif.) for generously providing the vivax-2 recombinant CS protein. This work was supported by contract DPE-0453-C-00-6060-00 from the U.S. Agency for International Development. LITERATURE CITED 1. Arnot, D. E., J. W. Barnwell, and M. J. Stewart. 1988. Does biased gene conversion influence polymorphism in CS gene of Plasmodium vivax? Proc. Natl. Acad. Sci. USA 85:8102-8106. 2. Arnot, D. E., J. W. Barnwell, J. P. Tam, V. Nussenzweig, R. S. Nussenzweig, and V. Enea. 1985. Circumsporozoite protein of Plasmodium vivax: cloning of the immunodominant epitope.
Science 230:815-818. 3. Ballou, W. R., S. L. Hoffman, J. A. Sherwood, M. R. Hollingdale, F. A. Neva, W. T. Hockmeyer, D. M. Gordon, I. Schneider, R. A. Wirtz, J. F. Young, G. F. Wasserman, P. Reeve, C. L. Diggs, and J. D. Chulay. 1987. Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet i:1277-1281. 4. Barr, P. J., J. L. Gibson, V. Enea, D. E. Arnot, M. R. Hollingdale, and V. Nussenzweig. 1987. Expression in yeast of a Plasmodium vivax antigen of potential use in a human malaria vaccine. J. Exp. Med. 165:1160-1171. 5. Egan, J. E., J. L. Weber, W. R. Ballou, M. R. Hollingdale, W. R. Majarian, D. M. Gordon, W. L. Maloy, S. L. Hoffman, R. A. Wirtz, I. Schneider, G. R. Woollett, J. F. Young, and W. T. Hockmeyer. 1987. Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development. Science 236:453-456. 6. Good, M. F., W. L. Maloy, M. N. Lunde, H. Margalit, J. L. Cornett, G. L. Smith, B. Moss, L. H. Miller, and J. A. Berzofsky. 1987. Construction of a synthetic immunogen: use of new T helper epitope on malaria circumsporozoite protein. Science 235:1059-1062. 7. Good, M. F., D. Pombo, I. A. Quakyi, E. M. Riley, R. A. Houghten, A. Menon, D. W. Ailing, J. A. Berzofsky, and L. H. Miller. 1988. Human T cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T cell domains map to the polymorphic regions of the molecule. Proc. Natl. Acad. Sci. USA 85:1199-1203. 8. Herrington, D. A., D. F. Clyde, G. Lonsonsky, M. Curtesia, J. R. Murphy, J. Davis, S. Baqar, A. M. Felix, E. P. Heimer, D. Gillesson, E. Nardin, R. S. Nussenzweig, V. Nussenzweig, M. R. Hollingdale, and M. M. Levine. 1987. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against P. falciparum sporozoites. Nature (London) 328:257-259. 9. Hoffman, S. L., C. N. Oster, C. Mason, J. C. Beier, J. A. Sherwood, W. R. Ballou, M. Mugambi, and J. D. Chulay. 1989. Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria. J. Immunol. 142:1299-1303. 10. Lal, A. A., V. F. de la Cruz, M. F. Good, W. R. Weiss, M. Lunde, W. L. Maloy, J. A. Welsh, and T. F. McCutchan. 1987. In vivo testing of subunit vaccines against malaria sporozoites using a rodent system. Proc. Natl. Acad. Sci. USA 84:86478651. 11. Mossman, T. 1983. Rapid colorimetric assay for cellular growth
578
NOTES
and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. 12. Nardin, E. H., P. J. Barr, E. Heimer, and H. M. Etlinger. 1988. Genetic restriction of murine humoral response to a recombinant Plasmodium vivax circumsporozoite protein. Eur. J. Immunol. 18:1119-1122. 13. Sinigaglia, F., M. Guttinger, J. Kilgus, D. M. Doran, H. Matile, H. Etlinger, A. Trzeciak, D. Gillessen, and J. R. L. Pink. 1988. A malaria T cell epitope recognized in association with most
INFECT. IMMUN. mouse and human MHC class II molecules. Nature (London) 336:778-780. 14. Vogel, J. M., H. C. Davis, D. M. McKinney, M. McMillan, W. J. Martin, and R. S. Goodenow. 1988. Molecular characterization of the C3HfB/HeN H-2Kkm2 mutation. Implications for the molecular basis of alloreactivity. J. Exp. Med. 168:1781-1800. 15. Zavala, F., J. P. Tam, P. J. Barr, V. Ley, R. S. Nussenzweig, and V. Nussenzweig. 1987. Synthetic peptide vaccine confers protection against murine malaria. J. Exp. Med. 166:1591-1596.
Vol. 58, No. 2
0019-9567/90/020575-04$02.00/0 Copyright C 1990, American Society for Microbiology
Identification of a T-Cell Epitope on the Circumsporozoite Protein of Plasmodium vivax F. W. GEORGE
IV,'* JUDY L.
LAW,2 KATHRYN A. RICH,'
AND
W. JOHN MARTIN'
Departments of Pathology' and Anatomy,2 University of Southern California School of Medicine, 2011 Zonal Avenue, Los Angeles, California 90033 Received 26 April 1989/Accepted 26 October 1989
A series of overlapping peptides, representing sequences in the vicinity of region II on the Plasmodium vivax circumsporozoite protein, was synthesized. One of the peptides (PV-23), a 20-mer containing the 6 C-terminal amino acids of region II, was found to evoke an in vitro T-celi proliferative response in spleen cells from C3Hf (H-2k12) mice immunized with the peptide. These results demonstrate that PV-23 contains a T-cell epitope. To our knowledge, this is the first report of a T-cell epitope on the circumsporozoite protein of P. vivax.
The last few years have seen a major expansion in worldwide efforts to develop an effective vaccine for human malaria. The variable results obtained with the in vivo testing of synthetic malaria vaccines containing portions of the circumsporozoite (CS)-protein repeat region (3, 5, 8, 10, 15) have intensified efforts to locate T-cell epitopes on malarial antigens. T-cell epitopes, those regions of a protein molecule which are specifically recognized by T lymphocytes, have been shown to significantly augment the antibody titers generated against the repeat region of Plasmodium falciparum CS protein (6, 13). Furthermore, a positive correlation between an in vitro proliferative response to such epitopes and resistance to malaria in individuals living in malaria-endemic regions has recently been reported (9). Although recent reports have described T-cell epitopes on the CS protein of P. falciparum (6, 7, 13), similar sequences have not yet been identified on the CS protein of Plasmodium vivax. We have screened the P. vivax CS protein for T-cell epitopes by using a murine model system. A recombinant DNA-derived CS protein of P. vivax has been shown to immunize mice of the H-2k haplotype (12). In an initial experiment, we sought to confirm that an in vitro proliferative response could be obtained with lymphoid cells derived from C3HfB/HeN (C3Hf) strain mice inoculated with P. vivax CS protein. C3Hf, a substrain of C3H/HeN, possesses a variant haplotype designated H-2k"? resulting from a mutation in the gene coding for the K molecule (14). C3Hf mice were inoculated with a highly purified, recombinant DNA-derived CS protein from the Belem strain of P. vivax, designated vivax-2 (4), encompassing amino acids (aa) 77 to 340 of the CS protein. Mice were inoculated subcutaneously at the base of the tail with 50 ,ug of CS protein or RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) emulsified in complete Freund adjuvant (CFA) and intraperitoneally with 100 jig of aqueous antigen or plain medium. On day 28, the spleens were removed and dissociated into medium. The lymphoid cells were isolated from erythrocytes by centrifugation on a Ficoll-Hypaque density gradient. The cells at the interface were washed and added at 2 x 105 cells per well to flat-bottom 96-well plates (Costar, Van Nuys, Calif.) in 0.2 ml of medium containing 10% fetal calf serum (GIBCO), 5 x 10' M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.), and gentamicin (50 jig/ml; Sigma) in the presence or absence of 100 jig of vivax-2 CS *
protein per ml. Cultures were incubated for 6 days. Proliferation was measured by adding 1.0 ,uCi of [3H]thymidine ([3H]TdR) (20 Ci/mmol; ICN Radiochemicals, Irvine, Calif.) to each microdilution plate well for the final 16 h of culture. At the end of the incubation period, the cells were harvested onto glass fiber filters and the levels of [3H]TdR incorporation were determined with a scintillation counter. A response to CS protein was observed in lymphoid cells from the CS protein-inoculated mouse (mean of triplicate wells the standard error of the mean, 28,582 322 cpm) but not in cells from the mouse receiving CFA alone (2,064 147 cpm). The responses of lymphoid cells cultured in the absence of CS protein were 2,706 490 and 1,629 298 cpm, respectively. Additional experiments (described below) with either spleen or lymph node cells from mice immunized in the same manner have confirmed this observation. To investigate the location of potential T-cell epitopes on the P. vivax CS protein, a series of overlapping synthetic peptides of 15 to 20 amino acids was constructed. Rather than synthesize peptides spanning the entire CS protein, we focused on sequences in the vicinity of region II, an area shown to contain several T-cell epitopes in the CS protein of P. falciparum (7). Peptides were synthesized by modified Merrifield chemistry on a model 430A automated peptide synthesizer (Applied Biosystems, Inc., Foster City, Calif.). They were chromatographed on Sephadex G-10 with 30% acetic acid, lyophilized, and stored at -20°C. Peptide solutions were prepared just before their use for inoculations and proliferative assays. The positions of these peptides on the P. vivax CS protein are diagrammed in Fig. 1. The peptide designations, positions, and sequences are as follows: PV-20 (aa 293 to 312), PNAKSVKEYLDKLETTVGTE; PV-21 (aa 298 to 317), VKEYLDKLETTVGTEWTPCS; PV-22 (aa 312 to 329), EWTPCSVTCGVGVRVRRR; PV-23 (aa 318 to 337), VTCGVGVRVRSRVNAANKKP; and PV-24 (aa 328 to 347), SRVNAANKKPEDLTLDDLET. To assess their immunogenicity, the peptides, in addition to CS protein, were used to immunize C3Hf mice as described above. After 17 to 36 days, the spleens or lymph nodes were removed and ±
±
±
±
dissociated into medium. Cultures were established in the presence or absence of 100 ,ug of either CS protein or peptide PV-22, PV-23, or PV-24 per ml, and proliferation was determined as described above. Pooled results from four experiments are summarized in Table 1. Lymphoid cells from uninoculated mice and from mice receiving only me-
Corresponding author. 575
576
INFECT. IMMUN.
NOTES
+NH 3
r-Region
295 (A)
r-Central Repeats-I
I-I
(LET)
325 310 r-Region II(S)
340
UVRVRRRVNAANKKPEDLTLDDLET -PNEKSVKEYLDKVRATVGTEWTPCSVTCGVGI I
-PV-20-
PV-21 PV-2 2 i
PV-2 3
I -PV-24FIG. 1. The location and amino acid sequence of synthetic peptides on P. vivax CS protein. The sequence presented is based on that published by Arnot et al. (1) and corresponds to the sequence for the vivax-2 CS protein (Chiron Corp.). Vivax-2 terminates at amino acid 340. Letters in parentheses denote amino acids which are different for the sequences of the designated synthetic peptides, whose sequences were based on findings published by Arnot et al. (2).
dium plus CFA were not stimulated by either CS protein or any of the peptides. In contrast, cells from the group of mice immunized with CS protein produced a significant proliferative response to CS protein (P < 0.05). The group of mice inoculated with PV-23 responded significantly to PV-23 (P < 0.006). Although CS protein-immunized mice as a group did not show a significant response to PV-23, the responses of some individual CS protein-immunized mice (two of four mice tested) were significant (t test for comparison of sample means, P < 0.01). Likewise, some PV-23-immunized mice gave individually significant responses to CS protein (two of five mice tested, P < 0.01). Mice inoculated with PV-24 did not respond to PV-24; one of four mice in this group did, however, give a significant response to PV-23 (P < 0.05). PV-22-inoculated mice produced no statistically significant responses to CS protein, PV-23, and PV-24. In other experiments, neither PV-20 nor PV-21 was found to immunize mice or to evoke a response in lymphoid cells from CS protein-immunized mice (data not shown). In some experiments, spleen cell responses were also analyzed by an alternative assay which detects cell proliferation and activation by monitoring the cleavage of the tetrazolium salt substrate MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] by mitochondrial enzymes. The assay used was a modification of that described by Mossman (11). In this assay, the amount of MTT solution converted from yellow to purple is proportional to the level of mitochondrial enzymes in a culture. Enzyme levels rise as a result of cellular activation or an increase in cell number. Mice were immunized, and in vitro cultures were initiated as detailed above. Two hours prior to the end of the 6-day incubation period, 0.1 ml of the medium from each well was removed and replaced with 0.1 ml of MTT solution (1 mg/ml TABLE 1. Proliferative Immunizing antigen
None CFA only CFA + CS protein CFA + PV-22 CFA + PV-23 CFA + PV-24
responses
in medium). After 2 h at 37°C, 0.1 ml of supernatant was removed from each well and 0.2 ml of dimethyl sulfoxide0.06 M HCl was added to each well to dissolve the blue formazan crystals which form as a product of MTT cleavage. The dimethyl sulfoxide was acidified to convert the phenol red in the medium to a yellow form not detected at the wavelength used to determine the MTT color change. The A540 in each well was then measured on an enzyme-linked immunosorbent assay plate reader (Biotek EL310). The results of the spleen cell assays as measured by this technique (Table 2) supported the findings obtained by [3H]TdR incorporation. For instance, the response of PV-23-inoculated mice to both PV-23 and CS protein was significant (P < 0.035). The responses of CS protein-immunized mice to CS protein and PV-23 were not significant by t test, but these values were more than three standard deviations above the mean response of mice inoculated with CFA alone. The responses of PV-22- and PV-24-inoculated mice to PV-23 were also more than three standard deviations above the mean response of CFA-treated mice under the same conditions. The data presented here demonstrate that the region of the P. vivax CS protein encompassed by peptide PV-23 is capable of immunizing mice of the H-2km2 haplotype and thus provide strong evidence that this sequence contains a T-cell epitope. To our knowledge, this is the first T-cell epitope described for the P. vivax CS protein. The crossreactivity in the responses to lymphoid cells from mice immunized with CS protein and from those receiving PV-23, as measured by the MTT assay (Table 2), suggests that this is a biologically relevant epitope recognizable by T cells after degradation of the CS protein by antigen-presenting cells. The positive responses of some PV-22 (Table 2)- and
of lymphoid cells from immunized mice to P. vivax synthetic peptides Mean of delta cpma + SEM in assays with:
CS protein
312 31 6,866 706 1,044 581
±
± ±
156 552 2,231 310 486 446
PV-22
-263 -543 -103 309 -349 -309
±
± ± ±
± ±
PV-23
180 161 108 146 41 255
-6 548 717 1,252 5,579 810
± ±
± ±
47 286 407 391 1,038 702
PV-24
-154 -115 135 -13 979 84
±
±
± ± ± ±
225 249 167 65 853 296
a Response of each mouse in a group. Values were calculated for each animal by taking the median value obtained from replicate (two to four) lymphoid cell cultures in the presence of antigen and subtracting from this the median value obtained for cultures without antigen. Each immunization group contained three to five mice. Values in boldface were found to be statistically significant compared with medium control values in the same group by using a two-tailed paired t test (P < 0.05 and P < 0.006, respectively). These values were also more than three standard deviations above the mean of responses to the same antigen by mice inoculated with CFA alone.
VOL. 58, 1990
NOTES
TABLE 2. MTT responses of lymphoid cells from immunized mice to P. vivax synthetic peptides Immunizing antigen
None CFA only CFA + CS protein CFA + PV-22 CFA + PV-23 CFA + PV-24
Mean delta A5401 (103) ± SEM in assays with: CS protein PV-22 PV-23 PV-24
7 0 134 -1 20 9
± 1 ± 3
+ 58b ± 1
±6 ± 5
-1 ± 2 4±2 -2 ± 4 -2 ± 4b -9 ± 2 23 ± 9 3 ± 7 23 ± 14b -11 ± 5 92 ± 25 -22 ± 6 31 ± job
-2 ± -2 ± 3± -4 ± 1± -3 ±
4 4 2 4 3 10
a MTT conversion. The response of each mouse in a group was determined by measuring color development due to MTT conversion in each lymphoid cell culture. Values were calculated for each animal by taking the median A540 obtained for replicate (two to four) cultures in the presence of antigen and subtracting from this the median value obtained for cultures without antigen. Each immunization group contained two to four mice. Values in boldface print were found to be statistically significant compared with the medium control values in the same group by using a two-tailed paired t test (P < 0.035) and were more than three standard deviations above the responses to the same antigen by mice inoculated with CFA alone. b Not significant by t test because of variance caused by the disparate response of one animal within each of the immunization groups. The mean values of these responses were, however, more than three standard deviations above the mean responses of animals inoculated with CFA alone.
PV-24 (Tables 1 and 2)-immunized mice to PV-23 indicate that the 10-amino-acid overlap regions between these peptides (aa 318 to 329 and 328 to 337) may contain portions of the epitope. The data also indicate that PV-23 was more effective than either PV-22 or PV-24 at eliciting an in vitro proliferative response and thus suggest that PV-22 and PV-24 may lack the amino acids within the epitope which are important for an optimal T-ceil response. Alternatively, this effect may be related to the ability of PV-23 to adhere to lymphoid cells, to the inability of PV-24 to do so (K. A. Rich et al., manuscript in preparation), and to our findings which suggest that the N-terminal portion of PV-22 may be immunosuppressive (data not shown). Further experimentation, utilizing a series of closely nested synthetic peptides, is necessary to identify the specific sequence required for maximal T-cell recognition and to determine any immunomodulatory effects of adjoining regions. The close correlation between the proliferation results presented in Table 1 and the data generated with the MTT assay (Table 2) is encouraging for two reasons. Firstly, since the MTT assay detects cellular responses by measuring parameters different from those analyzed in the [3H]TdR incorporation assay, the findings obtained with the MTT assays reinforce the [3H]TdR incorporation results. Secondly, these results suggest that the MTT assay could be utilized as a substitute for [3H]TdR in detecting lymphoid cell responses in malaria antigen assays when the use of a nonisotopic assay is preferable. Thus, the MTT assay could prove useful for conducting cellular-proliferation assays on malarial vaccine subjects in the field, where access to large equipment, such as scintillation counters, may be limited. That PV-23 may describe an important T-cell epitope is suggested by a recent report by Nardin et al. (12). They found that the antibody response to vivax-1 recombinant CS protein (aa 77 to 315) is haplotype restricted. In their discussion, however, they state that the response to vivax-2 is not restricted. While no data were presented in support of this statement, it is nevertheless interesting that 20 of the 25 C-terminal amino acids which make up the sequence difference between vivax-1 and vivax-2 are contained within PV-23. Thus, it is tempting to speculate that the epitope
577
contained within this peptide may play a role in minimizing Ir gene restriction. It is interesting that the epitope we have identified is similar in location to one of the human T-cell epitopes on the P. falciparum CS protein which Good et al. have described (7). This finding indicates that the area around region II may well contain T-cell epitopes in other malarial species. It also suggests that human T cells may recognize the epitope on PV-23. It will be important to determine directly the responsiveness of human lymphoid cells to the T-cell epitope contained within PV-23. Accordingly, studies are under way in our laboratory to test the in vitro response of human T cells, derived from donors previously infected with P. vivax malaria, to the various synthetic peptides. We thank Ian Bathurst of Chiron Corp. (Emeryville, Calif.) for generously providing the vivax-2 recombinant CS protein. This work was supported by contract DPE-0453-C-00-6060-00 from the U.S. Agency for International Development. LITERATURE CITED 1. Arnot, D. E., J. W. Barnwell, and M. J. Stewart. 1988. Does biased gene conversion influence polymorphism in CS gene of Plasmodium vivax? Proc. Natl. Acad. Sci. USA 85:8102-8106. 2. Arnot, D. E., J. W. Barnwell, J. P. Tam, V. Nussenzweig, R. S. Nussenzweig, and V. Enea. 1985. Circumsporozoite protein of Plasmodium vivax: cloning of the immunodominant epitope.
Science 230:815-818. 3. Ballou, W. R., S. L. Hoffman, J. A. Sherwood, M. R. Hollingdale, F. A. Neva, W. T. Hockmeyer, D. M. Gordon, I. Schneider, R. A. Wirtz, J. F. Young, G. F. Wasserman, P. Reeve, C. L. Diggs, and J. D. Chulay. 1987. Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet i:1277-1281. 4. Barr, P. J., J. L. Gibson, V. Enea, D. E. Arnot, M. R. Hollingdale, and V. Nussenzweig. 1987. Expression in yeast of a Plasmodium vivax antigen of potential use in a human malaria vaccine. J. Exp. Med. 165:1160-1171. 5. Egan, J. E., J. L. Weber, W. R. Ballou, M. R. Hollingdale, W. R. Majarian, D. M. Gordon, W. L. Maloy, S. L. Hoffman, R. A. Wirtz, I. Schneider, G. R. Woollett, J. F. Young, and W. T. Hockmeyer. 1987. Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development. Science 236:453-456. 6. Good, M. F., W. L. Maloy, M. N. Lunde, H. Margalit, J. L. Cornett, G. L. Smith, B. Moss, L. H. Miller, and J. A. Berzofsky. 1987. Construction of a synthetic immunogen: use of new T helper epitope on malaria circumsporozoite protein. Science 235:1059-1062. 7. Good, M. F., D. Pombo, I. A. Quakyi, E. M. Riley, R. A. Houghten, A. Menon, D. W. Ailing, J. A. Berzofsky, and L. H. Miller. 1988. Human T cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T cell domains map to the polymorphic regions of the molecule. Proc. Natl. Acad. Sci. USA 85:1199-1203. 8. Herrington, D. A., D. F. Clyde, G. Lonsonsky, M. Curtesia, J. R. Murphy, J. Davis, S. Baqar, A. M. Felix, E. P. Heimer, D. Gillesson, E. Nardin, R. S. Nussenzweig, V. Nussenzweig, M. R. Hollingdale, and M. M. Levine. 1987. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against P. falciparum sporozoites. Nature (London) 328:257-259. 9. Hoffman, S. L., C. N. Oster, C. Mason, J. C. Beier, J. A. Sherwood, W. R. Ballou, M. Mugambi, and J. D. Chulay. 1989. Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria. J. Immunol. 142:1299-1303. 10. Lal, A. A., V. F. de la Cruz, M. F. Good, W. R. Weiss, M. Lunde, W. L. Maloy, J. A. Welsh, and T. F. McCutchan. 1987. In vivo testing of subunit vaccines against malaria sporozoites using a rodent system. Proc. Natl. Acad. Sci. USA 84:86478651. 11. Mossman, T. 1983. Rapid colorimetric assay for cellular growth
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and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. 12. Nardin, E. H., P. J. Barr, E. Heimer, and H. M. Etlinger. 1988. Genetic restriction of murine humoral response to a recombinant Plasmodium vivax circumsporozoite protein. Eur. J. Immunol. 18:1119-1122. 13. Sinigaglia, F., M. Guttinger, J. Kilgus, D. M. Doran, H. Matile, H. Etlinger, A. Trzeciak, D. Gillessen, and J. R. L. Pink. 1988. A malaria T cell epitope recognized in association with most
INFECT. IMMUN. mouse and human MHC class II molecules. Nature (London) 336:778-780. 14. Vogel, J. M., H. C. Davis, D. M. McKinney, M. McMillan, W. J. Martin, and R. S. Goodenow. 1988. Molecular characterization of the C3HfB/HeN H-2Kkm2 mutation. Implications for the molecular basis of alloreactivity. J. Exp. Med. 168:1781-1800. 15. Zavala, F., J. P. Tam, P. J. Barr, V. Ley, R. S. Nussenzweig, and V. Nussenzweig. 1987. Synthetic peptide vaccine confers protection against murine malaria. J. Exp. Med. 166:1591-1596.
